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" micrornas mirnas have been reported to have important regulatory roles in the progression of several types of cancer including cervical cancer cc however the biological roles and regulatory mechanisms of mirnas in cc remain to be fully elucidated the aim of the present study was to examine the functions of mirnas in cc and the possible mechanisms using a microarray it was identified that mirna15a5p mir15a5p was one of the most downregulated mirnas in cc tissues compared with adjacent noncancerous tissues the low expression of mir15a5p was observed in cc tumor tissues with distant metastasis and in cc cell lines in addition the effects of mir15a5p upregulation on cell viability apoptosis invasion and migration of cc cells were investigated using cck‘ flow cytometry transwell and wound healing assays respectively it was demonstrated that upregulation of mir‘15a‘5p significantly suppressed the viability migration and invasion and promoted the apoptosis of siha and c33a cells furthermore yesassociated protein yap1 a well‘known oncogene was confirmed to be directly targeted by mir15a5p and was found to be negatively regulated by mir15a5p further correlation analysis indicated that mir15a5p expression was negatively correlated with yap1 expression in cc tissues notably overexpression of yap1 abrogated the tumor suppressive effects of mir15a5p in cc cells taken together these present findings indicated that the mir15a5pyap1 axis may provide a novel strategy for the clinical treatment of cccorrespondence to professor xu chen department of obstetrics and gynaecology huashan hospital north fudan university jingpohu road baoshan shanghai pr chinaemail xuchenccx163comcontributed equallykey words cervical cancer microrna15a5p cell viability migration invasion yesassociated protein introductioncervical cancer cc is a type of malignant tumor commonly presenting in women in cc cases are diagnosed each year and it accounts for of all female cancerassociated mortalities each year worldwide despite advances in the therapeutic strategies for cc including targeted therapies and immunotherapy the prognosis of cc remains poor due to the abnormal growth of epithelial cells thus it is imperative to clarify the molecular interactions occurring during the initiation and progression of ccmicrornas mirnas are a family of short noncoding rnas with an average length of nucleotides which negatively regulate target gene expression through either translation repression or rna degradation accumulating evidence has indicated that mirnas may function as oncogenes or tumor suppressors depending on their target mrna in various types of cancer including cc for example yang reported that mir214 inhibits the growth of cc cells by the regulation of its target enhancer of zeste homolog dong demonstrated a suppressive role of mir217 in the development of cc cells via targeting rhoassociated protein kinase chen reported that mir499a promotes the proliferation cell cycle progression colony formation migration and invasion of cc cells by targeting srybox transcription factor in addition several mirnas serve as diagnostic biomarkers in patients with cc such as mir152 and mir365 despite the aforementioned findings the roles of mirnas in the development of cc require further investigationin the present study a mirna microarray was performed to investigate the expression profiles of mirnas in cc tissues and the most downregulated mirna identified mir‘15a‘5p was selected for further analysis the potential role and underlying mechanism of mir15a5p in cc cells were also investigated the present results suggest that mir15a5p may serve as a therapeutic target for ccmaterials and methodspatients and samples in total paired cervical samples tumor tissues and adjacent noncancerous tissues were 0cchen mir15a inhibits cervical cancer cell growthobtained from female patients with cc who underwent cervical surgical resection without preoperative systemic therapy at the department of obstetrics and gynecology huashan hospital north of fudan university shanghai china between may and december the median age of the patients was years range years among all patients there were patients with metastatic cc and with nonmetastatic cc the matched nontumor adjacent tissue was obtained cm beyond the boundary of cc tissue all tissue samples were immediately snapfrozen in liquid nitrogen and stored at ‘Ëšc until use the experimental protocols were approved by the ethics committee of huashan hospital north of fudan university written informed consent for participation in the study was obtained from all patientsmirna expression profiling total rna from cc tissues three randomly selected paired tumor tissues and adjacent noncancerous tissues was extracted using mirneasy mini kit qiagen gmbh the samples were assessed using the mircury lna„¢ array v180 agilent technologies inc the procedure and imaging processes were performed as described previously cell culture human cc cell lines hela c33a caski and siha 293t cells and normal cervical epithelial cells ect1e6e7 were obtained from the american type culture collection all cells were cultured in dmem sigmaaldrich merck kgaa supplemented with vv fbs sigmaaldrich merck kgaa plus uml penicillinstreptomycin at ˚c with co2reverse transcription‘quantitative pcr rt‘qpcr total rna was extracted from tissues or cell lines using trizol reagent invitrogen thermo fisher scientific inc for mirna rt cdna was generated from ng total rna samples using taqman„¢ microrna reverse transcription kit applied biosystems thermo fisher scientific inc at ˚c for min for mrna rt cdna was synthesized using primescript rt reagent kit takara bio inc at ˚c for min qpcr for mirna and mrna was performed using the sybrgreen i realtime pcr kit applied biosystems thermo fisher scientific inc on an abi system applied biosystems thermo fisher scientific inc the reaction was performed under the following conditions ˚c for min followed by cycles at ˚c for sec and ˚c for sec and a final extension at ˚c for sec the primers for qpcr analysis were as follows mir15a5p forward 'aat gtt gcc cgt aat gcc3' and reverse 'ccc aag cgg aga aag gaa3' u6 forward 'gct tcg gca gca cat ata cta aaa t3' and reverse 'cgc ttc acg aat ttg cgt gtc at3' yesassociated protein yap1 forward 'cgg tcc act tca gtc tcc3' and reverse 'gag tgt ggt gga cag gta ctg3' and gapdh forward 'gtg gtg aag acg cca gtg ga3' and reverse 'cga gcc aca tcg ctc aga ca3' the expression levels of mir15a5p and yap1 were normalized to the expression of u6 and gapdh respectively the relative expression of each gene was calculated using the ‘ˆ†ˆ†cq method cell transfection the mir15a5p mimic mimic negative control nc mir15a5p inhibitor inhibitor nc yap1 overexpression plasmid pcdnayap1 and pcdnavector were all provided by guangzhou ribobio co ltd when c33a and siha cells 5x105 cellswell in 6well plates grew to confluence mir‘15a‘5p mimic nm mimic nc nm mir15a5p inhibitor nm inhibitor nc nm pcdnayap1 µg or pcdnavector µg were transfected into cells at ˚c for h using lipofectamine® invitrogen thermo fisher scientific inc the sequences were as follows mir15a5p mimic 'uag cag cac aua aug guu ugu g3' mimic nc 'uuc ucc gaa cgu guc acg utt3' mir15a5p inhibitor 'cac aaa cca uua ugu gcu gcu a3' and inhibitor nc 'cag uac uuu ugu gua gua caa3'in addition small interfering rna targeting yap1 si‘yap1 and the negative control targeting a non‘specific sequence siscramble were provided by thermo fisher scientific inc siha and c‘33a cells were transfected with the sirnas nmoll using lipofectamine invitrogen thermo fisher scientific inc the sequences of si‘yap1 and siscramble were as follows siyap1 'ctc agg atg gag aaa ttt a3' and siscramble 'ttc tcc gaa cgt gtc acg t3' at h posttransfection the cells were harvested for further analysis and the inhibition efficiency was determined by western blottingcell viability the c33a and siha cells were seeded in 96well plates at a density of 5x103well overnight following transfection the cell viability was measured using a cck8 assay briefly µl cck‘ solution was added to each well and cultured for h at ˚c the absorbance of the samples at nm was detected using a microplate reader biorad laboratories inccaspase‘ activity following transfection c33a and siha cells were harvested and the caspase3 activity was measured using a caspase3 activity assay kit beyotime institute of biotechnology according to the manufacturer's protocolcell apoptosis the apoptosis of c33a and siha cells was examined using flow cytometry following transfection c33a and siha cells were collected and the apoptotic cells were identified using an annexin v‘fitc apoptosis detection kit abcam according to the manufacturer's protocol after washing with cold pbs the cells were resuspended in binding buffer followed by staining with annexin v and propidium iodide for min in the dark at room temperature the fluorescence was measured using a facscan flow cytometer beckman coulter inc and then analyzed by flowjo v871 software flowjo llcimmunofluorescence assay following transfection c33a and siha cells were fixed in absolute ethyl alcohol for min at room temperature after washing twice with pbs the fixed cells were stained with primary antibody targeting cleavedcaspase3 cat no c ell signaling technology inc for h at room temperature subsequently an antirabbit conjugated antibody with fitc cat no f0382 sigmaaldrich merck kgaa was added for h in the 0cinternational journal of molecular medicine dark fluorescence images were obtained using an inverted fluorescence microscope magnification x200cell invasion assays transwell chambers 8µm pore bd biosciences coated with matrigel bd biosciences were used for the invasion assay briefly c‘33a and siha cells 8x104 were seeded in the top chamber with serumfree medium while the lower chamber contained culture medium with fbs following incubation for h the cells were fixed in paraformaldehyde solution beyotime institute of biotechnology for min and stained with crystal violet beyotime institute of biotechnology for min at room temperature images were captured with an inverted microscope olympus corporation magnification x100wound healing assay for the wound healing assay c33a and siha cells were seeded onto 12well plates 2x105 cellswell and h after transfection a scratch was made using a 10µl pipette tip in the confluent cell monolayer then cells were washed twice with pbs and incubated in dmem without fbs the wound healing images were captured at and h after scratching using an inverted light microscope olympus corporation magnification x100 the wound healing rate was calculated using imagej software v146 national institutes of healthdual‘luciferase reporter assay mirna target prediction tools including miranda httpmirandaorguk and targetscan httptargetscanorg were used to search for the putative targets of mir15a5p pgl3yap1 widetype or pgl3yap1 mutant type pgl3yap1mut promega corporation were cotransfected with mir15a5p mimics into 293t cells in 24well plates 2x105well using lipofectamine invitrogen thermo fisher scientific inc at h post‘transfection the luciferase activities were analyzed using the dualluciferase reporter assay system promega corporation with renilla luciferase activity as an internal control western blot analysis western blotting was performed as previously described briefly cells were lysed using radio immunoprecipitation assay buffer beyotime institute of biotechnology and the protein concentration was determined using the bicinchoninic acid assay total protein µglane was separated by sdspage and electrophoretically transferred onto a polyvinylidene difluoride membrane emd millipore subsequently membranes were blocked with skim milk for h at ˚c overnight each membrane was probed with primary antibodies against yap1 cat no and β‘actin cat no at ˚c overnight all primary antibodies were obtained from cell signaling technology inc subsequently the membrane was incubated with horseradish peroxidaseconjugated goat antirabbit igg cat no abcam at room temperature for h βactin served as the loading control and for normalization of protein expression the protein bands were developed using ecl kit ge healthcare and expression levels were quantified using imagej v146 national institutes of healthstatistical analysis all data are presented as mean ± standard deviation the correlation between mir15a5p and yap1 levels was evaluated using spearman's correlation analysis pairwise comparisons were performed by student's ttest and comparisons among groups were analyzed by oneway anova followed by tukey's posthoc test p005 was considered to indicate a statistically significant differenceresultsmir‘15a‘5p is downregulated in cc to examine the potential involvement of mirnas in the development of cc microarray analysis was performed to evaluate the mirna expression profiles between cc tissues and adjacent noncancerous tissues of differently expressed mirnas identified in the tumor group mirnas exhibited decreased expression and mirnas demonstrated increased expression compared with that in adjacent noncancerous tissues fig 1a among the aberrant mirnas the present study focused on mir15a5p for subsequent experiments due to its suppressive role in a variety of other cancer types such as endometrial cancer and chronic myeloid leukemia subsequently rtqpcr was performed to detect the expression of mir15a5p in pairs of tumor tissues and adjacent noncancerous tissues the results revealed that the level of mir15a5p was significantly lower in tumor tissues compared with that in adjacent noncancerous tissues fig 1b it was also observed that mir15a5p was expressed at a significantly lower level in tumor tissues with distant metastasis compared with in tumors tissues without distant metastasis fig 1c indicating that mir15a5p downregulation is associated with cc metastasis in addition rtqpcr was used to examine the mir15a5p level in four cc cell lines hela c33a caski and siha and the normal cervical epithelial cell line ect1e6e7 which was used as a control as expected mir‘15a‘5p was significantly lower in the four cc cell lines compared with ect1e6e7 cells fig 1d siha and c33a cells were selected for further experiments as they demonstrated the lowest expression of mir15a5p among all cell lines examinedupregulation of mir‘15a‘5p inhibits cell viability and promotes cell apoptosis in an attempt to understand the biological function of mir15a5p mir15a5p expression was upregulated or downregulated in the cultured siha and c33a cells by transfection with mir15a5p mimic or inhibitor respectively mir‘15a‘5p expression was significantly increased after mir15a5p mimic transfection whereas it was significantly decreased following mir‘15a‘5p inhibitor transfection in both siha and c33a cells fig 2a the present study then investigated the effect of mir15a5p expression on cell viability and the results demonstrated that the viability of siha and c‘33a cells was significantly inhibited by overexpression of mir15a5p whereas it was significantly enhanced by knockdown of mir‘15a‘5p compared with the negative control group fig 2b and c to assess the effects of mir15a5p upregulation on the apoptosis of siha and c33a cells caspase3 expression level and activity were analyzed by immunofluorescence and caspase‘ activity assays respectively as presented in fig 2d and e the expression of cleaved caspase3 and caspase3 activity was increased in siha and c33a cells transfected with 0cchen mir15a inhibits cervical cancer cell growthfigure mir‘15a‘5p is downregulated in cc tissues and cell lines a heat map presents significant differentially expressed mirnas in cc tissues and matched adjacent noncancerous tissues n3 green indicates downregulation and red indicates upregulation b mir15a5p expression was measured by rtqpcr in pairs of cc tissues and matched adjacent noncancerous tissues c mir15a5p expression was measured in tumor tissues with distant metastasis and tumors tissues without distant metastasis by rtqpcr d mir15a5p expression was detected in four cervical cancer cell lines hela c33a caski and siha and the normal cervical epithelial cells ect1e6e7 data are expressed at the mean ± standard deviation n3 of one representative experiment p001 vs ect1e6e7 cells mir microrna cc cervical cancer rtqpcr reverse transcriptionquantitative pcr mir15a5p mimic compared with the mimic nc groups furthermore the results of flow cytometry demonstrated that the extent of apoptosis was significantly increased after mir15a5p mimic transfection compared with the mimic nc groups fig 2f taken together these results indicate that overexpression of mir15a5p inhibits cell viability by inducing cell apoptosisupregulation of mir‘15a‘5p inhibits the invasion and migra‘tion of cc cells the present study further investigated whether overexpression of mir15a5p could reduce the invasiveness and migratory potential of cc cells using a transwell assay it was identified that the invasive capacities of siha and c‘33a cells were significantly inhibited by mir‘15a‘5p mimic whereas they were increased by mir15a5p inhibitor compared with the nc groups furthermore the wound healing assay results also demonstrated a significant reduction of cell migration in siha and c33a cells following mir15a5p overexpression however the migration of siha and c‘33a cells was significantly enhanced by mir15a5p inhibition fig 3c and d collectively the present data suggest that overexpression of mir15a5p suppresses the invasive and migratory abilities of cc cellsyap1 is a direct target of mir‘15a‘5p using the targetscan and miranda algorithms yap1 was found to have a putative target site of mir15a5p in its 'utr fig 4a to validate the possibility that yap1 is a direct target gene of mir15a5p a luciferase reporter assay was then performed the data revealed that mir‘15a‘5p mimic significantly inhibited the luciferase activity in the constructs containing the wildtype 0cinternational journal of molecular medicine figure overexpression of mir15a5p suppresses cell viability and promotes cell apoptosis siha and c33a cells were transfected with the mir15a5p mimic or inhibitor for h and then cells were used for analysis a transfection efficiency was assessed by reverse transcription‘quantitative pcr cell viability was measured by cck8 assay at indicated times for b siha and c c33a cells d the expression of cleaved caspase3 was determined by immunofluorescence assay magnification x200 e the caspase‘ activity was detected by a commercial caspase‘ activity kit f cell apoptosis was measured by flow cytometry data are expressed at the mean ± standard deviation n3 of one representative experiment p005 p001 vs mimic nc p005 p001 vs inhibitor nc mir microrna nc negative control od optical density pi propidium iodidebinding site of yap13'utr while it had no evident effects on the activity of yap13'utrmut by contrast mir15a5p inhibitor significantly increased luciferase activity without any evident effects on yap13'utrmut activity fig 4b subsequently to further detect the potential regulation of yap1 by mir15a5p the expression of yap1 protein was measured in cc cells by western blotting as presented in fig 4c the expression of yap1 was significantly decreased upon ectopic expression of mir15a5p suggesting that high expression of yap1 was partly due to the downregulation of mir15a5p in cc cells in addition it was identified that the mrna level of yap1 was significantly increased in cervical cancer compared with the control and inversely correlated with mir15a5p expression levels in cancer tissues fig 4d and e these results indicated that yap1 is a downstream gene of mir15a5p in cc 0cchen mir15a inhibits cervical cancer cell growthfigure overexpression of mir15a5p suppresses cell invasion and migration siha and c33a cells were transfected with the mir15a5p mimic or inhibitor for h and then cells were used for analysis invasion of a siha and b c‘33a cells was measured by a transwell assay magnification x200 the migration of c siha and d c33a cells was assessed by a wound healing assay the images were taken at and h after gaps were generated wound healing was quantified by the distance of the wounded region with an absence of cells data are expressed at the mean ± standard deviation n3 of one representative experiment p001 vs mimics nc p001 vs inhibitor nc mir microrna nc negative controlyap1 inhibition suppresses cell viability promotes cell apop‘tosis and inhibits invasion and migration previous evidence has shown that yap1 exerts an oncogenic function in several types of human cancer such as breast and lung cancer as the findings of the present study revealed that yap1 is upregulated in cc it was hypothesized that yap1 may act as an oncogenic gene in cc to confirm this hypothesis siha and c33a cells were transfected with siyap1 or siscramble western blot assay revealed that yap1 was notably downregulated following transfection with siyap1 fig 5a functionally yap1‘knockdown significantly suppressed the cell viability and induced cell apoptosis compared with the siscramble group fig 5b and c furthermore knockdown of yap1 significantly suppressed the invasive and migratory abilities of siha and c33a cells fig 5d and e suggesting that yap1 may play an oncogene role in the development of ccoverexpression of yap1 moderates the negative functions of mir‘15a‘5p on cell viability migration and invasion to ascertain whether yap1 is involved in the inhibitory effects of mir15a5p on cc cells the present study cotransfected pcdnayap1 andor mir15a5p mimic as well as their controls into siha and c33a cells the overexpression efficiency was verified by western blotting as shown in fig 6a yap1 was notably increased in siha and c33a cells after pcdna‘yap1 transfection subsequently the cell viability apoptosis invasion and migration were evaluated overexpression of yap1 significantly abolished the inhibitory effects of mir15a5p upregulation on the viability of siha and c33a cells fig 6b the increased apoptosis induced by mir15a5p overexpression was also reversed by overexpression of yap1 fig 6c furthermore overexpression of yap1 significantly reversed the inhibitory effects of mir‘15a‘5p on cell invasion and migration fig 6d and e in addition it was identified that overexpression of yap1 alone significantly promoted cc cell viability inhibited cell apoptosis and enhanced the invasion and migration compared with blank control group suggesting the oncogenic role of yap1 in cc cells these results indicate that mir15a5p exerts its tumor suppressive role in cc at least partially through yap1 0cinternational journal of molecular medicine figure yap1 is a direct target of mir15a5p a schematic of the yap1 'utr containing the mir15a5p binding sites b luciferase assay of 293t cells co‘transfected with firefly luciferase constructs containing the yap1 wt or mut '‘utrs and mir‘15a‘5p mimics mimics nc mir‘15a‘5p inhibitor or inhibitor nc as indicated n3 p001 c siha and c33a cells were transfected with the mir15a5p mimic and mimic nc for h and the expression levels of yap1 protein were determined by western blotting p001 vs mimic nc d yap1 expression was measured by reverse transcriptionquantitative pcr in cc tissues and matched adjacent noncancerous tissues n40 p001 e spearman's analysis was used to analyze the correlation between the expression of yap1 and the expression of mir15a5p expression in cervical cancer tissues r p001 data are expressed at the mean ± standard deviation n3 of one representative experiment yap1 yesassociated protein mir microrna 'utr 'untranslated region wt wildtype mut mutant nc negative controldiscussionin the present study mir15a5p was shown to be decreased in cc tissues and cell lines and associated with cc metastasis furthermore overexpression of mir15a5p inhibited the cc cell viability invasion and migration and promoted cell apoptosis while inhibition of mir15a5p demonstrated the opposite effects additionally yap1 was confirmed as a functional target of mir15a5p ectopic expression of which significantly reversed suppression of mir‘15a‘5p the present data indicated that mir15a5p may function as a tumor suppressor in cc progression by inhibiting yap1 expressiona number of studies have shown that mirnas participate in the development of cc for example xia reported that mir374b overexpression suppresses cell proliferative and invasive abilities via affecting forkhead box m1 expression yao also demonstrated that mir641 upregulation restricts cc cell growth in vitro and in vivo xu reported that mir2185p suppresses the progression of cc via the lynnfκb signaling pathway yuan demonstrated that overexpression of mir138 suppresses cc cell growth in vivo these findings suggest that targeting mirnas may be an effective therapeutic strategy for cc in the present study based on microarray expression data it was identified that mir15a5p is one of the most markedly downregulated mirnas in cc tissues notably previous studies have reported that mir15a5p functions as a tumor suppressor in several human cancer types although mir15a5p has been found to be downregulated in cc to the best of our knowledge the tumorigenic role and mechanism remain unknown therefore the present study focused on mir15a5p in cc for molecular analyses in the 0cchen mir15a inhibits cervical cancer cell growthfigure yap1 inhibition suppresses cell viability promotes cell apoptosis and inhibits invasion and migration siha and c33a cells were transfected with siyap1 or siscramble and then cells were harvested for further study a the expression of yap1 was measured by western blotting b cell viability was measured by cck‘ assay c the cell apoptosis was assessed by flow cytometry d cell invasion was measured by transwell assay e cell migration assessed by a wound healing assay data are expressed at the mean ± standard deviation n3 of one representative experiment p001 vs siscramble yap1 yesassociated protein mir microrna si small interfering rnafigure mir15a5p inhibits cell viability and induces cell apoptosis by targeting yap1 a siha and c33a cells were transfected with the pcdnayap1 plasmid for h and then the protein expression of yap1 was measured by western blotting subsequently siha and c33a cells were cotransfected with the pcdnayap1 plasmid and mir15a5p mimic for h and then cells were used for analysis b viability of siha and c33a cells was measured by cck8 assay at indicated times c the cell apoptosis was assessed by flow cytometry d cell invasion was measured by transwell assay e cell migration was measured by a wound healing assay data are expressed at the mean ± standard deviation n3 of one representative experiment p005 p001 vs blank group p001 mir microrna yap1 yesassociated protein microarray expression data the expression levels of numerous mirnas exhibited significant changes such as mir137 which demonstrated the most significant upregulation in cc tissues miao reported that mir137 upregulation inhibits cc cell invasion migration and epithelialmesenchymal transition by suppressing the tgfβsmad pathway 0cinternational journal of molecular medicine notably mir15a3p has also reported to exhibit differential expression and induce apoptosis in human cc cells although the present study did not detect the expression change of mir15a3p in the microarray expression data the expression of mir15a3p in four cc cell lines was examined and the results demonstrated that mir15a3p was also downregulated in cc cells compared with ect1e6e7 cells data not shown however the role and regulatory mechanisms of mir15a3p on invasion and migration remain unclear the function of more mirnas in cc will be investigated in the futureprevious studies have reported that mir15a5p has the potential to suppress cell growth and inhibit the progression of human cancers by regulating its downstream target genes for example luo demonstrated that overexpression of mir15a5p causes cellular growth inhibition and suppression of migration by targeting cyclin e1 in breast cancer wu and guo found that mir15a overexpression suppressed the cell proliferation and invasion by suppression of bmi1 translation in gastric cancer gc as well as pancreatic cancer pc of note several studies have reported aberrant expression of mir15a5p in cc tissues or cells however the role and mechanism of mir15a5p in cc remain largely unknown the present results demonstrated that overexpression of mir15a5p inhibited cell viability cell migration and invasion and induced cell apoptosis in siha and c33a cells while inhibition of mir15a5p demonstrated the opposite effects indicating that mir15a5p may serve as tumor suppressive role in cc yap1 a transcriptional coactivator and oncogene has been found to play an important role in different types of carcinoma for example liu reported that yap1 overexpression promotes the invasion migration and growth of colon cancer cells yu demonstrated that knockdown of yap1 causes a significant inhibition of the growth and migration of renal cell carcinoma cells in vitro and in vivo notably yap1 has been verified to target mir‘15a‘5p to suppress cell growth and metastasis in gastric adenocarcinoma and colon cancer however whether yap1 is a target of mir15a5p in cc remains unclear in the present study yap1 was confirmed to be a target of mir‘15a‘5p and its protein expression levels were negatively regulated by mir15a5p further investigation indicated that yap1 was significantly increased in cc tissues and inversely correlated with mir15a5p in cc tissues furthermore yap1 was confirmed to act as an oncogene gene in cc cells and its overexpression partly abrogated the inhibitory effect induced by enhanced expression of mir15a5p in cc cells taken together the present study demonstrates that mir15a5p exerts its tumor suppressive role in cc cells by targeting yap1due to the limitation in experimental conditions and funds further research in the future is required to investigate whether mir15a5p serves its role via other downstream targets in addition the present study investigated the cellular function of mir15a5p and its underlying mechanism in cc however in vivo studies and clinical trial data are required to validate the preliminary in vitro results obtained therefore the function of mir15a5p in cc needs to be further investigated in vivoin conclusion the present results demonstrated that mir15a5p suppresses the viability migration and invasion of cc cells by directly targeting yap1 based on these findings it is proposed that the mir15a5pyap1 axis may serve as a novel biomarker for new targets in cc therapyacknowledgementsnot applicablefundingfunding was received from the scientific research project of shanghai science and technology commission grant nos and availability of data and materialsthe datasets used andor analysed during the current study are available from the corresponding author on reasonable request authors' contributionsrc hl tz xy and sx performed the experiments contributed to data analysis and wrote the paper rc hl tz xy and sx analysed the data xc conceptualized the study design and contributed to data analysis and experimental materials all authors read and approved the final manuscriptethics approval and consent to participateall individuals provided written informed consent for the use of human specimens for clinical research the experimental protocols were approved by the ethics committee of huashan hospital north of fudan university patient consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interests references alldredge jk and tewari ks clinical trials of antiangiogenesis therapy in recurrentpersistent and metastatic cervical cancer oncologist tsikouras p zervoudis s manav b tomara e iatrakis g romanidis c bothou a and galazios g cervical cancer screening diagnosis and staging j buon fang j zhang h and jin s epigenetics and cervical cancer from pathogenesis to therapy tumour biol wang j liu y wang x li j we j wang y song w and zhang z mir1266 promotes cell proliferation migration and invasion in cervical cancer by targeting dab2ip biochim biophys acta mol basis dis zhu l zhu l s
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"colorectal cancer crc remains the third most prevalent cancer type and leading cause of cancerrelated deaths with million cases and deaths worldwide during the occurrence and progression of crc result from a wide array of cellular transformation processes which include genetic and epigenetic mutations that drive uncontrolled cell proliferation and escape from apoptosis2“ chemotherapy and surgery remain the major therapeutic treatment for crc patients5 fluoropyrimidinebased chemotherapy eg 5fluorouracil has been used as the firstline systemic chemotherapy of treating advanced crc for over a half century6 however most patients receiving chemotherapy finally develop drug resistance which is considered to be the major reason for crc therapy failure7 furthermore even though chemotherapy has significant antitumor activity the side effects can affect the quality of a patient's life which makes the new therapeutic approaches urgentdrug design development and therapy “ sun this work is published and licensed by dove medical press limited the full terms of this license are available at wwwdovepresscomtermsphp and incorporate the creative commons attribution “ non commercial unported v30 license httpcreativecommonslicensesbync30 by accessing the work you hereby accept the terms noncommercial uses of the work are permitted without any further permission from dove medical press limited provided the work is properly attributed for permission for commercial use of this work please see paragraphs and of our terms wwwdovepresscomtermsphp 0csun dovepresstraditional chinese medicines such as dendrobium have been shown to exert anticancer activity in many kinds of cancers89 erianin 2methoxy5[2345trimethoxy phenylethyl]phenol figure 1a a natural compound derived from dendrobium candidum shows various pharmacological activities and therapeutic potential to inhibit multiple cancers in vivo and in vitro10“ li demonstrated that erianin inhibited the proliferation of acute promyelocytic leukemia hl60 cells by regulating the expression of bcl2 and bax10 in addition erianin caused moderate growth delay in xenografted human hepatoma bel7402 and melanoma a37511 furthermore erianin induced cell cycle g2mphase arrest and apoptosis via the jnk signalling pathway in osteosarcoma and bladder cancer1213 erianin can also inhibit cell invasion metastasis and angiogenesis in lung cancer and breast cancer by the figure erianin inhibited crc cells growth a chemical structure of erianin b and c sw480 and hct116 cells were treated with indicated concentration b and time c of erianin cell viability was assessed by cck8 assay p ˂ p ˂ d and e ncm460 cells were treated with indicated concentration d and time e of erianin cell viability was assessed by cck8 assay f sw480 and hct116 cells were performed colony formation assay after being treated with indicated concentration of erianinsubmit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun regulation of ido mpp and timp expressions1415 interestingly besides the function on cell growth apoptosis and migration erianin was found to strongly affect the serum levels of cytokines and immune response in liver cancer16 more importantly in addition to the anticancer effects previous a study also suggested that erianin had no major anrelated toxicities12however to the best of our knowledge neither the mechanism nor the effect of erianin on colorectal cancer has been reported hence in this study we evaluate the antitumor potential and molecular mechanisms of erianin in human colorectal cancer sw480 and hct116 cells and provide a theoretical basis of erianin application for colorectal cancer therapymaterials and methodsmaterialsantibodies against cleaved parp cat bak cat bax cat bcl2 cat bclxl cat catenin cat cyclin d1 cat cmyc cat hdac2 cat and gapdh cat were purchased from cell signaling technologies danvers ma usa antibody against αtubulin cat t6199 was purchased from sigma aldrich co st louis mo usaerianin was purchased from shanghai yuanye bio technology co ltd china and dissolved in dmso wntcatenin signaling inhibitor wnt974 was purchased from medchemexpress monmouth junction nj usa and dissolved in dmsocell culturethe human colorectal cancer cell lines sw480 and hct116 were purchased from american type culture collection atcc manassas va usa cells were maintained in rpmi1640 medium supplemented with fbs thermo fisher scientific waltham ma usa uml penicillin and µgml streptomycin thermo fisher scientific and cells were cultured at °c with co2cell viability and colony formation assaycell viability was assessed with the cell counting kit cck8 dojindo japan according to the manufactorer™s instructionsfor the colony formation assay crc cells cells well were seeded in a sixwell plate and maintained in medium for “ days subsequently the colonies were fixed with paraformaldehyde and stained with crystal violet and the number of clones was counted using an inverted microscopekit quantitative realtime pcr qrtpcrtotal rna from crc cells was isolated using rna isolation kit omega norcross ga usa according to the manufacturer™s protocol total rna µg was used as the template for cdna synthesis by using iscripttm reverse transcription super mix biorad laboratories inc hercules ca usa before the samples were analyzed using sybr green master mix on a realtime pcr system biorad laboratories inc the primer sequences used were as follows cmyc forward 5ʹ aaacacaaacttgaaca gctac3ʹ reverse 5ʹ atttgaggcagtttacatt atgg3ʹ cyclin d1 forward 5ʹaggcggatgagaac aagcaga3ʹ reverse 5ʹcaggcttgactccagaag gg3ʹ cd47 forward 5ʹggcaatgacgaaggaggt ta3ʹ reverse 5ʹatccggtggtatggatgaga3ʹ and gapdh forward 5ʹcacccactcctccacctttg3ʹ and reverse 5ʹccaccaccctgttgctgtag3ʹ the 2δδcq method was used to calculate the relative expression levelswestern blottingfor western blotting μg cellular protein extracts were separated in sdspage gel and were then transferred to nitrocellulose membranes emd millipore burlington ma usa the membrane was blocked with nonfat milk and incubated with primary antibodies overnight at ° then the membranes were incubated with secondary antibody and the proteins were visualized using super signal west pico chemiluminescent substrate thermo fisher scientifictransit transfectionplasmid pegfpn1betacatenin was purchased from addgene watertown ma usa lipofectamine thermo fisher scientific carlsbad ca usa was used for transit transfection according to the instructionscatenin sirna was purchased from sigmaaldrich co lipofectamine rnaimax thermo fisher scientific was used for transfection according to the instructiondrug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepresscell cycle analysisafter treated with vehicle or indicated drugs crc cells were harvested by trypsinization fixed with ethanol and retained at ˆ’°c overnight after cells were centrifuged and washed with pbs they were resuspended in propidium iodide pi solution containing rnase μgml in the dark at room temperature for min and then studied in a flow cytometercaspase37 activity assayapoone„¢ homogeneous caspase37 assay promega corporation madison wi usa was used to measure caspase37 activity briefly apoone® homogeneous caspase37 reagent μlwell was added to a 96well plate and the plate was then placed on a shaker for five minutes “ rpm before incubating for h at room temperature the reading of each well was measured by spectrofluorometerapoptosis assay by annexin vannexin vfitc staining was used to detect the extent of apoptosis induced by erianin briefly crc cells were treated with erianin for h and were then collected and resuspended in μl annexin vbinding buffer and μl pi for minat room temperature in the dark then the cells were finally analyzed by the flow cytometry bd facs calibur with an emission filter of nm for pi red and “ nm for fitc greenapoptosis assay by dapithe effect of erianin on apoptosis induction was evaluated by dapi staining assay crc cells × were seeded in a 96well plate after treatment the cells were washed three times with pbs and paraformaldehyde was added to each well for fixation after permeabilization with triton x100 solution dapi solution was added the cells with condensed and fragmented chromatin were analyzed by echo fluorescence microscopycellular thermal shift assayfor cellular thermal shift assay crc cells were pretreated with μm mg132 for one hour and then incubated with erianin for four hours after washing with icecold pbs cells were aliquot into pcr tubes μl each and incubated at different temperatures for four minutes after being frozen and thawed twice using liquid nitrogen cells were centrifuged and proteins were analyzed by western blottingtopfop luciferase reporter assaythe transcriptional activity of catenin was assessed using the topfop dualluciferase reporter system dual glo„¢ luciferase assay system promega the renilla luciferase plasmid prltk promega which controls for transfection efficiency was cotransfected with catenin responsive firefly luciferase reporter plasmid topflash emd millipore or the negative control fopflash emd millipore using the lipofectamine thermo fisher scientific cells were harvested after h in culture and the luciferase activity was determined by the luciferase assay system promega using a microplate luminometer berthold bad wildbad germanyflow cytometry analysiserianin treated crc cells were washed and resuspended in μl facs buffer and stained with fitcconjugated anticd47 bd biosciences san jose ca usa antibodies all samples were incubated for minutes at °c and then washed twice with facs buffer flow cytometry analyses were performed on bd facs canto iiin vitro phagocytosis assayfor phagocytosis assay thp1 derived macrophages were seeded in a sixwell tissue culture plate erianintreated crc cells were washed and labeled with μm of carboxyfluorescein succinimidyl ester cfse thermo fisher scientific after incubating macrophages in serum free medium for two hours cfselabeled crc cells were added to the macrophages for another two hours at °c macrophages were then washed and imaged with an inverted microscope the phagocytosis efficiency was calculated as the number of macrophages containing cfse labeled crc cells per macrophageschromatin immunoprecipitation chip assaychip assays were performed using the simplechip® enzymatic chromatin ip kit cell signaling technologies according to manufacturer's instructions using the antibodies against h3k27ac immunoprecipitated dna was analyzed by qrtpcr using the following primers cd47 promoter fragment f ²aggatgaatgatgtggcctgt3² and r ² caaacaggcattagcagcgt3² fragment f submit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun ²ggggatgtgttggatacgct3² and r ² ctctg cgttcggctcgtcta3² fragment f ²agggaag agcagagcgagta3² and r ² ttgctttcactcc caccctc3² fragment f ²agagagaggacag tggggc3² and r ² ccagtcgcaggctccaga3² fragment f ² gccgcgtcaacagca3² and r ² aaaggcatcattcttggaaattgt3²with ¨° sw480 cells per mouse suspended in vivo xenograftnodscid shanghai slac laboratory animal co ltd china mice were injected subcutaneously in right flank in µl pbs and mixed with an equal volume of matrigel animals with tumors volume mm3 were divided into two groups n6 and treated with either placebo or mgkg erianin for continuously three weeks by intraperitoneal injection tumor size were measured at the indicated times all the animalrelated procedures were approved by the animal care and use committee of the changchun university of chinese medicine all animal experiments were conducted according to the nih guide for the care and use of laboratory animalsstatistical analysisdata were presented as mean ±sd from three independent experiments p value was determined using paired student™s ttest and a p value ˂ was deemed to indicate statistical significanceresultserianin inhibited crc cell growthfigure 1a illustrates the chemical structure of erianin to investigate the inhibitory effect of erianin on crc cell viability we treated two crc cell lines sw480 and hct116 with different concentrations of erianin and nm for and h as shown in figure 1b and c erianin treatment significantly inhibited the viability of crc cells in a dose and timedependent manner importantly erianin did not show cytotoxic effects on normal human colon mucosal epithelial cell line ncm460 figure 1d and e in addition consistent with the shortterm growth assay our colony forming unit assay also showed that erianin inhibited the colony formation ability of sw480 and hct116 cells figure 1ferianin elevated cell cycle arrest and apoptosisto verify the causal relation of cell viability inhibition the cell cycle distribution was analyzed erianin increased cell number at g2m phase but decreased cell number at s and g0g1 phases after 24h incubation with indicated concentration in sw480 and hct116 cells figure 2a and b to explore the effect of erianin on apoptosis we examined the activity of caspase the protein level of cleaved parp bax bak bcl and bclxl as shown in figure 2c“e the activity of caspase protein level of cleaved parp bak and bax pro apoptosis increased as the concentration of erianin increased in contrast the protein level of bcl2 and bclxl anti apoptotic decreased after erianin treatment figure 2e annexin v flow cytometry and dapi staining further confirmed that erianin could induce cell apoptosis figure 2f and gerianin inhibited catenin translocationincreasing evidence revealed that the wntcatenin pathway plays critical role in colorectal cancer tumorigenesis we hypothesized that erianin might have effect in modulating the wntcatenin pathway first we investigated the effect of erianin on catenin phosphorylation as shown in figure 3a no obvious change was observed on catenin phosphorylation level we then evaluated the effect of erianin on catenin translocation as shown in figure 3b“e catenin expression in cytoplasm was increased whereas expression in the nucleus was decreased with the treatment of erianin in a dose and timedependent manner to further explore the effect of erianin on catenin transcription activity we performed topfop dual luciferase assay we found that topfop relative luciferase activity was significantly decreased after erianin treatment both in sw480 and hct116 cells figure 3f and gerianin bound catenin directlysince erianin inhibited catenin translocation to the nuclear without changing its phosphorylation level we hypothesized that erianin might bind catenin directly to determine whether erianin physically binds catenin we performed a cellular thermal shift assay the results from this experiment indicated that erianin treatment increased the thermal stability of catenin when cells were pretreated with the proteasome inhibitor mg132 for one hour figure 4a and b in contrast erianin treatment had no effect on the thermal stability of gapdh a loading control figure 4a and b these results strongly suggested a specific physical interaction between erianin and catenindrug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressfigure erianin elevated cell cycle arrest and apoptosis a and b sw480 and hct116 cells were treated with erianin for h and then analyzed by pi staining to determine cell cycle phase distribution c sw480 and hct116 cells were treated with erianin for h the relative caspase37 activity was measured using apoone„¢ homogenous caspase37 assay p ˂ p ˂ d and e the protein level of cleaved parp1 bak bax bcl2 and bclxl were analyzed by western blotting after treated with indicated concentration of erianin f and g sw480 and hct116 cells were treated with erianin for h apoptosis was assessed using annexinv flow cytometry analysis f or dapi staining gsubmit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun figure erianin inhibited catenin translocation a the protein level of indicated proteins was analyzed by western blotting after being treated with indicated concentration of erianin for h b“e the protein level of catenin in cytosol and nucleus was analyzed by western blotting after treated with erianin for indicated concentration b and c and time d and e f and g sw480 and hct116 cells were treated with erianin for indicated concentration f and time g the transcriptional activity of catenin was assessed by topfop luciferase reporter assay p ˂ p ˂erianin inhibited the expression of cmyc and cyclin d1as cmyc and cyclin d1 are the direct targets of the wnt catenin pathway we then evaluated the mrna and protein level of cmyc and cyclin d1 unsurprisingly both mrna and protein level of these two proteins were significantly decreased after erianin figure 5a“c interestingly no synergetic effect was observed when combining erianin with wntcatenin signaling inhibitor wnt974 which indicated that erianin regulates cmyc treatment drug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressfigure erianin interacted with catenin a and b sw480 a and hct116 b cells were treated with μm mg132 for one hour followed by four hours incubation with nm erianin before performing thermal shift assay the lower panel shows the charts of percentages of nondenatured protein fractionand cyclin d1 via wntcatenin signaling figure 5d furthermore the inhibitory effect of erianin on cmyc and cyclin d1 expression and cell viability could be reversed by catenin overexpression figure 5e and f which indicated that erianin regulates crc cell growth via catenincd47 mediated phagocytosis we used an in vitro assay by coculturing thp1 derived macrophage with crc cell lines sw480 or hct116 as shown in figure 6g and h treatment of erianin markedly promote colorectal cancer cell phagocytosis by macrophages these results suggest that erianin treatment can attenuate cd47 expression and ultimately promote phagocytosis of crc cellserianin decreased cd47 expression and increased phagocytosisthe immune checkpoint protein cd47 is included in the list of wntcatenin target molecules with a role in immunity escape17 since catenin depletion by sirna inhibited the expression of cd47 figure 6a we then sought to know whether erianin regulates the expression of cd47 first we explored the effects of erianin on cd47 mrna protein and cell surface level in both sw480 and hct116 cells erianin treatment significantly decreased the mrna protein and cell surface level of cd47 figure 6b“d promoter analysis by ucsc genome browser demonstrates that h3k27 acetyl marks are enriched in cd47 promoter regions figure 6e next our chip assay demonstrated that h3k27ac enrichment specifically near promoter region f3f5 was significantly decreased with erianin treatment figure 6f to investigate the effect of erianin on erianin inhibited tumor growth in vivoto investigate the possibility of erianin as a potential therapy in crc we tested the function of erianin on tumor growth in a mouse model the mouse model was established by s c injection of sw480 cells into nodscid mice after three weeks treatment we analyzed the tumor size and weight as shown in figure 7a“c the tumor size and weight from the erianin treatment group were significantly lower than that from the control group in addition after days of bearing tumor the weight of the mice had no significant change figure 7dto examine the impact of therapy on catenin and its downstream signaling localization of catenin protein level of cd47 cmyc bcl2 and bax three representative tumors from each group were analyzed using western blotting as shown in figure 7e and f catenin expression in cytoplasm was increased whereas expression in nucleus was decreased with the treatment of erianin the submit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun figure erianin inhibited the expression of cmyc and cyclin d1 a“c after treated with indicated concentration and time of erianin mrna and protein level of cmyc and cyclin d1 were analyzed by qrtpcr and western blotting p ˂ d sw480 cells were treated with erianin orand wnt974 for h protein levels of cmyc and cyclin d1 were analyzed by western blotting e and f sw480 cells were treated with erianin for h followed by overexpression with catenin plasmid for h protein levels of cmyc and cyclin d1 were analyzed by western blotting e and cell viability was assessed by cck8 assay f p ˂protein level of cd47 cmyc and bcl2 decreased while bax increased after erianin treatment these data indicated that erianin inhibited tumor growth via catenin in vivodiscussioncrc is one of the most malignant and commonly diagnosed solid tumors all around the world18“ although crc incidence rates have declined somewhat chemotherapies are inefficient in most crc patients due to resistance2122 thus the development of acquired therapeutic drugs researching novel and safe treatment strategies is essential for improving the prognosis of crc patients in recent years natural medicinal plants are receiving more and more attention and considered to be important sources of treatment23 novel dendrobium is considered as one of the most important herbs in the orchidaceae family and shows diverse pharmacological functions including anticancer neuroprotective antidiabetic and immunemodulating activities24 erianin derived from dendrobium is one of the most for cancer drug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressfigure erianin decreased cd47 expression and increased phagocytosis a sw480 cells were transfected with nontarget nt or catenin sirna for h protein levels of indicated protein weres measured by western blotting b“d sw480 and hct116 cells were treated with erianin for indicated dose the mrna level b protein level c and cell surface cd47 d were detected by qrtpcr and flow e the ucsc genome browser revealed the enrichment of h3k27ac on cd47 promoter f the enrichment of h3k27ac on cd47 promoter f1f6 was detected by chip assay g and h sw480 and hct116 were treated with indicated concentration of erianin for h representative images showed the effect of erianin on phagocytosis g and bar graphs showed quantitative analysis of phagocytosis h p ˂ p ˂submit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun figure erianin inhibited tumor growth in vivo a typical photos of tumors from the control and erianin treated groups b and c erianin decreased tumor volume and weight p ˂ d mice body weight of control and erianin treated groups was measure at indicated time e the protein level of catenin in cytosol and nucleus in three representative tumors from mouse to mouse of each group were analyzed by western blotting f the protein level of indicated protein in three representative tumors from mouse to mouse of each group were analyzed by western blottingdrug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressnoteworthy constituents that have been used as an antipyretic and an analgesic in traditional chinese medicine25 recently several studies have proved that erianin shows significant antitumour activity in a variety of human cancer cells10ˆ’ consistent with literature in this study we found that erianin had a significant antiproliferative effect against crc cells the inhibitory effect caused by erianin may result from induction of apoptosis and arrest of cell cycle at g2m since the effect of erianin on crc cells has never been studied before we further confirm its antitumor activity in a mouse model which indicated that erianin significantly inhibited tumor growth in vivoseveral signaling pathways including egfrmapk pi3kakt or wntcatenin have been linked to crc genesis and progression26 as the aberrant activation is present in almost all crc cases wntcatenin signaling is prominent among these pathways27 inactivated mutations in the apc gene leads to stabilization and ensuing nuclear translocation of catenin to facilitate tcflef dependent transcription of wntcatenin signaling target genes such as cmyc and cyclin d1 to drive cell proliferation survival and metastasis28“ to understand the mechanisms of action of erianin we assessed the effect of erianin on wntcatenin pathway interestingly we found that erianin treatment had no effect on catenin phosphorylation but inhibited the translocation of catenin in the nucleus which suggested to us that erianin physically interacts with catenin our cellular thermal shift assay confirmed this hypothesis the thermal stability of catenin increased after erianin treatment as catenin downstream targets the expression level of cmyc and cyclin d1 significantly decreased after erianin treatmentcd47 a transmembrane glycoprotein expresses ubiquitously and mediates a œselfdonoteatme signal on normal cells however cd47 is often upregulated in tumor cells to evade innate immunity31“ anticd47 antibodies which block cd47 sirpα interactions and promote macrophage mediated phagocytosis of tumor cells has shown promise in several solid tumors31 in colorectal cancer cd47 promotes colon cancer cell migration and metastasis34 in addition upregulated immuneescape pathways such as cd47 sirpα are responsible for immune escape and survival in circulating tumor cells of colorectal cancer35 myc an oncogene identified as a wntcatenin target gene was reported to control cd47 transcription therefore mutations in components of the wntcatenin signaling pathway which induced
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glioma initiates from glial cells and contains several types such as astrocytoma and oligodendroglioma1 over a quarter of brain tumors are glioma which causes a large number of cancerrelated deaths every year around the world1 the current clinically therapeutic strategies are surgery combined with chemotherapy and radiotherapy2 however the prognosis of glioma patients remains not well post therapy3 hence it is urgently required to discover new molecular mechanism for glioma therapyboth long noncoding rna lncrna and microrna mirna belong to noncoding rnas which have no proteincoding ability lncrna is characterized with more than nucleotides while mirna is about nucleotides in length4 lncrna and mirna are involved in various cellular processes including cell division invasion and survival5 dysregulation of lncrna or mirna usually causes tumor initiation and progression67 for example lncrna linc00152 upregulation promotes gastric cancer growth and metastasis8 lncrna snhg6 overexpression facilitates lung cancer cell proliferation and metastasis9 mir3405p dysregulation promotes tumorigenesis of esophageal squamous cell carcinoma10 in addition mir126 cancer management and research “ du this work is published and licensed by dove medical press limited the full terms of this license are available at wwwdovepresscomtermsphp and incorporate the creative commons attribution “ non commercial unported v30 license httpcreativecommonslicensesbync30 by accessing the work you hereby accept the terms noncommercial uses of the work are permitted without any further permission from dove medical press limited provided the work is properly attributed for permission for commercial use of this work please see paragraphs and of our terms wwwdovepresscomtermsphpcorrespondence jun wu weiwen qiu email wwwwjjjj924163com weiwenqhotmailcomsubmit your manuscript wwwdovepresscomdovepresshttp102147cmars262279 0cdu dovepresssuppresses colon cancer cell survival and induces apoptosis11 besides lncrna has been identified as potential competing endogenous rna cerna for mirna to function in cancer12 the potential roles underlying lncrna and mirna still require much investigation and the relationship between lncrna and mirna also needs to be definedlinc00173 is an oncogene in lung cancer and breast cancer1314 the function of linc00173 in glioma is unclear in the current study we found that linc00173 was upregulated in glioma tissues linc00173 high expression was associated with a low survival rate linc00173 depletion suppressed proliferation migration and invasion of glioma cells linc00173 was discovered to sponge mir765 to elevate nutf2 expression taken together our findings supported that linc00173 plays essential oncogenic roles in glioma through activating mir765nutf2 pathwaymaterials and methodsclinical samplesthirtyseven glioma tissues and normal tissues were collected from lishui city people™s hospital patients received no radiotherapy or chemotherapy before surgery all tissues were stored in liquid nitrogen association between linc00173 expression and clinical characteristics in glioma tissues was analyzed in table written informed consent was obtained from every patient this study was approved by the ethics committee of lishui city people™s hospital no and the table association between linc00173 expression and clinical characteristics in glioma tissuesfeaturesage years‰gendermalefemalegradei“iiiii“ivtumor size cm‰low n19high n18pvalueexperiments were conducted in accordance with the declaration of helsinkicell culture and treatmentthe normal human astrocyte nha and glioma cell lines were purchased from institute of biochemistry and cell biology of the chinese academy of sciences shanghai china cells were cultured using pmi1640 medium invitrogen carlsbad ca usa supplemented with fetal bovine serum fbs invitrogen shrnas against linc00160 mir6293p mimics mir6293p inhibitors and negative controls were obtained from genepharma and transfected into glioma cells using lipofectamine invitrogen according to the manufacturer™s instructions efficiency was validated using qrtpcr after hqrtpcrtotal rna was extracted from tissues and cell lines using trizol invitrogen carlsbad ca primescript rt reagent kit rr047a takara holdings inc tokyo japan was used to generate cdna from rna template qpcr was completed through sybr premix ex taq„¢ ii takara japan gapdh was the normalized control relative expression was calculated through the ˆ’δδct methodluciferase reporter assaythe fragment of linc00173 or nutf2 containing indicated mir765 binding site was constructed into pmir report vector for luciferase reporter assay glioma cells were transfected with report vector and mir765 mimics after h the luciferase reporter activity was measured through the dualluciferase reporter assay system promega madison wiwestern blot assaycells were lyzed using radioimmunoprecipitation buffer beyotime shanghai china protein concentration was determined by a bca protein assay kit thermo fisher scientific ma then proteins were separated using sdspage and transferred onto pvdf membranes after blockage using bsa for h the membrane was incubated the primary antibodies at °c overnight after washed times using pbst the membranes were incubated with horseradish peroxidaselabeled second antibody followed by detection the enhanced chemiluminescence reagent emd millipore usathrough submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress du cck8 and colony forming assaysproliferation was measured using cck8 and colony formation assay cck8 assay was performed using the cck reagent dojindo kumamoto japan according to the manufacturer™s instructions and absorbance was determined at nm using a microplate reader biotek winooski vt for colony formation assay cells were seeded into 6well plates and cultured for days then the cells were fixed with methanol and stained with crystal violet for minutesedu assaycells were plated into 96well plates and incubated with edu μl at °c for h followed by detection using facstranswell migration and invasion assaystranswell plates corning ny were used to measure migration and invasion according to the manufacturer™s instructions in brief cells were suspended into μl serumfree medium and seeded into the upper chamber while the lower chamber was filled with µl of complete medium after incubation for cells in the lower chamber were fixed with methanol and stained with crystal violet for minutes migrated and invaded cells were counted through a light microscopestatistical analysisgraphpad prism graphpad ca usa was used to analyze results data were presented as means±standard deviation sd significant differences were analyzed using student™s ttest or oneway anova survival rate was analyzed by the kaplan“meier method and log rank test p005 was considered to be significantresultslinc00173 expression is elevated in gliomathe expression of linc00173 was firstly analyzed through qrtpcr we found that linc00173 level was elevated in glioma tissues compared with normal tissues figure 1a besides we found that linc00173 was also upregulated in glioma cell lines compared to nha cells figure 1b then according to the median value of linc00173 glioma tissues were classified into two groups after analysis we found that linc00173 high expression correlated with poor prognosis figure 1ctransfection of linc00173 enhanced glioma cell proliferation migration and invasionto explore the function of linc00173 u87 and u251 cells were chosen after shlinc00173 linc00173 expression was significantly downregulated figure 2a through cck8 assay we observed that linc00173 knockdown suppressed the proliferation capacity of glioma cells figure 2b and c which was validated by edu and colony formation assays figure 2d and e afterwards transwell assay was performed results indicated that linc00173 loss inhibited migration and invasion of glioma cells figure 2f and g thus linc00173 exerted oncogenic roles by affecting proliferation migration and invasionlinc00173 worked as the sponge for mir765linc00173 has been found to serve as cerna for several mirnas such as mir490 and mir2181314 to determine the mechanism of linc00173 in glioma we also figure linc00173 expression is elevated in glioma a the level of linc00173 in glioma tissues was measured b the expression of linc00173 in glioma cell lines and nhas c association between overall survival and linc00173 expression in glioma patients p005cancer management and research submit your manuscript wwwdovepresscom dovepress 0cdu dovepressfigure linc00173 enhanced glioma cell proliferation migration and invasion a qrtpcr analysis of linc00173 expression in u87 and u251 cells b“e proliferation ability was measured using cck8 edu and colony formation assays f and g migration and invasion capacity was evaluated after linc00173 knockdown in glioma cells p005suppressed the supporting their direct performed bioinformatics analysis using mirdb we identified that mir765 was the most potential candidate because it scored the highest to validate it we constructed luciferase reporters figure 3a followed by luciferase reporter assay results showed that mir765 activity of linc00173wt only figure 3b interaction pulldown assay further demonstrated their interaction figure 3c qrtpcr found that linc00173 overexpression suppressed the level of mir765 figure 3d next bioinformatics analysis using mirdb and targetsan implied that nutf2 is the most potential target of mir765 the corresponding luciferase reporters were further constructed figure 3e luciferase reporter assay also demonstrated the interaction between nutf2 and mir765 figure 3f besides nutf2 expression was suppressed by mir765 mimics figure 3g moreover nutf2 level was decreased after linc00173 knockdown while mir765 inhibitors reversed it figure 3h finally we found that mir765 level was negatively correlated with linc00173 or nutf2 in glioma tissues figure 3i and jlinc00173 promoted glioma progression through mir765nutf2 pathwaywe noticed that nutf2 expression was upregulated in glioma tissues figure 4a and b suggesting an oncogenic role to investigate whether linc00173 regulates glioma progression through mir765nutf2 we restored the expression of nutf2 in shlinc00173 transfected cells cck8 and transwell assays demonstrated that nutf2 restoration successfully rescued the capacities of proliferation migration and invasion in glioma cells transfected with shlinc00173 figure 4c“f therefore linc00173 submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress du figure linc00173 worked as the sponge for mir765 a bioinformatics analysis indicated the binding sites between linc00173 and mir765 b u87 cells were transfected with mir765 mimics or negative controls mirnc and luciferase reporter linc00173wt or linc00173mut then relative luciferase activity was determined c rna pulldown assay using biotinlabeled mirnas d relative expression of mir765 after linc00173 knockdown e bioinformatics analysis indicated the binding sites between mir765 and nutf2 f u87 cells were transfected with mir765 mimics or negative controls mirnc and luciferase reporter nutf2wt or nutf2mut then relative luciferase activity was determined g qrtpcr analysis for nutf2 expression h western blotting analysis for nutf2 protein level i and j correlation analyses of linc00173 mir765 and nutf2 in glioma tissues using pearson™s correlation coefficient p005contributes to glioma progression through mir765nutf2 pathwaydiscussionas the most malignant brain tumor glioma leads to a huge number of deaths patients with glioma display a poor prognosis therefore it is of great significance to reveal the mechanism underlying glioma progression in this study we found that linc00173 was upregulated in glioma tissues and cells linc00173 overexpression predicted a poor prognosis moreover linc00173 knockdown the proliferation migration and invasion of glioma cells linc00173 was also found to inhibit mir765 and promote nutf2 expression summarily our research discovered that linc00173 is an important oncogenic lncrna in gliomasuppressed the potential roles of lncrna in glioma have been researched for a long time many lncrnas have been identified to participate in glioma development for example lncrna nck1as1 enhances growth and metastasis of glioma through targeting mir13823p to activate β catenin signaling2 lncrna ccat2 contributes to glioma progression by activating vegfa pathway15 lncrna linc00467 upregulation promotes glioma development through repressing p53 level16 previous study showed that linc00173 downregulation promotes nonsmall cell lung cancer cell growth and survival17 however another study showed that linc00173 enhances chemoresistance and facilitates tumor progression in small cell lung cancer13 besides linc00173 contributes to breast cancer development14 yet how linc00173 works in glioma remains undermined in our study we found that linc00173 was upregulated in glioma tissues linc00173 knockdown inhibited the proliferation migration and invasion of glioma cells therefore our data discovered that linc00173 is a new oncogene in glioma for the first timecancer management and research submit your manuscript wwwdovepresscom dovepress 0cdu dovepressfigure linc00173 promoted glioma progression through mir765nutf2 pathway a and b nutf2 expression in glioma tissues and normal tissues according to tcga data using gepia tool and qrtpcr c and d proliferation was measured by cck8 assay e and f migration and invasion potential was determined by transwell assay p005lncrna has been found to serve as mirna sponge in tumor cells for instance lncrna ttnas1 sponges to promote breast cancer metastasis18 mir1405p lncrna cdkn2bas1 sponges mir3245p to regulate cellcycle progression in laryngeal squamous cell cancer19 previous studies also revealed linc00173 was a sponge for some mirnas such as mir4903p and mir2181314 in our study we did not observe linc00173 sponges above mirnas however through bioinformatics we identified linc00173 targeted mir765 in glioma we demonstrated their direct interaction and found that linc00173 overexpression inhibited mir765 expression mir765 has important roles in cancers mir765 was found to suppress tongue squamous cell carcinoma development20 mir765 also promotes myeloma and osteosarcoma progression2122 besides mir765 plays oncogenic or anticancer roles in gastric cancer and breast cancer2324 its role in glioma remains unclear our results suggested that mir765 was a tumor suppressor in gliomalncrnamirnamrna regulatory axis is widely observed in cancer for example linc00703mir181a klf6 axis suppresses the development of gastric cancer25 linc00312mir9cdh1 axis was found to promote breast cancer progression26 through bioinformatics we found that mir765 targeted nutf2 in glioma moreover we showed that nutf2 expression was regulated by linc00173mir axis the function of nutf2 in cancer is nearly unknown in our work we found that nutf2 expression was upregulated in glioma tissues compared to normal tissues moreover we found that nutf2 overexpression promoted the proliferation migration and invasion of glioma cells indicating nutf2 was an oncogene in gliomain conclusion our study showed that linc00173 acted as a sponge for mir765 to promote nutf2 expression and linc00173mir765nutf2 axis plays a critical function in promoting glioma progressionfunding this work was supported by zhejiang province analytical testing and experimental animal program lgd19h and zhejiang province welfare technology applied research project 2017c37111 disclosureall authors declare no conflicts of interest in this workreferences ostrom qt cioffi g gittleman h cbtrus statistical report primary brain and other central nervous system tumors diagnosed in the united states in neuro oncol 201921suppl 5v1“ v100 101093neuoncnoz150the of glioma huang l li x ye h et al long noncoding rna nck1as1 promotes sponging microrna13823p and activating the trim24wntbetacatenin axis j exp clin cancer res 101186s13046 tumorigenesis through chen w lei c liu p et al progress and prospects of recurrent glioma a recent scientometric analysis of the web of science in world neurosurg 2020134e387“e399 101016jwneu20 submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress du sun b meng m wei j wang s long noncoding rna pvt1 contributes to vascular endothelial cell proliferation via inhibition of mir190a5p in diagnostic biomarker evaluation of chronic heart failure exp ther med “ 103892etm20208599 feng s yao j chen y functional role of reprogrammingrelated long noncoding rna lincrnaror in glioma j mol neurosci “ 101007s120310140488z zhang d zhou h liu j mao j long noncoding rna asb16as1 promotes proliferation migration and invasion in glioma cells biomed res int sun l zhao m wang y neuroprotective effects of mir27a against traumatic brain injury via suppressing foxo3amediated neuronal autophagy biochem biophys res commun “ 101016jbbrc201612001 shi y sun h downregulation of lncrna linc00152 suppresses gastric cancer cell migration and invasion through inhibition of the erkmapk signaling pathway onco targets ther “ 102147otts217452 li k jiang y xiang x et al long noncoding rna snhg6 promotes the growth and invasion of nonsmall cell lung cancer by downregulating mir1013p thorac cancer wang x gu m ju y zhou j pik3c3 acts as a tumor suppressor in esophageal squamous cell carcinoma and was regulated by mir340 5p med sci monit 202026e920642 1012659msm923909 wei l chen z cheng n microrna126 inhibit viability of colorectal cancer cell by repressing mtor induced apoptosis and autophagy onco targets ther “ 102147 otts238348 chen y shen z zhi y long noncoding rna ror promotes radioresistance in hepatocellular carcinoma cells by acting as a cerna for microrna145 to regulate rad18 expression arch biochem biophys “ 101016jabb201803018 zeng f wang q wang s et al linc00173 promotes chemoresistance and progression of small cell lung cancer by sponging mir218 regulate etk expression oncogene “ to 101038s4138801909842 fan h yuan j li x et al lncrna linc00173 enhances triplenegative breast cancer progression by suppressing mir490 3p expression biomed pharmacother 1010 16jbiopha2020109987 sun sl shu yg tao my lncrna ccat2 promotes angiogenesis in glioma through activation of vegfa signalling by sponging mir424 mol cell biochem ““ 101007 s1101002003712y zhang y jiang x wu z et al long noncoding rna linc00467 promotes glioma progression through inhibiting p53 expression via binding to dnmt1 j cancer “ 107150 jca41942 yang q tang y tang c diminished linc00173 expression induced mir1825p accumulation promotes cell proliferation migration and apoptosis inhibition via agernfkappab pathway lung cancer am j transl res in nonsmallcell “ xue j zhang z li x ren q wang q long noncoding rna ttnas1 promotes breast cancer cell migration and invasion via sponging mir1405p oncol lett “ 1038 92ol201911222 liu f xiao y ma l wang j regulating of cell cycle progression by the lncrna cdkn2bas1mir3245prock1 axis in laryngeal squamous cell cancer int j biol markers “ 1011771724600819898489 ding j yang c yang s linc00511 interacts with mir765 and modulates tongue squamous cell carcinoma progression by targeting lamc2 j oral pathol med “ 101111 jop12677 long s long s he h chen g microrna765 is preregulated in multiple myeloma and serves an oncogenic role by directly targeting sox6 exp ther med “ 103892 etm20197473 lv db zhang jy gao k microrna765 targets mtus1 to promote the progression of osteosarcoma via mediating erkemt pathway eur rev med pharmacol sci “ 1026355eurrev_201906_18040 jiao y yuan c wu h li x yu j oncogenic microrna765 promotes the growth and metastasis of breast carcinoma by directly targeting ing4 j cell biochem yuan l ma t liu w et al linc00994 promoted invasion and proliferation of gastric cancer cell via regulating mir7653p am j transl res “ yang h peng m li y zhu r li x qian z linc00703 acts as a tumor suppressor via regulating mir181aklf6 axis in gastric cancer j gastric cancer “ 105230jgc2019 19e43 chen y qiu f huang l et al long noncoding rna linc00312 regulates breast cancer progression through the mir9cdh1 axis mol med rep “ 103892mmr201910895cancer management and research publish your work in this journal cancer management and research is an international peerreviewed open access journal focusing on cancer research and the optimal use of preventative and integrated treatment interventions to achieve improved outcomes enhanced survival and quality of life for the cancer patient the manuscript management system is completely online and includes a very quick and fair peerreview system which is all easy to use visit httpwwwdovepresscomtestimonialsphp to read real quotes from published authors dovepress submit your manuscript here wwwdovepresscomcancermanagementandresearchjournalcancer management and research submit your manuscript wwwdovepresscom dovepress 0c'
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" healthcare is an essential service at any time more so in the crisis like covid with increase in numberof cases and mortality from covid the primary focus is shifted to the management of the covid crisis and otherhealth emergencies thus affecting normal health services and routine treatment of other diseases like cancermethods this reviews the published literature and guidelines on covid and cancer and discusses them tooptimize the care of cancer patients during covid pandemic to improve treatment outcomesresults the results of the review of published literature show a twofold increase in probability of getting cov2infection by the cancer patients and a fourfold increase in chance of death on the other hand if left untreated a increase in cancer death is expected data further show that none of the medicines like remdesivir hydroxychloroquin dexamethasone or azithromycin improves survival and response to covid in cancer patients surgicalresults too show similar outcome before and after the pandemic though most of these report on highly selectedpatients populationss the covid pandemic places cancer patients in a very difficult situation wherein if they seektreatment they are exposing themselves to a risk of developing cov2 infection and if they do not the probabilityof dying without treatment increases hence for them it is a choice between the devil and deep sea and it is forthe healthcare providers to triage patients and treat who cannot wait even though the data from the carefullyselected cohort of patients show no increase in mortality or morbidity from treatment during covidkeywords covid cancer cov2 coronavirus treatment chemotherapy surgeryintroductionwith the onset of covid19 pandemic the situation forcancer patients has become a nightmare most of the patients who were on treatment in march had to miss outtheir further treatment due to lockdown and closure ofhospitals and all modes of transport both public and private those who developed cancer during lockdown toocould not reach doctors for the same reason surgerieswere postponed radiotherapy was postponed and there correspondence manojpandey66gmailcomdepartment of surgical oncology institute of medical sciences banarashindu university varanasi indiawas uncertainty as far as chemotherapy and immunotherapy were concerned every society and anization cameup with their own guidelines however none of these addressed the logistic problems that patients faced reachingthe treatment centers this lead to an increased pool ofuntreated patients an analysis from england estimatesadditional deaths from cancer over next monthsthat is increase proposed due to covid pandemic those who were willing to travel and take the risk ofgetting infected with covid19 too faced challenges dueto travel restrictions though the indian government issued special passes to allow travel for cancer treatment the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cchakraborty and pandey world of surgical oncology page of however the rider was that he or she has to travel withhis own private transport and not more than two peoplewere allowed in a four wheeler this meant that apartfrom the driver only the patient could travel withoutany caregiver if the patient does not own the car andcaregiver does not know how to drive these passeswere issued district or state wise as there was a ban oninterdistrict and interstate travel so the patients comingto bigger institutes like ours faced special problemswherein despite having a travel pass some of them hadto pay fines most of our patients come from neighboring states of bihar and madhya pradesh and even fromwest bengal and nepalapart from that at the beginning of the pandemicthings were not clear and there was no data on the incidence and mortality from covid19 in cancer patientsfurther though there were several guidelines [“] theywere not based on evidence as the evidence did not existat that time and it was not clear as to what treatmentmodalities are safe giving rise to uncertainties in themind of patients and health care workers the testing facilities were limited the availability of ppe was limitedand these were prioritized for suspected cases and contacts while other diseases were kept on hold or receivedlow priority this reviews the current literatureon various aspects of covid and cancermethodsa review of literature was carried out using the pubmedfor the s published using the string œcovid[allfields] and œcancer s[all fields] or œcancerated[all fields] or œcanceration[all fields] or œcancerization[all fields] or œcancerized[all fields] orœcancerous[all fields] or œneoplasms[mesh terms]or œneoplasms[all fields] or œcancer[all fields] orœcancers[all fields] following filters were applied clinical study clinical trial comparative study controlledclinical trial metaanalysis observational study pragmatic clinical trial randomized controlled trialthe s that discussed the incidence prevalencemortality treatment of cancer during covid or morbidity of treatment were included in the review as the obtained data was very heterogeneous no attempt atpooling or metaanalysis was doneresultsa total of s were extracted the of allthe s was read and after excluding reviews s were included in this review including one metaanalysis as for the prevalence the initial data from amulticentric study comparing cancer with noncancerpatients infected with covid found higher incidence ofsevere outcome in cancer compared to normal the outcome was worse in lung cancer and metastatic disease a study evaluating fatality in patients withcovid found a times risk of death in cancer patientscontracting covid this fear and uncertainty had affected the quality oflife and metal health of cancer patients caregivers andhealth care workers there were no socializing no outlets no markets or movies or dining out even the placesof worships were closed an italian survey of young cancer patients showed that they feel more vulnerable tocontract cancer and risk of higher complications ahigher burnout has been reported in healthcare workersworking in covid hospitals over a period of time things have started clearing upand preliminary data on incidence severity and complications of covid19 is now available a retrospectiveanalysis of patients across mainland china reported incidence of severe cases of these had at least one comorbidity with hypertension being most prevalent followed by diabetes in thisstudy the cancer was seen only in patients ofwhom had severe illness another study from chinaon patients with covid had patients with cancer they too found higher severity in cancer patientshowever the number of cancer patients in this study toowas very small similarly the study of chen had patients in total of covid19 patients andonly three of these had a severe disease similar resultshave been reported by others [ ]a pooled metaanalysis of studies reporting oncovid prevalence in cancer patients found a pooledprevalence of prevalence was higher at insmaller series reporting on less than patients between march and june over patients wereseen at our center of which undergoing surgery orthe interventions were tested for covid of these patients were found to be positive the prevalenceof cancer is high among covid19 patients in europehowever these do not seem to convert into higher mortality of cancer patients a study from uk reporting on deaths did not find increased mortality in cancer except in patients receiving chemotherapy however ametaanalysis involving patients thatincluded cancer patients showed a significant increase inhazard of death in cancer patients infected with covid hr the study also showed increased admission to icu hr however when patients abovethe age of were considered no difference in deathrate was observed this increase in cancer mortalityin covid19 has been attributed to advance age additional comorbidities diagnosis of lung cancer use ofchemotherapy etc [ “]recent data from the usa canada and spain fromthe covid19 and cancer consortium ccc19 database reported on patients with cancer of these 0cchakraborty and pandey world of surgical oncology page of patients had died advance age smokingmale gender more than one comorbidity and ecogmore than were found to be associated with increasedmortality these results and those detailed above clearlyshow that prevalence of covid in cancer is higher andmortality in these patients too is higher especially in patients with additional comorbidities and active diseaseand one needs to exercise full precaution while takingtreatment decisionsdiscussionprecautions taken at our center to prevent spread incancer patientsas cancer patients are at a higher risk of contractingcovid19 than the general population special precautionsare taken when patients are seen in the outpatient clinicopd and surgery no patient or the caregiver is allowedto enter the opd without a face mask all patients arescreened for body temperature and those found to havefever are immediately sent to covid opd this is followedby filling up of a symptom checklist and questionnairebased screening for all patients those with covid symptoms are referred for rtpcr testing before being allowedto see doctor and get the treatment this is similar to allothers coming to the hospital for treatment as there areno separate guidelines for cancer apart from that stricthand hygiene is followed and ppe has to be worn byhealth care workers those planning to undergo procedures and surgery are screened for covid19 using rtpcr testing they are kept in the holding area and afterbeing reported are shifted to wards any patient foundpositive is treated as per guidelinesplanned surgeryelective surgeries for cancer patients are avoided ifpossible this is usually a collective decision taken bythe oncologist in consultation with the patient if it isdecided to do a surgery a special covid19 consent istaken all emergency surgeries are performed withproper written covid consent and after covid testingfor even asymptomatic patients other than emergencies patients who cannot wait for weeks for surgeryas the disease may progress and become inoperableare considered for surgery other than that patientswho were started on neoadjuvant therapy before theonset of covid and have now completed neoadjuvanttherapy are also candidates for surgery as the opportunity of window period should not be lost any patient where the multidisciplinary team think can waitfor weeks is postponed patients undergoing surgeryare reported to be at higher risk than those treatedby radiation planned radiotherapyin the beginning all patients were postponed after discussion with patients or caregivers however as it is notpossible to postpone radiation forever selected patientsare now being taken up for radiation patients are informed that whatever little evidence that is available suggest higher chances of covid infection in this subset ofpatients recent guidelines have suggested omitting radiation for patients older than years using fast andfast forward omitting boost and hypofractionation[ ] recent s also suggest modification of thedelivery technique to reduce treatment time [ ]immunosuppressive therapythe current evidence does not support changing orwithholding chemotherapy targeted therapy and immunotherapy in cancer patients some of the studiessuggested stopping of immunotherapy for patients whohave a complete response or prolonged response ofmore than years a report on immune check pointinhibitors suggested that as there is not much immunecompromise and minimal hematological toxicities theycan be used with caution as patients are not devoid ofits benefits [ ] at our center we have continuedwith chemotherapy and immunotherapy and have notfound any increase in covid19 infection or mortality inthis sub groupstem cell transplantationcurrent guidelines recommend delaying the plannedallogenic stem cell transplant [ ] this is a uniquesituation where the donor and recipient both are at riskand a careful decision making keeping all factors into account be made no stem cell transplants took place atour center during this periodcoronavirus therapy in cancerthere is no evidence of benefit of using various therapies to treat covid19 in cancer patients remdesivir hasbeen approved by the fda for treatment of covid casesunder emergency use authorization the ccc19 studyfailed to show any benefit of azithromycin hydroxychloroquine alone or in combination in fact use of acombination of the two was found to be an independentfactor predicting mortality with hazard ratio of which was statistically significant there are occasional reports on the use of lopinavirritonavir [ ]in lung cancer patient with covid another dataset fromccc19 study reported on patients treated withhydroxychloroquine n azithromycin n remdesivir n highdose corticosteroids n tocilizumab n andother therapy n showed no benefit of anytreatment apart from slight activity of remdesivir no 0cchakraborty and pandey world of surgical oncology page of other drug showed any benefit [ ] patients also relowmolecular weightceived dexamethasone aspirinheparinabovetreatmentsanticoagulants besidesand otherresearch on covid2019 and cancera recent bioinformatic study using the geo databaseshowed that angiotensinconverting enzyme ace2 iselevated in uterine corpus endometrial carcinoma andrenal papillary cell carcinoma this increase also correlated with immune infiltrate present in the tumor theauthors suggested that these tumors may be more proneto covid19 infections however the authors failedto present any evidence to suggest this hypothesis similar bioinformatics study by fu also showed similarresults and they suggested that testes may also get affected by covid19 infections other tumors thatmay be affected are pancreas and colon howeverall these studies are based on geo databases and thereis no experimental evidence presented till datesthe literature on cancer and covid is fast expanding withmore and more information pouring in the literature sofar is clear in indicating that cancer patients are at highrisk of developing covid and when developed the severityand mortality is higher than the normal population therecent data also suggests a possible role of remdesivir inthe treatment of covid19 in cancer patients however theevidence to support this is very weak this absence of conclusive evidences has led to development of strategies totreat cancer safely most of these are based on reducingthe hospital visits and avoiding immune compromise theevidence is still evolving and more practice changes areexpected in the days to come and that may continue evenin post covid eraacknowledgementsnoneauthors™ contributionsmc prepared the draft and mp conceived and designed the and edited the final version the authors read and approved the finalmanuscriptfundingnoneavailability of data and materialsnot applicableethics approval and consent to participatethis does not report on human and animal experiments ethicalapproval and publication of consent is not applicableconsent for publicationnot applicablecompeting intereststhe authors declare that there are no conflicts of interestreceived june accepted august references wise j covid19 cancer mortality could rise at least because ofpandemic study finds bmj 2020369m1735 httpsdoi101136bmjm1735m1735civantos fj leibowitz jm arnold dj stubbs vc gross jh thomas gr sargiz casiano rr franzmann ej weed d perez c samuels m goodman kwgoodwin wj ethical surgical triage of head and neck cancer patientsduring the covid19 pandemic head neck coles ce aristei c bliss j boersma l brunt am chatterjee s hanna g jagsir kaidar po kirby a mjaaland i meattini i luis am marta gn offersen bpoortmans p rivera s international guidelines on radiation therapy forbreast cancer during the covid19 pandemic clin oncol r coll radiol “cortiula f pettke a bartoletti m puglisi f helleday t managing covid19in the oncology clinic and avoiding the distraction effect ann oncol “elkaddoum r haddad fg eid r kourie hr telemedicine for cancer patientsduring covid19 pandemic between threats and opportunities futureoncol “finley c prashad a camuso n daly c aprikian a ball cg bentley jcharest d fata p helyer l o'connell d moloo h seely a werier j zhongt earle cc guidance for management of cancer surgery during the covid pandemic can j surg 202063s2“finley c prashad a camuso n daly c earle cc lifesaving cancer surgeriesneed to be managed appropriately during the covid19 pandemic can jsurg 202063s1hanna tp evans ga booth cm cancer covid19 and the precautionaryprinciple prioritizing treatment during a global pandemic nat rev clinoncol “dai m liu d liu m zhou f li g chen z zhang z you h wu m zheng qxiong y xiong h wang c chen c xiong f zhang y peng y ge s zhen byu t wang l wang h liu y chen y mei j gao x li z gan l he c li zshi y qi y yang j tenen dg chai l mucci la santillana m cai h patientswith cancer appear more vulnerable to sarscov2 a multicenter studyduring the covid19 outbreak cancer discov “ deng g yin m chen x zeng f clinical determinants for fatality of patients with covid19 crit care “ casanova m pagani be silva m patriarca c veneroni l clerici ca spreaficof luksch r terenziani m meazza c podda m biassoni v schiavello echiaravalli s puma n bergamaschi l gattuso g sironi g massimino mferrari a how young patients with cancer perceive the covid19coronavirus epidemic in milan italy is there room for other fears pediatrblood cancer 2020e28318 wu y wang j luo c hu s lin x anderson ae bruera e yang x weis qian y a comparison of burnout frequency among oncologyphysicians and nurses working on the front lines and usual wardsduring the covid19 epidemic in wuhan china j pain symptommanag guan wj ni zy hu y liang wh ou cq he jx liu l shan h lei cl huidsc du b li lj zeng g yuen ky chen rc tang cl wang t chen pyxiang j li sy wang jl liang zj peng yx wei l liu y hu yh peng pwang jm liu jy chen z li g zheng zj qiu sq luo j ye cj zhu syzhong ns clinical characteristics of coronavirus disease in china nengl j med “ cai yc wang w li c zeng df zhou yq sun rh jiang h guo h wang sxjiang j treating head and neck tumors during the sarscov2 epidemic to sichuan cancer hospital head neck chen atc courafilho gb rehder mhh clinical characteristics of covid19in china n engl j med pii 101056nejmc2005203sa2 doi 1056nejmc200520310fong d rauch s petter c haspinger e alber m mitterer m infection rateand clinical management of cancer patients during the covid19pandemic experience from a tertiary care hospital in northern italy esmoopen 20205e000810 yu j ouyang w chua mlk xie c sarscov2 transmission in patients withcancer at a tertiary care hospital in wuhan china jama oncol 0cchakraborty and pandey world of surgical oncology page of zhang h xie c huang y treatment and outcome of a patient eith lungcancer infected with severe acute respiratory syndrome coronavirus2 jthorac oncol 202015e63“ rivera dr peters s panagiotou oa shah dp kuderer nm hsu cy rubinsteinsm lee bj choueiri tk de lima lg grivas p painter ca rini bi thompsonma arcobello j bakouny z doroshow db egan pc farmakiotis d fecher lafriese cr galsky md goel s gupta s halfdanarson tr halmos b hawley jekhaki ar lemmon ca mishra s olszewski aj pennell na puc mm revankarsg schapira l schmidt a schwartz gk shah sa wu jt xie z yeh ac zhu hshyr y lyman gh warner jl utilization of covid19 treatments and clinicaloutcomes among patients with cancer a covid19 and cancer consortiumccc19 cohort study cancer discov 2020cd0941 yang j li h hu s zhou y ace2 correlated with immune infiltration servesas a prognostic biomarker in endometrial carcinoma and renal papillary cellcarcinoma implication for covid19 aging albany ny “fu j zhou b zhang l balaji ks wei c liu x chen h peng j fu jexpressions and significances of the angiotensinconverting enzyme genethe receptor of sarscov2 for covid19 mol biol rep “ chai p yu j ge s jia r fan x genetic alteration rna expression and dnamethylation profiling of coronavirus disease covid19 receptor ace2in malignancies a pancancer analysis j hematol oncol “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations desai a sachdeva s parekh t desai r covid19 and cancer lessons from apooled metaanalysis jco glob oncol “ httpsdoi101200go2000097557559lee lyw cazier jb starkey t turnbull cd kerr r middleton g covid19mortality in patients with cancer on chemotherapy or other anticancertreatments a prospective cohort study lancet giannakoulis vg papoutsi e siempos ii effect of cancer on clinicaloutcomes of patients with covid19 a metaanalysis of patient data jcoglob oncol “ httpsdoi101200go2000225799808 gosain r abdou y singh a rana n puzanov i ernstoff ms covid19 andcancer a comprehensive review curr oncol rep “ addeo a obeid m friedlaender a covid19 and lung cancer risksmechanisms and treatment interactions j immunother cancer e000892 yang k sheng y huang c jin y xiong n jiang k lu h liu j yang j dongy pan d shu c li j wei j huang y peng l wu m zhang r wu b li ycai l li g zhang t wu g clinical characteristics outcomes and risk factorsfor mortality in patients with cancer and covid19 in hubei china amulticentre retrospective cohort study lancet oncol “ mehta v goel s kabarriti r cole d goldfinger m acunavillaorduna apradhan k thota r reissman s sparano ja gartrell ba smith rv ohri ngarg m racine ad kalnicki s perezsoler r halmos b verma a casefatality rate of cancer patients with covid19 in a new york hospitalsystem cancer discov “ meng y lu w guo e liu j yang b wu p lin s peng t fu y li f wang zli y xiao r liu c huang y lu f wu x you l ma d sun c wu p chen gcancer history is an independent risk factor for mortality in hospitalizedcovid19 patients a propensity scorematched analysis j hematol oncol“kuderer nm choueiri tk shah dp shyr y rubinstein sm rivera dr shetes hsu cy desai a de lima lg jr grivas p painter ca peters s thompsonma bakouny z batist g bekaiisaab t bilen ma bouganim n larroya mbcastellano d del prete sa doroshow db egan pc elkrief a farmakiotis dflora d galsky md glover mj griffiths ea gulati ap gupta s hafez nhalfdanarson tr hawley je hsu e kasi a khaki ar lemmon ca lewis clogan b masters t mckay rr mesa ra mans ak mulcahy mfpanagiotou oa peddi p pennell na reynolds k rosen lr rosovsky rsalazar m schmidt a shah sa shaya ja steinharter j stockerlgoldstein kesubbiah s vinh dc wehbe fh weissmann lb wu jt wulffburchfield exie z yeh a yu pp zhou ay zubiri l mishra s lyman gh rini bi warnerjl clinical impact of covid19 on patients with cancer ccc19 a cohortstudy lancet huang sh o'sullivan b su j ringash j bratman sv kim j hosni a bayleya cho j giuliani m hope a spreafico a hansen ar siu ll gilbert r irishjc goldstein d de aj tong l xu w waldron j hypofractionatedradiotherapy alone with gy per fraction for head and neck cancerduring the covid19 pandemic the princess margaret experience andproposal cancer “ vordermark d shift in indications for radiotherapy during the covid19pandemic a review of anspecific cancer managementrecommendations from multidisciplinary and surgical expert groups radiatoncol “ davis ap boyer m lee jh kao sc covid19 the use of immunotherapy inmetastatic lung cancer immunotherapy “kattan j kattan c assi t do checkpoint inhibitors compromise the cancerpatients' immunity and increase the vulnerability to covid19 infectionimmunotherapy “ bersanelli m controversies about covid19 and anticancer treatment withimmune checkpoint inhibitors immunotherapy “ ardura mi hartley dm dandoy c lehmann l jaglowski s auletta jjaddressing the impact of the coronavirus disease covid19 pandemic onhematopoietic cell transplantation learning networks as means for sharingbest practices biol blood marrow transplant mahmoudjafari z alexander m roddy j shaw r shigle tl timlin c culosk american society for transplantation and cellular therapy pharmacyspecial interest group position statement on pharmacy practicemanagement and clinical management for covid19 in hematopoietic celltransplantation and cellular therapy patients in the united states biol bloodmarrow transplant 0c"
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" the choice of treatment in patients with metastatic colorectal cancer mcrc is generally influenced by tumour and patient characteristics treatment efficacy and tolerability and quality of life better patient selection might lead to improved outcomesmethods this post hoc exploratory analysis examined the effect of prognostic factors on outcomes in the randomized double blind phase study of trifluridine tipiracil ftdtpi plus best supportive care bsc versus placebo plus bsc in patients with mcrc refractory to standard chemotherapies recourse trial patients were redivided by prognosis into two subgroups those with metastatic sites at randomisation low tumour burden and ‰¥ months from diagnosis of metastatic disease to randomisation indolent disease were included in the good prognostic characteristics gpc subgroup the remaining patients were considered to have poor prognostic characteristics ppcresults gpc patients n386 had improved outcome versus ppc patients n414 in both the trifluridinetipiracil and placebo arms gpc patients receiving trifluridinetipiracil n261 had an improved median overall survival vs months hr ci to p00001 and progression free survival vs months hr ci to p00001 than ppc patients receiving trifluridinetipiracil n273 improvements in survival were irrespective of age eastern cooperative oncology group performance status ecog ps kras mutational status and site of metastases at randomisation in the trifluridinetipiracil arm time to deterioration of ecog ps to ‰¥ and proportion of patients with ps0“ discontinuing treatment were longer for gpc than for ppc patients vs months and vs respectively low tumour burden and indolent disease were factors of good prognosis in late line mcrc with patients experiencing longer progression free survival and greater overall survivalintroductioninclusion of new therapeutic options into the current treatment landscape in metastatic colorectal cancer mcrc has led to an increased survival in the last couple of key questionswhat is already known about this subject –º the choice of treatment in patients with metastatic colorectal cancer mcrc is generally influenced by tumour characteristics and patient factors as well as treatment characteristics such as tolerability efficacy and quality of life effects trifluridinetipiracil is indicated in pretreated patients with mcrc based on results of the pivotal randomised double blind phase study of trifluridine tipiracil ftdtpi plus best supportive care bsc versus placebo plus bsc in patients with metastatic colorectal cancer refractory to standard chemotherapies recourse trial which demonstrated significantly improved overall survival os compared with placebo with a manageable safety profilewhat does this study add –º in recourse classification of patients as having good prognostic characteristics gpc defined as those with low tumour burden metastatic sites at randomisation and less aggressive disease ‰¥ months from diagnosis of first metastasis at randomisation identified a subgroup of patients with improved os and progression free survival with trifluridinetipiracil compared with patients with poor prognostic characteristics treated with trifluridinetipiracil and gpc patients treated with placebohow might this impact on clinical practice –º low tumour burden and indolent disease were shown to be factors of good prognosis in late line mcrc with these patients experiencing longer time on treatment and greater os this suggests that these patients could be candidates to receive further lines of therapy post trifluridinetipiracildecades1“ first line treatment of patients typically involves the use of vascular endothelial growth factor vegf or epidermal growth factor receptor egfr targeted agents eg bevacizumab cetuximab panitumumab to fluoropyrimidine based fluorouracil or tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accesscapecitabine chemotherapy regimens depending on the presence or absence of ras mutation positive disease2 in the usa immunotherapies nivolumab±ipilimumab or pembrolizumab are also recommended for the treatment of patients with mismatch repair deficient or microsatellite instability high disease4 in the second line setting vegf targeted treatments eg aflibercept ramucirumab can also be used in combination with chemotherapy2 the optimal chemotherapeutic regimen for use beyond third line remains unclear where resistantrefractory disease and residual toxicity potentially limit the treatment options with only two possible candidates at present5the general condition and performance status of a patient are strong prognostic and predictive factors for mcrc treatment2 fitter patients are typically assigned to a more intensive treatment approach ie a combination of “ cytotoxic agents with a biological agent than less fit patients2 the choice of treatment in the metastatic setting is generally influenced by tumour characteristics tumour burden localisation and biology patient characteristics age eastern cooperative oncology group performance status ecog ps an function and comorbidities and treatment characteristics efficacy toxicity profile administration and quality of life qol effects2the proportion of patients with mcrc receiving active treatment decreases from line to line leaving more than half of patients who received an active treatment in the first line without treatment in the third line setting even in randomised clinical trials in folfiri plus cetuximab versus folfiri plus bevacizumab as first line treatment for patients with metastatic colorectal cancer only of patients reached third line6 data from the usa indicate that only of patients receiving a first line of treatment move into the second line move to the third line and only will receive a fourth line of treatment7 being unable to receive a subsequent line of treatment therefore appears to have a negative impact on the patient™s survivalis trifluridinetipiracil ftdtpi lonsurf indicated for the treatment of adult patients with mcrc who have been previously treated with or are not considered candidates for available therapies including fluoropyrimidine based oxaliplatin based and irinotecan based chemotherapies anti vegf agents and anti egfr agents for eligible patient ras wild type combination of tipiracil hydrochloride with the nucleoside metabolic inhibitor trifluridine improves its bioavailability by inhibiting its catabolism by thymidine phosphorylase8 the relatively limited non haematological toxicity of trifluridinetipiracil makes it a good option in the third line and refractory settings2 in the pivotal phase iii randomised double blind phase study of trifluridine tipiracil ftdtpi plus best supportive care bsc versus placebo plus bsc in patients with metastatic colorectal cancer refractory to standard chemotherapies recourse trial conducted in patients with mcrc eligible for treatment in the third line and beyond treatment with trifluridinetipiracil versus placebo extended overall survival median os vs months hr p0001 and progression free survival median pfs vs months hr p000110 this effect was shown in all subgroups regardless of age ecog ps “ geographical region race and kras mutational status10 furthermore trifluridinetipiracil was well tolerated with few serious adverse events aes reported haematological toxicities were the most frequently observed aes10 also time to deterioration of ecog ps to ‰¥ was significantly improved median vs months hr p000110 with of patients treated with trifluridinetipiracil remaining at ps “ at discontinuation11 remaining at ecog ps “ is important as it could allow patients to further benefit from subsequent therapy and potentially extend their survival in recourse and of patients treated with trifluridinetipiracil remained alive at and months respectively in the refractory setting in the post hoc analysis described here we set out to explore other factors that could extend survival in the recourse population for the purposes of our exploratory analysis we defined the characteristics of good prognosis as low tumour burden metastatic sites by response evaluation criteria in solid tumors recist evaluation at randomisation and less aggressiveindolent disease ‰¥ months from diagnosis of first metastasis to randomisation which are known to be strong prognostic factors in patients with mcrc with good ecog ps12 our ultimate aim is to explore how clinicians can better predict individual treatment outcomes and support treatment selection through the continuum of carematerials and methodsstudy design and patientsthe study design and methodology of the recourse trial clinicaltrials gov number nct01607957 have been previously published10 in brief recourse was a phase iii randomised double blind placebo controlled study comparing the efficacy and safety of trifluridinetipiracil plus best supportive care with those of placebo plus best supportive care10 this study included patients with metastatic biopsy provendocumented adenocarcinoma of the colon or rectum who were previously treated with ‰¥ standard chemotherapy regimens or who had tumour progression within months of their most recent chemotherapy or who had clinically significant aes precluding readministration of standard chemotherapies patients were randomised to trifluridinetipiracil mgm2 two times a day on days “ and “ every weeks or matching placebo10 randomisation was stratified according to kras mutation status wild type vs mutant time from diagnosis of first metastasis to randomisation vs ‰¥ months and geographical region japan vs usa european union and australia10 all patients had adequate an function and were ecog ps tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0cof “ at inclusion10 the primary endpoint of the study was os and secondary endpoints included pfs objective response rate clinical benefit rate and safety10patient subgroupsin examining the effects of prognostic factors on treatment outcomes in the current analysis several subgroups of recourse patients were considered patients from recourse n800 were divided according to good prognostic characteristics gpc and poor prognostic characteristics ppc good prognosis was considered to be defined by low tumour burden metastatic sites by recist tumour evaluation at randomisation and less aggressiveindolent disease ‰¥ months from diagnosis of first metastasis to randomisation12 of the gpc subgroup n386 patients received trifluridinetipiracil and received placebo the remaining patients were included in the complementary ppc subgroup n414 of these received trifluridinetipiracil and received placeboanalysis outcomesos and pfs in the gpc subgroup were compared with those in the ppc subgroup these subgroups were then analysed according to other tumour and patient characteristics that is metastatic site at randomisation for those sites present in of the population liver lung lymph or peritoneum ecog ps vs kras mutation status wild type vs mutant and age vs ‰¥ years os and pfs with trifluridinetipiracil were compared with placebo and were analysed according to prognostic subgroups within each of the two arms finally the effect of prognostic classification of patients on ecog ps deterioration was analysed for all patients and subgroupsstatistical methodsdemographic and baseline characteristics of patients were summarised by treatment arm and subgroups using descriptive statistics n mean sd median minimum and maximum andor frequency distributions as appropriatethe differences in os pfs and time to ecog ps deterioration between trifluridinetipiracil and placebo patients or between subgroups of patients in a specific arm of treatment were assessed using the stratified log rank test stratification factors used for the randomisation from a cox proportional hazards model for each arm or each subgroup survival was summarised using kaplan meier curves and was further characterised in terms of the median with the corresponding two sided cisresultspatientsbaseline patient demographics and clinical characteristics were generally similar between gpc and ppc patients table in the trifluridinetipiracil arm slight imbalances were seen in ecog ps more gpc than ppc open accesspatients had an ecog ps of and kras status more gpc than ppc patients were kras wild type also more gpc than ppc patients had received ‰¥ prior regimens among the ppc group treated with trifluridinetipiracil of patients had ‰¥ months from diagnosis of first metastasis to randomisation but had ‰¥ metastatic sites and of patients had metastatic sites but months from diagnosis of first metastasis similar differences were observed in the placebo arm with the exception of kras status which was comparable in the gpc and ppc subgroupstreatmentamong trifluridinetipiracil treated patients those in the gpc group received more treatment cycles mean sd compared with patients in the ppc group mean sd online supplementary table s1 a higher proportion of gpc patients than ppc patients receiving trifluridinetipiracil had a dose delay vs respectively or dose reduction vs respectively which is consistent with a longer duration of treatment online supplementary table s1 however median dose intensity in the first four cycles was high ‰¥ and did not differ markedly between the groups cycle in the gpc group and the ppc group cycle and respectively cycle and respectively cycle and respectivelythe effect of good versus poor prognosis classifications on survivalsurvival curves for the gpc versus ppc subgroups are shown in figure median os was longer in the gpc subgroup than the ppc subgroup for both trifluridinetipiracil vs months hr ci to p00001 figure 1a and placebo vs months hr ci to p00001 figure 1b rates of month os and in gpc and and in ppc for trifluridinetipiracil and placebo respectively and month os and in gpc and and in ppc for trifluridinetipiracil and placebo respectively were also higher in gpc subgroups compared with ppc subgroups median pfs with trifluridinetipiracil was also longer in the gpc subgroup versus the ppc subgroup vs months hr ci to p00001 respective values for gpc versus ppc in the placebo arm were versus months hr p00699 pfs at and months in the ppc subgroup was and for trifluridinetipiracil and and for placebo respectively in the gpc subgroup these were and with trifluridinetipiracil and and with placebo respectivelyeffects of good prognostic factors on the relative efficacy of trifluridinetipiracilmedian os was prolonged with trifluridinetipiracil versus placebo in both subgroups but to a greater tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accesstable baseline patient demographics and clinical characteristics according to prognosistrifluridinetipiracilplacebogpc subgroup n261 ppc subgroup n273 gpc subgroup n125ppc subgroup n141 females male asian other years to years ‰¥ yearsmedian age yearspatient age n gender n ethnicity n ecog ps n kras status n time since diagnosis of metastasis n number of prior regimens n number of metastatic sites n site of metastatic lesion n   primary site of disease n liver lung lymph peritoneum ‰¥ “ ‰¥ mutant wild type months ‰¥ months colon rectum defined as metastatic sites and ‰¥ months since first metastasis only those in more than of the intent to treat population are included liver lung lymph and peritoneumecog ps eastern cooperative oncology group performance status gpc good prognostic characteristics ppc poor prognostic characteristicsextent in the gpc subgroup than in the ppc subgroup figure 2a similarly median pfs was prolonged with trifluridinetipiracil versus placebo in both subgroups with the greatest magnitude of benefit observed in the gpc patients figure 2banalysis of prognostic factorsthe effect of various prognostic factors on median os and pfs is shown in table their effect on month and month os and month month and month pfs is shown in online supplementary tables and for both tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accessfigure overall survival os for the good prognostic characteristics gpc and poor prognostic characteristics ppc subgroups in patients receiving a trifluridinetipiracil or b placebo ap0001 one sided bp0001 two sided ftdtpi trifluridinetipiracil mos median overall survival nr not reachedtrifluridinetipiracil and placebo the gpc subgroup had better median os and pfs than the ppc subgroup irrespective of patient age ‰¥ vs years ecog ps vs kras mutation status mutant vs wild type and liver metastases yes vs nowhen analysing the gpc subgroup the absence of liver metastasis at randomisation n153 representing of the gpc and of the intent to treat population was found to be the best factor of prognosis further information on this group of patients is available in online tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accessa overall survival os b progression free survival pfs and c time to eastern cooperative oncology group figure performance status ecog ps ‰¥ with trifluridinetipiracil versus placebo in the good prognostic characteristics gpc n386 and poor prognostic characteristics ppc n414 subgroups ftdtpi trifluridinetipiracil mos median overall survivalsupplementary table s4 and online supplementary figures s1 s3 among gpc patients treated with trifluridinetipiracil median os was months longer in patients with no liver metastases compared with those with liver metastases vs months table the month os rate in gpc patients treated with trifluridinetipiracil was in those without liver metastases and in those with liver metastases corresponding month os rates in these groups were and respectively online supplementary table s2 median os was also longer in patients with no liver metastases compared with those with liver metastases in the trifluridinetipiracil ppc subgroup vs months and both the gpc and ppc subgroups of the placebo arm vs months and vs months respectively table in the group of ppc patients treated with trifluridinetipiracil the month and month os rates were and respectively in those without liver metastases compared with and respectively in those with liver metastases online supplementary table s2 for the trifluridinetipiracil and placebo arms patients with baseline ecog ps had higher median os compared with ecog ps patients in both the gpc and ppc subgroups table in the trifluridinetipiracil arm age or ‰¥ years and kras status did not seem to affect the treatment outcome table similar results were found for pfs with an effect for all trifluridinetipiracil gpc and ppc subgroups with median pfs values ranging from to months table among gpc patients treated with trifluridinetipiracil the month pfs rate was in those with no liver metastases compared with in those with liver metastases corresponding month pfs rates in the ppc group of patients treated with trifluridinetipiracil were and respectively online supplementary table s3 no such effect was observed in the placebo arm with values ranging “ months whatever the prognosis at the outset for almost all subgroups median tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0ctable the effect of various prognostic factors on median overall survival os and progression free survival pfsnumber of patientsftdtpi placebomedian survival monthshr cinumber of patientsftdtpiplacebomedian survival monthshr ciopen accessosgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgrouppfsgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroup vs vs no liver metastasesn97n56n35n21no lung metastasesn89n25n54n24no lymph metastasesn208n93n116n53no peritoneal metastasesn242n119n203n100ecog ps0n158n77n143n70age yearsn137n72n163n76kras wild typen142n61n120n70 vs vs vs vs vs vs vs vs vs vs vs vs no liver metastasesn97n56n35n21no lung metastasesn89n25n54n24no lymph metastasesn208n93n116n53no peritoneal metastasesn242n119n203n100ecog ps0n158n77n143n70age yearsn137n72n163n76kras wild typen142n61n120n70 vs vs vs vs vs vs vs vs vs vs vs vs vs vs to to to to to to to to to to to to to to to to to to to to to to to to to to to to liver metastasesn164n69n238n120lung metastasesn172n100n219n117lymph metastasesn53n32n157n88peritoneal metastasesn19n6n70n41ecog ps1n103n48n130n71age‰¥ yearsn124n53n110n65kras mutantn119n64n153n71liver metastasesn164n69n238n120lung metastasesn172n100n219n117lymph metastasesn53n32n157n88peritoneal metastasesn19n6n70n41ecog ps1n103n48n130n71age ‰¥ yearsn124n53n110n65kras mutantn119n64n153n71 vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to good prognostic characteristics gpc were defined as metastatic sites at randomisation and ‰¥ months from first metastasis to randomisationftdtpi trifluridinetipiracil ppc poor prognostic characteristics ecog ps eastern cooperative oncology group performance statustabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accesstable effects of prognostic classification of patients on eastern cooperative oncology group performance status ecog psmedian time to deterioration to ecog ps ‰¥ monthsftdtpiplaceboitt population n80011good prognosis patients n386poor prognosis patients n414ftdtpi trifluridinetipiracil itt intent to treatpfs was longer and all hrs favoured treatment with trifluridinetipiracil table effects of prognostic classification of patients on ecog psdata relative to the effect of ecog ps are presented in table the proportion of gpc patients treated with trifluridinetipiracil with an ecog ps of “ at treatment discontinuation was among gpc patients with an ecog ps of at baseline had not deteriorated beyond a ps of “ at treatment discontinuation similarly among gpc patients with an ecog ps of at baseline had not deteriorated beyond a ps of “ at treatment discontinuation the median time to deterioration of ecog ps to ‰¥ in patients receiving trifluridinetipiracil was months in the gpc subgroup and months in the ppc subgroup figure 2ctolerability and safetythe most common aes in patients receiving trifluridinetipiracil were nausea anaemia neutropenianeutrophil count decrease diarrhoea fatigue and reduced appetite online supplementary table s5 the most common grade ‰¥ aes experienced by patients receiving trifluridinetipiracil were haematological anaemia neutropenianeutrophil count decrease white blood cell count decrease there was no evidence of a higher incidence of aes in patients with ppc versus gpc in the group receiving trifluridinetipiracil but there was a trend towards a higher incidence of aes in placebo recipients with ppc compared with gpc online supplementary table s5discussionthe results of our analysis show that patients in the gpc subgroup consistently performed better than those in the ppc subgroup in both the trifluridinetipiracil and placebo arms within the same subgroups patients treated with trifluridinetipiracil performed better than placebo trifluridinetipiracil has consistently been shown to provide a significant survival benefit to patients with mcrc refractory to standard therapy with a well tolerated safety profile in three large scale randomised clinical trials10 “ a previous subanalysis of recourse showed that trifluridinetipiracil was more effective than placebo in patients irrespective of region age racialhr ci to to to p valueecog ps “ at treatment discontinuation ftdtpiplaceboethnic differences or kras mutation status17 in the current analysis further categorisation of patients as having good prognosis using the criteria of metastatic sites by recist tumour evaluation at randomisation and ‰¥ months from diagnosis of first metastasis to randomisation12 identified a subgroup of patients with improved os and pfs with trifluridinetipiracil compared with poorer prognosis patients ie those with ‰¥ metastatic sites and months from first metastasis pfs and os were also improved in gpc patients treated with trifluridinetipiracil compared with gpc patients who received placebopatients with gpc received more cycles of treatment than patients with ppc because progression was delayed in this group which may have contributed to the better survival outcomes the difference cannot be explained by a difference in dose intensity since this was high and similar in both the ppc and gpc subgroups of patients receiving trifluridinetipiracil in addition there was no evidence for higher toxicity in the ppc than the gpc group in fact the haematological aes occurred at a slightly higher rate in gpc patients than in ppc patients who received trifluridinetipiracil which probably reflects a longer exposure to treatment in the gpc group more patients in the gpc than in the ppc subgroup had dose delays which suggests that grade ‰¥ haematological aes were appropriately managed during treatmentit is thought that the availability of more treatment options for mcrc has contributed to an improvement in os over the last years3 indeed a retrospective study in elderly patients aged ‰¥ years a patient population more prone to comorbidities poor performance status and the development of treatment related toxicity reported a correlation between os and the number of treatment lines received18 thus maintaining the general condition and performance status of a patient throughout the continuum of care is of great importance especially beyond the second line to ensure patients remain fit with good qol5 our analysis showed that the majority of patients in the gpc subgroup discontinued treatment with an ecog ps of “ at the time of disease progression suggesting that these patients could be candidates to receive further lines of therapy post trifluridinetipiracil this is important when sequencing through the continuum of care this is in line with other tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0canalyses indicating preservation of health related qol on treatment of patients with mcrc with trifluridinetipiracil19 while the post hoc nature of this analysis limits it to an exploratory analysis the relatively large number of patients analysed make these data a good tool to estimate the expected outcomes when treating patients with refractory mcrc with trifluridinetipiracil the smaller size of some of the subgroups may limit the s that can be drawn thus preventing an evaluation of other parameters that might impact on outcomes such as lactate dehydrogenase levels the exact definition of good and poor prognostic factors12 may require further validation in a prospective cohortthe current analysis shows that compared with poor prognosis patients treated with either trifluridinetipiracil or placebo and good prognosis patients treated with placebo patients with gpcs treated with trifluridinetipiracil adequate an function ecog ps “ metastatic sites by recist tumour evaluation at randomisation and ‰¥ months from diagnosis of first metastasis have an increased survival in terms of median os and month and month survival rates treatment with trifluridinetipiracil is effective and provides the majority of patients the opportunity to maintain ecog ps and the possibility to receive further treatment options through the continuum of careauthor affiliations1vall d™hebron institute of oncology uvic ucc medical oncology vall d'hebron hospital barcelona catalunya spain2vall d™hebron institute of oncology uvic ucc iob quironmedical oncology vall d'hebron hospital barcelona catalunya spain3medical oncology ospedale policlinico san martino istituto di ricovero e cura a carattere scientifico per l'oncologia genova liguria italy4department of medical oncology university hospital centre besançon besancon bourgogne franche comté france5kashiwa national cancer center hospital east kashiwa chiba japan6department of medical oncology dana farber cancer institute boston massachusetts usa7centre of excellence methodology and valorization of data centex mvd institut de recherches internationales servier suresnes france8global medical affairs les laboratoires servier sas suresnes île de france france9digestive oncology ku leuven university hospitals leuven leuven flanders belgiumacknowledgements the authors would like to thank andrea bothwell who wrote the first draft of this manuscript on behalf of springer healthcare communications this medical writing assistance was funded by institut de recherches internationales servier suresnes francecontributors jt and srmv contributed to the conception and design of the study all authors were involved in the acquisition analysis and interpretation of data and in writing andor revising drafts of the manuscript all authors have read and approved the final draft of the manuscript and accept responsibility for the finished article and the decision to submit the manuscript for publicationfunding the recourse study was funded by taiho oncology and taiho pharmaceutical co this analysis was funded by servier in partnership with taihocompeting interests jt has received personal fees from array biopharma astrazeneca bayer ag beigene boehringer ingelheim chugai genentech open accessgenmab as halozyme imugene limited inflection biosciences limited ipsen kura oncology eli lilly and company merck menarini merck serono merrimack pharmaceuticals merus molecular partners novartis peptomyc pfizer pharmacyclics proteodesign sl rafael pharmaceuticals f hoffmann la roche sanofi seattle genetics servier symphogen taiho pharmaceutical vcn biosciences biocartis foundation medicine haliodx sas pharmaceuticals and roche diagnostics ga has had an advisory role or received honoraria or travel grants from hoffmann la roche merck serono amgen sanofi bayer servier and bristol myers squibb afs has had an advisory role for amgen bayer celgene roche merck serono sanofi and servier and has attended a speakers™ bureau for amgen astrazeneca bayer bristol myers squibb celgene lilly merck serono roche sanofi and takeda evc has received research funding from amgen bayer boehringer ingelheim celgene ipsen lilly merck merck kga novartis roche sanofi and servier and has attended advisory boards for astellas astrazeneca bayer bristol myers squibb celgene lilly merck sharp dohme merck kgaa novartis roche and servier cb has attended advisory boards for roche servier and sanofi and has received a research grant from roche ao has received honoraria from ono bms chugai taiho eisai and amgen and has received research funding from bristol myers squibb an immediate family member of ao has been employed by celgene rjm declares no conflicts of interest lv and srmv are employees of servierpatient consent for publication not requiredethics ap
0
the periparturient period is one of the most challenging periods in dairy cows and encompasses the a0wk prior to and a0wk after parturition the nutrient requirements of dairy cows change greatly during this time largely due to the exponential growth of the gravid uterus and fetus followed by the demands of lactation nrc inflammation oxidative stress and adipose tissue mobilization lead to a reduction in dry matter intake dmi during the periparturient period this reduction in dmi leads to a negative nutrient balance nnb with a shortfall in the nutrient availability for the cow and fetus ingvartsen and andersen additionally this reduction in dmi also increases the risk of metabolic ketosis fatty liver milk fever and immunerelated disorders the risk of these diseases poor reproduction and low efficiency is greatly impacted by the degree and length of time during which these systems metabolism and immune response remain out of balance loor et a0al 2013a much of the research over the last decade have examined these biological interactions to identify the mechanisms behind the metabolic physiologic and immune adaptations that occur during the periparturient period loor et a0al 2013a 2013b roche et a0al bradford et a0al it is now known that nutrients such as amino acids aa serve functional roles outside of their use as building blocks for proteins and have immunomodulatory properties and interact through common biochemical pathways eg 1carbon metabolism figure a0 this concept has been well explored in nonruminant species li et a0 al sikalidis with nutritional management during the periparturient period continuing to be an active area of research it is important to develop a system understanding the potential immunometabolic role that dietary aa may play during this period thus the objective of this review is to provide an overview of physiological adaptations during the periparturient period the immune system and methods to assess immune function and oxidative stress this will be followed by a more specific discussion of the immunometabolic roles of specific aa and their potential effects in dairy cow during the periparturient period the potential effects of enhanced aa supply during the preweaning period will also be discussed brieflybiological adaptations in the transition cow”a brief overviewduring the nnb associated with the periparturient period biological mechanisms coordinate the mobilization of body reserves in order to support fetal growth and milk production bauman and currie insulin concentrations are reduced and the response of hormonesensitive lipase in adipose tissue eg low insulin high growth hormone and catecholamines or high glucocorticoid concentrations is greater to facilitate lipid mobilization this periparturient period is also characterized by a state of inflammation encompassing an increase in hepatic production of positive acutephase proteins app such as haptoglobin and serum amyloid a a0saa and a decrease in the production ingvartsen the authors published by oxford university press on behalf of the american society of animal sciencethis is an open access distributed under the terms of the creative commons attribution noncommercial license httpcreativecommonslicensesbync40 which permits noncommercial reuse distribution and reproduction in any medium provided the original work is properly cited for commercial reuse please contact spermissionsoupcoms175 0cs176 of animal science vol no suppl abbreviationsaa akt app asct2 bcaa bcat bcka bhmt bmec bsa cbs csad cth dmi gator1 gcl gclc gpx gsh gsr gss il inos kegg klh lps mpo mtor mtorc1 mtr nfe2l2 nfκb nnb nos pc pmn pmnl pmtor rns rom ros rpm saa sahh sam samtor sell shmt slc1a1 slc1a5 sod stat tag th tlr tnfα vldl amino acidsprotein kinase bacutephase proteinsalanineserinecysteine transporter branchedchain amino acidsbranchedchain amino transferasesbranchedchain αketoacidsbetaine homocysteine methyltransferasebovine mammary epithelial cellsbovine serum albumincystathionine synthasecysteine sulfinic acid decarboxylasecystathionine gammalyasedry matter intakegtpaseactivating protein activity towards rags glutamate cysteine ligaseglutamate cysteine ligase catalyticglutathione peroxidaseglutathioneglutathione reductaseglutathione synthaseinterleukininducible noskyoto encyclopedia of genes and genomeskeyhole limpet hemocyaninlipopolysaccharidemyeloperoxidasemechanistic target of rapamycinmtor complex 5methyltetrahydrofolatehomocysteine methyltransferasenuclear factor erythroid 2like2nuclear factor kappa bnegative nutrient balancenitric oxide synthasephosphatidylcholinepolymorphonuclear neutrophilspolymorphonuclear leukocytesphosphorylated mtorreactive nitrogen speciesreactive oxygen metabolitesreactive oxygen speciesrumenprotected metserum amyloid asadenosyl homocysteine hydrolasesadenosyl methioninesadenosylmethionine sensor upstream of mtorlselectinserine hydoxymethyltransferasesolute carrier family member solute carrier family member superoxide dismutasesignal transducer and activator of transcriptiontriacyglycerolthelpertolllike receptorstumor necrosis factorαverylowdensity lipoproteinsof negative app such as albumin bertoni et a0 al it has been well established that these responses are mediated by the proinflammatory cytokines interleukin il6 il1 and tumor necrosis factorα tnfα kindt et a0al additionally oxidative stress also occurs during this period and is driven by the imbalance between the production of reactive oxygen metabolites rom reactive nitrogen species rns and the neutralizing capacity of antioxidant mechanisms in tissues and blood some of the wellestablished cellular antioxidants include glutathione gsh taurine superoxide dismutase sod and vitamins a a0and e bernabucci et a0al when oxidative stress overwhelms cellular antioxidant capacity rom induce an inflammatory response that is controlled via changes in mrna abundance of transcription regulators eg signal transducer and activator of transcription [stat3] nuclear factorkappa b [nfκb] the increase in oxidative stress and inflammation during this period is also negatively associated with a reduction in liver functionality and measurement of app can provide a useful tool to assess liver function as well as inflammation bertoni and trevisi during the periparturient period aa metabolism is also altered with moderate carcass protein losses reported even when animals are fed to their predicted metabolizable protein requirements bell et a0 al additionally circulating aa concentrations change dramatically with favorable circulating profiles of many aa not being restored until d postpartum zhou et a0al 2016b furthermore the total concentration of aa in plasma reaches a nadir around day postpartum zhou et a0al 2016b which corresponds to a peak in total disease incidence during early lactation ingvartsen this decrease in circulating aa is likely associated with the increased use of aa for gluconeogenesis as well as for hepatic production of app thus it is important to investigate how supplemental aa during the periparturient period may modulate metabolism and immune responses to promote production and reduce susceptibility to diseasegeneral overview of the immune a0systemthe immune system consists of both the adaptive and innate immune responses which are linked together through signaling molecules such as cytokines the innate immune system is the first line of defense and includes physical barriers such as epithelial cell layers that express tight cell junctions and the mucus layer of the respiratory gastrointestinal and genitourinary tracts chaplin cells involved in the innate immune response include macrophages polymorphonuclear neutrophils pmn dendritic cells mast cells eosinophils natural killer cells and natural killer t cells turvey and broide the focus of the present review is on the effects of aa on phagocytic cells that is cells that engulf and kill such as macrophages and pmn macrophages not only phagocytose invading pathogens but also produce cytokines such as ils and tnfα which initiate innate and adaptive immune responses and recruit pmn to the site of infection chaplin the adaptive immune response consists of t lymphocytes b lymphocytes and humoral factors marshall et a0 al there are two types of t lymphocytes cytotoxic t cells cd8 cells and thelper th cells cd4 cells marshall et a0al cytotoxic t cells detect and eliminate infected cells while th cells produce ils and interferonγ ingvartsen and moyes the b lymphocytes are cells that produce antibodies that bind to antigens on the surface of pathogens to mark them for destruction marshall et a0al 0ccoleman et a0al s177aa immune function and oxidative a0stressduring the periparturient period the metabolic status is associated with the inflammatory regulation of peripheral blood mononuclear cells with a more pronounced inflammatory response in those cells during the nnb immediately postpartum agrawal et a0 al mann et a0 al this period is also associated with altered signaling of nutrientsensing kinases in immune cells such as the protein kinase b akt and mechanistic target of rapamycin mtor pathway which may modulate cytokine production mann et a0al aa can directly and indirectly alter the immune system besides being used in energy metabolism reactions and the synthesis of proteins aa are critical for the synthesis of other functional molecules such as the antioxidants gsh and taurine no histamine and hydrogen peroxide li et a0 al thus this section of the review will focus on the role that aa play in modulating immune function and oxidative stress in dairy cows during the periparturient period a a0special focus will be placed on aa involved in 1carbon metabolism as this represents an interconnected route through which aa could impact molecular events such as epigenetic regulation protein synthesis via mtor energy metabolism and antioxidant synthesis a a0summary of studies investigating the effects of aa on immune function and oxidative stress in dairy cows is provided in table a0 a a0summary model of how aa might alter immune function and oxidant status is depicted in figure a0methioninemethionine not only is essential for protein synthesis but also serves as a functional nutrient that is needed for the production of the antioxidants gsh and taurine atmaca and provision of methyl groups finkelstein these features are exemplified by the central role of met in 1carbon metabolism figure a0 in these pathways met is used to synthesize sadenosyl methionine sam which can be used to methylate dna and to support phosphatidylcholine pc and carnitine synthesis for fatty acid metabolism vance et a0al during the periparturient period triacyglycerol tag accumulate in the liver leading to mitochondrial dysfunction inflammation and reduced liver function li et a0al du et a0al pc is the main phospholipid component of verylowdensity lipoproteins vldl vance thus enhancing pc synthesis through greater met supply may help improve vldl synthesis and reduce hepatic tag accumulationas part of 1carbon metabolism homocysteine can be remethylated to met using betaine or folate as methyl donors bhmt and via betaine homocysteine methyltransferase 5methyltetrahydrofolatehomocysteine methyltransferase mtr respectively homocysteine is also used to synthesize cystathionine via cystathionine synthase cbs in the first reaction of the transsulfuration pathway banerjee et a0 al cystathionine is used to make cys which is utilized to synthesize the antioxidants taurine or gsh cbs is allosterically figure interrelationships among components of the 1carbon metabolism pathway methionine cycle folate cycle and transsulfuration pathway 5mthf 5methyltetrahydrofolate amd1 adenosylmethionine decarboxylase arg arginase b12 cobalamin b2 riboflavin b6 pyridoxal ²phosphate cbs cystathionine betasynthase dcsam decarboxylated sadenosylmethionine dhfr dihydrofolate reductase dnmt dna methyltransferase dtmp thymidine monophosphate dump deoxyuridine monophosphate ftcd formimidoyltransferase cyclodeaminase gnmt glycine nmethyltransferase mat methionine adenosyltransferase mthfr methylenetetrahydrofolate reductase mtrr 5methyltetrahydrofolatehomocysteine methyltransferase reductase odc1 ornithine decarboxylase pemt phosphatidylethanolamine nmethyltransferase sahh sadenosylhomocysteine hydrolase shmt serine hydroxymethyltransferase sms spermine synthase srm spermidine synthase tdh threonine dehydrogenase thf tetrahydrofolate tyms thymidylate synthetase 0cs178 of animal science vol no suppl activated by sam banerjee et a0 al therefore enhanced sam production with increased met supply can help enhance the flux of the transsulfuration pathway increasing taurine and gsh production to help reduce oxidative a0stressin dairy cattle low levels of serum met postpartum are associated with severe hepatic lipidosis shibano and kawamura and other than his met is the only aa for which net uptake by the liver increased pre and postpartum larsen and kristensen therefore enhancing postruminal met supply during the periparturient period is of interest to increase circulating concentrations of met for its functional roles in the body the work from dalbach et a0 al demonstrated that rumenprotected met rpm can be used to increase serum concentrations of met in the first 2wk postpartum which will enhance the availability of met for protein synthesis and metabolism via the 1carbon pathways in terms of production supplementation of met during the peripartal period concomitantly increases milk yield milk protein and milk fat soon after calving ordway et a0 al osorio et a0 al these responses are in large part driven by enhancing met availability and by the additional flux of met through the met cycle in the liver which consequently increases the production of downstream compounds such as sam pc gsh and taurine additionally work feeding rpm during the periparturient period has detected positive responses in maintaining consistent rates of dmi prepartum last d and faster and greater rates of dmi during the first to d after calving osorio et a0al zhou et a0 al 2016c batistel et a0 al the same milk production response was also observed when met was supplemented postpartum as the isopropyl ester of 2hydroxy4methylthiobutanoic acid stpierre and sylvester as described earlier the transient inflammatorylike status around parturition appears to be a œnormal aspect of the adaptations to lactation as cows approach parturition those with greater but still subclinical concentrations of circulating cytokines have greater inflammation and oxidative stress and lower liver function along with lower milk yield and lower postpartum dmi bertoni et a0 al in addition to their function in the immune system cytokines interferons and tnfα also elicit pathophysiological effects leading to œsickness table summary of studies in dairy cows investigating the effects of supplemental aa on immune function oxidative stress and inflammation tissuecellstreatmentmain outcomeplasmarpm for d prepartum and d postpartumimprovements in plasma biomarkers indicating reduced oxidative stress and inflammation and enhanced liver function increased neutrophil phagocytosis and oxidative burstreferencebatistel et a0al plasmaabomasal infusion of glu for first infusions of gln increased the abundance of cd4 tcells on day doepel et a0al d postpartumpostpartum and increased the abundance of monocytesmammary rpm for d prepartum and d methionine supply upregulated expression of genes involved in han et a0al glandplasmapostpartumantioxidant metabolism and increased activation of nfe2l2intravenous infusions of gln for glutamine infusion decreased plasma haptoglobin and increased jafari et a0al d postcalvinglpsbinding protein and saa subcutaneous rpm for d prepartum and d enhanced met supply increased mrna and protein abundance of liang et a0al 2019aadiposepmnlpostpartumenzymes related to gsh metabolismincubation with met andor supplemental met coupled with adequate choline enhanced gene lopreiato et a0al cholineexpression of pathogen recognition mechanisms methionine ameliorated the increased inflammation and oxidative stress observed when cells were incubated without choline plasma and protected gln for d postpartum increased total blood protein and albumin decreased plasma nemati et a0al milkwhole bloodrpm for d prepartum and d aspartate aminotransferase and milk somatic cell countincreased whole blood neutrophil phagocytosis on day osorio et a0al 2013apostpartumpostpartum with supplemental metliverrpm for d prepartum and d methionine supply altered flux through 1carbon metabolism via osorio et a0al 2014aliver and plasmaplasmapostpartumchanges in mrna to support antioxidant and met synthesisrpm for d prepartum and d methionine increased liver gsh and decreased concentrations of osorio et a0al 2014bpostpartum plasma biomarkers of inflammationrpm for d during midlactation increased proliferative ability of peripheral blood t lymphocytes soder and holden with supplemental metplasmarpm for d prepartum and increased antioxidant capacity of plasma and cd4cd8 t sun et a0al postpartumlymphocyte ratio with met supplywhole bloodrpm for d prepartum and d methionine damped the hyperresponse of il1 during an lps vailatiriboni et a0al plasmajugular infusion of arg and lps in arginine alleviated lpstriggered inflammation by decreasing il6 zhao et a0al 2018apostpartumchallenge through improvements in oxidative stressserumjugular infusion of arg and lps in infusion of arg promoted antioxidant mechanisms during lpszhao et a0al 2018bmidlactation cowsinducible nos and lpsbinding proteinmidlactation cowsrpm for d prepartum and d postpartumrpm for d prepartum and d triggered inflammation by increasing total antioxidant capacity and gsh peroxidase activity and decreasing malondialdehyde increased hepatic gsh and improved plasma biomarkers of liver function and inflammation with met neutrophil phagocytosis capacity and oxidative burst were also increased with metenhanced met supply increased mrna expression of genes zhou et a0al 2016azhou et a0al 2017bpostpartumassociated with pc and antioxidant synthesisrpm for d prepartum and d decreased expression of genes related to inflammation and zhou et a0al 2018bliver and plasmaliver pmnlpostpartumoxidative stress 0ccoleman et a0al s179figure the theoretical model of cellular aa utilization amp adenosine monophosphate atp adenosine triphosphate ctp cytidine triphosphate dttp deoxythymidine triphosphate gmp guanosine monophosphate imp inosine monophosphate no nitric oxide r5p ribose 5phosphate tca cycle tricarboxylic acid cycle ump uridine monophosphate utp uridine5²triphosphatebehaviors whose primary manifestation is satiety larson and dunn an example of this behavior in dairy cows is the reduction in dmi around calving in mice these cytokines have been shown to reduce meal size and duration as well as decrease meal frequency and prolong intermeal intervals platasalamán furthermore cytokines directly affect the hypothalamus il1 and ifn act directly and specifically on the glucosesensitive neurons in the brain œsatiety and œhunger sites platasalamán in addition to increases in dmi and milk production rpm supplementation during the periparturient period has been associated with positive health responses across four studies osorio et a0al 2014b sun et a0al zhou et a0al 2016a batistel et a0 al there have been consistent improvements in the concentrations of plasma biomarkers of inflammation where il1 and haptoglobin have decreased and albumin has increased summarized in table a0 improvements in biomarkers of oxidative stress have also been observed with enhanced met supply during the periparturient period in the study by batistel et a0al plasma concentrations of ferricreducing antioxidant power carotene tocopherol and total and reduced gsh were increased with rpm while rom were lower sun et a0al also observed an improvement in blood antioxidant status with rpm increasing total antioxidant capacity glutathione peroxidase gpx activity and vitamin e a0a a0similar effect was observed in liver tissue by osorio et a0 al 2014b where cows fed rpm had greater hepatic concentrations of total and reduced a0gshfrom a mechanistic standpoint changes in the mrna abundance of sadenosyl homocysteine hydrolase sahh mtr sod1 glutamate cysteine ligase catalytic gclc subunit and dna methyltransferase 3a suggested alterations in flux through 1carbon metabolism osorio et a0al 2014a importantly sahh the enzyme that makes homocysteine from sadenosylhomocysteine was upregulated with met which would support a supply of homocysteine to be used for antioxidant and met synthesis furthermore in the study by zhou et a0al 2016a greater met supply compared with rumenprotected choline increased antioxidant concentration in liver tissue despite a lower concentration of pc those responses were due to the greater abundance of phosphatidylethanolamine methyltransferase the enzyme that utilizes sam and phosphatidylethanolamine to make pc and cbs zhou et a0al 2017b additionally enhanced met supply during the periparturient period was observed to increase mrna and protein abundance of enzymes related to gsh metabolism in subcutaneous adipose tissue suggesting greater activation of those pathways liang et a0al 2019a a a0greater dietary supply of choline did not change the mrna abundance of bhmt and mtr in cows zhou et a0al 2017ba positive effect of met supplementation on mammary gland antioxidant mechanisms was observed by han et a0 al mrna abundance of gpx1 gclc glutamate cysteine ligase gcl modifier subunit malic enzyme ferrochelatase and ferritin heavy chain and genes involved in gsh and iron metabolism were upregulated protein abundance of phosphorylated nuclear factor erythroid 2like2 nfe2l2total nfe2l2 was also increased han et a0al nfe2l2is a regulator of transcription of antioxidant genes hence an increase in phosphorylated nfe2l2 suggested greater activation of antioxidant systems and is likely one of the mechanisms behind the changes in mrna abundance lastly across studies there has also been a consistent improvement in the concentrations of plasma biomarkers of liver function such as increases in paraoxonase and cholesterol with rpm osorio et a0al 2014b zhou et a0al 2016a batistel et a0 al which is likely linked to the reduction in inflammation and oxidative stress thus the consistent changes across studies in metabolites and plasma biomarkers as well as mrna abundance across tissues indicate that enhanced met supply during the periparturient period reduces oxidative stress and inflammation however more work is needed to verify the exact mechanisms behind the observed changes 0cs180 of animal science vol no suppl enhanced postruminal supply in the form of rpm during the periparturient period has been associated with improvements in immune cell function when rpm was provided for d prepartum and d postpartum whole blood neutrophil phagocytosis was increased compared with control cows at d postpartum osorio et a0 al in the study by zhou et a0 al 2016c rpm supplementation from day ˆ’ prepartum to day postpartum increased neutrophil phagocytosis capacity and oxidative burst activity zhou et a0 al 2016a this same improvement in neutrophil immune function was observed when rpm was supplied from day ˆ’ prepartum to day postpartum batistel et a0 al furthermore an increase in the proliferative ability of peripheral blood t lymphocytes was observed when rpm was supplemented to midlactation cows soder and holden zhou et a0 al 2018b isolated polymorphonuclear leukocytes pmnl and observed lower abundance of genes related to inflammation il1b tlr2 nfκb and stat3 and oxidative stress cbs gpx1 glutathione synthase [gss] and sod2 as well as an increase in plasma taurine with supplemental met suggesting a better redox and inflammatory status of those cells additionally those same cows were used for an ex vivo whole blood challenge with lipopolysaccharide lps to further investigate immune cell responses during this challenge a table summary of additional beneficial effects of feeding rpm during the transition period and early lactationbiomarkerresponse12sitebiological functionmetabolism carnitine cholesterolonecarbon metabolism cystathionine betasynthase activity cystathionine cystine homocysteineinflammation il1beta haptoglobin albumin oxidative stress rom†‘†‘†‘†‘†‘†‘†‘†“†“†“†‘†‘liveroxidation of fatty acidsplasmalipoprotein metabolismliverplasmaplasmaplasmaantioxidant synthesish2s biosynthesis redox statusredox statusmethylation reactionsplasmaproinflammatory cytokineplasmainflammation signalplasmaacutephase response†”†“plasmaperoxides gsh taurine antioxidant capacity paraoxonase†‘†‘†”†‘†”†‘†‘†‘superoxide ohradicalsliver blood antioxidantantioxidantplasmaplasmatotal antioxidants in bloodplasmaantioxidant enzyme†‘ beneficial increase †“ beneficial decrease †” no change in concentration2relative to a control or rumenprotected choline supplemented diet osorio et a0al 2014b zhou et a0al 2016a sun et a0al batistel et a0al vailatiriboni et a0al hyperresponse in il1 was observed around parturition which likely arose from oxidative stress vailatiriboni et a0 al however rpm supplementation dampened this hyperresponse likely through improvements in oxidative stress vailatiriboni et a0al the recent work by lopreiato et a0 al investigated the effects of incubating bovine pmnl with met andor choline and observed that supplemental met coupled with adequate choline enhanced gene expression of tlr2 and lselectin sell which are pathogen recognition mechanisms in the same experiment cells incubated without choline had greater mrna abundance of il1b il6 il10 and myeloperoxidase mpo glutathione reductase gsr gss cystathionine gammalyase cth and cysteine sulfinic acid decarboxylase csad indicating greater inflammation and oxidative stress this effect however was ameliorated by supplementing additional met lopreiato et a0 al thus the increased dmi and milk production observed when feeding rpm can be partly explained by a reduction in inflammation as it directly at the hepatic level and by dampening the immune cell overresponse and indirectly reducing oxidative stress decreases circulating proinflammatory cytokinesenhanced met supply during the periparturient period has also been associated with changes in mtor signaling mtor is a serinethreonine kinase that plays a central role in integrating environmental cues from growth factors nutrients particularly aa and energy powell et a0al in dairy cattle mtor has traditionally been studied in the context of its role in regulating protein synthesis however work from humans and nonruminant species has indicated that mtor is an important regulator of immune responses powell et a0 al jones and pearce when activated by aa mtor directs an activation of anabolic metabolism which allows growth proliferation and development this makes the activation of mtor in immune cells particularly important for maintaining proliferation and without proper activation cells may enter periods of growth arrest jones and pearce methionine specifically may interact with mtor via the production of sam specifically sam can bind to sadenosylmethionine sensor upstream of mtor samtor a protein that inhibits mtor complex mtorc1 by interacting with gtpaseactivating protein activity towards rags gator1 gu et a0al when sam binds to samtor it inhibits the association of samtor and gator1 allowing mtorc1 to be activated gu et a0al to our knowledge there is only one study investigating the expression of mtor signaling proteins in immune cells in dairy cattle and work is also lacking in nonruminant species in periparturient cows the activation of aktmtor signaling in immune cells was reduced postpartum compared with prepartum mann et a0al importantly agrawal et a0al also identified the expression of aa transporters and the kyoto encyclopedia of genes and genomes kegg pathways related to 1carbon metabolism and mtor in pmnl from peripartal cows a a0 list of these transporters and kegg pathways related to aa and 1carbon metabolism are summarized in table a0 together these studies support the potential importance of aa for nutrient signaling in immune a0cellsrecent work indicating that enhanced met supply activates mtor signaling in the mammary gland supports its role in enhancing protein synthesis for example in vitro enhancing met supply to immortalized bovine mammary epithelial cells bmec by varying the ratio of lys to met increased the concentration and utilization of essential aa particularly branchedchain aa bcaa dong et a0 al this change 0cwas potentially mediated by alterations in aa transporters controlled by mtor dong et a0al other studies with bmec have also revealed a potential for greater mtor activation when met supply is enhanced nan et a0al sala ma et a0al in vivo the effects of supplemental met during the periparturient period on mtor signaling were explored using cows from the study by batistel et a0al in the mammary gland cows receiving rpm had lower protein abundance of total mtor and phosphorylated mtor pmtor compared with control cows on day postpartum but the ratio of pmtortotal mtor was not different suggesting that there was no difference in mtor activation between treatments ma et a0 al however changes in aa transporters and insulin signaling indicated that insulin sensitivity of the mammary gland was enhanced with supplemental met ma et a0al in subcutaneous adipose tissue the mrna and protein abundance of some aa transporters and pmtor were upregulated with enhanced met supply while changes in insulin signaling and plasma glucose also indicated that met helped improve insulin sensitivity liang et a0 al 2019b thus enhancing met supply during the periparturient period may lead to mtor activation in immune cells as well as improved nutrient uptake which could help to support proliferation and development additional work in ruminants and nonruminants is needed to understand whether met modulates mtor signaling in immune cellscysteinecysteine can be synthesized endogenously from homocysteine as described earlier and is needed to synthesize gsh and taurine gsh is synthesized via two enzymes gcl and gss lushchak to synthesize taurine cysteine sulfinic acid is first synthesized from cysteine by cysteine dioxygenase and can then be utilized by csad to produce taurine park et a0al dietary cys has been explored as a way to improve health in nonruminant species and humans under a variety of conditions including type2 diabetes cardiovascular disease and liver disease to name a few yin et a0 al across these studies increased dietary cys increased the concentrati
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" appendectomy for acute appendicitis aa is considered one of the most common emergencysurgeries however emergency appendectomy accompanied with complex lesions such as extensive abscessformation is not recommended in most cases therefore nonoperative management followed by intervalappendectomy ia for aa has been tried herein we present three aa cases with specific etiology that underwentinterval appendectomycase presentation case a 68yearold man was diagnosed aa with intestinal malrotation and intraabdominalabscesses he initially treated with conservative therapy and underwent laparoscopic ia after detailed preoperativeexaminationcase a 22yearold man had been under treatment for pancolitistype ulcerative colitis uc also bothered byrightlower abdominal pain several times a year the appendix always appeared swollen on every ct taken duringsymptoms he underwent laparoscopic ia pathological finding revealed typical uc histological features in theresected appendix after the surgery he never suffered from terrible right lower abdominal paincase a 69yearold woman complaining a right lower abdominal pain had undergone ct examination whichrevealed aa with appendiceal mass irregular wall thickness of the cecum and mediastinal and paraaortic lymphnode swelling the operation was carried out after conservative therapy the pathological diagnosis revealed brafmutated colorectal carcinoma she had received systematic chemotherapy after the surgery and all metastaticlesions have completely disappeared interval appendectomy provided us with much clearer anatomical information and precise therapeuticstrategies avoiding technical and general operative complications and also induced fast recovery and short lengthof hospital stay interval appendectomy is a reasonable procedure and could be recommended in case of aa withsome different etiologykeywords interval appendectomy malrotation ulcerative colitis appendiceal carcinoma correspondence kasagisurg2medkyushuuacjp1department of surgery national hospital anization fukuoka higashimedical center koga japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 0ckasagi surgical case reports page of appendectomy for aa is considered the most commonemergency surgeries and prompt appendectomy haslong been a standard treatment for aa [“] currentlylaparoscopic appendectomy la becomesthe firsttherapeutic choice for aa la is a safe and effectiveprocedure for the treatment of simple appendicitis andthis approach is superior to open appendectomy oa interms of postoperative wound infections analgesia requirement length of hospital stay return to work andoverall recovery [ ]immediate appendectomy is technically demandingwith distorted anatomy adhesive loops of bowel anddifficulty to close the appendiceal stump because of theinflamed tissues then early la for aa with suchconditions may be converted to oa ileocecal resectionor right hemicolectomy therefore nonoperative management followed by iafor aa has been tried in many hospitals initially aamay be managed in an elective nonsurgical manner including intravenous antibiotics and selective percutaneous drainage and then carrying out operation mostlyla [“] this management has been performed especially in pediatric patients however the validity ofia is still controversial in adult patients [ ]herein we present three cases of aa with specific etiology who eventually underwent interval appendectomydiscussing beneficial effects of interval appendectomy foraa with different etiologycase presentationcase a 68yearold man complaining a leftlower abdominalpain had visited our hospital ct revealed the cecum located in the left lower side of the abdomen and a swollen blindend structure with intraabdominal abscessesfig 1a finally he was diagnosed as aa with intestinalmalrotation he received conservative treatment withfig a ct findings the cecum was located in the left lower side of the abdomen the swollen appendix was surrounded by intraabdominalabscesses arrow heads b the clinical course of this patient he was discharged at day and underwent an interval appendectomy at day cthe gastrografin enema findings the cecum arrow located in leftlower side of the abdomen d the trocar placement of the intervalappendectomy note the mirror image trocar placement against an ordinal laparoscopic appendectomy e intraoperative findings iliocecalstructures were located in the left lower abdomen 0ckasagi surgical case reports page of intravenous antibiotics which ameliorated his symptomand inflammatory findings fig 1b he left the hospitalat day afterwards we carried out detailed preoperative examination on his outpatient visit the gastrografinenema confirmed that the cecum was located in the leftlower side revealing the presence of intestinal malrotation fig 1c we underwent a laparoscopic ia using amirrorimage trocar placement fig 1d the iliocecalstructures were found at the left side of the abdomenfig 1e he was discharged from the hospital at thepostoperative day case a 22yearold man had been under the treatment forpancolitistype ulcerative colitis uc fig 2a b and hasbeen also bothered by rightlower abdominal pain several times a year the appendix always appeared swollenon every ct taken during symptoms fig 2c on everyonset of symptoms he received intravenous followed byoral antibiotics which always ameliorated his symptomsendoscopy examination revealed that the appendicealorifice was almost normal fig 2d both uc and aamight cause his right lower abdominal pain he wastreated with conservative treatment at first he underwent a laparoscopic ia in order to obtain the accuratediagnosis there were no operative complications andhe left the hospital days after the operation thepathologicalfindings showed not only aa featuresinflammatory cell infiltration but also histological characteristics typical of uc crypt distortion in the resectedappendix fig 2ecase a 69yearold woman complaining a right lower abdominal pain undergone ct examination which revealedruptured aa with appendiceal mass and irregular wallthickness of the cecum fig 3a which made us suspectcolon cancer moreoverthere were mediastinal andparaaortic lymph node swelling suspecting malignantlymphoma or lymph node metastases serum soluble il2r cea and ca199 were uml normal range“ ngml normal range “ and uml normal range respectively and those serumfindings made us suspect malignant lymphoma or epithelial neoplasm conservative therapy with intravenousantibiotics for aa was started seven days after the initiation of conservative therapy her general condition andinflammatory signs were significantly improved fig 3bshe underwent ileocolectomy ie extended appendectomy because there were strong suspicions of malignancy for making a pathological diagnosis she left thehospital days after the operation without any complications fig 3b the pathological features concludedpoorly differentiated adenocarcinoma with peripherallymph node metastases fig 3c d the genotype analyses revealed rightside colorectal carcinoma with braffig a b colonoscopic findings continuous ulcerative lesions and erythema were observed c ct fingings ct revealed a swollen appendix onevery symptomatic course arrow heads d colonoscopic findings the appendiceal orifice was almost normal arrow heads e pathologicalexamination the pathological finding revealed inflammatory cells infiltration and crypt distortion in the resected appendix 0ckasagi surgical case reports page of fig a ct findings ct revealed acute appendicitis arrow heads with abdominal abscess and irregular wall thickness of the cecum circle bthe clinical course of this patient she underwent interval operation at day and left the hospital at postoperative day c a macrofinding ofthe resected an the arrow heads indicate the appendix the oval indicates the tumor lesion d pathological examination pathological featuresrevealed poorly differentiated adenocarcinomamutation and liver metastasis lesion appeared later shereceived chemotherapy bevacizumab 5fufolinateoxaliplatin cpt11 months later either mediastinaland paraaortic lymph node swelling and all metastaticlesions had completely disappeared she was kept on acomplete response cr at the final visit year and months after the operationdiscussionthere are still controversies over the efficacy of ia foracute appendicitis [ ] we presented here three interesting cases of aa who underwent ia which eventually proved to be a very effective therapeutic choice sofar there have been case reports with a single case regarding the efficacy of ia the current manuscript contains three cases with different unique etiologies wecan contrast one case with another in order to understand how the managements are different some reportssuggested that the recurrence rate of aa during thewaiting time for ia is “ and the complication rateof surgery for recurrent aa is as high as “ however especially in a phlegmon or appendiceal massia may have some advantages for example providing adefinite diagnosis ruling out any underlying malignancyand avoiding unwanted injury to the surrounding tissue[ ] and also the advantage of ia is to performthe operation at a time when the peritonitis has resolvedpotentially resulting in fewer intraoperative andor postoperative complications there are some analysesabout costeffectiveness of ia [ ] however costbenefits of ia remain controversial ia requires onemore additional admission”one for conservative treatment and one for surgical therapy ia might requiremuch more medical resources nevertheless ia providesa lot of benefits accurate preoperative informationavoiding technical and general operative complicationsand improves patient™s qol fast recovery and shortlength of postoperative hospital stay particularly forcases with some characteristic etiology like we presentedin the current manuscriptinterval operation also 0ckasagi surgical case reports page of provides us with precise therapeutic strategies that leadto well results and prognosis therefore we advocateinterval operation for aa with unique etiologiesin case aa with appendiceal mass was initially treatedwith conservative therapy which enabled further examinations for intestinal malrotation the anatomical information was extremely useful making the operation safe andavoiding technical complications ia seemed to induce hisfast recovery and short length of hospital stay those results seemed to be one of the greatest benefits of iarecent several reports have shown a significant negative association between appendectomy and uc [“]however the majority of the studies deal with the history of appendectomy before the development of uc[ ] there are a few published or unpublished dataabout the course of uc when appendectomy is performed after uc diagnosis those reports suggested thatthe disease seems to become milder after appendectomy[ ] case underwent ia electively after which henever suffered from terrible right lower abdominal painalthough there were no direct objective testimonies tothe symptomatic improvements such as blood tests andpathological findings in this case ia seemed to ameliorate his uc conditionthere have been considerable studies on appendicealadenocarcinoma in a recent report about primary appendiceal carcinomas with an average age of years old were t3t4 tumors and of them had lymph nodemetastasis more than were poorly differentiated stageiv disease represented of the cases and 5year overall survival was for all stages [ ] such information reinforces the indication of any surgical interventionappendectomyileocolectomy required to treat appendiceal inflammatory mass after conservative treatment in patients with metastatic disease they often have peritoneuminvolvement patients who were submitted to surgicalcytoreduction had a median recurrencefree survival of years and an overall survival of years was achievedwhen patients could undergo a complete cytoreductionunfortunately complete cytoreduction was achieved onlyin of the patients in case t3 tumor was accompanied with peripheral and distant lymph nodes liver metastasis we had undergone a complete cytoreduction afterthe conservative therapy for aa and were able to obtainthe accurate diagnosis without any postoperative complications furthermore in spite of the poor prognosis withbraf gene mutation those therapeutic strategies enabledthe induction of precise early systematic chemotherapyresulting in a well prognosisthe ideal interval is thought to be approximately “months needless to say appendectomy can be performed easily once the inflammation abates the interval ofcase was ideal in this regard however the intervals ofcase and case were short in case he had repeatedlysuffered abdominal pains caused by appendicitis whichwere always alleviated by a short course of antibioticsthere were no abscesses around the appendix when anappendectomy may be performed easily such short intervalcan be acceptable in case there were strong suspicionsof malignancy and the appendix was perforated thereforewe performed the ileocolectomy as soon as the inflammation around the appendix somewhat abated in order topromptly start appropriate therapy against malignancyalthough the beneficial role of ia for aa is still controversial there are some advantages for selected aa caseswith specific etiology we present representative three aacases with intestinal malrotation inflammatory bowel disease and colorectal malignancy in these cases interval appendectomy provided us with much clearer anatomicalinformation and precise therapeutic strategies avoidingtechnical and general operative complications moreoverinterval appendectomy also induced postoperative fast recovery and short length of hospital stayinterval appendectomy is a reasonable procedure andcould be recommended in case of acute appendicitis suspiciously having some different etiologyabbreviationsaa acute appendicitis ia interval appendectomy uc ulcerative colitisla laparoscopic appendectomy oa open appendectomy cr complete responseacknowledgementsnone declaredauthors™ contributionsyk and hu wrote the manuscript yk and yn performed the surgery and etand sk participated in the surgery yk kn ta yn mi and hu participated inthe study design and coordination kt developed histological staining anddiagnosed findings hu and yk performed total anization of writing themanuscript all authors read and approved the final manuscriptfundingthis manuscript was not funded externallyavailability of data and materialsdata sharing is not applicable to this as no datasets were generatedor analyzed during the current studyethics approval and consent to participatethe present study was conducted in accordance with the ethical standardsof our institutionconsent for publicationconsent was obtained from the patient and patient™s family for thepublication of this case report and accompanying imagescompeting interestswe have no competing interestsauthor details1department of surgery national hospital anization fukuoka higashimedical center koga japan 2department of pathology nationalhospital anization fukuoka higashi medical center koga japan 0ckasagi surgical case reports page of received june accepted august referencesquartey b interval appendectomy in adults a necessary evil j emergtrauma shock “ditillo mf dziura jd rabinovici r is it safe to delay appendectomy inadults with acute appendicitis ann surg “udgiri n curras e kella vk nagpal k cosgrove j appendicitis is it anemergency am surg “frazee rc roberts jw symmonds re snyder sk hendricks jc smith rw a prospective randomized trial comparing open versus laparoscopicappendectomy ann surg “guller u hervey s purves h muhlbaier lh peterson ed eubanks s laparoscopic versus open appendectomy outcomes comparison based ona large administrative database ann surg “ahmed i deakin d parsons sl appendix mass do we know how to treatit ann r coll surg engl “oliak d yamini d udani vm lewis rj arnell t vargas h initialnonoperative management for periappendiceal abscess dis colon rectum“skoubokristensen e hvid i the appendiceal mass results of conservativemanagement ann surg “hoffmann j lindhard a jensen he appendix mass conservativemanagement without interval appendectomy am j surg “ adalla sa appendiceal mass interval appendicectomy should not be therule br j clin pract “lai hw loong cc chiu jh chau gy wu cw lui wy intervalappendectomy after conservative treatment of an appendiceal mass worldj surg “ vons c barry c maitre s pautrat k leconte m costaglioli b amoxicillin plus clavulanic acid versus appendicectomy for treatment ofacute uncomplicated appendicitis an open label noninferiorityrandomized controlled trial lancet “ hori t machimoto t kadokawa y hata t ito t kato s laparoscopicappendectomy for acute appendicitis how to discourage surgeons usinginadequate therapy world j gastroenterol “ corfield l interval appendicectomy after appendiceal mass or abscess inadults what is œbest practice surg today “ andersson re petzold mg nonsurgical treatment of appendiceal abscess orphlegmon a systematic review and metaanalysis ann surg “lare s raminder n brandon b richard n interval appendectomy findingthe breaking point for costeffectiveness j am coll surg “ hung wl che cl chew ww wing yl watchful waiting versus intervalappendectomy for patients who recovered from acute appendicitis with tumorformation a costeffectiveness analysis j chin med assoc “ gent ae hellier md grace rh swarbrick et coggon d inflammatorybowel disease and domestic hygiene in infancy lancet “ rutgeerts p d™haens g hiele m geboes k vantrappen g appendectomyprotects against ulcerative colitis gastroenterology “ duggan ae usmani i neal kr logan rf appendicectomy childhoodhygiene helicobacter pylori status and risk of inflammatory bowel diseasea case control study gut “ russel mg stockbrugger rw is appendectomy a causative factor inulcerative colitis eur j gastroenterol hepatol “ekbom a appendicectomy and childhood hygiene different sides of thesame coin gut benedix f primary appendiceal carcinoma epidemiology surgery andsurvival results of a german multicenter study ejso “ nitecki ss the natural history of surgically treated primary adenocarcinomaof the appendix ann surg “lieu ch systemic chemotherapy and surgical cytoreduction for poorlydifferentiated and signet ring cell adenocarcinomas of the appendix annoncol “ darwazeh g cunningham sc kowdley gc a systematic review ofperforated appendicitis and phlegmon interval appendectomy or waitandsee am surg “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"
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"Marek™s disease MD is a chicken neoplastic disease which brings huge economic losses to theglobal poultry industry The wild type p53 a tumor suppressor gene plays a key role in blocking cell cyclepromoting apoptosis and maintaining the stability of the genome However the mutant p53 losses its tumorinhibitory role and become an oncogene when a mutation has happenedResults The mutation rate of p53 was in the experimentally and naturally infected chickens The mutationsincluded pointmutations and deletions and mostly located in the DNAbinding domain The mutated p53 wasexpressed in various tumor tissues in an infected chicken The mutant P53 proteins were notably accumulated inthe cytoplasm due to the loss in the function of nuclear localization Unlike the study on human cancer theconcentrations of P53 in the serums of MD infected chicken were significantly lower than the control groupConclusions The p53 mutations were apparent in the development of MD P53 and P53 antibody level in serumcould be a useful marker in the diagnosis and surveillance of MDKeywords Marek™s disease p53 P53 antibodyBackgroundMarek™s disease MD is a lymphoproliferative neoplasticdisease caused by the chicken Marek™s disease virusMDV or named Gallid alphaherpesvirus The infection caused by this virus may lead to lymphocyte proliferation tumor formation immunosuppression paralysisand mononuclear cell infiltration in peripheral nervesgonads and immune ans [ ] As one of the mosthighly contagious tumor diseases in chickens the data ofOIE [] reported by about half of the world has shownthat this disease accounts for the loss of up to “ billionUS dollars annually in the global poultry industry [] Correspondence liusidsdaueducn1College of Animal Science and Veterinary Medicine Shandong AgriculturalUniversity Daizong Street Taian Shandong ChinaFull list of author information is available at the end of the Described often as the œguardian of the genome [] andthe œcellular gatekeeper [] p53 is the most relevantand important tumor suppressor gene with the highestmutation frequency in human and animal tumor diseases [] The chicken p53 gene has a fulllength reading frame ™ and ™ untranslated regions and a polyadenylation signal which encodes amino acidsThese amino acids share a homology to the aminoacids of human P53 []p53 is divided into the wild type and the mutant typeThe wild type is a normal tumor suppressor gene whilethe mutant p53 is an oncogene transformed from atumor suppressor gene due to spatial conformationalchange As a result the mutant P53 protein losses itsability in regulating cell growth apoptosis and DNA repair [] which often a prerequisite for tumorigenesis and The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cZhang BMC Veterinary Research Page of disease progression [] The proportion of p53 mutations in tumor tissue varies between “ []The wild type P53 has a very short halflife while themutant P53 protein is prolonged This abnormality ofP53 can lead to the accumulation of P53 antibody inserum as well as the P53 protein in tumor tissue []Several studies have demonstrated that the P53 antibodyplays a predictive role in tumorigenesis and its manifestation in serum is an early event in the development ofmalignant tumors in humans [“] The level of P53antibody in serum correlates significantly with commonclinical neoplastic diseases but it has been barely detectable in the serum of healthy subjects [] This correlation may also exist in chicken and currently there is aknowledge gap concerning the role of mutant p53 inMarek™s disease in chicken Therefore this study aimedto investigate the role of p53 as a tumor marker inassisting the clinical diagnosis and prognosis of MDResultsHistopathological and immunohistochemical analysis ofp53 expressionHistopathological examination revealed the evidence ofliver cells undergoing necrosis in the infected SPFchicken Such liver tissues demonstrated focal infiltration and hyperplasia of lymphoid tumor cells Fig 1aIn addition the lymphoid follicle in the bursa of Fabricius was atrophied diffuse infiltration and proliferationof lymphoid tumor cells between the follicles were observed Fig 1b In the livers of the clinically infectedchickens the cytoplasm of the lymphoid tumor cellsunderwent multifocal proliferation which was stainedbrown by the immunohistochemicalFig 2aPositive staining was also detected in the tumor cells inIHCthe spleen and the bursa of Fabricius tissues Fig 2b and cIn contrast no positive stained cells were observed in thecontrol group Fig 2dMutations in the p53The mutation rate of p53 was in the infectedpoultry There were two types of p53 mutations deletions and point mutations detected which was consistent with the results reported in a previous study []Among the mutated p53 genes the base sites withhigh mutation frequency were and There was no mutation found in the control groupMost of the mutations were located in the core domainand the Cterminal domain The mutation analyses wereshown in Table P53 antigen and P53 antibody levels for MDThe concentrations of the P53 antigen of the clinicaland experimental MDVinfected group were significantlylower than the control group However the P53 antibody levels in the experimental infection group were significantly higher than the control group These analyseswere shown in Fig DiscussionThe human P53 is widely acknowledged as an intranuclear phosphorylated protein The wide type P53 normally exists in the nucleus for an extremely short period[] On the other hand the mutated P53 has a prolonged halflife to “ h as it is not digested quicklyand therefore accumulates inside tumor cells [] Thisallows the detection of mutant P53 protein via IHC forFig Histopathological observation of diseased chickens infected with MDV a Different sizes and shapes of focal infiltration and hyperplasia oflymphoid tumor cells were observed in the liver tissues HE — b Diffuse infiltration and proliferation of lymphoid tumor cells between thefollicles were observed in the bursa of Fabricius HE — 0cZhang BMC Veterinary Research Page of Fig Immunohistochemical staining of p53 in infected chickens a Liver lymphoid tumor cells with cytoplasm expression HE — b Spleen lymphoid tumor cells with cytoplasm expression HE — c Bursa of Fabricius lymphoid tumor cells with cytoplasm expression HE —d Liver Negative controls were incubated with PBS HE —which IHC has now become an important modality forthe detection of various tumor biomarkers P53 proteinrepresents an effective substitute marker for TP53 mutation status [] but at present P53 IHC is mainly applied for tumor diseases in humans P53 IHC allows theassessment of the stage and grade of cancers with only afew studies reported in poultry tumor diseasesIn this study the immunohistochemical analysis revealed that P53 was present in the liver spleen and thebursa of Fabricius of infected chicken with MDV Interestingly P53 was notably expressed in the cytoplasmThe potential explanation of this phenomenon could bethat the mutated P53 protein lost its function in nuclearlocalizationtheaccumulatedinandeventuallycytoplasm Furthermore p53 has been regarded as theœhotspots given that p53 mutations have been frequently detected in a variety of tumors [] In thisstudy several types of p53 mutations were demonstratedin natural and experimental infections of MDV The fivemost frequent mutation sites were and The altered codons of mutant p53 comparedwith the wildtype were ACGACA GCAGCC CGCCGG GCCGCA and ACCACA but these were all synonymous mutationsIn our study a short form of p53 transcript was detected in a clinical case The deleted sequence was located at the “ bp in the reading frame of thereference sequence resulting in missense mutations fromTable Primers used toamplify chicken p53 cDNAPrimerp531Nucleotide sequence™GTGGCCGTCTATAAGAAATCAGA3™™AAAAAGGGGGCGTGGTCAGT3™p532Annealing temperature „ƒSize of fragments amplifiedlocation bp 0cZhang BMC Veterinary Research Page of Fig Levels of p53 antigen and antibody in the serum of study groups The Yaxis represented the concentrations of antigen or antibody andthe Xaxis represented the different groups of samples Each group consisted of seven samples Statistical significance was designated as p or p the position to the termination The short form ofp53 transcript was also found in the cases infected byavian leukosis virus [] A series of missense mutationsfrom the position to the termination due to a base deletion was found in an experimentally infected chicken Inaddition this study found that the p53 was mutated in of poultry oncology with the mutation region concentrated mainly in the DBD The DBD allows the specificrecognition of target sequences [] Once the p53 is deleted or point mutations in this region occursit mayaffect the formation of tetramers which in turn leads tothe conversion of wild type P53 into a mutant type resulting in the loss of normal functionThe conformation of mutant P53 extends its halflifeto several hours in humans The accumulated mutantP53 then acts as a target antigen which elicits an autoimmune response [] However in our study the levelsof serum P53 antigen in clinical and experimentalMDVinfected groups were significantly lower than thecontrol group contrary to the findings in human cancerresearch Only the serum P53 antibody concentrationsof the experimentalinfected group were significantlyhigher than the control group This might be that P53as a tumor suppressor was largely consumed in response to the occurrence of MD Moreover tumorigenesis promoted mutation of p53 and further reduced P53concentrationConclusionsOur study revealed p53 was mutated and expressed inMDV infection which suggested that these mutationswere playing an important role in the development ofMD The mutant p53 was expressed in the tumor cellsof various tissues of the infected chicken Mutant P53protein lost its nuclear localization function and transferred from the nucleus to the cytoplasm Unlike studiesin human cancer the concentration of P53 was significantly lower in the natural and experimental MDV infectionnovelinnovations for the diagnosis and monitoring of MD inpoultryresults maygroup OurprovideMethodsNatural infection of MDV in chickensSeven 160dayold egglaying hens were obtained from alocal flock These hens were confirmed to have acquiredMDV infection naturally with the absence of other common viral diseases by PCR detection Their serums werecollected for the detection of the P53 antigen and antibody Their tissue samples including liver spleen pancreas and bursa of Fabricius were collected and fixedwith formaldehyde for h before histopathologicaland immunohistochemical studiesExperimental infection of chickens with MDVThe number of experimental animal was referred to thenumber of clinical samples Twenty 1dayold SPF chickens were obtained from the Shandong Academy of Agricultural Sciences Poultry Institute SPF Chicken ResearchCenter Jinan China They were randomly divided intotwo equal groups as an infection group and a controlgroup The two groups of chickens were raised separately in isolators with filtered air under positive pressureOn the first day of the experiment each of the chickenin the infection group was inoculated intraabdominallyia with plaqueforming units PFU of vvMDV 0cZhang BMC Veterinary Research Page of GX0101 which normally cause tumors at the tenthweek The chickens in the control group were inoculatedwith PBS In the tenth week serums were collected fromseven chickens in each group The experiment endedafter ten weeks and all chickens were put into a euthanasia box Thirty percent of carbon dioxide CO2 by volume was infused into the euthanasia box per minuteuntil all chickens lost their consciousness Then theCO2 flow rate was increased to for one minuteand the euthanasia box was kept airtight for ten minutesWhen all chickens were confirmed dead they were dissected and their livers and spleens were collected forhistopathology and immunohistochemistry studyHistopathology and ImmunohistochemistryThe fixed liver spleen pancreas and bursa of Fabriciuswere dehydrated waxed and cutinto µm slicesfollowed by Haematoxylin and eosin HE staining for ahistopathology examination In IHC processing tissueswere cut into sections of µm thickness and mountedon microslides treated with polyLlysine The sections were deparaffinized in xylene and rehydrated in agraded series of ethanol solutions into PBS then werepretreated in citrate buffer molL pH °Cfor antigen retrieval by microwaving and cooled at roomtemperature for min [] Endogenous alkaline peroxidase was quenched with hydrogen peroxide solutionin methanol for min [] Nonspecific antibody binding sites were obliterated by incubating the sections with fetal bovine serum for min at room temperatureFollowing this the antiP53 polyclonal antibody BOSTER China as a primary antibody was diluted into and immersed overnight at °C in a black humidchamber The secondary antibodies were HRP horseradish peroxidaseconjugated goatIgGCWBIO China Immunoreactivity was then visualizedwith diaminobenzidine DAB staining The sectionswere counterstained with hematoxylin and mountedNegative controls were incubated with PBS instead ofthe primary antibody in the immunohistochemicalanalysisantimouseTotal RNA isolation and reverse transcriptionThe total RNA ofliver and spleen tissue samples mgtissue were isolated by using TRIzol reagentTakara Japan according to the manufacturer™s instructions Extracted total RNA 1ug was reverse transcribedto cDNA with PrimeScript„¢ RT Reagent Kit RocheSwitzerland According to the published complementaryDNA cDNA sequence of the chicken p53 GeneBankaccession number nm205264 specific primers were designed to amplify the DNAbinding domain DBD ofp53 as shown in Table Using PCR Polymerase ChainReaction techniques p53 cDNA genes sequences wereamplified The PCR reaction was performed by using athermal cycler Takara Japan under the following conditions „ƒ for min followed by cycles of „ƒfor s „ƒ for s „ƒ for s and a final „ƒTable Mutations of p53 genes in MDSample InfoNCMutation analysisBase mutation sitesNDMD CMD CMD CMD CMD CMD CMD EMD EMD EMD EMD EMD E TC AC C G AG AC gA CG CA AC CG CA CG CA delete TC TC AC CG CG CA“ delete CA CA TG AC CG CA gA CA CA AG AC CG CA CT gA AC CG CA gA gT CT CG CA gANC was SPF chicken without diseases E was experimental chicken C was clinical chickenAmino acid mutationsSite from toNDMutation area Y A E G R HNDND Stop StopNDND N S T I G DND R L V M E Y H YCore domainCterminal domainCore domainCore domainCore domainCore domainCterminal domainCore domainC terminal domain 0cZhang BMC Veterinary Research Page of for min extension cycle DNA templates that were acquired from the MDV positive chickens were amplifiedusing primer pair p5312 The SPF chickens wereregarded as negative controls DNA fragments were successfully amplified with sizes of bp The target fragment was gel extracted and connected to the pEASYT1vector Then the recombinant plasmid was transformedinto bacteria and sequencing analysis was made Finallythe sequencing results were compared with the wild typep53 cDNA sequences reported previouslyEnzymelinked immunosorbent assay ELISA for P53 andP53 antibodyThe P53 antigen and antibody levels of chicken weremonitored by ELISA Mlbio China In order to eliminate subjective interference the assessors were blinded tothe background of the samples The detection rangeswere “ pgml for P53 antigen and pgmlfor P53 antibody Samples were diluted five time with aspecial diluent and all processes were implemented inaccordance with the instructions The absorbance ODof each sample was finally measured at a wavelength of nm The sample concentration was calculated depending on the standard curveStatistical analysisStatistical analyses were conducted with GraphPadPrism Version San Diego CA USA The differences in the levels of P53 antigen and antibody were antwotailed Student™s Ttestalyzed by usingStatistical significance was designated as p or p theAbbreviationscDNA Complementary DNA DAB Diaminobenzidine DBD DNAbindingdomain ELISA Enzymelinked immunosorbent assay HE Haematoxylin andeosin IHC Immunohistochemical HRP Horse radish peroxidasePCR Polymerase chain reaction PFU Plaque forming unitsAcknowledgementsNot applicableAuthors™ contributionsHXZ carried out laboratory work wrote the manuscript and performed thedata analyses MDL designed the experiment wrote the manuscript andperformed the data analyses HZ SLC YL SNJ and YNS carried out laboratorywork SDL conceived and supervised this work revised manuscript All authorsread and approved the final manuscriptFundingThe work was supported by grants from the National natural sciencefoundation project NO The funding body was solely involved infunding and had no role in the design of the study the collection analysisand interpretation of the data or in writing the manuscriptEthics approval and consent to participateThe sample were collected and handled in accordance with the goodanimal practices required by the Animal Ethics Procedures and Guidelines ofthe People™s Republic of China Informed consent to participate wasobtained from the chicken farmer owner All animal protocols andprocedures were performed according to the Chinese Regulations ofLaboratory Animals and were approved by the Animal Ethics Committee ofShandong Agricultural UniversityConsent for publicationNot applicableAvailability of data and materialsThe data supporting the findings are included in the manuscriptCompeting interestsThe authors declare that they have no competing interestsAuthor details1College of Animal Science and Veterinary Medicine Shandong AgriculturalUniversity Daizong Street Taian Shandong China 2China AnimalHealth and Epidemiology Center Nanjing Road QingdaoShandong ChinaReceived January Accepted August ReferencesJarosinski KW Tischer BK Trapp S Marek™s disease virus lytic replicationoncogenesis and control Expert Rev Vaccines “Reddy MS Book Review Marek™s Disease An Evolving Problem Vet Pathol“Boodhoo N Gurung A Sharif S Behboudi S Marek™s disease in chickens areview with focus on immunology Vet Res Lane DP p53 guardian of the genome Nature “Bertzbach LD Kheimar A Ali FAZ Kaufer BB Viral Factors Involved inMarek™s Disease Virus MDV Pathogenesis Curr Clin Microbiol Rep “Levine AJ p53 the cellular gatekeeper for growth and division Cell “Soussi T B¨gue A Kress M Stehelin D May P Nucleotide sequence of acDNA encoding the chicken p53 nuclear oncoprotein Nucleic Acids ResZilfou JT Lowe SW Tumor suppressive functions of p53 Review ColdSpring Harb Perspect Biol 20091a001883Stiewe T Haran TE How mutations shape p53 interactions with thegenome to promote tumorigenesis and drug resistance Drug Resist Updat“K¶bel M Piskorz AM Lee S Lui S LePage C Marass F Rosenfeld N MesMasson AM Brenton JD Optimized p53 immunohistochemistry is anaccurate predictor of TP53 mutation in ovarian carcinoma J Pathol Clin Res“ Zekiye H B¼lent T Taner E p53 antibody is it an indicator ofdedifferentiated thyroid cancer Ann Nucl Med “ Balogh GA Mailo D Nardi H Corte MM Vincent E Barutta E Lizarraga GLizarraga P Montero H Gentili R Serological levels of mutated p53 proteinare highly detected at early stages in breast cancer patients Exp Ther Med“Sabapathy K Lane DP Understanding p53 functions through p53antibodies J Mol Cell Biol “Kunizaki M Fukuda A Wakata K Tominaga T Nonaka T Miyazaki TMatsumoto K Sumida Y Hidaka S Yasutake T Sawai T Hamamoto RNanashima A Nagayasu T Clinical Significance of Serum p53 Antibody inthe Early Detection and Poor Prognosis of Gastric Cancer Anticancer Res“ Yue Q Yulong G Liting Q Shuai Y Delong L Yubao L Lili J Sidang LXiaomei W Mutations in and Expression of the Tumor Suppressor Gene p53in EggType Chickens Infected With Subgroup J Avian Leukosis Virus VetPathol “ Bykov VJN Eriksson SE Bianchi J Wiman KG Targeting mutant p53 forefficient cancer therapy Nat Rev Cancer “ Yemelyanova A Vang R Kshirsagar M Lu D Marks MA Shih IeM Kurman RJImmunohistochemical staining patterns of p53 can serve as a surrogatemarker for TP53 mutations in ovarian carcinoma an immunohistochemicaland nucleotide sequencing analysis Mod Pathol “ 0cZhang BMC Veterinary Research Page of Takagi M Ohashi K Morimura T Sugimoto C Onuma M The presence ofthe p53 transcripts with truncated reading frames in Marek™s diseasetumorderived cell lines Leuk Res “ Arlt C Ihling CH Sinz A Structure of fulllength p53 tumor suppressorprobed by chemical crosslinking and mass spectrometry Proteomics “Thierry S Analysis of p53 Gene Alterations in Cancer A Critical View Years of p53 Research “Stiasny A Freier CP Kuhn C Schulze S Mayr D Alexiou C Janko C Wiest IDannecker C Jeschke U Kost BP The involvement of E6 p53 p16 MDM2and Gal3 in the clinical outcome of patients with cervical cancer OncolLett “Shen FX Ma GP Cheng AC Wang MS Li CF Sun KF Chang H Zhu DK JiaRY Chen XY Sun T Development and application of an indirectimmunohistochemical method for the detection of duck plague virusvaccine antigens in paraffin sections and localization in the vaccinatedduckling tissues Poult Sci “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"
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LncRNA SNHG15 predicts poor prognosis in uveal melanoma and its potential pathwaysXue Wu123 XiaoFeng Li123 Qian Wu4 RuiQi Ma123 Jiang Qian123 Rui Zhang123·Basic Research·1Department of Ophthalmology Eye ENT Hospital of Fudan University Shanghai China2NHC Key Laboratory of Myopia Fudan University Shanghai China 3Laboratory of Myopia Chinese Academy of Medical Sciences Shanghai China4Department of Pathology West China Hospital Sichuan University Chengdu Sichuan Province ChinaCofirst authors Xue Wu and XiaoFeng LiCorrespondence to Rui Zhang Department of Ophthalmology Eye ENT Hospital of Fudan University Fen Yang Road Shanghai China zhangrui936163comReceived Accepted Our research suggests that SNHG15 may play a vital role as a potential marker in UM that predicts poor prognosis Besides GSEA indicates the underlying signaling pathways enriched differentially in SNHG15 high expression phenotype KEYWORDS SNHG15 uveal melanoma the Cancer Genome Atlas pathology prognosis Gene Set Enrichment Analysis1018240ijo20200804Citation Wu X Li XF Wu Q Ma RQ Qian J Zhang R LncRNA SNHG15 predicts poor prognosis in uveal melanoma and its potential pathways Int J Ophthalmol Abstract— AIM To evaluate the role of long noncoding RNA lncRNA SNHG15 and its potential pathways in uveal melanoma UM METHODS The SNHG15 mRNA expression level and corresponding clinicopathological characteristics of patients with UM were obtained from the Cancer Genome Atlas TCGA database and further analyzed The SPSS statistical software package was used for statistical analyses To investigate the potential function of SNHG15 in UM we conducted indepth research on Gene Set Enrichment Analysis GSEA— RESULTS The univariate analysis revealed that the age tumor diameter pathological type extrascleral extension cancer status and high expression of SNHG15 were statistical risk factors for death from all causes The multivariate analysis suggested that the mRNA expression level of SNHG15 was an independent risk factor for death from all causes as was age and pathological type KaplanMeier survival analysis confirmed that UM patients with high SNHG15 expression might have a poor prognosis In addition SNHG15 was significantly differentially expressed in the different groups of tumor pathologic stage metastasis and living status Besides the logistic regression analysis indicated that high SNHG15 expression group in UM was significantly associated with cancer status pathologic stage metastasis and living status Moreover the GSEA indicated the potential pathways regulated by SNHG15 in UM INTRODUCTIONU veal melanoma UM the most common intraocular cancer in adult worldwide[] is a malignant tumor that originates in melanocytes of the choroid plexus ciliary body and iris of the eye At present despite definitive radiotherapy or removal of the primary lesion numerous patients eventually develop metastases and subsequently prognosis is significantly poor[] In addition UM tends to metastasize to liver through hematogenous pathway a distant site relative to their origins in the eye[] There is an incubation period between the enucleation of the primary tumor and the emergence of metastasis which can range from a few months to several decades[] Despite the advancement of UM management there are currently no effective therapy once the metastases occurred[] Therefore close followup and further research on the pathogenesis and novel makers exploration of UM are of great significance for accurate diagnosis appropriate therapy and prognosis prediction Long noncoding RNA lncRNA is a class of noncoding transcripts with a length of larger than nucleotides[] which has been involved widely in biological processes of different cancers including cell cycle apoptosis cell differentiation[] In the development of UM lncRNA is also reported to play a vital role in cell cycle cell proliferation apoptosis invasion and autophagy[] For example silencing of lncRNA PVT1 prevents the development of UM by impairing microRNA173pdependent MDM2 upregulation[] ZNNT1 can suppress Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomathe progression of UM by inducing the expression of crucial autophagy gene[] The lncRNA RHPN1AS1 facilitates the tumorigenesis of UM by influencing cell proliferation and migration[] However the study of vital lncRNAs in UM still remains to be exploredSNHG15 a novel lncRNA located on chromosome 7p13[] is identified to play a key role in many types of human tumors such as osteosarcoma[] papillary thyroid carcinoma[] pancreatic ductal adenocarcinoma[] colorectal carcinoma[] hepatocellular carcinoma[] prostate cancer[] and breast cancer[] To our knowledge the potential impact of SNHG15 on the tumorigenesis of UM seems unclear recently Thus the purpose of this study was to evaluate the pivotal role of SNHG15 in the progression of UM In addition the relationship between SNHG15 expression and clinicopathologic characteristics in UM was preliminarily demonstrated To explore the underlying mechanisms of the biological pathways involved in UM we conducted a research on Gene Set Enrichment Analysis GSEA MATERIALS AND METHODSEthical Approval The study protocol was approved by the Ethics Committee of the Eye ENT Hospital of Fudan University and all procedures were complied with the principles of the Declaration of Helsinki All datasets of our present study were downloaded from an database TCGA so there was no written informed consent from participantsRNASequencing Patient Data and Bioinformatics Analysis The RNASeq gene expression level and clinicopathological characteristics including cases were obtained from the official website of the Cancer Genome Atlas TCGA UM project portalgdccancergov Patients with UM were classified as two groups based on the median SNHG15 expression level cutoff value794 FPKM Finally patients with UM were retained and their clinicopathological characteristics were further analyzed including the detailed information of age gender tumor diameter thickness pathological type extrascleral extension cancer status pathological stage metastasis living status SNHG15 expressionGene Set Enrichment Analysis GSEA is a common bioanalysis used to interpret and analyze microarray and other similar data and to speculate related pathways that can significantly enrich regulatory genes[] Through TCGA UM project we obtained the RNASeq gene expression level of UM patients And the analysis was conducted using GSEA v30 software In this study according to the association with SNHG15 expression the ordered gene list was generated firstly by GSEA Subsequently GSEA was conducted to clarify statistically significant differences between the two groups with high and low SNHG15 expression A total of permutations were performed The SNHG15 expression level was identified as a phenotype label The related pathways statistically enriched in each phenotype were selected with the nominal P005 and an false discovery rate FDR Statistical Analysis The SPSS statistical software package SPSS Inc USA was used for statistical analyses Both the univariate and multivariate analyses using Cox analysis were performed to demonstrate independent prognostic biomarkers for UM patients The survival curve was generated by conducting KaplanMeier method To compare the significant differences in overall survival OS the logrank test was conducted The plot chart was performed to visualize the difference of SNHG15 expression level for diverse variables through Graphpad The relationship between the SNHG15 expression and clinicopathological characteristics were analyzed using logistic regression The median value of SNHG15 expression was selected as the cutoff value P005 was considered statistically significantRESULTSPatient Characteristics The records of primary UM with both RNASeq gene expression level and clinicopathological characteristics were obtained from TCGA database The mean age of UM patients was years old including males and females The mean value of tumor diameter and thickness were and mm respectively In our study cohort the pathological type of UM included epithelioid cell dominant type and spindle cell dominant type of tumors were epithelioid cell dominant and were spindle cell dominant There were cases without extrascleral extension and cases with extrascleral extension The cancer status included tumorfree cases and cases with tumor Pathologic stage II was found in cases and stage IIIIV in cases And of cases had metastases of cases had no metastases Of cases cases died of all causes Survival Outcomes and Multivariate Analysis Prognostic factors of UM were analyzed using univariate and multivariate Cox regression The univariate analysis suggested that high SNHG15 expression was a risk factor for death from all causes Other clinicopathologic variables related to poor prognosis included age tumor diameter pathological type extrascleral extension cancer status Table In a multivariate analysis SNHG15 was an independent risk factor for death from all causes as was age and pathological typeSNHG15 Expression Associated with Clinical Pathological Characteristics A total of UM cases with SNHG15 expression data and clinicopathologic characteristics were analyzed from TCGA KaplanMeier survival analysis 0cTable Prognostic parameters in UM were analyzed using univariate and multivariate Cox regressionDeath from all causesParametersnmeanUnivariate analysisPHR95CIAge yGenderFemaleMaleTumor diameter mmThickness mmPathological typeEpithelioid cell dominantSpindle cell dominantExtrascleral extensionNoYesCancer statusTumor freeWith tumorPathological stageIIIIIIVSNHG15HighLowUM Uveal melanomaMultivariate analysisPHR95CIdemonstrated that high SNHG15 expression group had a worse prognosis when compared to low SNHG15 expression group Figure 1A P005 As shown in Figure 1B1D SNHG15 was statistically differentially expressed in diverse groups of the tumor pathologic stage stage II vs IIIIV P00257 metastasis P00071 living status P00017 To clarify the clinicopathologic impact of SNHG15 we also used logistic regression and concluded that the SNHG15 expression based on median value of FPKM as a categorical variable was statistically related to clinicopathologic features Table High SNHG15 expression was significantly related to cancer status pathologic stage metastasis living status in UM all P005 Table These results demonstrated that UM with high SNHG15 expression were prone to progress to cancer status of survival with tumor a more advanced stage metastasis and poor living status when compared to the low SNHG15 expression group However there was no statistically significant difference in age gender tumor diameter thickness pathological type extrascleral invasionMain Enriched Pathways in UM Tissues with High SNHG15 Expression To explore the SNHG15related potential signaling pathways activated in UM GSEA was performed In the current study based on the association with SNHG15 expression the gene list was generated firstly Figure The SNHG15 expression was associated with clinical pathological characteristics A Patients with high SNHG15 expression had a shorter OS when compared with the low SNHG15 expression group P002 BD The expression of SNHG15 was statistically different in diverse groups of the tumor pathologic stage P00257 metastasis P00071 living status P00017 aP005 bP001by GSEA To clarify the statistically significant differences between high and low SNHG15 expression groups GSEA was Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomaTable Association between SNHG15 expression and clinicopathologic variables using logistic regressionParametersAge yGenderFemaleMaleTumor diameter mmThickness mmPathological typeEpithelialNonepithelialExtrascleral invasionNoYesCancer statusTumor freeWith tumorPathological stageIIIIIIVMetastasesNoYesLiving statusAliveDeadnmeanSNHG15 expressionLowHighPOR95CINESNominal PvalESTable Enriched pathways for differential SNHG15 expression in UMNameSpliceosomeCell cyclePyrimidine metabolismDNA replicationNucleotide excision repairRNA degradationHomologous recombinationMismatch repairUM Uveal melanoma ES Enrichment score NES Normal enrichment score FDR False discovery rateFDR Qvalconducted subsequently The results indicated that there were significant differences in spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair among patients with high SNHG15 expression phenotype Figure Table DISCUSSIONAccumulating evidences indicate that SNHG15 plays a dual role in the tumorigenesis and development of different tumors[] Previously SNHG15 has been demonstrated as a carcinogenic lncRNA which is usually upregulated in tumor tissues compared with normal tissues[] It exerts 0cFigure Enrichment plots from GSEA Spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair are enriched significantly in SNHG15 high expression phenotypean oncogenic effect via various epigenetic mechanisms[] For example it can suppress the expression of miR3383p and facilitate the proliferation of colorectal cancer cells[] It plays a carcinogenic role by affecting miR3383pFKBP1A axis in prostate cancer[] It can also enhance hepatocellular carcinoma progression by negative regulation of miR1413p[] However there are reports that SNHG15 has a tumor suppressive effect suggesting that low SNHG15 expression is related to poor prognosis in thyroid cancer and upregulating expression of SNHG15 can significantly suppress cell proliferation[] At present the impact of SNHG15 on UM is still unclear Therefore vital roles and potential biological mechanism of SNHG15 in UM needs to be elucidated In this study we revealed that high SNHG15 expression was related to clinicopathologic features in UM Through RNASeq gene expression level and clinicopathological characteristics obtained from the TCGA UM project we analyzed the relationship among SNHG15 expression clinicopathological features and prognosis of UM The univariate analysis demonstrated that SNHG15 expression level age tumor diameter pathological type extrascleral extension and cancer status were risk factors for death from all causes The multivariate analysis suggested that high SNHG15 expression along with age and pathological type was an independent risk factor for death from all causes Therefore the results demonstrated that high SNHG15 expression was an independent predictor of poor prognosis in UM through univariate and multivariate analysis KaplanMeier survival analysis also indicated that high SNHG15 expression group had a worse prognosis when compared to low SNHG15 expression group in UM In addition an analysis was conducted to further explore the relationship between SNHG15 and clinicopathological features The SNHG15 expression was statistically different in diverse groups of the tumor pathologic stage metastasis and living status Besides high SNHG15 expression based on median expression value of FPKM in UM was associated with cancer status of survival with tumor advanced pathologic stage metastasis and living status It demonstrated that high SNHG15 expression in UM was strongly related to poor prognosis The mechanisms of SNHG15 dysregulation in malignant tumors are quite complex and are far from being completely understood Previous studies have suggested that SNHG15 is involved in diverse pathological and physiological processes of many tumors through their abnormal expressions including cell proliferation invasion migration and autophagy[] To explore the biological mechanism of SNHG15 in UM GSEA was conducted It indicated that spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair were all enriched differentially in SNHG15 high expression phenotype Alternative splicing is essential for gene regulation and abnormal splicing plays a vital role in inactivating tumor suppressor genes or activating oncogenes[] SNHG15 may have an impact on the invasion Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomaand migration of UM cells by affecting spliceosomal related factors The abnormal cell proliferation of tumor is related to the lack of checkpoint control over the cell cycle which is the basis of genetic instability[] Evidence shows that the lack of homologous recombination may facilitate the disturbance of cell cycle the instability and accumulated mutations of genome during the progression and development[] Mismatch repair proteins have an significant role in DNA hypermethylation alteration and tumorigenesis[] SNHG15 is closely related to DNA replication and mismatch repair demonstrating that SNHG15 may promote the occurrence of UM by affecting DNA replication and DNA mismatch repair It indicated that SNHG15 may be identified as a novel marker of diagnosis therapeutic and prognosis prediction in UM However the related mechanism needs to be further elucidated This research also has some limitations The most important one is the limited number of patients and time of followup In addition some patient characteristics such as ciliary body involvement were not completely recorded in the database In fact ciliary body involvement plays a critical role in UM[]In conclusion this study aims to demonstrate the vital role of SNHG15 in UM and the potential relationship between SNHG15 expression and clinical parameters SNHG15 expression may be a valuable biomarker for poor survival in UM Moreover we have preliminarily explored the crucial pathway associated with SNHG15 in UM However further experimental validation is needed to be performed for clarifying the significant impact of SNHG15 And it is of great significance to further identify its independent prognostic value in a largescale standardized researches on UMACKNOWLEDGEMENTSFoundations Supported by the National Natural Science Foundation of China No81970835 No81800867 Conflicts of Interest Wu X None Li XF None Wu Q None Ma RQ None Qian J None Zhang R NoneREFERENCES van Raamsdonk CD Griewank KG Crosby MB Garrido MC Vemula S Wiesner T Obenauf AC Wackernagel W Green G Bouvier N Sozen MM Baimukanova G Roy R Heguy A Dolgalev I Khanin R Busam K Speicher MR O€™Brien J Bastian BC Mutations in GNA11 in uveal melanoma N Engl J Med Carvajal RD Sosman JA Quevedo JF Milhem MM Joshua AM Kudchadkar RR Linette GP Gajewski TF Lutzky J Lawson DH Lao CD Flynn PJ Albertini MR Sato T Lewis K Doyle A Ancell K Panageas KS Bluth M Hedvat C Erinjeri J Ambrosini G Marr B Abramson DH Dickson MA Wolchok JD Chapman PB Schwartz GK Effect of selumetinib vs chemotherapy on progressionfree survival in uveal melanoma a randomized clinical trial JAMA Shain AH Bagger MM Yu R Chang D Liu SS Vemula S Weier JF Wadt K Heegaard S Bastian BC Kiilgaard JF The genetic evolution of metastatic uveal melanoma Nat Genet Bagger M SmidtNielsen I Andersen MK Jensen PK Heegaard S Andersen KK Friis S Kiilgaard JF Longterm metastatic risk after biopsy of posterior uveal melanoma Ophthalmology Kujala E Mäkitie T Kivelä T Very longterm prognosis of patients with malignant uveal melanoma Invest Ophthalmol Vis Sci Chandran SS Somerville RPT Yang JC Sherry RM Klebanoff CA Goff SL Wunderlich JR Danforth DN Zlott D Paria BC Sabesan AC Srivastava AK Xi LQ Pham TH Raffeld M White DE Toomey MA Rosenberg SA Kammula US Treatment of metastatic uveal melanoma with adoptive transfer of tumourinfiltrating lymphocytes a singlecentre twostage singlearm phase study Lancet Oncol Mendell JT Targeting a long noncoding RNA in breast cancer N Engl J Med Lan Y Xiao XW He ZC Luo Y Wu CF Li L Song X Long noncoding RNA OCC1 suppresses cell growth through destabilizing HuR protein in colorectal cancer Nucleic Acids Res Cao CH Sun JY Zhang DY Guo XJ Xie LW Li X Wu DH Liu L The long intergenic noncoding RNA UFC1 a target of microRNA 34a interacts with the mRNA stabilizing protein HuR to increase levels of βcatenin in HCC cells Gastroenterology 20151482415426e18 Wang P Xue YQ Han YM Lin L Wu C Xu S Jiang ZP Xu JF Liu QY Cao XT The STAT3binding long noncoding RNA lncDC controls human dendritic cell differentiation Science Zheng XL Tang HW Zhao XF Sun YM Jiang YF Liu YH Long noncoding RNA FTH1P3 facilitates uveal melanoma cell growth and invasion through miR2245p PLoS One 20171211e0184746 Lu QK Zhao N Zha GP Wang HY Tong QH Xin SH LncRNA HOXA11AS exerts oncogenic functions by repressing p21 and miR in uveal melanoma DNA Cell Biol Lu LN Yu XY Zhang LL Ding X Pan H Wen XY Xu SQ Xing Y Fan JY Ge SF Zhang H Jia RB Fan XQ The long noncoding RNA RHPN1AS1 promotes uveal melanoma progression Int J Mol Sci Wu S Chen H Han N Zhang CX Yan HT Long noncoding RNA PVT1 silencing prevents the development of uveal melanoma by impairing MicroRNA173pdependent MDM2 upregulation Invest Ophthalmol Vis Sci Li P He J Yang Z Ge SF Zhang H Zhong Q Fan XQ ZNNT1 long noncoding RNA induces autophagy to inhibit tumorigenesis of uveal melanoma by regulating key autophagy gene expression Autophagy Dong YZ Meng XM Li GS Long noncoding RNA SNHG15 indicates poor prognosis of nonsmall cell lung cancer and promotes 0ccell proliferation and invasion Eur Rev Med Pharmacol Sci SNHG15 serves as an oncogene and predicts poor prognosis in epithelial ovarian cancer Onco Targets Ther Liu K Hou Y Liu YK Zheng J LncRNA SNHG15 contributes to proliferation invasion and autophagy in osteosarcoma cells by sponging miR141 J Biomed Sci Wu DM Wang S Wen X Han XR Wang YJ Shen M Fan SH Zhang ZF Shan Q Li MQ Hu B Lu J Chen GQ Zheng YL LncRNA SNHG15 acts as a ceRNA to regulate YAP1Hippo signaling pathway by sponging miR200a3p in papillary thyroid carcinoma Cell Death Dis Guo XB Yin HS Wang JY Evaluating the diagnostic and prognostic value of long noncoding RNA SNHG15 in pancreatic ductal adenocarcinoma Eur Rev Med Pharmacol Sci Sun XT Bai Y Yang C Hu SY Hou ZL Wang GX Long noncoding RNA SNHG15 enhances the development of colorectal carcinoma via functioning as a ceRNA through miR141SIRT1Wntβcatenin axis Artif Cells Nanomed Biotechnol Ye JF Tan LD Fu Y Xu HJ Wen LJ Deng Y Liu K LncRNA SNHG15 promotes hepatocellular carcinoma progression by sponging miR1413p J Cell Biochem Zhang JH Wei HW Yang HG Long noncoding RNA SNHG15 a potential prognostic biomarker for hepatocellular carcinoma Eur Rev Med Pharmacol Sci Zhang YL Zhang DH Lv J Wang S Zhang Q LncRNA SNHG15 Acts as an oncogene in prostate cancer by regulating miR3383pFKBP1A axis Gene Kong QL Qiu M Long noncoding RNA SNHG15 promotes human breast cancer proliferation migration and invasion by sponging miR2113p Biochem Biophys Res Commun Wang TQ Sun HB Bao Y En R Tian YJ Zhao W Jia LZ POM121 overexpression is related to a poor prognosis in colorectal cancer Expert Rev Mol Diagn Shuai Y Ma ZH Lu JW Feng JF LncRNA SNHG15 a new budding star in human cancers Cell Prolif 2020531e12716 Qu C Dai CM Guo YH Qin R Liu JB Long noncoding RNA Ma YW Xue YX Liu XB Qu CB Cai H Wang P Li ZQ Li Z Liu YH SNHG15 affects the growth of glioma microvascular endothelial cells by negatively regulating miR153 Oncol Rep Li M Bian ZH Jin GY Zhang J Yao SR Feng YY Wang X Yin Y Fei BJ You QJ Huang ZH LncRNASNHG15 enhances cell proliferation in colorectal cancer by inhibiting miR3383p Cancer Med Liu YC Li JL Li F Li M Shao Y Wu LP SNHG15 functions as a tumor suppressor in thyroid cancer J Cell Biochem Liu YC Li JL Li M Li F Shao Y Wu LP microRNA5105p promotes thyroid cancer cell proliferation migration and invasion through suppressing SNHG15 J Cell Biochem Li YW Guo HY Jin CJ Qiu CP Gao M Zhang L Liu ZJ Kong BH Spliceosomeassociated factor CTNNBL1 promotes proliferation and invasion in ovarian cancer Exp Cell Res Williams GH Stoeber K The cell cycle and cancer J Pathol Yu B Ding YM Liao XF Wang CH Wang B Chen XY Overexpression of PARPBP correlates with tumor progression and poor prognosis in hepatocellular carcinoma Dig Dis Sci Maiuri AR Peng M Podicheti R Sriramkumar S Kamplain CM Rusch DB DeStefano Shields CE Sears CL O€™Hagan HM Mismatch repair proteins initiate epigenetic alterations during inflammationdriven tumorigenesis Cancer Res Berry D Seider M Stinnett S Mruthyunjaya P Schefler AC Ocular Oncology Study Consortium Relationship of clinical features and baseline tumor size with gene expression profile status in uveal melanoma a Multiinstitutional study Retina Jiang ZM Yu FH Li M Upregulation of BCL2 kD proteininteracting protein BNIP3 is predictive of unfavorable prognosis in uveal melanoma Med Sci Monit Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0c'
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breast cancer bc is the most common malignant tumour in women worldwide and one of the most common fataltumours in women deltanotchlike epidermal growth factor egfrelated receptor dner is a transmembraneprotein involved in the development of tumours the role and potential mechanism of dner inepithelial“mesenchymal transition emt and apoptosis in bc are not fully understood we find that dner isoverexpressed in bc tissue especially triplenegative breast cancer tnbc tissue and related to the survival of bc andtnbc patients in addition dner regulates cell emt to enhance the proliferation and metastasis of bc cells via thewntcatenin pathway in vitro and in vivo moreover the expression levels of catenin and dner in bd tissue arepositively correlated the simultaneously high expression of dner and catenin contributes to poor prognosis in bcpatients finally dner protects bc cells from epirubicininduced growth inhibition and apoptosis via the wntcatenin pathway in these results suggest that dner induces emt and prevents apoptosis by the wntcatenin pathway ultimately promoting the malignant progression of bc in our study demonstrates thatdner functions as an oncogene and potentially valuable therapeutic target for bcintroductionbreast cancer bc is the most common malignanttumour in women worldwide and one of the most common fatal tumours in women12 bc treatments can beused to improve patient outcome3 however tumourrecurrence and metastasis and chemotherapeutic resistance are the most common causes of cancer treatmentfailure therefore the need to screen and identify keyregulatory factors in the process of tumour recurrenceand metastasis for the treatment of bc is urgentcorrespondence si sun karensisi126com or shengrong sun sun137sinacom1department of breast and thyroid surgery renmin hospital of wuhanuniversity wuhan hubei china2department of pathophysiology wuhan university school of basic medicalsciences wuhan hubei chinafull list of author information is available at the end of the these authors contributed equally zhong wang zhiyu liedited by s taittumour emt is a multifactorial and complex event inwhich epithelial properties and the ability to adhere toadjacent cells are lost and mesenchymal and stem cellphenotypes are eventually obtained4“ emt a crucialregulatory mechanism by which tumours acquire invasiveand metastatic abilities and the ability to resist apoptosisplays an irreplaceable role in the development of malignant tumours8“ recent studies upon activation of theclassical wntcatenin pathway catenin enters andaccumulates in the nucleus which induces the transcription and translation of downstream target genes thusaccelerating emt10 therefore maintaining cateninactivity is important for the wntcatenin pathway andtumour progressiondner a neuronspecific transmembrane protein foundin a variety of peripheral cells11“ is a member of theatypical notch ligand family and binds to notch1 receptor1115 dner is expressed at abnormally high levels in the authors open access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate ifchanges were made the images or other third party material in this are included in the ™s creative commons license unless indicated otherwise in a credit line to the material ifmaterial is not included in the ™s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this license visit httpcreativecommonslicensesby40official of the cell death differentiation association 0cwang cell death and disease page of various cancer tissues16 and promotes the proliferationmigration and invasion of cancer cells1617 but has aninhibitory effect on cell proliferation in glioma14 nevertheless the precise function and underlying molecularmechanisms of emt and chemosensitivity in bc areunclearin this study we have revealed the previously unrecognized role of dner in cancer progression emt andthe apoptosis of bc cells furthermore we investigatedthe expression of dner and its relationship with survivalin bc and tnbc patients in addition we have providedevidence for the correlation between dner and cateninand the prognostic value of the highlevel expression ofdner and catenin in bc patients finally the crucial roleof catenin in dnerinduced emt and the inhibitoryeffect of dner on apoptosis have been revealed takentogether our results elucidate the potential functions andmechanism of dner in emt and apoptosis in bc cellsand provide a new therapeutic pathway for the recurrence metastasis and chemotherapy resistance of bcmaterials and methodsethics statementtwo groups of the same human tissue specimens wereacquired from patients of renmin hospital of wuhanuniversity who were diagnosed with bc from to one group of specimens was promptly stored atˆ’ °c for western blotting and pcr analysis the othergroup of specimens was fixed in formalin and paraffinizedfor immunohistochemistry ihc all patients did notreceive chemotherapy radiotherapy or immunotherapythis research was approved by the ethics committee ofrenmin hospital of wuhan university and informedconsent was obtained from all patientscell culture and reagentshuman bc cell lines mcf7 and mdamb468 cellswere obtained from american type culture collectionand incubated by their corresponding recommendedmethod all celllines were mycoplasmafree by morphological examination and verified for their authenticities by str profiling epirubicin was purchased frompfizer pharmaceutical co ltd wuxi china and dissolved in physiological saline chir catenininhibitor and xav939 catenin agonist were purchased from selleck shanghai china and dissolvedin dmso “ “ and the stainingintensity was evaluated as follows no staining weak staining moderate staining and strongstaining the final protein staining score was the percentage score multiplied by the intensity score finalprotein staining scores were divided into three categoriesas follows negative “ low expression and “ high expressionsirna and plasmid transfectionscrambledner sirna ²gcuuugccaguccaagauuttsirna ²uucuccgaacguguandcacgutt were synthesized from genepharma coshanghai china flagdner and flagnc werepurchased from genechem co shanghai china whencells in a sixwell plate had grown to the appropriatedensity sirna and plasmids were transiently transfectedwith lipofectamine3000 invitrogen usa and rnaimax invitrogen usa respectively according to themanufacturer™s instructions after h of transfection thecells were used for subsequent experimentsqrtpcrtotal rna from tissue specimens and cell samples wasextracted by using trizol invitrogen usa according tothe protocol and then reverse transcribed to cdna usinga transscript firststand cdna synthesis kit takarajapan qrtpcr was implemented by using sybr greenmastermix takara japan with an abi 7900ht realtime pcr system usa the primer sequences areshown in supplemental table cell counting kit cck8 assayafter a series of interventions equal numbers of bccells were plated into 96well plates and cultured for days ten microlitres of cck8 ck04 dojindo japansolution was added to each well and the cells wereincubated at °c for h the absorbance was determined at nmwound healing assayafter intervention the cells were seeded into sixwellplates when the cell density exceeded the cells werewashed twice with pbs and scratches were made with ayellow plastic pipette tip cells were cultured in serumfree medium for h and photographed under amicroscopeimmunohistochemical staininginvasion assayihc staining was performed as previously described18the results of ihc staining were evaluated by two independent pathologists and scored according to the percentage of positive tumour cells and staining intensitythe percentage of positive cells was scored as follows after a series of treatments × cells in serumfreemedium were plated in the upper chambers of a transwell apparatus with matrigel corning ny usa medium in the bottom chambers containing fbs servedas an attractant after h of incubation cells that passedofficial of the cell death differentiation association 0cwang cell death and disease page of through the chamber membrane were fixed with precooled formaldehyde and stained with crystal violetc0121 beyotime the cells were counted and photographed under a microscopewestern blottingthe prepared tissue and cell samples were separated byprotein sdspage and transferred to a nitrocellulosenc membrane the membrane was blocked in skimmilk powder for h at room temperature and immunoblotted with primary antibody at °c overnight afterincubation with secondary antibody at room temperaturefor h protein expression was detected with corresponding protein development instrument and quantifiedby imagej software w s rasband image j nih theantibodies used are listed in supplementary table nuclear and cytoplasmic protein extractionnuclear and cytoplasmic extraction reagent p0027was purchased beyotime biotechnology the nuclear andcytoplasmic proteins were extracted according to theinstructions and then used for subsequent experimentsflow cytometry to detect apoptosisa fitc annexin v apoptosis detection kit i bdpharmingen usa was used to detect cell apoptosis the cellswere seeded in sixwell plates after a series of interventionscells were processed following the manufacturer™s protocolfig dner is upregulated in bc tissues and correlated with poor prognosis in bc and tnbc patients a the expression levels of dner inluminal a and tnbc tumour tissues compared with adjacent tissue by ihc magnification × b the mrna levels of dner in luminal a and tnbctumour tissues compared with adjacent tissue c the dner protein expression in bc tissues and adjacent tissues by western blotting d thekaplan“meier analysis showed the rfs of bc and tnbc patients with dner high expression or dner low expression e the staining of dner ecadherin and ncadherin in bc tissue by ihc magnification × f correlation analyses of protein expression levels between ecadherin ncadherinand dner p p vs the control groupofficial of the cell death differentiation association 0cwang cell death and disease page of and the cell fluorescence was measured with a facscan flowcytometer facscan becton dickinsontable clinicopathological associations of dnerexpression in breast canceranimal experimentsto acquire mdamb468 cells with dner stablyknocked down and mcf7 cells stably overexpressingdner cells were transfected with dner knockdown andoverexpression lentivirus genechem shanghai chinaand then selected with puromycin when the transfectionefficiency approached the dner protein level wasdetected with western blotting all experimental procedures were conducted according to the regulations ofexperimental animal administration issued by the animal committee of wuhan university the mice wererandomly divided into two groups a total of × stable cells in μl pbs were subcutaneously inoculatedinto the right iliac fossa of to 5weekold femaleathymic nude mice balbc after a certain period ofintervention the mice were sacrificed by anaesthesia andxenografts were removed for weighing and photographing the expression of relative proteins was detected bywestern blotting and ihcfor mammaryfatpad tumour assays we establishedmdamb231 cells with dner stably knocked downthe mice were randomly divided into two groups × stable cells were resuspended in a mixture of pbs andmatrigel and then injected into the fourth mammaryfat pad on the same side of nude mice to observe lungmetastasis tumours were excised by surgical operationwhen they reached about mm3 ten days after theoperation the mice were sacrificed by anaesthesia and thenumber of metastatic tumours per lung were determinedthe entire lung tissues were fixed with formalin andsectioned for haematoxylin and eosin he staining todetermine the presence of lung metastasis the entirelung tissues were fixed with formalin and sectionedfor haematoxylin and eosin he staining to determinethe presence of lung metastasisimmunofluorescenceimmunofluorescence staining was performed as previously described19 in brief after corresponding treatments the cells fixed with paraformaldehyde wereperforated by tritonx for min and blockedwith bsa for h next the cells were incubated withcatenin dilution overnight at °c and thenincubated for min with 488conjugated antibodyinvitrogen a11034 finally the slides were stained withdapi for min the images of sample were analyzed bylaser confocal microscopy zeiss lsm statistical analysisstatisticalspss software spss inc chicago il and graphpadanalyses were performed usingofficial of the cell death differentiation associationvariableslown highn p valueage at diagnosis years‰¤gradewellmoderatelypoorlytumour size cm‰¤lymph node metastasisnegativepositivevascular invasionnegativepositiveernegativepositiveprnegativepositiveher2negativepositiveki67 ‰¥ recurrencenoyes p values calculated by logrank testing bold if statistically significant p er oestrogen receptor pr progesterone receptor her2 human epithelial growthfactor receptor2prism graphpad software la jolla ca usa all datawere analyzed with at least three independent experiments and are presented as the mean ± sd a survivalcurve was prepared by kaplan“meier analysis and thelogrank test was used to compare survival differencesbetween groups pearson™s correlation method was used 0cwang cell death and disease page of table clinicopathological associations of dnerexpression in triple negative breast cancervariableslown highn p valueage at diagnosis years‰¤gradewellmoderatelypoorlytumour size cm‰¤lymph node metastasisnegativepositivevascular invasionnegativepositiveki67 ‰¥ recurrencenoyes p values calculated by logrank testing bold if statistically significant p to analyze the correlation between dner and catenina chisquare test was used to analyze associationsbetween dner expression levels and clinical characteristics oneway anova was used to compare differencesin three or more groups differences in which p were considered statistically significantresultsdner is upregulated in bc tissues and correlated withpoor prognosis in bc and tnbc patientsto determine the role of dner in development of bcwe first measured the expression levels of dner in bctissue and matched adjacent normal breast tissue by ihcthe expression level of dner in bc tissue was markedlyhighertheexpression in tnbc was higher than that in luminal a bcfig 1a we also detected the expression of dner in bctissue by pcr the results of which were consistent withthose of ihc experiments fig 1b to further verifytissue moreoverthan thatin adjacentofficial of the cell death differentiation associationdner expression in bc we utilized western blotting todetect dner protein expression in bc and adjacent tissues as expected compared with dner expression inadjacent tissues dner expression in bc tissues wassignificantly elevated fig 1c furthermore the highestdner expression level was found in tnbc tissue theclinicopathological characteristics with different expression of dner in all bc and tnbc patients were shown intables and kaplan“meier analysis of rfs showed thatthe group expressing high levels of dner had a worseprognosis than the group expressing low levels of dnerthe results of survival analysis of tnbc patients were thesame as that of bc patients and tnbc patients had ashorter rfs than bc patients fig 1d next to verifywhether the poor prognosis of bc patients caused bydner is related to emt we detected the correlationbetween dner and emtrelated markers the resultsshowed that dner expression was negatively correlatedwith the expression of ecadherin while positively correlated with ncadherin expression fig 1e f in addition we found that high expression of mesenchymalmarkers was significantly associated with high expressionof dner in bc through the tcga database httpgepiacancerpkucn although the negativecorrelationbetween ecadherin and dner in tcga database wasnot significant it also presented a negative trend supplementary fig 2a the results therefore suggested thatdner is highly expressed in bc and that elevated dnerprotein expression contributes to the progression of bcespecially tnbcdner increases the biological functions of bc cells in vitroto evaluate the effect of dner on bc cell proliferationmigration and invasion we used sirna to suppressdner expression in both mcf7 and mdamb468cells compared with dner expression in the control andscramble sirna groups dner was silenced by almost and in mcf7 and mdamb468 cells transfected with sirna respectively fig 2a b as shown infig 2c dner knockdown visibly downregulated thegrowth rate of bc cells by cck8 assay next a woundhealing assay was used to evaluate cell migration capacitycompared with wound closure in the scramble sirnagroup dner knockdown significantly inhibited woundclosure after h in bc cells fig 2d in addition thetranswell assay revealed that dner knockdown clearlyreduced bc cell invasion fig 2e these results suggestthat dner acts as a cancerpromoting gene in bc cellsto further confirm the role of dner in bc progressiondner was overexpressed by transfection with the flagdner plasmid for h as shown in supplementary fig1a dner was successfully overexpressed in the two bccell lines in striking contrast with the effects of dnerknockdown the ability of cell proliferation migration and 0cwang cell death and disease page of fig dner knockdown inhibits cell proliferation and metastasis of bc cells a b the knockdown efficiency of dner in mcf7 and mdamb cells c cell growth was measured by cck8 assay after dner knockdown in two bc cell lines d wound healing assay was used to determine themigratory ability of bc cells with dner knockdown e the invasion capacity of bc cells with knockdown of dner was confirmed by transwell assaydown quantitative analysis of invasion ratio was shown the values are the mean ± sd from three independent experiments nsp p p p p vs the control groupinvasion was markedly enhanced after dner overexpression supplementary fig 1b“e taken togetherthese results indicated that dner plays a crucial role inbc growth and metastatic potentialdner induces emt in bc cellstumour cell emt promotes the malignant progressionand metastasis of tumour cells10 we next examinedwhether dner has a regulatory effect on bc cell emtto assess this function we detected emtrelated proteinexpression by western blotting dner knockdown significantly upregulated epitheliallike marker ecadherinexpression and downregulated mesenchymal marker ncadherin vimentin snail expression fig 3a b conversely overexpression of dner dramatically shown theopposite effect fig 3c d these results indicate thatdner drives emt in bc cells to provide further evidence of this effect of dner on emt we suppresseddner expression and then transfected cells with theflagdner plasmid to restore the dner protein levelwe then determined whether dner overexpression couldreverse changes in the expression of emtrelated proteins as shown in fig 3e f dner knockdown alone hadan inhibitory effect on emt whereas dner knockdownand flagdner transfection suppressed the effect ofdner knockdown on ecadherin and partially restoredthe expression of ncadherin vimentin and snail theseresults suggest that dner plays a pivotal role in inducingemt in bc cellsofficial of the cell death differentiation association 0cwang cell death and disease page of fig dner induces emt in bc cells a b emtrelated proteins ecadherin ncadherin vimentin and snail were detected by western blotting indner knockdown cells right quantitative analysis of the optical density ratio of ecadherin ncadherin vimentin and snail compared with actinare shown c d emtrelated protein levels were measured by western blotting after dner overexpression in bc cells right quantitative analysis ofthe optical density ratio of ecadherin ncadherin vimentin and snail compared with actin are shown e f dner was overexpressed in dnerknockdown cells and then western blotting detected the expression of emtrelated proteins the values are the mean ± sd from three independentexperiments p p p vs the corresponding groupdner activates the wntcatenin signalling pathway andis positively correlated with cateninprevious reports have shown that the wntcateninsignalling pathway plays a crucial role in cancer cellmetastasis and emt2021 therefore we examined whether dner mediates the canonical wntcatenin signalling pathway as shown in fig 4a b compared withcontrol cells in dner knockdown cells the protein levelsof notch1 pgsk3 and catenin were increased andthose of gsk3 were unchanged conversely dneroverexpression dramatically shown the opposite effectnext we investigate whether there is a relationshipbetween notch signal and catenin in the case of dneroverexpressioncells wein dneroverexpressingknocked down notch1 and found that catenin expression was decreased compared with dner overexpressionalone supplementary fig 2b notch1 functioned as animportant role in the wntcatenin pathway and theactivation of notch1 was positively related to the nucleartranslocation of catenin22 theaccumulation ofcatenin in the nucleus plays an important role in themalignant progression of tumours we assessed the effectof dner knockdown on nuclear catenin accumulationby western blotting and observed that upon the knockdown of dner the levels of nuclear catenin and snailwere reduced in bc cell lines fig 4c and supplementaryfig 2c the nuclear location of catenin detected byimmunofluorescence showed the same results as thoseofficial of the cell death differentiation association 0cwang cell death and disease page of fig dner activates the wntcatenin signalling pathway and is positively correlated with catenin a b western blotting detected theexpression of notch1 pgsk3 gsk3 and catenin after dner knockdown or dneroverexpressing in bc cells c total proteins catenin andsnail nuclear proteins catenin and snail in dner knockdown cells were assayed with western blotting d the mrna levels of survivin cmyc andlef1 were detected by qrtpcr e the staining of dner and catenin in bc tissue by ihc magnification × f correlation analyses of proteinexpression levels between dner and catenin g kaplan“meier survival analysis of bc patients was performed with dnerhighcateninhigh anddnerlowcateninlow expression the values are the mean ± sd from three independent experiments p p vs thecorresponding groupdetermined by western blotting supplementary fig 2dto further confirm the decrease in nuclear cateninaccumulation following dner knockdown we examinedthe expression levels of catenin downstream targetgenes in bc cells by pcr consistent with the westernblotting results the mrna expression levels of survivincmyc and lef1 were significantly downregulated upondner knockdown fig 4d these data indicated thatdner knockdown can inhibit nuclear translocation andtranscriptional activity of catenin thereby controllingthe wntcatenin signalling pathwayto verify the relationship between dner and cateninwe measured the protein expression levels of dner andcatenin in bc tissues ihc showed that catenin washighly expressed when dner was overexpressed whilecatenin levels were low when dner was knocked downfig 4e interestingly correlation analyses showed thatcatenin expression was positively correlated with theexpression of dner fig 4f we also found a strongpositive correlation between dner expression andnuclear catenin expression supplementary fig 2efurthermore immunofluorescence analysis showed thatdner overexpression promoted more nuclear accumulation of catenin in bc cells supplementary fig 2ffinally kaplan“meier analysis showed that the prognosisof bc patients with high levels of dner and cateninwas worse than the prognosis of bc patients with lowlevels of both dner and catenin fig 4g in additionofficial of the cell death differentiation association 0cwang cell death and disease page of table clinicopathological associations of both dnerand catenin expression in breast cancervariableslown highn p valueage at diagnosis years‰¤gradewellmoderatelypoorlytumour size cm‰¤lymph node metastasisnegativepositivevascular invasionnegativepositiveernegativepositiveprnegativepositiveher2negativepositiveki67 ‰¥ recurrencenoyes p values calculated by logrank testing bold if statistically significant p er oestrogen receptor pr progesterone receptor her2 human epithelial growthfactor receptor2we continued to show the correlation between the highlevel expression of both dner and catenin and bcpatient clinicopathologic features as shown in table these data suggest a strong correlation between theexpression of dner with that of catenin and high levelsof dnercatenin with poor prognosis in bcofficial of the cell death differentiation associationthe wntcatenin signalling pathway is involved in dnerinduced emt and prometastatic phenotypesto determine whether the wntcatenin pathwayfunctions in dnerinduced emt we assessed whetherchir a specific wntcatenin pathway activator23 and xav939 a wntcatenin pathway inhibitor24 could reverse the effect of dner overexpressionand dner knockdown in bc cells catenin levels in thetwo bc cell lines were significantly elevated after chir treatment and markedly suppressed after xav939treatment fig 5a b compared with dner knockdownalone levels of the emtrelated proteins were dramatically exhibited the opposite effect after of the treatment ofdner knockdown cells with chir fig 5a thetreatment of dneroverexpressing cells with xav939clearly show similar results fig 5b these findingsindicated that chir partly rescued the inhibitoryeffect of dner knockdown on emt progression and thatxav939 suppressed the activation of emt induced bydner overexpression to investigate the role of the wntcatenin pathway in dnermediated cell proliferationmigration and invasion we performed rescue experimentsby activating or inhibiting catenin in dner knockdownor dneroverexpressing cells respectively consistentwith the effects of wntcatenin pathway activation andinhibition on emt in the presence of chir theproliferation migration and invasion of dner knockdown cells were clearly elevated fig 5c e f similarlyinhibition ofin dneroverexpressing cells distinctly decreased metastatic ability as shown by changes in cell growth migration andinvasion fig 5d g h altogether these data suggestedthat catenin is indispensable for dnerinduced bc cellemt and prometastatic phenotypescatenin by xav939dner enhances the tumorigenic and metastatic ability ofbc cells in vivoto verify our results in vitro we next examined the roleof dner in vivo to that end mdamb468 cells inwhich dner was stably knocked down and mcf7 cellsstably overexpressing dner were successfully establishedto use to establish xenograft models in mice fig 6a b fg after a period of time the xenografts were removedphotographed and weighed dner knockdown significantly inhibited tumour size and weight comparedwith those in nc group fig 6c d consistent with theeffect of dner knockdown xenografts from dneroverexpressing group were larger and heavier than thosefrom nc group more importantly xav939 reversedchanges in the size and weight of xenografts fig 6h ithe dner catenin cmyc and snail protein levels inxenograft tissue were measured to confirm the upregulation and downregulation by western blotting fig 6e jsupplementary fig 3a moreover ihc results found 0cwang cell death and disease page of fig the wntcatenin signalling pathway is involved in dnerinduced emt and metastasis a b the expression of emtrelated proteinsand catenin were detected by western blotting in dner knockdown or dneroverexpressing cells with chir μm h or xav939 μm h treatment respectively c d cell growth was measured by cck8 in bc cells treated as described above e g wound healing assay was used toexamined migration ability in bc cells treated as described above f h transwell assay showed the cell invasion abilities in bc cells treated asdescribed above right quantitative analysis of invasion ratio was shown the values are the mean ± sd from three independent experiments p p vs the corresponding groupthat dner knockdown reduced nuclear location ofcatenin while dner overexpression promoted thisnuclear translocation effect supplementary fig 3c inaddition as shown in supplementary fig 3a c thewestern blotting and ihc results showed that dnerimpacted the tumour growth in vivo was related to thelevel of ki67 which is consistent with the positive correlation between dner expression and ki67 expression inbc patients of tcga database supplementary fig 3bto explore the role of dner in bc metastasis to lungmdamb231 cells with stably dner knockdown wassuccessfully established fig 6k as shown in fig 6l theofficial of the cell death differentiation association 0cwang cell death and disease page of fig dner enhances the tumorigenic ability of bc cells in vivo a f k the transfection efficiency of dner knockdown or expression in mdamb468 mcf7 or mdamb231 cells respectively b g the knockdown or overexpression efficiency of dner in mdamb468 cells or mcf7 cellsrespectively c h the xenograft pictures of shdner and ncdner in mdamb468 cells n d i comparison of tumour weights from variousgroups e j the expression of dner and catenin in xenograft tissue by western blotting h the xenograft pictures of ncdner group oednergroup and oedner treated with xav939 group in mcf7 cells n l schematic diagram of in vivo experimental procedure for lung metastasispotential in situ of bc m bright imaging of the lungs metastasis left and quantification of the metastases tumour right generated by mdamb231cells n p vs the corresponding groupofficial of the cell death differentiation association 0cwang cell death and disease page of fig see legend on next pageofficial of the cell death differentiation association 0cwang cell death and disease page of see figure on previous pagefig dner reduces the chemosensitivity of bc cells to epirubicin in vitro a cell proliferation was detected by cck8 after treated withdifferent concentrations of epirubicin in two bc cell lines b c dner was analyzed by western blotting in bc cells treated as described above rightquantitative analysis of the optical density ratio of dner compared with actin are shown d expression of epirubicininduced dner was detectedby pcr e cell viability was assessed by cck8 after dner knockdown treated with epirubicin or not f analysis of apoptosis with facs in mdamb cells treated as described in e right quantitative analysis of apoptosis ratio g the expression of parp was detected by western blotting in bccells treated as described above right quantitative analysis of the optical density ratio of cparp compared with actin are shown h cell growthwas measured by cck8 after dner overexpression treated with epirubicin or not i analysis of apoptosis with facs in mdamb468 cells treated asdescribed in h right quantitative analysis of apoptosis ratio j the expression of parp was detected by western blotting in bc cells treated asdescribed above right quantitative analysis of the optical density ratio of cparp compared with actin are shown the values are the mean ± sdfrom three independent experiments p p p vs the corresponding groupcorresponding treated mdamb231 cells were injectedinto the fourth mammary fat pad and tumours wereexcised when they reached about mm3 lung metastasis was observed in each group after days brightfieldpicture demonstrated that more lung metastasis wasfound in the ncdner group compared with the shdner group fig 6m similar trends were observable inhe staining analysis supplementary fig 3e moreoverihc results of xenografts showed that dner knockdownobservably upregulated ecadherin expression anddownregulated ncadherin expres
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" ethnopharmacological relevance herba patriniae has been used for thousands of years in china as a traditional chinese medicine with heatclearing and detoxicating effects it is applied widly for the treatment of rheumatoid arthritis diarrhea acute hepatitis pelvic inflammatory disease and ulcerative colitis in clinic two species namely patrinia scabiosaefolia fisch ps and patrinia villosa juss pv from the caprifoliaceae family are considered as herba patriniae in the pharmaceutical industry aim of the review this paper aims to comprehensively outline the traditional uses botanical description phytochemistry pharmacology toxicology quality control pharmacokinetics and patents of herba patriniae and elaborate the samedifferent characteristics between ps and pv materials and methods detailed information of herba patriniae was collected from various online databases pubmed web of science google schola china national preproof 0c knowledge infrastructure database national intellectual property administration prc national medical products administration and those published resources msc thesis and books results a total of compounds have been identified in herba patriniae including triterpenoid saponins flavonoids anic acids irids and volatiles a very distinct difference was observed that ps is rich in triterpenoid saponins and volatiles while pv contains more flavonoids two source species of herba patriniae gave similar pharmacological effects on anticancer antiinflammatory antioxidant antimicrobial sedative and hypnotic effects but there were no reports were on antipruritic proangiogenic and antidiarrheal effects for ps and no studies on antidiabetic effects for pv generally herba patriniae showed nontoxic in the clinical dose but mild side effects such as temporary leukopenia dizziness and nausea could be found when large and excessive dosage is used a variety of compounds have been quantified for the quality control of ps and pv the variety growth environment growth time and harvest time not only affected the contents but also the pharmacological activities of the bioactive compounds in the past year patents for compositions containing pv and ps have been filed mainly involving human health hygiene agriculture and animal husbandry unfortunately the research on pharmacokinetics is insufficient only the prototype components and metabolites were repored after intragastric administration of total flavonoids extract from pv in rats herba patriniae has displayed a significant medicinal value in clinic but the differences in phytochemistry pharmacological effects and the content of compounds have been found between two official recorded species about side effects and pharmacokinetic characteristics the differeces between two species have not been well studied for a better clinical use of herba patriniae it is urgent to establish systematic pharmacology quality control pharmacokinetics and clinical researches on the samedifferent characteristics between ps and pv keywords herba patriniae traditional uses phytochemistry pharmacology quality control preproof 0c cells a549 human lung cells cancer aspartate polysaccharide mixture ast list of abbreviations 3t3l1 preadipocytes 5fuhct8 human ileocecal adenocarcinoma cells a2780 human ovarian cancer cells a375s2 human melanoma cells a498 human renal abts carcinoma 'azinobis3ethylbenzothiazoline6sulphonic acid ags human gastric cancer cells akt protein kinase b alt alanine aminotransferase ap acute pancreatitis ap3 aminotransferase bax bcl2associated x protein bcl2 bcell lymphoma2 bclxl b cell lymphoma factor xl bel7402 human hepatoma cells bv2 mouse microglia cells caco2 human colon cancer cells cox2 cyclooxygenase2 crc colorectal cancer dai disease activity index dpph 22diphenyl1picrylhydrazyl ec50 half maximal effective concentration emt epithelialmesenchymal transition fak focal adhesion kinase gcms gas chromatographymass spectrometer glut4 glucose transporter gsh glutathione h2o2 hydrogen peroxide hela human cervical cancer cells hepg2 human hepatoma cells hl60 human promyelocytic leukemia cells ho1 heme oxygenase1 hplc high performance liquid chromatography hsp heat shock proteins hsp heat shock proteins ht1080 human fibrosarcoma cells ht29 human colon carcinoma cells huvecs human umbilical vein endothelial cells ic50 inhibitory concentration icam1 intercellular adhesion molecule icr institute of cancer research il1 interleukin1 beta il6 interleukin il8 interleukin inos inducible nitric oxide synthase irs insulin receptor substrate k562 human malignant myeloid cells ldh lactate dehydrogenase lps lipopolysaccharides mcf7 human breast cancer cells mda malondialdehyde mdamb231 human breast cancer cells mpo myeloperoxidase mrna messenger ribonucleic acid nfκb nuclear factor κb ngf nerve growth factor no nitric oxide nqo1 quinine oxidoreductase nrf2 nuclear factor erythroid 2related factor o2 superoxide anion oh hydroxyl radical pcna proliferating cell nuclear antigen pid pelvic inflammatory disease ps patrinia scabiosaefolia fisch pv patrinia villosa juss raw2647 mouse leukemic monocyte macrophage ros reactive oxygen species rsv respiratory syncytial virus sars severe acute respiratory syndrome sd sprague dawley sgc7901 human gastric cancer cells smmc7721 hepatocellular carcinoma cells stat3 signal transducer and activator of transcription sw480 human colon carcinoma cells tc50 half toxic concentration tcm traditional chinese medicine tgf transforming growth factor beta ti drug treatment index tnfα tumor necrosis factor alpha taoc total antioxidant capacity tsod total superoxide dismutase u14 mice cervical cancer cells u266 human multiple myeloma cancer cells u937 human lymphoma cells uc ulcerative colitis uv ultraviolet preproof 0c table of contents introduction traditional uses botany phytochemistry triterpenoid aglycones and triterpenoid saponins flavonoids anic acids irids volatiles other compounds pharmacology anticancer effect antiinflammatory effect antioxidant effect antimicrobial antiviral and antifungi effects sedative and hypnotic effects others toxicity quality control pharmacokinetics patented formulations and perspectives acknowledgements conflict of interest author contribution references preproof 0c introduction herba patriniae as known as œbai jiang cao in chinese is a traditional chinese medicine tcm originally recorded in œshen nong™s herbal classic as a middle grade medicinal material which has been used for thousands years besides korean ancient pharmacopaea œdonguibogam also record its medical value and it has been used for more than years in korea jeon et al it possesses the tcm properties of pungent and bitter in flavor and slightly cold in nature and has been classified to the stomach large intestine and liver meridians xiao two official species of patrinia scabiosaefolia fisch ps and patrinia villosa juss pv figure were considered as herba patriniae in chinese pharmacopoeia edition and chinese provincial pharmacopoeias these two plants have been widely used for more than years with good biological activities of clearing heat and detoxification eliminating carbuncle and expelling pus dispelling blood stasis and relieving pain through an analysis of ancient and modern literatures herba patriniae was mostly used in intestinal carbuncle lung carbuncle gynecological epigastric pain postpartum blood stasis and eczema in ancient times chen and han modern pharmacological studies have found that it has effects of anticancer antiinflammation antipathogenic microanisms antioxidation sedation and hypnosis wang et al 2019a nowadays herba patriniae is widely used in the respiratory system digestive system genitourinary system gynecology dermatology and other multidisciplinary diseases in clinical practice zhu and jiang and the number of applied patents increases every year httppsssystemcnipagovcn in view of its high content of amino acids vitamins minerals and other nutrients herba patriniae is not only regarded as a potherb with healthy value but also processed into tea products su et al zeng et al zhong et al in the past decades an increasing number of scholars have studied the chemical constituents and pharmacological effects of herba patriniae interestingly based on these studies we found that there are many differentsame characteristics between ps and pv both of them are official species for herba patriniae but differentiated clinical uses of them in different diseases may be better for the clinical outcome unfortunately we cannot found a comprehensive and updated review on the samedifferent characteristics of the two sources of herba patriniae and actually these two species also have not been differentiated in clinical uses therefore this review aims to systematically summarize the similarities and differences from the preproof 0c aspects of the traditional uses botanical description phytochemistry pharmacology and quality control of these two species of herba patriniae as well as being evidences for their clinical application and further research figure two species of herba patriniae a patrinia villosa juss b patrinia scabiosaefolia fisch a httpwwwcvhaccnspmcshcsh0005548 b httpwwwcvhaccnspmsyaufsyauf010108 traditional uses herba patriniae has a wide geographical distribution mainly in east asia and north america he et al some plants such as sonchus arvensis l sonchus asper vill sonchus oleraceus l etc may be confused as herba patriniae lu and hence these adulterants of herba patriniae should be exclude when clinical use traditionally according to records of œshen nong™s herbal classic 神农本草经 œcompendium of materia medica 本 草 纲 目 and œsynopsis of the golden chamber 金匮要略 œtai ping sheng hui fang 太平圣惠方 œpu ji fang 普 济方 œsheng ji zong lu 圣济æ»å½• œqian jin yi fang 千金翼方 and œqian jin fang 千金方 ancient doctors have used the whole herbs and roots of herba patriniae for disease treatment such as the stomach intestine liver gallbladder and gynecological diseases tian and tian zhu and jiang herba patriniae was recorded in chinese pharmacopoeia edition for the treatments of appendicitis dysentery enteritis hepatitis conjunctivitis postpartum blood stasis abdominal pain swollen wellingabscess and clove sores pharmacopoeia committee of the ministry of health of p r china in addition herba patriniae is also preproof 0c recorded in the standards of traditional chinese medicine in many provinces of china table in miao nationality herba patriniae is also called œjia jiang le and used to treat rheumatoid arthritis colds and diarrheal qiu wang in dong medicine lu yi medicine drug control institute of yunnan chuxiong health bureau and dai medicine shi ps is called œnyangt ngeec liongc bail jangl œshe wei long and œpa hong respectively its whole herb is used to treat infantile diarrhea schizophrenia and infantile tinea capitis respectively ps is also called ba gai bao in zhuang medicine and its root is used to treat icteric hepatitis furuncles and snakebites shi pv is called œbitter vegetable by she nationality biological products identification institute of the ministry of health and œpao zi tong by tujia nationality peng and guan its whole herb can be used to treat appendicitis intestinal febrile symptoms constipation mammary abscess blister carbuncle and qi stagnation pv is also called œba gai lan and œhong pa in zhuang medicine biological products identification institute of the ministry of health and dai medicine shi respectively and its root is used to treat jaundice hepatitis furuncle local ulceration caused by snake injury and infantile convulsion moreover in korea people usually use the roots or whole plants of ps and pv as a traditional herbal medicine to treat appendicitis inflammation wound healing edema abscesses endometritis and abdominal pain after childbirth kang et al yang et al in recent years herba patriniae has been extensively applied in clinical practice in china especially in gynecology such as postpartum pain mastitis dysmenorrhea and tubal obstructive infertility liu 2019a it is noteworthy that herba patriniae is one of the most important ingredients in many prescriptions of tcm which is effective in diarrhea he acute hepatitis song pelvic inflammatory disease zhang 1997a typhoid fever paratyphoid fever sun ulcerative colitis liu anal cryptitis shi pelvic endometriosis yan and qiu acute pancreatitis he et al 2019b itching wang and wang gastroesophageal reflux disease benign prostatic hyperplasia rhinosinusitis mumps and phlebitis kong and zhao zhu and jiang a powder composed of coicis semen radix aconiti lateralis preparata and herba patriniae is a classic prescription for treating intestinal carbuncle in the œsynopsis of the golden chamber which is clinically used to treat chronic appendicitis chronic pelvic inflammatory disease and chronic prostatitis ji in addition a powder containing herba preproof 0c patriniae in the prescription is also used to treat sinusitis acute purulent tonsillitis and recurrent upper respiratory tract infection qin and diao zhu and jiang moreover it showed significant efficacy in the treatment of psoriasis vulgaris yan et al keshan disease scientific research cooperation group of herba patriniae in yan'an city for the prevention and control of keshan disease and chronic pelvic inflammation jia in the form of tablets in the chinese pharmacopoeia edition there are chinese herbal medicine prescriptions containing herba patriniae among which kangfu xiaoyan shuan and yifei qinghua gao are used to treat gynecological diseases and respiratory diseases respectively while longqing pian nankang pian niaosaitong pian and qianliexin jiaonang are used to treat genitourinary diseases state pharmacopoeia commission of p r china a summary of the traditional and traditional and clinical preparation of herba patriniae in china is given in table the tender stems and leaves of herba patriniae are rich in nutrients fresh in taste and grow in the mountains without environmental pollution it is a highquality vegetable that urban and rural residents like to eat pv tea is also abundant in hubei province and fujian province jiang 2019a xu et al herba patriniae is not only used in human health but also in agriculture fishery and animal husbandry interplanting herba patriniae in the newly reclaimed tea garden can increase the natural vegetation and reduce soil erosion and surface runoff caused by rainstorm erosion in the rainy season chen the combination of herba patriniae and other medicinal plants can be used to treat poisoned wound of cattle by agkistrodon acutus bitting crawling bee disease liver and skin diseases of turtle and fish and postpartum abdominal pain in cattle chen li shi zhao preproof 0c table the information of herba patriniae in national and local standards in china standards standard of traditional chinese medicine in hunan province standard of traditional chinese medicine in shandong province standard of traditional chinese medicine in heilongjiang province standard of traditional chinese medicine in liaoning province standard of traditional chinese medicine in sichuan province standard of traditional chinese medicine in guizhou province chinese pharmacopoeia edition application acute appendicitis diarrhea enteritis hemorrhagic leucorrhea red eye pterygium postpartum abdominal pain boils and carbuncles appendicitis dysentery enteritis hepatitis conjunctivitis postpartum blood stasis abdominal pain boils and carbuncles acute appendicitis diarrhea hemorrhagic leucorrhea postpartum blood stasis abdominal pain swelling and pain of eye hepatitis boils and carbuncles acute appendicitis diarrhea dysentery postpartum blood stasis abdominal pain conjunctivitis boils and carbuncles acute appendicitis and its abdominal pain postpartum blood stasis abdominal pain boils and carbuncles appendicitis dysentery enteritis hepatitis conjunctivitis postpartum blood stasis abdominal pain boils and carbuncles appendicitis dysentery enteritis hepatitis conjunctivitis postpartum blood stasis abdominal pain boils and carbuncles dosage g standardsetting department hunan food and drug administration g g g g g g shandong medical products administration heilongjiang medical products administration liaoning food and drug administration sichuan food and drug administration guizhou medical products administration pharmacopoeia committee of the ministry of health of p r china preparation name yiyi fuzi baijiang san 薏苡附子败酱散 machixian heji 马齿苋合剂 aiye san 艾叶散 baijiang san 败酱散 baijiang san 败酱散 baijiang tang 败酱汤 baijiang tang 败酱汤 table traditional and clinical preparation of herba patriniae in china formulation main compositions powder coicis semen aconiti lateralis radix praeparata herba patriniae decoction portulacae herba isatidis folium arnebiae radix herba patriniae persicae semen carthami flos paeoniae radix rubar powder powder artemisiae argyi folium angelicae sinensis radix paeoniae radix alba dipsaci radix achyranthis bidentatae radix herba patriniae herba patriniae angelicae sinensis radix chuanxiong rhizoma paeoniae radix alba cinnamomi cortex powder herba patriniae moutan cortex cinnamomi cortex siphonostegiae herba aucklandiae radix decoction herba patriniae notopterygii rhizoma et radix dianthi herba aurantii fructus cinnamomi cortex persicae semen decoction herba patriniae reference jin gui yao lüe ãŠé‡‘匮要略㋠zhang 1997b surgery of chinese medicine ãŠä¸­åŒ»å¤–科学㋠beijing traditional chinese medicine hospital tai ping sheng hui fang ãŠå¤ªå¹³åœ£æƒ æ–¹ã‹ volume wang pu ji fang ãŠæ™®æµŽæ–¹ã‹ volume zhu tai ping sheng hui fang ãŠå¤ªå¹³åœ£æƒ æ–¹ã‹ volume wang sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao qian jin yi fang ãŠåƒé‡‘翼方㋠volume sun preproof 0cpreparation name baijiang tang 败酱汤 baijiang tang 败酱汤 baijiang yin 败酱饮 changyong tang 肠痈汤 decoction chenzhou sheyao pian 郴州蛇药片 chure jili wan 除热蒺藜丸 tablets pills danggui xi tang 当归洗汤 danggui yin 当归饮 ganyan chongji 肝炎冲剂 decoction decoction electuary formulation main compositions decoction herba patriniae rhei radix et rhizoma persicae semen decoction herba patriniae cinnamomi cortex siphonostegiae herba moutan cortex aucklandiae radix decoction herba patriniae angelicae sinensis radix bambusae caulis in taenias rehmanniae radix moutan cortex glycyrrhizae radix et rhizoma herba patriniae zingiberis rhizoma recens poria coicis semen platycodonis radix liriopes radix salviae miltiorrhizae radix et rhizoma paeoniae radix alba rehmanniae radix pv tribuli fructus rhei radix et rhizoma herba patriniae cinnamomi cortex ginseng radix et rhizoma aconiti lateralis radix praeparata coicis semen coptidis rhizoma astragali radix abri herba angelicae sinensis radix aurantii fructus immaturus paeoniae radix alba tetrapanacis medulla angelicae sinensis radix angelicae pubescentis radix angelicae dahuricae radix sanguisorbae radix herba patriniae angelicae sinensis radix herba patriniae dipsaci radix paeoniae radix alba rehmanniae radix bambusae caulis in taenias bupleuri radix angelicae sinensis radix paeoniae radix alba paeoniae radix rubra citri reticulatae pericarpium aurantii fructus curcumae radix cyperi rhizoma salviae miltiorrhizae radix et rhizoma scrophulariae radix artemisiae scopariae herba isatidis radix herba patriniae huangdan tang 黄疸汤 decoction jiedu dihuang wan 解毒地黄丸 pills gualou san powder artemisiae scopariae herba gardeniae fructus lonicerae japonicae flos forsythiae fructus herba patriniae isatidis radix paeoniae radix rubra paeoniae radix alba bupleuri radix perillae caulis platycodonis radix sojae semen germinatum rehmanniae radix astragali radix trichosanthis radix scutellariae radix liriopes radix mantidis ootheca rhei radix et rhizoma ginseng radix et rhizoma gardeniae fructus cistanches herba peucedani radix cimicifugae rhizoma paeoniae radix alba anemarrhenae rhizoma vaccariae semen polygalae radix herba patriniae jujubae fructus trichosanthis semen herba patriniae asari radix et rhizoma zingiberis rhizoma magnoliae officinalis cortex platycodonis radix ginseng radix et rhizoma saposhnikoviae radix reference sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao qian jin fang ãŠåƒé‡‘方㋠volume （sun ） gu jin ming fang ãŠå¤ä»Šåæ–¹ã‹ yan and liu qian jin fang ãŠåƒé‡‘方㋠volume （sun ） qian jin fang ãŠåƒé‡‘方㋠volume （sun ） sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao study on the treatment of common diseases with traditional chinese medicine ãŠå¸¸è§ç—…的中 医 æ²» 疗 研 究 ㋠teaching and research group of traditional chinese medicine the first affiliated hospital of xi'an medical college lin zheng yi an yi fang ãŠä¸´è¯åŒ»æ¡ˆåŒ»æ–¹ã‹sun sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume preproof 0c preparation name 栝蒌散 lanwei xiaoyan wan 阑尾消炎丸 lanweiyan heji 阑尾炎合剂 formulation main compositions pills lonicerae japonicae flos isatidis folium herba patriniae taraxaci herba spatholobi caulis toosendan fructus rhei radix et rhizoma aucklandiae radix persicae semen paeoniae radix rubra scutellariae radix decoction lonicerae japonicae flos taraxaci herba herba patriniae forsythiae fructus rhei radix et rhizoma paeoniae radix rubra toosendan fructus aucklandiae radix persicae semen lanweiyan tang 阑尾炎汤 lanwei yihao xiaoyan wan 阑尾ä¸å·æ¶ˆç‚Žç‰‡ lenge xiaoji tang 棱莪消积汤 lishi zhiyang pu yao 理湿止痒扑药 lidan tuihuang tang 利胆é黄汤 neibu wuxiang wan 内补五香丸 qianliexian tang 前列腺汤 qumai wan 瞿麦丸 decoction pills decoction powder decoction pills decoction pills yinqiao hongjiang jiedu tang decoction rhei radix et rhizoma moutan cortex persicae semen paeoniae radix alba salviae miltiorrhizae radix et rhizoma bupleuri radix lonicerae japonicae flos forsythiae fructus herba patriniae coicis semen lonicerae japonicae flos isatidis folium herba patriniae taraxaci herba toosendan fructus rhei radix et rhizoma aucklandiae radix persicae semen paeoniae radix rubra scutellariae radix talci pulvis sargentodoxae caulis sparganii rhizoma curcumae rhizoma salviae miltiorrhizae radix et rhizoma paeoniae radix rubra corydalis rhizoma moutan cortex persicae semen coicis semen sargentodoxae caulis herba patriniae kochiae fructus bombyx batryticatus dictamni cortex angelicae dahuricae radix schizonepetae spica artemisiae scopariae herba herba patriniae alumen glycyrrhizae radix et rhizoma talcum cinnabaris artemisiae scopariae herba herba patriniae isatidis radix curcumae radix gardeniae fructus aquilariae lignum resinatum olibanum aucklandiae radix caryophylli flos dipsaci radix rehmanniae radix praeparata paeoniae radix alba magnoliae officinalis cortex herba patriniae ginseng radix et rhizoma poria cervi cornu salviae miltiorrhizae radix et rhizoma lycopi herba paeoniae radix rubra persicae semen carthami flos olibanum myrrha vaccariae semen citri reticulatae pericarpium toosendan fructus foeniculi fructus angelicae dahuricae 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coicis semen angelicae sinensis radix atractylodis rhizoma chuanxiong rhizoma cyperi rhizoma alismatis rhizoma corydalis rhizoma httpswwwyaozhcom nmpa preproof 0c formulation main compositions reference preparation name 康妇炎胶囊 manshenning heji 慢肾宁合剂 nankang pian 男康片 decoction tablets astragali radix cinnamomi ramulus epimedii folium rehmanniae radix asini corii colla poria alismatis rhizoma scutellariae radix herba patriniae moutan cortex leonuri herba httpswwwyaozhcom nmpa paeoniae radix rubra rehmanniae radix praeparata cistanches herba glycyrrhizae radix et rhizoma taraxaci herba pyrolae herba phellodendri chinensis cortex carthami flos houttuyniae herba epimedii folium fubi fructus atractylodis macrocephalae rhizoma astragali radix cuscutae semen violae herba herba patriniae chrysanthemi indici flos angelicae sinensis radix sophorae fructus notoginseng radix et rhizoma sophorae flavescentis radix bletillae rhizoma cnidii fructus artemisiae argyi folium herba patriniae lonicerae 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"Despite the biological link between thyroid hormones and breast cancer cell proliferation shown inexperimental studies little is known about the association between hyperthyroidism and breast cancer as well asits association with the most common mammographic and genetic risk predictors for breast cancerMethods This study estimates the incidence rate ratios IRRs of breast cancer among women diagnosed withhyperthyroidism compared to those who are not using two cohorts a Swedish national cohort of the generalfemale population n “ and the Karolinska Mammography Project for Risk Prediction of BreastCancer KARMA n “ We used logistic regression to estimate the odds ratios ORs ofhyperthyroidism according to the mammographic and genetic risk predictors for breast cancerResults An increased risk of breast cancer was observed in patients in the national cohort with hyperthyroidismIRR CI “ particularly for toxic nodular goiter IRR CI “ Hyperthyroidismwas associated with higher body mass index early age at first birth and lower breastfeeding duration Highermammographic density was observed in women with toxic nodular goiter compared to women withouthyperthyroidism Additionally among genotyped women without breast cancer in the KARMA cohort N hyperthyroidism was associated with a high polygenic risk score PRS for breast cancer overall OR CI “ and for estrogen receptorpositive specific PRS OR CI “Conclusion Hyperthyroidism is associated with an increased risk of breast cancer particularly for patients withtoxic nodular goiter The association could be explained by higher mammographic density among these womenas well as pleiotropic genetic variants determining shared hormonalendocrine factors leading to the pathology ofboth diseasesKeywords Breast cancer Hyperthyroidism Mammographic density Genetic pleiotropy Correspondence haominyangkise1Department of Epidemiology and Health Statistics School of Public HealthFujian Medical University Xuefu North Road University Town Fuzhou China2Department of Medical Epidemiology and Biostatistics Karolinska InstitutetSE17177 Stockholm SwedenFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cYang BMC Medicine Page of BackgroundBreast cancer is the most common cancer diagnosed amongwomen worldwide and the leading cause of cancer deathsamong women [] Breast cancer is usually regarded as ahormonerelated cancer with approximately “ ofcases being estrogen receptorpositive [] Experimental studies show that thyroid hormones can stimulate cell proliferation in breast tissue [ ] At high serum concentrationsthyroid hormones can have estrogenlike effects [“] whichinduce the expression of progesterone receptors [] and canenhance estradiolmediated effects on cell proliferation [ ]Several populationbased studies have reported hyperthyroidism excessive production of thyroid hormones tobe associated with breast cancer [“] while others havefound no association [“] The disparity of findingsmay be explained by differences in study design smallsample sizes or using a specific subgroup of womenMoreover no study to date has examined the impact ofdifferent subtypes of hyperthyroidism on breast cancerrisk which may differ in their pathological processAlthough it is shown that thyroid hormones are associated with breast tissue proliferation and a subsequent increase in breast cancer risk [ ] the association betweenhyperthyroidism and mammographic features of breasttissue is less studied [] Mammographic density refers tothe percentage of radiologically dense epithelium stromaand connective tissues identified in a mammogram Mammographic density is a widely used mammographic featurefor breast cancer risk prediction and is considered an intermediate phenotype for breast cancer [ ] It can therefore be a powerful proxy when investigating the associationbetween hyperthyroidism and breast cancerBesides the direct effect of thyroid hormones on breastcancer risk the association between hyperthyroidism andbreast cancer could also be explained by genetic pleiotropicpathways leading to both hyperthyroidism and breastcancer [] A recent study has shown an associationbetween thyroidrelated single nucleotide polymorphismsSNPs and breast cancer risk [] Howeverit is stillunclear whether a genetic predisposition to breast cancer isassociated with hyperthyroidism which is important forexamining a potential pleiotropic genetic effectIn this study we investigated mechanisms behind the association between hyperthyroidism and breast cancer Specifically whether a genetic susceptibility to breast cancermeasured using polygenic risk score and mammographicdensity could explain some of this association while beingable to account for important factors such as menopausalstatus body mass index BMI and reproductive healthMethodsStudy populationsTo study the association between hyperthyroidism andbreast cancer risk we used two cohorts a Swedishnational cohort of the general female population and a mammographic screeningbased cohort KarolinskaMammography Projectfor Risk Prediction of BreastCancer KARMA Additional file Fig S1The national cohort included all women above age years who lived in Sweden between and N Having a main diagnosis of hyperthyroidismwas retrieved from the Swedish Patient Register andclassified according to the International Classification ofDiseases ICD code E05 Types of hyperthyroidism weredefined by the ICD codes E050 Graves™ disease E051“E052 toxic nodular goiter and E053“E059 other orunspecified types Followup of the cohort ended on thedate of breast cancer diagnosis date of death date ofemigration or end of followup December whichever came first Information on breast cancer diagnosis death and emigration was obtained by usingunique personal identification numbers to link the cohort to the Swedish Cancer Register Swedish Cause ofDeath Register and the Swedish Migration Register TheSwedish Cancer Register was founded in The completeness and accuracy of this register are estimated at and respectively [] The ICD7 code wasused to identify breast cancer diagnoses in the cancerregister and to exclude breast cancer cases before includingquestionnaireKARMA is a screeningbased cohort formed throughan invitation to all women participating in a mammographic screening or clinical mammography in one offour hospitals in Sweden between January andMarch [] During the recruitment period women consented to join the study with a participationrate of Aside from mammographic imaging andblood sample collection participants also answered awebbaseddemographicanthropometric reproductive lifestyle and familial riskfactors related to breast cancer This cohort was linkedto the Swedish Patient Register to retrieve diagnoses ofhyperthyroidism Breast cancer cases in the cohort wereidentified through linkage to the Swedish Cancer Register For consistency with the national cohort followupof the KARMA cohort also started from and endedwith the same criteria as the national cohort except foran extension of the followup time until Dec We therefore excluded women with hyperthyroidism orbreast cancer before ending with the final studypopulation of women Characteristics of womenin the national and the KARMA cohort are shown inAdditional file Table S1Polygenic risk scoreBlood samples from a subset of women who didnot have breast cancer when they joined the KARMAcohort were genotyped These women were part of theBreast Cancer Association Consortium and were 0cYang BMC Medicine Page of iSelectcustom Illuminarandomly selected as controls for the genomewideassociation studies GWAS [ ] Genotyping wasperformed using aarrayiCOGS comprising SNPs [] or OncoArraycomprising of SNPs [] Details of the arraydesign sample handling quality control processes andimputation of nongenotyped variants are described elsewhere [ ] Hyperthyroidism cases were defined aswomen who were diagnosed with hyperthyroidism from to while the controls were women without adiagnosis of hyperthyroidism To assess whether hyperthyroidism is associated with a genetic predisposition ofbreast cancer we selected genomewide significantSNPs reported in a recent metaanalysis of breast cancerGWAS for constructing polygenic risk scores for breastcancer overall and by estrogen receptor ER status []For all individuals a weighted polygenic risk score PRSwas calculated using the following formulaPRS ¼ βx1 þ βx2 þ ¦Î²kxk þ βnxnwhere β is the perallele log odds ratio OR of breastcancerassociated risk allele for SNP k xk is the numberof alleles for the same SNP and n is the totalnumber of the breast cancer SNPs included in the profile The SNPs and corresponding betas used to derivethe PRS are summarized in Additional file Table S2For the analyses PRS was categorized into quartilesMammographic densityFullfield digital mammograms from mediolateral oblique views of the left and right breasts in the most recent screening before were used For the KARMAcohort we used the areabased STRATUS method tomeasure mammographic density [] Percent densitywas calculated by dividing the dense area by the totalbreast area in the mammogram and further categorizedinto quartiles For this part of the study we excludedwomen with any cancer diagnosis as well as those whohad a breast enlargement reduction or surgery resulting in a final study population of Only womendiagnosed with hyperthyroidism before the latest screening were considered as casesThe study was approved by the Regional Ethical Review Board in Stockholm Sweden Dnr and Statistical analysisFor both cohorts the incidence rate ratio IRR of breastcancer among hyperthyroidism patients was calculatedusing Poisson regression using attained age as the timescale and adjusting for calendar period 3year categories For these analyses hyperthyroidism was treated as atimevarying exposurein which the exposed persontime was counted from months after the hyperthyroidism diagnosis index date and the unexposed persontimewas counted from Jan and ended on the date ofbreast cancer diagnosis date of death date of emigrationindex date or end of followup whichever came first Theanalysis was further stratified by menopausal status ageand time since hyperthyroidism diagnosis and type ofhyperthyroidism In the analyses Model adjusted forcalendar period and Model further adjusted for BMIage at menarche number of births family history of breastcancer oral contraceptive use hormone replacementtreatment and having had a benign breast diseaseThe association between hyperthyroidism and variouslifestyle risk factors for breast cancer were assessed amongKARMA women using logistic regression models Theserisk factors included having had a benign breast diseasenumber of births age at first birth breastfeeding durationBMI family history of breast cancer oral contraceptiveuse hormone replacement therapy use age at menarcheand menopausal status Two models were conducted forthis analysis a univariable model and a multivariablemodel adjusting for all these risk factors simultaneouslyGiven that mammographic density is causally relatedto breast cancer risk we also tested the associationbetween having a prior diagnosis of hyperthyroidism andmammographic density among KARMA women usinglogistic regression models Model was the univariablemodel Model adjusted for age at mammogram andBMI and Model further adjusted for age at menarchenumber of births family history of breast cancer oralcontraceptive use hormone replacement treatment andhaving had a benign breast disease Linear regressionmodels were also used to test the effect of hyperthyroidism on mammographic density square roottransformed as a continuous variableTo test the association between hyperthyroidism andhaving a genetic predisposition to breast cancer we usedlogistic regression models to calculate the ORs of hyperthyroidism among KARMA women who had beengenotyped by quartiles of PRS for breast cancer overallfor ERpositive cancers and for ERnegative cancersThese associations were adjusted for age the first threeprinciple components and for genotyping array We further adjusted for age at menarche number of birthsfamily history of breast cancer BMI menopausal statusoral contraceptive use hormone replacement treatmentand having had a benign breast diseaseStatistical analyses were performed using SAS version SAS Institute Inc Cary NC USA and Stata softwareversion Stata Corporation College Station TXResultsIn the national cohort cases of breast cancerpatients were observed during person years 0cYang BMC Medicine Page of following a diagnosis of hyperthyroidism correspondingto an incidence rate of person years Patientswith hyperthyroidism experienced a increased riskof breast cancer IRR CI “ In theKARMA cohort there were cases of breast cancerafter hyperthyroidism diagnosis during a followup timeof person years corresponding to an incidence rateof person years KARMA women with adiagnosis of hyperthyroidism also had a increasedrisk of breast cancer although the association was notstatistically significantin the multivariable adjustedmodel IRR CI “ In both cohortswomen diagnosed with hyperthyroidism aged youngerthan years had a significantly increased risk of breastcancer while there was no strong difference for the riskof breaststatusTable Analyses by type of hyperthyroidism in thenational cohort indicated a stronger association betweenbreast cancer and toxic nodular goiter IRR CI “according to menopausalcancerAmong the major risk factors for breast cancer a diagnosis of hyperthyroidism was significantly associatedwith obesity BMI OR CI “which was predominantly driven by toxic nodular goitersAdditional file Table S3 Other reproductive factorsincluding early age at first birth breastfeeding for lessthan a month and menopausal status were also associated with hyperthyroidism Table When investigating the association between hyperthyroidism and mammographic density no overall statistically significant association was observed Table However a prior diagnosis of toxic nodular goiter wassignificantly associated with high mammographic densityOR for the highest quartile CI “ p for continuous even after adjustingfor all other potential risk factors for breast cancerAdditional file Table S4Among KARMA women with genotyped data a higherPRS for breast cancer was associated with hyperthyroidism OR for the highest quartile CI “ This association was mainly driven by the association between hyperthyroidism and ERpositive breastcancer OR for the highest quartile CI “ Table There was no association betweenhyperthyroidism and PRS for ERnegative breast cancerDiscussionWomen diagnosed with hyperthyroidism had an increased risk of breast cancer compared to the generalpopulation This finding was more pronounced amongTable Risk of breast cancer in women in the national and KARMA cohorts with a diagnosis of hyperthyroidism compared towomen without hyperthyroidismSwedish national cohort N “IRR CI Model No HT “ “ “No BCKARMA cohort N “No HTNo BCIRR CI Model “ “ “IRR CI Model “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “OverallPremenopausalPostmenopausalBy age years “ By years sincehyperthyroidismdiagnosis““By types of hyperthyroidismGraves™ diseaseToxic nodular goiterOthers or unspecified “ “ “ “ “ “ “ “ “Hyperthyroidism patients were identified by the main diagnosis given in the inpatient and outpatient registers IRRs were calculated by comparinghyperthyroidism patients to women without hyperthyroidism using Poisson regression using age as the underlying time scale In the analyses stratified bymenopausal status women with age younger than in the national cohort were considered as premenopausal women while in the KARMA cohort the exactage at menopause was used Model adjusted for calendar period and Model further adjusted for BMI age at menarche number of births family history ofbreast cancer hormone replacement therapy use oral contraceptive use and benign breast disease Statistically significant results are boldedHT hyperthyroidism BC breast cancer IRR incidence rate ratio CI confidence interval 0cYang BMC Medicine Page of Table The association between hyperthyroidism and major breast cancer risk predictors in KARMA cohort N Variable namesBenign breast diseaseNo of nonhyperthyroidismOR2 CINo of hyperthyroidismOR1 CINoYesNumber of births Age at first birthyears “‰¥ Breastfeeding duration months “ BMI kgcm2 category ““ Family history of breast cancerNoYesOral contraceptive useNoYesHormone replacement therapy useNoYesAge at menarche years “ Menopausal statusPremenopausalPerimenopausalPostmenopausalHistory of irregular menstrual periodsNoYes REF “ REF “ “ “ REF “ “ REF “ “ “ REF “ “ REF “ REF “ REF “ REF “ “ REF “ REF “ “ “ REF “ “ REF “ “ “ REF “ “ REF “ REF “ REF “ REF “ “ REF “ “ REF “ “ REF “ REF “OR1 was calculated using a univariate model while OR2 was calculated with a multivariate model including all risk predictors simultaneously The analysis wasrestricted to KARMA women who had not had a breast cancer diagnosis when they entered the studyOR odds radio BMI body mass indexAnalyses for age at first birth and breastfeeding duration were limited to parous women 0cYang BMC Medicine Page of Table The association between hyperthyroidism and mammographic density among KARMA women without a cancer diagnosisn Mammographic densityQ1Q2Q3Q4P for square root continuousNoYesOR CIModel REF “ “ “Model Model REF “ “ “ REF “ “ “Model is the univariate model Model adjusted for age and BMI Model further adjusted for age at menarche number of births family history of breastcancer hormone replacement therapy use oral contraceptive use and benign breast diseasethose diagnosed at a younger age and with a toxic nodular goiter Hyperthyroidism was also associated withbreast cancer risk predictors such as BMI age at firstbirth and duration of breastfeeding The association between mammographic density and hyperthyroidism wasonly observed in women with a diagnosis of toxic nodular goiter Additionally having hyperthyroidism wasassociated with a high PRS for breast cancer particularlyfor ERpositive breast cancerOur estimates for an increased risk of breast canceramong women diagnosed with hyperthyroidism were thesame when analyzing data from the national andKARMA cohorts which is also consistent with previousin Denmark and Taiwan [ ] ThisestimatesTable The association between hyperthyroidism and breast cancer polygenic risk scores PRS among women in the KARMAcohort N PRS for BC overallQ1Q2Q3Q4P for trendStandardized continuousPRS for ER BCQ1Q2Q3Q4P for trendStandardized continuousPRS for ER ˆ’ BCQ1Q2Q3Q4P for trendStandardized continuousNoNo caseOR1 CIOR2 CI REF “ “ “ “ REF “ “ “ “ REF “ “ “ “ REF “ “ “ “ REF “ “ “ “ REF “ “ “ “Analysis was performed among KARMA women without breast cancer and who had genotyped data OR1s were adjusted for age array of genotyping andprinciple components OR2s were additionally adjusted for BMI menopausal status age at menarche number of births family history of breast cancer HRT useoral contraceptive use and benign breast disease Statistically significant results are boldedNo case the number of hyperthyroidism patients No the number of women without hyperthyroidism OR odds ratio CI confidence interval ER estrogen receptor 0cYang BMC Medicine Page of association however was not statistically significant inthe KARMA cohort probably due to a limited numberN The associationof breast cancer patientsbetween hyperthyroidism and breast cancer was furthersupported by an increased risk of breast cancer amongwomen with high serum thyroxine T4 [ ] Despitethis the effect of serum triiodothyronine T3 on breastcancer risk is still conflicting in different studies [ ]which might be influenced by nonthyroidalillnesssyndrome in cancer patients []Several biological pathways may exist between hyperthyroidism and breast carcinogenesis T3 could activatethe PI3K pathways mediated by integrin αvβ3 andstimulate breast cancer cell invasion [] In additionT4 induces MAPKmediated nuclear ERα phosphorylation and promotes cell proliferation to an extent comparable to the effect of estradiol [] which could beinhibited by the T4 analog tetrac []In addition to the known association between thyroidhormones and breast cancer risk it has been hypothesized that I131 radioactive iodine treatment for hyperthyroidism may also increase the risk of breast cancerHowever majority of the studies did not find a significant association between I131 treatment and breastcancer [“] After surgery or radioactive iodine treatment hyperthyroidism patients may subsequently reacha hypothyroid state and thus be prescribed thyroidreplacement medication eg levothyroxine [] Despite this a recent metaanalysis did not find an increasedrisk of breast cancer after subsequent hypothyroidism[] nor following treatment with levothyroxine []Moreover considering the potential late effect of treatment on breast cancer the observed association betweenhyperthyroidism and breast cancer in our study particularly for short term risk cannot be explained by theeffect of treatmentIn this study hyperthyroidism was associated withseveral risk factors for breast cancer Both Graves™disease and toxic nodular goiter are strongly influencedby pregnancy [ ] which could result in early cessation of breastfeeding and supports our finding of anassociation between reduced breastfeeding duration andhyperthyroidism The strong association between hyperthyroidism and breast cancer observed among womenaged below years in both the national and KARMAcohortsfurther suggests that closer surveillance forbreast cancer may be useful among women diagnosedwith hyperthyroidism during their reproductive yearsWe found a significantly higher BMI among patientswith hyperthyroidism despite previous knowledge that reduced weight is associated with hyperthyroidism [] Thisdisparity could be explained by treatments for hyperthyroidism which might eventually result in excess weightgain for patients [ ] Moreoverthe associationbetween hyperthyroidism and BMI was mainly driven bytoxic nodular goiters This finding is consistent withprevious studies indicating that obesity is associated withthyroid nodules [ ]Independent of BMI toxic nodular goiters were associated with higher mammographic density While oneprevious study did not find a significant association between thyroid disorders including hyperthyroidism andthyroid nodules and mammographic densitythis islikely due to having a limited sample size [] Thisfinding in the KARMA cohort was consistent with thepronounced association between breast cancer and toxicnodular goiter in the national cohort Unlike Graves™disease which is an autoimmune disease toxic nodulargoiters are likely to be hormonerelated and thereforemore closely linked to breast cancer Nodular thyroiddiseases are associated with benign breast disease andbreast cancer [ ] and some shared pathways are involved in the proliferation of thyroid and breast tissues[ ] Hence the association between hyperthyroidism and breast cancer could result from both a hormonal effect and tissue proliferation and therefore possiblymediated through mammographic densityAdditionally we found a significant association between breast cancer PRS and hyperthyroidism Amongthose GWAS significant SNPs for breast cancer previous studies show that one SNP in the ABO gene is associated with thyroid function [ ] Another geneoverlapping the GWAS for thyroidstimulating hormoneand breast cancer is IGFBP5 [ ]indicating theshared growth hormoneinsulinlike growth factor GHIGF pathways between thyroid function and breast cancer Although insignificant at the GWAS level one SNPin the DIO1 gene has been associated with both free T4and breast cancer [] suggesting the role of deiodinaseactivity in the association between thyroid function andbreast cancer [] Overall given the genetic associationwas only significant for the ERpositive breast cancerPRS and not for ERnegative PRS we hypothesize thatgenetic pleiotropy between hyperthyroidism and breastcancer is involved in the shared hormonalendocrinepathways for both diseasesConsidering the phenotypic and genetic associations between hyperthyroidism and breast cancer as well as thebeneficial effect of introduced euthyroid hypothyroxinemia in the setting of breast cancer [] hyperthyroidismshould be considered in the evaluation of women™s risk ofbreast cancer Despite concerns of possible overdiagnosisand anxiety caused by mammographic screening [ ]better adherence to the scheduled breast cancer screeningprogram is recommended for patients with hyperthyroidism in order to detect breast cancer at an early stageThe main strength of our study is having a large sample size and a populationbased cohort design including 0cYang BMC Medicine Page of both Swedish health registers and a mammographicscreening cohort Other strengths include the KARMAcohort containing rich lifestyle mammographic andgenetic data which allowed us to explore underlyingmechanisms for the associations investigated We alsoacknowledge severallimitations The validity of ICDcodes in the Swedish patient register is about “[] which indicates potential misclassification Therefore we tried to minimize any potential misclassificationby using main diagnoses only Due to increased medicalsurveillance of patients with hyperthyroidismthesewomen may be diagnosed earlier with breast cancer thanwomen without hyperthyroidism While this possiblebias might slightly shift the temporal risk pattern amongwomen with hyperthyroidism it would not strongly influence the overall association An older age at breastcancer diagnosis among patients with hyperthyroidismsee Additional file Table S1 further argues againstthis surveillance bias In the national cohort we werenot able to adjust for breast cancer risk factors Nevertheless in the KARMA cohort which contains detailedinformation on these confounders multivariable adjustment did not change the point estimates indicating aweak confounding effect ofthese breast cancer riskfactors In the analysis of breast cancer risk using theKARMA cohort we started the followup before womenentered the KARMA study which could have introducedsurvivor bias However we believe the effect of any suchbias would be minimal given that hyperthyroidism isnot a deadly disease and the same estimates were foundusing the national cohortConclusionIn conclusion we found that women diagnosed withhyperthyroidism were associated with an increasedrisk of breast cancer and major predictors for breastcancerincluding mammographic density and polygenic risk score These findings may partly be explained byfactorsbetween these two diseases and may contribute to amorecomprehensive understanding of hormonalendocrinal factors contributing to breast cancer riskshared geneticand hormonalSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s1291602001690yAdditional file Table S1 Characteristics of women in the national andKARMA cohorts by hyperthyroidism status Table S2 Single nucleotidepolymorphisms SNPs used for constructing the polygenic risk score PRS forbreast cancer Table S3 The association between hyperthyroidism and majorbreast cancer risk predictors among the KARMA cohort by type ofhyperthyroidism N Table S4 The association betweenhyperthyroidism and mammographic density among KARMA women withoutcancer by type of hyperthyroidism n Figure S1 Sample attritionfor the analyses using the KARMA cohortAbbreviationsBMI Body mass index CI Confidence interval ER Estrogen receptWAS Genomewide association studies ICD International Classification ofDiseases IRR Incidence rate ratio KARMA Karolinska Mammography Projectfor Risk Prediction of Breast Cancer OR Odds ratio PRS Polygenic risk scoreSNPs Single nucleotide polymorphisms T3 Triiodothyronine T4 ThyroxineAcknowledgementsWe thank all the participants in the KARMA studyAuthors™ contributionsHY had full access to all of the data in the study and takes responsibility forthe integrity of the data and the accuracy of the data analysis KC NH andHY conceived and designed the study All authors acquired analyzed orinterpreted the data HY and NH drafted the manuscript All authors criticallyrevised the manuscript for important intellectual content HY performed thestatistical analysis KC and HY obtained the funding All authors read andapproved the final manuscriptAuthors™ informationNot applicableFundingThis work was supported by the Swedish Research Council [grant no ] Swedish Cancer Society [grant no CAN190266] and FORTE [grant no] We would like to also acknowledge Märit and Hans Rausing™s Initiative against Breast Cancer Haomin Yang is supported by Startup Fund forhighlevel talents of Fujian Medical University [grant no XRCZX2020007] andStartup Fund for scientific research Fujian Medical University [grant no2019QH1002] access funding provided by Karolinska InstituteAvailability of data and materialsThe KARMA data used in the present study are available from thecorresponding author upon reasonable request The registerbasednationwide cohort datasets linked and analyzed in the current study are notpublicly available due to Swedish law but are available by applying throughthe Swedish National Board of Health and Welfare and Statistics SwedenDetailed information on data application can be found using the followinglinks httpsbestalladatasocialstyrelsensedataforforskning and httpswwwscbsevaratjansterbestallamikrodataEthics approval and consent to participateThe study was approved by the Regional Ethical Review Board in StockholmDnr and KARMA participantsconsented to participate in the study with written informed consentConsent for publicationAll authors approved the manuscript and consented to its publicationCompeting interestsThe authors declare no competing interestAuthor details1Department of Epidemiology and Health Statistics School of Public HealthFujian Medical University Xuefu North Road University Town Fuzhou China 2Department of Medical Epidemiology and BiostatisticsKarolinska Institutet SE17177 Stockholm Sweden 3Department of OncologySouth General Hospital SE11883 Stockholm SwedenReceived February Accepted June ReferencesGlobal Burden of Disease Collaboration Global regional and nationalcancer incidence mortality years of life lost years lived with disability anddisabilityadjusted lifeyears for cancer groups to a systematicanalysis for the global burden of disease study JAMA Oncology “Allred DC Brown P Medina D The origins of estrogen receptor alphapositive and estrogen receptor alphanegative human breast cancer BreastCancer Res “ 0cYang BMC Medicine Page of Hall LC Salazar EP Kane SR Liu N Effects of thyroid hormones onhuman breast cancer cell proliferation J Steroid Biochem Mol Biol““Conde I Paniagua R Zamora J Blanquez MJ Fraile B Ruiz A Arenas MIInfluence of thyroid hormone receptors on breast cancer cell proliferationAnn Oncol “Dinda S Sanchez A Moudgil V Estrogenlike effects of thyroid hormone onthe regulation of tumor suppressor proteins p53 and retinoblastoma inbreast cancer cells Oncogene “Nogueira CR Brentani MM Triiodothyronine mimics the effects of estrogenin breast canc
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" inflammatory bowel disease ibd is the collective term for chronic immunemediated diseases ofunknown multifactorial etiology arising from the interplay between genetic and environmental factors andincluding two main disease manifestations ulcerative colitis uc and crohn™s disease in the last few decadesnaturally occurring alkaloids have gained interest because of their substantial antiinflammatory effects in severalanimal models of disease studies on mouse models of ibd have demonstrated the antiinflammatory action of themain tobacco alkaloid nicotine in addition anatabine a minor tobacco alkaloid also present in peppers tomatoand eggplant presents antiinflammatory properties in vivo and in vitro in this study we aimed to evaluate theantiinflammatory properties of nicotine and anatabine in a dextran sulfate sodium dss mouse model of ucresults oral administration of anatabine but not nicotine reduced the clinical symptoms of dssinduced colitisthe result of gene expression analysis suggested that anatabine had a restorative effect on global dssinducedgene expression profiles while nicotine only had limited effects accordingly map findings revealed that anatabinereduced the colonic abundance of dssassociated cytokines and increased il10 abundances our results support the amelioration of inflammatory effects by anatabine in the dss mouse modelof uc and suggest that anatabine constitutes a promising therapeutic agent for ibd treatmentkeywords plantderived alkaloid mouse model nicotine colitis correspondence pedroantonioruizcastropmicomblainephillipspmicom juliahoengpmicom pedro a ruiz castro ulrike kogel giuseppe lo sasso blaine w phillips andalain sewer contributed equally to this work1philip morris international rd philip morris products sa quai jeanrenaud neuch¢tel switzerland2philip morris international research laboratories pte ltd science parkroad the kendall science park ii singapore singapore the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cruiz castro of inflammation page of crohn™s disease cd and ulcerative colitis uc the mainclinical phenotypes of inflammatory bowel diseases ibdare chronic relapsing inflammatory disorders that affectthe gastrointestinal tract ibd are thought to result froman inappropriate and continuing inflammatory responseto commensal microbes in a genetically susceptible hostenvironmental triggers such as increased hygiene druguse stress smoking and diet influence the onset of thedisease over the past decades naturally occurring alkaloids from plant or medicinal herb sources have sparkedconsiderable interest because of their significant antiinflammatory and antioxidant properties [“]alkaloids”a class of natural bioactive compoundsderived from amino acids that contain one or moreheterocyclic nitrogen atoms”are produced by a widerange of living anisms such as bacteria fungi plantsand animals in plants alkaloids are produced assecondary metabolites in response to environmentalbiotic or abiotic interactions and they confer protectionthrough a range of insecticidal antimicrobial and pharmacological attributes the antiinflammatory activities ofplantderived alkaloids have been documented in severalanimal models of disease including respiratory distress spinal cord injury hepatic fibrosis cancer and ibd [ ] the protective effects of alkaloids havebeen attributed to amelioration of inflammatory responsesand colonic oxidative stress [ ] promotion of epithelial barrier function and positive regulation of gutmicrobiota pyridine alkaloids present in tobacco nicotiana tabacum have been the subject of intensive research nicotinethe major alkaloid in tobacco accounts for approximately of the total alkaloid content of tobacco while thestructurally related nornicotine and anatabine are themost abundant minor pyridine alkaloids accounting for to of total alkaloids other pyridine alkaloids intobacco such as anabasine anabaseine and cotinine arepresent in smaller amounts nicotine and all minortobacco alkaloids have been shown to be pharmacologically active upon binding to several nicotinic acetylcholinereceptors nachrs tobacco nachr agonists suchas nicotine anatabine anabasine anabaseine and cotininedisplay protective effects in animal models of several inflammatory conditions including sepsis parkinson™sdisease alzheimer™s disease and ibd several in vitro and in vivo studies have shown a clearnicotinedependent positive effect on inflammatory processes [ ] in a previous study oral nicotine administration reduced the severity of dssinduced colitis andreduced colonic tnfα synthesis while subcutaneous injection or minipump infusion had no effect highlightingthe crucial role of administration route for the protectiveeffects of nicotine in dss colitis nicotine has alsobeen shown to attenuate the severity of dss colitis andexpression of il6 in cd4t cells a recent studysuggested that nicotine ameliorates dssinduced inflammation through inhibition of signaltransducer andactivator oftranscription stat3 in gutinfiltratedlymphocytes and intestinal epithelial cells recentlynicotine was shown to cause a decrease in leukocyte recruitment disease activity index dai and histologicalscore in dss colitis and block tnfmediated expressionof mucosal vascular addresin cell adhesion molecule1 inendothelial cells these authors concluded that nicotinesuppressesinflammation through downregulation ofadhesion molecules in the gut nonetheless clinicalstudies on the efficacy and tolerability of nicotine haveshown thattherapeutic application of nicotine fortreatment of uc is limited because of frequent adverseeffects and nicotine inconsistent efficacy in maintainingremission in uc patients [ ]anatabine is found in plants of the solanacea familywhich includes tobacco peppers tomato and eggplant little is known about the biological properties of anatabinealthough several studies have suggested that this alkaloid is apotential candidate compound for antiinflammatory drugdevelopment [ ] anatabine was marketed in the us asa dietary supplement under the name anatabloc in aninternetbased survey approximately of all users ratedthe effect of anatabine supplementation as good or excellentfor joint pain stiffness and functionality anatabine hasbeen shown to inhibit lipopolysaccharide lpsinducedproinflammatory gene expression as well as nfκb andstat3 phosphorylation in human neuroblastoma shsy5y hek293 human microglia and human bloodmononuclear cells as well as in the brain and spleen ofmouse models of autoimmune encephalomyelitis andalzheimer™s disease in shsy5y cells anatabine alsoreduced the expression of betasecretase ”the rate limitingenzyme for amyloid peptide production which is a majorhallmark of alzheimer™s disease”through inhibition of nfκb activation in this study we aimed to assess the antiinflammatoryeffects of the tobacco alkaloids nicotine and anatabine inthe established dss mouse model of uc our resultsshow that oral administration of anatabine but notnicotine ameliorates the clinical manifestations of dsstreatment in mice the results of gene expression analysisindicated that anatabine had a partial restorative effect onglobal dssinduced gene expression profiles while nicotineonly had minimal effects moreover multianalyte profilingmap showed that anatabine but not nicotine suppressesthe production of il6 il1α tnfα granulocytecolonystimulating factor gcsf and keratinocyte chemoattractant kc while increasing il10 expression in the colon ofdsstreated mice for an overview of the study conceptand analytical procedures see œonline resource  0cruiz castro of inflammation page of resultsanatabine has a protective effect in the dss mousemodel of colitisto study the antiinflammatory properties of nicotineand anatabine c57bl6 mice were provided with nicotine or anatabine in drinking water for a total of dayscolitis was induced by oral administration of dssin drinking water ad libitum during days “ fig 1adsstreated mice developed colitis as evident from thebody weight loss fig 1b increased colon weightlengthratio fig 1c increased dai fig 1d increased stooloccult blood score fig 1e increased intestinal bleedingscore fig 1fincreased diarrhea score fig 1g andincreased colon inflammation score fig 1h in micefig clinical parameters of dsstreated mice administered nicotine or anatabine a mice were orally administered nicotine or anatabine or mgkg for a total of days colitis was induced by oral administration of dss in drinking water ad libitum during days “ b bodyweight changes in mice during the colitis induction and recovery phases body weight changes were calculated as percentage relative to thestarting day of dss treatment day c colon weightlength ratio are represented as mgcm of colon d dai was calculated according to weightloss colon weightlength ratio and intestinal bleeding e stool occult blood score f intestinal bleeding score g diarrhea score h coloninflammatory status data are shown as mean ± sem p p nic nicotine mgkg nic nicotine mgkg ana anatabine mgkg ana anatabine mgkg 0cruiz castro of inflammation page of not subjected to dss treatment nicotine and anatabine administration had no significant effect on these parametershowever mice treated concomitantly with anatabine anddss showed a decrease in colitis severity in particular inmice that received concomitant anatabine and dss treatment anatabine improved body weight recovery at mgkg p fig 1b caused a decrease in global dai relative to that in dsstreated mice at daily dose of p and mgkg p fig 1c and reduced the stooloccult blood score at mgkg p fig 1e interestingly nicotine but not anatabine improved the intestinalbleeding score relative to that in dssadministered mice at mgkg p fig 1f the diarrhea score fig 1gand the colon inflammation score fig 1h were notaffected by nicotine or anatabine no variation in waterconsumption was registered across the different experimental groups online resource anatabine reduces dssinduced gene expression changesin the distal colon on a global levelto complement these pathological findings we analyzed the colon transcriptome dsselicited lesions inmost mouse inbred strains including c57bl6 micehave been shown to be more pronounced in the distalcolon [ ] therefore we focused our study on thatportion of the large intestine principal componentanalysis clearly captured the effect of dss treatmenton the first principal component explainedvariance fig 2a while nicotine treatment did notproduce a pronounced effect on the first principalcomponent in the dsstreatment context anatabinetreatmentin combination with dss produced aresponse more similar to that observed in the watercontrols no dssindicating that anatabine had anameliorating effect on dssinduced gene expressionchanges the results of our differential gene expressionanalysis suggested substantial perturbation of the colontranscriptomefdr fig 2b“c application of dss in drinking watercaused significant changes in nearly genes incolon tissues addition of nicotine to dsscontainingdrinking water slightly increased the number of differentially expressed genes in contrast addition of anatabine decreased the number of differentially expressedgenes upon dss treatment by approximately fig 2b and c this alleviating effect of anatabinetreatment on dssinduced gene expression profileswas also apparent in the global gene expression heatmap which showed a global reduction of expressionfold changes upon anatabine treatment fig 2d and eof note in the absence of dss anatabine treatmentdid not result in differential expression of genes andnicotine treatment alone had minor effects fdr pvalue online resource upon dsstreatmentanatabine reduces dssinduced inflammatory geneexpression in the distal colonto gain a more mechanistic understanding of the effectsof nicotine and anatabine treatment we further investigated gene expression changes at the level of functionalgene sets and a ucrelevant causal network model gsaofthe reactome database showed changes acrossmultiple functional categories fig 3b the effects onfunctional categories in dsstreated mice administerednicotine appeared rather similar to those in mice treatedwith dss alone whereas administration of anatabineresulted in a general repression of dssmediated effectswe also evaluated the interaction terms betweennicotineanatabine and dss treatment to more directlyfocus on the modulating effect of anatabine and nicotinetreatment on dss effects fig 3a online resource the following six biological categories showed significant interaction terms upon anatabine and dss treatmentsupporting asignificant suppression of dssinduced effects in thepresence of anatabine œimmune system œextracellularmatrix anization œprotein localization œmetabolism œhemostasis and œsignal transduction fig 3bof these we further explored œimmune system œextracellular matrix anization and œsignal transductionas the most relevant categories in ibdana 20dss fdr allfiginteractiontermsp the hierarchical anization of the reactome database allowed us to investigate the underlying functionalchanges in more detail the œimmune system categoryincludes œadaptive immune system œcytokine signaling in immune system and œinnate immune systemin particular within these immune categories œcytokinesignaling egincluding il6 family signaling andœtolllike receptor cascades were modulated withsignificant3conline resource online resource within thereactome œsignal transduction category œsignaling byreceptor tyrosine kinasesby rhogtpases œsignaling by transforming growth factortgfbeta family members œmapk family signalingcascades œintegrin signaling œsignaling by erythropoietin and œsignaling by gpcr were found to be significantly impacted online resource online resource within the œextracellular matrix anization category almost all subcategories were perturbed includingœdegradation of the extracellular matrix œelastic fiberformation and œextracellular matrix proteoglycansonline resource online resource œsignalingœdegradation ofthe extracellular matrix includesbiological processes such as activation of matrix metalloproteinases mmps and collagen degradationto further follow up on the effects of nicotine andanatabine on inflammatory signaling we evaluatedthe perturbation of the ucrelevant tlril1rtnfr 0cruiz castro of inflammation page of fig transcriptomics results of colon biopsy samples a principal component pc analysis the plot displays all samples in the reduced pc1“pc2plane covering of the total data variance it allows us to examine the relationships between the various groups and treatmentsremarkably pc1 captured the pure dss effect black arrow while pc2 captured the pure exposure effects of anatabine and nicotine blue andred arrows respectively b volcano plots for individual gene differential expressions the horizontal axis represents the log2 differential expressionœfold changes and the vertical axis represents its statistical significance as log10 fdr c number of differentially expressed genes for theselected pairwise comparisons the bar plot displays the number of genes with positive or negative fold changes with corresponding statisticallysignificant fdr ‰¤ note that the lower fdr values observed for the œana dss comparisons do not necessarily prefigure a reduction ofbiological effects because the statistics underlying the fdrp values also depend on the gene expression variance within the experimentalgroups d highlevel heatmap of the statistically significant differentially expressed genes for the selected pairwise comparisons in order toprovide a comprehensible display of the large — foldchange matrix we first normalized its rows to their maximum absolute values andthen reordered them by hierarchical clustering complete linkage method on the basis of their euclidean distances e scatter plot from thecomparisons between the pure and exposuremodified dss effects by using the differential gene expression results the horizontal axis of thescatter plots represents the fold changes of the pure dss effect œwater dss vs water pairwise comparison whereas the vertical axis containsthe corresponding values in case of anatabine or nicotine exposure œanatabinenicotine dss vs water pairwise comparisons in an idealcase included as a reference [first plot] the bestfitted straight line coincides with the diagonal indicated in green and its slope is equal to the transcriptomics effects of anatabine or nicotine exposure were quantified by the slope of the bestfitted straight lines indicated on each plotnic nicotine mgkg nic nicotine mgkg ana anatabine mgkg ana anatabine mgkgnetwork model by using an established networkenrichment approach we inferred the activationstates of the network nodes on the basis of the observedgene expression changes and calculated the overall network perturbation amplitude for each group comparisonfig 3c dss treatment had a strong activating effect onthis signaling network including activation of several coresignaling nodes such as il1rassociated kinase irak1irak4 and myeloid differentiation primary response myd88 online resource œheatmap leadingnodes of note the network perturbation induced bydss treatment was reduced only in the presence ofanatabine with this the results of network analysisfurther supported the ameliorating effect of anatabineon dssmediated inflammation whereas no similareffect was apparent upon nicotine treatment 0cruiz castro of inflammation page of fig biological interpretation of the transcriptomics results a schematic representation of the effects of anatabinenicotine exposure as amodification of the pure dss effect the measured combined œanatabinenicotine dss effect captured by the pairwise comparisons œanatabinenicotine dss vs water can be viewed as the sum of the pure effects of the two factors œdss treatment and œanatabinenicotine exposuretogether with the adjusting twofactor interaction term œanatabinenicotinedss anatabinenicotine exposure is synergistic with dss treatmentif the interaction term has the same sign as the pure dss effect eg both are positive as in the schema in contrast the relationship isantagonistic if the interaction term has an opposite sign to the pure dss effect b gsa results for the top reactome pathway categories theheatmap displays the gsa scores normalized rowwise to their maximum absolute values as well as their competitive q1 statistical significancethe fdrs were calculated only among the top reactome pathway categories which are sufficiently distinct in their gene content to assumeindependence of their enrichment results c hierarchical representation of the gsa results for all pathways contained in the top reactomeœimmune system category and for the twofactor œana 20dss interaction the color map corresponds to the normalized gsa scores containedin the interval [ˆ’ ] whereas their statistical significance competitive q1 p values ‰¤ is indicated by black circles around the nodes theactual labels of the nodes ie the reactome pathway names can be found in online resource the treelike structure of the reactomepathway collection enables topdown investigation within the relevant pathway categories by identifying increasingly specific biologicalprocesses ie involving fewer and fewer genes along the longest paths connecting the statistically significant pathways d npa results for thetlr“il1r“tnfr network model the bar plot displays the npa values for the selected contrasts and their statistical significance is indicated bythe three colored asterisks note that by definition the positive npa values depend on the square of the input genelevel fold changes andtherefore might amplify the differences between the contrasts without preserving the additive relationships among them nic nicotine mgkg nic nicotine mgkg ana anatabine mgkg ana anatabine mgkgexpressionvalues”from theto validate the observations obtained by microarraytranscriptomics we quantified the expression of six œleadingedge genes”genes of the gene set with the highestœimmunedifferentialsystem œextracellular matrix anization and œsignaltransduction reactome categories using realtime quantitative pcr qpcr selected genes included il33 œsignaltransduction and œimmune system categoriesil6œsignal transduction and œimmune system categoriesmmp13 œextracellular matrix anization categoryserpine1 œsignal transduction and œextracellular matrixanization categories thbs1 thrombospondin œsignal transduction and œextracellular matrix anizationcategories and timp1 tissue inhibitor of metalloproteinase œextracellular matrix anization and œimmunesystem categories qpcr results showed a clear decreasein dssinduced expression of il33 il6 mmp13 serpine1thbs1 and timp1 expression in the presence of anatabineat mgkg thereby confirming the findings obtainedfrom the microarray data fig 0cruiz castro of inflammation page of fig validation of microarray transcriptomic results using qpcr a boxandwhisker plots for the distribution of the qpcr expression levels δcqof the selected genes the boxes represent the quartiles while the whiskers extend to the most extreme data point which is no more than times the interquartile range from the box the horizontal brackets indicate the statistical significance of the corresponding comparisons mean pvalue smaller than respectively b scatter plots comparing the mouse colon differential expression values obtained bymicroarray horizontal axis and qpcr vertical axis the following similarity metrics were indicated œbeta is the slope of the best interceptfreelinear fit between microarray and qpcr values œr2 is the coefficient of determination measuring the œgoodnessoffit and œpval is the pvalueassociated to the null hypothesis beta nic nicotine mgkg ana anatabine mgkganatabine decreases dssinduced proinflammatorycytokine production and promotes il10 expressionnext we sought to evaluate the impact of nicotine andanatabine on the expression of colonic inflammatorycytokines by mapin line with the previous dataanatabine but not nicotine significantly reduced theabundance of dssassociated inflammatory cytokines including il6 kc tnfα il1α and gcsf whereas itincreased the levels of the antiinflammatory cytokineil10 at a daily dose of mgkg fig interestinglyanatabine also increased the abundance of il21 andshowed a clear tendency towards increasing colonic il 0cruiz castro of inflammation page of fig map results for colon biopsies statistical assessment of the differences observed in the abundance of selected cytokines between thestudy experimental groups and water control fold changes in the treatment groups nicotine and anatabine at and mgkg dss relative towater control are illustrated with colors ranging from blue decrease to red increase statistically significant differences on the basis of raw pvalues no adjustment has been made for multiple testing grey cells highlight missing estimates on the observed differences due to lackof cytokine quantifications nic nicotine mgkg nic nicotine mgkg ana anatabine mgkg ana anatabine mgkg1 levels fig taken together the map data confirmthe antiinflammatory effects of anatabine in dssinduced colitisdiscussionour study shows that oral administration of anatabinebut not nicotine reduces the clinical manifestations ofdssinduced colitis in a mouse model in line with thesefindings anatabine demonstrated a global downregulatory effect on dssinduced gene expression changes inthe colon whereas the effects of nicotine were morelimited in particular the results of gene expression profiling further supported the reduction of inflammatorysignaling processes upon anatabine treatment includingsuppression of il6 signaling as shown by gsa findingsand tlr signaling as shown by the results of networkperturbation analysis and gsa map also showed asignificant decrease in the abundance of il6 kc tnfαil1α and gcsf and an increase in the expression of il and the antiinflammatory cytokine il10several studies have reported on the antiinflammatoryeffects of nicotine on the dss mouse model of uc subcutaneous administration of nicotine was shown toameliorate tissue injury in dss colitis and il6 expression in cd4t cells via α7nachrs nicotine wasalso shown to decrease the activation of stat3 throughinduction of mir124 in gutinfiltrated lymphocytes andintestinal epithelial cells strikingly alsharari 0cruiz castro of inflammation page of observed that oral but not subcutaneous injection ofnicotine ameliorated intestinalinflammation and colonic tnfα expression in dsstreated mice in spiteof the reported beneficial effects our results do not support a protective effect of orally administered nicotineon dss colitis of note we found that nicotine significantly reduced dssassociated intestinal bleeding whichwas the only clinical parameter affected by this tobaccoalkaloid the vasoconstrictor effects of nicotine are wellstablished in the gut mucosa nicotine decreasesblood flow and cigarette smoking decreases rectalblood flow to normal levels in patients with uc however how changes in blood flow affect the pathophysiology of uc is still unclear the possible therapeuticuse of nicotine to induce remission in uc patients hasbeen evaluated in five clinical studies [“] and twometaanalyses [ ] these studies have demonstrated avariable efficacy of nicotine therapy in induction of remission with several studies showing no effect [ ]moreover a high frequency of adverse events increasedthe withdrawal rate in the nicotine group in some studiesthus limiting the therapeutic benefit of nicotine nucleotidebindingto the best of our knowledge the present study isthe first to assess the impact of anatabine on experimental colitis gene expression analysis of distal colonbiopsy specimens highlighted the antiinflammatoryproperties of anatabine in multiple functional categories including œimmune responses œsignal transduction and œextracellular matrix anization which inturn encompassed several tlr and cytokine signalingpathways genes contributing to the downregulation oftlr cascades included tlr2 tlr4 and tlr6 and anumber of downstream signaling factors shared byseveral tlrs including myd88 ripk2 irak3 irak4and nod1oligomerizationdomaincontaining protein as well as the nuclearfactors elk1 fos cyclic adenosine monophosphatecamp response elementbinding protein creb1activating transcription factor atf1 and atf2 seeonline resource of the respective gene lists for thecytokine signaling pathways the contributing genes included il6 and il6 receptor α ifnγ il4 cxcl10il22 receptor α2 il2 receptor α tnf receptor superfamily member 1a il17α and il17 receptor α jak1jak2 stat3 and stat4 members of the nfκbsignaling pathway also contributed to the overall reducincluding nfκb p65tion in inflammatory cascadesp105 and p100 subunits iκbα and the nfκb activating protein tab3 in agreement with the results oftranscriptional analysis map findings showed a significant decrease in dssassociated il6 kc tnfα il1αand gcsf protein expression and an increase in il10expression in the presence of anatabine strikinglywhile tlr downstream factors modulated by anatabineare shared by most tlrs the majority of cytokineassociated signaling molecules are specific for eachpathway the seemingly pleiotropic regulatory effects ofanatabine suggest that this alkaloid reduces inflammation by inhibiting the expression of several factors involved in different proinflammatory signaling cascadesprevious studies using in vitro and in vivo diseasemodels have demonstrated the antiinflammatory effectsof anatabine [ ] paris showed that this pyridine alkaloid reduced the plasma levels of il1 il6and il17a as well as the expression of il1 infγ andtnfα in the spleen of experimental autoimmune encephalomyelitis mice these authors also showedthat anatabine suppressed stat3 and nfκb phosphorylation in the spleen and brain of these mice anatabine also prevented lps and tnfαinduced nfκb andstat3 phosphorylation in shsy5y and hek cellshuman microglia and human blood mononuclear cells additionally anatabine prevented lpsinduced il1 expression in human whole blood as well as il1il6 and tnfα production in the plasma kidney andspleen in the lps mouse model phosphorylatedstat3 tnfα and il6 were also downregulated in thepresence of anatabine in a transgenic mouse model ofalzheimer™s disease our results on the effects of anatabine in the dssmouse model of uc are also in line with the findings ofa substantial number of studies demonstrating the protective effects of natural alkaloids in several animalmodels of colitis [ ] intraperitoneal administrationof the minor tobacco alkaloid and nicotinic receptoragonist anabaseine was shown to reduce tissue damagemyeloperoxidase activity and colonic tnfα expressionin a tnbs mouse model of colitis these mice alsoshowed reduced nfκb activation in lamina propriamononuclear cells while mice administered a nicotinicreceptor antagonist presented worse colitis symptomsthan those treated with tnbs alone the algaealkaloid caulerpin reduces dss colitis by suppressingnfκb activation and subsequently inhibiting the colonicproduction of tnfα ifnγ il6 ifnγ and il17 oral administration of berberine also ameliorates dssinduced colitis and downregulates the expression oftnfα ifnγ kc and il17 in colonic macrophages the plantderived alkaloid nmethylcytisine andthe tea alkaloid theophylline mitigate colitis by downregulating tnfα il1 and il6 expression in dss andacetic acid models of colitis respectively [ ] induction ofthe antiinflammatory cytokine il10 in thepresence of natural alkaloids has also been reportedthus indirubin ameliorates dssinduced colitis by suppressing the expression of colonic tnfα ifnγ and il2and upregulating il10 additionally indole alkaloids caulerpin and isatin have been shown to increase 0cruiz castro of inflammation page of the expression of il10 in dss and tnbs models of ibdrespectively [ ] of note our results show anincrease in the abundance of il21 and a tendencytowards increase in colonic il1 levels in the presenceof anatabine although il21 expression is increased inmany chronic inflammatory disorders genetic deficiencyof il21 is associated with ibd and inhibition of il21in the early phases of some inflammatory disorders exacerbates disease development suggesting the dual role ofil21 in the control of immune responses il21also promotes il22 expression in mucosal tcellsthrough a mechanism involving stat3 retinoidrelatedorphan receptor γt and aryl hydrocarbon receptorthereby helping protect immunodeficient mice from dsscolitis interestingly il21 has been recently shownto induce il1 production in dendritic cells through astat3dependent but nfκbindependent mechanismthereby suggesting a link between il21 and il1 mounting evidence suggests that alkaloids”in particular isoquinoline alkaloids present in traditional medicineherbs”exertthroughregulation of nfκb and stat3 signaling pathways forexample sanguinarine and cavidine suppress the expression of nfκb p65 subunit thereby reducing colonictnfα and i
0
"Accumulating evidence has revealed the critical role of long noncoding RNAs lncRNAs in cellularprocesses during tumor progression As documented in cancerrelated literatures LINC00992 expression isassociated with cancer progression whereas its function in tumors including prostate cancer has not beencharacterized yetMethods Data from GEPIA database suggested LINC00992 expression in prostate cancer tissues The expressionlevels of RNAs were monitored via qRTPCR Western blot evaluated the levels of proteins The proliferationapoptosis and migration of prostate cancer cells were assessed by CCK8 EdU TUNEL Transwell and woundhealing assays Luciferase reporter RNA pull down and RIP assays were applied to detect the interplays amongLINC00992 miR3935 and GOLM1Results Elevated levels of LINC00992 and GOLM1 were detected in prostate cancer tissues and cells LINC00992exerted facilitating functions in prostate cancer cell proliferation and migration Mechanically LINC00992 interactedwith and negatively regulated miR3935 to elevate GOLM1 expression in prostate cancer cells In addition thein vitro suppressive effect of silenced LINC00992 on prostate cancer cell proliferation and migration was reversed byGOLM1 upregulation Likewise LINC00992 depletion restrained tumor growth in vivo was offset by enhancedGOLM1 expressionConclusions LINC00992 competitively bound with miR3935 to elevate GOLM1 expression and therefore facilitatethe oncogenic phenotypes of prostate cancer cells implying a potential LINC00992targeted therapy for prostatecancerKeywords INC00992 miR3935 GOLM1 Prostate cancer Correspondence engineyangsinacom5Department of Urology the Second Affiliated Hospital of Bengbu MedicalCollege Hongye Road Bengbu Anhui ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cChen BMC Cancer Page of BackgroundClinically prostate cancer manifests as a dominatingcause of malerelated death worldwide and is characterized as the most continually occurred tumor amongmen in the United States [] The biggest challenge isthe detectable bone metastases in roughly advancedprostate cancer [] Virtually all prostate cancer patientsduring years™ androgen deprivation treatment inevitably undergo castrationresistance which contributes tothe poor clinical consequences in prostate cancer []However the mechanism underlaid prostate cancer remains mostly unknownThe widely studied long noncoding RNAs lncRNAsare transcribed from nonproteincoding human genomeand have more than nt in length [] LncRNAs are increasingly functionally identified and experimentally consolidated to be related to tumor neoplasia and progressionin diverse cancers [] Additionally lncRNAs with dysregulation can functionally modulate tumor developmentfrom multiple pathological aspects such as cell proliferation drugresistance and metastasis [“] For examplelncRNA A1BGAS1 inhibits cell proliferation and invasionin hepatocellular carcinoma via targeting miR216a5p []LncRNA LOC730100 sponges miR760 from FOXA1 toaccelerate cell proliferation and invasion in glioma []LncRNA SNHG16 functions as an oncogene in hepatocellular carcinoma [] Long intergenic nonprotein codingRNA LINC00992 is a novel lncRNA that has beenpreviously revealed to be elevated in tumors and substantiated as a master regulator for chemoresistance [] Besides LINC00992 has been uncovered as an elevatedlncRNA in prostate cancer [] which is consistent withthe detection from GEPIA database Despite that no previous study has given a comprehensive explanation aboutthe precise function or detailed mechanism of LINC00992in prostate cancerIn past decades the fact that lncRNAs function in tumors depending on their secondary or tertiary structureshas been reported in many cancerlinked studies For instance in the nucleus lncRNAs are entitled to work asmolecular scaffolds or alternative splicing assistants [] On the contrary lncRNAs dispersing in cytoplasm influence downstream mRNA translation or degradationthrough serving as miRNA sponges [ ] For exampleTNFαinduced lncRNA LOC105374902 promotes themalignant behaviors of cervical cancer cells by acting as asponge of miR12853p [] LncRNA TTNAS1 promotes papillary thyroid cancer tumorigenesis by regulatingmiR1533pZNRF2 axis[] Nevertheless whetherLINC00992 could exert its functions in prostate cancer viaits sponging role of certain miRNA remains unknownWe conducted this research aiming to explore thefunction or probable mechanism of LINC00992 in prostate tumor which might enrich the understanding interms of prostate tumor pathology and contribute to awider applied scopeMethodsTissue samplesThe prostate cancer tissue samples and matched peritumor tissue samples were collected from patientsdiagnosed with prostate cancer under the approval ofthe Ethics Committee of the First Affiliated Hospital ofKunming Medical University Each participant did notreceive radiotherapy and chemotherapy prior to tissuecollection and signed the written informed consentsbefore this study All samples were snapfrozen in liquidnitrogen and then stored at °C until required forfurther analysisCell cultureThe prostate epithelial cell line RWPE1 CRL11609and prostate cancer cellsincluding PC3 CRL1435LNCaP CRL1740 C4“ CRL3314 and DU145HTB81 were all purchased from American TypeCulture Collection ATCC Manassas VA USAinOctober All cells were cultured as recommendedin Dulbecco™s modified Eagle™s medium containing FBS GIBCO MA USA under the condition of a cellincubator with CO2 at °C Before using in thisstudy all cell lines were authenticated by STR profilingand tested for mycoplasma contamination in June Cell transfectionLINC00992 shRNA or negative control shRNA andpcDNA31LINC00992 pcDNA31GOLM1 or its emptycontrol pcDNA31 plasmid were chemically synthesizedand provided by Gene Pharma Shanghai China MiR mimics miR3935 inhibitor and theirrelatednegative controls NCmimics NCinhibitor were allpurchased for upregulating or downregulating miR3935from Ribobio Guangzhou China In line with the directions of LipofectamineTM RNAiMAX TransfectionReagent Thermo Fisher Scientific transfection of theseplasmids into DU145 PC3 and RWPE1 cells wasconducted and qRTPCR checked the transfection efficiency The sequences were as follows shNC ²CCGGTAGTAATTGACAACCATTATACTCGAGTATAATGGTTGTCAATTACTATTTTTG3²shLINC009921²CCGGATTATCCAAGAGTATTAACATCTCGAGATGTTAATACTCTTGGATAATTTTTTG3² shLINC0 ²CCGGTGTTAGATGATCATTGAGGTGCTCGAGCACCTCAATGATCATCTAACATTTTTG3² s²CCGGTTACCTAATCAGTAGAThLINC009923GCAGCTCGAGCTGCATCTACTGATTAGGTAATTTTTG3² NCmimics ²UCAGGUAGGGCUCAAACCAACC3² miR3935 mimics ²UGUAGAUACGAGCACCAGCCAC3² NCinhibitor²CUGGCUUUAG 0cChen BMC Cancer Page of GGUGCCACUUAG3² miR3935 inhibitor ²GUGGCUGGUGCUCGUAUCUACA3²Quantitative realtime PCR qRTPCROn the basis of the instructions of Trizol reagent Invitrogen USA RNA extraction was executed in prostatecancer cells After the examination of RNA purity withspectrophotometry cDNA was obtained from aboveRNA with reverse transcription kit ThermoFisher Scientific shanghai China qRTPCR analysiswas devised with the aid of a BioRad CFX96 system andSYBR green was applied for investigating the RNA levelsThe internal reference for LINC00992 and mRNAs wasGAPDH whereas that for miRNAs expression was U6Relative expression was assessed based on the method ofˆ’ΔΔCtab97779Western blotProtein content in cells was determined by western blotanalysis RIPA lysis buffer Beyotime Shanghai Chinawas adopted for cell lysing followed by the evaluation ofthe protein concentration with BCA Protein Assay KitP0011 Beyotime Tech SDSPAGE gel was applied for separating proteins μg protein per sampleand then proteins were transferred onto μm PVDFmembranes BioRad Hercules CA USA Antibodiesincluding antiGOLM1 ab109628 AbcamCambridge UK antiPCNA ab92552 AbcamantiCDK2 ab32147 Abcam antiCyclin D1ab40754 Abcam antiBax ab32503 Abcam antiBcl2 ab32124 Abcam antiMMP2antiMMP9ab38898 Abcam antipSrc ab40660 Abcam antiSrc ab47405 Abcam antipFAKab81298 Abcam antiFAK ab131435 Abcam antiGAPDH ab8245 Abcam andantiTubulin ab7291 Abcam were applied toprobe the membranes overnight at °C After that themembranes were further incubated for h with HRPconjugated secondary antibody Santa Cruz Co LtdSant Cruz CA USA atroom temperature ECLSubstrates Millipore Billerica MA USA was utilizedfor the visualization of signals followed by exposure toXfilm Kodak Rochester NY USA The quantificationof immunoblots was conducted with the aid of imageJsoftware National Institute of Health Bethesda MDUSA with GAPDH or Tubulin as the normalizer asneeded AbcamLuciferase reporter assayFragments of fulllength LINC00992 with wildtype ormutant binding sites for miR3935 and sequences ofGOLM1 ™UTR containing wildtype or mutated miR binding sites were inserted into the pmirGLOvectors Promega Madison WI USA for the construction of reporters LINC00992WT LINC00992MUTGOLM1WT GOLM1MUT Then the four reportersand miR3935 mimics or miR3935 inhibitor GenePharma were cotransfected into DU145 and PC3 cellsapplying lipofectamine2000 Invitrogen as neededFortyeight hours later DualLuciferase Reporter AssaySystem Promega was employed for the examination ofthe luciferase activity GloMax® Discover Multimode Microplate Reader Promega assessed the ratio of FireflyRenilla luciferase activity and the activity of Renilla wasthe normalized controlRNA immunoprecipitation RIP assayAccording to the direction for usage of Magna RIP„¢RNA Binding Protein Immunoprecipitation Kit “Millipore RIP assay was strictly performed RIP lysisbuffer was firstly applied to treat the transfected DU145and PC3 cells Afterwards the obtained cell lysates wereprocessed with magnetic beads integrated with humanantiAgo2 antibodies ab32381 Abcam MA USA orantiIgG AP162KC Millipore Following the recoveryof antibody by the protein AG beads qRTPCR detected the levels of LINC00992 miR3935 and GOLM1mRNA in the precipitates IgG worked as the negativecontrol for the normalization of RNAIPsRNA isolation of nuclear and cytoplasmic fractionsThe dispersion of LINC00992 in the prostate cancercells was assayed as described previously [] The isolation of cytosolic and nuclear sections was executed followingAM1921Invitrogen RNA levels of U1 nuclear control GAPDHcytoplasmic control and LINC00992 were all estimatedby qRTPCR analysisPARIS„¢ KittheprotocolofFluorescence in situ hybridization FISH assayIn line with the recommendation of Ribo„¢ FISH KitC10910 Ribobio Guangzhou China FISH analysiswas implemented for testing the presence of LINC00992in prostate tumor cells Ribobio Company synthesizedthe LINC00992 probes labeled by Cy3 fluorescent dyeFollowing the fixation by paraformaldehyde and Triton X100 permeabilization DU145 and PC3 cellswere subsequently blocked in prehybridization bufferblocking solution Then incubation of cells with probehybridization buffer was later performed Next day afterrinsing and Hoechst staining the fluorescence was measured under a confocal laser scanning microscope ZeissGermanyCell counting kit8 CCK8 assayFor the viability assessment in DU145 PC3 and RWPE cells CCK8 assay was implemented as described 0cChen BMC Cancer Page of previously [] Cell viability was monitored at and h In short after being seeded onto 96well platesand cultured for indicated times cells were processedwith μl of CCK8 solution Then a microplate readerexamined the absorbance values at the wavelength of nm²ethynyl2²deoxyuridine EdU incorporation assayCell proliferation was examined through EdU assay asdescribed previously [] by using ClickiT EdU AlexaFluor Imaging Kit C10086 Invitrogen After hoftransfection EdU staining was carried out asinstructed The observation and calculation of EdUpositive cells was proceeded under the fluorescencemicroscopyTransferasemediated dUTP nick end labeling TUNELstainingTUNEL assay was carried out as described previously[] for probing DU145 and PC3 cell apoptosis with theassistance of an In Situ Cell Death Detection Kit Roche Mannheim Germany TUNELpositivecells were recorded under a light microscope × from visual fields which were chosen at randomTranswell migration assayThe application of transwell chambers with pore size μm Corning Costar Cambridge MA USA was aimedfor detecting cell migration in strict line with the instructions Cells that were previously suspended inserumfree RPMI1640 media were seeded into theupper chamber RPMI1640 medium containing FBS was supplemented in lower chamber as a chemoattractant Cells in the filters following h incubationwere immobilized in methanol and went through crystal violet staining The images of cells migratedthrough the filters were obtained and counted under themicroscopeWound healing assayThe DU145 PC3 and RWPE1 cells × cellswellwere prepared on glass culture dishes and cultivated at °C for a whole night to allow cells adhered to theplates followed by the straight scratch made with a plastic pipette tip after cell samples reached confluenceLater cells were rinsed in PBS to clear the detachedcells Finally the wounds at and h were imaged viaa light microscopy Olympus Tokyo JapanIn vivo experimentSixteen sixweekold male BALBC athymic nude micewere commercially available from the National Laboratory Animal Center Beijing China and maintained inSPFgrade animal lab All animalrelated protocols wereapproved by the Animal Research Ethics Committee ofthe First Affiliated Hospital of Kunming Medical University The in vivo experiment was undertaken via subcutaneous injection of × DU145 cells into the nudemice while the DU145 cells injected into indicated fourshgroups of mice were transfected with shNCLINC009921shLINC009921 pcDNA31 orshLINC009921 pcDNA31GOLM1 Tumor volume wasmonitored every days 28day after injection nudemice were sacrificed via cervical dislocation and thentumor samples were carefully dissected for weight assessment and hematoxylin and eosin HE stainingImmunohistochemistry IHCThe tumor samples collected from in vivo experimentswere treated with PFA dehydrated and embedded inparaffin Afterwardsthe paraffinembedded sections μm were prepared for IHC assay as described previously [] by use of the antiKi67 and antiPCNA antibodies AbcamStatistical analysisSPSS statistical software SPSS Armonk NY USAwas employed in the processing of data from threebiological replicates and data were expressed as mean ±SD Significance of difference within two groups wasdetermined using Student™s ttest while that among noless than two groups was tested via oneway or twowayANOVA P was considered as the threshold ofsignificanceResultsLINC00992 is overexpressed in prostate cancer andregulates cell proliferation apoptosis and migrationLINC00992 expression pattern in prostate cancer wasacquired from online GEPIA database As a resultLINC00992 was considerably upregulated in PRADprostate adenocarcinomatissues relative to normalones Fig 1a After detecting LINC00992 expression intissue samples obtained from patients with prostate cancer we observed that LINC00992 expression was higherin prostate cancer tissues than that in peritumor tissuesFigure S1A Moreover clinical data showed that higherexpression of LINC00992 in prostate cancer patientswas associated with lower survival rate Figure S1BFurthermore LINC00992 expression in the prostate cancer cells and RWPE1 cells was evaluated by qRTPCRConsequently higher level of LINC00992 was exhibitedin prostate cancer cells than that in RWPE1 cells Fig1b which was completely consistent with the result presented in previous discovery [] Particularly DU145and PC3 cells expressed the highest level of LINC00992and was thereby chosen for the later assays For silencingLINC00992 special shRNAs targeting LINC00992 was 0cChen BMC Cancer Page of Fig LINC00992 was overexpressed in prostate cancer and regulates cell proliferation apoptosis and migration a GEPIA database demonstratedthe overexpression of LINC00992 in tumor tissues in contrast to adjacent normal ones b LINC00992 expression was detected by qRTPCR in fourprostate cancer cell lines and control RWPE1 cells c LINC00992 expression was monitored by qRTPCR in DU145 and PC3 cells after transfectionwith shRNAs targeting LINC00992 shNC was used as the negative control d The viability of DU145 and PC3 cells was estimated through CCK8assay following LINC00992 depletion e The proliferation of DU145 and PC3 cells was investigated after LINC00992 depletion via EdU assay Scalebar μm f The apoptosis of DU145 and PC3 cells transfected with shLINC0099212 or shNC was estimated via TUNEL assay Scale bar μm g Western blot analysis was applied to examine the expression of apoptosisrelated proteins hi The migration of DU145 and PC3 cellswas analyzed via Transwell migration assay scale bar μm and wound healing assay scale bar μm after inhibiting LINC00992expression The fulllength images for blots in Fig 1g were presented in Supplementary figure P p transfected into DU145 and PC3 cells and the efficiencywas corroborated in qRTPCR Fig 1c And then thedata from CCK8 assay revealed that LINC00992 depletion suppressed the proliferation of DU145 and PC3cells Fig 1d As expected a declined proportion ofEdU positive cells was observed after knocking downLINC00992 Fig 1e suggesting the suppressive effect ofLINC00992 deficiency on prostate cancer cell proliferation Additionally the expression levels of proliferationrelated proteins PCNA CDK2 and Cyclin D1 were allreduced by silenced LINC00992 Figure S1C On thecontrary TUNEL assay uncovered that LINC00992knockdown facilitated cell apoptosis Fig 1f Meanwhile western blot analysis revealed that LINC00992knockdown promoted the apoptosis of DU145 and PC3cells as Bax protein level was increased whereas Bcl2protein level was decreased after LINC00992 was silenced in these two cells Figs 1g Figure S1D FurtherTranswell and wound healing assays indicated that themigration of DU145 and PC3 cells was retarded byLINC00992 depletion Fig 1hi Likewise the expression of migrationrelated molecular markers MMP2MMP9 pSrc and pFAK was decreased by LINC00992inhibition Figure S1E To further verify the biological 0cChen BMC Cancer Page of role of LINC00992 in prostate cancer we carried outgainoffunction assays in RWPE1 cells After overexpressing LINC00992 in RWPE1 cells Figure S2A cellproliferation was promoted Figure S2BC As expectedthe expression of PCNA CDK2 and Cyclin D1 wasdecreased by upregulation of LINC00992 Figure S2DSimilarly LINC00992cellIn addition upregulatingmigration Figure S2EFLINC00992 resulted in the elevated protein levels ofMMP2 MMP9 pSrc and pFAK Figure S2G All thesedata elucidated that LINC00992 could facilitate cell proliferation and migration whereas suppress cell apoptosisin prostate cancerupregulationfacilitatedMiR3935 is targeted by LINC00992Given the high correlation of the sublocalization ofLINC00992 with its functional mechanism the predication of LINC00992 presence in cells was performed viaLncLocatorhttpwwwcsbiosjtueducnbioinflncLocator Result predicted that LINC00992 located mainlyin cytoplasm Fig 2a Likewise FISH assay and RNAisolation of nuclear and cytoplasmic fractions furtherverified the abundance of LINC00992 in the cytoplasmof prostate cancer cells Fig 2bc highlighting a posttranscriptional control of LINC00992 in such cellsHence we speculated that LINC00992 might act as aceRNA in prostate cancer regulation According toDIANAlncBase the top three potential miRNAs possessing the binding capacity with LINC00992 were listedFig 2d To targetthe highlymatched miRNA toLINC00992 qRTPCR analysis was conducted to testthe expression changes of these miRNAs following eitherLINC00992 depletion or augmentation The resultsdemonstrated that only miR3935 expression was increased by LINC00992 depletion Fig 2e but reducedby LINC00992 overexpression in the meantime Fig 2fThus miR3935 was chosen for further analysis Afterwards RNA pull down assay was implemented and theresult depicted that LINC00992 was pulled down byBiomiR3935WT Fig 2g which indicated the bindingof LINC00992 and miR3935 Later we observed thesatisfactory efficiency of miR3935 overexpression andmiR3935 inhibition through qRTPCR analysis Fig2h Thereafter RIP assay applying antiAgo2 was executed Results illustrated that LINC00992 and miR3935were highly enriched in antiAgo2 group in comparisonwith control antibody Fig 2i certifying the associationof LINC00992 with miR3935 in the RNAinduced silencing complexes RISCs To further explore the interaction between LINC00992 and miR3935 the bindingsites between LINC00992 and miR3935 were predictedat first and then data from luciferase reporter assayrevealed that miR3935 upregulation decreased theluciferase activity of LINC00992WT reporter whereasmiR3935 inhibition increased the luciferase activity ofLINC00992WT reporter Fig 2j Altogether LINC0 combined with miR3935 to act as a miRNA decoyin prostate cancerLINC00992 regulates the expression of GOLM1 a targetof miR3935Present evidence has suggested that miRNAs can bindwith downstream target genes to inhibit their expressionHerein we searched the miR3935 target genes andeight mRNAs were found out Subsequently we detectedtheir expression in prostate cancer cells and normalcells Interestingly we found that only Golgi membraneprotein GOLM1 was highly expressed in four prostate cancer cell lines relative to normal controls Fig 3aFurther we discovered that GOLM1 expression wasmarkedly upregulated in prostate cancer tissues according to data from GEPIA database Fig 3b SimilarlyGOLM1 expression was much higher in prostate cancertissue samples than in peritumor samples Figure S3AIn addition the mRNA and protein levels of GOLM1were overexpressed in prostate cancer cells in contrastto RWPE1 cells Fig 3c Figure S3B Besides GOLM1has been previously revealed as a prostate cancer facilitator and was metastasisrelated in prostate tumor [“] Thus we hypothesized that GOLM1 might act asthe downstream of LINC00992miR3935 signaling inprostate cancer Through TargetScan httpwwwtargetscanvert_72 the binding site between GOLM1and miR3935 was predicted Fig 3d After conductingluciferase reporter assay in DU145 and PC3 cells weobserved that upregulation of miR3935 specifically decreased the luciferase activity of GOLM1WT reporterFig 3e confirming the interaction between miR3935and GOLM1 relied on the putative binding sites Thenwe unveiled that GOLM1 mRNA and protein levels wereboth reduced by LINC00992 inhibition or miR3935upregulation according to qRTPCR and western blotanalyses Fig 3fg Figure S3C Moreover data fromRIP assay unveiled the binding of miR3935 to GOLM1in the RISCs Fig 3h Further we demonstrated thatthe decreased mRNA and protein levels of GOLM1 induced by LINC00992 depletion could be restored afterinhibiting miR3935 expression Fig 3ij Figure S3DAll the results showed that LINC00992 upregulatedGOLM1 expression via directly binding to miR3935LINC00992 promotes prostate cancer cell proliferationand migration via elevating GOLM1 expressionTo test whether LINC00992 affected prostate cancer cellproliferation apoptosis and migration via regulatingmiR3935targeted GOLM1 we executed the rescue experiments with the upregulation of GOLM1 To beginwiththe efficiency of overexpressing GOLM1 was 0cChen BMC Cancer Page of Fig MiR3935 was targeted by LINC00992 a LncLocator predicted LINC00992 subcellular location b FISH analysis of LINC00992 distribution inprostate cancer cells Scale bar μm c RNA isolation of nuclear and cytoplasmic fractions assayed the subcellular distribution of LIN00992 inprostate cancer cells d Top three miRNAs which might interact with LINC00992 were predicted by DIANAlncBase e After transfection ofLINC00992silencing plasmids the expression of miR31575p miR11783p and miR3935 was examined via qRTPCR f Following LINC00992upregulation qRTPCR tested the levels of miR31575p miR11783p and miR3935 in DU145 and PC3 cells g RNA pull down assay wasimplemented to testify the binding capacity between LINC00992 and miR3935 h miR3935 overexpression efficiency and inhibition efficiencywere examined by qRTPCR i RIP assay disclosed the binding of miR3935 to LINC00992 in the antiAgo2 group j The potential binding sitebetween LINC00992 and miR3935 was shown And the luciferase activity of LINC00992WT or LINC00992MUT reporter was assessed vialuciferase reporter assay in DU145 and PC3 cells after transfection with miR3935mimics miR3935inhibitor NCinhibitor or NCmimics P p p analyzed through qRTPCR and western blot analysesand the outcome turned out to be satisfactory Fig 4abFigure S3E Then we observed that overexpression ofGOLM1 could significantly elevate the mRNA and protein expression of GOLM1 in shLINC009921transfected cells Figure S3F Afterwards data from CCK8revealed that the viability of DU145 cells was firstly hindered due to LINC00992 depletion while subsequentGOLM1 elevation reversed the inhibitory trend onDU145 cell viability Fig 4c Results from EdU assayalso exposed similar trends that GOLM1 upregulationimpactposedthesuppressivecountervailedbyLINC00992 downregulation on DU145 cell proliferationFig 4d Similarly the restraining effect of silencedLINC00992 on the expression of proliferationrelatedproteins could be reversed by GOLM1 upregulationFigure S3G Later TUNEL assay revealed that cellapoptosis rate was elevated by LINC00992 depletion andthen overexpressing GOLM1 reduced the increasedapoptosis rate of LINC00992depleted cells Fig 4eLikewise western blot analysis uncovered that overexpressing GOLM1 could offset the effect of LINC00992 0cChen BMC Cancer Page of Fig LINC00992 regulated the expression of GOLM1 a target of miR3935 a The expression of eight mRNAs in four prostate cancer cell linesand RWPE1 cells was detected by qRTPCR b GOLM1 was overexpressed in prostate cancer tissues according to GEPIA database c The mRNAand protein levels of GOLM1 were evaluated in prostate cancer cell lines and RWPE1 cell line by qRTPCR and western blot respectively d Thebinding sites between GOLM1 and miR3935 were predicted via TargetScan e Luciferase reporter assay presented the inhibited luciferase activityof GOLM1WT reporter in the presence of miR3935 mimics not NCmimics fg GOLM1 expression in transfected cells was tested by qRTPCR andwestern blot analyses h The combination of GOLM1 with miR3935 in the antiAgo2 group was validated by RIP assay ij The mRNA and proteinlevels of GOLM1 in different groups were examined via qRTPCR and western blot The fulllength gels for western blot data in Fig 3c g and jwere presented in Supplementary Figure P p p downregulation on the expression of apoptosisrelatedproteins Fig 4f Figure S3H Moreover Transwellmigration and wound healing assays illuminated thatthe retarding influence of silenced LINC00992 on cellmigration could be rescued by GOLM1 overexpression Fig 4gh As expected the inhibitory effect ofLINC00992 depletion on the expression of migrationrelated molecular markers MMP2 MMP9 pSrc andpFAK could be countervailed by GOLM1 overexpression Figure S3I Collectively GOLM1 was requiredcancercellular processesLINC00992regulatedinprostateLINC00992 contributes to tumor growth via upregulatingGOLM1 expressionAfter the in vitro exploration of LINC00992 performance in prostate cancer we applied the in vivo assays tofurther validate above findings As shown in Fig 5a tumors derived from LINC00992silenced DU145 cellswere smaller with the growth rate quite slower thanthose from control cells more importantly such blockage on tumor growth was obviously countervailed afterGOLM1 overexpression Besides elevating GOLM1 expression could recover the lessened tumor volume anddeclined tumor weight induced by LINC00992 deficiency 0cChen BMC Cancer Page of Fig LINC00992 promoted prostate cancer cell proliferation and migration via elevating GOLM1 expression ab GOLM1 mRNA and proteinlevels in DU145 cells transfected with pcDNA31 or pcDNA31GOLM1 were detected via qRTPCR and western blot pcDNA31 served as thenegative control c The viability of DU145 cells was determined via CCK8 following transfection of different plasmids d The proliferation oftransfected cells was evaluated via EdU assay e The apoptosis of transfected cells was monitored via TUNEL assay Scale bar μm f Theprotein levels of Bax and Bcl2 in different groups were detected via western blot gh The migration of transfected cells was measured viaTranswell migration assay scale bar μm and wound healing assay scale bar μm The fulllength gels for western blot data in Fig 4band f were presented in Supplementary Figure P p Fig 5bc Of note we discovered decreased level ofLINC00992 and enhanced level of miR3935 in tumorsfrom latter three groups compared to control group whilethe lowered expression of GOLM1 in tumors withLINC00992 inhibition was normalized under GOLM1overexpression Fig 5d In addition the inhibitory impactof silenced LINC00992 on the positivity of proliferationassociated proteins PCNA and Ki67 could be reversedby upregulation of GOLM1 Fig 5e Taken togetherLINC00992 promoted the tumorigenesis of prostate cancer through upregulating GOLM1 expressionDiscussionAs documented the aberrant regulation of lncRNAs is afrequent event in diversified tumor types Besides thecorrelation between abnormallncRNA expression andprostate cancer oncogenesis has also been extensivelyexplored For example lncRNA SNHG7 facilitates prostate cancer carcinogenesis via cyclin D1 by spongingmiR503 [] LncRNA SChLAP1 aggravates prostatecancer cell proliferation and metastasis by targetingmiR198 [] LncRNA PCAT1 contributes to prostatecancer tumorigenesis through modulating FSCN1 andsponging miR1455p [] In our work LINC00992 wasrevealed to be highly expressed in prostate cancer tissuesand cells but unlike former investigations our studygave a precise explanation about its role in prostate cancer Our study unveiled that LINC00992 promoted cellproliferation and migration whereas suppressed cellapoptosis in prostate cancer Abovementioned data 0cChen BMC Cancer Page of Fig LINC00992 contributes to tumor growth via upregulating GOLM1 expression a Representative images and the growth curves of tumorsfrom indicated groups bc The volume and weight of tumors from above groups d The expression of LINC00992 miR3935 and GOLM1 intumors from different groups was detected via qRTPCR analysis e The staining of PCNA and Ki67 in different groups was measured via IHCScale bar μm p 0cChen BMC Cancer Page of validated that LINC00992 elicited a tumorpromotingfunction in prostate cancerPresently accumulating evidence has indicated thatcytoplasmic lncRNAs assisted the expression of downstream miRNAtargeted mRNAs via sponging the specific miRNAs Before exploring LINC00992mediatedmechanism in prostate cancer herein we firstly discovered its subcellular distribution in prostate cancer cellswith both aids from online prediction tool LncLocatorand experimental data FISH and RNA isolation of nuclear and cytoplasmic fractions Our study for the firsttime uncovered that LINC00992 located mainly in thecytoplasm of prostate cancer cells Besides our studyalso completed LINC00992modulated mechanism bydisclosing the downstream target miR3935 The directinteraction between LINC00992 and miR3935
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" tumor microenvironment tme plays an important role in malignant tumors our study aimed toinvestigate the effect of the tme and related genes in osteosarcoma patientsmethods gene expression profiles and clinical data of osteosarcoma patients were downloaded from the targetdataset estimate algorithm was used to quantify the immune score then the association between immune scoreand prognosis was studied afterward a differential analysis was performed based on the high and lowimmunescores to determine tmerelated genes additionally cox analyses were performed to construct two prognosticsignatures for overall survival os and diseasefree survival dfs respectively two datasets obtained from the geodatabase were used to validate signaturesresults eightyfive patients were included in our research the survival analysis indicated that patients with higherimmune score have a favorable os and dfs moreover genes were determined as tmerelated genes theunsupervised clustering analysis revealed two clusters were significantly related to immune score and t cells cd4memory fraction in addition two signatures were generated based on three and two tmerelated genesrespectively both two signatures can significantly divide patients into low and highrisk groups and were validatedin two geo datasets afterward the risk score and metastatic status were identified as independent prognosticfactors for both os and dfs and two nomograms were generated the cindexes of os nomogram and dfsnomogram were and respectively tme was associated with the prognosis of osteosarcoma patients prognostic models based on tmerelated genes can effectively predict os and dfs of osteosarcoma patientskeywords tumor microenvironment osteosarcoma prognosis immune features nomogram osteosarcoma is the most common bone tumor especiallyin children and adolescents it was reported that approximately of patients are between and yearsold and osteosarcoma is considered as the second leadingcause of death in this age group currently surgery and correspondence 407404159qqcom4wenzhou medical university wenzhou chinafull list of author information is available at the end of the chemotherapy are still major treatments for osteosarcomapatients and these therapies are constantly improving inrecent years however due to the susceptibility of localaggressiveness and lung metastasis in osteosarcoma patients the prognosis of osteosarcoma remains unfavorable previous studies indicated that the 5years survivalrates were and in metastatic and nonmetastaticpatients respectively thereforeit is necessary to the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0chu bmc cancer page of investigate the mechanism of pathogenesis and progressionof osteosarcoma and accurately classify the risk of patientsrecently an increasing number of diagnostic and prognostic biomarkers of osteosarcoma patients have beenidentified for example chen reported that tumorsuppressor p27 is a novel biomarker for the metastasis andsurvival status in osteosarcoma patients moreover huang discovered that dysregulated circrnas serve asprognostic and diagnostic biomarkers in osteosarcomapatients and the relative potential mechanism mainly attributes to the regulation of downstream signaling pathwaysby sponging microrna in addition lncrna microrna and many clinical data were also identified asprognostic biomarkers for osteosarcoma patients however osteosarcoma is one of the malignant cancers entitiescharacterized by the high level of heterogeneity in humanstherefore it is necessary to find accurate biomarkers forosteosarcomain recent years researchers have paid more and moreattention to the role of the tumor microenvironmenttme in malignant tumors the function of tme inthe tumorigenesis progression and therapy of tumorshave been initially understood [ ] more importantly estimation of stromal and immune cells in malignant tumor tissues using expression data estimate an algorithm to quantify the score of immune cellsand stromal cells by analyzing the gene expression datawas developed in based on the algorithm theprognostic value of immune and stromal cells in bladdercancer acute myeloid leukemia gastric cancer cervicalsquamous cell carcinoma adrenocortical carcinomaclear cell renal cell carcinoma hepatocellular carcinomathyroid cancer and cutaneous melanoma have beenreported [“] generally the above research indicatedthat tme can serve as the prognostic biomarker in tumorsand many tmerelated genes were determined as the prognostic genes however the role of tme and tmerelatedgenes in osteosarcoma patients remains unclearin the present study gene expression data and corresponding clinicopathologic data were obtained from thetherapeutically applicable research to generate effectivetreatments target dataset then the estimatealgorithm was performed to quantify the immune score ofosteosarcoma and the tmerelated genes were identifiedby the differential expression analysis subsequently theprognostic value of tme and tmerelated genes weredetermined by a series of bioinformatics methodsmethodsgene expression datasetslevel data of gene expression profiles and correspondingclinical data of osteosarcoma patients were downloadedfrom target dataset ocgcancergovprogramstarget accessed on oct the correspondingclinicopathologic data included in the present study wereage gender race ethnicity tumor site and metastaticstatus after data were extracted from the public domainthe estimate an algorithm inferring tumor puritystromal score and immune cell admixture from expression data was performed to evaluate the immune score byusing the estimate package in r software version meanwhile the messenger rnamrna expressionprofiles and clinical data ofincludinggse21257 and gse39055 were obtained fromthe gene expression omnibus as external validationcohortstwo cohortssurvival analysis and correlation analysisafter scores were obtained patients were divided intohighscore group and lowscore group according to themedian of the immune score the kaplanmeier survivalanalysis with logrank test was performed to estimatethe differences of overall survival os and diseasefreesurvival dfs between high and lowscore cohorts inaddition the association between clinicopathologic dataand tme score was also studied mannwhitney signedrank test was performed to compare the differences ofimmune score between each clinical group all statisticalanalyses in the present study were performed using rsoftware except for the special instructions p value twoside was identified as statistically significantin the present studydegexpressed genedifferentially expressed gene analysisdifferentiallyanalysis wasperformed by comparing the proteincoding genesexpression between the lowimmune score group andthe highimmune score group the limma package in rsoftware was used to perform the differential analysisand genes with log fc and adjusted pvalue qvalue were identified as degs to further understand the function of degs identifiedin the present study gene ontology goincludingbiological processes bp molecular functions mf andcellular componentscc and kyoto encyclopedia ofgenes and genomes kegg analysis were performedby clusterprofiler package in r software evaluation of association with immune cellsto further investigate the association between degs andimmune cells the cibersort package was used toestimate the relative proportions of types of immunecells meanwhile the œconsensusclusterplus package was used to cluster in an unbiased and unsupervisedmanner based on the overlapping degs cumulative distribution function cdf and relative change inarea under the cdf curve were used to determine theoptimal number of clusters k then mannwhitney 0chu bmc cancer page of signedrank test was performed to study the differenceof immune cells proportion between the clusters and theviolin plot was established to show the differences ofimmune cells among clusters survival analysis of degsbased on the degs the univariate cox analysis was performed to determine the prognostic value of immunerelated genes then the osrelated genes were validatedin the gse21257 dataset while the dfsrelated geneswere validated in the gse39055 dataset only genes successfully validated were selected for further analysis afterward based on the validated genes the multivariate coxanalysis was performed to establish the prognostic signature for predicting the prognosis of osteosarcoma patientsthe risk score for each patient was calculated based onthe coefficient from the multivariate cox analysis and thecorresponding gene expression meanwhile all patientswere divided into the high and lowrisk groups accordingto the median of the risk score the survival receiver operating characteristic roc curve was used to show the discrimination of signatures and the kaplanmeier survivalcurve with the logrank test was generated to show thedifferences of os and dfs between high and lowriskgroups in addition the risk score of patients in the validation cohort was also calculated according to the aforementioned risk signature the kaplanmeier survivalcurve and survival roc curve were generated to show thepredictive ability of the signature in the validation cohortdevelopment of a nomogram for osteosarcoma patientsnomogram is a tool to visualize the predictive model andconvenient for clinical practice therefore we attemptedto develop a nomogram based on the tmerelated genessignature and clinicopathologic data to predict the prognosis of osteosarcoma patients firstlythe univariatecox analysis was performed to filter prognostic variableswhich will be further included in the multivariate coxanalysis secondly based on independent prognostic variables two nomograms were established for predicting theos and dfs respectively the cindex was used to assessthe discriminatory performance of the nomogram whichrange from to a cindex of means agreement by chance and a cindex of represents perfectdiscriminatory performance the higher value of the cindex the better performance of the nomogram is furthermore the calibration curves of and 3year weredeveloped to evaluate the effectiveness of nomogramsresultsimmune significantly associated with the prognosis ofosteosarcoma patients osteosarcoma patients were included in the presentstudy including males and females the immunescore of the cohort range from ˆ’ to tostudy the relationship between the immune score and theprognosis of osteosarcoma patients patients wereincorporated into the lowimmune score group while theremaining patients were incorporated into the highimmune score group the survival analysis indicated thatpatients with higher immune score had a favorable osand dfs fig 1a and b after adjusted age tumor siteand metastatic status the immune score still was a prognostic variable for both os and dfsfig 1a and b inaddition the relationship between immune score and clinical features was also investigated however there was nosignificant relationship between immune score and clinicalvariables supplementary figure 1a1cdifferential expression analysisaccording to the median of the immune score patients were divided into highscore n and lowfig association between immune score and prognosis in osteosarcoma patients a kaplanmeier survival analysis of overall survival for patientswith high vs low immune score b kaplanmeier survival analysis of diseasefree survival for patients with high vs low immune score 0chu bmc cancer page of score group n there were differentiallyexpressed genes between two groups which include upregulated genes and downregulated genesfig 2a b and supplementary table to furtherunderstand the function of degs go analysisand kegg analysis were performed the top significant results of go analysis among three types wereillustrated in fig 2c interestingly we can find that theresults of go analysis are mostly associated with immunity which further verify that the immunerelated degsare associated with immune features in addition the results of kegg also confirmed it such as œphagosomeœautoimmune thyroid disease œantigen processing andpresentation œb cell receptor signaling pathway œintestinal immune network for iga production œinflammatorybowel disease œprimary immunodeficiency œth1 andth2 cell differentiation œth17 cell differentiation œnatural killer cell mediated cytotoxicity and œnfˆ’kappa bsignaling pathway fig 2dconsensusunsupervisedevaluation of degs and immune cellsto further understand the molecular heterogeneity ofosteosarcomaanalysis wasperformed to divide patients into subgroups to explorewhether immunerelated genes presented discernable patterns based on the consensus matrix heat map patientswere clearly divided into two clustersfig 3a in additionby comprehensively analyzing the relative change in areaunder the cumulative distribution function two clusterswere determined fig 3bc the immune score betweentwo clusters was significantly different fig 3d in additionthe proportion of types of immune cells in osteosarcomapatients was illustrated in a barplot fig 3e interestinglywe can see that the t cells cd4 memory activated ofcluster is significantly higher than cluster fig 5fprognostic value of tmerelated genesprevious studies indicated that tmerelated genes canserve as the prognostic biomarker for tumor patientsfig differentially expressed genes with the immune score in osteosarcoma patients a heatmap of significantly differentially expressed genesbased on immune score b the volcano figure to show the upregulated and downregulated genes c go analysis of differentially expressedgenes d kegg of differentially expressed genes go gene ontology kegg kyoto encyclopedia of genes and genomes 0chu bmc cancer page of fig the immune landscape of the tumor microenvironment ac unsupervised clustering of all samples based on the overlapping degs dcomparison of immune score between two clusters e the distribution of types of immune cells in osteosarcoma patients f the comparisonof types of immune cells between clusters deg differentially expressed genehence we performed the univariate cox analysis toidentify prognostic degs the results showed that and genes were identified as os and dfsrelateddegs respectively supplementary table and afterward five osrelated genes were successfully validated inthe gse21257 data set and five dfsrelated genes were successfully validated in the gse39055 cohort furthermoremultivariate cox analysis was performed and two prognostic signatures were generated for predicting the os anddfs respectively the risk score for predicting the os wasasrisk score fcgr2b0766 gfap0702 mpp70387 in addition the risk score for predicting thedfs was as follows risk score cyp2s10574 icam3 the auc values of osrelated signature were follows 0chu bmc cancer page of and in and 3year respectively fig 4aand the auc values of dfsrelated signature were and in and 3year respectively fig 5amoreover survival curves showed that patients in the highrisk group had worse os and dfs compared with the lowrisk patients figs 4b and 5b heat maps risk score plotsand survival status were generated to show the distinctionbetween highrisk patients and lowrisk patients figs 4ceand 5ce then both signatures were validated in independent cohorts for os signature the auc values ofvalidation cohort were and at and3year fig 4f for dfs signature the auc values ofvalidation cohort were and at and3year fig 5f additionallyin both validation cohortssurvival curves showed that lowrisk patients were favorableprognosis than highrisk patients figs 4g and 5gheat maps risk score plots and survival status of validation cohorts were also generated to show the distinction between highrisk patients and lowrisk patientsfigs 4hj and f 5hjdevelopment of a nomogram for osteosarcoma patientsto generate a nomogram for clinical use the cox analysiswas performed to select the clinical prognostic variables infig establishment and validation of the prognostic model for overall survival based on significant degs a receiver operating characteristiccurves of prognostic signature in the training cohort b the survival curve showed the different overall survival status between high and lowriskpatients c the heat map showed the expression of prognostic genes in the training cohort d the risk curve of each sample reordered by riskscore e the scatter plot showed the overall survival status of osteosarcoma patients in the training cohort f receiver operating characteristiccurves of prognostic signature in validation cohort g the survival curve showed the different overall survival status between high and lowriskpatients h the heat map showed the expression of prognostic genes in the validation cohort i the risk curve of each sample reordered by riskscore j the scatter plot showed the overall survival status of osteosarcoma patients in the validation cohort 0chu bmc cancer page of fig establishment and validation of the prognostic model for diseasefree survival based on significant degs a receiver operatingcharacteristic curves of prognostic signature in the training cohort b the survival curve showed the different diseasefree status between highand lowrisk patients c the heat map showed the expression of prognostic genes in the training cohort d the risk curve of each samplereordered by risk score e the scatter plot showed the diseasefree status of osteosarcoma patients in the training cohort f receiver operatingcharacteristic curves of prognostic signature in validation cohort g the survival curve showed the different diseasefree status between high andlowrisk patients h the heat map showed the expression of prognostic genes in the validation cohort i the risk curve of each sample reorderedby risk score j the scatter plot showed the diseasefree status of osteosarcoma patients in the validation cohortthe univariate cox analysis risk score and metastatic status were identified as both os and dfsrelated variablesfig 6a and e afterward risk score and metastatic statuswere determined as both independent os and dfsrelated variables in the multivariate cox analysis fig 6band f based on independent variables two nomogramswere established for predicting the os and dfs in osteosarcoma patients respectively fig 6c and g the cindexvalues were and in os nomogram and dfsnomogram respectively the results of cindex mean thatboth two nomograms have good discrimination meanwhile to evaluate the calibration of nomograms six calibration curves were generated and the results showed thatthe predictive curves were close to the ideal curve fig 6dand h which indicated a good calibrationdiscussionthe relationship between tme and tumor have beenwidely studied in recent years in the present study estimate algorithm was utilized to quantify the immunescore based on gene expression profiles in osteosarcomapatients from target database we confirmed that thetme is significantly associated with the prognosis ofosteosarcoma patientsinadditionfunctional enrichment analyses of tmerelated genes indicated that immunerelated processesincluding os and dfs 0chu bmc cancer page of fig nomograms based on the tumor microenvironment related genes for osteosarcoma patients a univariate cox analysis of overall survivalrelated variables b multivariate cox analysis of overall survivalrelated variables c nomogram for predicting the overall survival in osteosarcomapatients d1 and 3year calibration curves of overall survival nomogram e univariate cox analysis of diseasefree survivalrelated variables fmultivariate cox analysis of diseasefree survivalrelated variables g nomogram for predicting the diseasefree survival in osteosarcoma patientsh1 and 3year calibration curves of diseasefree survival nomogramknown to contribute to tumor progression more importantly degs based on the tme were identified asimportant prognostic biomarkers for osteosarcoma patients and two nomograms were developed for predicting the os and dfs of osteosarcoma patientsrespectivelyin recent years an increasing number of studiesfocused on the carcinogenesis and progression of tumorsbased on the tme and the estimate algorithm is oneof the most important quantitative tools for this researchfield based on the estimate algorithm the association between the prognosis and tme has been initially 0chu bmc cancer page of elucidated in some tumors such as cervical squamouscell carcinoma gastric cancer cutaneous melanomaacute myeloid leukemia bladder cancer and clear cellrenal carcinoma [ “] however previousstudies indicated that tme scores serve as a differentrole in different tumors for example for hepatocellularcarcinoma gastric cancer acute myeloid leukemiabladder cancer and clear cell renal carcinoma patientswith high immune score have a worse prognosis [ “] however for cervical squamous cell carcinoma adrenocortical carcinoma and cutaneous melanoma patients with high immune score have a favorableprognosis [ ] therefore we can find great heterogeneity among different tumors from the perspectiveof tme for osteosarcoma patients the present studyindicated that patients with higher immune score had abetter os and dfs hence the present study indicatedthat immune cells infiltrating tumor tissue may play animportant role in suppressing tumor progressionin our research tmerelated genes were identified by comparing the highscore and lowscore osteosarcoma patients the functional enrichment includinggo and kegg analyses showed that tmerelated geneswere mainly involved in the immune features such asregulation of leukocyte activation mhc protein complex mhc protein and complex binding more importantly the unsupervised cluster analysis based on degswas performed and all patients were divided into twoclusters immune score and t cell cd4 memory activated fraction were significant difference between twoclusters which further elucidated the relationship between degs and immune featuresdue to the poor prognosis of osteosarcoma patientsidentifying robust prognostic biomarker is very importantthe tumor immune microenvironment is closely relatedto the prognosis of bone tumor patients emilie etal performed the first genomewide study to describe therole of immune cells in osteosarcoma and found thattumorassociated macrophages are associated with reduced metastasis and improved survivalin highgradeosteosarcoma recently the prognostic signature based ontmerelated genes have been established for many tumors [ ] but only one study focused on osteosarcoma patients compared with the study performedby zhang we think that our research have someadvantages firstly our signatures were established basedon several validated genes and both two signatures weresuccessfully validated in independent cohorts secondlythe outcome of dfs was not reported in the previousstudy as reported in published studies tumor recurrenceis a terrible medical problem for osteosarcoma patientsand the 5year survival rate for osteosarcoma patients withmetastasis or relapse remains disappointing [ ]hence the dfs nomogram can improve the managementof osteosarcoma patients finally two nomograms incorporated tmerelated signature and clinical variables wereestablished in our research which further facilitated theclinical application of our findingsin our research five genes were incorporated into thefinal prognostic signatures fcgr2b gfap and mpp7were identified and validated as osrelated biomarkerswhile cyp2s1 and icam3 were dfsrelated biomarkersthe role of these genes in tumor prognosis had beenwidely reported in previous studies [“] fcgr2bhas been confirmed as an immunerelated gene previously although the relationship between fcgr2band prognosis in sarcoma patients had not been reported the prognostic value of fcgr2b had been widelyconfirmed in other cancerssuch as hepatocellularcarcinoma and glioblastoma [ ] in addition newm etal demonstrated that mpp7 is novel regulatorsof autophagy which was thought to be responsible forthe prognosis of pancreatic ductal adenocarcinomacyp2s1 described as cytochrome p450 family subfamily s member was reported significantly associatedwith colorectal cancer in primary colorectal cancercyp2s1 was present at a significantly higher level ofintensity compared with normal colon more importantly the presence of strong cyp2s1 immunoreactivity was associated with poor prognosis the roleof icam3 in cancer was also widely reported in published studies and the akt pathway plays an importantrole in the impact of icam3 on tumors yg kim etal reported that icam3 can induce the proliferationof cancer cells through the pi3kakt pathway additionally jk park etal showed that the icam3 can enhancethe migratory and invasive potential of human nonsmall celllung cancer cells by inducing mmp2 andmmp9 via akt pathway showed that the icam3can enhance the migratory and invasive potential ofhuman nonsmall celllung cancer cells by inducingmmp2 and mmp9 via akt pathwayalthough the role of tme and tmerelated genes inosteosarcoma patients have been initially studied by bioinformatic and statistical analyses in our research somelimitations should be elucidated firstly the treatmentinformation cannot be obtained from the target database which may influence the prognosis of osteosarcomapatients secondly two nomograms were generated andshowed good performance in our study however externalvalidation by a large cohort is needed thirdly many independent prognostic genes for osteosarcoma patients wereidentified in the present study but the potential mechanism to influence osteosarcoma remains unclear finally inthe training cohort and degs were identified asos and dfsrelated degs respectively however onlyfive os and five dfsrelated genes were identified in thevalidation cohort the different age structures smaller 0chu bmc cancer page of sample sizes and the platform covering only part of thegenes may contribute to this resultreceived february accepted july in tme plays an important role in osteosarcoma patients and related with the progression of thetumor moreover tmerelated genes can serve as prognostic biomarkers in osteosarcoma patients howeverfurther researches are needed to study the potentialmechanism and validate the nomogram that developedin our present studysupplementary informationsupplementary information accompanies this paper at doi101186s12885020072162additional file additional file additional file additional file abbreviationstme tumor microenvironment deg differentially expressed genesos overall survival dfs diseasesfree survival roc receiver characteristiccurve estimate estimation of stromal and immune cells in malignanttumor tissues using expression data target therapeutically applicableresearch to generate effective treatments go gene ontology bp biologicalprocesses mf molecular functions cc cellular components kegg kyotoencyclopedia of genes and genomes cdf cumulative distribution functionacknowledgementsnoneauthors™ contributionsc h l y sq t c l and yh w conceived of and designed the study c h r sand c l performed literature search r s l y and b c generated the figuresand tables l y hl r x y and jy l analyzed the data c h wrote themanuscript and sq t and l y critically reviewed the manuscript l ysupervised the research all authors have read and approved the manuscriptfundingwe received no external funding for this studyavailability of data and materialsthe data of this study are from target and geo databaseethics approval and consent to participatethe research didn™t involve animal experiments and human specimens noethics related issuesconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of joint surgery the affiliated hospital of qingdao universityqingdao china 2department of medical oncology the first hospital ofchina medical university shenyang china 3department of nursing sir runrun shaw hospital affiliated to zhejiang university hangzhou china4wenzhou medical university wenzhou chinareferencesjaffe n bruland os bielack s pediatric and adolescent osteosarcoma vol new york springer science business media vander rg osteosarcoma and its variants orthopedic clin north am “biermann js adkins d benjamin r brigman b chow w conrad eu 3rdfrassica d frassica fj gee s healey jh bone cancer j natl comprcancer netw “simpson s dunning md de brot s grauroma l mongan np rutland cscomparative review of human and canine osteosarcoma morphologyepidemiology prognosis treatment and genetics acta vet scand chen x cates jm du yc jain a jung sy li xn hicks jm man tkmislocalized cytoplasmic p27 activates pak1mediated metastasis and is aprognostic factor in osteosarcoma mol oncol “huang x yang w zhang z shao z dysregulated circrnas serve as prognosticand diagnostic markers in osteosarcoma by sponging microrna to regulatethe downstream signaling pathway j cell biochem “liu m yang p mao g deng j peng g ning x yang h sun h long noncoding rna malat1 as a valuable biomarker for prognosis in osteosarcoma asystematic review and metaanalysis int j surg “xu k xiong w zhao s wang b microrna106b serves as a prognosticbiomarker and is associated with cell proliferation migration and invasionin osteosarcoma oncol lett “zheng w huang y chen h wang n xiao w liang y jiang x su w wens nomogram application to predict overall and cancerspecific survival inosteosarcoma cancer manag res kahlert c kalluri r exosomes in tumor microenvironment influence cancerprogression and metastasis j mol med “ binnewies m roberts ew kersten k chan v fearon df merad m coussenslm gabrilovich di ostrandrosenberg s hedrick cc understanding thetumor immune microenvironment time for effective therapy nat med“ yoshihara k shahmoradgoli m martínez e vegesna r kim h torresgarcia wtreviño v shen h laird pw levine da inferring tumour purity and stromaland immune cell admixture from expression data nat commun yang s liu t nan h wang y chen h zhang x zhang y shen b qian pxu s comprehensive analysis of prognostic immunerelated genes inthe tumor microenvironment of cutaneous melanoma j cell physiol “ deng z wang j xu b jin z wu g zeng j peng m guo y wen z miningtcga database for tumor microenvironmentrelated genes of prognosticvalue in hepatocellular carcinoma biomed res int zhao k yang h kang h wu a identification of key genes in thyroid cancermicroenvironment med sci monit xu wh xu y wang j wan fn wang hk cao dl shi gh qu yyzhang hl ye dw prognostic value and immune infiltration of novelsignatures in clear cell renal cell carcinoma microenvironment agingalbany ny chen b chen w jin j wang x cao y he y data mining of prognosticmicroenvironmentrelated genes in clear cell renal cell carcinoma a studywith tcga database dis markers li x gao y xu z zhang z zheng y qi f identification of prognostic genesin adrenocortical carcinoma microenvironment based on bioinformaticmethods cancer med “ pan xb lu y huang jl long y yao ds prognostic genes in the tumormicroenvironment in cervical squamous cell carcinoma aging albany ny wang h wu x chen y stromalimmune scorebased gene signature aprognosis stratification tool in gastric cancer front oncol huang s zhang b fan w zhao q yang l xin w fu d identification ofprognostic genes in the acute myeloid leukemia microenvironment agingalbany ny yan h qu j cao w liu y zheng g zhang e cai z identification ofprognostic genes in the acute myeloid leukemia immunemicroenvironment based on tcga data a
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" tumor microenvironment tme plays an important role in malignant tumors our study aimed toinvestigate the effect of the tme and related genes in osteosarcoma patientsmethods gene expression profiles and clinical data of osteosarcoma patients were downloaded from the targetdataset estimate algorithm was used to quantify the immune score then the association between immune scoreand prognosis was studied afterward a differential analysis was performed based on the high and lowimmunescores to determine tmerelated genes additionally cox analyses were performed to construct two prognosticsignatures for overall survival os and diseasefree survival dfs respectively two datasets obtained from the geodatabase were used to validate signaturesresults eightyfive patients were included in our research the survival analysis indicated that patients with higherimmune score have a favorable os and dfs moreover genes were determined as tmerelated genes theunsupervised clustering analysis revealed two clusters were significantly related to immune score and t cells cd4memory fraction in addition two signatures were generated based on three and two tmerelated genesrespectively both two signatures can significantly divide patients into low and highrisk groups and were validatedin two geo datasets afterward the risk score and metastatic status were identified as independent prognosticfactors for both os and dfs and two nomograms were generated the cindexes of os nomogram and dfsnomogram were and respectively tme was associated with the prognosis of osteosarcoma patients prognostic models based on tmerelated genes can effectively predict os and dfs of osteosarcoma patientskeywords tumor microenvironment osteosarcoma prognosis immune features nomogram osteosarcoma is the most common bone tumor especiallyin children and adolescents it was reported that approximately of patients are between and yearsold and osteosarcoma is considered as the second leadingcause of death in this age group currently surgery and correspondence 407404159qqcom4wenzhou medical university wenzhou chinafull list of author information is available at the end of the chemotherapy are still major treatments for osteosarcomapatients and these therapies are constantly improving inrecent years however due to the susceptibility of localaggressiveness and lung metastasis in osteosarcoma patients the prognosis of osteosarcoma remains unfavorable previous studies indicated that the 5years survivalrates were and in metastatic and nonmetastaticpatients respectively thereforeit is necessary to the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0chu bmc cancer page of investigate the mechanism of pathogenesis and progressionof osteosarcoma and accurately classify the risk of patientsrecently an increasing number of diagnostic and prognostic biomarkers of osteosarcoma patients have beenidentified for example chen reported that tumorsuppressor p27 is a novel biomarker for the metastasis andsurvival status in osteosarcoma patients moreover huang discovered that dysregulated circrnas serve asprognostic and diagnostic biomarkers in osteosarcomapatients and the relative potential mechanism mainly attributes to the regulation of downstream signaling pathwaysby sponging microrna in addition lncrna microrna and many clinical data were also identified asprognostic biomarkers for osteosarcoma patients however osteosarcoma is one of the malignant cancers entitiescharacterized by the high level of heterogeneity in humanstherefore it is necessary to find accurate biomarkers forosteosarcomain recent years researchers have paid more and moreattention to the role of the tumor microenvironmenttme in malignant tumors the function of tme inthe tumorigenesis progression and therapy of tumorshave been initially understood [ ] more importantly estimation of stromal and immune cells in malignant tumor tissues using expression data estimate an algorithm to quantify the score of immune cellsand stromal cells by analyzing the gene expression datawas developed in based on the algorithm theprognostic value of immune and stromal cells in bladdercancer acute myeloid leukemia gastric cancer cervicalsquamous cell carcinoma adrenocortical carcinomaclear cell renal cell carcinoma hepatocellular carcinomathyroid cancer and cutaneous melanoma have beenreported [“] generally the above research indicatedthat tme can serve as the prognostic biomarker in tumorsand many tmerelated genes were determined as the prognostic genes however the role of tme and tmerelatedgenes in osteosarcoma patients remains unclearin the present study gene expression data and corresponding clinicopathologic data were obtained from thetherapeutically applicable research to generate effectivetreatments target dataset then the estimatealgorithm was performed to quantify the immune score ofosteosarcoma and the tmerelated genes were identifiedby the differential expression analysis subsequently theprognostic value of tme and tmerelated genes weredetermined by a series of bioinformatics methodsmethodsgene expression datasetslevel data of gene expression profiles and correspondingclinical data of osteosarcoma patients were downloadedfrom target dataset ocgcancergovprogramstarget accessed on oct the correspondingclinicopathologic data included in the present study wereage gender race ethnicity tumor site and metastaticstatus after data were extracted from the public domainthe estimate an algorithm inferring tumor puritystromal score and immune cell admixture from expression data was performed to evaluate the immune score byusing the estimate package in r software version meanwhile the messenger rnamrna expressionprofiles and clinical data ofincludinggse21257 and gse39055 were obtained fromthe gene expression omnibus as external validationcohortstwo cohortssurvival analysis and correlation analysisafter scores were obtained patients were divided intohighscore group and lowscore group according to themedian of the immune score the kaplanmeier survivalanalysis with logrank test was performed to estimatethe differences of overall survival os and diseasefreesurvival dfs between high and lowscore cohorts inaddition the association between clinicopathologic dataand tme score was also studied mannwhitney signedrank test was performed to compare the differences ofimmune score between each clinical group all statisticalanalyses in the present study were performed using rsoftware except for the special instructions p value twoside was identified as statistically significantin the present studydegexpressed genedifferentially expressed gene analysisdifferentiallyanalysis wasperformed by comparing the proteincoding genesexpression between the lowimmune score group andthe highimmune score group the limma package in rsoftware was used to perform the differential analysisand genes with log fc and adjusted pvalue qvalue were identified as degs to further understand the function of degs identifiedin the present study gene ontology goincludingbiological processes bp molecular functions mf andcellular componentscc and kyoto encyclopedia ofgenes and genomes kegg analysis were performedby clusterprofiler package in r software evaluation of association with immune cellsto further investigate the association between degs andimmune cells the cibersort package was used toestimate the relative proportions of types of immunecells meanwhile the œconsensusclusterplus package was used to cluster in an unbiased and unsupervisedmanner based on the overlapping degs cumulative distribution function cdf and relative change inarea under the cdf curve were used to determine theoptimal number of clusters k then mannwhitney 0chu bmc cancer page of signedrank test was performed to study the differenceof immune cells proportion between the clusters and theviolin plot was established to show the differences ofimmune cells among clusters survival analysis of degsbased on the degs the univariate cox analysis was performed to determine the prognostic value of immunerelated genes then the osrelated genes were validatedin the gse21257 dataset while the dfsrelated geneswere validated in the gse39055 dataset only genes successfully validated were selected for further analysis afterward based on the validated genes the multivariate coxanalysis was performed to establish the prognostic signature for predicting the prognosis of osteosarcoma patientsthe risk score for each patient was calculated based onthe coefficient from the multivariate cox analysis and thecorresponding gene expression meanwhile all patientswere divided into the high and lowrisk groups accordingto the median of the risk score the survival receiver operating characteristic roc curve was used to show the discrimination of signatures and the kaplanmeier survivalcurve with the logrank test was generated to show thedifferences of os and dfs between high and lowriskgroups in addition the risk score of patients in the validation cohort was also calculated according to the aforementioned risk signature the kaplanmeier survivalcurve and survival roc curve were generated to show thepredictive ability of the signature in the validation cohortdevelopment of a nomogram for osteosarcoma patientsnomogram is a tool to visualize the predictive model andconvenient for clinical practice therefore we attemptedto develop a nomogram based on the tmerelated genessignature and clinicopathologic data to predict the prognosis of osteosarcoma patients firstlythe univariatecox analysis was performed to filter prognostic variableswhich will be further included in the multivariate coxanalysis secondly based on independent prognostic variables two nomograms were established for predicting theos and dfs respectively the cindex was used to assessthe discriminatory performance of the nomogram whichrange from to a cindex of means agreement by chance and a cindex of represents perfectdiscriminatory performance the higher value of the cindex the better performance of the nomogram is furthermore the calibration curves of and 3year weredeveloped to evaluate the effectiveness of nomogramsresultsimmune significantly associated with the prognosis ofosteosarcoma patients osteosarcoma patients were included in the presentstudy including males and females the immunescore of the cohort range from ˆ’ to tostudy the relationship between the immune score and theprognosis of osteosarcoma patients patients wereincorporated into the lowimmune score group while theremaining patients were incorporated into the highimmune score group the survival analysis indicated thatpatients with higher immune score had a favorable osand dfs fig 1a and b after adjusted age tumor siteand metastatic status the immune score still was a prognostic variable for both os and dfsfig 1a and b inaddition the relationship between immune score and clinical features was also investigated however there was nosignificant relationship between immune score and clinicalvariables supplementary figure 1a1cdifferential expression analysisaccording to the median of the immune score patients were divided into highscore n and lowfig association between immune score and prognosis in osteosarcoma patients a kaplanmeier survival analysis of overall survival for patientswith high vs low immune score b kaplanmeier survival analysis of diseasefree survival for patients with high vs low immune score 0chu bmc cancer page of score group n there were differentiallyexpressed genes between two groups which include upregulated genes and downregulated genesfig 2a b and supplementary table to furtherunderstand the function of degs go analysisand kegg analysis were performed the top significant results of go analysis among three types wereillustrated in fig 2c interestingly we can find that theresults of go analysis are mostly associated with immunity which further verify that the immunerelated degsare associated with immune features in addition the results of kegg also confirmed it such as œphagosomeœautoimmune thyroid disease œantigen processing andpresentation œb cell receptor signaling pathway œintestinal immune network for iga production œinflammatorybowel disease œprimary immunodeficiency œth1 andth2 cell differentiation œth17 cell differentiation œnatural killer cell mediated cytotoxicity and œnfˆ’kappa bsignaling pathway fig 2dconsensusunsupervisedevaluation of degs and immune cellsto further understand the molecular heterogeneity ofosteosarcomaanalysis wasperformed to divide patients into subgroups to explorewhether immunerelated genes presented discernable patterns based on the consensus matrix heat map patientswere clearly divided into two clustersfig 3a in additionby comprehensively analyzing the relative change in areaunder the cumulative distribution function two clusterswere determined fig 3bc the immune score betweentwo clusters was significantly different fig 3d in additionthe proportion of types of immune cells in osteosarcomapatients was illustrated in a barplot fig 3e interestinglywe can see that the t cells cd4 memory activated ofcluster is significantly higher than cluster fig 5fprognostic value of tmerelated genesprevious studies indicated that tmerelated genes canserve as the prognostic biomarker for tumor patientsfig differentially expressed genes with the immune score in osteosarcoma patients a heatmap of significantly differentially expressed genesbased on immune score b the volcano figure to show the upregulated and downregulated genes c go analysis of differentially expressedgenes d kegg of differentially expressed genes go gene ontology kegg kyoto encyclopedia of genes and genomes 0chu bmc cancer page of fig the immune landscape of the tumor microenvironment ac unsupervised clustering of all samples based on the overlapping degs dcomparison of immune score between two clusters e the distribution of types of immune cells in osteosarcoma patients f the comparisonof types of immune cells between clusters deg differentially expressed genehence we performed the univariate cox analysis toidentify prognostic degs the results showed that and genes were identified as os and dfsrelateddegs respectively supplementary table and afterward five osrelated genes were successfully validated inthe gse21257 data set and five dfsrelated genes were successfully validated in the gse39055 cohort furthermoremultivariate cox analysis was performed and two prognostic signatures were generated for predicting the os anddfs respectively the risk score for predicting the os wasasrisk score fcgr2b0766 gfap0702 mpp70387 in addition the risk score for predicting thedfs was as follows risk score cyp2s10574 icam3 the auc values of osrelated signature were follows 0chu bmc cancer page of and in and 3year respectively fig 4aand the auc values of dfsrelated signature were and in and 3year respectively fig 5amoreover survival curves showed that patients in the highrisk group had worse os and dfs compared with the lowrisk patients figs 4b and 5b heat maps risk score plotsand survival status were generated to show the distinctionbetween highrisk patients and lowrisk patients figs 4ceand 5ce then both signatures were validated in independent cohorts for os signature the auc values ofvalidation cohort were and at and3year fig 4f for dfs signature the auc values ofvalidation cohort were and at and3year fig 5f additionallyin both validation cohortssurvival curves showed that lowrisk patients were favorableprognosis than highrisk patients figs 4g and 5gheat maps risk score plots and survival status of validation cohorts were also generated to show the distinction between highrisk patients and lowrisk patientsfigs 4hj and f 5hjdevelopment of a nomogram for osteosarcoma patientsto generate a nomogram for clinical use the cox analysiswas performed to select the clinical prognostic variables infig establishment and validation of the prognostic model for overall survival based on significant degs a receiver operating characteristiccurves of prognostic signature in the training cohort b the survival curve showed the different overall survival status between high and lowriskpatients c the heat map showed the expression of prognostic genes in the training cohort d the risk curve of each sample reordered by riskscore e the scatter plot showed the overall survival status of osteosarcoma patients in the training cohort f receiver operating characteristiccurves of prognostic signature in validation cohort g the survival curve showed the different overall survival status between high and lowriskpatients h the heat map showed the expression of prognostic genes in the validation cohort i the risk curve of each sample reordered by riskscore j the scatter plot showed the overall survival status of osteosarcoma patients in the validation cohort 0chu bmc cancer page of fig establishment and validation of the prognostic model for diseasefree survival based on significant degs a receiver operatingcharacteristic curves of prognostic signature in the training cohort b the survival curve showed the different diseasefree status between highand lowrisk patients c the heat map showed the expression of prognostic genes in the training cohort d the risk curve of each samplereordered by risk score e the scatter plot showed the diseasefree status of osteosarcoma patients in the training cohort f receiver operatingcharacteristic curves of prognostic signature in validation cohort g the survival curve showed the different diseasefree status between high andlowrisk patients h the heat map showed the expression of prognostic genes in the validation cohort i the risk curve of each sample reorderedby risk score j the scatter plot showed the diseasefree status of osteosarcoma patients in the validation cohortthe univariate cox analysis risk score and metastatic status were identified as both os and dfsrelated variablesfig 6a and e afterward risk score and metastatic statuswere determined as both independent os and dfsrelated variables in the multivariate cox analysis fig 6band f based on independent variables two nomogramswere established for predicting the os and dfs in osteosarcoma patients respectively fig 6c and g the cindexvalues were and in os nomogram and dfsnomogram respectively the results of cindex mean thatboth two nomograms have good discrimination meanwhile to evaluate the calibration of nomograms six calibration curves were generated and the results showed thatthe predictive curves were close to the ideal curve fig 6dand h which indicated a good calibrationdiscussionthe relationship between tme and tumor have beenwidely studied in recent years in the present study estimate algorithm was utilized to quantify the immunescore based on gene expression profiles in osteosarcomapatients from target database we confirmed that thetme is significantly associated with the prognosis ofosteosarcoma patientsinadditionfunctional enrichment analyses of tmerelated genes indicated that immunerelated processesincluding os and dfs 0chu bmc cancer page of fig nomograms based on the tumor microenvironment related genes for osteosarcoma patients a univariate cox analysis of overall survivalrelated variables b multivariate cox analysis of overall survivalrelated variables c nomogram for predicting the overall survival in osteosarcomapatients d1 and 3year calibration curves of overall survival nomogram e univariate cox analysis of diseasefree survivalrelated variables fmultivariate cox analysis of diseasefree survivalrelated variables g nomogram for predicting the diseasefree survival in osteosarcoma patientsh1 and 3year calibration curves of diseasefree survival nomogramknown to contribute to tumor progression more importantly degs based on the tme were identified asimportant prognostic biomarkers for osteosarcoma patients and two nomograms were developed for predicting the os and dfs of osteosarcoma patientsrespectivelyin recent years an increasing number of studiesfocused on the carcinogenesis and progression of tumorsbased on the tme and the estimate algorithm is oneof the most important quantitative tools for this researchfield based on the estimate algorithm the association between the prognosis and tme has been initially 0chu bmc cancer page of elucidated in some tumors such as cervical squamouscell carcinoma gastric cancer cutaneous melanomaacute myeloid leukemia bladder cancer and clear cellrenal carcinoma [ “] however previousstudies indicated that tme scores serve as a differentrole in different tumors for example for hepatocellularcarcinoma gastric cancer acute myeloid leukemiabladder cancer and clear cell renal carcinoma patientswith high immune score have a worse prognosis [ “] however for cervical squamous cell carcinoma adrenocortical carcinoma and cutaneous melanoma patients with high immune score have a favorableprognosis [ ] therefore we can find great heterogeneity among different tumors from the perspectiveof tme for osteosarcoma patients the present studyindicated that patients with higher immune score had abetter os and dfs hence the present study indicatedthat immune cells infiltrating tumor tissue may play animportant role in suppressing tumor progressionin our research tmerelated genes were identified by comparing the highscore and lowscore osteosarcoma patients the functional enrichment includinggo and kegg analyses showed that tmerelated geneswere mainly involved in the immune features such asregulation of leukocyte activation mhc protein complex mhc protein and complex binding more importantly the unsupervised cluster analysis based on degswas performed and all patients were divided into twoclusters immune score and t cell cd4 memory activated fraction were significant difference between twoclusters which further elucidated the relationship between degs and immune featuresdue to the poor prognosis of osteosarcoma patientsidentifying robust prognostic biomarker is very importantthe tumor immune microenvironment is closely relatedto the prognosis of bone tumor patients emilie etal performed the first genomewide study to describe therole of immune cells in osteosarcoma and found thattumorassociated macrophages are associated with reduced metastasis and improved survivalin highgradeosteosarcoma recently the prognostic signature based ontmerelated genes have been established for many tumors [ ] but only one study focused on osteosarcoma patients compared with the study performedby zhang we think that our research have someadvantages firstly our signatures were established basedon several validated genes and both two signatures weresuccessfully validated in independent cohorts secondlythe outcome of dfs was not reported in the previousstudy as reported in published studies tumor recurrenceis a terrible medical problem for osteosarcoma patientsand the 5year survival rate for osteosarcoma patients withmetastasis or relapse remains disappointing [ ]hence the dfs nomogram can improve the managementof osteosarcoma patients finally two nomograms incorporated tmerelated signature and clinical variables wereestablished in our research which further facilitated theclinical application of our findingsin our research five genes were incorporated into thefinal prognostic signatures fcgr2b gfap and mpp7were identified and validated as osrelated biomarkerswhile cyp2s1 and icam3 were dfsrelated biomarkersthe role of these genes in tumor prognosis had beenwidely reported in previous studies [“] fcgr2bhas been confirmed as an immunerelated gene previously although the relationship between fcgr2band prognosis in sarcoma patients had not been reported the prognostic value of fcgr2b had been widelyconfirmed in other cancerssuch as hepatocellularcarcinoma and glioblastoma [ ] in addition newm etal demonstrated that mpp7 is novel regulatorsof autophagy which was thought to be responsible forthe prognosis of pancreatic ductal adenocarcinomacyp2s1 described as cytochrome p450 family subfamily s member was reported significantly associatedwith colorectal cancer in primary colorectal cancercyp2s1 was present at a significantly higher level ofintensity compared with normal colon more importantly the presence of strong cyp2s1 immunoreactivity was associated with poor prognosis the roleof icam3 in cancer was also widely reported in published studies and the akt pathway plays an importantrole in the impact of icam3 on tumors yg kim etal reported that icam3 can induce the proliferationof cancer cells through the pi3kakt pathway additionally jk park etal showed that the icam3 can enhancethe migratory and invasive potential of human nonsmall celllung cancer cells by inducing mmp2 andmmp9 via akt pathway showed that the icam3can enhance the migratory and invasive potential ofhuman nonsmall celllung cancer cells by inducingmmp2 and mmp9 via akt pathwayalthough the role of tme and tmerelated genes inosteosarcoma patients have been initially studied by bioinformatic and statistical analyses in our research somelimitations should be elucidated firstly the treatmentinformation cannot be obtained from the target database which may influence the prognosis of osteosarcomapatients secondly two nomograms were generated andshowed good performance in our study however externalvalidation by a large cohort is needed thirdly many independent prognostic genes for osteosarcoma patients wereidentified in the present study but the potential mechanism to influence osteosarcoma remains unclear finally inthe training cohort and degs were identified asos and dfsrelated degs respectively however onlyfive os and five dfsrelated genes were identified in thevalidation cohort the different age structures smaller 0chu bmc cancer page of sample sizes and the platform covering only part of thegenes may contribute to this resultreceived february accepted july in tme plays an important role in osteosarcoma patients and related with the progression of thetumor moreover tmerelated genes can serve as prognostic biomarkers in osteosarcoma patients howeverfurther researches are needed to study the potentialmechanism and validate the nomogram that developedin our present studysupplementary informationsupplementary information accompanies this paper at doi101186s12885020072162additional file additional file additional file additional file abbreviationstme tumor microenvironment deg differentially expressed genesos overall survival dfs diseasesfree survival roc receiver characteristiccurve estimate estimation of stromal and immune cells in malignanttumor tissues using expression data target therapeutically applicableresearch to generate effective treatments go gene ontology bp biologicalprocesses mf molecular functions cc cellular components kegg kyotoencyclopedia of genes and genomes cdf cumulative distribution functionacknowledgementsnoneauthors™ contributionsc h l y sq t c l and yh w conceived of and designed the study c h r sand c l performed literature search r s l y and b c generated the figuresand tables l y hl r x y and jy l analyzed the data c h wrote themanuscript and sq t and l y critically reviewed the manuscript l ysupervised the research all authors have read and approved the manuscriptfundingwe received no external funding for this studyavailability of data and materialsthe data of this study are from target and geo databaseethics approval and consent to participatethe research didn™t involve animal experiments and human specimens noethics related issuesconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of joint surgery the affiliated hospital of qingdao universityqingdao china 2department of medical oncology the first hospital ofchina medical university shenyang china 3department of nursing sir runrun shaw hospital affiliated to zhejiang university hangzhou china4wenzhou medical university wenzhou chinareferencesjaffe n bruland os bielack s pediatric and adolescent osteosarcoma vol new york springer science business media vander rg osteosarcoma and its variants orthopedic clin north am “biermann js adkins d benjamin r brigman b chow w conrad eu 3rdfrassica d frassica fj gee s healey jh bone cancer j natl comprcancer netw “simpson s dunning md de brot s grauroma l mongan np rutland cscomparative review of human and canine osteosarcoma morphologyepidemiology prognosis treatment and genetics acta vet scand chen x cates jm du yc jain a jung sy li xn hicks jm man tkmislocalized cytoplasmic p27 activates pak1mediated metastasis and is aprognostic factor in osteosarcoma mol oncol “huang x yang w zhang z shao z dysregulated circrnas serve as prognosticand diagnostic markers in osteosarcoma by sponging microrna to regulatethe downstream signaling pathway j cell biochem “liu m yang p mao g deng j peng g ning x yang h sun h long noncoding rna malat1 as a valuable biomarker for prognosis in osteosarcoma asystematic review and metaanalysis int j surg “xu k xiong w zhao s wang b microrna106b serves as a prognosticbiomarker and is associated with cell proliferation migration and invasionin osteosarcoma oncol lett “zheng w huang y chen h wang n xiao w liang y jiang x su w wens nomogram application to predict overall and cancerspecific survival inosteosarcoma cancer manag res kahlert c kalluri r exosomes in tumor microenvironment influence cancerprogression and metastasis j mol med “ binnewies m roberts ew kersten k chan v fearon df merad m coussenslm gabrilovich di ostrandrosenberg s hedrick cc understanding thetumor immune microenvironment time for effective therapy nat med“ yoshihara k shahmoradgoli m martínez e vegesna r kim h torresgarcia wtreviño v shen h laird pw levine da inferring tumour purity and stromaland immune cell admixture from expression data nat commun yang s liu t nan h wang y chen h zhang x zhang y shen b qian pxu s comprehensive analysis of prognostic immunerelated genes inthe tumor microenvironment of cutaneous melanoma j cell physiol “ deng z wang j xu b jin z wu g zeng j peng m guo y wen z miningtcga database for tumor microenvironmentrelated genes of prognosticvalue in hepatocellular carcinoma biomed res int zhao k yang h kang h wu a identification of key genes in thyroid cancermicroenvironment med sci monit xu wh xu y wang j wan fn wang hk cao dl shi gh qu yyzhang hl ye dw prognostic value and immune infiltration of novelsignatures in clear cell renal cell carcinoma microenvironment agingalbany ny chen b chen w jin j wang x cao y he y data mining of prognosticmicroenvironmentrelated genes in clear cell renal cell carcinoma a studywith tcga database dis markers li x gao y xu z zhang z zheng y qi f identification of prognostic genesin adrenocortical carcinoma microenvironment based on bioinformaticmethods cancer med “ pan xb lu y huang jl long y yao ds prognostic genes in the tumormicroenvironment in cervical squamous cell carcinoma aging albany ny wang h wu x chen y stromalimmune scorebased gene signature aprognosis stratification tool in gastric cancer front oncol huang s zhang b fan w zhao q yang l xin w fu d identification ofprognostic genes in the acute myeloid leukemia microenvironment agingalbany ny yan h qu j cao w liu y zheng g zhang e cai z identification ofprognostic genes in the acute myeloid leukemia immunemicroenvironment based on tcga data a
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Mukonal is an active member of carbazole alkaloids isolated from Murraya koenigii It has been shown to possess remarkable biological and pharmacological activities including anticancer activity Therefore the aim of current investigation was to explore antibreast cancer activity of mukonal and to explore the underlying mechanism Results indicate that mukonal has potential to induce antiproliferative effects against MDAMB231 and SKBR3 cells with an IC50 of µM No significant toxicity of mukonal was observed in case of normal breast cells The antiproliferative effects of mukonal were found to proceed via apoptosis which was further supported by increased cleavage of PARP and caspase3 and reduced expression of Bcl2 Mukonal induced autophagic cells death in breast cancer cells as was evidenced by formation of autophagosomes and enhanced expressions of Beclin1 LC3BI and LC3BII proteins In vivo examination of antibreast cancer property of mukonal indicated that it could potentially reduce tumor weight and volume in xenografted mice models In mukonal has a remarkable potential of inhibiting breast cancer via induction of apoptosis and autophagy Mukonal also inhibited xenografted tumors models in a dosedependent manner Therefore mukonal may prove lead molecule in breast cancer drug discovery and treatment provided further investigations are recommendedKeywords Breast cancer Alkaloids Carbazole alkaloids Mukonal Autophagy ApoptosisIntroductionBreast cancer is the most frequent and devastating disease prevailing in females worldwide Alone in the year of more than a0million deaths were recorded globally due to this lethal malignancy in women Sun et a0 al Every year approximately of all cancers over a0million diagnosed in women are breast cancer United States registered over of all cancers in women were due breast cancer in the year of Siegel et a0al Metastatic nature of breast cancer leads to easy transfer of disease to distant places in the body wherein it develops Correspondence wangcqy2020163comDepartment of Thyroid Breast Surgery Gong An County People™s Hospital Gong An Jingzhou Hubei Chinaindividually like brain lung liver and bones Early detection of the disease may lead with better overall survival chances and prognosis Screening of breast cancer is widely performed by mammography which has been proved fruitful in lowering the mortality rate effectually Several risk factors have been linked to enhance the possibility of breast cancer development like that of genetic mutations family history estrogen levels aging sex and poor lifestyle Majeed et a0al Primary development of breast cancer takes place from ductal hyperproliferation and then maturing into metastatic or benign tumors Thus far significant advances have been achieved in theoretical and clinical investigations of breast cancer The current strategies for effective management of breast cancer incorporate biologicalprevention chemoprevention The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 0cWang a0et a0al AMB Expr Page of and screening Smith and Henderson Besides recent advances and effective management breast cancer remains major cause of cancer related deaths in females of age “ a0 years Therefore there is a pressing need for novel and capable chemopreventive drugs that can assists us with better results in overcoming this disastrous malignancy Since time immemorial natural products have revealed health promoting effects in human beings Natural products are mostly the secondary metabolites synthesized by the plants to survive and adapt to harsh environmental conditions Williams et a0 al Alkaloids are a major class of naturally occurring secondary metabolites in plants with huge medicinal and biological properties including antidiabetic analgesic antiinflammatory antihypertensive antimalarial and anticancer Mukonal molecule is an active member of carbazole alkaloids found in Murraya koenigii Bhattacharyya and Chakraborty This molecule has shown significant antioxidant antimicrobial and anticancer activity Samanta et a0al Mukonal has been reported to show anticancer activities against different human cancer cell lines in a0vitro including laryngeal AMCHN8 cancer cells and nasopharyngeal CNE1 carcinoma cells It has shown a remarkable potential of autophagy initiation apoptosis induction modification of mitochondrial membrane potential cell cycle arrest blocking of MEKERK and PI3KAKT signalling pathways Li et a0al Guo et a0al Therefore current investigation was designed to unveil the antibreast cancer potential of mukonal along with its effects of autophagy and apoptosis inductionMaterials and a0methodsReagents chemicals and a0cell culturesMukonal with of purity by HPLC and other chemicals were bought from SigmaAldrich Darmstadt Germany unless otherwise mentioned Breast cancer cell lines MDAMB231 CAMA1 MDAMB436 and SKBR3 and normal breast cell line MB157 were procured from Type Culture Collection of Chinese Academy of Sciences Shanghai China All cell cultures were grown in RPMI1640 media maintaining fetal bovine serum Thermo Fisher Scientific Inc Waltham United States and penicillin G a0Uml and streptomycin a0µgml as suitable antibiotics Allinclusive cell cultures were placed and maintained within a humid environment of CO2 concentration and a temperature of a0°CDetermination of a0cellular proliferationThe cellular proliferation of MDAMB231 CAMA MDAMB436 and SKBR3 and a normal breast MB157 cell line were estimated after mukonal exposure by 345dimethylthiazol2yl25diphenyl tetrazolium bromide MTT assay In brief both cancer and noncancer cells were placed with a concentration of — cells per well of 96well plates and precultured for a0h in a humid environment of CO2 concentration and a temperature of a0 °C Thereafter each well plate was supplemented with different mukonal doses viz and a0µM and left untouched on incubation for a0h Mukonal treated cancer and noncancer breast cells were washed twice with phosphate buffered saline PBS prior to staining with a0µl of MTT stock solution of concentration a0 mgml for a0 h The formazan crystals then evolved were dissolved with dimethyl sulphoxide followed by colorimetric analysis Finally absorbance was taken at a0nm to determine the optical density using microplate spectrophotometer BioRad Laboratories Inc Hercules United States Experiments for individual mukonal concentrations were repeated thriceColony formation assayMDAMB231 and SKBR3 were plated onto 6well plates with cells per well After a0h of preculturing the cells treatment with variant mukonal doses of and a0µM was instigated Thereafter cancer breast cells were left on incubation for days devoid of any physical and chemical disturbance After days of incubation cell colonies were stained with crystal violet Finally MDAMB231 and SKBR3 cell colonies were totaled under a light microscope OLYMPUS Japan and only colonies with cells were considered for countingAnnexin VPI staining assayAnnexin VPI dual staining assay was performed to monitor and quantify apoptosis in mukonal treated cancer MDAMB231 and SKBR3 breast cells These cancer cells were placed onto 6well plates and subjected to variant mukonal doses viz and a0µM for a0h After that cells were washed in PBS fixed in of formaldehyde and yet again washed in PBS Finally these cells were stained with annexin VPI dual staining solution and eventually analyzed through flow cytometryTransmission electron microscopyMukonal treated MDAMB231 and SKBR3 cells at variant doses and a0µM were subjected to electron microscopy for autophagy assessment Mukonal treated cancer cells were fixed in the solution of glutaraldehyde bearing a0m of sodium cacodylate Afterwards post fixation of treated cells was carried out by of osmium tetroxide followed by moisture removal using alcohol Then cells were prepared for implantation over Epon for sectioning and finally investigated under Zeiss CEM electron microscope Oberkochen Germany 0cWang a0et a0al AMB Expr Page of Assessment of a0protein expressions by a0western blottingThe expressions levels of apoptosis and autophagy allied proteins were evaluated by western blotting Mukonal treated and a0 µM and untreated controls tumerous breast cells MDAMB231 and SKBR3 were lysed with RIPA buffer for protein collection Each lysate was subjected to bicinchoninic acid assay for quantification of proteins and a0µg of proteins from each sample were run on SDSPAGE Thereafter proteins were transferred to PVDF membranes and these membranes were blocked with nonfat milk at room temperature for a0h blocked membranes were subjected to suitable primary antibodies of dilution anticaspase3 antiPARP antiBAX antiBcl2 antiLC3BI antiLC3BII and antiBeclin1 Santa Cruz Biotechnology Inc Dallas United States overnight at a0°C Afterwards secondary antibody treatment was instigated with dilutions of horse radish peroxidaseconjugated antirabbit secondary antibody Santa Cruz Biotechnology Inc Dallas United States for a0h at room temperature Finally the protein bands were visualized using ECL Advanced Western Blot Detection kit GE Healthcare Life Sciences SwedenXenograft studyXenograft studies were carried out by strictly obeying the ethical guidelines permitted by animal ethics committee Gong An County People™s Hospital Gong An Jingzhou Hubei China Immunodeficient nude mice of 6weeks weighing “ a0 g were placed in sterile steel cages These cages were placed in an environment of a0 h cycle of lightdark relative humidity of and a moderate temperature of °C These mice were given subcutaneous injections of SKBR3 cells — on their left flank As the tumor became superficially apparent treatment with mukonal was instigated by intraperitoneal insertion with of DMS dissolved mukonal and diluted normal saline at doses of and a0mgkg body weight This treatment procedure was repeated each second of a week and only of DMSO was injected to the control mice The tumor volume and weight was monitored after every week and the procedure lasted for weeks The mice were then sacrificed for this noble cause by inhaling of deep anesthesia with isoflurane The tumor volume was determined using the following formula V W — W — L2 where ˜V™ is the volume ˜W™ is the width and ˜L™ is the length of the tumor The study was approved by the animal ethics committee of Gong An County People™s Hospital Gong An Jingzhou Hubei China under approval number GACPH022019Statistical analysisStatistical analysis of data collected by execution of each individual experiment in triplicates was performed via analysis of variance ANOVA and followed by Tukey™s posthoc test Entire data were represented as mean ± SE standard error considering p as statistically significantResultsMukonal inhibited proliferation of a0breast cancer cellsThe proliferation rate of four different cancer breast cell lines MDAMB231 CAMA1 MDAMB436 and SKBR3 and a normal breast MB157 cell line was determined using MTT assay after treatment with variant Mukonal a0µM Results revealed that mukonal significantly retarded the proliferation of these cancer breast cells with an IC50 value ranging from a0 µM to a0µM Table a0 Higher efficiency with lower IC50 value was recorded in case of MDAMB231 and SKBR3 cells Fig a0 1a Therefore rest of the studies was carried on these two cell lines The viability in case of MDAMB231 cells reduced from to almost Fig a01b and that of SKBR3 cells from to nearly with enhanced doses of mukonal from to a0µM Fig a01c The mukonal induced no significant cytotoxicity against normal MB157 breast cells and a high IC50 of a0µM was obtained Fig a0 1d Enhanced cytotoxicity was observed in case of normal MB157 cells at higher mukonal doses Clonogenic assay was used to monitor the impact of mukonal treatment over colony generation propensity of MDAMB231 and SKBR3 cells On treatment of MDAMB231 and SKBR3 cells with mukonal remarkable suppression of their colonies was observed in comparison to controls The numbers of MDAMB231 colonies left over were nearly and those of SKBR3 cells were nearly after days long treatment at a0µM of mukonal concentration Fig a0 Therefore mukonal induced remarkable toxicity against MDAMB231 and SKBR3 cells in comparison to MB157 cells which indicates specificity in anticancer activity of mukonalTable Effects of a0 Mukonal on a0 the a0 viability of a0 breast cancer cells as a0 depicted by a0 MTT assay and a0 presented as a0 IC50S noBreast cancer cell linesIC50 µMMDAMB231CAMA1MDAMB436SKBR3MB157The experiments were performed in triplicate 0cWang a0et a0al AMB Expr Page of Fig Mukonal inhibits the growth of breast cancer cells a Chemical structure of mukonal molecule b The viability of SKBR3 breast tumor cells after being subjected varying mukonal concentration as indicated SKBR3 cells were exposed to mukonal for h and then subsequently stained with MTT solution for calorimetric analysis c The viability of MDAMB231 breast tumor cells after being subjected varying mukonal concentration as indicated d The viability of normal MB157 breast cells after being subjected varying mukonal concentration as indicated All the experiments were executed three times and data was shown as mean ± SE standard error The p value of was taken as a statistical significant figureFig Clonogenic analysis of MDAMB231 and SKBR3 cells after being exposed to indicated mukonal doses MDAMB231 and SKBR3 breast tumor cell lines were treated with mukonal left untouched for days and stained with crystal violet to calculate the number of colonies generated provided considering colonies with number of cells for calculation All the experiments were executed three times and data was shown as mean ± SE standard error The p value of was taken as a statistical significant figure 0cWang a0et a0al AMB Expr Page of Mukonal induced apoptosis in a0breast cancer cellsThe annexin VPI assay was employed to determine the percentage of apoptosis in MDAMB231 and SKBR3 cells Results indicated that mukonal could potentially exhibit proapoptotic effects against both MDAMB231 as well as SKBR3 cells in a dosereliant fashion In comparison to control group the number of early apoptotic late apoptotic and necrotic cell percentage enhanced significantly in treated groups The percentage of apoptotic cells was raised to about in case of treated SKBR3 cells Fig a03a The apoptotic cell percentage of MDAMB231 cells at controls was but it enhanced to nearly at a0 µM of mukonal concentration Fig a03b Further western blotting analysis indicated that Mukonal enhanced the cleavage of caspase3 and PARP levels Moreover the expression of Bcl2 decreased and Bax increased with increase in the dosage of mukonal in case of SKBR3 cells Fig a03c The expressions of cleaved caspase3 and cleaved PARP enhanced significantly and Bcl2 PARP and Bax decreased with enhancing doses of mukonal in case of MDAMB231 cells Fig a03d Therefore the results from annexin VPI staining and western blotting analysis indicated that antiproliferative effects of mukonal could be mediated via its apoptosis inducing propensityMukonal induced autophagy in a0breast cancer cellsThe impact of mukonal exposure on cellular autophagy in MDAMB231 and SKBR3 cells was analyzed through transmission electron microscopy and western blotting assay Results indicated that mukonal potentially induce proautophagic effects in both of these cancer breast cell lines In comparison to control groups autophagosomes are clearly visible in both mukonal treated SKBR3 Fig a04a and MDAMB231 Fig a04b cells After exposure to variant doses “ a0µM of mukonal MDAMB231 and SKBR3 cell lines showed enhanced expression levels of Beclin1 LC3BI and LC3BII proteins Which indicated molecular features of autophagic cell death in mukonal treated MDAMB231 and SKBR3 cancer cells Fig a04c d Therefore it may be concluded that mukonal exhibits antiproliferative effects mediated via its autophagy inducing potentialIn vivo inhibition of a0tumor growth by a0mukonalNude mice xenografts were used to determine in a0 vivo anticancer effects of mukonal The results showed that SKBR3 tumor growth was remarkably retarded by mukonal administration in comparison to control group At the end of 6weeks the average tumor volume and weight in untreated control group was substantially advanced than mukonal treated groups Moreover the in a0 vivo anticancer effects of mukonal were dose and timedependent manner Fig a05a bDiscussionDespite significant advancements in cancer research management it still remains the most prevalently diagnosed cancer and cause of death in women globally Better approaches are needed to understand and recognize this disease at molecular levels Due to timely diagnoses of the disease North America has registered over 5year survival rate among the breast cancer patients DeSantis et a0al High frequency of occurrence late diagnosis and disastrous side effects of present day chemopreventives creates an emergency for novel therapeutic drugs that can deliver better results with higher efficacy Waks and Winer Collignon et a0 al Mukonal is a carbazole alkaloid and has revealed to possess remarkable pharmacological and biological propensities It has been reported to induce anticancer effects against different human cancer cell lines Li et a0al Guo et a0al Therefore the current study was undertaken due to accumulative evidences suggesting that mukonal has a great propensity to act as an anticancer agent The results revealed that mukonal decreased the proliferation rate of five variant breast cancer cell lines but remarkable results with IC50 of a0µM was recorded against breast cancer MDAMB231 and SKBR3 cancer cell lines Mukonal showed a minuscule toxicity against normal breast MB157 cell line indicating some specificity of inducing toxicity against cancerous breast cell lines Mukonal induced potential inhibition of colony generation by MDAMB231 and SKBR3 cells Further chemopreventive drugs target several cellular processes to induce cytotoxicity against cancer cells Apoptosis has been a focal target of chemopreventives and is often termed as typeI PCD programmed cell death Khursheed et a0 al Apoptosis remains dormant in normal cells but in case of injury malfunction aging and macromolecule accumulation in cells it plays a vital role of elimination Bonofiglio et a0al Herein mukonal induced apoptotic cell death in MDAMB231 and SKBR3 cell lines in a dosereliant fashion as suggested by annexin VFITC assay Similar results have been reported previously wherein mukonal induced apoptosis in laryngeal cancer cells Mukonal stimulated apoptosis was further supported by enhanced expression levels of cleaved PARP cleaved caspase3 and Bax and retarded expression levels of caspase3 PARP and Bcl2 proteins in both the cancerous cell lines after mukonal exposure Autophagy is another process that remains conserved in multicellular anisms activated under stressful conditions like starvation Levy et a0 al Autophagy is often termed as typeII PCD and 0cWang a0et a0al AMB Expr Page of Fig a Flow cytometric analysis of mukonal treated SKBR3 cells SKBR3 cells were cultured in 6well plates and then subjected to indicated mukonal doses Afterwards staining with annexin VPI was performed to analyze apoptosis flow cytometrically b Flow cytometric analysis of mukonal treated MDAMB231 cells MDAMB231 cells were cultured in 6well plates and then subjected to indicated mukonal doses Afterwards staining with annexin VPI was performed to analyze apoptosis flow cytometrically c Western blotting analysis was performed to assess the activity of apoptosis allied proteins in SKBR3 cells Results indicated that mukonal enhanced activity of proapoptosis proteins wherein blocking of antiapoptotic protein expression d Western blotting analysis was performed to assess the activity of apoptosis allied proteins in MDAMB231 cells Results indicating that mukonal enhanced activity of proapoptosis proteins in MDAMB231 cells wherein blocking of antiapoptotic protein expression All the experiments were executed three times and data was shown as mean ± SE standard error The p value of was taken as a statistical significant figure 0cWang a0et a0al AMB Expr Page of Fig a TEM analysis of SKBR3 cells after being exposed to indicated doses of mukonal Results revealed formation of autophagic vesiclesautophagosomes in treated groups as compared to control group Autophagosomes have been shown by arrows in the picture b TEM analysis of MDAMB231 cells after being exposed to indicated doses of mukonal Results revealed formation of autophagic vesiclesautophagosomes in treated groups as compared to control group Autophagosomes have been shown by arrows in the picture c Western blotting results of SKBR3 cells after being exposed to mukonal at indicated doses In treated groups enhanced activity of Beclin1 LC3BI and LC3BII was observed with increasing concentrations of mukonal d Western blotting results of MDAMB231 cells after being exposed to mukonal at indicated doses In treated groups enhanced activity of Beclin1 LC3BI and LC3BII was observed with increasing concentrations of mukonalalso remains as focal target of chemopreventives PoilletPerez et a0al Autophagy plays a key role in the degradation of marcomolecules and damaged anelle This process is completely hallmarked by the formation of autophagosomes which on maturation turns into autolysosomes Garc­aPrat et a0 al Mukonal was observed to induce autophagic cell death in both MDAMB231 and SKBR3 cell lines Similar results have been reported previous wherein mukonal induced autophagic cell death in human nasopharyngeal cancer cells Both of these cancerous cell lines showed enhanced expressions levels of LC3BI LC3BII and Beclin1 proteins indicating autophagy initiation at molecular levelsThe in a0 vivo investigation of mukonal revealed that it noticeably retarded growth of MDAMB231 and SKBR3 breast tumor growth in comparison to untreated control group and no apparent toxicity was detected The tumor weight and volume in mice models were observed to decline comparative to increased doses of mukonalIn the results of this investigation revealed that mukonal could potentially induce antibreast cancer effects both in a0 vitro and in a0 vivo against MDAMB231 and SKBR3 cell lines The antibreast cancer effects of mukonal were observed to mediate through induction of autophagy and apoptosis These studies point towards the potential of Mukonal in the treatment of breast cancer However further in a0vitro and in a0vivo studies are required to fully establish it as a lead molecule in the development of breast cancer chemotherapy 0cWang a0et a0al AMB Expr Page of Received June Accepted July ReferencesBhattacharyya P Chakraborty A Mukonal a probable biogenetic intermediate of pyranocarbazole alkaloids from Murraya koenigii Phytochemistry “Bonofiglio D Giordano C De Amicis F Lanzino M Ando S Natural products as promising antitumoral agents in breast cancer mechanisms of action and molecular targets MiniRev Med Chem “Collignon J Lousberg L Schroeder H Jerusalem G Triplenegative breast cancer treatment challenges and solutions Breast Cancer Targets Therapy DeSantis CE Fedewa SA Goding Sauer A Kramer JL Smith RA Jemal A Breast cancer statistics convergence of incidence rates between black and white women CA Cancer J Clin “Garc­aPrat L Mart­nezVicente M Perdiguero E Ortet L Rodr­guezUbreva J Rebollo E RuizBonilla V Gutarra S Ballestar E Serrano AL Sandri M Autophagy maintains stemness by preventing senescence Nature “Guo Y Hao Y Guan G Ma S Zhu Z Guo F Bai J Mukonal inhibits cell proliferation alters mitochondrial membrane potential and induces apoptosis and autophagy in human CNE1 nasopharyngeal carcinoma cells Med Sci Mon Int Med J Exp Clin Res Khursheed A Rather MA Rashid R Plantbased natural compounds and herbal extracts as promising apoptotic agents their implications for cancer prevention and treatment Adv Biomed Pharma “Levy JM Towers CG Thorburn A Targeting autophagy in cancer Nat Rev Cancer Li L Huizhi L Binu W Xinxin D Longjun W Liping Y Yingying Z Anticancer activity of mukonal against human laryngeal cancer cells involves apoptosis cell cycle arrest and inhibition of PI3KAKT and MEKERK signalling pathways Med Sci Mon Int Med J Exp Clin Res Majeed W Aslam B Javed I Khaliq T Muhammad F Ali A Raza A Breast cancer major risk factors and recent developments in treatment Asian Pac J Cancer Prev “PoilletPerez L Xie X Zhan L Yang Y Sharp DW Hu ZS Su X Maganti A Jiang C Lu W Zheng H Autophagy maintains tumour growth through circulating arginine Nature “Samanta SK Kandimalla R Gogoi B Dutta KN Choudhury P Deb PK Devi R Pal BC Talukdar NC Phytochemical portfolio and anticancer activity of Murraya koenigii and its primary active component mahanine Pharmacol Res “Siegel RL Miller KD Jemal A Cancer statistics CA Cancer J Clin “Smith G Henderson IC New treatments for breast cancer Semin Oncol “Sun YS Zhao Z Yang ZN Xu F Lu HJ Zhu ZY Shi W Jiang J Yao PP Zhu HP Risk factors and preventions of breast cancer Int J Biol Sci Waks AG Winer EP Breast cancer treatment a review JAMA “Williams DH Stone MJ Hauck PR Rahman SK Why are secondary metabolites natural products biosynthesized J Nat Prod “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsFig Mukonal inhibits MDAMB231 and SKBR3 tumor growth in vivo a Tumor volume and b tumor weight were measured at indicated time intervals and doses of mukonal molecule All the experiments for separate drug concentration were executed three times and data was shown as mean ± SE standard error The p value of was taken as a statistical significant figureAcknowledgementsAll the author of this manuscript is thankful to Gong An County People™s Hospital Gong An Jingzhou Hubei China to conduct the presented protocolAuthors™ contributionsWW and ZJ designed the protocol of the study WW ZZ XZ LC and SB performed the experimental work and collect the data for presented study WW and ZJ involve in the statistical analysis ZJ supervised the work and drafted the manuscript although all author contributed for the preparation of manuscript All authors read and approved the final manuscriptFundingNot applicableAvailability of data and materialsNot applicableEthics approval and consent to participateThe study was approved by the animal ethics committee by Gong An County People™s Hospital Gong An Jingzhou Hubei China under approval number 721GACPH022019Consent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interests 0c'
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cancer is a complex and heterogeneous disease with many possible genetic and environmentalcauses the same treatment for patients of the same cancer type often results in different outcomes in terms ofefficacy and side effects of the treatment thus the molecular characterization of individual cancer patients isincreasingly important to find an effective treatment recently a few methods have been developed to constructcancer samplespecific gene networks based on the difference in the mrna expression levels between the cancersample and reference samplesmethods we constructed a patientspecific network with multiomics data based on the difference between areference network and a perturbed reference network by the patient a network specific to a group of patients wasobtained using the average change in correlation coefficients and node degree of patientspecific networks of thegroupresultsnetworks with multiomics data the main differences of our method from previous ones are as follows networksare constructed with multiomics mrna expression copy number variation dna methylation and micrornaexpression data rather than with mrna expression data alone networks are constructed with bothnormal samples and cancer samples of the specified type to extract cancerspecific gene correlations and bothpatient individualspecific networks and patient groupspecific networks can be constructed the results of evaluatingour method with several types of cancer show that it constructs more informative and accurate gene networks thanprevious methodss the results of evaluating our method with extensive data of seven cancer types show that thedifference of gene correlations between the reference samples and a patient sample is a more predictive feature thanmrna expression levels and that gene networks constructed with multiomics data show a better performance thanthose with single omics data in predicting cancer for most cancer types our approach will be useful for finding genesand gene pairs to tailor treatments to individual characteristicskeywordsindividualspecific gene network groupspecific gene network cancer multiomics datacorrespondence khaninhaackr1department of computer engineering inha university incheon southkoreafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriatecredit to the original authors and the source provide a link to the creative commons licence and indicate if changes weremade the images or other third party material in this are included in the ™s creative commons licence unlessindicated otherwise in a credit line to the material if material is not included in the ™s creative commons licence and yourintended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directlyfrom the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creativecommons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data madeavailable in this unless otherwise stated in a credit line to the data 0clee bmc medical genomics 13suppl page of for the past years we have witnessed the rapid development of targeted cancer therapy targeted therapies forcancer work by targeting specific genes proteins or tissues that contribute to cancer growth and survival manytargeted therapies are effective only for patients with specific genetic alterations known as driver mutations thathelp cancer cells form and grow [ ] thus identifying genetic mutations specific to individual patients is ofutmost importance to determine targeted therapies thatcan effectively cure cancer patients while minimizing sideeffects motivated by a massive amount of data generated byhighthroughput technologies several cancer studies usedgene networks to explore gene expression characteristics [“] however constructing a patientspecific genenetwork with a single sample obtained from a patient isdifficult because a gene network requires many samples tocompute genegene relationsrecently a few methods have been proposed to construct cancer samplespecific gene networks based onthe difference in the mrna expression levels betweenthe cancer sample and reference samples for exampleliu proposed a method to construct a samplespecific network by computing the difference between areference network from multiple reference samples anda network perturbed by a new sample however a slightchange to the reference samples can result in a significantly different samplespecific network for the samesample due to the small number of reference samples furthermore their samplespecific networks cannot reflectposttranslational modification and epigenetics becausethe networks are built using mrna expression data onlythis paper presents a new method for constructingcancer patientspecific and groupspecific gene networkswith multiomics data using a samplespecific networkand network propagation method network propagation strategies are widely used in recent cancerrelatedresearch li presented a synergy prediction algorithm using network propagation and predicted the drugsynergy in various cancers zhang introduceda propagation algorithm which learns the mutated subnetworks underlying tumor subtypes using a supervisedapproach and classified tumors to known subtypes onbreast and glioblastoma tumors peng identifiedbladder cancerrelated genes by propagating informationfrom seed genes to candidate genes the primary focusof our method is to construct a gene correlation network specific to cancer with multiomics data thus it isdifferent from a typical gene coexpression network thatrepresents coexpression relations between genes frommrna expression data our gene network is not a generegulatory network because our network does not showregulatory relations between genesthe main differences of our method from previous onesare as follows networks are constructed with multiomics mrna expression copy number variation dnamethylation and microrna expression data rather thanwith mrna expression data alone networks are constructed with both normal samples andcancer samples of the specified type to extract cancerspecific gene correlations and both patient individualspecific networks and patient groupspecific networks canbe constructed as shown later in this paper the resultsof evaluating our method with several types of cancershow that it constructs more informative and accuratetargetspecific networks than previous methodsmethodsat the top level our method consists of the followingsteps data processing constructing individualspecific gene networks and constructing a groupspecific gene networks a highlevel description of themethod is given in fig data collection and preprocessingfrom the broad institute tcga gdac firehose we obtained multiomics data of cancer samples of seventypes breast invasive carcinoma brca colon adenocarcinoma coad head and neck squamous cell carcinomahnsc pankidney cohort kipan liver hepatocellular carcinoma lihc lung adenocarcinoma luad andlung squamous cell carcinoma luscthe multiomics data used in this study includemrna expression mrnaseq copy number variationcnv dna methylation and mature mirna expression mirseq data the mrnaseq data were processedusing quartile normalized rsem and then log2transformed the segmented cnv data were convertedto genelevel data using the ensembl api and thecntools package of bioconductor the methylationdata were filtered to select the probe with the mean signal values for each gene the mirseq data were processedby rpm and log2transformed mrnas and mirnas thatwere not expressed in more than of the total sampleswere excluded in further analysis missing expression values of mrnas and mirnas were replaced by the smallestpositive normalized floatingpoint number realmin ofmatlab the number of samples and genes used in thisstudy are available in additional file individualspecific gene networkin each group of tumor samples and normal samples wefirst computed genegene relations by the pearson correlation coefficient pcc selected highly correlated genepairs ie those with pcc and constructed twosample networks one for each group from the tumorsample network we removed edges common to both 0clee bmc medical genomics 13suppl page of fig overview of constructing an individualspecific network and a groupspecific network with multiomics datatumor and normal sample networks and obtained a template reference network for cancer fig 2a the templatereference network consists of highlycorrelated gene pairsthat are specific to cancerwith n reference samples which may be different fromtumor samples used in the template network we computed pcc for every pair of genes in the template reference network and constructed a reference network forthe reference samples for a patient of interest we constructed a network which is a perturbed network byadding a single sample of the patient to the n reference samples a patientspecific network was obtainedby subtracting the reference network from the perturbednetwork 01pcc pccn1 ˆ’ pccnwe computed the difference in the absolute value ofpcc between the perturbed reference network and reference network by eq we also carried out a ztest ofpccn1ˆ’pccn by eq for a large n we can approximatethe mean μ and standard deviation σ of pccn1 ˆ’pccn as and ˆ’ pcc2nn ˆ’ respectively pccchange pccn1 ˆ’ pccnz ˆ’ score pccchange ˆ’ μpccchangeσ pccchange pccchangeˆ’pcc2nnˆ’the edges of the patientspecific network were classified into four types correlationgained edges fene pairs whose pccs are increased from the referencenetwork to the patientspecific network correlationlost edges for gene pairs whose pccs are decreased fromthe reference network to the patientspecific network correlationreversed edges for gene pairs whose signs ofpccs are changed from positive to negative or negativeto positive and correlationinvariant edges for genepairs with little change in pccs between the reference andpatientspecific networks ie those with zscore fig 2bthe edges were classified in the following way we firstselected gene pairs with zscore as correlationinvariant type and then selected gene pairs which havedifferent signs of pccs between the reference networkand the patientspecific network as correlationreversedtype the remaining gene pairs were classified into eithercorrelationgained or correlationlost type depending onwhether their pccs are increased correlationgained ordecreased correlationlost from the reference network tothe patientspecific network thus zscore ‰¥ in bothcorrelationgained and correlationlist gene pairsgroupspecific gene networka groupspecific gene network is useful when analyzinga large number of patientspecific gene networks afterconstructing patientspecific gene networks we obtained 0clee bmc medical genomics 13suppl page of fig process of constructing a patientspecific gene network a template of the cancer reference network obtained by removing edges common toboth networks b patientspecific gene network and four types of edges in the network edges with a zscore represent correlationinvariantgene pairs and edges with zscore ‰¥ and different signs of pccn and pccn1 represent correlationreversed gene pairs edges with zscore ‰¥ and 01pcc are correlatedgained gene pairs and those with zscore ‰¥ and 01pcc are correlationlost gene pairsa gene network specific to a group of patients based onthe average 01pcc and node degree of the patientspecificnetworks fig if the dominant type for a particular edge is ˜correlationgained™ positive 01pcc in thepatientspecific networks the edge is represented in redin the groupspecific network in contrast if the dominant type for a particular edge is ˜correlationlost™ negative 01pcc in the patientspecific networks the edgeis represented in blue in the groupspecific network inthe groupspecific network only the dominant type isshown for each edge if nondominant types are shownin addition to the dominant type for each edge the network becomes cluttered and unreadable the node sizeof a groupspecific gene network is proportional to theaverage degree of the nodeintegration of multiomics datato integrate multiomics data we first computed intergene correlations by pcc with four different types ofsingle omics data mrna expression cnv dna methylation and mirna expression separately and selectedsignificant intergene correlations only in mrna expression cnv and dna methylation data we select thetop pcc with pvalue in mirna expression data we selected the top pcc with pvalue due to a smaller number of mirnas inthe data the intergene correlations selected in eachsingle omics data are represented in four correlationmatrices mexpr mcnv mmethyl and mmirna andnormalizedusing the proteinprotein interactions ppis of thestring database we constructed separate weightednetworks from each omics data by eq in eq wexprwcnv and wmethyl denote the weighted networks andppiexpr ppicnv and ppimethyl are subnetworks of a ppinetwork consisting of genes present in each omics datasince the ppi network does not contain information onmirna a weighted network for mirna was not constructedcid3wexpr ˆ’ cid2 ˆ’ ppiexprwcnv ˆ’ ˆ’ mcnv × ˆ’ ppicnv wmethyl ˆ’ cid2 ˆ’ ppimethylwe then integrated the multiomics data by linear xmethylregression using eq in eq yi xcnvand xmirnadenote gene i™s expression level cnv levelmethylation level and mirna regulator expression levelrespectively βcnvdenote the regression coefficients of gene i™s expression level on cnv and methylation respectively βmirnais the regression coefficientof gene i™s expression level on its mirna regulator j™sexpression leveland βmethyl ˆ’ mexpr ˆ’ mmethylcid3 × cid2cid3 × cid2cid3iiijiiijyi βcnvii βmethylxcnvixmethyli ncid4j1βmirnaijxmirnaij 01 0clee bmc medical genomics 13suppl page of fig process of constructing a groupspecific gene network from multiple patientspecific gene networks in the groupspecific gene network thenode size and node color are proportional to the average degree and average 01pcc of the node respectively if the dominant type for a particularedge is ˜correlationgained™ positive 01pcc in the patientspecific networks the edge is represented in red in the groupspecific network if thedominant type for a particular edge is ˜correlationlost™ negative 01pcc in the patientspecific networks the edge is represented in blue in thegroupspecific networkfrom the regression coefficients and the weighted networks a weight matrix w was derived and normalizedinto w eqs and the weight matrix w is symmetricso wij wji w11 w22 w33 and w44 represent wexprwcnv wmethyl and mmirna respectively the submatrices w21 and w31 contain regression coefficients βcnvand βmethylfor every gene i respectively w41 representsiβmirnaij the submatrices w32 w42 and w43 are emptyiŽ¡Ž¢Ž¢Ž£w w11 w12 w13 w14w21 w22 w23 w24w31 w32 w33 w34w41 w42 w43 w44Ž¤Ž¥Ž¥Ž¦eq and updated iteratively by eq in this iterative process the influence of the seed is propagated tothe neighbors until a mean squared error of st and stˆ’‰¤ × ˆ’Ž§ŽªŽªŽªŽªŽ¨ŽªŽªŽªŽªŽ©if v is a nonseed nv ‰¥ αif v is a nonseed nv αif v is a seednvnvenvˆ’α × nvnvdv st λ × stˆ’ × w ˆ’ λ × d wheres1 d where nv is the number of neighbors of node v and nv isthe number of seeds in the neighbors the parameter αwhich is a threshold for nv was set to and λ was set to genes with the top st were used in findingcancerrelated genes and in classifying tumor samples andnormal samplesw i j w i jcid11cid12cid12cid13mcid4k1w i k × mcid4k1w k jin network propagation seed genes have greater impactthan nonseed genes on their neighbors thus only thegenes with a high average 01pcc were selected as seedgenes for a groupspecific network and their mirnasregulators extracted from mirtarbase were used asseed mirnas we calculated the cancerrelevance st ofeach gene to reflect the effect of the seed genes and mirnas on neighbors the initial score d was calculated byresultspatientspecific and groupspecific gene networksin this study we constructed patientspecific genenetworks for seven cancer types additional file foreach cancer type we also constructed groupspecific genenetworks as an example fig shows a groupspecificgene network derived from lung squamous cell carcinoma lusc patients 0clee bmc medical genomics 13suppl page of there are three distinct subnetworks in the networkfor the lusc group the subnetwork enclosed in box aof fig contains many hub genes large green nodesthe subnetwork in box b consists of correlationgainededges dark red edges whereas the subnetwork in box ccontains many correlationlost edges dark blue edgescomparison of multiomics data and singleomics datawe performed leaveoneout cross validation loocvto evaluate cancerrelevance score st of a gene and thecontribution of multiomics data to finding cancerrelatedgenes for comparison the cancerrelevance scores werecomputed with multiomics data and single omics dataseparately each seed gene was regarded as a nonseed anda new cancerrelevance score was calculated for the geneseed genes and nonseed genes were considered as positive and negative respectively seed genes included in thetop n genes were considered as true positives and seedgenes not included in the top n genes were consideredas false negatives similarly nonseed genes included infig groupspecific gene network for lung squamous cell carcinoma lusc patients a subnetwork of multiple hub genes large greennodes b subnetwork of correlationgained edges dark red edges c subnetwork with many correlationlost edges dark blue edges 0clee bmc medical genomics 13suppl page of the top n genes and nonseed genes not included in thetop n genes were considered as false positives and truenegatives respectivelywe carried out loocv with different ratios of seedgenes to nonseed genes figure shows the receiver operating characteristic roc curve and the area under thecurve auc of loocv of the cancer relevance of geneson data of breast cancer samples with various seedratios ranging from to enlarged plots of fig are available in additional file for comparative purposes we also computed the cancer relevance of geneswith single omics data as shown in fig multiomicsdata consistently exhibited better performance than singleomics data with any seed ratio between to forlater analysis the seed ratio was set to by default theaverage 01pcc and class label of each gene are available inadditional file indeed the superiority of multiomics data over singleomics data in determining the cancer relevance scoreof genes was observed in all seven types of canceradditional file in seven types of cancer the cancerrelevance score of genes computed with multiomics dataexhibited a good performance auc ˆ¼ thecancer relevance score of genes computed with mrnaexpression data showed the second best performanceauc ˆ¼ in particular the cancer relevancescore computed with mrna expression data showed avery similar performance to that with multiomics inbreast cancer brca the performance of the cancer relevance score computed with cnv auc ˆ¼and dna methylation data auc ˆ¼ alonewas lower than that with mrna expression data auc ˆ¼fig roc curves of the leaveoneout cross validation of the cancer relevance score st of genes with different ratios of seed genes breastcancer samples were used in the leaveoneout cross validation the performance of multiomics data is always better than that of single omics data 0clee bmc medical genomics 13suppl page of evaluation of gene correlations and networksmany networkbased approaches to cancer research havefocused on finding genes that show differential expressions between tumor samples and normal samples genegene correlations ie intergene correlations may bemore helpful than individual genes because intergenecorrelations depend on the expression of neighbor genesin a gene regulatory network to compare the effect ofusing individual genes to that of intergene correlationsie 01pcc we constructed a support vector machinesvm model for classifying cancer samples and normalsamples the svm model was implemented using csvcand rbf kernel and the parameter values of the modelwere determined by the grid search algorithm mrnaexpression levels and 01pccs were used as features ofthe svm models for rigorous validation the test dataused in testing the models were not used in training themadditional file as shown in fig 6a 01pcc showed a better performance than mrna expression levels for six cancertypes except lusc the classification model with 01pccshowed mcc above in six cancer types except hnscwe also examined the effect of different networks on individualspecific networks in the workby liu ppi data with high confidence scoresin the string database were used to construct a network however the ppi data of stringdoes not reflect cancer typespecific characteristicsfigure 6b shows the performance of the classificationmodel with two different networks network from ppi data of string the approachby liu and cancer network ourapproach 01pcc was used as a feature of the classification model except for coad the performance ofthe classification model with the cancer network was better than the model with the string reference network in particular the classification model showed a significant difference for breastcancer brca mcc of vs mcc of detailed results of the classification model are available inadditional file discussionin the analysis of finding cancerrelated genes and genepairs we focused on a subnetwork of genes with a 01pcctable shows the top genes with a high average 01pcc in each groupspecific network of seven cancertypes in breast invasive carcinoma brca fam171a1showed the highest average 01pcc in the groupspecificnetwork fam171a1 is known as a potential biomarkerin triplenegative breast cancer foxc1 is involvedin tumor development and metastasis and associated withpoor prognosis in basallike breast cancer il33 isoverexpressed in various cancers and the serum concentration of il33 is a valuable indicator of poor prognosis inbreast cancer mamdc2 is significantly correlatedwith diseasefree survival of breast cancer patients mterfd1 is closely related to breast cancer recurrencefig results of evaluating features and networks by a validation set a comparison of mrna expressions of genes and 01pcc of genepairs b comparison of the cancer network with the network from ppi data 0clee bmc medical genomics 13suppl page of table top genes with a high average 01pcc in a groupspecific network for seven cancer typesbrcakipanlihccoadhnscfam171a1foxc1il33mamdc2mterfd1hoxa7cttnbp2znf204pjam3frem1gfra2scnn1bddx27rnps1ube2imdficctnnbl1cdk5rap1esf1trmt6cyp2j2barx2znf135ppfia1parlfaddcst6cobloraov1xpo7tfcp2l1kcnq1arl15kctd1oaz2tmem45bhpcal1tmem91sema5begln3slc19a3ecm1cyp2b6fbp1gpaa1f9pahagxt2hsd17b6rnase4luadcldn18adamts8pecam1sftpa1gimap6akr1c1mmeatad2cyp3a5chaf1bluscnsun2cct5fbxo45gpr116slc39a8fxr1inmtveph1crtamwdr53 and hoxa7 plays a critical role in regulating theproliferation of erpositive cancer cells in colon adenocarcinoma coad gfra2 showed thehighest average 01pcc in the groupspecific network itis known to be crucial for enteric neuron survival scnn1b and ddx27 are significantly related to colorectal cancer [ ] no direct relation of rnps1 withcolorectal cancer is known but rnps1 is essential tononsensemediated mrna decay that plays complexfunctions in cancer knockdown of sumo conjugating enzyme ube2i also known ubc9 or e2 inhibitsmaintenance and selfrenewal of colorectal cancer stemcell while overexpression of ube2i increases colorectalcancer cell stemness among the top genes with a high average 01pccin lung adenocarcinoma luad several genes such ascldn18 adamts8 pecam1 and sftpa1 have beenknown to be associated with luad in previous studies[“] no direct relation of nsun2 and slc39a8 withlung squamous cell carcinoma lusc has been known sofar however recent studies [ ] reported that nsun2is correlated with survival in other types of squamous cellcarcinomas gao also showed that the epigeneticsilencing of slc39a8 expression by dna methylation isinvolved in the acquisition of resistance against cadmiumin lung cells and the relation between cadmium andlung cancer has received much attention many othergenes in table found in the groupspecific networksfor head and neck squamous cell carcinoma hnscpankidney cohort kipan and liver hepatocellular carcinoma lihc are also directly or indirectly related tocancerin addition to individual genes we identified genepairs of the same type ie either correlationgained orcorrelationlost in most patientspecific networks of thesame type table shows the most frequent gene pairs in breast cancer samples the most frequent gene pairsin other types of cancer are listed in additional file it isinteresting to note that all the gene pairs shown in table include at least one gene in the gene pair mamdc2hoxa7 and that they are correlationgained edges in thegroupspecific network for breast cancer figure showsa subnetwork containing mamdc2 and hoxa7 in thegroupspecific network of breast cancer the subnetworkwas obtained by selecting the edges for which the proportion of the same edge type ie correlationgained or lostis above in the total individualspecific networks ofbreast cancer patients it is interesting to note that all thegene pairs in table are included in the subnetworkto date the actual role of the mamdc2 gene in cancer is not clear but meng reported mamdc2as one of three genes mamdc2 tshz2 and cldn11that are significantly correlated with diseasefree survival of breast cancer patients mamdc2 is known as atarget of mir196a in head and neck squamous cell carcinoma as a member of the family of homeoboxgenes hoxa7 is associated with cell proliferation nerveinvasion distant metastasis and degree of tumor differentiation in several cancers [ “] hoxa7 is regulatedtable the most frequent gene pairs in breast cancersamples all the gene pairs are of a correlationgained type thegenes of table are shown in bold the proportion represents theratio of the gene pairs of the same type ie correlationgained orlost to the total number of patientspecific networksgene pairgene pairsproportion of the gene pairsin total cancer samplesmamdc2hoxa7mamdc2ccl14mamdc2znf204pmamdc2klmamdc2svep1mamdc2coro2bhoxa7meox2hoxa7hoxa9mamdc2sobpmamdc2hoxa9 0clee bmc medical genomics 13suppl page of fig a subnetwork of the groupspecific network of brca which contains mamdc2 and hoxa7 genes in the most frequent gene pairs shown intable are enclosed by a circleby several mirnas including mir196 [“] thusboth mamdc2 and hoxa7 are related with mir196but a clear relation among them is to be uncoveredso far most approaches to constructing individualspecific gene networks have been constructed based onthe differential expressions between a small number ofreference samples and a sample of interest howeversuch networks cannot reflect posttranslational modification and epigenetics and are not reliable because a slightchange to the reference samples can result in a significantly different samplespecific network for the samesamplein this paper we presented a new approach to constructing cancer patientspecific and groupspecific networks with multiomics data the main differences ofour method from previous ones are as follows gene networks are constructed with multiomics mrnaexpression copy number variation dna methylationand microrna expression data rather than with mrnaexpression data alone networks can beconstructed with cancer samples of the specified type and both patient individualspecific networks and patientgroupspecific networks can be constructed the resultsof testing our method with several cancer types showedthat it constructs more informative and accurate genenetworks than existing methodsevaluation of our method with extensive data of sevencancer types showed that changes in gene correlations 01pcc between the reference samples and a patient sample is a more predictive feature than mrna expressionlevels and that gene networks constructed with multiomics data are more powerful than those with singleomics data in predicting cancer for most cancer typesmore work is required to validate the genes and genepairs identified in the cancer patientspecific and groupspecific networks however the method for constructingnetworks specific to individual patients or patient groupswith multiomics data should be useful aids in determining effective treatments to individual characteristicssupplementary informationsupplementary information accompanies this paper athttpsdoi101186s12920020007367additional file number of samples and genes in types of canceradditional file roc curve and auc of the cancerrelevance score ofbrca by various seed ratiosadditional file average 01pcc and class label of each gene in typesof canceradditional file roc curve of the cancerrelevance score of each cancertype with the seed ratio of additional file performance of classification of tumor samples andnormal samplesadditional file top gene pairs for each cancer type 0clee bmc medical genomics 13suppl page of abbreviationsauc area under the curve brca breast invasive carcinoma cnv copynumber variation coad colon adenocarcinoma gdac genome dataanalysis center hnsc head and neck squamous cell carcinoma kipan pankidney cohort lihc liver hepatocellular carcinoma loocv leaveoneoutcross validation luad lung adenocarcinoma lusc lung squamous cellcarcinoma mcc matthews correlation coefficient pcc pearson correlationcoefficient ppi proteinprotein interactions roc receiver operatingcharacteristic svm support vector machine tcga the cancer genome atlasacknowledgementsthe authors thank the anonymous reviewers for the constructive commentsthat contributed to improving the final version of the paperabout this supplementthis has been published as part of bmc medical genomics volume supplement selected s from the 15th international symposiumon bioinformatics research and applications isbra19 medical genomicsthe full contents of the supplement are available online at httpsbmcmedgenomicsbiomedcentralcomssupplementsvolume13supplement6authors™ contributionswl designed and performed all about this study and prepared the initialmanuscript dh helped integrating multiomics data of breast cancer khsupervised the work and wrote the manuscript all authors read and approvedthe final manuscriptauthors™ informationwook lee is currently working toward his phd degree at the department ofcomputer engineering inha university korea deshuang huang is a directorof the institute of machines learning and systems biology tongji universitychina kyungsook han is a professor at the department of computerengineering inha university koreafundingthis work was supported by the national research foundation of korea nrfgrant funded by the ministry of science and ict nrf2017r1e1a1a03069921and inha university research grant publication of this was funded bythe nrf grant nrf2017r1e1a1a03069921 the funders had no role in thedesign of the study data collection and analysis or writing the manuscriptavailability of data and materialsadditional files are available at httpbclabinhaackrcancernetworkethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of computer engineering inha university incheon southkorea 2institute of machine learning and systems biology school ofelectronics and information engineering tongji university shanghaichinareceived may accepted june published august references widakowich c de castro g de azambuja e dinh p awada a reviewside effects of approved molecular targeted therapies in solid cancersoncologist “liu s kurzrock r toxicity of targeted therapy implications for responseand impact of genetic polymorphisms cancer treat rev “verma m personalized medicine and cancer j personalized med“barabasi al gulbahce n loscalzo j network medicine a networkbasedapproach to hum
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this study hypothesizes that bromelain bl acts as radiosensitizer of tumor cells and that it protects normal cells from radiation effects in vitro and in vivo studies have been carried out to prove that assumption in vitro mtt cell proliferation assay has shown that the irradiated ehrlich ascites carcinoma eac cell line could be sensitized by bl pretreatment in vivo animals were randomly divided into groups group control pbs ip for days group ehrlich solid tumor est bearing mice group est Îradiation fractionated dose gy — group est bl mgkg ip daily for days group est bl for days followed by Îirradiation gy — the size and weight of tumors in gammairradiated est bearing mice treated with bl decreased significantly with a significant amelioration in the histopathological examination besides bl mitigated the effect of Îirradiation on the liver relative gene expression of poly adp ribose polymerase1 parp1 nuclear factor kappa activated b cells nfκb and peroxisome proliferatoractivated receptor α pparα and it restored liver function via amelioration of paraoxonase1 pon1 activity reactive oxygen species ros content lipid peroxidation lpo and serum aspartate transaminase ast alanine transaminase alt and albumin alb it is concluded that bl can be considered as a radiosensitizer and radioprotector suggesting a possible role in reducing radiation exposure dose during radiotherapykeywordsbromelain tumor Îradiation radiosensitizer radioprotectorsubmitted april revised july accepted july introductionradiotherapy has been used for a long time in treating cancer1 however from the clinical perspective radiotherapy provides inadequate success due to the radioresistance of many tumors as well as the high risk of recurrence and effects on normal cells may occur23 radioresistance occurs as the microenvironment of solid tumors is hypoxic compared with normal tissue4 in addition some tumors have either an intrinsic resistance to ionizing radiation or can attain this property through accumulation of genetic mutations causing an increased survival and proliferation5 thus strategies to improve radiation therapy could include increasing resistance of normal tissues to radiation andor increasing sensitivity of the tumor cells6radiosensitizing agents increase the sensitivity of tumor cells via enhancing the generation of reactive oxygen species ros increasing lipid peroxidation depletion of glutathione which leads to dna damage inhibition of dna repair inhibition of dna synthesis induction of cell cycle arrest induction of apoptosis and inhibition of proliferation7 numerous nutritive cancer chemopreventive compounds having antioxidant properties have been recognized to potentiate radiation therapyinduced cytotoxic 1drug radiation research department national centre for radiation research and technology egyptian atomic energy authority nasr city cairo egypt2biochemistry department alazhar university cairo egyptcorresponding authorhanan a fahmy drug radiation research department national centre for radiation research and technology atomic energy authority p o box nasr city cairo egypt email fahmyhananyahoocomcreative commons non commercial cc bync this is distributed under the terms of the creative commons attributionnoncommercial license creativecommonslicensesbync40 which permits noncommercial use reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages ussagepubcomenusnamopenaccessatsage 0c integrative cancer therapies effects on cancer cells inversely decreasing its toxicity on normal adjacent tissues89 in this regard much research has aimed to develop numerous antioxidant drugs of both natural and synthetic origin tested in both in vitro and in vivo models and also human clinical trials to overcome injuries caused by ir exposure and to induce killing of cancer cells at the same time previous studies have reported that phytochemical soy isoflavones genistein daidzein and glycitein which exhibit anticarcinogenic properties through their antioxidant activities could be used as potent radiosensitizers to enhance the efficacy of radiotherapymediated suppression of the growth and metastatic ability of cancers1011 along parallel lines resveratrol and piperine which possess antitumor activity have been shown to augment ionizing radiation irinduced apoptosis and loss of mitochondrial membrane potential in murine colon carcinoma and melanoma cells via enhancing irinduced ros generation12 moreover pentoxifylline ptx a methylxanthine that possesses antioxidant properties is known for improving tumor tissue oxygenation in murine hypoxic tumors and inhibiting post radiation induced normal tissue injury in mice1314 consequently searching for a natural product possessing anticancer activity that increases radiosensitivity of tumor cells and radioresistance of normal cells may lead to a potential future drug in cancer therapyamong the natural products bromelain bl extract attracts interest due to its anticancer antioxidant as well as antiinflammatory effects1517 bl an extract from pineapple stem ananas comosus belongs to a group of protein digesting enzymes it is a mixture of diï¬erent thiol endopeptidases and other components like phosphatase glucosidase peroxidase cellulase escharase calcium and several protease inhibitors1819 the anticancer activity of bl has been examined in various types of gastrointestinal and breast cancers cell lines in in vivo models bl has shown antimetastatic effect and reduction in local tumor growth2023 it is also used for reducing the severity of such radiation therapy side effects as mucositis skin reactions and dysphagia in patients24 hence this study was aimed to evaluate the radiosensitizing and radioprotective effect of bl using in vivo and in vitro approachesmaterials and methodin vitro studiesmtt cell proliferation assay the growth and viability of ehrlich ascites carcinoma eac cell line were tested in vitro by 345dimethylthiazol2yl25diphenyltetrazolium bromide mtt assay according to freimoser and buch 2526 to verify the antitumor and radiosensitizing effect of bl two plates were designed for this study the first one contained eac cells maintained by serial subculturing at the national cancer institute egypt incubated for hour before irradiation irr gy alone and with different concentrations of bromelain bl in phosphate buffer saline pbs the second one contained eac cells serving as a control and eac with different concentrations of bl each test was seeded in triplicate into a plate at concentration of — cellswell containing rpmi media with fbs nahco3 uml penicillin and µgml streptomycin and each plate was incubated for hours at °c in co2 and humidity atmosphere then μl mtt reagent bio basic inc canada was added over the cells in each well and the plate was incubated in the dark for to hours until a purple precipitate was seen and the absorbance was measured at nm the amount of color produced was directly proportional to the number of viable cells viable cell a samples ˆ’ a blanka control ˆ’ a blank — the inhibitory concentration ic50 is the dose of a drug which reduces the viability to and was calculated using nonlinear regression analysisfree radical scavenging assay the antioxidant activity of bromelain was evaluated by 1diphenyl2picrylhydrazyl dpph radical assay and its scavenging power was compared with some antioxidants naringin polyphenolic antioxidant garlic oil and glutathione sulfur containing antioxidants about µl of samples mgml dissolved in dist water was added to µl of a solution of dpph g100 ml dissolved in vv methanol after minutes incubation at room temperature in the dark the absorbance was read at nm against a blank µl dist water µl dpphmethanol solution the experiments were done in triplicate according to the method of braca 27 glutathione mgml was used as a standard antioxidant the scavenging percentage of dpph was calculated according to the followscavengin ing equation where b was the absorbance of the blank and a was the absorbance of samples or standard ec50 is defined as concentration of sample that causes dpph loss there values were calculated using nonlinear regression analysisˆ’b ab\uf8ee\uf8ef\uf8f0\uf8f9\uf8fa —\uf8fbin vivo studiesradiation processing whole body Îirradiation of mice was carried out using gamma cell40 137cesium manufactured by the atomic energy of canada limited ontario canada installed in the national center for radiation research and technology ncrrt cairo egypt the dose rate was gymin during the experimental period daily correction for humidity barometric pressure and temperature were madeanimals adult female swiss albino mice weighing to g obtained from the breeding unit of ncrrt cairo egypt all animal procedures were performed in accordance with the committee of scientific ethics at faculty of 0cmekkawy table sequences of primers for realtime quantitative pcrgeneparp1 nm0074152nfκb nc0000696pparα nc0000816βactin nc0000716forward primerreverse primer²ccatcgacgtcaactacga3²²caatggctacacaggacca3²²actccacctgcagagcaacca3²²gcgtggggacagccgcatctt3²²gtgcgtggtagcatgagtgt3²²cactgtcacctggaaccaga3²²tagatctcctgcagtagcggg3²²atcggcagaaggggcggaga3²pharmacy alazhar university egypt following the guidelines for animal use the animals were housed in colony cages micecage under proper environmental conditions that is hours darklight cycle good ventilation condition and temperature to humidity at the ncrrt animal house fed with standard diet pellets and provided with water ad libitum animals were left week for acclimatization on the lab environment before starting the experimenttumor transplantation the eac cell line was supplied by serial subculturing at the national cancer institute cairo university egypt it was implanted in each donor female swiss albino mice by ip injection of — cells22 g b wt and allowed to multiply28 the ehrlich solid tumor est was obtained by the intramuscular inoculation of ml of — viable eac in the right lower limb of each mouse29 mice with a palpable solid tumor diameter mm3 that was maintained within to days after inoculation were used in the studyanimal grouping animals were randomly divided into groups mice each group control not bearing tumor received pbs ip for days group ehrlich solid tumor est bearing mice received pbs ip for days group est Îirradiation gy — fractionated doses starting days after tumor appearance mm3 and lasting for days group est bearing mice receiving freshly prepared bl dissolved in pbs mgkg ip daily for days according to pilot study starting once est becomes mm3 bl was purchased from merck kgaa co darmstadt germany group est bearing mice received bl as in group hours before Îirradiation as in group mice were anesthetized days after last irradiation dose using urethane mgkg30 blood samples were collected through cardiac puncture and divided into parts edta coated and plain vials at that time they were euthanized by cervical dislocation liver and tumor tissues were dissected out rinsed with icecold saline dried on a filter paper and weighed then homogenized in icecold pbs ph and stored at ˆ’°c until used for subsequent biochemical analysisestimation of total body tumor and liver weights animals in each group were checked daily for any adverse clinical symptoms and deaths after to days post inoculation with eac body weights were recorded so body weight change could be estimated tumor and liver weights were measured during sample collection and then the tumor inhibitory ratio was calculated by the following formula inhibition ratio aˆ’ba — where a is the tumor weight average of the control and b is that of the treated group also relative liver weight was calculated as liver weighttotal body weight — histopathological examination three tumors of each group were collected and fixed in neutral buffered formalin the specimens were dehydrated in ascending grades of ethyl alcohol cleared in xylene and embedded in paraffin wax four micron thick paraffin sections were mounted on clean slides stained with ehrlich™s hematoxylineosin he31 and examined using an olympus microscope bx41 hamburg germany histopathological evaluation was done by assessment of necrosis and calculation of tumor area percentage using image analysis software image j 146a nih usa through the following equation of tumor area area of tumortotal area of the field — molecular analyses the mrna levels of poly adpribose polymerase parp1 nuclear factor kappa b nfκb and peroxisome proliferatoractivated receptors pparα genes and of the housekeeping gene βactin were measured by real time polymerase chain reaction rtpcr total rna was isolated from liver tissues using qiagen tissue extraction kit qiagen usa in accordance with the manufacturer™s instructions the extracted rna μg was used for cdna conversion using high capacity cdna reverse transcription kit fermentas usa and μl reaction volume sybr chemistry in applied biosystems thermal cycler usa to amplify pcr under the following conditions °c for denaturation then °c to °c for annealing using primers mentioned in table and °c for elongationresults were expressed using the comparative ˆ†ˆ†ct method for relative mrna quantification of target genes normalized to an endogenous reference βactin and a relevant control equal to ˆ’ˆ†ˆ†ct ˆ†ˆ†ct is the difference between the mean ˆ†ctsample and mean ˆ†ctcontrol where ˆ†ctsample is the difference between the mean ctsample and the mean ctβactin and ˆ†ctcontrol is the difference between the mean ctcontrol and the mean ctβactin 0c integrative cancer therapies estimation of lipid peroxidation lpo reactive oxygen species ros and paraoxonase pon1 in liver homogenate liver lipid peroxidation was estimated by measurement of malondialdehyde mda formation using the thiobarbituric acid method of yoshioka 32 a modified technique of vrablic 33 was used to measure the generation of ros by the intracellular conversion of nitro blue tetrazolium nbt to formazan by the action of superoxide anion paraoxonase activity was estimated by using fluorometric assay enzchek® kit invitrogen uk for the anophosphatase activity of paraoxonase based on the hydrolysis of a fluorogenic anophosphate analog34hematological and biochemical analyses whole blood was immediately analyzed for complete blood count with platelet count using the fully automated analyzer abx cobas micros roche germany estimation of serum alanine aminotransferase alt aspartate aminotransferase ast and albumin alb assays follow the recommendations of the international federation of clinical chemistry ifcc but were optimized for performance and stability using the rochehitachi cobas c 311systemstatistical analysis the statistical analysis was performed using oneway analysis of variance anova and the groups were compared by tukeykramer test viability percentage at different concentrations and body weight change analyzed by twoway anova followed by bonferroni™s posttest graphs were sketched using graph pad prism isi® software usa version software data were presented as mean ± standard error se and p values considered significantresultsin vitro studieseffect of bromelain and gammairradiation blirr on tumor cell growth and viability the radiosensitizing effect of bl on eac cells was determined by performing mtt assay eac cells exposed to gy Îradiation showed high cell viability percentage reflecting a radioresistance of eac cell line while bl treatment showed in vitro cytotoxic activity with ic50 value of mgml however the maximum cytotoxic effect appeared when the eac cells were subjected to bl then Îradiation gy compared to control or irradiated group with ic50 mgml table effect of bromelain and some natural compounds as free radical scavengers the inhibitory percentage of each compound is shown in figure the ec50 value concentration of sample that causes dpph activity loss is a reliable way for estimation of the radical scavenging activity the ec50 value of glutathione referenced antioxidant is mgml while table cytotoxic activity of blirr against eac cell line bromelain concentration mgmleac bl mgmlic50 mgmlviability non irradiated eac irradiated eac48ab59ab60ab69a787a10ab158ab18ab27ab52ab ± ± each value indicates the mean of records statistical analysis carried out by twoway anova followed by bonferroni posttests a significant versus control ehrlich ascites carcinoma eac group where b significant versus irradiated eac group at p ic50 ± se values were calculated by using nonlinear regression analysisbromelain and garlic oil ec50 are almost equal and mgml respectively however the naringin phenolic antioxidant is the least potent one ec50 mgml in this comparisonin vivo studieseffect of bromelain and gammairradiation blirr on tumor weight and volume table shows a significant decrease in tumor weight in groups treated with bl andor Îirradiation as compared to the est nontreated group the more drastic decrease in the tumor weight ratio observed in the combined therapy group bl irr compared with the estirradiated group as well as est group indicates that combination therapy is more significantly effective than single agent therapy the photograph of est xenografts at the time of sacrifice shows the synergistic effect of bl and irr on tumor volume figure effect of bromelain and gammairradiation blirr on tumor histopathological features of est bearing mice histopathological examination of solid tumor sections revealed typical malignant features including sheets of malignant cells infiltrating adjacent muscular tissue the malignant cells show pleomorphism hyperchromatism and mitotic activity while the necrotic cells appear as nonviable homogenous structureless material with degenerated or karyorrhectic nuclei untreated est bearing group shows a deeply stained tumor cells arrow head and areas of necrosis arrow figure 3a — also it displays intact cancer cells arrow and giant cells arrow figure 3b and c — the estirradiated group shows muscle fibers invaded by deeply stained tumor cells arrow head and large areas of necrosis arrow figure 3d — also displays a notable necrosis of cancer cells n figure 3e — the bl treated group shows a 0cmekkawy figure dpph 1diphenyl2picrylhydrazyl reduction curve for glutathione bromelain naringin and garlic oil each value represents mean ± se all experiments were replicated timestable tumor weight and inhibition ratio of ehrlich solid tumor estbearing mice treated with gammairradiation irr gy — andor bromelain bl mgkggroupsestest irrest blest bl irrtumor weight g ± ± 004a ± 005a ± 005abtumor inhibitory ratio ” ± 24a ± 14ab ± 204ababbreviations est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationthe values shown are mean ± se of data a significant versus est group where b significant versus estirradiated group at p figure a photograph of ehrlich solid tumor est xenografts at the time of scarification showing the effect of bromelain and gammairradation blirr on tumor volume e ehrlich solid tumor e ir ehrlich solid tumor irradiation e br ehrlich solid tumor bromelain e ir br ehrlich solid tumor bromelain irradiation 0c integrative cancer therapies figure photo micrograph of ehrlich solid tumor est xenografts in different animal groups est sections show the degree of tumorogenesis necrosis n regression of tumor by appearance of muscle fibers m — and — a b c est ehrlich solid tumor d e est irr ehrlich solid tumor irradiation f g est bl ehrlich solid tumor bromelain h i est bl irr ehrlich solid tumor bromelain irradiationwide area of necrosis arrow and n few groups of cancer cells arrow head and muscle fiber m figures 3f — and 4g — however combined treatment bl irr displays muscle fiber m significant regression of tumor or wide areas of necrotic cancer cells n and few groups of intact cancer cells arrow figure 3h — and 3i —the tumor area percentage per total tissue area could determine the degree of proliferation as seen in figure there is a great regression of tumor area in the group treated with bl alone or bl and irr compared with untreated est or estirradiated group indicating that combination therapy significantly more effective than single agent therapy 0cmekkawy bl irr shows nonsignificant group change additionally bl irr group significantly upregulated pparα expression compared with est and estirradiated groups indicating that bl might have a hepato as well as radioprotective effect figure effect of bromelain and gammairradiation blirr on the hepatic lipid peroxidation lpo level reactive oxygen species ros content and paraoxonase1 pon1 activity of ehrlich solid tumor est bearing mice lpo in liver tissues significantly increased in all est bearing groups compared to the control group except the combined treated group irr bl succeeded in returning mda lpo measured as mda malondialdehyde level to the normal level however liver ros significantly increased only in untreated and Îirradiated est bearing groups when compared to the control group while a significant decrease in liver ros showed in estirradiated mice treated with bl in comparison with both est untreated and estirradiated groups pon1 activity in liver homogenate was significantly decreased in est untreated and estirradiated groups when compared with the control group bl treated groups revealed significant increases in pon1 when compared with both est untreated and estirradiated groups showing that bl might have a hepato and radioprotective effect figure effect of bromelain and gammairradiation blirr on hematological measurements wbcs and plts were significantly elevated while hgb and hct significantly decreased in the untreated estbearing mice in comparison with control mice however Îirradiation resulted in a significant decrease in wbcs rbcs plt hgb and hct compared with the control mice treatment of the estbearing mice with bl shows a significant amelioration in wbcs plt and hct compared to est untreated mice combined treatment bl irr shows an enhancement in wbcs plt and hct compared to est untreated and gammairradiated est bearing mice table effect of bromelain and gammairradiation blirr on the serum alanine transaminase alt aspartate transaminase ast and albumin alb to investigate the cytoprotective effects of bl against irradiation the levels of serum alt ast and alb were measured figure it was found that alt and ast significantly increased and conversely alb significantly decreased in est untreated and estirradiated groups compared with the control group however estbearing mice treated with bl alone show nearly the same result of alt and alb as control values estbearing mice treated with bl in combination with irradiation initiated a significant decrease in ast and alt as compared with estirradiated group which may reflect a potential hepatic radioprotective effect of blfigure percentage of tumor areatotal tissue area of ehrlich solid tumor est bearing mice treated with gammairradiation irr gy — andor bl mgkg the values shown in the plotted area are mean of records from animals ± se significant versus est group where significant versus estirradiated group at p effect of bromelain and gammairradiation blirr on body weight change and relative liver weight regarding the day by day documented recording of body weight bwt illustrated in figure there is almost no change in bwt of bl treated group while it increases significantly in the untreated est group conversely estirradiated groups with or without bl treatment show a significant decrease in bwt when compared with the control group table relative liver weight was compared after normalization to mg body weight untreated estbearing group shows a significant increase in liver weight by hepatomegaly while nonsignificant changes were observed in bl treated groups compared to the normal group table effect of bromelain and gammairradiation blirr on the poly adpribose polymerase parp1 nuclear factor kappa b nfκb and peroxisome proliferatoractivated receptors pparα relative gene expression of ehrlich solid tumor est bearing mice to test the possibility that bl reduces radiation damage to the liver mrna gene expression of parp1 nfκb and pparα was measured in the liver homogenates of est bearing mice and compared to control pbs treated mice the results illustrated in figure show that irr causes significant increases in parp1 and nfκb expression compared to the control group however combined treatment bl irr shows a significant increase in parp1 and nfκb expression compared to control group and a significant attenuation compared to estirradiated groupmoreover all est bearing groups show significant decreases in hepatic pparα relative gene expression compared to the control group except the combined therapy 0c integrative cancer therapies figure effect of bromelain and gammairradiation blirr on body weight during experiment period each value represents the mean of records ± se significant versus control group where significant versus est group and significant versus estirradiated group at p est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationtable change in body weight and relative liver weight of control mice and ehrlich solid tumor est bearing mice treated with gammairradiation irr gy — andor bromelain bl mgkggroupscontrolestest irrest blest bl irrbody weight change ± ± 101aˆ’ ± 217abˆ’ ± 201bˆ’ ± 341abrelative liver weight ± ± 026a ± 022a ± 026a ± 026ababbreviations est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationeach value represents the mean ± se a significant versus control group where b significant versus est group at p body weight changes percent are related to the initial weight of animalsdiscussionresistance of tumor cells to chemoradiotherapy as well as the damaging effects to nearby normal tissues remains a major obstacle to successful cancer management therefore the current study has been conducted to estimate the effect of bromelain bl as a tumor radiosensetizer and to show to what extent it can protect normal tissue from radiation hazardsradiosensitizers are compounds that when combined with radiation therapy achieve greater cytotoxicity they can be determined in vitro by the mtt assay2635 the present study has found that the radioresistant eac cells could be sensitized when incubated with bl before irradiation it was known previously that in vitro treatment with bl on mouse tumor cell lines resulted in inhibition of cell growth and invasion capacities3637 the anticancer property of bl has been mainly attributed to the protease component through digestion and diffusion in tumor cells38 it may also be due to the bl enhancement of p53 expression as well as another activator of apoptosis eg bax39 in addition it decreases the activity of cell survival regulators such as akt and erk it also deactivates aktdependent proapoptotic regulator foxo3a thus promoting apoptotic cell death in tumors40it is well known that during cancer and radiotherapy excessive energy is used from the host41 ultimately contributing to mechanisms that promote loss of weight as shown in the present study which also showed that bl could return body weight to a normal level by decreasing tumor weight and volume currently the combined therapy bl irr has been shown to be more effective than single agent therapy in reducing tumor volume and weight indicating that bl could possess a radiosensitizing effect in addition the combined therapy has revealed a drastic decrease in tumor area percentage wide areas of necrotic cancer cells and presence of muscle fiber in the histopathological examination compared with the control est and estirradiated groups this seems to be in agreement with other findings of the role of bl in reducing metastasis and local tumor growth2342 in chemically induced mouse skin papillomas topical application of bl reduced tumor formation tumor volume and caused apoptotic cell death39 bl is a hydrolytic enzymatic complex which shows an efficient digestion and diffusion in tumor cells through attacking the glycosidic linkages and hence denatures glycoproteins thus it protects against tumor growth37 another study has demonstrated the use of controlled proteolytic activity on tumor as a successful strategy to increase therapeutic efficacy43 0cmekkawy figure effect of bromelain and gammairradiation blirr on relative gene expression of liver a parp1 b nfκb and c pparα each value represents the mean of records ± se significant versus control group where significant versus est group and significant versus estirradiated group at p est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationthe aim of the radiotherapy protocols is to achieve the maximum curative effect on tumor cells with minimal damaging effect on normal cells hence antioxidants and other nutrients which do not interfere with therapeutic modalities for cancer may enhance the killing property decrease side effects and protect normal tissue44for estimation of the antioxidant ability of bl dpph assay was conducted in vitro and the free radical inhibitory action of bl was compared with some antioxidant compounds it was found that bl has a powerful free radical scavenging power bl belongs to thiol proteases in which the catalytic nucleophile is sulfhydryl groups of cysteine residues which in turn accounts for its antioxidant activity45the involvement of ros mda and pon1 are important mechanisms that play a vital role during radiation toxicity the use of antioxidants is an important preventive to decrease the toxic and pathological effects associated with oxidative stress caused by radiation46 the attained results show a hepatic impairment on the third day from exposure to Îradiation elevation of lpo and ros levels and inhibition of pon1 activity compared to normal mice however treatment with bl revealed an amelioration in hepatic damage caused by irradiation these results were in accordance with liu 47 who described the effect of radiation induced ros generation which in turn might attack cell membrane phospholipids and circulating lipids and thus increases production of mda48 lpo acts as a sensitive biomarker for oxidative stress that occurs as part of the pathogenesis of irradiation49 bl has sulfhydryl groups consequently accounting for its antioxidant activity45 thus it could act as ros scavengermeasurement of pon1 postradiotherapy could be an effective clinical biomarker of hepatic and systemic oxidative stress and may be used as an index of the usefulness of radiotherapy50 it has been demonstrated to catalyze hydrolysis of lipid hydroperoxides and lactones51 pon1 protects serum hdl and ldl ps against lipid peroxidation52 in the present study the decreased activity of pon1 upon radiation exposure might be due to its super saturation of lipid hydroperoxides and lactones upon treatment with bl the activity of pon1 was restored near to the normal level hence the pon1
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Supraclavicular Recurrence inCompletely Resected ypN2NonSmall Cell Lung CancerImplications for PostoperativeRadiotherapyLiang Liu   Zhiqin Zheng   Juan Li Yuan Li and Jianjiao Ni Department of Radiation Oncology Fudan University Shanghai Cancer Center Shanghai China Department of OncologyShanghai Medical College Fudan University Shanghai China Department of Radiation Oncology Minhang BranchHospital Fudan University Shanghai Cancer Center Shanghai China Department of Pathology Fudan University ShanghaiCancer Center Shanghai Chinatarget volume CTVBackground The clinical value and delineation of clinicalof postoperative radiotherapy PORTin completely resected ypN2 nonsmall celllung cancer NSCLC remain controversial Investigations specifically focusing on thecumulative incidence and prognostic significance of initial disease recurrence at thesupraclavicular region SCR in this disease population are seldom reportedMethods Consecutive patients with curatively resected ypN2 NSCLC who receivedadjuvant chemotherapy from January to December at our cancer center wereretrospectively examined Disease recurrence at the surgical margin ipsilateral hilumandor mediastinum was defined as locoregional recurrence LRR Disease recurrencebeyond LRR and SCR was defined as distant metastasis DM Overall survival OS1 andOS2 were calculated from surgery and disease recurrence to death of any cause in theentire cohort and in patients with recurrent disease respectivelyResults Among the patients enrolled PORT without elective supraclavicularnodal irradiation ESRT was performed in patients and neoadjuvant chemotherapywas administered in patients With a median followup of months patientsdeveloped recurrent disease including SCRs among which were without DMand involved the ipsilateral supraclavicular region The and 5year cumulativeincidence of SCR were and respectively Chosen DM as a competingevent cN2 ypN2 not receiving lobectomy and negative expression of CK7 weresignificantly associated with SCR using the univariate competing risk analysis whileypN2 was identified as the only independent risk factor of SCR p PORTsignificantly reduced LRR p and prolonged OS1 p but didn™timpact SCR p Pattern of failure analyses indicated that the majority of LRRsdeveloped within the actuarial or virtual CTV of PORT and of the ipsilateralSCRs could be covered by the virtual CTV of proposed ESRT In terms of OS2patients who developed SCR but without DM had intermediate prognosis comparedwith those who had DM p and those who had only LRR p Edited byStephen V LiuGeetown University MedicalCenter United StatesReviewed byTim KruserNorthwestern Medicine United StatesHeloisa De Andrade CarvalhoUniversity of S£o Paulo BrazilCorrespondenceJianjiao Ninijianjiao8sinacom These authors have contributedequally to this workSpecialty sectionThis was submitted toThoracic Oncologya section of the journalFrontiers in OncologyReceived May Accepted July Published August CitationLiu L Zheng Z Li J Li Y and Ni J Supraclavicular Recurrence inCompletely Resected ypN2NonSmall Cell Lung CancerImplications for PostoperativeRadiotherapy Front Oncol 103389fonc202001414Frontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLCConclusions SCR is not uncommon and has important prognostic significance incompletely resected ypN2 NSCLC The clinical value of PORT and ESRT in suchpatients need to be further investigatedKeywords supraclavicular recurrence postoperative radiotherapy nonsmall cell lung cancer overall survivalclinical target volumeINTRODUCTIONStage III nonsmall cell lung cancer NSCLC is a heterogeneousdisease and surgical resection with or without neoadjuvanttherapy could be carried out in selected patients Aftercurative resection disease recurrence poses a considerablethreat and it has been demonstrated that platinumbasedadjuvant chemotherapy could significantly reduce postoperativerecurrence and improve 5year survival Howeveralthough numerous retrospective studies and several populationbased investigations “ have suggested a beneficial role ofpostoperative radiotherapy PORT in reducing locoregionalrecurrence LRR prolonging diseasefree survival DFS andeven improving overall survival OS among patients withcompletely resected ypN2 NSCLC the clinical valueof PORT is still controversial due to a lack of convincing datafrom large randomized clinical trials Moreover there is no definite agreement on the delineationof clinical target volume CTV during PORT for completelyresected ypN2 NSCLC and it varies between diï¬erentinstitutions and clinical trials The rationales of CTVdelineation are mostly based on the patterns of disease recurrencein surgical resected patients who don™t receive PORT and patternsof treatment failure in those who receive PORT In these studiescumulative incidence anatomic locations and risk factors of LRRwere extensively examined However the definitions of LRR arediï¬erent some of which include the initial disease recurrencedeveloped in the supraclavicular region SCR whileothers don™t Investigations specifically focused on SCR areseldom reported and elective supraclavicular nodal irradiationESRT is not routinely performedIn the current study we investigated the cumulative incidencerisk factor and prognostic significance of SCR in completelyresected ypN2 NSCLC Additionally our recent study findscrucial prognostic value of routine immunohistochemical IHCmarkers in completely resected NSCLC Hence besidescommon clinicpathological variables a list of routine IHCmarkers were examined when investigating the risk factorsof SCRMATERIALS AND METHODSPatientsLung cancer patients who received surgery at Fudan Universityfrom January toShanghai Cancer Center FUSCCreviewed PatientsDecemberwho underwentresection withpathologically confirmed N2 disease and received standardretrospectively werecompletesurgicaladjuvant chemotherapy were included in the study Patientsreceived PORT or not as well as neoadjuvant chemotherapyor not were both allowed to be included Exclusion criteriaincluded a second primary tumor compromised resectionpositive surgical margins neoadjuvant radiotherapy receivingno adjuvant chemotherapy death due to surgical complicationsand postoperative follow up monthsinvasion perineuralFor each patient common clinicpathological parameterswere gathered from the electronic medical records including agesex smoking history the Eastern Corporative Oncology GroupECOG performance score clinical TNM stage pathologicalTNM stage primary tumor size tumor diï¬erentiation tumorlymphovascular invasion visceralhistology tumor locationpleuralinvasion and type of surgeryPathologic TNM stage was in accordance with the eighth editionLung Cancer Stage Classification Tumor diï¬erentiationand tumor histology were determined on the basis of the World Health anization Classification of Tumors of the LungPleura Thymus and Heart Besides the expression statusof IHC markers ie HER2 TTF1 ERCC1 CK20 CK56CK7 P63 NapsinA Syn RRM1 EGFR and Ki67 were collectedThe IHC staining and evaluation were routinely performedin the Immunohistochemistry Diagnostic Laboratory of ourcancer center Our study followed The Declaration of HelsinkiThe institutional review board of FUSCC approved the studyInformed consent was waived by the institutional review boardbecause this was a retrospective studyTreatmentPretreatment evaluation generally included clinical assessmentblood test bronchoscopy contrastenhanced chest computedtomography CT scan ultrasonographic examination or CTthe abdomen brain magnetic resonance imagingscan ofMRI and bone scans Patients with mediastinallymphnode enlargement cm in the short axis on CT scan orpathologically proven to be malignant were defined as harboringclinical N2 cN2 disease Of note positron emission tomographyPETCT as well as invasive staging of the mediastinum wasstrongly recommended for patients with cN2 disease at ourcancer centerNeoadjuvanttherapy generally consisted of “ cyclesof platinumbased doublet regimen and surgicaltreatmentincluded lobectomy sublobectomy and pneumonectomy withsystematic multilevel mediastinallymph node dissection oradequate mediastinal sampling no N2 stations must includethe subcarinal station PORT was performed according toour institutional protocol using the intensitymodulatedradiation therapy technique employing a linear accelerator withFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLC6MV Xrays Briefly the CTV for left lung cancers included thebronchial stump and 2R 2L 4R 4L and 11L lymphnode stations while the CTV for right lung cancers included thebronchial stump and 2R 4R and 11R stations ESRT wasnot performed The total radiation dose prescibed to the planningtarget volume PTV was generally Gy administered daily at Gy per fraction days per weekFollow UpFollowups were at the discretion of the treating physicians andwere generally scheduled at regular intervals every monthsafter surgery in the first years every months for the next years and annually thereafter During followup blood testschest CT scans and CT scans or ultrasonographic examination ofabdominal and cervical regions were routinely performed whilebrain MRI and bone scans were not mandatory Telephone callswere also implemented when necessaryPostoperative recurrence was diagnosed considering allthe evidence provided by imaging scans and pathologicconfirmation Initial disease recurrence in the supraclavicularregion was defined as SCR and first relapse developed at thesurgical marginipsilateral hilum andor mediastinum wasconsidered LRR Initial disease recurrence beyond LRR and SCRwas categorized as distant metastasis DMPattern of Failure AnalysesFor patients with LRR the PTVs were restored for thosewho received PORT and virtual PTVs were created for thosewho didn™t receive PORT by independent radiation oncologistaccording to ourinstitutional protocol mentioned aboveMeanwhile for patients with SCR individual virtual PTVs werecreated for ipsilateral ESRT PTVsc by independent radiationoncologist according to the CT atlas proposed by Lynch et al Then we plotted the sites of LRRs andor SCRs and overlaidthem with restored or created PTVs Coverage of the LRRs andSCRs by the PTVs were investigatedStatistical AnalysesRecurrence free survival RFS was calculated from surgery toinitial disease recurrence Overall survival OS1 was calculatedfrom surgery to death of any cause in the entire cohort andOS2 was calculated from initial disease recurrence to deathof any cause in patients with recurrent disease Diï¬erences andbetween clinical parameters were compared using the χFisher™s exact tests The predictors of SCR were selected usingcompeting risk methodology and Stata version softwareStataCorp College Station TX USA The associations betweenclinicpathological parameters and OS were identified using theCox proportional hazard regression model The hazard ratioHR and the confidence interval CI were calculatedusing coefficients from the model Kaplan“Meier methodwas used to estimate survival and diï¬erences among groupswere investigated by the logrank test Statistical analysis wasperformed using SPSS SPSS Chicago IL USA Allassessment is considered to be significant when twosided pvalueis RESULTSPatients CharacteristicsA total of patients were finally enrolled and a flowchartfor patient selection was presented in Supplementary Figure Detailed baseline disease characteristics of the patientswere summarized in Table The majority of patients had ahistology of nonsquamous NSCLC and received lobectomyTABLE Disease characteristicsVariablesNumber of patients Age at diagnosis years‰¤SexFemaleMaleSmoking historyEver smokerNever smokerECOG performance scoreClinical N stagecN0“cN2Neoadjuvant chemotherapyYesNoSurgery typeSublobarLobectomyPneumonectomyPathological T stagepT0“pT3“Lymphovascular invasionAbsentPresentVisceral pleural invasionAbsentPresentTumor locationLeft lower lobeLeft upper lobeRight lower lobeRight middle lobeRight upper lobeHistologySquamous cell carcinomaNonsquamous nonsmall cell lung cancerECOG Eastern Corporative Oncology Group Frontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLCFIGURE Patterns of supraclavicular recurrence A Venn diagram demonstrating the distribution of initial postoperative recurrence B Pie chart demonstrating thedistribution of SCR SCR supraclavicular recurrence LRR locoregional recurrence DM distant metastasisFIGURE Cumulative incidence and dynamics of supraclavicular recurrence A Cumulative incidence of supraclavicular recurrence in the entire cohort andstratified by pathological status ypN2 vs pN2 B The dynamics of hazard ratio of supraclavicular recurrenceThe positive rate of HER2 TTF1 ERCC1 CK20 CK56 CK7P63 NapsinA Syn RRM1 and EGFR was and respectivelyAdditionally Ki67 ‰¥ was detected in of the patientsPretreatment PETCT was performed in patients andinvasive staging of the mediastinum was underwent in patients One hundred and sixtyfour patients were found tohave cN2 disease among whom patients receivedpretreatment PETCT and patients had invasivestaging of the mediastinum A total of patientsreceived neoadjuvant chemotherapyCumulative Incidence and Risk Factors ofSCRPost surgery patients received PORT and with a medianfollow up of range “ months patients developedrecurrent disease including SCRs Of note of the SCRswere pathologically confirmed and the rest were diagnosed byclinical assessments and radiographic findings The and year RFS were and in patients without PORTrespectively and were and in patients withPORT respectively Among the patients with SCR patients developed SCR without DM Figure 1A and patients developed SCR involving the ipsilateral supraclavicularregion Figure 1B Moreover among the patients with leftlung cancer who developed SCR seven were ipsilateral threebilateral and two contralateral Among the patients with rightlung cancer who developed SCR nine were ipsilateral threebilateral and three contralateralThe and 5year cumulative incidence of SCR were and respectively Figure 2A and the dynamicof hazard ratio of SCR was presented in Figure 2B ChosenDM as a competing event cN2 disease ypN2lobectomyand CK7 were identified as significant risk factors of SCRFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLCTABLE Competing risk analyses of clinicalpathological variables associatedwith supraclavicular recurrenceVariablesUnivariate Analyses Multivariate AnalysesHR 95CIpHR 95CIpAge vs ‰¤ “ Sex Male vs Female “ Smoking Never vs Ever “ ECOG vs cN2 vs “ “ “ pT stage T3“ vs T0“ “ pN1 vs “ “ Multiple levels of pN2 vs “ “ Histology SCC vs NonSCC “ Differentiation P vs WM “ LVI vs “VPI vs “PNI vs “ypN2 vs pN2 “ “ “ “ “ Tumor Location Left vs Right “ Tumor Lobe Upper vs Others “ TLN ‰¥ vs “ PLN ‰¥ vs “ LNR ‰¥ vs “ Surgery Others vs Lobectomy “ “ PORT vs “ERCC1 vs “Her2 vs “ “ “ “ Ki67 ‰¥ vs “ TTF1 vs “CK20 vs “CK7 vs “CK56 vs “P63 vs “ “ “ “ “ “ “ NapsinA vs “ “ Syn vs “RRM1 vs “EGFR vs “ “ “ “ HR hazard ratios CI confidence intervals SCC squamous cell carcinoma LVILymphovascular invasion VPI Visceral pleuralinvasion ECOGthe Eastern Corporative Oncology Group TLN total lymph node examined PLN positivelymph node LNR positive lymph node ratio PORT postoperative radiotherapy WMwellmoderate P poor Bold values indicates statistical significantinvasion PI perineuralusing the univariate competing risk analysis Table Sincethere was a significant association between cN2 disease and testreceiving neoadjuvant chemotherapy p χwe excluded cN2 disease and included the otherthreesignificant risk factors in the multivariate competing riskanalyses The result showed that only ypN2 were identifiedas an independent risk factor of SCR Table The and 5year cumulative incidence of SCR were and among ypN2 patients respectively and were and among pN2 patients respectivelyFigure 2APattern of Failure AnalysesIn the entire cohort of the patients who receivedPORT developed LRR while of the patientswho did not receive PORT developed LRR PORT significantlyreduced the risk of LRR Figure 3A Among the six patients whoreceived PORT and subsequently developed LRR five developedLRR only within the PTV and the rest one developed LRRboth within and outside the PTV Among the patients whodid not receive PORT and subsequently developed LRR developed LRR only within the proposed PTV three developedLRR both within and outside the proposed PTV and the restone developed LRR outside the proposed PTV That patienthad adenocarcinoma in the middle lobe of right lung withpathologically proven metastatic lymph node in the right hilumand station but developed recurrent disease at mediastinallymph node stations and On the other hand of the patients who receivedPORT developed SCR while of the patients whodid not receive PORT developed SCR in the entire cohort PORTwithout ESRT didn™t reduce the incidence of SCR Figure 3BFifteen of the ipsilateral SCRs could be covered by theproposed PTVsc and the ipsilateral parts of the six bilateral SCRscould all be covered by the proposed PTVscSurvival AnalysesBy the time of data cutoï¬ patients had died and the medianOS1 was 95CI “ months PORT was found tosignificant prolong OS1 in the entire cohort Figure 3C Agesex ECOG scorelymphovascular invasion total number ofpositive lymph node positive lymph node ratio PORT and Ki67were found to be significantly associated with OS1 in univariateCox analyses while age ECOG score PORT and Ki67 wereidentified to be independent indicators of OS1 in multivariateCox analyses Table Among the patients with recurrentdisease the median OS2 was 95CI “ monthsAge sex ECOG score and DM were revealed to be significantlyassociated with OS1 in univariate and multivariate Cox analysesTable In order to investigate the prognostic significance of SCRpatients with recurrent disease were further divided into threegroups Group A consisted of patients who had DM n Group B consisted of patients who did not have DM but have SCRn and Group C consisted of patients who only had LRR n In terms of OS2 patients in Group B had an intermediateprognosis when compared with patients in Group A and GroupC Figure 3DDISCUSSIONTo the best of our knowledge this is the first comprehensivestudy specifically focusing on SCR in completely resected ypN2NSCLC with a relatively large sample size in the era of modernradiation technique SCR was not uncommon and had imperativeprognostic significance indicating that treatment modalities ableto reduce the incidence of SCR may be beneficial AdditionallyPORT without ESRT significantly reduced LRR and prolongedOS but did not decrease SCR in our study suggesting that theFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLCFIGURE Prognostic significance of postoperative radiotherapy and supraclavicular recurrence The impact of postoperative radiotherapy PORT on locoregionalrecurrence LRR A supraclavicular recurrence SCR B overall survival OS1 C in the entire cohort Kaplan“Meier survival curve stratified by the diseaserecurrence patterns among patients with recurrent disease Dclinical value of ESRT may be reconsidered in selected patientswith high risks of SCRSCR is not uncommon in completely resected ypN2 NSCLCespecially among those with extra risk factors Although therewas limited historical data published that could be directlycompared the incidence of SCR in our study was reliable sincethe overall recurrence rate and the percentage of SCR amongpatients with recurrent disease were in accordance with previousfindings The cumulative incidence of postoperative recurrencein the PORT group and nonPORT group were generallycomparable with recent studies Furthermorestudies from our institution and others had reported asimilar percentage of SCR among patients with recurrent disease“ in the literature in our study Compared withtheir counterpart patients staged cN2 or ypN2 generally had amore advanced and aggressive disease and thus it was reasonablefor them to have a higher risk developing disease recurrenceincluding SCR “ Compared with those receivinglobectomy patients receiving pneumonectomy generally hada higher tumor burden and those receiving sublobectomycommonly had unfavorable prognostic factors such as morecomorbidities and poorer preoperative lung functions that madethem unsuitable for lobectomy Therefore patients whodidn™t receive lobectomy were also at a higher risk developingpostoperative recurrence which is generally consistent with arecent retrospective study using the SEER database Inaddition two recent studies found that positive expression ofCK7 were associated with more advanced disease and shorteroverall survival In our study distant metastasis waschosen as a competing event and negative expression of CK7was identified as a risk factor of SCR which need to befurther verifiedCompared with patients developing only LRR and thosedeveloping DM patients developing SCR but without DM hadintermediate OS2 highlighting the vital prognostic significanceof SCR in curatively resected ypN2 NSCLC The TNMstaging system is one ofindicators ofpatient™s prognosis in NSCLC among which patients havingsupraclavicular lymph node metastasis N3 generally haveintermediate prognosis when compared with those having distantmetastasis M1 and those harboring metastatic tumor lesionslimited to the ipsilateral hilar N1 or mediastinal N2 lymphthe most powerfulFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLCTABLE Cox analyses of clinicalpathological variables associated with overallsurvival OS1TABLE Cox analyses of clinicalpathological variables associated with OS2 inpatients with recurrent diseaseVariablesUnivariate Analyses Multivariate AnalysesVariablesUnivariate AnalysesMultivariate AnalysesHR 95CIpHR 95CIpHR 95CIpHR 95CIpAge vs ‰¤ “ “ Sex Male vs Female “ “ Smoking Never vs Ever “ ECOG vs cN2 vs “ “ “ “ pT stage T3“ vs T0“ “ pN1 vs “ “ Multiple levels of pN2 vs “ “ Histology SCC vs NonSCC “ Differentiation P vs WM “ LVI vs “VPI vs “PNI vs “ypN2 vs pN2 “ “ “ “ “ Tumor Location Left vs Right “ Tumor Lobe Upper vs Others “ TLN ‰¥ vs “ Age vs ‰¤ “ “Sex Male vs Female “ “Smoking Never vs Ever “ECOG vs cN2 vs “ “ “ “pT stage T3“ vs T0“ “pN1 vs “ “Multiple levels of pN2 vs “ “Histology SCC vs NonSCCDifferentiation P vs WM “ “LVI vs “VPI vs “PNI vs “ypN2 vs pN2 “ “ “ “Tumor Location Left vs Right “Tumor Lobe Upper vs Others “TLN ‰¥ vs “PLN ‰¥ vs “LNR ‰¥ vs “PLN ‰¥ vs “ “ Surgery Others vs Lobectomy “LNR ‰¥ vs “ “ Surgery Others vs Lobectomy “ PORT vs “ERCC1 vs “Her2 vs “ “ “ “ “ Ki67 ‰¥ vs “ “ TTF1 vs “CK20 vs “CK7 vs “CK56 vs “P63 vs “ “ “ “ “ “ NapsinA vs “ “ Syn vs “RRM1 vs “EGFR vs “ “ “ “ HR hazard ratios CI confidence intervals SCC squamous cell carcinoma LVILymphovascular invasion VPI Visceral pleuralinvasion ECOGthe Eastern Corporative Oncology Group TLN total lymph node examined PLN positivelymph node LNR positive lymph node ratio PORT postoperative radiotherapy WMwellmoderate P poor Bold values indicates statistical significantinvasion PI perineuralnodes Similarly SCR represented an unfavorable sign ofsubsequent disease metastasis to distant ans and thus wasreasonable to have worse prognosis when compared with thosewho had only LRR On the other hand when compared withthose who already had DM patients who had recurrent diseaselimited to the thoracic region ie LRR and SCR could beconsidered as harboring locoregional disease and may benefitfrom aggressive locoregional treatment as well as systematictherapies and thus may still have a chance of longterm survival In fact among the patients with SCR but without DMthe 3year survival rate exceeded in our study Figure 3DHowever due to the advancement of adjuvant chemotherapy andPORT vs “DM vs “ERCC1 vs “Her2 vs “ “ “ “ “ “Ki67 ‰¥ vs “TTF1 vs “CK20 vs “CK7 vs “CK56 vs “P63 vs “NapsinA vs “Syn vs “RRM1 vs “EGFR vs ““ “ “ “ “ “ “ “ “OS overall survival HR hazard ratios CI confidence intervals SCC squamouscell carcinoma LVI Lymphovascularinvasion PIperineural invasion ECOG the Eastern Corporative Oncology Group TLN total lymphnode examined PLN positive lymph node LNR positive lymph node ratio PORTpostoperative radiotherapy DM distant metastasis WM wellmoderate P poor Boldvalues indicates statistical significantinvasion VPI Visceral pleuralPORT the number of patients who developed localized recurrentdisease ie LRR and SCR was small patients in group Band patients in group C although a total of patients wereenrolled and followed up for a median of months Hencethe prognostic significance of SCR needed to be interpreted withcaution and future investigations with larger sample size andprospective design are warrantedThe clinical value of PORT in completely resected ypN2NSCLC was demonstrated again in our study but the delineationof CTV remain controversial In the current study PORTsignificantly reduced LRR and improved OS1 which havebeen demonstrated in various studies “ However since ESRT was not routinely performed in our cancerFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLCindicating thatinstitution PORT failed to reduce SCRthe majority of SCRs represented the outgrowth of subclinicaltumor lesions already in the supraclavicular region and werenot originated from the locoregional recurrent disease throughlymphatic metastasis In fact of the patientswith SCR had no LRR in the current study These dataindicated a potential role of ESRT in selected patients withhigh risks Actuallyfor locally advanced NSCLC receivingchemoradiotherapy there is no significant diï¬erence of patient™ssurvival between those with or without N3 disease highlighting that the treatment efficacy of chemoradiotherapyin locally advanced NSCLC was largely dependent on theintrinsic biology of the tumor and the prognosis of patients withor without macroscopic supraclavicular tumor lesions seemedsimilar PORT with adjuvant chemotherapy has been repeatedlyshown to significantly reduce LRR indicating the beneficial roleof adjuvant chemoradiotherapy in treating microscopic N1N2disease It is possible that adjuvant chemoradiotherapy ieadjuvant chemotherapy in combination with ESRT may alsoplay a role in reducing SCR and subsequently improve patient™ssurvival Furthermore nearly of the ipsilateral SCRs couldbe covered with the virtual CTV of ESRT in our study Howeverthere are also evidence against the use of ESRT for patients withcompletely resected NSCLC Elective irradiation of mediastinalcontralateral hilar and supraclavicular lymph nodes failed toimprove patient™s survivalin unresectable stage III NSCLCwithout clinical N3 disease And pattern of failure analyses ofa prospective trial of PORT without ESRT suggested that the useof limited CTV including only the involved lymph node stationsand those with a risk of invasion was associated withacceptable risk of geographic miss Taken together PORTwithout ESRT provided significant clinical benefit for patientswith completely resected ypN2 NSCLC and the clinical valueof ESRT in highly selected patients for example those withpersistent N2 ypN2 disease after neoadjuvant chemotherapyneed to be further investigatedOur study also has some limitations Firstly since ESRT is notroutinely performed in our cancer center we could not directlyexamine the clinical value and prognostic significance of ESRTSecondly as this was a retrospectively study treatment decisionsand followup strategies were at the discretion of the treatingphysicians Diï¬erent neoadjuvant and adjuvant chemotherapyregimens were used and the protocols of followup were notidentical Moreover since brain MRI and bone scans were notmandatory asymptomatic brain andor bone metastasis may beunderestimated Despite these limitations we believe our studyprovided valuable information about the cumulative incidenceand prognostic significance of SCR in completely resected ypN2NSCLC which may guide better design of adjuvant treatmentmodalities and individualized surveillance strategiesDATA AVAILABILITY STATEMENTThe raw data supporting the conclusions of this will bemade available by the authors without undue reservationETHICS STATEMENTThe studiesinvolving human participants were reviewedand approved by the institutional review board of FudanUniversity Shanghai Cancer Center Written informed consentfor participation was not required for this study in accordancewith the national legislation and the institutional requirementsAUTHOR CONTRIBUTIONSLL ZZ and JN conceptualization LL and ZZ methodologyvalidation and writing”original draft preparation LL ZZ andJL formal analysis and investigation LL ZZ and YL resourcesand data curation LL and JN writing”review and editing Allthe authors have approved the final manuscriptFUNDINGThis study was supported by the National Natural ScienceFoundation of China No to JN and grant provided bythe Shanghai Municipal Health Commission No 20194Y0501to JNSUPPLEMENTARY MATERIALThe Supplementary Materialonline202001414fullsupplementarymaterialfor this can be foundhttpswwwfrontiersins103389foncatSupplementary Figure Flowchart of patient enrollment NSCLC nonsmallcell lung cancerREFERENCES Eberhardt WE De Ruysscher D Weder W Le Pechoux C De Leyn PHoï¬mann H et al 2nd ESMO Consensus Conference in Lung Cancerlocally advanced stage III nonsmallcell lung cancer Ann Oncol “ 101093annoncmdv187 Tan WL Chua KLM Lin CC Lee VHF Tho LM Chan AW et alAsian thoracic oncology research group expert consensus statement onoptimal management of stage III NSCLC J Thorac Oncol “ 101016jjtho201910022 Kenmotsu H Yamamoto N Yamanaka T Yoshiya K Takahashi T Uenostudy of pemetrexed plus cisplatintoT et al Randomized phase IIIversus vinorelbine plus cisplatin for completely resected stage IIIIIA nonsquamous nonsmallcell“ 102139ssrn3460654lung cancerJ Clin Oncol Kato H Tsuboi M Kato Y Ikeda N Okunaka T Hamada C Postoperativeadjuvant therapy for completely resected earlystage nonsmall cell lungcancer Int J Clin Oncol “ 101007s101470050493x Herskovic A Mauer E Christos P Nagar H Role of postoperativeradiotherapy in pathologic stage IIIA N2 nonsmall cell lung cancer in aprospective nationwide oncology outcomes database J Thorac Oncol “ 101016jjtho201609135 Deng W Xu T Xu Y Wang Y Liu X Zhao Y et al Survival patterns for patientswith resected N2 nonsmall cell lung cancer and postoperative radiotherapya prognostic scoring model and heat map approach J Thorac Oncol “ 101016jjtho2018082021Frontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLC Feng W Fu XL Cai XW Yang HJ Wu KL Fan M et al Patternsof localregional failure in completely resected stage IIIAN2 nonsmallcellimplications for postoperative radiation therapyclinical target volume design Int J Radiat Oncol Biol Phys “ 101016jijrobp201312048lung cancer cases Dai H Hui Z Ji W Liang J Lu J Ou G et al Postoperative radiotherapy forresected pathological stage IIIAN2 nonsmall cell lung cancer a retrospectivestudy of cases from a single institution Oncologist “ 101634theoncologist20100343 Zou B Xu Y Li T Li W Tang B Zhou L et al A multicenter retrospectiveanalysis of survival outcome following postoperative chemoradiotherapy innonsmallcell lung cancer patients with N2 nodal disease Int J Radiat OncolBiol Phys “ 101016jijrobp200905044 Billiet C De Ruysscher D Peeters S Decaluwe H Vansteenkiste J Dooms Cet al Patterns of locoregional relapses in patients with contemporarily stagedstage IIIN2 NSCLC treated with induction c
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coronavirus disease COVID19 pandemic access to surgical care for patients with head and neck cancer HNC is limited and unpredictable Determining which patients should be prioritized is inherently subjective and difficult to assess The authors have proposed an algorithm to fairly and consistently triage patients and mitigate the risk of adverse outcomes METHODS Two separate expert panels a consensus panel participants and a validation panel participants were constructed among international HNC surgeons Using a modified Delphi process and RAND CorporationUniversity of California at Los Angeles methodology with consensus rounds and meetings groupings of highpriority intermediatepriority and lowpriority indications for surgery were established and subdivided A pointbased scoring algorithm was developed the Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancer SPARTANHN Agreement was measured during consensus and for algorithm scoring using the Krippendorff alpha Rankings from the algorithm were compared with expert rankings of case vignettes using the Spearman rank correlation coefficient RESULTS A total of indications for surgical priority were rated Weights for each indication ranged from ˆ’ to scale range ˆ’ to The response rate for the validation exercise was The SPARTANHN demonstrated excellent agreement and correlation with expert rankings Krippendorff alpha [ CI ] and rho [ CI ] S The SPARTANHN surgical prioritization algorithm consistently stratifies patients requiring HNC surgical care in the COVID19 era Formal evaluation and implementation are required Cancer American Cancer Society LAY SUMMARY ¢ Many countries have enacted strict rules regarding the use of hospital resources during the coronavirus disease COVID19 pandemic Facing delays in surgery patients may experience worse functional outcomes stage migration and eventual inoperability¢ Treatment prioritization tools have shown benefit in helping to triage patients equitably with minimal provider cognitive burden¢ The current study sought to develop what to the authors™ knowledge is the first cancer“specific surgical prioritization tool for use in the COVID19 era the Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancer SPARTANHN This algorithm consistently stratifies patients requiring head and neck cancer surgery in the COVID19 era and provides evidence for the initial uptake of the SPARTANHN KEYWORDS coronavirus disease COVID19 delivery of health care head and neck cancer health priorities patient selection surgical procedures waiting listsCorresponding Author John R de Almeida MD MSc Division of Surgical Oncology Department of Otolaryngology“Head and Neck Surgery 8NU883 Toronto General Hospital University Health Network Elizabeth St Toronto ON M5G 2C4 Canada Johndealmeidauhnca Division of Surgical Oncology Department of Otolaryngology“Head and Neck Surgery Princess Margaret Cancer Center University Health Network University of Toronto Toronto Ontario Canada Institute of Health Policy Management and Evaluation Dalla Lana School of Public Health University of Toronto Toronto Ontario Canada Division of Otolaryngology“Head and Neck Surgery Dalhousie University Halifax Nova Scotia Canada Division of Otolaryngology“Head and Neck Surgery McMaster University Hamilton Ontario Canada Department of Otolaryngology“Head and Neck Surgery Western University London Ontario Canada Department of Otolaryngology“Head and Neck Surgery Memorial Sloan Kettering Cancer Center New York New York Head and Neck“Endocrine Oncology Moffitt Cancer Center Tampa Florida Department of Otolaryngology“Head and Neck Surgery Medical University of South Carolina Charleston South Carolina Department of Otolaryngology“Head and Neck Surgery Stanford University Palo Alto California Department of Otolaryngology“Head and Neck Surgery University of Michigan Ann Arbor Michigan Department of Otolaryngology“Head and Neck Surgery The University of Texas MD Anderson Cancer Center Houston Texas Head and Neck Unit The Royal Marsden Hospital London United Kingdom Department of Otolaryngology“Head and Neck Surgery Icahn School of Medicine at Mount Sinai New York New York Department of Otolaryngology“Head and Neck Surgery Sunnybrook Health Sciences Centre University of Toronto Toronto Ontario Canada Department of Otolaryngology“Head and Neck Surgery Sinai Health System University of Toronto Toronto Ontario CanadaThe first authors contributed equally to this Additional supporting information may be found in the online version of this 101002cncr33114 Received June Revised June Accepted June Published online Month in Wiley Online Library wileyonlinelibrarycomCancer Month 0cOriginal INTRODUCTIONOn March the World Health anization declared a global pandemic due to the novel coronavirus severe acute respiratory syndrome coronavirus SARSCoV2 and the resulting coronavirus disease COVID191 As a result in many jurisdictions operating room capacity has been limited to only emergent or urgent surgical procedures2 Several advisory bodies have issued recommendations to safeguard access to oncologic surgery while still acknowledging that treatment delays may be necessary The American College of Surgeons has recommended postponing elective surgery including for patients with lowrisk cancers while recommending that other urgent cancer surgeries proceed34 Cancer Care Ontario has issued similar guidance recommending that hospitals include cancer surgery in their care delivery plan5The time from the diagnosis of head and neck cancer HNC to surgery is a metric with prognostic importance with treatment delays portending poorer oncologic outcomes68 In a recent systematic review evaluating delays in time from diagnosis to treatment initiation of studies demonstrated a decrease in survival to be associated with treatment delays68 These data support the urgency of initiating treatment for patients with HNC but to our knowledge do not inform a stratification schema when operating room access is not available for all patientsAs a result of these new imposed constraints difficult decisions regarding prioritization for cancer surgery are obligatory and require the consideration of broader principles regarding scarce resource allocation9 Key among these is the need for consistency and transparency to achieve fairness and to avoid engendering disparities in both access and outcomes1011 Prioritization on a casebycase basis using expert clinical judgment can be logistically challenging carries a cognitive burden and is susceptible to the biases of practitionersSurgical prioritization tools or algorithms offer decisionmaking transparency and provide equitable and timesensitive access to care to the patients who need it most1213 Although tools for surgical prioritization in the era of COVID19 continue to emerge to our knowledge oncology patients have not been explicitly considered14 Herein we have presented the development and validation of a novel algorithm Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancer SPARTANHN for the prioritization of surgery for patients with HNCMATERIALS AND METHODSThe current study was granted a waiver from the research ethics board at the University Health NetworkParticipants and SettingFor instrument development a group of expert HNC surgeons JRD DPG RG JCI DBC DB AE DJE KMH EM and IJW from institutions University Health Network Sinai Health Systems and Sunnybrook Health Sciences Centre at the University of Toronto participated in the consensus process consensus panel At the time of the consensus process all institutions were operating under significant resource constraints with limited availability of operating room time For instrument validation a group of participants JRD CWN DF DPG and EM completed the scoring algorithm designed after the consensus process Fifteen external head and neck surgeons HZ ACN RJW MAC CM EMG VD AGS AJR CML EYH JM VP BM and EG from institutions across Canada institutions the United States institutions and the United Kingdom institution participated in a ranking exercise of clinical vignettes validation panelScopeThe scope of variables considered in the prioritization algorithm was established and vetted by the consensus panel see Supporting Information All indications for prioritization were presented to the consensus panel using an online survey platform Google Forms httpsdocsgooglecomforms With exceptions survey respondents were asked to consider each of the indications in isolation For wait times panel members were asked to also consider histologic grade Similarly for surgical site the panel was asked to simultaneously consider extent of surgery Related indications were presented sequentially to facilitate pairwise comparison eg stage I and II vs stage III and IV were presented in sequence AJCC 8th edition The list of indications was pilot tested by surgeons JRD DPG EM and RG for sensibility readability content validity language and comprehensibilityConsensus ProcessThe consensus panel participated in a Delphi consensus process with rounds of rating see Supporting Information The first rounds aimed to achieve consensus regarding the priority grouping high intermediate or low High priority was defined as an indication to Cancer Month 0cproceed to surgery within weeks The second rounds of rating involved ranking each indication less important neutral or more important within their respective priority grouping Two teleconference meetings were conducted between the first and second rounds and between the third and fourth rounds with anonymized results from the prior round presented for discussion and to address inconsistencies and misinterpretationsA modification of the RANDUniversity of California at Los Angeles UCLA method was used to achieve consensus15 This methodology typically is used to determine the appropriateness of an intervention but in this setting was used to determine surgical priority We used a scale ranging from to in rounds and to indicate the decision to not operate or low priority scores intermediate priority scores or high priority scores For rounds and we used a scale from to to rate each indication compared with other indications within each of the priority groupings as either less important neutral or more important Consensus was determined based on RANDUCLA criteria15 For the first rounds to determine surgical priority a hierarchical logic was adopted to determine consensus regarding whether surgery should be performed and to then determine the priority of surgery based on the given indication Agreement on the decision to not operate was defined as a minimum of of the panelists rating a given indication with a zero score If there was no agreement to avoid surgery agreement for surgical priority then was defined as ‰¤ panelists rating the indication outside the 3point range containing the median as per RANDUCLA guidelines15 For rounds and any indication that failed to achieve consensus was classified as being of intermediate priority and for rounds and any indication failing to achieve consensus was classified as neutral within the priority groupingDevelopment of the SPARTANHNThe algorithm uses a pointbased system to assign a total score based on the sum of the individual indication scores see Supporting Information with higher scores corresponding to higher priority Scoring weights were based on consensus from both sets of rounds such that highpriority indications were assigned scores ranging from to intermediatepriority indications were assigned scores ranging from ˆ’ to and lowpriority indications were assigned scores ranging from ˆ’ to ˆ’ Within each priority grouping 3point range the scores were assigned based on the consensus ratings from the third and fourth rounds For any patients with the same total score the SPARTANHNde Almeida et alpatient with the longer surgical wait time was assigned the higher priority rankClinical VignettesTwelve clinical vignettes were constructed see Supporting Information after the consensus rounds to validate the SPARTANHN The vignettes described a variety of clinical scenarios incorporating multiple prioritization indications and additional clinical information Experts were asked to consider only the patientlevel information provided to them and not their own unique clinical and community practice environments Twelve scenarios were selected for diversity of cases The number was considered appropriate while avoiding the excessive cognitive burden associated with ranking too many scenariosStatistical AnalysisAgreementAgreement between raters during the Delphi process was calculated at each round and within each priority grouping using the Krippendorff alpha Kalpha Because typical coefficients of reliability are not suitable for coded data agreement for the rank orders generated by coders JRD CWN DF EM and DPG applying the SPARTANHN algorithm to the clinical vignettes was assessed using Kalpha calculated with bootstrap samples16 The Kalpha allows for estimation of reliability for any number of raters and categories and may be used when there are missing data17Validity of the SPARTANHN AlgorithmConvergent validity of the median rankings from the coders of each of the vignettes using the SPARTANHN and the expert panel rankings were assessed using the Spearman rank correlation coefficient The strength of the correlation was considered weak if the rho was moderate if the rho was between and and strong if the rho was In addition to SPARTANHN a second algorithm using a decisionmaking flowchart was developed SPARTANHN2 The tool and associated performance characteristics are included in Supporting Information Sample Size ConsiderationsFor determination of an adequate sample size for the expert panel we assumed that for model validity there was a strong correlation between the model rank order and expert rank order ie rho ‰¥ an alpha of power of and a nonresponse rate of Therefore the calculated sample size requirement was participantsCancer Month 0cOriginal All analyses were 2sided and statistical significance was set at P\xa0‰¤\xa0 Analyses were conducted using SAS University Edition statistical software SAS Institute Inc Cary North CarolinaRESULTSEstablishing Consensus Priority Groupings First Consensus RoundsAfter the first rounds the panel failed to achieve consensus for any indications that would result in a decision to not operate More than respondents indicated that they would not operate for the following indications the availability of alternative nonsurgical treatment with a similar prognosis respondents poor performance status ie Eastern Cooperative Oncology Group [ECOG] performance status of respondents and very severe comorbidity as indicated by a non“cancerspecific survival rate of at year respondents In the first round consensus was achieved for indications for surgical prioritization of which were considered high priority of which were considered intermediate priority and of which were considered low priority After review of firstround results consensus was achieved for an additional indications indications were rated as being of intermediate priority and indications were rated as low priority Table Establishing Ranking Within Each Priority Grouping Second Consensus RoundsOf the lowpriority indications consensus for the importance of factors was achieved for scenarios both of which were deemed less important Table Of the intermediatepriority indications consensus for the importance of factors was achieved for of scenarios Of highpriority factors consensus for the importance of factors was achieved for scenarios all of which were deemed to be more importantAgreement during consensus rounds was found to be weak to moderate for all rounds ranging from to The agreement was similar when measured as per priority grouping in which the Kalpha ranged from to Table SPARTANHN Surgical Prioritization Scoring SystemPriority weights for each indication ranged from ˆ’ to spanning a 9point range and translated from the rounds of priority groupings into categories Four indications were assigned a weight of based on consensus that these factors were both high priority and more important Supporting Information Table All other highpriority indications were assigned a weighted score because there was no consensus that they were either less or more important For intermediatepriority indications a weighted score of was assigned for of the indications deemed to be more important by consensus The other indication deemed to be more important thyroid cancer with tracheal invasion was assigned a score of because of the fact that this indication can be associated with lowgrade histology which is assigned a negative weighted score Three intermediatepriority indications that were rated as more important were resource use indications which generally are colinear As such the decision was made to assign a maximum score of for the presence of any or all of these indications One intermediatepriority indication was deemed to be less important by consensus and was assigned a score of ˆ’ All other intermediatepriority indications were assigned scores of For the lowpriority indications those deemed to be less important were assigned a weight of ˆ’ and all other indications were assigned a weight of ˆ’ The total scale score ranged from ˆ’ to Fig Reliability and Validity AssessmentAgreement between the coders for the SPARTANHN was excellent Kalpha Agreement between the expert raters was moderate Kalpha Convergent validity was demonstrated by a strong correlation between the rank orders generated by the SPARTANHN and external experts rho CI [P\xa0 a0 ] Agreement between expert rankings and SPARTANHN rankings for the vignettes is shown in Figure DISCUSSIONIn the setting of the COVID19 pandemic in which the availability of operating room time as well as hospital and intensive care unit beds is limited the prioritization of surgical oncology cases is imperative to mitigate downstream adverse outcomes1920 The current methodology was adopted based on expert consensus In the current study we have proposed the SPARTANHN with the objective of providing transparency and facilitating surgical prioritization for treatment providersCreating COVID19“era allocation schemas that are ethically sound is both critical and challenging Emanuel et al have advocated ethical principles with which to Cancer Month 0cSPARTANHNde Almeida et ali ytidbrom lanoitcnufi tnacifings laitnetoP fi ytilibareponi rohtw romut fi tnemriapmi citem etaredom laitnetoPsoc ro lanoitcnuf htiw recnac doryhTiinosavni laehcarthtw romut inossergorp esaeisd citamotpmySENEtsil tiawno e lihwTR suoverPi dedeecxe emit tiaW dedeecxe emit tiaWihgh rof kw‰¥ yb liygootsh edargihgh rof kw yb liygootsh edarg igngami ro lacniilC hpmyl decnavdAegats gncnavdai ei inossergorpi cpocsorcam roN ge esaesd edoni esaesdV inoitceserI ot III egatSnoitceser enob htiw recnac ytivac larOnoitide ht CCJA yregrus fo htgneL latot ro latotraen gniriuqer recnac ytivac larO yats fo htgnel latipsoHh tinu erac evsnetni ioNtinu nwodpets rod yregrus larosnart htiw recnac laegnyrahporOymotcessogllymotoubdnam htiiw recnac laegnyrahporO ymotcegnyral latot htiw recnac laegnyrahpopyHymotcegnyral latot gniriuqer recnac laegnyraLi cpocsodne gniriuqer recnac laegnyrahposaNymotcegnyrahp laitrapdna odne gniriuqer recnac suns lasanarap roi lasaNymotolli xamgniriuqer recnac laegnyrahposaNnoitceser cpocsinoitceseri noitceser nks gniriuqer recnac nks decnavdAinoitcurtsnocer palf lanoger dnai edon hpmyl detimil htiw renac kcen dna daeH edon hpmyl on htiw recnac kcen dna daeHycnangilam enob laropmeTesaesdiII ot I egatSy egAy egAy egAesaesdi SPGOCE y ‰¥ egAycnangil amditorap edarghgHinoitcurtsnocer palfeerf gniriuqer recnac nks decnavdAi laitrapgniriuqeryregrus laegnyral recnac laegnyraL gniriuqer recnac laegnyrahpopyHnyrahpognyral latotymotcegriuqer recnac sunsi roiretna nepogn i lasanarap ro lasaN ‰¥i lacgrus lacafonarciili ygootsh edargwol rof dedeecxe emit tiaWkwnoitceser eussittfos htiw recnac ytivac larOi rof gnhcaorppa tubdedeecxe ton em it tiaW dedeecxe emit tiaWliygootsh edarghghiwoliygootsh edargl rof kw deecxe ton emit tiaWromoc ereves yreV SP GOCE ei sutats ecnamrofrep rooP ypareht evitanretlAlebaliava ni dehcaorppa tub edargwol rof kw ditorap edargwoLycnangilam edon hpmyl htiw recnac doryhTiesaesdilygootshi recnacnonge ytidb i ta i lavvrusy srotcaF ytiroirPhgHisrotcaF ytiroirPetademretniIsrotcaF ytiroirPwoLˆ’ˆ’ˆ’ˆ’i gnknaR fo sdnuoR retfA serocSdna snoitacdn iI noitazitiroirP ELBATCancer Month 0cOriginal TRj tnavuda dna esaesddecnavda hti iw tneitaPderiuqer tinu nwodpets ro tinu erac evsnetniIderiuqer ebut ymotsoehcart oNderiuqer palf eerf oNd yats fo htgnel latipsoHh yregrus fo htgneLd yats fo htgnel latipsoHderiuqer palf eerFh yregrus fo htgneLnoitpo na si SPGOCEˆ’ˆ’ˆ’ˆ’srotcaF ytiroirPhgHisrotcaF ytiroirPetademretniIsrotcaF ytiroirPwoLdeunitnoC ELBATyparehtodar iTRi nosnetxe ladonartxe ENE sutats ecnamrofreppu ygoocnO evitarepooCnretsaE l SPGOCE snoitaverbbAiTABLE Agreement Between Experts During the Delphi ProcessRoundOrdinal ScaleaLCL UCLPer Priority GroupLCL UCLAbbreviations LCL lower confidence limit UCL upper confidence limitThere were raters and agreement was measured using the Krippendorff alphaaœOrdinal scale refers to the scale of to used to rate priority of surgery and œPer Priority Group refers to the lowpriority mediumpriority and highpriority groups related to the scoring scaleguide the allocation of scarce resources maximizing the benefits produced by scarce resources treating people equally promoting and rewarding instrumental value and giving priority to those patients who are worst off9 These have been contextualized for cancer care more broadly and are manifest in the SPARTANHN algorithm21 The highpriority indications implicitly embrace an underlying premise of saving the most lives andor preserving the most lifeyears Many procedures for patients with HNC are aerosolgenerating and increase the risk to health care workers and other hospitalized patients22 Our process accounted for these by giving consideration to these factors during the consensus process although indications associated with potential exposure to health care workers did not emerge as lowpriority ones Indications associated with lower resource use did achieve consensus for higher importance This may help to avoid the opportunity cost of treating fewer patients with longer surgeriesAnecdotal and institutionspecific prioritization schemas for patients with HNC and general otolaryngology have been suggested213 These parallel similar efforts for general surgery cardiac surgery and orthopedic surgery12132328 In many of these patients are prioritized by the scoring of several criteria and summing of the scores to achieve a total patient score Many of these systems have been validated against expert rankings of surgical priority2728We used a methodology for developing a pointbased prioritization system similar to those previously described29 To the best of our knowledge pointbased surgical prioritization systems have been very well studied to date Hansen et al previously proposed a methodology for developing a pointbased prioritization system using the following steps ranking patient case vignettes Cancer Month 0cSPARTANHNde Almeida et alFIGURE The Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancer SPARTANHN scoring system ECOG indicates Eastern Cooperative Oncology Group ENE extranodal extensionusing clinical judgment drafting the criteria and categories within each criteria pretesting the criteria and categories consulting with patient groups and other clinicians determining point values for criteria and categories checking the testretest reliability and face validity and revising the points system as new evidence emerges29 Our approach to the development of the SPARTANHN was similar However given the relatively expedited nature of the process we did not directly involve patientsOne method proposed for establishing the priority of all indications in a pointbased scoring system is known as Potentially All Pairwise Rankings of all Alternatives PAPRIKA30 In the current study we chose to use the RANDUCLA process instead of pairwise comparison to minimize computational burden We established Cancer Month 0cOriginal FIGURE External validation rank results A total of experts were asked to rate the scenarios provided shown on the xaxis and the results were compared with the rank generated by models and shown on the yaxis Green shading reflects high priority ranked yellow shading indicates medium priority ranked and red shading indicates low priority ranked Asterisk denotes ties from the algorithm SPARTANHN indicates Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancerindications for surgical prioritization that would create an enormous computational burden using pairwise comparison methodology One problem inherent in the PAPRIKA methodology is the assumption that all indications are not equal and can be ranked However clinically certain indications may be equivalent in priority Furthermore pairwise comparisons assume mutual exclusivity of each of the indications which is not always the case Use of the RANDUCLA consensus process avoids the need for multiple pairwise comparison and allows for consideration of each factor in isolation The goal of the consensus rounds was not to establish a rank order for all indications but mainly to understand which indications result in high intermediate or low priorityThe SPARTANHN algorithm has demonstrated preliminary reliability and validity We demonstrated good agreement between raters and the SPARTANHN algorithm suggesting minimal interpretive error Many of the highpriority indications accounted for some component of interpretation because raters were forced to consider imminent disease progression that may result in an adverse outcome Despite the subjective decisions that must be made as part of SPARTANHN agreement remained high In fact true interrater reliability was found to be higher because the Kalpha is a conservative measure of reliability Other measures of reliability such as the Kendall coefficient of concordance tend to overestimate reliability and cannot be applied to missing data31 Perhaps most important the SPARTANHN correlated highly with expert rankings With established validity this algorithm may be ready for preliminary clinical use although further testing against realworld data to validate it with other cancer outcomes such as survival is neededThe results of the current study must be interpreted within the context of the study design Although externally validated by other surgeons across North America and the United Kingdom the criteria for which consensus was achieved for prioritization were not vetted by patients advocacy groups or other stakeholders such as medical or radiation oncologists The latter groups represent essential providers in the multidisciplinary care of patients with HNC and may have important insight into the availability and effectiveness of nonsurgical treatments1920 Nonetheless the actual prioritization of surgical waitlists remains the sole responsibility of surgeons and their practice partners In addition the SPARTANHN algorithm is intended to assist in making difficult prioritization decisions and is not intended to make recommendations for the time frame in which patients should receive treatment Instead established guidelines should be adhered to for treatment targets Patient wait times as they relate to those targets should be considered when using the SPARTANHN algorithm The validation process in the current study used expert opinion as the gold standard of prioritization which is potentially biased and reflected the opinions of surgeons practicing in academic medical centers from resourcerich nations Subsequently use of the SPARTANHN algorithm in other geographic regions Cancer Month 0cand health care systems requires additional investigation because local treatment paradigms and risk factors may vary substantiallyThe current study has presented the development and validation of a novel algorithm for the prioritization of surgery for patients with HNC Further evaluation of its implementation in various practice settings will be obligatory However the results of the current study have provided data with which to inform realworld use as the current pandemic has obviated our ability to more rigorously study the instrument prior to making necessary and difficult realtime allocation decisionsFUNDING SUPPORTNo specific funding was disclosedCONFLICT OF INTEREST DISCLOSURESEvan M Graboyes has received grants from the National Cancer Institute and the Doris Duke Charitable Foundation for work performed outside of the current study Vinidh Paleri offers his services as a proctor for a transoral robotic surgery proctoring program run by Intuitive Surgical and has been remunerated for his time Antoine Eskander has received a research grant from Merck and acted as a paid consultant for BristolMyers Squibb for work performed outside of the current study Ian J Witterick has stock in Proteocyte Diagnostics Inc and has received honoraria from Sanofi Genzyme and Medtronic Canada for work performed outside of the current study The other authors made no disclosuresAUTHOR CONTRIBUTIONSJohn R de Almeida Study idea and design writing and editing Christopher W Noel Study design writing data analysis and editing David Forner Study design writing data analysis and editing Han Zhang Data acquisition and editing Anthony C Nichols Data acquisition and editing Marc A Cohen Data acquisition and editing Richard J Wong Data acquisition and editing Caitlin McMullen Data acquisition and editing Evan M Graboyes Data acquisition and editing Vasu Divi Data acquisition and editing Andrew G Shuman Writing data acquisition and editing Andrew J Rosko Data acquisition and editing Carol M Lewis Data acquisition and editing Ehab Y Hanna Data acquisition and editing Jeffrey Myers Data acquisition and editing Vinidh Paleri Data acquisition and editing Brett Miles Data acquisition and editing Eric Genden Data acquisition and editing Antoine Eskander Data acquisition and editing Danny J Enepekides Data acquisition and editing Kevin M Higgins Data acquisition and editing Dale Brown Data acquisition and editing Douglas B Chepeha Data acquisition and editing Ian J Witterick Data acquisition
2
" IBDFecal calprotectinEndoscopic activityIBD noninvasive managementThe term IBD is usually used for referring to a group of ‚ammatory gastrointestinal diseases mainly Crohn'sdisease and ulcerative colitis Accordingly IBD arises as a result of inappropriate immune response to intestinalcommensal anisms among genetically susceptible individuals Performing colonoscopy and histopathologicevaluation on an ‚amed bowel biopsy specimen are currently considered as gold standards for diagnosis andmanagement of IBD Correspondingly these techniques are known to be invasive and costly In recent decadesfecal calprotectin as a biomarker has received much attention for the diagnosis and noninvasive managementof IBD Up to now many studies have investigated the efficacy of fecal calprotectin in the areas of IBD diï¬erentiation from IBS prediction of endoscopic and histologic activities of IBD and prediction of disease recurrenceAlthough some of these studies have reported promising results some others have shown significant limitationsTherefore in this paper we reviewed the most interesting ones of these studies after a brief discussion of thelaboratory measurement of fecal calprotectin Moreover we attempted to provide an answer for the question ofwhether fecalcalprotectin could be considered as a potential surrogate marker for colonoscopy IntroductionInflammatory bowel disease IBD is a long life disease with remission and relapse periods IBD arises as a result of inappropriateimmune response to intestinal commensal anisms in individualswith genetic predisposition and consequently causes ‚ammation andintestinal ulcers [] In addition IBD has a complex pathogenesis andmany factors such as dysbiosis oxidative stress and epigenetics thatmay also be involved in disease pathogenesis [“] Ulcerative colitisUC and Crohn's diseases CD are known as two main forms of IBDAccordingly these diseases cause intestinal ulcers and some annoyingsymptoms such as diarrhea abdominal pain and rectal bleeding Occasionally the severity of these symptoms is very high which can leadpatients to be hospitalized In this regard therapeutic approaches totreat these diseases mainly focus on prolonging remission and are almost similar however diï¬erential diagnosis can also help to treat thedisease in a more eï¬ective way For example 5ASA which is acommon drug in the treatment of IBD is less eï¬ective on maintainingremission in CD patients On the other hand antibiotic therapy is notrecommended for the treatment UC but it can be eï¬ective on CD patients [][] Diï¬erential diagnosis is a serious challenge because CDand UC have significant similarities in terms of their clinical endoscopic and histological features However there are some diï¬erencesbetween UC and CD which are summarized in Table1 In addition tointestinal complications UC and CD also have significant extraintestinal manifestations For example it was shown that UC is significantly associated with Primary sclerosing cholangitis and CD is alsoassociated with cholelithiasis especially in cases that the ileum is involved [] Furthermore CD can cause fistulas to the urinary systemwhich leads intestinal bacteria to enter the urethra and recurrent urinary tract infections [] Both CD and UC can cause several disorderssuch as arthritis Erythema nodosum pyoderma gangrenosum andanemia which are known as the most important extraintestinal manifestations of IBD [][] The latest statistics showed that the globalŽ Corresponding author at Department of Clinical Biochemistry and Laboratory Medicine Faculty of Medicine Tabriz University of Medical Sciences DaneshgahStreet PO Box Tabriz IranEmail address vagharimtbzmedacir M VaghariTabari101016jcca202008025Received July Received in revised form August Accepted August Available online August Elsevier BV All rights reserved 0cF KhakiKhatibi et alTable1Clinical endoscopic and histological features of CD and UCClinical FeaturesFeaturesRectal bleedingAbdominal painFeverMucus defectionIntestinal obstructionPerineal diseasePostoperative recurrenceASCA positiveANCA positiveEndoscopic FeaturesCDOccasionallyFrequentlyFrequentlyOccasionallyYESYESYESFrequentlyNot commonUCFrequentlyOccasionallyNot commonFrequentlyNONONONot commonFrequentlyFeaturesCDUCLocationMucosal involvementDepth of ulcerationfistulaCobblestone appearanceAphthous ulcerationMucosal friabilityHistological featuresFeaturesGranulomasCrypt abscessesPatchinessAny part of GI tractDiscontinuousDeepYesYESFrequentlyNot commonCDFrequentlyNot commonFrequentlyColon and rectumContinuoussuperficialNONOOccasionallyFrequentlyUCRareFrequentlyNot commonprevalence of IBD currently is on the rise and it is not an exaggerationif we consider it as a global serious health problem [] According to areport published in IBD has the highest prevalence rate inEurope and its prevalence in the newly industrialized countries of AsiaAfrica and South America also appears to be increased over the pastthree decades []Unfortunately the peak of the disease is at the young age of“ years old [] therefore in addition to the suï¬ering from ‚icts on the patients it also has many negative eï¬ects on societyMoreover many financial burdens are annually imposed on countriesfor controlling and treating this chronic disease The invasive diagnosticand therapeutic measures are currently undertaken to diagnose andmanage IBD which are unpleasant for patients as well as having thehigh associated costs Now the gold standard method for diagnosingIBD and monitoring patient status is performing colonoscopy examination and histopathologic evaluation which are invasive measures[] Therefore in recent years many studies have been conducted tofind a suitable laboratory marker with sufficient sensitivity and specificity for the purpose of diagnosing and noninvasive management ofIBD A high proportion of these studies have investigated the efficacy offecal calprotectin in diagnosing and monitoring patients Althoughsome of these studies reported auspicious results there are still somedoubts on the eï¬ectiveness of fecal calprotectin on diagnosing andmonitoring IBD patients So in this review we addressed the advantages and limitations of fecal calprotectin for the diagnosis andmanagement of IBD The role of fecal calprotectin in diagnosis and management ofIBDThe efficacy of fecal calprotectin as an laboratory marker in various areas of IBD diagnosis and management have been studied including IBD diï¬erentiation from irritable bowel syndrome IBS evaluation of endoscopic activity of the disease evaluation of histologicalactivity of the disease and prediction of disease recurrence andClinica Chimica Acta “response to treatment In following after a brief introduction andmentioning the important points regarding laboratory measurement offecal calprotectin we reviewed the most interesting findings in all ofthe abovementioned areas Calprotectin A clinically valuable proteinCalprotectin is an antimicrobial protein mainly secreted by neutrophils This protein competes with bacteria over zinc thus kills thebacteria However this is not the only contribution that it has to antimicrobial activity Moreover this protein has many potential clinicalapplications such as the elevated serum levels that have been observedunder various immunological and immunopathological conditionsSerum calprotectin levels rapidly increase in response to bacterial infections in the kidney and heart or during transplant rejection At theearly stages of ‚ammation of the lung serum calprotectin can also beconsidered as a reliable marker besides plasma levels of calprotectinappear to be useful in reflecting disease activity in ‚ammation of thejoints [] In addition it has been demonstrated that serum calprotectin levels are increased in patients with bacterial sepsis so it can beconsidered as a reliable biomarker [] In Neonatal Sepsis the serumlevel of calprotectin increases as well as a sensitivity of and aspecificity of that have been reported for serum calprotectin indiagnosis of Neonatal Sepsis [] It has been recently shown thatserum calprotectin levels increase in patients with aneurysmal subarachnoid hemorrhage and higher levels in the first of onset areassociated with a poor prognosis at the first three months [] Serumcalprotectin levels also increase in patients with rheumatoid arthritisand even in patients with a moderate to high disease activity who havenormal or low CRP levels so they appear to be more efficient at reflecting disease activity []Some studies have also investigated the efficacy of serum calprotectin in the diagnosis of cancers Correspondingly in one of thesestudies it was shown that serum calprotectin levels significantly increased in patients with laryngeal carcinoma compared with healthyindividuals and those with benign laryngeal pathologies Moreover inthis study a direct relationship was also observed between serum levelsof calprotectin and stage of cancer [] Another study showed that theserum level of calprotectin increased in patients with papillary thyroidcarcinoma but it significantly decreased after operation [] Alsoregarding the efficacy of serum and saliva calprotectin for the diagnosisof IBD impressive results have been reported [][] A study onpatients with IBD both UC and CD have shown that serum calprotectinlevels were directly correlated with fecal calprotectin levels and weremore potent in IBD diagnosis compared to CRP and albumin This studyalso indicated that the combination of serum calprotectin with CRP oralbumin can be helpfulin the prediction of treatment escalationespecially in patients with CD [] However no significant correlationwas observed between serum calprotectin and fecal calprotectin levelsin patients with CD and UC as well as a slight correlation betweenserum calprotectin level and CRP that was observed only in patientswith UC [] Another study showed that the serum level of calprotectin was significantly higher in patients with CD compared to healthyindividuals In addition although a significant correlation was observedwith the clinical activity of the disease no significant correlation wasfound between the level serum calprotectin and endoscopic activity ofthe disease [] The efficacy of salivary calprotectin in the diagnosisof IBD has also been studied which showed that salivary calprotectinsignificantly increased in patients with IBD compared to healthy individuals In this study AUC values for unstimulated saliva and stimulated saliva to distinguish IBD patients from healthy individualswere reported to be and respectively [] However thepopularity of calprotectin is mainly due to the use of fecal calprotectinin the diagnosis and management of IBD that is discussed in the following 0cF KhakiKhatibi et alClinica Chimica Acta “ Laboratory measurement and reference intervalFecal calprotectin is a stable protein that remains stable for “ daysat room temperature [] This property is an excellent advantage for alaboratory marker Also it seems that keeping the specimen at refrigerated temperature °C can increase the stability of fecal calprotectin [] However evidence has been obtained regarding thatthe stability of this protein decreases after staying for three days atroom temperature On the other hand it is not also recommended tokeep samples in the refrigerator for more than days [] It seemsthat fecal calprotectin remains stable up to one year at ˆ’ °C []Measurement of fecal calprotectin can be done both qualitatively andquantitatively Accordingly in the qualitative measurement monoclonal antibodies are used to detect fecal calprotectin and the positiveresults are characterized by the appearance of colored lines on the testcassette However in the qualitative one only positive or negative results are reported and despite of sensitivity test specificity in theevaluation of disease activity was reported to be only It seems thatthe main application of this test is to diï¬erentiate healthy individualsfrom IBD patients rapidly however some studies have shown that it isnot accurate enough in this case as well [][] Nevertheless asignificant concordance has been reported between home test resultsIBDoc and fecal calprotectin laboratory measurement results whenQuantum Blue calprotectin ELISA kit was used Notably the agreements between results were and depending on the selectedcutoï¬s [] Several commercial kits are also available for fecal calprotectin qualitative test known as rapid calprotectin These tests reportpositive results ranged from to µgg There are also severalcommercial kits that can be used for the quantitative measurement offecal calprotectin These kits are usually designed in terms of the ELISAmethod and some have a measurement range between and µgg Moreover the chemiluminescence immunoassays CLIAmethod can also detect values between and µgg Fluoro enzyme immunoassays FEIA and particle enhanced turbidimetric immunoassays PETIA can also be used for the measurement of fecalcalprotectin In this regard one of the most serious challenges to thelaboratory evaluation of fecal calprotectin is the determination of theupper limit in healthy individuals Among healthy adults there is asignificant agreement on µgg as an upper limit One study suggested values up to µgg in people over years old and up to µgg in children aged between and years old as referenceranges of fecal calprotectin in healthy individuals []Fecal calprotectin levels appear to be higher in healthy infants andchildren under four years old than in adults and further studies areneeded in this regard to determine the acceptable upper limit for diagnosis of pediatric IBD [] Table lists the median levels of fecalcalprotectin in healthy individuals with diï¬erent ages reported in somestudies According to these reports age can aï¬ect fecal calprotectinlevels Fecal calprotectin and IBD diagnosisOnly a small percentage of patients complaining of abdominal painand diarrhea have IBD In many cases IBS as a functional gastrointestinal disorder is known as the cause of such clinical symptomsPatients with IBS have normal colonoscopy results while IBD patientsindicate abnormal colonoscopy results and have intestinal ulcersUnfortunately the significant prevalence of IBS and the overlap between clinical symptoms and IBD can increase the colonoscopy rateTherefore a noninvasive diagnostic marker can be very helpful in thisregard Notably the first evidence of the efficacy of fecal calprotectin inthe diagnosis of IBD was obtained in the 1990s Røseth et al in proposed a method for measuring Calprotectin in stool specimens []One of the first and most interesting studies regarding fecal calprotectinutility in IBD diagnosis was the study by Røseth et al published in In this study patients with ulcerative colitis were studied and according to their results fecal calprotectin levels are higher in patientswith ulcerative colitis compared to healthy controls This study havealso shown that even patients with low disease activities had higherlevels of fecal calprotectin compared to healthy individuals []Subsequent studies somehow confirmed and complemented the findings of this study In another study published in AUC values of CI “ were reported for fecal calprotectin in thediagnosis of colorectal ‚ammation [] Moreover in a study onchildren with IBD it was shown that the level of fecal calprotectin washigher in these patients compared to healthy children so it can beconcluded that it is also directly correlated with ESR levels [] In astudy published in Kolho et al reported AUC values of CI “ for fecal calprotectin in the diagnosis of pediatric IBD [] In a study on patients with Crohn disease a sensitivity of and a specificity of at cutoï¬ of μgg have been reportedfor fecal calprotectin in diagnosis of the disease [] The results of ourrecent study along with other studies showed that fecal calprotectin ispreferred over traditional ‚ammatory biomarkers such as CRP andESR in the diagnosis of IBD [][] Diamanti et al reported a sensitivity of and a specificity of for fecal calprotectin at a cutoï¬ of μgg in IBD diagnosis [] In our recent study a sensitivityof and a specificity of at a cutoï¬ of μgg were observed for fecal calprotectin in the diagnosis of IBD however oursample size was and the majority of patients were in the active phaseof the disease []In another study conducted on patients with ulcerative colitis asensitivity of and a specificity of at cutoï¬ of μgg havebeen reported in this regard [] In one study it was shown that fecalcalprotectin in cutoï¬ of μgg is able to distinguish patients withIBD from patients without IBD patients with diseases other than IBDpatients with IBS and healthy persons with sensitivity and specificity [] Caviglia et al in their study reported a sensitivity of and a specificity of at a cutoï¬ of μgg for fecalcalprotectin in diï¬erentiating between IBS and IBD [] Howeversome studies have reported significantly lower values Accordingly in astudy on patients with ulcerative colitis Kalantari et al reported asensitivity of and a specificity of at a cutoï¬ of μgg []Besides there is a considerable agreement between fecal calprotectinand capsule endoscopy findings in patients with Crohn's disease Asensitivity of and a specificity of have also been reported at acutoï¬ of mgkg for fecal calprotectin in predicting CE findings anddiagnosis of Crohn's disease [] In another study lower sensitivityand specificity rates sensitivity specificity were reportedfor fecal calprotectin in this regard [] Furthermore in one studythat examined the efficacy of fecal calprotectin in predicting wirelesscapsule endoscopy findings a sensitivity of and a specificity ofTable Reported median levels of fecal calprotectin in healthy individuals of diï¬erent agesAgesMedian levels of fecal calprotectin range µggNumber of subjectsUsed kitUp to monthChildren “ yearsChildren “ yearsAdultsOver years “ “ “ “ “Bühlmann ELISABühlmann ELISACALPRO® Calprotectin ELISA Test ALPPhiCalPhicalReference[][][][][] 0cF KhakiKhatibi et al were reported for this biomarker at μgg in the diagnosis ofsmall bowel ‚ammation in Crohn's disease [] Given these findings it seems that fecal calprotectin has no ideal sensitivity and specificity for the diagnosis of IBD where the small intestine is involvedBesides there are some preanalytical limitations which are explainedin the next sections Therefore optimistically speaking fecal calprotectin measurement can eliminate the need for colonoscopy Howeverin a metaanalysis performed to evaluate the efficacy of fecal calprotectin and some other ‚ammatory markers to diï¬erentiate betweenIBD and IBS the probability of IBD was less than at fecal calprotectin values lower than µgg or CRP values lower than mgdL[] Therefore it seems that fecal calprotectin can be helpful at leastin ruling out the possibility of IBD in patients with IBSlike symptoms aswell as reducing the rate of colonoscopy Moreover it should be notedthat although a systematic review has reported pooled sensitivity andspecificity above for fecal calprotectin to diï¬erentiate between IBDand IBS it emphasized more on the possibility of falsepositive resultsin low cutoï¬ points [] Hence performing extensive studies indiï¬erent countries on the healthy population and the IBD patient is beneeded to determine a suitable cutoï¬ with maximum sensitivity andspecificity and minimum falsepositive resultsTable summarizes the results of various clinical investigationsregarding fecal calprotectin utility in the diï¬erential diagnosis of IBDfrom IBS and Table4 summarizes some metaanalysis results in thisregard As shown in Table the most important limitation of the majority of clinical studies conducted to date is the small sample size Alarge global study may be helpful in providing a more precise evaluation of fecal calprotectin clinical value in discrimination between IBDand nonIBD diseases Fecal calprotectin and endoscopic and histologic activity evaluationUndoubtedly one of the most serious challenges in the managementof IBD is evaluating the endoscopic and histologic activities of thedisease Nowadays colonoscopy and histopathologic examinations arethe routine tools for the assessment of mucosal healing in patients withIBD As noted earlier several scoring systems have been devised toscore disease activity based on the findings of colonoscopy and histopathologic examinations In recent years many promising results havebeen reported regarding the correlation between these scores and fecalcalprotectin levels In addition many studies have been performed inthe last decade all of which cannot be reviewed in this article The firstevidence of a link between fecal calprotectin and disease endoscopicactivity was obtained in the late 1990s In one of the first studiesRoseth et al found a significant correlation between fecal calprotectinlevels and endoscopic and histologic activities in patients with ulcerative colitis [] Furthermore in another study they observed that IBDpatients who were in remission clinically and had normal fecal calprotectin levels less than mgL had normal colonoscopy results[] These interesting findings indicate that fecal calprotectin can beconsidered as a biomarker in the evaluation of endoscopic activity andClinica Chimica Acta “Table4summarized results of some metaanalysis regarding the utility of fecal calprotectin in discrimination between patients with IBD and without IBDSample sizePooled SensitivityPooled SpecificityReferences[][][][][]mucosal healing in IBD patients Also these studies were the startingpoint of extensive studies that have been conducted up to now In astudy conducted on patients with Crohn's disease Sipponen et alinvestigated the sensitivity and specificity of fecal calprotectin in predicting endoscopic activity of Crohn's disease [] Correspondinglythe researchers used the Crohn's Disease Endoscopic Index of SeverityCDEIS scoring system in their study to evaluate the endoscopic activity of Crohn's disease As a result they found that there was a significant correlation between the endoscopic activity of the disease andthe level of fecal calprotectin Besides the findings of this study demonstrated that fecal calprotectin at µgg cutoï¬ can predict theendoscopic activity of Crohn's disease with sensitivity and specificity In another study CDEIS and Mayo Disease Activity IndexMDAI were used to evaluate the endoscopic activity of Crohn's diseaseand ulcerative colitis respectively According to the results of thatstudy on IBD patients there was a significant correlation between fecalcalprotectin levels and disease endoscopic activity [] Another studyshowed that fecal calprotectin is more strongly correlated with theendoscopic activity of the disease in ulcerative colitis compared to theRachmilewitz clinical activity index In addition in this study theoverall accuracy of fecal calprotectin for endoscopically active diseaseidentification was obtained as []Some studies have also shown the superiority of fecal calprotectinover traditional ‚ammatory markers like CRP Besides one studyfound that fecal calprotectin was more strongly correlated with theSimple Endoscopic Score for Crohn's disease SESCD compared to theCRP and even Crohn's disease activity index CDAI [] The modifiedBaron Index was also used in another study to evaluate the endoscopicactivity of ulcerative colitis As a result it was shown that calprotectinis more strongly correlated with the endoscopic activity of ulcerativecolitis compared to CRP and clinical activity of the disease [] In thisregard similar results were also observed in our recent study in whichthe Ulcerative Colitis Endoscopic Index of Severity UCEIS and SESCDwere used [] Therefore fecal calprotectin appears to be superior totraditional ‚ammatory markers in the prediction of IBD endoscopicactivity The high values of sensitivity and specificity that were mentioned earlier have raised the hope that using fecal calprotectin canreduce colonoscopy rate for patients™ monitoring However severalrecent studies have reported some significantly lower values Accordingly in a recent study in which Mayo Endoscopic Score [MES] wasused to evaluate the endoscopic activity of ulcerative colitis aTable Summary of the results of some studies regarding the utility of fecal calprotectin in discrimination between patients with IBD and without IBDNumber of IBD patientsAge groupLocationCut oï¬SensitivitySpecificity CD and UC CD and UC CD and UC and unclassified68CD and UC CD and UC and unclassified CD and UC CD and UC UC CD UCAdultsAdultsAdultsBoth adult and pediatricpediatricAdultspediatricAdultsAdultsBoth adult and pediatricTaiwanChinaItalySpainFinlandIranItalyIranDenmarkIndia48µgg µgg150µgg150µgg595µgg784µgg160µgg164µgg150µgg188µggAUCReferences[][][]SPSrefidbib60[][][][][][][] 0cF KhakiKhatibi et alClinica Chimica Acta “Table Summary of the results of some studies regarding the correlation of fecal calprotectin with endoscopic activity in IBD patientsAge groupStudylocationUsedendoscopicactivity indexCorrelationcoefficientrReferenceNumberof IBDpatients CD UC UC CD UCAdultsAdultsAdultsAdultsAdultsFinlandIranSwitzerlandSwitzerlandSwitzerland ModifiedCDEISUCEISRachmilewitzSESCD UC CDAdultsAdults UC CD UC CD CD UC UCAdultsAdultsAdultsAdultsAdultsAdultsAdultsBaron ScoreRachmilewitzSESCDGermanyUSA andCanadaJapanItalyItalyBrazilFranceFranceSouth Korea UCEISMattsSESCDMayo scoreSESCDCDEISMayo score[][][][][][][][][][][][][][]sensitivity of and a specificity of were reported for fecalcalprotectin at µgg to diï¬erentiate active endoscopic from inactiveMES or from MES or [] In another study the sensitivityand specificity of fecal calprotectin at a cutoï¬ of µgg for diï¬erentiating MES ‰¤ in patients with ulcerative colitis were and respectively [] Overall as presented in Table several studiesperformed in diï¬erent countries reported the correlation between fecalcalprotectin and IBD endoscopic activity Although some of these studies reported a strong correlation some others reported a relativelyweak correlation As noted earlier there are significant diï¬erencesbetween the reports on the sensitivity and specificity of fecal calprotectin to predict the endoscopic activity of IBD Undoubtedly a widerange of factors from sample size and the inclusionexclusion criteriato preanalysis variables and indexes used to evaluate the endoscopicactivity may also contribute to these diï¬erences However fecal calprotectin does not appear to be a very reliable marker for the predictionof IBD endoscopic activity so currently it seems a bit optimistic toconsider fecal calprotectin as a reliable alternative for colonoscopy Inthis regard further studies are still needed However under some certain circumstances such as pregnancy or pandemics the use of fecalcalprotectin to evaluate IBD endoscopic activity can be helpfulPregnant patients with IBD have serious limitations for colonoscopyexamination and it has been recommended that colonoscopy should beonly performed in the second trimester of pregnancy and where there isa strong indication [] Therefore noninvasive markers such as fecalcalprotectin can be helpful during pregnancy In one study physicianglobal assessment [PGA] which is a clinical symptombased criterionwas used to evaluate IBD activity and subsequently the associationbetween fecal calprotectin and this criterion was investigated in pregnant women with IBD The results of this study showed a significantcorrelation between fecal calprotectin and PGA levels at prepregnancyduring pregnancy and postpartum stages [] In another study asignificant association was reported between fecal calprotectin levelsand clinical activity of IBD in pregnant women Moreover it was shownthat stool calprotectin at a cutoï¬ of mgkg had a sensitivity between and as well as a specificity between and in the assessment of IBD clinical activity at diï¬erent stages ofpregnancy [] A recently published systematic review has also confirmed the conclusions obtained from these studies [] According tothese results it seems that fecal calprotectin is not aï¬ected by physiological changes during pregnancy however it is significantly correlatedwith IBD clinical activity during pregnancy Therefore from the viewpoint of relatively acceptable sensitivity and specificity in predictingthe endoscopic activity of IBD fecal calprotectin may be considered as anoninvasive biomarker for the evaluation of IBD endoscopic activity inpregnant women In addition under pandemic conditions fecal calprotectin can be very helpful Following the COVID19 pandemicwhich began in late and is still ongoing healthcare systems indiï¬erent countries were forced to impose significant limitations oncolonoscopy Therefore noninvasive IBD management and fecal calprotectin as a noninvasive laboratory marker have become moreimportant than before The combination between disease clinical activity and fecal calprotectin has been recommended as a noninvasiveapproach that can help in making decisions on treatment duringCOVID19 pandemic [] Therefore it seems that fecal calprotectincan be considered as an alternative for colonoscopy used for IBD endoscopic activity evaluation during pandemic Fecal calprotectin appears to be associated with IBD histologic activity as well Given thedifficulty in the evaluation of the histologic activity of Crohn's disease[] some studies have been focused on the ulcerative colitis andmany scoring systems have been devised so far Correspondingly thesesystems score the disease's histologic activity based on histologic observationsTherefore for this purpose a biopsy of the intestinal tissue is required which can be prepared by colonoscopy and then sent to thelaboratory In this regard one of these histologic scoring systems isRobert™s score that was used in one of our recent studies where weobserved a significant correlation between the level of fecal calprotectinand the histologic activity of ulcerative colitis which was calculatedbased on the Robert™s scoring system [] Theede et al also used themodified Harpaz Index and performed some interesting studies in thisregard In one of their studies fecal calprotectin was found to be significantly associated with the histologic activity of the ulcerative colitisand it was shown that it could predict histological mucosal healingAUC CI95 “ Sensitivity Specificity andCutoï¬ mgkg [] In another study on patients with endoscopically inactive ulcerative colitis Mayo endoscopic score the researchers showed that patients with ulcerative colitis who were inendoscopic remission but had histologically active disease had higherlevels of fecal calprotectin compared to patients with no histologicallyactive disease versus mgkg P Also despite thehigh specificity the sensitivity of fecal calprotectin in theprediction of score of histological activity was achieved as at mgkg [] In a recent study the Geboes
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"cellular recognition of microbial dna is an evolutionarily conserved mechanism by which the innate immunesystem detects pathogens cyclic gmpamp synthase cgas and its downstream effector stimulator of interferongenes sting are involved in mediating fundamental innate antimicrobial immunity by promoting the release oftype i interferons ifns and other inflammatory cytokines accumulating evidence suggests that the activation ofthe cgassting axis is critical for antitumor immunity the downstream cytokines regulated by cgasstingespecially type i ifns serve as bridges connecting innate immunity with adaptive immunity accordingly a growingnumber of studies have focused on the synthesis and screening of sting pathway agonists however chronicsting activation may lead to a protumor phenotype in certain malignancies hence the cgassting signalingpathway must be orchestrated properly when sting agonists are used alone or in combination in this review wediscuss the dichotomous roles of the cgassting pathway in tumor development and the latest advances in theuse of sting agonistskeywords cgassting innate immunity type i interferon sting agonists antitumor response cancerdevelopmentintroductionthe discovery of phagocytosis in advanced the understanding of innate immunity the first line of host defenses protection againston patternrecognition receptors prrs which recognize microbialproducts coordinate antimicrobial defenses and activateinfection dependsagainstinfection byvarious pathogens correspondence zqliucsueducn juyan zheng and junluan mo contributed equally to this work1department of clinical pharmacology hunan key laboratory ofpharmacogenetics and national clinical research center for geriatricdisorders xiangya hospital central south university changsha people™s republic of china2institute of clinical pharmacology engineering research center for appliedtechnology of pharmacogenomics of ministry of education central southuniversity changsha people™s republic of chinafull list of author information is available at the end of the adaptive immunity abnormal rna or dna rnadna hybridization and cyclic dinucleotides derived frommicrobes are usually considered pathogenassociated molecular patterns pamps [ ] cells associated with innate immunity recognize different microbial pampsthrough specific prrs thereby playing key roles in hostresistance to microbial infection the pathways governing rna recognition such as retinoid acid induciblegene i rigilike receptors have been reviewed elsewhere and will not be covered herein in the case of dnarecognition one of the best known prrs is tolllike receptor tlr9 which senses extracellular cpg hypomethylated dna that has entered the cytosol through thephagosomelysosome system in addition the aim2like receptor aim2 inflammasome can be triggered afterthe entry of doublestranded dna dsdna into the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czheng molecular cancer page of cytosolic compartment which induces the proteolyticmaturation of proinflammatory cytokines such as il1and il18 and the activation of gasdermin d leading topyroptosis [“] nevertheless the most notable prr iscgas a direct cytosolic dsdna sensor which was identified by dr chen™s group in once cgas bindsto dsdna the cgassting pathway is activated to further induce the expression of type i ifns and other inflammatory cytokinesthus triggering innate immuneresponses mounting evidence suggests that cgassting signaling not only plays pivotal roles in the hostdefense against microbialinfection but also modulatestumorigenesis hence in this review we summarize themechanism of cgassting activation and elaboratefindings regarding its dual effects on tumor developmentcurrent advances in the use of sting agonists as a novelstrategy for antitumor therapy are also reviewedinsights into the cgassting signal transductioncascadecgas is an innate immune sensor that identifies variouscytosolic dsdnaincluding dna with viral bacterialmitochondrial micronuclei and retroelement originswhich can be mainly divided into pathogenderiveddna and selfdna table in the cytoplasm cgas isactivated by interacting with dsdna in a sequence[“]independent butstructural and biochemical analyses have revealed thatthe cterminal lobe of cgas contains a conserved zinclengthdependent mannerionbinding module that mediates dna binding andcgas dimerization [ ] dna ligands promotecgas activation primarily by inducing conformationalchanges around the catalytic site and in the dnabinding structures of cgasthe gscontaining loopundergoes conformational change to maintain stabilitywhich is a major mechanism of cgas activation bydna in addition to the primary dnabinding sitementioned above the secondary site located beside theprimary site is a helix formed between strands 78and several surfaceexposed loops the proximity ofthe two dnabinding sites in cgas leads to a cgasdna complex assembly in which two cgas moleculesembrace two molecules of dsdna [ ] the cgasdimers are anized in œheadtohead alignment nextto the dna and thus form stable œladderlike networks between one long curved dsdna helix or two independent dsdna strands [ ] in this way eachindividual cgasdsdna complex can be cooperativelystabilized and can lead to stronger enzymatic activitywhich may provide a possible explanation for longerdsdna as more likely to activate cgas in additionlong dna is more efficient than short dna in drivingthe liquidliquid phase separation of cgas and the formation ofcriticallydependent on the concentration of cgas and dna inthe cytoplasm cgas and dsdna are spatially concentratedcgasdimerization and activation [“] once cgas andcgas liquidlike dropletsin liquiddropletsistofacilitatetable classification of the cytosolic dsdna that activates the cgassting signaling axisclassificationselfdnasource of dsdnamicronucleipossible mechanismsrupture of the micronuclei membrane leads to exposureof chromatin dna that is recognized by cgas whichactivates the cgassting pathwayreferences mitochondrionnuclear rnapathogenderived dnadna virushsv1 hsv2 kshv adenovirus vacciniavirus cytomegalovirus papillomavirusmurine gammaherpesvirus retrovirushiv siv murine leukemia virusrna viruswest nile virus dengue virus vsvsarscov2bacterialisteria monocytogenes mycobacteriumtuberculosis listeria shigella francisellachlamydia and neisseriamitochondrial stress induces mtdna leakage into thecytosol thus activating the sting pathway and inducingproduction of cytokinesfacilitated by endogenous retroelements nuclear rnacan be reversely transcribed into dna that activatescgassting signaling dna viruses invade host cells and release pathogenderiveddna to induce sting activation[“]dna intermediates generated from reverse transcription maybe recognized by cgas to stimulate downstream stingsignaling infection with rna viruses might cause cellular damage andcell death which results in the release of cellular dna andfurther activation of the cgassting axis sarscov2 bindingto ace2 can lead to excessive angiotensin ii signaling thatactivates the sting pathway in mice[“]bacteria produce cdns such as cyclic digmp and cyclicdiamp which can directly bind to and activate sting[ “]hsv1 herpes simplex virus hsv2 herpes simplex virus kshv kaposi sarcoma“associated herpesvirus hiv human immunodeficiency virus siv simianimmunodeficiency virus vsv vesicular stomatitis virus cdns cyclic dinucleotides and sarscov2 severe acute respiratory syndrome coronavirus 0czheng molecular cancer page of dsdna interacts structural switches rearrange the catalytic pocket to enable cgas to catalyze the synthesis of²²cyclic gmpamp ²²cgamp with atp andgtp as substrates the first step in this process is theformation of a linear dinucleotide ²pppg ²²pawith atp serving as the donor and ²oh on gtp serving as the acceptor then the intermediate product flipsover in the catalytic pocket placing gtp at the donorposition and amp at the acceptor position to form asecond ²² phosphodiester bond [ ] notablyalthough dsrna or singlestrand dna ssdna is ableto bind to cgas neither can rearrange the catalyticpocket which may explain the exclusive activation ofcgas by dsdna ultimately cgamp acts as a secondmessenger to bind to and activate sting a small endoplasmic reticulum erlocated protein kd withfour putative transmembrane domains [ ] normally in a resting state sting is retained in the er byinteracting with the ca2 sensor stromalinteractionmolecule stim1 the cytosolic ligandbindingdomain lbd of sting exists as the most functionalunit capable of integrating with ²² cgamp or cdnscyclic dinucleotides such as cdiamp cdigmp or ²²cgamp from bacteria upon interaction the obviousclosure of the ligand binding pocket in the lbd is observed which is related to the activation of sting next sting transforms into a tetramer through a highorder oligomerization reaction and is translocated fromthe er to the perinuclear area facilitated by cytoplasmiccoat protein complex ii copii and adpribosylationfactor arf gtpases [ ] in the golgi sting ispalmitoylated atcys88 andcys91 a posttranslational modification necessary forsting activation modified sting recruits thekinase tankbinding kinase tbk1 in turn the cterminal domains of sting are phosphorylated bytbk1 and then phosphorylated sting recruits interferon regulatory factor irf3 which is also phosphorylated by tbk1 and dimerizes ultimately dimerizedirf3 enters the nucleus and exerts its function in thetranscription of type i ifns and interferonstimulatedgenes isgs in parallel sting can also bind toand stimulate iκb kinase ikk to mediate the production of nuclear factorκb nfκbdriven inflammatorygenes upon signal transduction termination sting istransferred to endolysosomes for degradation considering that cgamp can be transferred through gapjunctions or delivered in viralexosome packages cgassting signaling may be activated in the cytoplasmwithout dsdna [ ] moreover newly produced typei ifns activate heterodimer interferon receptors ifnar1 and ifnar2 through paracrine signaling and thusinduce the transcription of isgs [ ] in summaryonce virusderived dna and selfdna are located intwo cysteine residuesthe cytoplasm they can be sensed by cgas and a cgasdsdna complex is formed to catalyze the synthesis of ²²cgamp with atp and gtp then ²²cgamp and bacteriaderived cdns induce sting activation and mediate the release of downstream type iifns tnfα and il6 which are prerequisites for antimicrobial defense and antitumor effects the wholeprocess shows that the dsdnacgassting axis canlead to the activation of both innate and adaptive immunity fig the antitumor functions of the cgasstingsignaling pathwayrecent evidence has revealed the close association of thecgassting pathway with cancer development thissignaling pathway is generally regarded as a potent regulator of cancer immunity a stingmediated immunesupportive microenvironment can hamper malignancyoccurrence stressbytumor cell cytosolic dsdna induces sting activationunder normal circumstances dna is strictly unaffiliatedwith the cytoplasm in eukaryotic cells to avoid autoimmunity however dna leaks aberrantly in tumorcells [ ] cancer cells share common features including genome instability tumor suppressor gene mutation or deletion oxidativeand vigorousmetabolism under these intense states nuclear andmitochondrial dna is fragile and easily damaged whichleads to eventual dna leakage in the forms of micronuclei chromatin fragments andor free telomeric dna[ ] chromosomal instability cin is the primary source of cytoplasmic dna in malignant cells andis generally associated with tumor progression distantmetastasis and therapeutic tolerance excessive proliferation of cancer cells results in unstable genomes usuallychromosomal missegregation during mitosis due to defects in segregation lagging chromosomes generate micronuclei in a cellcycledependent manner the vulnerable membraneof micronuclei easily exposes the inner dna to the cytoplasm and activates the cgassting signaling axis exogenous stimuli such as chemotherapy and irradiation can also cause dna damage in addition to leakednuclear dna oxidative stressinduced mitochondrialdna leakage is another crucial initiator of sting pathway activation several anticancer treatments that precisely attack mitochondrial membranes result in effluxand cell death therefore the permeabilization of mitochondria membranes provides a reasonable explanationfor mitochondrial dna escape [ ] other sourcessuch as apoptotic cellderived dna exosomal dnaexodna and transposable elements have also beencharacterized 0czheng molecular cancer page of fig the cgassting dna sensing signaling pathway various dna derived from virus dying tumor cells or nucleus and mitochondria binds toand activates the cytosolic dna sensor cgas cgas catalyzes the synthesis of ²²cgamp in the presence of atp and gtp then ²²cgamp bindsto the er adaptor sting which also can be activated by cdns derived from bacteria upon activation sting translocates from er to golgicompartments where it activates tbk1 and ikk which phosphorylate irf3 and iκbα respectively then irf3 and iκbα dimerize and enter nucleusinitiating the transcription of type i ifn tnf and il6 the primary roles of these cytokines are reflected in host defense inflammation andantitumor immunitydemonstrated to evoke cgas“sting activation intumor cells [ ]type i ifns mediators of sting and adaptive antitumoreffectscgassting signaling exerts antitumor functions incancer cells both in an autonomous and nonautonomousmanner on the one hand dna damage can provokeacute sting signal transduction and induce cellularsenescence an irreversible cell cycle arrest state whichthwarts the aberrant proliferation of tumor cells throughacquisition ofsecretoryphenotype sasp which is associated with the releaseof abundantinflammatory mediators proteases andgrowth factors [ ] in contrast to undergoingsenescence tumor cells also directly propel apoptosisprocesses by upregulating proapoptosis protein bcl2associated x bax and downregulating the bcl2 apoptosis on the other hand stingsenescenceassociatedtheregulatoractivation in tumor cells not only facilitates the transcription of downstream type i ifns to induce dendriticcell maturation but also recruits supportive immunecells for direct nonspontaneous tumor elimination sting activation in nonmalignant cells causes tumorsuppressive effects as well sting signaling protectsagainst colitisassociated carcinomas cacs induced byazoxymethane aom and dextran sulfate sodiumdss which induce dna damage in intestinal epithelialcells and further trigger sting activation downstreamcytokines of sting signaling such as il1 and il18prevent neoplastic transformation by facilitating woundrepair more importantly sting signaling can also provoke cytotoxic t cell responses to control tumorigenesis necrotic cancer cells are commonly engulfed byantigenpresenting cells especially the basic leucine zippertranscription factor atflike batf3drivenlineage of dendritic cells dcs batf3 dcs take intumorassociated antigens and migrate towardsthe 0czheng molecular cancer page of tumordraining lymph node via the lymphatic systemwhere they crossprime tumorspecific cd8 t cellsthen cd8 t cells undergo activation and clonal expansion in the lymph nodes and are trafficked throughblood vessels to kill tumor cells in turn damaged cancercells release more antigens that are further captured bydcs the whole process forms a positive feedback loopcalled the cancerimmunity cycle tumor eradication can be achieved by multiple processes in thecancerimmunity cycle including tumor antigen captureand presentation and t cell priming and activation withtumor antigenspecific t cell priming and activationrelying on dcs and type i ifn release the involvement of type i ifns in innate immune sensing and adaptive immunity provides a reasonable hypothesis forexploring candidate prr pathways as potential immunomodulators mice lacking tlr9 myeloid differentiationprimary response gene myd88 cytosolic rna sensor mavs or the purinergic receptor p2x7r maintainintact antitumor immunity responses whereas mice deficient in sting or irf3 present with impaired cd8 tcell priming and activation [ ] in fact dying tumorcells can release multiple damageassociated molecularpatterns damps to trigger innate immune responsesin dcs among these released stimuli tumor cellderiveddna is a pivotal inducer in general the phagocytosis ofapoptotic cells causesimmune silence because ofdnasebased degradation nevertheless tumor cellreleased dna can be preserved in the dc endolysosomal compartment through an unknown mechanism cgas recognizes dna invading the cytoplasm andinduces the activation of sting cascades excretion oftype i ifns and expression of isgs additionally undersome physiological conditions such as hypoxia andacidic environments nuclear or mitochondrial dnamight be packaged in exosomes exosomal dnaexodna animates sting signaling once it is absorbedby tumorinfiltrating dcs finallytumor cellderived cgamp can also be transferred to host dcs bythe folate transporter slc19a1 and then directly bindsto sting activating it in dcs a recent study moredirectly demonstrated that cellautonomous sting promoted the maintenance of stem celllike cd8 t cellsand augmented antitumor t cell responses and mechanistically cgasstingmediated type i interferon signaling reinforced the stem cell“like cd8 t celldifferentiation program mainly by restraining akt activity immune cellderived type i ifns have crucial functions in antitumor immunity control on the one handtype i ifns boost cross presentation by various mechanisms first they stimulate the maturation of dcs secondthey slow the endosomelysosome acidificationprocess to prevent engulfed tumor antigen clearance andelevate the expression of mhc i molecules on the cellsurface [ ] finally they accelerate dc migrationtowardslymph nodes where they can crossprimetumorspecific cd8 t cells on the other handtype i ifns drive the expression of multiple chemokinessuch as cxcl9 and cxcl10 both of which are necessary for cytotoxic t lymphocyte ctl transfer and infiltration similarly type i ifns restrain the defaultimmune suppressive action of regulatory t treg cellsby downregulating phosphodiesterase pde4 and upregulating cyclic amp camp consequently typei ifns serve as bridges linking the cgassting pathway with cd8 t cellmediated antitumor immunitythe antitumor mechanisms of the cgassting signaling axis are illustrated in fig indeed previous studies revealed that sting activation can stimulate antitumor immune responses inleukemia melanoma glioma and hepatocellular carcinoma [“] additionally sting expression is downregulated in a wide variety of tumor tissues and celllines according to a pancancer analysis with a smallproportion of tumors approximately bearing silent sting expression lower sting expressionwas found in hepatic carcinoma and gastric cancer compared with its level in corresponding normal tissues andthis lower expression level was correlated with highertumor stage and poorer prognosis [ ] consistentlycompared with that in the mcfg10a mammary epithelial cell line lower sting expression was detected inmalignant breast cancer cellincluding mcf7hbl100 and t47d cells as well as human melanomacell lines and colorectal adenocarcinoma lines [ ] collectivelythat cgassting signaling might act as a tumor suppressor in certain types of cancersthese findings suggestlinessting pathway agonists as cancer therapeuticsthe immunostimulatory potential of the cgasstingpathway makes it an attractive pharmacological targetsince its activation in the tumor microenvironmenttme can induce efficient crosspriming oftumorspecific antigens and facilitate the infiltration of effectort cells recent drug research has focused on the development of sting agonists because of their potential inanticancer therapy [ ] to date various kinds ofsting agonists have been discovered and they aremainly divided into the following categories cyclic dinucleotides and their derivates dmxaa and its analogsand small molecular agonists in addition some conventional antitumor therapeutics can also indirectly activatesting such as chemotherapy radiotherapy rt andtargeted therapy in addition sting agonists areable to enhance the efficacy of other anticancer therapeutic agents when used in combination sting 0czheng molecular cancer page of fig the antitumor immunity effect of the cgassting pathway dna damage leads to the formation of dsdna in tumor cells upon itsstimulation sting signaling is activated and promotes the release of type i ifn which is crucial for dc maturation sting signaling activation indcs is the core step of the whole cancerimmunity cycle which can be initiated through engulfment of dyingdamaged tumor cells exosometransfer and cgamp gap junctions then dcs migrate towards the tumordraining lymph node and crossprime tumor specific cd8 t cells withthe help of type i ifns finally t cells undergo clonal expansion and traffic through the blood vessel to conduct tumor killingagonists and their synergistic use with other remedies isfurther explored in detail belowcyclic dinucleotides cdnscdns constitute a main type of sting agonist whichmainly originate from bacteria the known naturalcdns consist of exogenous cyclic digmp cdigmpcdiamp ²²cgamp and endogenous ²²cgampamong these cdigmp cdiamp and ²²cgampare synthesized by bacteria and identified as secondarymessengers that mediate sting signal transduction inprokaryotic cells while ²²cgamp functions as theinitiator of sting in mammalian cells the antitumor potential of these natural dinucleotides was firstproven by the finding that cdigmp could inhibit theproliferation of human colon cancer cells in vitro andbasal cell proliferation of human cecal adenocarcinomah508 cells was inhibited with μm cdigmp intraperitoneal ip injection of highdose cdigmpdirectly activated caspase3 and triggered t1 tumoripcell apoptosis in vitro nmol of cdigmp reduced thegrowth of t1 tumor cells in vitro by and nmreduced it by while lowdose cdigmp nmol accelerated the adaptive t cell response by converting a subgroup of myeloidderived suppressor cellsmdscs into immune stimulatory cells producing il12injection of ²²cgamp consistentlymgkg expedited dramatic leukemic elimination in eltcl1 transgenic mice bearing chronic lymphocyticleukemia cll and promoted tumor shrinkage of multiple myeloma in vivo from the perspective of endogenous cdns ²²cgamp mgkg was alsoshown to restrain tumorigenesis and improve the survival rate of mice bearing ct26 colon adenocarcinomain a dosagedependent manner relying on dc activationand t cell crosspriming more recently ohkurit further demonstrated that intratumoral it injection of ²²cgamp μg25 μldose on and days after the injection of tumor cells significantly mitigated tumor growth and prolonged the survival of breast 0czheng molecular cancer page of cancer t1luc squamous cell carcinoma mscc1colon cancer ct26 and melanoma b16f10 mousemodels notably the it injection of ²²cgampinhibited not only tumor growth but also lung metastases in mice bearing b16f10 cellderived tumors suggesting that cgampinduced cd8 tcell priming can drivesystemic antitumor immunity to control local and distant tumor growth termedvaccinestingvaxconsidering the superior properties of sting signaling in activating adaptive immunityit is rational toutilize sting agonists such as cdns as cancer vaccineadjuvants to increase tumor immunogenicity fu investigated the in vivo therapeutic efficacy of acancercomprisinggranulocytemacrophage colonystimulating factor gmcsf and bacteriaderived or synthetic cdns theyobserved that after it injection of stingvax with μg of cdns per vaccine dose the volume of b16melanoma tumors was dramatically reduced in a dosedependent manner compared to mice receiving gmcsf cancer vaccine alone stingvaxtreated mice hadmore infiltrating cd8 ifnγ t cells in the tumormicroenvironment the in vivo antitumor effect of stingvax was also verified in models of colon carcinomact26 pancreatic carcinoma panc02 and upper aerodigestive squamous cell carcinoma sccfvii although natural cdns are able to produce robust antitumor immunity their chemical features might hindertheir future application in the clinical setting first native cdns are easily degraded by enzymes inside the cellor in the bloodstream second their negatively chargedproperty hydrophilicity and phosphate moieties severelyimpede cdns from penetrating cell membranes to activate cytosolic sting leading to low bioavailability andpoor retention of the cdns in specific cells and tissuesthird unintentional toxicities and narrow therapeuticwindows are also unavoidable thus new strategies toimprove therapeutic efficacy and reduce adverse effectsare urgently needed including drug delivery carrier engineering original structural modification and nonnucleotide agonist screening regarding agonistdelivery smith reported that biopolymer implantscodelivering cdigmp μg and chimeric antigen receptor t cart cells resulted in significant tumor regression in mice bearing pancreatic tumors moreoveriv administration of cdigmpysk05lip equivalent to μg of cdigmp aysk05liposome delivery system encapsulating cdigmp led to a tremendous decrease in metastatic lesionsin a b16f10 mouse melanoma model with nearly ofthe injected mice showing resistance against tumor relapsethe adaptive immune responsememory was successfully induced chen alsofound thatliposomalindicating thatinjection ofintravenousintravenousivnanopdelivered cgamp cgampnp could activate the sting axis more effectively than solublecgamp and converted the immunosuppressive tme toa tumoricidal state in a transplanted b16f10 cell melanoma model and in a genetically engineered triplenegative breast cancer model moreover a recentstudy creatively suggested that modified bacteria mightbe exploited as a selective carrier of sting agonistsintroduction of a dinucleotide cyclasecoding gene intothe escherichia coli nissle strain was an attempt at realizing this effect however advancements to the systemare needed tobysnakeapartdigestionresistancecompoundatoms the modifiedfrom improving delivery methods cdnswith superior properties are currently being synthesized and tested for instance to prevent enzymatichydrolysis of cgamp the nonbridging oxygen atomsin cgamp phosphodiester linkages were replaced by²²sulfurcgsasmp showed resistance against degradation byenpp1 a major ²²cgamp hydrolasetherebyleading to a longer halflife and sustained high affinity for human sting hsting syntheticdithio mixedlinkage cdns with both rp rp r rand rp sp r s dithio diastereomers possessed notonlyvenomphosphodiesterase but also enhanced affinityforsting a novel superior modified product ml rrs2 cda also termed adus100 had the potencyto activate all hsting variants and mouse stingmsting adus100 had higher efficiency in activating sting signaling than endogenous or exogenous cdns mainly because of its enhanced stabilityand lipophilicity its powerful tumor elimination effect was extensively demonstrated in multiple murinemodelsincluding b16 melanoma t1 breast cancer and ct26 colon cancer with all treated animalsshowing significant and durable tumorregressionafter itinjection of adus100 three mg doseswhen tumor volumes reached mm3 theremarkableforhsting laid the foundation for its clinical use related clinicaltrials of adus100 are outlined intable in addition to adus100 some other novelsting agonists have been well designed iacs8779and iacs8803 are two highly potent ²²thiophosphate cdn analogs that induced striking systemicantitumorin a b16 melanoma murineinjection μg on and daysmodel after itposttumor implantation compared with adus100or cgamp the characteristics and preclinicalapplications of all these mentioned cnds are summarized in table because of the structural modification and optimization of delivery strategiestheapplication range and efficacy of cdns have beenand high affinityresponsescurativeeffect 0czheng molecular cancer page of table characteristics and preclinical applications of different sting agonistsclassificationcharacteristicsapplicationmodelsnatural cdnagonistscdigmppoor membrane permeabilitysuitable for various codeliverytechnologiescolon cancer h508cells t1 metastaticbreast cancertreatmentinformation μm nmol ip nmol ip nmol ip²²cgamp²²cgamphigher binding affinity formsting than for hstinghigher affinity for hsting thanits lineage isomers binds tovarious sting nucleotidepolymorphisms observed inhumans easily degraded byphosphodiesteraseimpermeable to the cellmembranechronic lymphocyticleukemia mgkg ipmultiple myeloma mgkg ipct26 colonadenocarcinoma mgkgbreast cancer t1lucsquamous cellcarcinomasmscc1 μg25 μldose it μg25 μldose itcolon cancer ct26 μg25 μldose itmelanoma b16f10 μg25 μldose ittherapeutic effects references[ ] [ ]inhibitsproliferation tumorregression tumorregressionaccelerates tcellresponseleukemiaeliminationsuppressesgrowthrestrainstumorigenesisimproves survivalratedelays tumrowthdelays tumrowthdelays tumrowthdelays tumrowthstingvaxsyntheticcdnagonistspotent in vivo antitumor efficacyin multiple therapeutic modelsof established cancercgampnpsbiopolymer scaffolds cdigmp and car t cellscdigmpysk05lip²²cgsasmpadus100iacs8779iacs8803noncdnagonistsfaaliposomal nanops npsdeliver cgamp intracellularlymore effectively than realizedwith soluble cgamperadicates tumors moreeffectively than systemicdeliveryysk05 is a lipid that can efficientlydeliver cdigmp to the cytosolpossesses high fusogenic activitywhich enhances endosomalescapemore resistant to degradation byenpp1 tenfold more potent atinducing ifn secretion potentialuse as a cancer vaccine adjuvantimproves stability and lipophilicityhigher affinity for hsting thannatural cdn agonists capable toactivate all hsting variants andmstingstimulates a superior systemicantitumor response thanadus100 and cgampcauses hemorrhagic necrosisfailed in a phase i clinical trialdue to species specificity μg cdns itreduces tumorvolume b16 melanomacolon carcinomact26pancreaticcarcinoma panc02b16f10 melanomaivtnbccreates atumoricidal state pancreatic cancer μg cdigmptumor regression b16f10 mousemelanoma μg cdigmp ivdecreasesmetastasisthp1 monocytesb16 melanomathree mg doses it t1 breast cancerthree mg doses itmc26 colon cancerthree mg doses itdurable tumorregressiondurable tumorregressiondurable tumorregression b16 melanoma μg on day and posttumor implantationantitumorresponse murine colontumorsextensive tumorrejection[ ]dmxaafirst discovered as a vascularrat mammary mgkg iphigh anticancer[ 0czheng molecular cancer page of table characteristics and preclinical applications of different sting agonists continuedclassificationcharacteristicsapplicationmodelstreatmentinformationinduces proinflammatory cytokinesin a stingdependent mannerhuman fibroblastsantiviral activity selectively induces stingdependentsynthesis and secretion of bioactiveifns no evidence of binding directlyto stingactivates sting in œopenconformation submicromolarlevels induce sting activationand ifn productionhuman fibroblastsantiviral activity colon tumors mgkg iv of a treatedgroup remainedtumor free faa flavone acetic acid dmxaa 56dimethylxanthenone4acetic acid cma 10carboxymethyl9acrid
0
"Purpose The aim of this prospective study was to evaluate whether [18F]FDG-PET/CT performed within two weeks of starting erlotinib therapy can predict tumor response defined by RECIST 1.1 criteria after 8 weeks of treatment in patients with inoperable (stage IIIA to IV) non-small cell lung cancer patients. Patients and Methods Three [18F]FDG-PET/CT scans were acquired in 12 patients before (5±4 days) and after 9±3 days (early PET) and 60±6 days (late PET) of erlotinib therapy. Conventional evaluation including at least chest CT (baseline versus after 8 weeks of treatment) was performed according to RECIST 1.1 criteria. Change in [18F]FDG uptake was compared with conventional response progression-free survival (PFS) and overall survival (OS). Results By using ROC analysis the Area Under the Curve for prediction of metabolic non-progressive disease (mNP) by early PET was 0.86 (95% CI 0.62 to 1.1; P?=?0.04) at a cut-off of 21.6% reduction in maximum Standardized Uptake Value (SUVmax). This correctly classified 11/12 patients (7 with true progressive disease; 4 with true non-progressive disease; 1 with false progressive disease). Non-progressive disease after 8 weeks of treatment according to RECIST 1.1 criteria was significantly more frequent in patients classified mNP (P?=?0.01 Fisher's exact test). mNP patients showed prolonged PFS (HR?=?0.27; 95% CI 0.04 to 0.59; P<0.01) and OS (HR?=?0.34; 95% CI 0.06 to 0.84; P?=?0.03). Late PET analysis provided concordant results. Conclusion Morphologic response PFS and OS survival in non-small cell lung cancer patients can be predicted by [18F]FDG-PET/CT scan within 2 weeks after starting erlotinib therapy. The authors have no support or funding to report. Introduction Lung cancer is the leading cause of cancer-related death in both Europe[1] and the United States of America.[2] The most common forms of lung cancer are non-small cell lung cancer (NSCLC) histological subtypes. Systemic chemotherapy has contributed to a significant improvement in NSCLC therapy but progress appears to be stagnating.[3] [4] Over the last decade a better knowledge of cellular pathways has allowed the development of new therapies based on NSCLC-driving genetic abnormalities. Targeted therapies have been developed to block pathological cellular pathways involved in cancer cell survival proliferation and metastasis. Epidermal Growth Factor Receptor (EGFR) is overexpressed in NSCLC[5] and has been extensively studied as a potential therapeutic target. Two EGF Receptor blockers gefitinib and erlotinib have been demonstrated to be effective in front-line therapy in patients with inoperable NSCLC harboring EGFR-activating mutations.[6] [7] Erlotinib is also authorized after failure of previous chemotherapy and as maintenance therapy.[8] [9] In clinical practice evaluation of tumor response is based on changes in tumor size according to criteria proposed by the World Health anization[10] or RECIST criteria.[11] [12] This morphological evaluation may lead to underestimation of the efficacy of cytostatic therapeutic agents such as erlotinib that stabilize the disease in non-mutated patients whereas conventional cytotoxic drugs induce shrinkage of tumor dimensions in the case of tumor response. NSCLC tumor size evaluation can also be difficult due to atelectasis of normal lung. The major limitations to morphological imaging methods are their inability to assess response to therapy at an early stage and their inability to identify cancer in residual masses after treatment. In patients with NSCLC [18F]FDG-PET has been recognized as an adequate staging tool[13] [14] and several studies also suggest that the standardized uptake value (SUV) has a prognostic value in NSCLC.[15] [16] The value of SUV for evaluation of tumor response to targeted therapy is currently being investigated. We designed a preliminary study to evaluate tumor response in NSCLC patients eligible for erlotinib therapy. The aim of this prospective study was to determine whether [18F]FDG-PET/CT performed several days after starting erlotinib therapy could predict tumor response defined by RECIST 1.1 criteria and [18F]FDG-PET/CT after 8 weeks of treatment. Materials and Methods Patients Twelve consecutive eligible patients with stage IIIA to IV NSCLC (7 adenocarcinomas 3 large cell carcinomas 2 squamous cell carcinomas) in whom erlotinib therapy was indicated were studied at the Angers University Hospital France. Screening for EGF receptor mutations was carried out (patient characteristics are shown in ). Eligibility criteria were: histologically or cytologically proven NSCLC; unresectable stage III/IV disease or recurrent disease after surgery; age over 18 years; measurable disease according to RECIST 1.1 criteria; Eastern Cooperative Oncology Group (ECOG) performance status between 0 to 2; adequate bone marrow function liver function and renal function. Patients were not included if they had previous lung diseases such as interstitial pneumonitis or lung fibrosis identified by chest Computed Tomography (CT) scan or diabetes mellitus that could artefact PET imaging. Life expectancy was predicted to be longer than 12 weeks. Erlotinib was administered orally in a dosage of 150 mg/day on an empty stomach until clinical disease progression unacceptable toxicity or patient refusal. The medical ethics committee of the CHU of Angers approved the study protocol. All patients gave informed written consent before inclusion according to local medical ethical committee regulations and in accordance with the guidelines established by the World Medical Association Declaration of Helsinki. .0087629.t001 Clinical characteristics of the study population. Patients Male 6 (50) Female 6 (50) Total 12 (100) Histology Adenocarcinoma 7 (58) Large cell carcinoma 3 (25) Squamous cell carcinoma 2 (17) Clinical stage IIIA or IIIB 2 (17) IV 10 (83) Smoking status Current 5 (42) Former 2 (17) Never 5 (42) EGFR mutation status Presence 2 (17) Absence 10 (83) Previous chemotherapy Yes 10 (83) No 2 (17) Size of primary tumor (cm) 1.0“2.0 4 (33) 2.1“3.0 3 (25) 3.1“5.0 5 (42) >5.1 1 (8) Metastasis Lymph nodes 12 (100) Lung 4 (33) Liver 2 (17) Bone 4 (33) Adrenal glands 0 Work Plan (study design) [18F]FDG PET/CT imaging Three [18F]FDG PET/CT scans were planned: PET1 before starting therapy PET2 within 2 weeks after starting therapy and a third [18F]FDG PET/CT scan (PET3) 8 weeks after starting erlotinib therapy. PET/CT examinations were obtained in 2D mode from the vertex to mid-thighs (5 minutes of emission scan per bed position with an average of 7 bed positions at 15 cm intervals) (Discovery-ST GE Healthcare France). Patients were instructed to fast for at least 6 hours prior to scanning. Unenhanced CT scan was performed from the skull base to the upper thighs. CT parameters were 120 kVp 100 mAs 0.8 second rotation 3.27 mm slice collimation and Pitch 1.5. CT data were used for attenuation correction and PET images were reconstructed by clinical standard 2D-iterative algorithm (ordered subset expectation maximization using 4 iterations and 16 subsets; zoom 100%; image matrix size: 128—128; and Gaussian post-smoothing of 5 mm in full width at half maximum). No corrections for partial volume effect lean body mass or blood glucose levels were applied. Conventional evaluation Conventional staging and follow-up were performed according to standards of care.[11] [12] Conventional evaluation included at least clinical examination plus CT scan performed before (CT1; 7±6 days) and 8 weeks after (CT2; 58±8 days) starting erlotinib therapy. None of the patients underwent additional CT scanning during the 2 weeks after starting erlotinib therapy. Chest abdomen and pelvis CT scans (Brillance 64 PHILIPS Medical System® France) were acquired from the lung apex to the symphysis pubis after an intravenous embolus of 130 mL of iodinated contrast agent (Xenetix350®). Helical scanning parameters were 130 kVp 120 mAs 1 second rotation 4 mm slice collimation 8 mm/s bed speed and 3 mm section width. Image analysis and response evaluation CT data were interpreted by two experienced physicians specialists in thoracic oncology blinded to PET/CT results according to the Response Evaluation Criteria in Solid Tumors (RECIST 1.1 criteria[12]) by comparison of baseline CT scan (CT1) and final CT scan (CT2). Therapeutic response evaluation was defined as: 1) complete response (CR: disappearance of all target lesions); 2) partial response (PR: at least 30% decrease in the sum of the longest diameter of five target lesions); 3) progressive disease (PD: at least a 20% increase in the sum of the longest diameter of five target lesions); and 4) stable disease (SD: neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD). Patients were then classified in the progressive disease (P) group or the non-progressive disease (NP) group including CR PR and SD therapeutic response. [18F]FDG PET interpretation was performed on an Imagys® workstation (Keosys Saint-Herblain France) qualitatively and semi-quantitatively by two experienced nuclear medicine physicians blinded to clinical and conventional evaluation results. Any focus of increased [18F]FDG uptake over background not located in areas of normal [18F]FDG uptake and/or [18F]FDG excretion was considered to be positive for tumor. For semi-quantitative analyses of [18F]FDG uptake 3D regions of interest (VOIs) were placed over all lesions considered to be positive for tumor by using Imagys® software (Keosys France). The maximum standardized uptake value (SUVmax) was calculated using the single hottest pixel inside the tumor VOI. SUV peak was also calculated using a 1.2 cm diameter spherical VOI containing the SUVmax. Bone lesions were not taken into account as they were considered to be non-measurable lesions. For patients with more than one tumor lesion the sum of SUVmax and SUVpeak were calculated and used for evaluation of changes between PET1 and PET2. PET measurements were performed in up to a maximum of five measurable target lesions. All SUVs were normalized to the injected dose and patient body weight. The percentage changes in SUV between PET1 and PET2 were finally calculated as follows: ?SUV?=?(SUV1?SUV2)/SUV1. The same protocol was used for PET1 and PET3. Statistical analysis Data are expressed as mean±SD excepted for survival data that were expressed as the median. The primary endpoint of the study was comparison of changes in tumor [18F]FDG uptake on PET2 versus PET1 PET3 versus PET1 and subsequent CT scan evaluation at 8 weeks after initiation of erlotinib therapy. Friedman test was used for non-parametric comparison of repeated measures. The secondary endpoints were to determine the Receiver Operating Characteristic (ROC) analysis for [18F]FDG changes with regard to predicting response to erlotinib therapy. The relationship between metabolic response (patients stratified according to the median value of SUV variations) and clinical response was analyzed by Fisher's exact test. Progression-free survival (PFS) and overall survival (OS) were determined by standard Kaplan-Meier survival analysis and between-group comparison was performed by log-rank test. PFS was defined as the time interval from the date of enrolment in the study until the first signs of progression. OS was calculated from the date of enrolment until death from any cause. All analyses were performed using Graphpad prism version 4.0 b 2004 (Graphpad Software San Diego CA). The limit of significance was set at 0.05. Results Population Twelve eligible patients with NSCLC 6 women (50%) and 6 men (50%) with a mean age of 60±13 years were included. Two patients presented tumors harboring an activating Epidermal Growth Factor Receptor mutation (2573T>G substitution (p.Leu858Arg) in exon 21 in one patient; deletion (L747_E749del) in exon 19 in the other patient). Patient characteristics are described in . The median duration of erlotinib therapy was 75 days. Due to rapid progression and death PET3 and CT3 could not be performed in 2 patients. Tumor 18F-FDG uptake The three [18F]FDG PET/CT scans were acquired as follows: PET1 5±4 days before starting therapy PET2 9±3 days after starting therapy and PET3 60±6 days after starting erlotinib therapy. Scanning started 68±17 min (PET1) 71±16 min (PET2) and 64±13 min (PET3) after [18F]FDG injection of 271±53 MBq (PET1) 270±61 MBq (PET2) and 263±54 MBq (PET3). Blood glucose level was less than 1.5 g/L for all PET examinations i.e. 1.1±0.1 g/L for PET1 1.1±0.2 g/L for PET 2 and 1.1±0.2 g/L for PET3. Non-parametric Friedman tests did not show any significant difference between PET1 PET2 and PET3 for FDG uptake time injected FDG dose or blood glucose. Fifty-five lesions were described on PET1 before treatment and 45 lesions were defined as target lesions for PET evaluation of response to treatment (up to five most hypermetabolic lesions per patient; mean 3.8 lesions/patient). The mean tumor SUVmax of the most [18F]FDG“avid lesion (SUVmax) was 10.0±4.7 for PET1 and did not vary significantly over time with a mean of 10.1±6.6 for PET2 and a mean of 9.1±5.6 for PET3 (P?=?0.97). The SUVpeak was 8.6±4.3 for PET1 8.1±5.4 for PET2 and 7.1±4.6 for PET3 and did not vary over time (P?=?0.60). No variation over time was observed for the sums of SUV. The mean sum of tumor SUVmax of all target lesions was 30.1±19.5 for PET1 27.5±17.7 for PET2 and 28.3±22.4 for PET3 (P?=?0.83). Sums of SUVpeak of all target lesions were 22.7±14.3 for PET1 20.6±13.4 for PET2 and 22.2±18.6 for PET3 (P?=?0.44). [18F]FDG-PET response versus conventional evaluation CT scan data were interpreted by chest physicians blinded to PET/CT scan results (Table 2). Evaluation of response to treatment according to RECIST 1.1 criteria demonstrated 7 patients with progressive disease (group P) and 5 patients with non-progressive disease (group NP) including 4 cases of stable disease (SD) and 1 partial response (PR). .0087629.t002 Table 2 CT and PET assessments of response rates OS and PFS. Patient PET2 versus PET1 PET3 versus PET1 RECIST 1.1 Evaluation PFS OS New lesion ? SUVmax * ? SUVpeak * ? SUVmax * ? SUVpeak * Response to Treatment Progressive (P) or not (NP) days days on PET3 #1 ?21.6 ?17.6 18.6 ?1.5 SD NP 267 915 ? #2 25.9 26.9 70.3 77.4 PD P 57 316 + #3 9.0 7.6 23.4 23.3 PD P 216 447 + #4 ?18.6 ?15.0 ?3.2 ?2.6 PD P 67 414 + #5 ?20.3 ?11.1 42.1 51.1 PD P 53 152 + #6 ?56.7 ?59.9 ?72.1 ?70.6 PR NP 190 296 ? #7 ?22.0 ?26.0 ?31.3 ?24.3 SD NP 727 1249 + #8 ?32.0 ?25.1 3.9 ?3.9 SD NP 317 1146 ? #9 16.4 7.8 ?5.4 ?10.8 SD NP 77 359 ? #10 2.1 4.4 MD MD PD P 37 92 MD #11 36.1 20.0 30.3 25.7 PD P 104 734 ? #12 ?7.2 ?10.5 MD MD PD P 61 71 MD * For patient with more than one tumor lesion the sum of SUVmax and of SUVpeak were calculated and used for the evaluation of changes between PET1 and PET2 (or between PET1 and PET3). Missing data are indicated as MD. On ROC analysis the AUC for prediction of non-progressive disease by PET2 was 0.86 (95% CI 0.62 to 1.1; P?=?0.04) corresponding to a maximum specificity of 0.80 and sensitivity of 0.86 for non-progressive disease at a cut-off of 21.6% reduction in SUVmax (Figure 1) and a positive predictive value (PPV) of 0.86 a negative predictive value (NPV) of 0.80 an accuracy of 0.83 and a maximum Youden index of 0.65. The use of this SUVmax cut-off value correctly classified 11/12 patients (7 with true progressive disease (Figures 2 and 3); 4 with true non-progressive disease (Figures 4 and 5); 1 with false progressive disease (Figure 6). Non-progression after 2 months of treatment was significantly more frequent in patients with an early decrease in SUVmax of 21.6% or more (P?=?0.01 Fisher's exact test). The only misclassified patient (patient #9 false progressive disease on PET2 versus PET1) displayed a 16.4% increase of SUVmax but metabolic progression was not confirmed on PET3 with a 5.4% decrease of SUVmax compared to PET1. Similar results were observed for SUVpeak as non-progressive disease after 2 months of treatment was significantly more frequent in patients with a decrease in SUVpeak of at least 17.6% on PET2 (P?=?0.01 Fisher's exact test). Similar results were also obtained in terms of AUC sensitivity specificity PPV NPV and accuracy and with the same classification of patients (7 with true progressive disease; 4 with true non-progressive disease; 1 with false progressive disease). .0087629.g001 Figure 1 Percentage change in SUVmax on 18F-FDG PET/CT (cut-off: ?21.6%) within 2 weeks of starting erlotinib therapy in relation to conventional imaging response. Each red or green bar represents a patient NP or P respectively."
1
"Research has shown a strong association between a high lung cancer susceptibility score derived from family history of cancer the 20 SNPs COPD history (Auckland formula) and the development of lung cancers whereas healthy smokers matched for age gender and lifetime smoking habits had a relatively low score (n?=?446 lung cancer subjects 484 healthy current smokers). The odds ratio for lung cancer risk varied from 0.2-3.2 depending on the genetic risk (p?<?0.001) [910]. The accuracy of the Auckland formula in estimating lung cancer risk for a score of >4 was: sensitivity 90% specificity 45% ( which also includes scores for 52 subjects who developed cancer from a six year prospective study of 1212 smokers and ex-smokers). The score for prediction of non-cancer was conducted with a follow up of just six years. It means that 45% of non-cancer subjects have a low cancer score and 55% have some degree of increased score. The 55% with increased scores have simply not been followed up long enough for lung cancers to develop yet. Notwithstanding this limitation there is now a 20 SNP gene test for prediction of lung cancer in smokers under the trade name Respiragene. Research that established the respiragene test. Distribution of the Respiragene score in a cross-sectional study of 484 control smokers (blue) and 446 with lung cancer (red) (Total?=?930) and from the prospective study of 52 lung cancer cases (green). Reference [8]. Two case-control studies showed a 5-10% increase in cessation with a single gene test of small effect [1112]. A small smoking cessation pilot study using spirometry results and explanations using the Fletcher-Peto diagram to explain risk demonstrated that patients find this is an acceptable method and the quit rate at 12 months was 27% [13]. In a randomised control trial patients were given either a full explanation of the results of spirometry testing including an estimation of lung age or just their forced expiratory volume in the first second (FEV1) without explanation (control group). The group of patients who were given the full explanation had a 7.2% higher quit rate than the control group [14]. Data from a hospital outpatient cohort in Auckland suggest a larger increase in quit rate with Respiragene test and the Auckland formula (). Subjects who were current smokers in the pre-contemplative and contemplative stage were randomised into either the test group or control group and only the test group had the Respiragene test. Counselling and follow-up was done by telephone. Using Auckland formula to incorporate the results of the Respiragene test clinical data and family history a score ranging 1-12 with associated risk level (moderate risk high risk very high risk) was calculated and explained to test subjects. Neither group were involved in any formal smoking cessation programme. Indeed of the 13 subjects that had managed to stop smoking (28% of the gene-tested group) 48% quit without any medical assistance and only 52% had nicotine replacement therapy [1516]. When compared with previous studies using telephone counselling alone [17] () there is a 20-25% improvement in smoking cessation with the Respiragene test (). The improvement in intention to quit increases from 56% before testing to 67% in smokers with an average smokers risk of lung cancer or 89% in smokers with a high risk of lung cancer [18]. Respiragene study in Auckland NZ (n?=?43) Cancer susceptibility scale (compared with normal lifetime risk) Cancer susceptibility score Estimated lifetime risk of lung cancer Initial intention to quit Proportion that stopped smoking at 2-4 weeks Proportion still not smoking at 6 months Expected result for telephone counselling () - - 15% 41% 10-20% 9-12% Telephone counselling?+?Respiragene test 1-2.3 (10-35% risk) 2.3-6.7 (35-65% risk) 6.7-8 (65%-80% risk) 27 had average risk score 15% 67% 8/27 (30%) 8 (30%) 16 had high or very high risk score 30-50% (4-10 times average risk) 89% 10/16 (63%) 6 (37.5%) Smoking cessation after Respiragene testing and estimation of lung cancer risk with telephone counselling in a small pilot study in Auckland NZ (n?=?43) compared with expected quit rate. Efficacy of the variety of smoking cessation strategies. Percent increase of success for six months over unaided attempts for each type of quitting (chart from West & Shiffman based on Cochrane review data). Totally unaided smoking cessation has a 3-6% success rate. Therefore telephone support () increases success rate by 6% = 9-12% quit rate. A large hospital trial using Respiragene for calculating lung cancer susceptibility is currently underway in the USA [19] but there are no planned UK investigations. This study fills that gap and uses the NHS framework for smoking cessation. Other studies have taken place looking at how lung functioning testing in COPD might motivate smokers to quit suggesting that it is feasible to conduct this sort of study [131420]. This protocol describes a trial to evaluate a gene-based risk test (using genetic and clinical data) as a smoking cessation motivator in smokers wishing to participate in an NHS primary care smoking cessation clinic (in the action stage of change) alongside the usual counselling and prescribing protocol. It will differ from previous studies using gene testing as a motivator however in that the NHS primary care counselling and prescribing protocol will include several other motivators (CO breath testing saliva cotinine testing and intensive counselling) whereas the Auckland trial using the same gene test had none of these. Also the method of recruitment will differ in that primary care subjects will of necessity be different from the Auckland hospital outpatient cohort [1516]. Research question Can the Respiragene test combined with an estimation of lung cancer susceptibility be used to increase the uptake adherence to and success rate in an established smoking cessation programme in subjects who want to quit in a National Health Service United Kingdom (NHS UK) setting? Hypothesis Genetic testing and estimation of lung cancer susceptibility should increase œsmoking cessation outcomes at six months to >30% (or 1.5-2 fold greater than usual care) irrespective of the risk scores assigned to subjects [11]. Method/Design This protocol has been approved by Surrey Research Ethics Committee at the Royal Surrey County Hospital Guildford Surrey UK. 1/Recruitment Focus groups A number of focus groups of different aged smokers will be held to enable them to contribute to the design of the study 2/ Recruitment Subjects will be recruited from a large general practice in Surrey (practice population=?>?30000). Smokers aged 20-70 years will be identified from the practice records and contacted by post by their GP. Patients who reply stating that they wish to stop smoking will be randomised (stratified randomisation to ensure equivalent age and gender mix) to two clinics () only one of which will include the gene-based test. Previous trials of genetic testing in association with smoking cessation achieved 83-100% of participants opting for the test depending on the method of recruitment [821]. There will be two mailings with SAEs for recruitment with the aim of recruiting at least 30 subjects per clinic (see Power calculations under heading œstatistics). In the first letter the patient™s GP asks the patient to give permission for the researcher to contact him/her to ask about taking part in smoking cessation research (with possible genetic risk testing) and encloses fact sheet 1 and a stamped addressed envelope (SAE) for reply. Consort 2010 flow diagram for GeTTS recruitment. Mailing 2. The principal investigator mails patient with Letter 2 to ask him/her if they would like to attend an 8-week smoking cessation clinic and asks if they would be willing to have a test for genetic susceptibility to development of lung cancer and encloses SAE for reply. Mailing 3. The principal investigator mails Group B subjects and Group A test-concordant subjects to confirm dates of the smoking cessation sessions and full patient information leaflet and consent form enclosed. The information sheet will be slightly different for group A and B. Non-test concordant subjects within group A will be invited to attend the practice nurse for smoking cessation. 3/Inclusion and exclusion criteria i. Inclusion criteria: Aged 20-70 years smoking more than 10 cigarettes daily. ii. Exclusion criteria: Aged under 20 years or over 70 years smoking less than 10 cigarettes daily history of major depression and other psychiatric conditions dementias and serious or terminal illness (cancers etc.). Patients on warfarin would be excluded due to interactions between warfarin and varenicline as varenicline will be used as the modern treatment of choice for smoking cessation. Patients who smoke less than 10 cigarettes/day and patients who did not wish to have a genetic test or do not wish to take part in a research study will be referred to the practice nurse for smoking cessation. 4/Smoking cessation clinics For group A subjects only subjects who have expressed an interest in having a genetic test and gene-based estimation of susceptibility to lung cancer in mailing 2 will be invited to participate (see referral for decliners above). For group B subjects all subjects willing to participate are invited to do so. Uptake into smoking cessation programme (i.e. proportion of invitees who accept invitation and attend clinic of those mailed invitation) will be recorded. All subjects who attend the first session of the research clinic will be asked by the principal investigator JN to sign a consent form and will be invited to raise any concerns about the protocol (as explained in the full information sheet). The consent form will then be countersigned by JN. Group A clinics and Group B clinics will be held on different weekdays at the same health centre premises. Test Subjects who attend Clinic A will be offered a fact sheet on the health risks of smoking (including lung cancer) and the option of the gene-based test for calculation of lung cancer susceptibility whilst subjects who attend Clinic B will be given the same fact sheet on the health risks of smoking (including lung cancer) but without any reference to the gene-based test. The principal investigator will be responsible for handing out the fact sheets and administering the gene-based test in Clinic A and for handing out and explaining the fact sheet in Clinic B. NHS Surrey™s Smoking Cessation Practitioners will lead in-house smoking cessation clinics A and B using the NHS smoking cessation guidelines [22] under the supervision of the principal investigator at the medical centre. There will be: ¢Introductory session which includes a new near patient test for salivary cotinine (nicotine metabolite) “ trade name SmokeScreen [23]. ¢At session 2 patients will be given advice on therapies for smoking cessation. We expect that most patients will opt for a course of varenicline and they will be advised to contact their GP for a prescription. ¢This is followed by seven more weekly sessions and a follow-up session at six months (Figure 4). Uptake and adherence to smoking cessation will be monitored by weekly carbon monoxide exhalation measurements (breath test). The principal investigator will be involved in clinic A administering the gene-based test and determining if subjects have COPD from practice records and history in session 1. Participants who are heavy smokers have a smokers cough and use a salbutamol inhaler can be judged to have COPD even if this is not entered in their GP records (all Group A & B subjects will have spirometry at their 6-month follow-up). Subsequently the principal investigator will report back to clinic A patients with estimated lung cancer risks (session 3). To ensure balance in the control clinic the principal investigator will also attend Clinic B sessions 2 and 3 (see Figure 5 flow charts). ¢At the eight week clinic and the 6-month follow-up clinic smoking cessation status and carbon monoxide breath test score will be recorded and a feedback questionnaire used to assess efficacy of various components will be administered. Mailing 4. Telephone calls followed by letters to patients with invitation to 6-month follow-up session with NHS Smoking Cessation Practitioners and the principal investigator when cessation rate will be assessed and verified by repeating the carbon monoxide breath and salivary cotinine tests. “ for further details see Figure 5: flow charts Figure 4 Timeline of project. Figure 5 Flow chart for the duration of the trial. a. Flow chart of project from start to week 12. b. Flow chart of project to week 36. We anticipate good attendance at the eight week free smoking cessation clinic as would be expected if it were a regular NHS smoking cessation clinic but the attendance at the 6-month follow-up clinic may be more challenging. We consider this attendance essential and as attendance will take up an evening of their time study participants should be paid for their travel expenses (£20) and will receive up to three reminders. Group B subjects attending at the 6-month follow up who have been unable to quit will be offered the gene-based test at this stage. Technique for taking the respiragene test The test requires a Buccal swab and the subjects should not eat or drink within 15 minutes prior to supplying a sample (if has eaten or taken a drink within 15 minutes then rinse mouth with tap water). The nurse taking the sample should wear latex or plastic gloves and take care to avoid contact with the buccal swab collection tip to avoid DNA (deoxyribonucleic acid) contamination. Then: 1. Open buccal swab package at the handle end and carefully remove the swab. 2. Holding handle end of swab stick scrape the collection tip firmly against the inside of the cheek 5-6 times (about 10 seconds) being careful not to press the plunger that ejects the tip. 3. After taking the sample eject the swab tip into a labelled 2 ml microcentrifuge tube by firmly pressing the plunger at the end of the handle. 4. Complete and affix the sample tube label onto the microtube. The sample label requires the anonymised trial code for the subject. Storage of the respiragene test After sample collection tips can be kept at room temperature if they are posted immediately. If storage is necessary freeze the tubes containing the tips at -20°C. Packaging instructions for return of samples to Lab21 Ltd 1. Place absorbent material around the tube and then place tube in the plastic bag provided with the kit. Seal the plastic back as per the instructions on the bag 2. Place the plastic bag containing the sample tube into the shipping box. 3. Seal the box with the security seal supplied. 4. Using the Freepost service provided send the samples to: Lab 21 184 Cambridge Science Park Cambridge CB4 0GA. Patients will be asked to sign a disclaimer form that explains clearly that this test can only give an estimation of cancer risk and is a test that is still under development (one copy of form for investigators and one for patient). Interpretation of result of respiragene test Lung cancer susceptibility is calculated using the Respiragene test Auckland formula [7]: Lung cancer score?=?(number of susceptible genotypes) - (number of protective genotypes)?+?3 (for positive family history)?+?4 (for past history of COPD)?+?4 (for age?>?60 years old). The laboratory reports include the scores with an explanation of how the scores relate to a risk category (see ). When the subject is aged <60 years the report will also include the score and risk category that would apply if the subject is still a smoker at age 60 years or over. Follow-up questionnaires The questionnaires will be slightly different for groups A (questionnaire 2a) and B (questionnaire 2b) as only 2a will contain a direct reference to the gene-based test. Patients who fail to attend at eight weeks and six months will be contacted by telephone to remind them to complete their questionnaires and hand them in to the practice manager. They are designed to determine which subjects have quit smoking or cut down and which subjects who have failed to quit still plan to do so. There is a section that asks about general motivators and components of the smoking cessation programme. The subjects will be asked to score these motivators and smoking cessation aids for their efficacy in helping them to quit. The questions in this section are almost identical to a validated questionnaire [24]. There are also further questions on whether the subject would recommend the Respiragene test to a relative or friend and an open ended question for subjects to add their own comments about the concept of a test that predicts susceptibility to lung cancer in a smoker. Data quality assurance The study has been designed and will be reported in accordance with CONSORT (Consolidated Statement of Reporting Trials) [25]. Data will be controlled in accordance with data protection legislation institutional protocols of Sussex NHS Research Consortium and NHS policies for research and information governance for ensuring patient confidentiality [26]. Data will be analysed in SPSS (Statistical Package for Social Sciences) version 15 using an intention to treat approach. Outcome measures Primary endpoint Comparison of smoking cessation rates (7 day point abstinence and continuous abstinence) in Clinic A and Clinic B at 8 weeks and six months. Secondary endpoints A. Personal data: 1. Number of smokers still smoking who state that they still plan to stop. 2. Daily cigarette consumption of those still smoking. 3. Mean scores for ranking of smoking cessation aids (gene-based test - Clinic A only salivary cotinine lung cancer facts - controls in Clinic B only and general counselling from NHS smoking counsellors). B. Analyse questions about whether subjects would recommend the test to a member of family or a friend. C. Analyse last (open ended) question using qualitative research methodology. Statistics Primary end point The difference between smoking cessation between Clinic A and Clinic B will be estimated from the four week and six month follow up for the primary endpoint (smoking status confirmed by carbon monoxide breathalyser and salivary cotinine tests). If there is the expected higher rate of smoking cessation for Clinic A compared with Clinic B statistical significance will be demonstrated by the ?2 test. Since there are as yet no case-control studies that compare quit rate following the gene-based test versus quit rate without the test the expected difference in quit rate between Clinic A and Clinic B is difficult to estimate. Two case-control studies showing only a 5-10% increase in smoking cessation involved just a single gene of small effect [1112]. In a randomised control trial patients were given either a full explanation of the results of spirometry testing including an estimation of lung age or just the FEV1 without explanation (control group). The group of patients who were given the full explanation had a 7.2% higher quit rate than the control group. However data from Auckland suggest a larger uplift of quit rate with Respiragene. This can be explained by the superior predictive power of a 20-gene test combined with clinical history (personal history of COPD and family history of lung cancer) to give a rather more impressive estimate of cancer risk than anything previously available. The adequacy of sample size was tested using data from smoking cessation trials that showed: ¢30-40% smoking cessation at 6-months with similar protocols [2728]. ¢A 48% quit rate at 2-4 weeks in subjects with high and very high lung cancer risk scores but this difference shrinks to 27% at 6 months. ¢Data from Young et al [1518] (independently verified by McBride et al [11]) that even being given an average score for lung cancer susceptibility increases smoking cessation by approximately 10%. Therefore with a minimum sample sizes of 30 per group the following calculations based on these estimated quit rates apply (Table 2). Statistical power of 87.1% is generally acceptable for publication (for alpha error of 5% - i.e. 5% probability of incorrectly rejecting the null hypothesis that there is no difference in the percentage values). For further detailed statistical analysis refer to Additional file 1. Table 2 Summary of values from which the power of the study are estimated Control group expected quit rate as %ge Respiragene group expected quit rate as %ge ? 2 calculated from four-some table P value based on ? 2 Power calculations* 8 weeks Sample size 30/30* 70% 94% 5.9 <0.05 79.3% Sample size 60/60* 11.7 <0.01 96.9% 6 months Sample size 30/30** 35% 52% 1.7 NS 36.5% Sample size 60/60** 6.2 <0.05 87.1% *Telephone (alone) quit rate (see ) assumed to be 20%. **Telephone (alone) quit rate (see ) assumed to be 10-15%. Secondary outcome measures Similarly the significance of secondary endpoints on intention to stop smoking cigarette consumption uptake of invitation to cessation adherence to cessation course and self-reported smoking cessation will be calculated by the ?2 test but the p value for the ranking scores for information on lung cancer risk and other smoking cessation aids and motivators will be estimated from the unpaired student t-test. The open ended question: œHow do you feel now about having had a genetic test that estimates the probability that you will develop lung cancer at some future date? will have to be analysed by qualitative analysis to determine the main recurrent themes in responses. Discussion Overview Smoking cessation is one of the most cost effective interventions that can be achieved in primary care [29]. However many smokers are very reluctant to commit to a smoking cessation programme (precontemplative and contemplative) and about half of those that attend for smoking cessation intervention (action stage of change) are likely to drop out or give up trying. Therefore any methodology that increases motivation in both unmotivated and motivated smokers could be very valuable. The gene-based test we are offering has shown promise as a smoking cessation motivator in precontemplative-contemplative smokers in a hospital outpatient setting [1518] and now needs to be tested out as a motivator for improving adherence in a primary care smoking cessation clinic using a randomised controlled study. Strengths The main strengths of this study are that it is being carried out on subjects from a large primary care population and should therefore be more representative of the general population than previous studies recruited from hospital patients and other special groups. We also have the advantage of being able to carry out this research within the established framework of the local stop smoking service. Limitations and assumptions Although we have estimated based on previous smoking cessation work using this gene-based test that the primary endpoint will show that having the test improves quit rate by 20-25% this was based on a cohort of hospital outpatients in Auckland New Zealand and subjects recruited from primary care may respond differently. Although we plan to recruit a minimum of 60 subjects this may not be enough to balance unexpected and unknown confounding factors. What we might find We aim to recruit a minimum of 60 subjects to randomise 30 into group A (test group) and 30 into Group B (control group). The normal experience in NHS smoking cessation clinics is a drop-out rate of 40-50% [30-32]. We need therefore to attempt to recruit about 120 subjects in order to get a statistically significant result based on the assumptions in our power calculations. We may however have underestimated the 6-month quit rate using the NHS local stop smoking guidelines [22] which typically involves a multi-interventional programme which includes combinations of varenicline prescriptions breath carbon monoxide monitoring and intensive counselling giving a quit rate of 70-80% at 6-weeks.There are however no Surrey data for 6-month quit rate which we assume on the basis of similar smoking cessation data to be about half the 6-week figure [33] ? 35%. An unknown and unpredictable factor that could skew results significantly is the possibility that our multi-interventional approach could help to reinforce the health risk message equally for subjects in both groups. Also the Auckland study design involved recruitment of precontemplative-contemplative smokers from a hospital outpatient setting compared to this study that will involve primary care subjects who have volunteered to participate in a smoking cessation programme (ie smokers in the action stage of quitting)."
1
purpose pancreatic ductal adenocarcinomas exhibit a high degree of desmoplasia due to extensive extracellular matrix deposition encasement of mesenteric vessels by stroma in locally advanced pancreatic cancer lapc prevents surgical resection this study sought to determine if the addition of a monoclonal antibody to connective tissue growth factor pamrevlumab to neoadjuvant chemotherapy would be safe and lead to improved resectability in this surgically adverse patient populationmethods in this phase iii trial patients with lapc were randomised to gemcitabinenab paclitaxel plus arm a n24 or minus arm b n13 pamrevlumab those who completed six cycles of treatment were assessed for surgical eligibility by protocol defined criteria resection rates progression free and overall survival were evaluatedresults eighteen patients in arm a and seven in arm b completed six cycles of therapy with similar toxicity patterns in arms a and b carbohydrate antigen “ response as defined by ‰¥ decline from baseline occurred in and respectively sixteen per cent of patients were radiographically downstaged by national comprehensive cancer network criteria in arm a and in arm b positron emission tomography normalised in vs of patients in arm a vs arm b respectively and correlated with surgical exploration eligibility for surgical exploration was vs p00019 and resection was achieved in vs of patients in arm a vs arm b p01193 respectively postoperative complication rates were not different between armss neoadjuvant chemotherapy with pamrevlumab holds promise for enhancing resection rates in patients with lapc without added toxicity this combination merits evaluation in a larger patient cohortintroductionpancreatic cancer is currently the third leading cause of cancer death in the usa1 and by it will likely become the second leading cause of cancer related death after key questionswhat is already known about this subject –º pamrevlumab is anti ctgf1 inhibitor highly safe when given to patients with pancreatic cancer and potentially adds to the activity of gemcitabine and erlotinib when given in metastatic diseasewhat does this study add –º this study examines a the safety and activity of pamrevlumab when added to gemcitabine and nab paclitaxel in locally advanced pancreatic cancer b the impact of this regimen and surgical resection and postoperative safety c explores the utility of a novel study design for locally advanced pancreatic cancerhow might this impact clinical practice –º this study has the potential to lead to practice changing activity in locally advanced pancreatic cancer via the eventual approval of pamrevlumab for use in this situation the promulgation of a new study design for locally advanced pancreatic cancer and increased potential for surgical resection and thus prolonged os curability in locally advanced pancreatic cancerlung cancer surpassing breast and colon cancer2 surgical resection is generally necessary for treatment with curative intent or to extend life expectancy3 however only of patients have disease amenable to upfront curative resection at the time of diagnosis4 approximately “ of patients are diagnosed with locally advanced disease5 determined surgically unresectable per national comprehensive cancer network nccn guidelines6 patients with locally advanced pancreatic cancer lapc have a prognosis similar to those with metastatic disease with a historical median overall survival os of picozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen access“ months with recent trials demonstrating median os of months7 recent single institution retrospective studies have reported the potential for resection of lapc with neoadjuvant therapy irrespective of imaging findings with promising results8 however these are limited by significant selection bias lack of strict radiographic classification and chemotherapy standardisation current prospective trials have documented resection rates of lapc in the range of to therefore novel approaches are needed to improve patient outcomesthe tumour biology inherent to pancreatic ductal adenocarcinoma pdac significantly contributes to the poor outcomes seen in this disease notably pdac exhibits a high degree of desmoplasia often in association with elevated connective tissue growth factor ctgf expression12 ctgf appears to play a central role in the biology of pancreatic cancer affecting both its cellular biology and its extracellular matrix composition this leads to many simultaneous biological effects that promote pancreatic cancer growth including increased cellular proliferation and differentiation increased cellular adherence and migration antiapoptosis vascular permeability angiogenesis and suppression of tumour immunological responses13 this stroma may also contribute to the radiographic imaging findings of mesenteric vessel involvement or encasement that is used to determine resectability of pancreatic tumours executing a pharmacological intervention on the pancreatic cancer stromal environment is therefore a major goal of the development of novel pancreatic cancer therapeuticspamrevlumab is a human monoclonal antibody that targets ctgf preclinical studies showed that ctgf overexpression is associated with both desmoplasia and gemcitabine resistance in the kpc pancreatic cancer mouse model14 when pamrevlumab was used in combination with gemcitabine sensitivity to gemcitabine was enhanced which correlated with inhibition of xiap an antiapoptotic protein15 when tested in patients with advanced pancreatic cancer stage iv and locally advanced stage iii treated with gemcitabine and erlotinib in a phase iii study n75 pamrevlumab displayed multiple favourable outcomes16we hypothesised that through inhibition of the downstream effects of ctgf overexpression on tissue adhesion and other mechanisms pamrevlumab may influence resectability of pdac tumours with this in mind this novel phase iii randomised multicentre trial was designed to explore the safety and efficacy of pamrevlumab in combination with gemcitabinenab paclitaxel in lapc with special emphasis on surgical eligibility and safetymethodsstudy designthis was a phase iii randomised trial of safety and efficacy in patients with lapc who received gemcitabine and nab paclitaxel with or without pamrevlumab as neoadjuvant therapy the randomisation was preplanned and blinded to the investigator the study was approved by individual institutional review boards at nine us institutions and conducted according to the declaration of helsinki the trial was registered at clinicaltrials gov as nct eligibilitykey protocol eligibility requirements included biopsy proven diagnosis of pdac radiographic staging consistent with locally advanced unresectable disease as defined nccn guidelines v2 clinical stage confirmed by diagnostic laparoscopy radiographically measurable disease per response evaluation criteria in solid tumors recist v11 eastern cooperative oncology group ecog performance status of or adequate haematological renal and hepatic function no prior therapy for pdac and no concomitant cancer diagnosis within the past yearsstudy schemaeligible patients were randomised to arm a or arm b to receive a total of six treatment cycles “ weeks of therapy figure patients in arm a received pamrevlumab mgkg by intravenous infusion on days and of each day cycle with an additional dose given on day in the first cycle patients in both arms a and b received gemcitabine mgm2 by intravenous infusion on days and of each day treatment cycle nab paclitaxel mgm2 by intravenous infusion on days and of each day cycle doses for gemcitabine and nab paclitaxel were modified for haematological and non haematological toxicity as per standard of care soc15 patients remained on therapy for six treatment cycles “ weeks unless they had disease progression an intolerable adverse event ae or toxicity withdrew consent or were withdrawn at the investigator™s discretion all patients were followed for drug toxicity until days after the last drug dose patients undergoing surgery were followed for days following hospital discharge for surgical complications ctgf levels were obtained prior to treatment from all patients plasma samples for pamrevlumab level determination were obtained from all patients receiving this drug after all protocol specified therapy was completed patients were followed for disease progression survival and additional oncological therapy postoperative complications including day readmissions and day mortality were notedresponse assessmentpatients were evaluated for response by the following measures carbohydrate antigen ca “ measured at baseline first day of each cycle and end of treatment eot recist v11 read based on full body ct imaging high resolution dual phase fine cut ct imaging at baseline and every weeks thereafter fluorodeoxyglucose fdg positron emission tomography pet imaging and nccn v2 resectability criteria at baseline and eotpicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen accessfigure patient flow and surgery outcomes in arm a four of the eligible subjects had their surgeries cancelled 1portal vein thrombosis 3medical issues precluding surgery in arm a four eligible subjects underwent surgery but resection was not achieved 3metastatic disease discovered 1extensive sma encasement in arm b one eligible subject underwent surgery but resection was not achieved 1extensive vascular encasement sma superior mesenteric arterysurgical assessmentsubjects who finished six cycles of combination chemotherapy were evaluated for eligibility for surgical exploration per protocol pp defined criteria given that patients included in the trial were determined to be initially unresectable by radiographic imaging and nccn criteria objective criteria were developed to standardise attempts at surgical resectionpatients were deemed eligible for surgery if one or more of the following criteria were met reduction in plasma ca “ level by ‰¥ at eot compared with baseline reduction in fdg pet maximum standardised uptake value suvmax by ‰¥ at eot compared with baseline radiological tumour response per recist of partial response pr or complete response cr at eot or met the definition of resectable or borderline resectable per nccn guidelines subjects were classified as ineligible for surgical exploration if any of the following occurred development of distant metastases or local progression on ct scan tumour anatomy precluding vascular reconstruction unreconstructible local complications preventing surgery eg portal vein pvsplenic vein thrombosis pancreatitis or decline in performance status to a karnofsky score ‰¤ or picozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668absolute contraindication to surgery from comorbidity eg recovery from myocardial infarction or uncontrolled diabetes the final decision regarding whether resection was to be performed was made by the treating surgeonendpointssafety endpoints included serious adverse events sae during neoadjuvant therapy and surgical complications postresection the efficacy endpoints included surgical eligibility r0 resection r0r1 resection median os progression free survival pfs and year survival rate all patients were followed and data analysis was stratified by pp population and intention to treat itt cohortstatistical considerationsthe comparison between selected clinical characteristics toxicity profiles and eligibility for surgical exploration or completed surgical resection was performed using the χ² test exact cis for the point estimates as well as the treatment difference were obtained from the sas proc freq procedure with the exact option the two treatment arms were compared using the cochran mantel haentzel test controlling for baseline factors tnm stage ecog ca “ pet suvmax 0copen accesssuperior mesenteric artery sma involvement coeliac abutment and so on as prespecified in the protocol all cause mortality was used in determining os which was analysed by the kaplan meier method survival status was updated within month before the data cut off date data from patients who were alive at the cut off date were censored for survival analysis all statistical tests were performed at the significance level of α005 using two sided testsresultspatient characteristics and dispositionthirty seven patients were randomised to study treatment to arm a pamrevlumabgemcitabinenab paclitaxel and to arm b gemcitabinenab paclitaxel alone patient characteristics at baseline are summarised in table all patients enrolled were unresectable by nccn criteria patients had tumour arterial involvement sma encasement ° coeliac abutment table patient characteristicsbaseline demographics “ years “ years ‰¥ years median male femaleage group sex bmi kgm2 mean sd median min maxecog grade grade tnm stage t3 n0 m0 t3 n1 m0 t4 n0 m0 t4 n1 m0 t4 nx m0location of the tumour in the pancreas non resectability per nccn criterion head body tail median tumour size mm ° sma encasement any coeliac abutment inferior vena cava invasion or encasement unreconstructible smvportal occlusion aortic invasion and encasementarm agnppn24 arm bgnpn13 totaln37 to to to · ok as isnot mutually exclusivebmi body mass index ecog eastern cooperative oncology group g gemcitabine n number of subjects nccn national comprehensive cancer network np nab paclitaxel p pamrevlumab pv portal vein sma superior mesenteric artery smv superior mesenteric veinpicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen accesswithout gastroduodenal artery involvement or aortic involvement inferior vena cava invasion and unreconstructible pvsuperior mesenteric vein smv occlusion a higher percentage of patients with sma encasement ° were randomised to arm a vs arm b patient disposition is summarised in figure twenty four patients in arm a received gemcitabinenab paclitaxel and pamrevlumab patients completed six treatment cycles six patients discontinued treatment early due to progressive disease three patients aes two patients or physician decision one patient thirteen patients in arm b received gemcitabinenab paclitaxel patients completed six treatment cycles six patients discontinued treatment early due to progressive disease two patients aes two patients or patientphysician decision two patientssafetysaes are summarised in table forty one per cent of patients had a treatment emergent sae arm a arm b no individual toxicity category occurred with frequency except systemic infection patients there was no demonstrable increase in any toxicity with the addition of pamrevlumab to gemcitabinenab paclitaxel chemotherapytable summary of treatment emergent serious adverse eventssystem organ classpreferred term ascites nausea pancreatitis vomiting device occlusion drug withdrawal syndrome feverno of patients with any treatment emergent saeblood and lymphatic disorders haemolytic uremic syndrome lymphadenopathycardiac disorders cardiac failure supraventricular tachycardiagastrointestinal disorders general disorders and administrative site conditions hepatobiliary disorders infections sepsis cellulitis urinary tract infectioninjury poisoning and procedural complications respiratory thoracic and mediastinal disorders skin and subcutaneous disorders cholangitis hyperbilirubinaemia craniocerebral injury pneumonitis pulmonary embolism rasharm an24n arm bn13n overalln37n · ok as ispicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen accessresponse to therapyin arm a had ‰¥ ca “ decline at eot response by recist pr ‰¥ decline in pet suvmax and were radiographically downstaged by nccn criteria during the treatment period the median ca “ decline was patients were non secretors seven out of patients had best objective recist response crpr some patients had ˜exceptional™ responses defined as normalisation or ‰¥ decline of ca “ patients or normalisation pet suvmax in in arm b had ‰¥ ca “ decline at eot response by recist pr ‰¥ decline in pet suvmax and were radiographically downstaged by nccn criteria four out of patients had best objective recist response cr pr in arm b of patients had an œexceptional ca “ response and had an ˜exceptional™ pet response as defined by either ‰¥ normalized ca response normalized suv max andorradiographic downstaging post therapy completion surgical evaluationoverall of the total study patients were eligible for surgical exploration using protocol defined criteria arm a arm b p00019 resection was completed in of the patients arm a arm b p01193 details of the nine resected patients are shown in table in arm a of the patients were eligible for surgical exploration in the itt population and of the patients were eligible in the pp population patients who completed six cycles of treatment in arm a out of eligible patients ultimately underwent surgical exploration for resection five operations were cancelled four due to drug toxicitymedical comorbidity sepsis gallbladder perforation declined performance status and fever and one patient declined eight out of patients in arm a were resected r0 r1 the remaining four patients who were explored were not resected due to progression or unresectable disease intraoperatively in arm b of the patients were eligible for surgical exploration in the itt population and were eligible in the pp population of the two subjects found to be surgically eligible only one was resected as the other patient had local progressionpredictors of resectionhigh ca “ response ‰¥ decline andor normalisation was contributive to surgical eligibility vs p03 normalisation versus non normalisation of pet suvmax was predictive of surgical eligibility vs p0013 and successful resection vs p0002 combining these two criteria was highly predictive for surgical eligibility vs p0003 and completed vs p0002 resection all nine successful resections were identified by one or both of these criteria table summary of resected patientssitesubject idtreatmentarmresponse to treatmentnccnbaselinenccnend of treatmentresection status“““““““““aaaaaaaab unresectablecoeliacunresectablesma smvunresectablecoeliacunresectablecoeliacunresectablesmvunresectablesmaunresectablesma smv coeliacunresectablesmaunresectablecoeliacunresectablecoeliacunresectablesma smvunresectablecoeliacborderline resectableunresectablesmvunresectablesmaunresectablecoeliacunresectablesmaunresectablecoeliacr0r1r0r0r1r1r1r0r0protocol defined criteria ca “ decrease fdg pet suvmax decrease ‰¥ recist v11 response pr or cr nccn resectable or borderline resectable criteriaca carbohydrate antigen cr complete response fdg fluorodeoxyglucose nccn national comprehensive cancer network pet positron emission tomography pr partial response recist response evaluation criteria in solid tumors sma superior mesenteric artery smv superior mesenteric vein suvmax maximum standardised uptake valuepicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0cconversely radiographic features of response did not correlate with operative potential neither recist response nor radiographic downstaging per nccn criteria statistically correlated with completed resectionsurgical complicationspostoperative complications were summarised according to the clavien dindo classification posthoc analysis ischaemic gastritis and ulceration and right lower lung lobe collapse were reported for one patient in arm a grade ii there was one episode of clinically significant pancreatic leak in each arm grade iiia no reoperations and no day or day surgical mortality were noted one patient in arm b had a delayed gastric perforation following a distal pancreatectomy with coeliac axis resection likely due to thermal injury and was treated non operatively grade iiib no wound complications or superficial site infections were noted in either group four out of patients and out of patients in arm a and b respectively were readmitted within days and the difference between treatment arms was not clinically or statistically significantsurvivalas of the data cut off date of evaluable patients were known to be alive with a median length of follow up at approximately months pfs was months ci to and months ci to in arm a and arm b respectively one year survival and median os were and months ci open accessto in arm a and and months ci nr in arm b the median os for all patients who were eligible for surgical exploration arm a arm b vs ineligible arm a arm b was months ci nr vs months ci to p00766 the median os for resected arm a arm b vs non resected patients arm a arm b was not reached ci nr vs months ci to p00141 figure discussionthe treatment of lapc with neoadjuvant therapy remains challenging and there is no established soc several high volume centres have reported their single centre experiences with varying neoadjuvant chemotherapy and chemoradiation strategies8 the combination of more active regimens delivered over an extended period and surgeons™ comfort with resecting and reconstructing major mesenteric vessels has led to an increase in resection rates a meta analysis of studies using folfirinox has demonstrated resection rates ranging from to in lapc17 one of the larger studies including patients with lapc reported a resection rate of that was more dependent on duration of therapy months than chemotherapy regimen folfirinox or gemcitabine based18 recently a single institution and single arm prospective study of neoadjuvant folfirinox and losartan with selective use of radiation in patients with lapc reported an r0 resection rate of figure overall survival resected vs non resected patientspicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen access however the lack of randomisation makes it difficult to determine what aspect of therapy was responsible for this effect and only of resected patients needed any type of vascular reconstruction perhaps suggesting more favourable outcome than usually seen in locally advanced disease these retrospective studies contain significant interpretative challenges including selection bias treatment variables including radiation and the use of different definitions of lapcthe anti ctgf mechanism of action with respect to gemcitabine based therapy a recent large scale prospective trial of patients with lapc treated with induction gemcitabine with or without erlotinib followed by radiation only patients were able to undergo resection10 furthermore the addition of erlotinib to gemcitabine did not improve any downstaging capacity and there was no difference in survival with the addition of radiation more recently the la pact trial examining the role of induction gemcitabine nab paclitaxel found that only out of patients with lapc were able to undergo surgery following neoadjuvant therapy with combination gemcitabine and nab paclitaxel for six cycles by investigator™s choice11 last although folfirinox has been the most studied induction combination chemotherapy regimen in this population recent randomised data from european patients who received neoadjuvant folfirinox versus gemcitabinenab paclitaxel prior to resection20 showed no clear difference with respect to r0r1 to resection rate vs p0135 or os vs months p0268given for pamrevlumab its use in the neoadjuvant setting has the potential to impact tumour regression and modulate the desmoplastic niche and possibly affect tumour margins allowing for improved resection rates previous studies have looked at the ability of gemcitabinenab paclitaxel to reduce cancer associated fibroblasts resulting in a ˜softening™ of tumours by endoscopic ultrasound elastography21 this stromal depletion also translated into a decrease of suv uptake on pet22 in the study reported herein we combined gemcitabine and nab paclitaxel with pamrevlumab to explore its effect in terms of therapeutic response the impact on eligibility for surgical exploration and improved resection rates in locally advanced patientsthe protocol specified therapeutic response criteria ca “ pet suvmax recist and nccn criteria were used as criteria to determine eligibility for surgical exploration in lapc this is a novel approach specific to the protocol and allows participating patients to be explored for resection when otherwise they may not qualify by current treatment standards nccn criteria for example by nccn conversion alone ie converted from unresectable to borderline resectable only of patients in arm a would have been eligible for surgical exploration however by protocol criteria of patients in arm a were eligible for surgical exploration a higher percentage of patients were eligible for surgical exploration by the above criteria in arm a vs arm b vs respectivelyoverall the rate of successful resections in the pamrevlumab treated group was higher than in the control group but this did not reach statistical significance most probably due to small sample size of the nine subjects that were successfully resected in this trial only one was converted by nccn criteria to borderline resectable prior to surgical exploration despite this phenomenon the data support the hypothesis that pamrevlumab a human monoclonal antibody with anti ctgf mechanism of action could alter tumour characteristics allowing resection in otherwise unresectable patients this hypothesis needs to be confirmed and patients should be stratified by coeliac andor sma involvementthe most common predictive factors for eligibility for surgical exploration and resection were ca “ decline and pet suv max response which are indicators of tumour response to treatment the combination of these two factors proved to be a highly sensitive objective readout for prediction of potential surgical success both the ability of ca “ response and the inability of radiographic response recist and nccn criteria of resect ability to predict surgical outcome has been observed by others3 and these observations deserve further examination in subsequent clinical trials in the mpact study both ca “ and pet response correlated to improved survival in metastatic patients treated with gemcitabine and nab paclitaxel23 recent surgical series of patients with borderline resectable and lapc have also corroborated their impact in the localised setting25 correlation of clinical response with plasma levels of endogenous ctgf and pamrevlumab exposure as shown in the prior study by picozzi et al16 may provide added prognostic and predictive insightwith regard to safety no major incremental toxicity in any category was noted with the addition of pamrevlumab to gemcitabinenab paclitaxel in addition a higher number of patients were able to complete six cycles of the three drug combination including pamrevlumab when compared with gemcitabinenab paclitaxel alone pamrevlumab is well tolerated and considered safe compared with the soc drugs for patients with pdac these observations represent a very favourable attribute when considering potential neoadjuvant chemotherapy in a patient population with the frequency of medical problems typically seen in lapc in addition there were no signals of increased surgical morbidity or wound healing problems with ctgf blockade by pamrevlumab in fact there were only two clinically significant pancreatic leaks one in each arm which is comparable to national outcome data from high volume pancreatic surgery centres similarly readmissions following resection were comparable between arms and reflected the complexity of this challenging patient populationfinally while survival data are not yet mature both patients who were eligible for surgery and those that picozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0cwere ultimately resected had longer pfs and os highlighting the importance of surgical resection of the tumour therefore more investigation into newer agents targeting lapc and increased consideration of candidacy for surgery in those patients who do not progress on therapy or suffer toxicity should be of utmost importance to improve outcomes in this diseasein this is the first prospective randomised multicentre trial examining the role of neoadjuvant therapy in lapc with prespecified criteria for surgical exploration the use of pamrevlumab in combination with gemcitabine and nab paclitaxel showed a potential to enhance tumour response and increase resection rates further evaluation of this drug combination in the neoadjuvant treatment setting for lapc is warranted and a larger phase iii trial with resection and survival endpoints is ongoingcontributors fibrogen inc was the study sponsor that designed the study in consultation with the principal investigator vp and surgical co investigator fgr all authors except those of the sponsor contributed patients to the study fibrogen was responsible for data collection and analysis all authors reviewed the manuscript and signed off on its accuracyfunding the study was funded by fibrogen inc san francisco cadisclaimer the corresponding author has the right to grant on behalf of all authors and does grant on behalf of all authors an exclusive licence or non exclusive for government employees on a worldwide basis to the bmj publishing group ltd and its licensees to permit this article if accepted to be published in esmo open editions and any other bmjpgl products to exploit all subsidiary rights as set out in our licencecompeting interests mc mz sp ek and ec are employees of fibrogen and hold stock andor stock options in fibrogenpatient consent for publication not requiredprovenance and peer review not commissioned externally peer revieweddata availability statement data are available on reasonable request all data relevant to the study are included in the article or uploaded as supplementary information data will be available as presented in this manuscriptopen access this is an open access article distributed in accordance with the creative commons attribution non commercial cc by nc license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited any changes made are indicated and the use is non commercial see a0http creativecommons org licenses by nc orcid idewa a0carrier http orcid org references american cancer society cancer facts figures available httpswww cancer org content dam cancer org research cancer facts and statistics annual cancer facts and figures cancer facts and figures pdf rahib l smith bd aizenberg r et a0al projecting cancer incidence and deaths to the unexpected burden of thyroid liver and pancreas cancers in the united states cancer r
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"objectives to describe the benefits and limitations of using individual and combinations of linked english electronic health data to identify incident cancersdesign and setting our descriptive study uses linked english clinical practice research datalink primary care cancer registration hospitalisation and death registration dataparticipants and measures we implemented case definitions to identify first site specific cancers at the most common sites based on the first ever cancer diagnosis recorded in each individual or commonly used combination of data sources between and we calculated positive predictive values and sensitivities of each definition compared with a gold standard algorithm that used information from all linked data sets to identify first cancers we described completeness of grade and stage information in the cancer registration data setresults gold standard cancers were identified positive predictive values of all case definitions were ‰¥ and ‰¥ for the four most common cancers breast lung colorectal and prostate sensitivity for case definitions that used cancer registration alone or in combination was ‰¥ for the four most common cancers and ‰¥ across all cancer sites except bladder cancer using cancer registration alone for case definitions using linked primary care hospitalisation and death registration data sensitivity was ‰¥ for the four most common cancers and ‰¥ for all cancer sites except kidney oral cavity and ovarian cancer when primary care or hospitalisation data were used alone sensitivities were generally lower and diagnosis dates were delayed completeness of staging data in cancer registration data was high from minimum in and in for the four most common cancerss ascertainment of incident cancers was good when using cancer registration data alone or in combination with other data sets and for the majority strengths and limitations of this study –º this is the first study to present comprehensive information on the implications of using different individual and combinations of linked electronic health data sources in england to identify cases of the most common incident cancers –º using a gold standard algorithm that combined all available data from multiple sources as a comparator we were able to estimate both positive predictive values and sensitivity values for a range of pragmatic case definitions –º we described similarities and differences in values between age groups sexes and calendar years the impact of choice of sources on diagnosis dates and mortality rates and completeness of stage and grade in cancer registration data –º a key limitation was that our gold standard algorithm is not validated and may be affected by differences in clinical diagnosis and coding of invasive cancers between data sourcesof cancers when using a combination of primary care hospitalisation and death registration dataintroductionthe clinical practice research datalink cprd provides de identified primary care data linked to additional secondary health data sources under a well governed framework1 use of linked data helps researchers to answer more epidemiological questions and increase study quality through improved exposure outcome and covariate classification2 in the field of cancer epidemiology cprd primary care data linked to hospital episode statistics admitted patient care strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access data hes apc office of national statistics ons mortality and national cancer registration and analysis service ncras cancer registration data are used to analyse factors contributing to the risk of cancer and the consequences of cancer and its treatment use of linked data reduces the sample to the common source population and data coverage period for each included data set and has cost and logistical implications which are greatest for ncras data research teams therefore commonly choose not to use all available linked data3 cancer epidemiology studies can also be conducted using ncras and hes apc data provided by national health service nhs digital and public health england phe without linkage to cprd primary care data4 this provides national coverage at the expense of the detailed health data that are available in primary care recordsvalidation studies assessing concordance between cprd gold hes apc and ncras data have estimated high positive predictive values ppvs for cprd gold data and varying proportions of registered cancers that are not captured in cprd gold and hes apc5“ the most up to date analysis by arhi et al included the five most common cancers and all papers focussed on concordance between cprd gold only and ncras existing evidence therefore does not provide a complete assessment of the benefits and limitations of using different combinations of data sources within the context of practical study designs national data are available describing completeness of data fields within the cancer registry data in each collection year9 and over time for all cancers combined4 missingness for individual years has been associated with age comorbidities and clinical commissioning groups10 we aim to describe and compare the benefits and limitations of using different combinations of linked cprd primary care data hes apc ons mortality and ncras cancer registration data for conducting cancer epidemiology studies our analyses focus on incident cancer ascertainment as it is a common and important outcome in cancer epidemiology and it is more difficult to distinguish between secondary recurrent and primary cancers at a second site in these data sets we have compared definitions of the most common cancers based on the first ever cancer recorded in individual or combinations of data sets with a gold standard definition comparing information from all four data sets we also describe the availability of stage grade and treatment variables over time in the cancer registration data for the cprd linked cohort this reflects real life study design and will help researchers to decide which combination of data sources to use for future studiesmethodsstudy design and settingwe completed a concordance study using linked2 english cprd gold hes apc ons mortality and ncras data cprd gold data were extracted from the january monthly release and the 13th update to cprd™s linked data the study period was from january to december with december matching the end of the ncras coverage periodthe cprd gold database includes de identified records from participating general practices in the uk who use vision software1 general practice staff can record cancer diagnoses using read codes or in free text comments boxes though the latter are not collected by cprd diagnoses will typically be entered duringfollowing a consultation or from written information that is returned to the practice from secondary care cprd gold data are linked to hes apc ons mortality and ncras through a trusted third party for english practices that have agreed to participate in the linkage programme2 hes apc data are collected by nhs digital to co ordinate clinical care in england and calculate hospital payments12 admissions for and related to cancer diagnoses are recorded using international classification of diseases version icd10 codes national cancer registration data are collected by ncras which is part of phe in accordance with the cancer outcomes and services data set13 which has been the national standard for reporting of cancer in england since january data include icd10 codes to identify the cancer site and more detailed information such as stage and grade ons mortality data includes dates and causes of deaths registered in england recorded using icd10 codesparticipants exposures and outcomesour underlying study population included male and female patients registered in cprd gold practices who were eligible for linkage to hes apc ncras and ons mortality data and had at least days of follow up between january and december start of follow up was defined as the latest of the current registration date within the practice and the cprd estimated start of continuous data collection for the practice up to standard date end of follow up was determined as the date the patient left the practice ons mortality date of death or practice last collection dateidentification and classification of cancer codeswe used code lists to classify cancer records in each of cprd gold hes apc and ons mortality data as one of the most common sites other specified cancers history of cancer secondary cancers benign tumours administrative cancer codes unspecified and incompletely specified cancer codes https org data incompletely specified cancer codes could be mapped to cancer site eg icd10 code c689 œmalignant neoplasms of urinary organ unspecified was considered consistent with both bladder and kidney cancer for ncras we accessed coded records for the most common cancers we included cancers recorded in the clinical or referral file for cprd gold cancers recorded in any diagnosis field for hes apc and the underlying or strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure gold standard algorithm to identify incident site specific cancers using all data sources hes hospital episode statistics ncras national cancer registration and analysis service ons office of national statisticsmost immediate cancer cause of death in ons mortality datacancer case definitions based on individual sources and combinations of sourceswe developed alternative cancer case definitions mirroring those commonly used in epidemiology studies based on identifying the first malignant cancer excluding administrative codes and benign tumours recorded in various combinations of data sources ncras alone ncras and hes apc all sources cprd gold hes apc and ons mortality cprd gold alone hes apc alone multiple malignant cancers recorded on the index date in cprd gold or hes apc were reclassified as multiple site cancer and were not considered as individual site cancer records for positive predictive value and sensitivity calculations multiple codes recorded in different sources on the same date were reclassified as the site identified in the ncras data if available and as multiple site cancer if not for each case definition we only examined the first malignant cancer per individual where this occurred within the study period and at least year after the start of follow upgold standard cancer case definitionwe developed a gold standard algorithm that classifies incident records of the most common cancers by comparing the first malignant cancer identified in each individual source figure cancers recorded in ncras alone with no contradictions ie records for first cancers at different sites were considered true cases whereas cancers recorded in hes apc alone or gold alone required internal confirmation within that source in the form of another code for cancer consistent with the same site or with site unspecified within months and no contradictory codes eg for cancers at other sites in this period where cancer records were present in data source we considered a site specific cancer to be a true case a if it was recorded as the first cancer in ncras and the total number of data sources with records for cancer at that site was equal to or greater than the number of data sources with contradictory records ie records for first cancers at different sites or b where the cancer was not present in ncras if there were more data sources in total with records for cancer at that site than data sources with contradictory recordswe used ncras data to identify stage grade and treatment where available in the cancer registry only cohort binary surgery chemotherapy and radiotherapy variables were derived using individual records of treatment from the first year after diagnosisstatistical analysisfor each cancer site and each individual or combined data source we combined our applied study definitions strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access with our gold standard definition to classify each applied study definition as a true positive false positive or false negative recordwe used these categories to calculate sensitivity and positive predictive value overall and stratified by age categories to and calendar year and sex we calculated differences in diagnosis dates for true positives by subtracting the gold standard index date from the index date for each source and combination of sourceswe used kaplan meier methods to describe mortality over time for cancers identified using each definition the ons mortality death date was used for these analyseswe used the ncras only definition to calculate proportions of patients with complete stage and grade and recorded cancer treatment modalities over timepatient public involvementpatients and the public were not involved in conceiving designing or conducting this study and will not be consulted regarding the dissemination of study resultsresultsof research quality patients in the cprd gold january build were eligible for linkage to hes ons mortality and ncras data in set were excluded due to unknown sex of the remainder and had at least year of follow up between january and december and were included in the study population using the gold standard algorithm incident cases of cancer were identified the number of patients identified with each cancer is presented in online supplementary appendix table half n82 of these patients were male aged to aged to and aged or olderfigure presents ppvs for each case definition comparing the first recorded cancer in each combination of data sources with the gold standard algorithm when using ncras data alone to of cancers were confirmed by the algorithm for out of cancer sites the ncras only case definition gave the highest ppv case definitions using data sources not including ncras generally had lower ppvs ranging from to for individual cancer sites for the four most common cancers breast lung colorectal and prostate ppvs were at least for all case definitions minimal differences in ppvs were observed between age groups years and sexes online supplementary appendix figures “figure presents sensitivity values for each case definition sensitivity was generally higher for the case definitions that included ncras data ranging from to for individual cancer sites except bladder cancer identified using ncras data alone and ‰¥ for the four most common cancers breast lung colorectal and prostate sensitivity was also generally high for definitions using a combination of cprd gold hes apc and ons mortality data ranging from to ‰¥ for the four most common cancers sensitivity was lower for case definitions that used cprd gold alone range to for individual cancer sites or hes apc alone range to sensitivity values for cprd gold alone and hes apc alone increased slightly in younger patients and more recent years no differences were observed between men and women online supplementary appendix figures “ post hoc analysis suggested that the low sensitivity of cprd gold only definitions for kidney cancer sensitivity n false negatives was driven by missing n1136 or incompletely specified urinary organ cancer codes n1108 in cprd gold rather than contradictory information about the first cancer record n625 these incompletely specified codes are less likely to be used for bladder cancers n85 than kidney cancers n1108 bladder cancers that were not recorded in ncras data n3445 were commonly recorded in both hes apc and cprd gold n2228 or in hes apc only with a subsequent unspecified or bladder cancer record in hes apc within months n995 table describes the number of days median iqr and 5th95th percentile lag between the date of incident cancers from the gold standard definition and the date of cancer arising from each case definition ie the first record within the specific combinations of data sources used case definitions using ncras alone and combinations of ‰¥ data sources captured cancers close to the gold standard date median lag ‰¤ days for all cancer sites whereas median lags were generally longer for the case definitions using cprd gold alone and hes apc alonefigure describes mortality over time following incident cancer diagnoses ascertained from each case definition minimal differences in mortality were observed between cancers identified from different case definitions where variability was observed cancers identified using cprd gold only had the lowest mortality rates eg kidney cancer and cancers identified using hes apc only or ncras only had higher mortality rates eg prostate cancer and bladder cancer respectivelyfigure describes completeness of grade and stage for cancers identified using ncras only recording of grade was highly variable between cancers with gradual increases in completeness over time completeness of staging information was low in earlier calendar years but improved substantially from around especially for the four most common cancers minimum in and in post hoc logistic regression models adjusted for year and cancer site indicated that completeness of stage and grade were associated with each other and these variables were least complete in patients aged stage data was more complete for higher grade tumours whereas grade data was more complete for lower stage tumours online supplementary appendix figure online supplementary appendix figure describes recording of treatment modalities identified using ncras strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure positive predictive value of cancer diagnoses for each combination of sources when compared with the main gold standard algorithm percentage of incident cancers defined using the first ever record in each combination of sources confirmed by a gold standard algorithm that considers confirmatory and contradictory data from each source cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system nhl non hodgkin's lymphoma cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service ons office of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access figure sensitivity of cancer diagnoses for each combination of sources when compared with the main gold standard algorithm percentage of incident cancers identified using the main gold standard algorithm that considers confirmatory and contradictory data from each source that are identified using the first ever record in each combination of sources cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system nhl non hodgkin's lymphoma cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service onsoffice of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0celitnecrep ht“htrqi inademelitnecreprqi inadem ht“htelitnecrep ht“ht inademrqielitnecrep ht“htcpaseh dlogdrpc ytil atromsnodna cpaseh dlogdrpc cpasehdnasarcn secruos fonoitanbmoc hcae nii iidrocer reve tsrfi ot etad ssongaddradnats dog nammorf syad n ili emti l ebatopen access recnac lanoitan sarcn atad erac tneitapdetti mda scitsitats edospei latipsoh cpaseh knil atadhcraeser ecitcarp lacniilc drpc metsys suovren lartnec sncl tluafed ybnoitinfieddradnats dog eht sa emas eht si etad ssongad sa nwohs tonnoitinfied secruos ll iia setis recnac nommoc tsom ruofdcii nosrev sesaesdi fonoitacfissacliscitsitats lanoitan rof ecfifo snoi atadnoitartsgerrecnac ecvres ssyanadna liinoitartsgeri lanoitanretni eht imorf sedoc gndnopserroc ot gndrocca deredro era setis recnaci snoitinfiedde ilii ppa dna etad ssongaddradnats dog nam neewteb syadil fo rebmun ot ot ““ ot ot ot cl amoeym epitluml ot ci ameakuel““““““““““““ ot ot ot ot ot ot ot ot ot ot ot ot “ot “““““““ ot ot ot ot ot ot ot ““““““““““““““““““““ ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ““““““““““““““ˆ’““““““ ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ““““““““““““““““““ inademrqi ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot elitnecrep ht“ht““““““““““““““sarcn inademrqi ot ot ot ot ot ot ot ot ot ot ot ot ot ot “ ot ot ot ot “““““c ytivac laroc laegahposeoc hcamots cc latcerooclrecnac amonaeml tnangilamc saercnapc gnulc suretuc etatsorpc seiravoc yendkic tsaerbci xvreccc sncnarbic reddablc amohpmyl s'inkgdo hnonc idoryhtc revlistrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access figure mortality following first ever record of cancer in each combination of sources cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service nhl non hodgkin's lymphoma ons office of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure completeness of grade and stage for cancers identified using ncras data only cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites grading information is not applicable to braincns sarcoma or haematological cancers and not required by in the national data standard cosd for prostate cancer core staging is not applicable to haematological and gynaecological cancers other types of staging are recommended by cosd cns central nervous system cosd cancer outcomes and services data set ncras national cancer registration and analysis service nhl non hodgkin's lymphomastrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access only missing records may indicate that the patient did not receive that treatment modality or that the treatment modality was not recordeddiscussionstatement of principal findingswe investigated the use of different sources of electronic health record data to identify incident cancers for all case definitions using individual or combined data sources a minimum of of incident site specific cancers were confirmed using the gold standard algorithm this rose to of the four most common cancers use of cancer registration data alone or in any combination of data sources captured at least of site specific cancers identified by the gold standard algorithm excepting bladder cancer and of cases for the four most common cancers combining cprd gold hes apc and ons mortality data captured at least of site specific cancers excepting kidney oral cavity and ovarian cancers and captured of cases for the four most common cancers sensitivity was much more variable when using primary care or hospital data alone and dropped to when identifying bladder cancers using cancer registration data alone use of primary care or hospital data alone resulted in a small lag in identifying cancers of interest compared with the gold standard dates but other case definitions captured cancers close to the gold standard date finally while we observed minimal changes in ppvs and sensitivities between and completeness of ncras cancer registration stage and grade data increased markedly from onwards for specific cancer types demonstrating that initiatives to improve data can have a profound impact on the quality of the data4 completeness of cancer treatment recording was difficult to assess due to the absence of a missing categorystrengths and weaknesses of the studythe main strength of this study is that we have developed a gold standard algorithm using the entirety of the evidence available from cprd to demonstrate the impact of choice of data sets in identifying incident cancers for real life studies we have also assessed the value of using ncras cancer registration data to measure stage grade and cancer treatment modalitiesa limitation of the study is that our gold standard algorithm is not validated we feel that we were justified in pre weighting ncras data as more reliable that other data sources as ncras is a highly validated data set that matches merges and quality checks data from multiple sources4 we did not consider ncras to be the outright gold standard as it is plausible that ncras does not identify all tumours diagnosed and treated in primary and secondary care for most cancer sites our gold standard algorithm identified a small proportion of cancers that are recorded in hes apc cprd gold or ons mortality data but not in ncras these tumours may have been diagnosed and coded as invasive in primary or secondary care but not by ncras been incorrectly coded in hes apc cprd gold or ons mortality data not have been notified to ncras eg tumours treated in private hospitals or be the result of linkage errors between the data sets the proportion of cancers identified in hes apc but not in ncras is particularly high for bladder cancer this is likely to be the result of difficulties inconsistencies and changes in the pathological definition and coding of cancers over time in ncras which are greatest for bladder cancer4 this explanation is supported by the higher mortality rates that we observed in bladder cancer cases identified in ncras compared with other data sources to identify incident cancers we required months of research quality follow up in cprd gold prior to inclusion in the study previous research has demonstrated that historic data is generally incorporated within the patient record with this time frame15 the identification of first ever cancers will also have been affected by different lengths of follow up data available in linked data sources as ncras data collection started in hes apc in and ons mortality data in and by the inclusion of all diagnostic codes in hes apc assuming that the first ever primary or secondary record identified incident cancer reassuringly ppvs for liver and brain cancer were high for all individual and combinations of data sets suggesting that these were not unduly misclassified as primary incident cancers despite being common sites for metastases requiring internal confirmation within months for cancers recorded in cprd gold alone in our gold standard definition is more likely to discount cancers with poorer prognoses and those recorded in the last months of follow up our data cut only included ncras data for the top cancers earlier cancers at other sites will have been missed in this studyit is also important to note that as the gold standard algorithm uses data recorded after the first record of the cancer site in any source index date it cannot be used to identify outcomes in applied studies and follow up of cohort studies with cancer as an exposure would need to start at least months after diagnosis our first ever cancer record in any source definition would be more appropriate for most studiesstrengths and weaknesses in relation to other studies discussing important differences in resultsthe most up to date study describing concordance between linked cprd gold hes apc and ncras data sets demonstrated that to of the five most common cancers recorded in cprd gold are not confirmed in either hes apc or cancer registration data and to of registered cancers are not recorded in cprd gold8 for cancers recorded in both sources the diagnosis date was a median of to days later in cprd gold than in the cancer registration data using cprd gold alone to identify these strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0ccancers marginally over represented younger healthier patients and identified to fewer deaths in the first years after diagnosis use of hes apc only identified a higher proportion of patients with the correct diagnosis date than cprd gold but over represented older patients and those diagnosed through the emergency route the majority of registered cancers were picked up using both cprd gold and hes apc ranging from for lung cancer to for breast cancer previous research demonstrated similar results with substantial differences between cancer types5 additionally a study using data from to found that using hes data in addition to ncras data identified an additional and of surgically treated colorectal lung and breast cancer cases respectively16our study is consistent with these results and provides more complete and practical evidence of the strengths and limitations of using individual and combinations of linked data sets to identify and characterise the most common incident cancerswe have also demonstrated the added value of using cancer registration data to measure stage and grade of incident cancers from about onwards levels of data completeness of staging information in the cprd extract in were similar to those reported by the united kingdom and ireland association of cancer registries ukaicr9meaning of the study possible explanations and implications for clinicians and policymakersuse of ncras cancer registration data maximised the proportion of cases confirmed as true positive based on all available linked information and captured the highest proportion of true positive cases highly complete staging and grading information is available from this source from approximately case definitions based on a combination of cprd gold hes apc and ons mortality data also had acceptable validity for the majority of cancer sites including the four most common cancersthese findings should be considered when deciding which data sources to include in research studies and which sources to use to define cancer exposures outcomes and covariatesunanswered questions and future researchfurther research is required to investigate the validity of cancer recorded in cprd gold and hes apc that are not recorded in the ncras data and to understand differences in cancer data recording with cprd gold and cprd aurum cprd™s recently launched primary care database based on records from practices that use emis software17 further investigation would be required to confidently identify subtypes of cancer either using codes available in each data set eg colon and rectal cancer or additional information available in hes apc or ncras data use of ncras™s recently open accesslaunched systemic anti cancer therapy sact18 and national radiotherapy data sets will also improve ascertainment of therapies for futu
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the heterogeneity of cancer cells is generally accepted and astem celllike subpopulation that is called œcancer stem cellscscs has been identified in various types of malignanttumors although the lack of consensus on the definitioncscs are widely recognized as a small subpopulation amongcancer cells with the properties of selfrenewal and tumor initiation as cscs play a critical role in the recurrence andmetastasis of cancer targeting the cscs is thought to bea promising approach for curing cancera large number of past studies have tried to identify andcharacterize the cscs as normal tissuespecific stem cellsare considered as the main origin of cancer the cscsare also thought to be inherited at least partially the characterization of normal tissuespecific stem cells thereforemany studies on the identificationpurification of cscs havesimply shared markers of hematopoietic stem cells includingthe most popularly used cell surface markers of cd44 andcd133 [ ] cd44 is a type i transmembrane glycoproteinthat is expressed on hematopoietic fibroblastic and glial cellsand functionally known to mediate cellcell and cellmatrixinteractions previous studies have demonstrated that thecd44 is not only a biomarker but also plays critical roles inthe maintenance of cscs the resistance to various therapiesstresses and the metastasis of cancer cells [“]cd133 is originally identified as protein expressing on thecell surface of hematopoietic stem cells and has subsequently been found to be critical in the maintenance ofœstemness of stem cells in various tissues [“] cd133has also been found in some csc [“] which contributesto therapeutic resistance through the activation of akt bcl2and mapkpi3k signaling pathways [“] although theexpressions of cd44 and cd133 in cancer cells likely associate with the resistances to radiotherapy chemotherapy andvarious stresses the diï¬erent significance between cd44and cd133 has not yet been well understoodin this study we investigated whether the expression ofcd44 and cd133 in human colorectal cancer cells hct8diï¬erently contributed to drug resistance our data indicated 0cstem cells internationalthat the expression of cd133 rather than cd44 closely associated with doxorubicin dxr resistance at least partiallythrough drug excretion and redox regulation materials and methods cell culture human colorectal cancer hct8 cells werecultured in rpmi medium fujifilm wako purechemical japan supplemented with fbs gibco°thermo fisher scientific ma usa at c in a humidifiedatmosphere of air and co2 separation of cd44 and cd133positive cells fromhct8 cells we separated the parent hct8 cells intocd44positive cd44 and cd133positive cd133 cellsby a twostep magnetic cell sorting method as described previously [ ] briefly hct8 cells were collected as a singlecell suspension by trypsinization and then incubated withmagnetic microbeadconjugated antihuman cd44 antibodymiltenyi biotec germany for min after washing cellswere separated into cd44 and cd44 subpopulations byusing the automacs„¢ pro separator miltenyi biotecaccording to the manufacturer™s instruction the purifiedcd44 cells were further expanded and then harvested as asinglecellsuspension to be incubated with magneticmicrobeadconjugated antihuman cd133 antibody miltenyibiotec for min after washing the cd44cd133 andcd44cd133 subpopulations were separated as describedabove this twostep isolation enabled us to obtain a sufficientnumber of cd44 cd44 cd44cd133 and cd44cd133 cells for our experimentsto verify the purity of each subpopulation isolated cellswere stained with pelabelled mouse antihuman cd133clone ac133 miltenyi biotec and fitclabelled mouseantihuman cd44 clone db105 miltenyi biotec according to the supplied protocols flow cytometry analysis wasperformed using a facscalibur becton dickinson asdescribed previously mouse igg1pe miltenyi biotecand mouse igg1fitc miltenyi biotec were used as a negative control cytotoxicity assays cells were seeded in 96well cultureplates at a density of — cells per well and cultured overnight the cells were then treated with various concentrationsof dxr fujifilm wako pure chemical in the absence orpresence of verapamil fujifilm wako pure chemicalcytotoxicity assays were performed using the cell proliferation kit i mtt roche applied science germany asdescribed previously the absorbance was measured at nm using a microplate reader multiskan fc thermofisher scientific analysis on the expression of abc transporters theexpressions of the atpbinding cassette subfamilies of bmember abcb1 or g member abcg2 were analyzedby flow cytometry briefly cells were incubated with mouseprimary antibodies against human abcb1 and abcg2bd biosciences ca usa and then labeled by fitcconjugated antimouse igg bd biosciences according tothe manufacturer™s instruction respective isotype controlswere used as a negative control after washing flow cytometry analysis was performed using a facscalibur analysis of cellular accumulation of dxr the intracellular accumulation of dxr was analyzed by flow cytometrybriefly cells were treated by μm dxr for hr in theabsence or presence of μm verapamil or μm buthionine sulfoximine bso sigmaaldrich mo usa cellswere then collected as a singlecell suspension and washedtwice with icecold phosphatebuï¬ered saline the accumulation of dxr within cells was evaluated by the intracellularfluorescence intensity using a facscalibur the nucleusaccumulation of dxr was analyzed by using cell pelletstreated with triton x100pbs as assay material asdescribed previously expressionlevelsanalysisimmunoblot detection of intracellular ros the intracellular roslevel based on the oxidation of ²²dichlorodihydrofluorescein diacetate h2dcfda molecular probes thermofisher scientific was measured to form the fluorescent compound ²²dichlorofluorescein dcf using a facscaliburofphosphorylatedp38 map kinase phosphop38mapk totalp38mapk and nuclear factor erythroid 2related factor nrf2 in the cells were estimated by immunoblotting brieflycell lysate μg of total protein was separated by sodiumdodecyl sulfate“polyacrylamide gel electrophoresis sdspage gel transferred to pvdf membranes biorad causa and then incubated with primary antibodies cell signaling technology ma usafollowed by appropriatehrplabeled secondary antibodies dako agilent pathologysolutions ca usa blots were developed by enhancedchemiluminescence using an ecl kit ge healthcare lifesciences pa usa semiquantitation was done by measuringthe density of bands using the image quant las minibiomolecular imager ge healthcare life sciences asdescribed previously sirna treatment smallinterfering rna sirnaspecific targeting to cd133 on targetplus sirnaand a scramble sirna on targetplus sirna negativecontrol were obtained from dharmacon horizon discovery cambridge uk cells were seeded in 6well plates — cellswell and incubated for hr transfectionswere performed using dharmafect sirna transfectionreagents dharmacon according to the manufacturer™sinstructions analyses were done at hr after sirnatransfection statistical analysis all of the results are presented as themeans ± sd statistical significance was determined by oneway analysis of variance anova followed by tukey™s testdr spss ii chicago il diï¬erences were considered significant when p results hct8 cells were separated into various subpopulationsbased on their expressions of cd44 and cd133 first we 0cstem cells internationalparent cellshlffl1hdccd44hlfhlfcd44ˆ’fl1hcd44fl1hahlfhlfcd44cd133ˆ’fl1hcd44cd133fl1hparent cd44ˆ’cd44 cd44133ˆ’ cd44133enilesab fo noitarefilorp llecparent cd44ˆ’ cd44 cd44ˆ’cd44bcfigure continued 0cstem cells internationalenilesabstnuo0ccd44cd133ˆ’ ± stnuo0ccd44cd133 ± fl2hfl2hsyad stnuo0c ± fl2hstnuo0cd ± fl2hfigure the separation of hct8 colorectal cancer cells into diï¬erent subpopulations based on the expression of cd44 and cd133 arepresentative dot plots of flow cytometry analysis show the purities of each subpopulation of isolated cells quantitative data in the dotplots are presented as the percentages of positive cells from three independent experiments b representative photos of morphologicalproperties upper and mtt assay on cell growth lower at hr after the initiation of culture data are presented as the mean ± sd fromthree independent experiments c d representative histograms of flow cytometry analysis showed the expressions of cd44 c andcd133 d at baseline and days after cell culture the dotted vertical lines through histograms indicate the diï¬erence in the expressionpeaks between the baseline and at days after culture quantitative data in the histograms are presented as the mean fluorescentintensity from three independent experimentsseparated the hct8 cells into cd44 and cd44 subpopulations and compared their sensitivity to anticancer drugs ofdxr and cisplatin cisdiaminedichloroplatine cddphowever no diï¬erence in the sensitivity to the two drugswas observed between cd44 and cd44 cells data notshown we further tried to purify a small population ofcd133 cells from these cd44 cells cd44 cells almostnegatively expressed with cd133 figure 1a as a resultwe separated hct cells into diï¬erent subpopulationsincluding cd44 cd44 cd44cd133 and cd44cd133 cells the purities of isolated cells in each subpopulation were confirmed to be around by flow cytometryfigure 1aandphenotype changein diï¬erent growthsubpopulations of cells the morphology and proliferationof these cells could not be found obviously diï¬erent amongsubpopulations figure 1b the expression of cd44 in allsubpopulations kept stable within days of reculturingfrom the frozen cells that stocked immediately after isolationinterestingly the expression of cd44 was a tendency todecrease with culture time in cd44 fluorescence intensity ± at baseline vs ± at days p figure 1c and cd44cd133 cells fluorescence intensity ± at baseline vs ± at days p figure 1c but still kept stable in cd44cd133 cells at days after reculturing fluorescence intensity ± at baseline vs ± at days p figure 1cthe expression of cd133 in cd44cd133 cells kept verystable fluorescence intensity ± at baseline vs ± at days p figure 1d and cd44cd133cells did not turn to express cd133 within days of reculturing fluorescence intensity ± at baseline vs ± at days p figure 1d therefore we usedthe cells within days after reculturing from the frozenstocked cells in subsequent experiments dxr resistance of cd44cd133 cells next we evaluated the sensitivity of cells to dxr by mtt assay withthe addition of μm of dxr in medium we foundthat the survival of cd44cd133 cells was significantlyhigher than all other subpopulations of cells after hr of 0cstem cells international ytilibaiv llecŽŽŽŽŽðœ‡mparent cellscd44ˆ’ cellscd44 cellscd44133ˆ’ cellscd44133 cellsdxrbso003hct8doxb24 hfl3h006hct8 cd44ˆ’doxb24 hfl3h009hct8 cd44doxb24 hsllec tnerapstnuocsllec ˆ’dcstnuocsllec dcstnuocdxr001hct8dox24 hfl3hstnuocadxrverapamil002hct8doxv24 hstnuocfl3h004hct8 cd44ˆ’dox24 h005hct8 cd44ˆ’doxv24 hstnuocstnuocfl3hfl3h007hct8 cd44dox24 h006hct8 cd44doxv24 hstnuocstnuocfl3hfl3hsllec ˆ’dcsllec dc006hct8 cd44cd133ˆ’dox24 h006hct8 cd44cd133ˆ’doxv24 hstnuocstnuocstnuocfl3hfl3h00hct8 cd44cd133dox24 h00hct8 cd44cd133doxv24 hstnuocfl3hstnuocstnuocfl3hbfigure continuedfl3h012hct8 cd133ˆ’doxb24 hfl3h015hct8 cd44cd133doxb24 hfl3h 0cstnuocstem cells internationalabcg2abcb1stnuocfl1hparent cellscd44ˆ’cellscd44cellscd44133ˆ’ cellscd44133 cells ± ± ± ± ± parent cellscd44ˆ’cellscd44cellscd44133ˆ’ cellscd44133 cellscfl1h ± ± ± ± ± figure dxr resistance of diï¬erent subpopulations of cells a mtt assay was done to evaluate the cytotoxicity of dxr data are expressedas the percentile of baseline before dxr treatment from three independent experiments ˆ—p vs all other subpopulations brepresentative histograms of flow cytometry analysis show the accumulation of dxr in cells hr after the treatment with μm dxrin the absence or presence of μm verapamil and μm bso the dotted vertical lines through histograms indicated the mean levels ofdxr accumulation in cd44cd133 cells for comparing with other subpopulations of cells the results were reproducible in threeindependent experiments c representative histograms of flow cytometry analysis show the expression of the abcb1 or abcg2 indiï¬erent subpopulations of cells quantitative data in the histograms are presented as the mean fluorescent intensity from threeindependent experimentsculture p vs other groups at diï¬erent dxr concentrations figure2ato understand the relevant mechanism we measured theintracellular accumulation of dxr in cells by flow cytometrythe accumulation of dxr in cd44cd133 cells wasdetected as the lowest among these subpopulations at hrafter the exposure to μm dxr figure 2b we furtherfound that the intracellular accumulation of dxr in cd44cd133 cells was obviously increased by the treatment withverapamil an inhibitor for drug efflux cell membrane transporters of abcb1 and abcg2 figure 2b however theintracellular accumulation of dxr in cd44cd133 cellsdid not change by the treatment with bso a glutathione synthesis inhibitor that indirectly regulates drug efflux throughabcc1 figure 2b we also confirmed that the expressionof abcb1 p vs other groups but not abcg2 wasenhanced in cd44cd133 cells figure 2c suggestingthe probable role of abcb1 on dxr resistance in cd44cd133 cellsto further confirm the causal relationship between theenhanced drug efflux and dxr resistance we evaluatedthe cytotoxicity of dxr in the presence or absence of verapamil unexpectedly verapamil only partially enhanced thecytotoxicity of dxr in either cd44cd133 or cd44cd133 cells figure 3ait is well known that dxr interacts with nuclear dna toinhibit macromolecular biosynthesis therefore we alsoestimated the eï¬ect of verapamil on the nuclear accumulation of dxr the nuclear accumulation of dxr wasobserved obviously less in cd44cd133 than cd44cd133 cells but tended to have comparable levels withverapamil treatment figure 3b cd44cd133 cells showed better stress tolerance thancd44cd133 cells it is well known that the stress responsekinase p38mapk can be activated by various extracellularstresses and plays critical roles in cell survival and apoptosis although the basal level of phosphorylated p38mapkwas detected very similar between cd44cd133 andcd44cd133 cells p figure lower expressionwas observed in cd44cd133 than cd44cd133 cellsafter dxr exposure even under verapamiltreatmentp figure this suggests a better tolerance tostress of cd44cd133 cells independent on the accumulation of dxr 0cstem cells internationalˆ’ limaparev ytilibaiv llec limaparev ytilibaiv llec hoursŽŽŽŽ 𝜇mŽŽŽŽðœ‡mcd44cd133 cellscd44cd133ˆ’ cellsa hoursŽŽŽ ˆ’ limaparevstnuoccd44133ˆ’ cd44133𝜇mfl3hŽŽŽŽðœ‡m limaparevstnuocfl3hbfigure dxr resistance and nuclear dxr accumulation in cd44cd133 and cd44cd133 cells in the absence or presence of drug effluxinhibitor a mtt assay was done to compare the cytotoxicity of dxr in cd44cd133 and cd44cd133 cells with or without theaddition of μm verapamil data were expressed as a percent of baseline before dxr treatment from three independent experiments ˆ—p vs cd44cd133 cells b representative histograms of flow cytometry analysis showed the nuclear accumulation of dxr incells hr after the treatment by μm dxr with or without the addition of μm verapamil the results were reproducible in threeindependent experimentsverapamil ˆ’verapamil phosphop38 mapk totalp38 mapk noisserpxe evitalercd133 ˆ’p p p ˆ’ ˆ’ ˆ’ ˆ’ control hours dxr treatmentp p p ˆ’ˆ’control ˆ’ ˆ’ ˆ’ hours dxr treatmentfigure diï¬erent expression of phosphorylated p38mapk between cd44cd133 and cd44cd133 cells representative blots andsemiquantitative data on the expression of phosphorylated p38mapk and total p38mapk in cells treated with μm dxr in theabsence or presence of μm verapamil the quantitative data are normalized to total p38mapk data are expressed as relative values tocd44cd133 cells without dxr treatment and presented as the mean ± sd from three independent experiments 0cstem cells internationaldxr ˆ’dxr ˆ’ limaparevstnuoc limaparevstnuoccd44133cd44133ˆ’stnuocfl1hfl1hstnuocfl1hafl1hverapamil ˆ’verapamil nrf2𝛽tubulin noisserpxe evitalercd133 ˆ’p p p ˆ’ ˆ’ ˆ’ ˆ’ controldxr treatment hours bp p ˆ’ˆ’control ˆ’ ˆ’ ˆ’ dxr treatment hours figure diï¬erent antioxidant capacity between cd44cd133 and cd44cd133 cells a representative histograms of flow cytometryanalysis show the intracellular ros levels hr after the treatment by μm dxr in the absence or presence of μm verapamil theresults were reproducible in three independent experiments b representative blots and semiquantitative data on the expression of nrf2in cells treated with μm dxr in the absence or presence of μm verapamil the quantitative data are normalized to βtubulin dataare expressed as relative values to cd44cd133 cells without dxr treatment and presented as the mean ± sd from three independentexperiments cd44cd133 cells showed higher antioxidantcapacity than cd44cd133 cells it is also well known thatdxr generates ros and oxidative stress due to ros generation may induce the activation of p38mapk therefore weestimated the ros levels in cells with or without dxr exposure we observed a lower level of ros in cd44cd133 0cstem cells internationalstnuocstnuocstnuocstnuocstnuoc0nm ± stnuocfl2h5nm ± stnuocfl2h10nm ± stnuocfl2h15nm ± stnuocfl2h25nm ± stnuocfl2h0nm ± fl2h25nm ± ytilibaiv llecfl2h50nm ± fl2h75nm ± fl2h100nm ± fl2hŽŽŽŽðœ‡m cd44133ˆ’ cd44133 cd44133 control sirna cd44133 cd133 sirna abfigure continued 0cstnuo0cstnuo0cexpression of abcb10nm ± 0nmstnuo0c ± fl1h5nm ± fl1h50nmstnuo0c ± fl1hfl1h25nmstnuo0c ± stnuo0cfl1h100nm ± fl1hstem cells internationalaccumulation of dxrcontrol sirna 5nm ± fl3hcd133 sirna 5nm ± stnuo0cstnuo0cfl3hcdfigure the eï¬ect of silencing cd133 expression on dxr resistance of cd44cd133 cells a representative histograms of flowcytometry analysis on the expression of cd133 in cd44cd133 cells after silencing by diï¬erent dosages of targeted sirna quantitativedata in the histograms are presented as the mean fluorescent intensity from three independent experiments b mtt assay was done toevaluate the cytotoxicity to dxr cells were treated with nm sirna for hr followed by dxr treatment for another hr data areexpressed as a percent of baseline before dxr treatment from three independent experiments ˆ—p vs cd44cd133 cells crepresentative histograms of flow cytometry analysis on the expression of abcb1 in cells after silencing by diï¬erent dosages of targetedsirna quantitative data in the histograms are presented as the mean fluorescent intensity from three independent experiments drepresentative histograms of flow cytometry analysis on the accumulation of dxr quantitative data in the histograms are presented asthe mean fluorescent intensity from three independent experimentsthan cd44cd133 cells especially under dxr exposurebut verapamil did not obviously change the ros levelsfigure 5a based on these findings we speculated thatthe enhanced antioxidant capacity in cd44cd133 cellsmight help to maintain a lower level of phosphorylatedp38mapknrf2 a transcription factor that is well known to beactivated by oxidative stress such as ros and electrophilicsubstances can protect cells against various stresses we alsocompared the expression level of nrf2 between cd44cd133 and cd44cd133 cells western blotting showeda higher expression of nrf2 in cd44cd133 than cd44cd133 cells especially under dxr exposure p figure 5b and the enhanced expression of nrf2 in cd44cd133 cells was not cancelled by verapamil treatmentp figure 5b sirna treatment to further confirm the regulatory roleof cd133 in drug resistance we tried to silence cd133expression in cd44cd133 cells by sirna and then estimated cytotoxicity of dxr although the decrease ofcd133 expression was clearly observed by targeted sirnap vs nm figure 6a dxr resistance of cd44cd133 cells only partially improved figure 6b unexpectedlythe silencing of cd133 did not change theexpression of abcb1 in cd44cd133 cells even using 0cstem cells internationalexcessive concentrations of cd133 sirna p vs nmfigure 6c we also confirmed that the silencing of cd133did not aï¬ect the accumulation of dxr in cd44cd133cells p vs control sirna figure 6dthis suggests that beyond the drug excretion and redoxregulation other complex mechanisms are also likelyinvolved in the dxr resistance in cd44cd133 cells discussionby using the wellrecognized cell surface markers of cd44and cd133 for csc identification we tried to separate thehct8 human colon cancer cells into cd44 cd44 cd44cd133 and cd44cd133 subpopulations and then investigated how the expressions of cd44 and cd133 associatedwith drug resistance actually we checked several cancer celllines on the expression of cd44 and cd133 including helacells and a549 cells however both hela cells and a549 cellsshowed almost expression of cd44 only the hct8cells showed a partial expression of cd44 about anda rare expression of cd133 therefore we only isolateddiï¬erent subpopulations from hct8 cells for this studyfirst we found that the expression level of cd44 keptvery stable in the cd44cd133 cells but gradually declinedin cd44cd133 cells during a cell passaging process on theother hand some of cd44 cells shifted to express cd44 during a cell passaging process figure 1c these findings suggested the plasticity of cd44 expression in hct8 cellsactually ohata et al reported that cd44 highexpressedcells from human intractable colon cancer patients can diï¬erentiate into cd44 lowexpressed cells and a fraction of cd44lowexpressed cells can also generate cd44 highexpressedcells in a xenograft mouse model however it is unclearwhy the cd44cd133 cells but not cd44cd133 cellsstably maintain the expression level of cd44 unlike theextensive expression of cd44 with high plasticity the expression of cd133 was only observed in very few of the hct8cells with poor plasticitya number of previous studies have demonstrated thatcscs are likely resistant to chemotherapeutic drugs thecd44cd133 cells but not the cd44 and cd44cd133cells showed dxr resistance figure 2a according to thisdata the expression of cd133 but not cd44 seems to beclosely associated with drug resistance actuallythesecd44cd133 cells showed the enhanced expression ofabcb1 and the decreased intracellular accumulation ofdxr figures 2b and 2c liu et al reported that nonsmallcell lung cancer cells treated with lowdose cddp aresufficient to enrich cd133 cells and upregulate abcb1expression through notch signaling which thereforeincreases the crossresistance to dxr however theinhibition of abcb1 by verapamil only partially improvedthe dxr resistance of cd44cd133 cells in this studyto find other potential mechanisms involving in thedxr resistance of cd44cd133 cells we investigatedseveral interesting aspects including the stress protectionand redox regulation we found that p38mapk one of themost popular protein kinases known to be activated byinflammatory cytokines lipopolysaccharide osmotic shockultraviolet light and other stresses was more obviouslyinduced by dxr in cd44cd133 cells than cd44cd133cells figure moreover the activation of p38 mapk wasnot dependent on the intracellular accumulation of dxrfigure dxr is known to insert between the base pairs of dna oftumor cells and exhibits antitumor eï¬ects by suppressing thebiosynthesis of both dna and rna through the inhibitionof dna polymerase rna polymerase and topoisomeraseii reactions furthermore it is believed that dxr has theability to generate sufficient ros to raise oxidative stressindeed we observed dxrinduced ros generation in bothcd44cd133 and cd44cd133 cells butthe dxrinduced ros generation was detected even higher in cd44cd133 than cd44cd133 cells independent on the intracellular accumulation of dxr figures 5a 2b and 3bsuggesting the enhanced antioxidant capacity in cd44cd133 cellsthe keap1nrf2 control system plays a central role in theantioxidant defense mechanisms nrf2 is known as a transcription factor to activate various genes involving in biological defense mechanisms it has been reported that nrf2 isconstantly expressed in many cancer cells [“] moreover the enhanced expression of nrf2 has been confirmedto associate with poor prognosis of cancer patients [“]in our study nrf2 expression was detected higher in cd44cd133 than cd44cd133 cells and the diï¬erence in nrf2expression was observed even clearer between cells with dxradministrationindependent on the dxr accumulationfigure 5b these findings also clearly indicate theenhanced antioxidant capacity in cd44cd133 cellsalthough the absence of direct evidence by interferenceexperiment pathways involving in the stress protection andredox regulation might at least partially contributed to thedxr resistance of cd44cd133 cellsvery strangely our data showed that the silencing ofcd133 expression in cd44cd133 cells by sirna couldonly partially increase the cytotoxicity of dxr figure 6bbut did not change the expression of abcb1 and the intracellular accumulation of dxr figure 6c other unknownmechanisms beyond the drug excretion and redox regulationare asked to be defined on the dxr resistance of cd44cd133 cellsbased on data from the present study the expression ofcd133 rather than cd44 more closely associated with theresistance of cancer cells to anticancer drug as complexmechanisms including the drug excretion and redox regulation are likely involved in the drug resistance of cscs multiple approaches may be needed to overcome the big problemof drug resistance in cancer patientsabbreviationsabcb1abcg2atpbinding cassette subfamily b member 1pglycoproteinmultidrug resistance protein1mdr1atpbinding cassette subfamily g member2breast cancer resistanceproteinbcrpcd388 0cabcc1bsocscsdxrmttatpbinding cassette subfamily c member1multidrug resistanceassociated protein1mrp1buthionine sulfoximinecancer stem cellsdoxorubicinadriamycin345dimethylthiazol2yl25diphenyltetrazolium bromidenuclear factor erythroid 2related factor nrf2p38mapk p38 map kinaserossirnareactive oxygen speciessmall interfering rnadata availabilitythe data that support the findings of this study are availablefrom the corresponding author upon reasonable requestdisclosurethe funder played no role in the study design data collectionand analysis decision to publish or preparation of themanuscriptconflicts of interestthe authors indicate no potential conflicts of interestacknowledgmentsthis work was supported by a grantinaid for the ministryof education science sports culture and technology ofjapan grant numbers and 16k15622 and thecollaborative research program of the atomic bomb diseaseinstitute of nagasaki universityreferences r c elble œthe role of cancer stem cells in relapse of solidtumors frontiers in bioscience vol e4 no pp “ j e visvader œcells of origin in cancer 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development vol no pp “ u m gehling s erg¼n u schumacher et al œin vitro diï¬erentiation of endothelial cells from ac133positive progenitorcells blood vol no pp “ m peichev a j naiyer d pereira et al œexpression ofvegfr2 and ac133 by circulating human cd34 cellsidentifies a population of functional endothelial precursorsblood vol no pp “ n uchida d w buck d he et al œdirect isolation of humancentral nervous system stem cells proceedings of the nationalacademy of sciences of the united states of america vol no pp “ b j cummings n uchida s j tamaki et al œhuman neuralstem cells diï¬erentiate and promote locomotor recovery inspinal cordinjured mice proceedings of the national academy of sciences of the united states of america vol no pp “ b bussolati s bruno c grange et al œisolation of renal progenitor cells from adult human kidney the american journalof pathology vol no pp “ l riccivitiani d g lombardi e pilozzi et al œidentification and expansion of human coloncancerinitiating 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avlonitis et al œcd133 cancerstemlike cells in small cell lung cancer are highly tumorigenicand chemoresistant but sensitive to a novel neuropeptideantagonist cancer research vol no pp “ q zhang s shi y yen j brown j q ta and a d le œasubpopulation of cd133 cancer stemlike cells characterized in human oral squamous cell carcinoma confer resistanceto chemotherapy cancer letters vol no pp “ s ma t k lee b j zheng k w chan and x y guanœcd133 hcc cancer stem cells confer chemoresistance bypreferential expression of the aktpkb survival pathwayoncogene vol no pp “ s bao q wu r e mclendon et al œglioma stem cells promote radioresistance by preferential activation of the dnadamage response nature vol no pp “ c yan l luo c y guo et al œdoxorubicininduced mitophagy contributes to drug resistance in cancer stem cells fromhct8 human colorectal cancer cells cancer letters vol pp “ s goto y ihara y urata et al œdoxorubicininduced dnaintercalation and scavenging by nuclear glutathionestransferase ϝ the faseb journal vol no pp “ h ohata t ishiguro y aihara et al œinduction of the stemlike cell regulator cd44 by rho kinase inhibition cont
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" nlr plr and lmr have been associated with pancreatic ductal adenocarcinoma pdac survivalprognostic value and optimal cutpoints were evaluated to identify underlying significance in surgical pdac patientsmethods nlr plr and lmr preoperative values were available for pdac patients who underwent resectionbetween and os rfs and survival probability estimates were calculated by univariate multivariable andkaplanmeier analyses continuous and dichotomized ratio analysis determined bestfit cutpoints and assessed ratiocomponents to determine primary driversresults elevated nlr and plr and decreased lmr represented and of the cohort respectively osp and rfs p were significantly decreased in resected pdac patients with nlr ‰¥ compared to thosewith nlr optimal prognostic os and rfs cutpoints for nlr plr and lmr were and respectivelylymphocytes alone were the primary prognostic driver of nlr demonstrating identical survival to nlrs nlr is a significant predictor of os and rfs with lymphocytes alone as its primary driver weidentified optimal cutpoints that may direct future investigation of their prognostic value this study contributes tothe growing evidence of immune system influence on outcomes in earlystage pancreatic cancerkeywords neutrophil lymphocyte ratio platelet lymphocyte ratio lymphocyte monocyte ratio pancreatic cancerbiomarker correspondence mokengemalafamoffitt1department of gastrointestinal oncology h lee moffitt cancer center andresearch institute usf magnolia dr tampa fl usafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cpointer bmc cancer page of pancreatic ductal adenocarcinoma pdac is the thirdleading cause of cancerrelated death in the us with anestimated deaths in and a 5year overall survival os rate of among newly diagnosed pdacpatients only to present with resectable diseasewith resection as the only chance for cure prognosis isgenerally poor with reported 5year os of “ afterresection [“] ajcc tnm staging is the only widelyaccepted indicator of prognosis for resectable pancreaticcancer however its performance in earlystage diseasehas been questioned additionally controversy regarding initial treatment of earlystage pancreatic cancerpersists yielding no uniform treatment algorithm giventhe wide variation in the biological behavior of pdacand treatment algorithms for this disease there is an unmet need for enhanced prognostic biomarkers biomarkers derived from easily obtainable laboratory valueshave shown potential to meet this need and may help tostratify patients with earlystage pancreatic cancer andguide future treatment plansconventionally survival outcomes among cancer patients have been determined by the disease stage and receipt of treatment more recently howeverincreasedattention has been directed toward the role of inflammation and immune response in the tumor microenvironment and their effects on tumor behavior quantifyingthe systemic inflammatory response by creactive protein and various nutritional parameters has shown prognostic significance in gastrointestinal gynecological andthoracic cancers additionally inflammatory indicesand immunologic ratios including ratios comprised ofintratumoral or circulating neutrophils plateletslymphocytes and monocyte counts have been proposed tobe prognostic biomarkers for a wide range of malignancies [“]the neutrophil to lymphocyte ratio nlr platelet tolymphocyte ratio plr and lymphocyte to monocyte ratio lmr are among the many surrogate biomarkers forinflammation that have been associated with outcomesin gastrointestinal cancers although these ratios havebeen reported to have promising prognostic value fewstudies have examined the effect of these inflammatoryratios in us surgical cohorts [“] moreover manysingleinstitution studies have reported inconsistentprognostic outcomes for these surrogate biomarkers wepreviously reported an inverse association between survival and nlr in patients with borderline resectable disease to expand the scope of our previous analysiswe evaluated the prognostic significance of the nlrplr and lmr in a cohort of patients with resectedpdac who were treated at a highvolume cancer centerfurthermore we aimed to establish optimal nlr plrand lmr cutpoints for determining os and recurrencefree survival rfs and define the primary factor drivingthe prognostic value of these ratios for survival outcomes we hypothesized that preoperatively increasednlr and plr and decreased lmr were associated withworse os in patients with resectable pdacmethodsa retrospective review was conducted using our institutional prospective pancreatic cancer database as part ofour ongoing outcomebased study the study was approved by our institutional review board mcc16446and patient consent was unable to be obtained as thisstudy was conducted retrospectively on deidentified dataposing less than minimal risk patients diagnosed withpdac who underwent curativeintent resection for thetreatment of their disease were identified resectable andborderline resectable pdac patients were defined and included on the basis of the nccn guidelines applied at thetime of diagnosis pancreatic resection included open orminimally invasive pancreaticoduodenectomy total pancreatectomy and distal pancreatectomy performed at ourinstitutionpatient characteristics were summarized using descriptive statistics including median and range for continuous measures and proportions and frequenciesforcategorical measures kaplanmeier plots were made todetermine os and rfs for the nlr plr and lmrsurvival probability estimates were calculated using thekaplanmeier method univariate and multivariable coxproportionalhazard models for os and rfs were runfor each ratio as continuous predictors and dichotomized forms the nlr plr and lmr were calculatedby dividing the absolute neutrophil count by thelymphocyte count the platelet count by the lymphocytecount and the lymphocyte count by the monocytecount respectively dichotomized analyses included neutrophil and lymphocyte counts and percentages whichwere defined as the proportion of neutrophils or lymphocytes to all white blood cells in the sample valuesused for these calculations were part of the last completeblood count and differential obtained after neoadjuvanttherapy and before operative intervention cutpoints of and were used for nlr plr and lmr respectively nlr cutpoints were determined on the basisof values used in previously published studies [ ]cutpoints for plr and lmr were not well establishedtherefore the medians of the observed data were usedoptimal nlr plr and lmr cutpoints for the prediction of os and rfs were determined using maximallyselected rank statistics based on the logrank method the resulting cutpoint for each ratio provided thebest separation of the responses into groups in whichthe standardized rank statistics take their maximumthe p value approximation was based on the improved 0cpointer bmc cancer page of bonferroni inequality variables were evaluated inrelation to os and rfs for predetermined cutpoints andnewly identified bestfit cutpoints all analyses were performed using r software version resultsa total of patients treated at our institution between and were eligible for this study two hundredseventyseven patients with complete data met the inclusion criteria and were included in the analysis the meanage was ± years of whom were maletwentyfive percent of patients had a charlson comorbidity index cci ‰¤ had a cci of to and had a cci ‰¥ medicare with a private supplement wasthe largest represented insurance provider among patients sixtyfour percent of our cohort was classified as resectable and treated with upfront resection and received neoadjuvant systemic therapy marginnegative r0 resection was achieved in of our patients with and demonstrating lymphovascularand perineural invasion respectively table mean preoperative nlr plr and lmr was ± ± and ± respectively additional file using the predetermined cutpoints described above and of patients demonstrated preoperative nlr ‰¥ plr ‰¥ and lmr ‰¤ respectivelyos was significantly shorter among patients with annlr ‰¥ than patients with an nlr in univariatehr [ ci “] p and multivariable hr [ ci “] p analysestable neither the plr nor lmr demonstrated a significant association with os table and fig patients with a high nlr also demonstrated significantlyworse rfs in univariate hr [ ci “]p and multivariable hr [ ci “] p analyses table and fig this wasnot observed with plr or lmr in multivariable analyses pathologic t stage presence of grade complications cci ‰¥ nlr node positivity and perineuralinvasion were found to be significant predictors of osand rfs tables and maximally selected rank analyses of nlr plr andlmr were performed to identify optimal cutpoints forpredicting os and rfs os optimal cutpoints for nlrplr and lmr were and respectively forrfs cutpoints were and respectively because neutrophil percentage is highly correlated with nlrwe found the corresponding cutpoint for determining ahigh neutrophil percentage to be resulting in patients being above the cutpoint similarly lymphocytepercentage was highly negatively correlated with nlrwith a corresponding cutpoint percentage of thecomponents of nlr was analyzed separately to evaluatetheir prognostic importance the lymphocyte percentagealone yielded a survival curve that was identical to that ofthe nlr whereas the neutrophil percentage km plot wasnot statistically significant additional file discussionwe demonstrated a statistically significant associationbetween preoperative nlr and both os and rfs inpdac patients who underwent curativeintent resectionat a highvolume cancer center plr and lmr failed todemonstrate any correlation with survival in additionwe identified optimal cutpoints for immunologic ratiosurvival analyses on the basis of our cohort data finallywe identified the lymphocyte component of nlr to bethe primary driver of survival prognosis to our knowledge this is the largest us cohort utilized to analyzeimmunologic ratio biomarkerassociated outcomes andperform dichotomized analyses for the purpose of identifying the prognostic driver of the nlr in surgical pdacpatientsinflammation and the inflammatory response have beendiscussed extensively in the literature in relation to tumorigenesis progression and metastasis furthermorelinkshave been established between the inflammatory responseand oncogenic signaling pathway interactionstumormicroenvironment analyses and use of immunetargetedtherapies surrogate biomarkers of inflammation haveproven useful in predicting disease progression recurrenceand overall prognosis across a wide range of malignancies[ “] in a metaanalysis evaluating the role of thesystemic immuneinflammation index zhong showedthat an elevated systemic immuneinflammation index isassociated with worse os in hepatocellular carcinomaurinary cancers gastrointestinal cancers and smallcell lungcancer in a review of patients with gastrointestinalmalignancies nora demonstrated nlr and plr to besignificant predictors of lymph node positivity metastaticdisease and recurrence especially when used in combination the use of the nlr plr and lmr have shownpromise in pancreatic adenocarcinoma demonstratingprognostic value in both resectable and palliative populations [ ]the nlr has shown substantial potential for prognostic utility in pancreatic adenocarcinoma patients in alarge retrospective analysis of surgical pdac patients alow nlr was associated with longer median survival vs months p and an nlr ‰¥ independently predicted poor prognosis hr [ ci“] p giakoustidis further explored pretreatment nlr in surgical pdac patients andidentified decreased os rates to be associated with a highnlr in univariate analyses which maintained independent prognostic significance in multivariable analyses two recent metaanalyses including a total of patients have also suggested an association between 0cpointer bmc cancer page of table descriptive statistics of study cohortsnlr demographicsn “overalln “age median range ynlr ‰¥ n “plr n pvalue “plr ‰¥ n “lmr ‰¤ n pvalue “lmr n “sex no femalemalerace no blackotherwhite bmi median range“““ ““ ““ “““ ““ “ “cci no ““‰¥ tumor sizepathologic stage no t0t1 no t2 no t3preoperative resectabilityno neoadjuvant therapy nonoyesmargin no negativepositivelymphovascular invasionno pvalue borderlineresectable noyes perineural invasion no noyes complication 34a no noyes completion of adjuvanttherapy no 0cpointer bmc cancer page of table descriptive statistics of study cohorts continuednlr ‰¥ demographicsn nlr n overalln nopvalueplr n plr ‰¥ n pvaluelmr ‰¤ n lmr n pvalueyes aclaviendindo classification of surgical complicationsabbreviations bmi body mass index nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte ratio cci charlesoncomorbidity indexnlr and os in which elevated nlr carried poor prognoses zhou found elevated nlr to be associatedwith shorter rates of os hr [ ci “]p and diseasefree survival hr [ ci“] p evaluating os alone mowbray also demonstrated that significantly shorter rates ofos were associated with elevated nlr hr [ci “] p we corroborated these results in our own resected pdac patients and similarlydemonstrated that decreased rates of os were associatedwith an nlr ‰¥ in multivariable analyses additionallywe showed a significant association between hightable univariate and multivariate cox proportional hazard models for overall survivalvariablep valueunivariate analysishr cimultivariable analysishr ciap valuegenderfemalemaleage‰¤ pathologic staget0t1t2t3cci“nlr ‰¥ plr ‰¥ lmr ‰¥ perineural invasionnoyesnananananananananana reference “ reference “ reference “nanananananananananananananacomplication grade “4bpositive nodesamodel includes age gender pathologic stage cci complication score nlr nodal and perineural invasion status b claviendindo classification ofsurgical complicationsabbreviations cci charlson comorbidity score nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte rationananana reference “ reference “ reference “ “ “ reference “ reference “nananana reference “ “ “nananana 0cpointer bmc cancer page of fig kaplanmeier plot demonstrating overall survival in a nlr b plr and c lmrpreoperative nlr and a decrease in rfs our study further supports the nlr as a valid prognostic biomarkerfor earlystage pdacalthough a cutpoint of has been widely used to define highlow nlr variations in cutpoints exists withsome groups using values ranging from to [ “] with no clearly defined cutpoint we chose to perform a continuous analysis to identify an optimal cutpoint for the nlr in relation to survival based solely onthe data from our cohort optimal cutpoints of foros and for rfs were obtained our study supportsthe prognostic value of the commonly used nlr cutpoint of as the nlr was the only significant ratio inour cohort we elucidated its prognostic driver by analyzing the components of the ratio the denominatorthe lymphocyte count percentage alone yielded a survival curve identical to the nlr whereas the numeratorthe isolated neutrophil count percentage was not statistically significant suggesting that lymphocyte count percentages have equal prognostic value and perhaps offera simpler alternative to the nlr biomarker this findingis supported by those from previous studies that showedlow lymphocyte counts to be poor prognostic indicatorsin pancreatic and colorectal cancers [“] the finding also has immunotherapeutic implications which corroborate basic science findings on a population level[“]in contrast to our study other studies have found noprognostic significance of the nlr in some pdac patient populations recently chawla described a cohort of resectable pdac patients whose nlr atdiagnosis did not correspond to os jamieson similarly reported patients who underwent pdacresection and found no relationship between nlr and 0cpointer bmc cancer table univariate and multivariate cox proportionalhazard models for recurrencefree survivalvariablep valueunivariate analysishr cimultivariable analysishr ciapage of p value reference “ reference “ “ “ reference “ reference “nananana reference “ “ “nanagenderfemalemalepathologic staget0t1t2t3cci“nlr ‰¥ plr ‰¥ lmr ‰¥ perineural invasionnoyesnananananananana reference “ reference “ reference “nanananananananacomplication grade “4bpositive nodesabbreviations cci charlson comorbidity score nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte ratioa model includes age gender pathologic stage cci complication score nlr nodal and perineural invasion statusb claviendindo classification of surgical complicationsnanananasurvival similar findings have been reported byother groups [ ] the reasons for this variability include diverse patient populations differences in ratiocutpoints timing of blood collections and receipt ofneoadjuvant therapy in the current study of patients received neoadjuvant therapy before pancreatic resection which may havecellpopulationsinfluenced immuneincreased monocyte presence in the tumor microenvironment or in circulation has been implicated inangiogenesis tumor growth and poor prognosis in cancer patients circulating monocytes are commonlyquantified by the lmr which has demonstrated an inverse association with survival and prognosis in solidtumor malignancies few studies have investigatedthis parameter in surgical pdac patients in a large review and metaanalysis of patients li reported a favorable prognosis associated with elevatedlmr in pooled analyses hr [ ci “]p although this study included a range oflmr cutpoints and both resected and nonoperablepdac patients a prognostic value of the lmr was observed in surgical patients in subgroup analyses sierzega reported a series of resectable pdacpatients demonstrating prolonged median survival vs months p in the lmr ‰¥ group anlmr was an independent predictor of poor prognosis hr [ ci “] p in contrastaldemonstrated no association between lmr and os ordiseasefree survival in a large retrospective analysis ofthe prognostic effects of patientspecific nutritional andimmunologic factors in resected pdac patients we also did not show a prognostic value of lmr in ouranalyses of resected pdac patients differences in prognostic outcomes were likely due to the paucity of dataevaluating lmr and survival inconsistency in evaluatedpatient cohorts and variation of cutpoint delineationto studies previously discussed abeet 0cpointer bmc cancer page of fig kaplanmeier plot demonstrating recurrencefree survival in a nlr b plr and c lmrwe used mean values for lmr cutpoints in our analysesbecause of the variation of cutpoints reported in the literature an optimal cutpoint analysis of lmr for osand rfs was performed to clarify the reporting of lmrassociated outcomessurvival outcomes have similarly been linked to elevated plr in solid tumor malignancies comparedto other commonly described ratios the application ofplr to pdac is less clear with mixed outcomes reported giakoustidis also investigated pretreatmentplr in surgical pdac patients and identified decreasedos with high plr in univariate analyses the plrdid not maintain independent prognostic significance inmultivariable analysis interestingly patients with concurrently high nlr and plr experienced significantlydecreased os when compared to those with normalnlr and plr or those with an elevation of either ratio respectively p in a subsequentanalysis of resected and inoperable pdac patients stotz found no association between os hr [ci “] p and plr hr [ ci“] p in either cohort similarly nodemonstrable association between plr and os was observed in several separate resected pdac patient series[ ] consistent with the literature discussedabove our study did not find a significant correlation between survival os or rfs and plr in resected pdacpatientshowever some authors have demonstrated the plr tobe an important predictor of survival smith and 0cpointer bmc cancer page of watanabe reported elevated plrs as the most significant determinant of survival in their resected pdaccohorts of and patients respectively [ ]reasons for inconsistent results may have included differing plr cutpoint values small patient cohorts andvariations in multidisciplinary treatments of these patients with complex pdac furthermore the plr wassynthesized using surrogates that are fundamental tomany biologic functions ie coagulation cascade whichmay explain the variability of correlation in oncologicoutcomes in our study mean values were initially usedfor plr cutpoints because of the variation reported inthe literature again an optimal plr cutpoint analysiswas performed to provide clarity and consistency in thereporting of plrassociated factorsthereforsettingis potentialthe limitations of this study include those inherent inreviewing retrospective data although our data set wasrobust and associated with an electronic medical recordthe potential for selection bias exists additionally although all blood specimens were collected in the preoperativevariationregarding the date and time blood draws were done inrelation to the surgery date the present study did notstratify patients based on receipt of neoadjuvant therapythis stratification was previously investigated by ourgroup who reported significantly decreased rates of osamong patients with increased nlr after neoadjuvanttherapy when compared to those with stable nlr finally we did not analyze pretreatment immunologicratios in patients who received neoadjuvant chemotherapy therefore we were not able to determine whetherchemotherapy significantly altered preoperative valuesthere continues to be little doubt about the importanceof inflammation and immunity in cancer biology thenlr and other immunologic ratios are derived from easily obtainable standard laboratory values with littleadded expense when obtained in the preoperative setting the nlr is a biomarker with the potential to guidetreatment algorithms in earlystage pdac patients andprovide clarity on common unresolved management dilemmas routinely debated today given their demonstrable poor outcomes patients with high nlr maybenefitfrom neoadjuvant systemic therapy variationmore detailed preoperative staging or stratification inclinical trials additionally consistent with the findingsof developing research on the tumor microenvironmentand immunotherapy lymphocytes alone may be significant drivers of survival in the context of improving outcomes ourtargeting inflammatorypathways may be relevant in chemoprevention prospective trials would serve to elucidate the provided prognostic information and provide insightinto alternativesuggestresultstreatment algorithms that can improve outcomes amongpatients with pdacsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020071829additional file summary statistics of immunologic ratiosadditional file kaplanmeier plot demonstrating overall survival osin dichotomized nlr values a neutrophil and lymphocyte bpercentageabbreviationscci charlson comorbidity index lmr lymphocyte to monocyte rationlr neutrophil to lymphocyte ratio os overall survival pdac pancreaticductal adenocarcinoma plr platelet to lymphocyte ratio r0 marginnegative resection rfs recurrencefree survivalacknowledgmentseditorial assistance was provided by the moffitt cancer center™s scientificediting department by dr paul fletcher daley drucker no compensationwas given beyond their regular salaries this work was presented as a posterat the ahpba meeting and the pancreas club meeting theabstract of this work was previously published in hpb journalauthors™ contributionsdp conception and design acquisition of data analysis and interpretation ofdata drafting of original critical revision gave final approval ofcompleted manuscript dr conception and design acquisition of dataanalysis and interpretation of data drafting of original critical revisiongave final approval of completed manuscript bp conception and designacquisition of data analysis and interpretation of data critical revision gavefinal approval of completed manuscript gm conception and designacquisition of data critical revision gave final approval of completedmanuscript se conception and design acquisition of data critical revisiongave final approval of completed manuscript zt statistical analysis andinterpretation of data critical revision gave final approval of completedmanuscript ms statistical analysis and interpretation of data critical revisiongave final approval of completed manuscript ph conception and designanalysis and interpretation of data critical revision gave final approval ofcompleted manuscript jp conception and design analysis andinterpretation of data critical revision gave final approval of completedmanuscript jf conception and design analysis and interpretation of datacritical revision gave final approval of completed manuscript mmconception and design primary investigator supervision analysis andinterpretation of data critical revision gave final approval of completedmanuscriptfundingthis work was supported by the h lee moffitt cancer center researchinstitute nci cancer center support grant p30ca076292 the funders hadno role in study design data collection and analysis decision to publish orpreparation of the manuscriptavailability of data and materialsthe data that support the findings of this study are available from thecorresponding author upon reasonable requestethics approval and consent to participatethis study was approved by the moffitt cancer center institutional reviewboard mcc because of the retrospective nature of this studypatient consent was not required no personally identifiable data for anypatients were included the study was performed in accordance with thedeclaration of helsinkiconsent for publicationthis study was approved by the moffitt cancer center institutional reviewboard mcc due to the retrospective nature of this study patientconsent was not required 0cpointer bmc cancer page of competing intereststhe authors have no conflicts of interest to declareauthor details1department of gastrointestinal oncology h lee moffitt cancer center andresearch institute usf magnolia dr tampa fl usa2department of surgery university of texas southwestern dallas tx usa3department of biostatistics and bioinformatics h lee moffitt cancer centerand research institute tampa fl usareceived april accepted july referencessiegel rl miller kd jemal a cancer statistics ca cancer j clin “ryan dp hong ts bardeesy n pancreatic adenocarcinoma n engl j med“katz mh wang h fleming jb longterm survival aftermultidisciplinary management of resected pancreatic adenocarcinoma annsurg oncol “neoptolemos jp palmer dh ghaneh p comparison of adjuvantgemcitabine and capecitabine with gemcitabine monotherapy in patientswith resected pancreatic cancer espac4 a multicentre openlabelrandomised phase trial lancet “oettle h neuhaus p hochhaus a adjuvant chemotherapy withgemcitabine and longterm outcomes among patients with resectedpancreatic cancer the conko001 randomized trial jama “chen dt davisyadley ah huang py prognostic fifteengenesignature for early stage pancreatic ductal adenocarcinoma plos one2015108e0133562helm j centeno ba coppola d histologic characteristics enhancepredictive value of american joint committee on cancer staging inresectable pancreas cancer cancer “proctor mj morrison ds talwar d a comparison of inflammationbased prognostic scores in patients with cancer a glasgow inflammationoutcome study eur j cancer “bindea g mlecnik b tosolini m spatiotemporal dynamics ofintratumoral immune cells reveal the immune landscape in human cancerimmunity “ hong x cui b wang m yang z wang l xu q systemic immuneinflammation index based on platelet counts and neutrophillymphocyteratio is useful for predicting prognosis in small cell lung cancer tohoku jexp med “ zhong jh huang dh chen zy prognostic role of systemic immuneinflammation index in solid tumors a systematic review and metaanalysisoncotarget “templeton aj mcnamara mg seruga b prognostic role of neutrophiltolymphocyte ratio in solid tumors a systematic review and metaanalysisj natl cancer inst 20141066dju124 giakoustidis a neofytou k costa neves m identifying the role ofneutrophiltolymphocyte ratio and plateletstolymphocyte ratio asprognostic markers in patients undergoing resection of pancreatic ductaladenocarcinoma ann hepatobiliary pancreatic surg “ glazer es rashid om pimiento jm hodul pj malafa mp increasedneutrophiltolymphocyte ratio after neoadjuvant therapy is associated withworse survival after resection of borderline resectable pancreatic ductaladenocarcinoma surgery “sierzega m lenart m rutkowska m preoperative neutrophillymphocyte and lymphocytemonocyte ratios reflect immune cellpopulation rearrangement in resectable pancreatic cancer ann surg oncol“li w tao l zhang l xiu d prognostic role of lymphocyte to monocyteratio for patients with pancreatic cancer a systematic review and metaanalysis oncotargets ther “ abe t nakata k kibe s prognostic value of preoperative nutritionaland immunological factors in patients with pancreatic ductaladenocarcinoma ann surg oncol “ quigley da dang hx zhao sg genomic hallmarks and structuralvariation in metastatic prostate cancer cell “ e759 halazun kj aldoori a malik hz elevated preoperative neutrophil tolymphocyte ratio predicts survival following hepatic resection for colorectalliver metastases eur j surg oncol “lausen b schaumacher m maximally selected rank statistics biometrics“lausen b sauerbrei w schumacher v classification and regression treescart used for the exploration of prognostic factors measured on differentscales in university of essex research repository p “ mantovani a allavena p sica a balkwill f cancerrelated inflammationnature “ giakoustidis a neofytou k khan az mudan s neutrophil to lymphocyteratio predicts pattern of recurrence in patients undergoing liver resectionfor colorectal liver metastasis and thus the overall survival j surg oncol“li c wen tf yan ln postoperative neutrophiltolymphocyte ratioplus platelettolymphocyte ratio predicts the outcomes of hepatocellularcarcinoma j surg res “ nora i shridhar r huston j meredith k the accuracy of neutrophil tolymphocyte ratio and platelet to lymphocyte ratio as a marker fastrointestinal malignancies j gastrointest oncol “ ye s bai l comparison and validation of the value of preoperativeinflammation markerbased prognostic scores in resectable pancreaticductal adenocarcinoma cancer manag res “ zhou y wei q fan j cheng s ding w hua z prognostic role of theneutrophiltolymphocyte ratio in pancreatic cancer a metaanalysiscontaining patients clin chim acta “ mowbray ng griffith d hammoda m shingler g kambal a alsarirehb a metaanalysis of the
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increasing relevancy of geospatial technologies such as geographic information system GIS inthe public health domain particularly for the infectious disease surveillance and modelling strategies Traditionally thedisease mapping tasks have faced many challenges” authors rarely documented the evidence that were used to createmap before evolution of GIS many errors aroused in mapping tasks which were expanded extremely at global scalesand there were no fidelity assessment of maps which resulted in inaccurate precision This study on infectious diseasesgeosurveillance is divided into four broad sections with emphasis on handling geographical and temporal issues to help inpublic health decisionmaking and planning policies geospatial mapping of diseases using its spatial and temporalinformation to understand their behaviour across geography the citizen™s involvement as volunteers in giving healthand disease data to assess the critical situation for disease™s spread and prevention in neighbourhood effect scientificanalysis of healthrelated behaviour using mathematical epidemiological and geostatistical approaches with capacitybuilding program To illustrate each theme recent case studies are cited and case studies are performed on COVID19 todemonstrate selected modelsKeywords Geospatial technology 01 Citizen Science 01 Public health 01 COVID19 01 Mathematical epidemiologyIntroductionThe public health sector™s increasing demand for mappinganalytics and visualization had started a date back in thelast years which has resulted in a growing informationage technology for communicable disease surveillance andepidemiology Baker Bos and Blobel Friede Friede Khan Reeder Yu and Edberg This continuous publichealth burden with advances in information technology Sameer SaransameeriirsgovinPriyanka SinghPriyankaiirsgovinVishal KumarVishalkumariirsgovinPrakash ChauhanprakashiirsgovinIndian Institute of Remote Sensing Indian Space Researchanisation Kalidas Road Dehradun Indiacombined with spatial data led to the development ofvarious tools and systems that provides visualization ofdisease data in space and time Dredger Kothari Robertson and Nelson Schriml et alThe first integral definition of public health was given byWinslow as ˜˜science and art of preventing diseaseprolonging life and promoting health through the anized efforts and informed choices of society anizations public and private communities and individuals™™The American Public Health Association APHA mentioned public health as a practice of preventing the spreadof disease and an aim of promoting good health from smallcommunities to across the world Turnock Advances in information technology and spatial features resultedin geospatialtechnology which is acute for mappingsurveillance predicting outbreaks detecting clustering andanalysing spread patterns of infectious diseases with epidemic or pandemic potential in communities and acrossterritories AvRuskin Carpenter Castronovo Dominkovics Gao 0c Heymann and Brilliant Hills Klompas Reis Geospatial technology has provided visualization and analytical tools topublic health professionals and decision makers to executediseases control programs in affected andor suspectedregions and make analysis and predictions possible thatwas once technologically out of reachGeospatial technology includes geographical information systems GIS global positioning systems GPS andsatellitebased technologies such as remote sensing RSGIS is known for geographic data capture input updatemanipulation transformation analysis query modellingand visualization of all forms of geographically referencedinformation through the set of computer programs BonhamCarter GPS provides positioning navigationand timing PNT services by capturing data from satellitesand providing it to users Eldredge and RS isan earth observation instrumentthat delivers regionalinformation on climatic factors and landscape featuresTherefore GPS and RS provide regional and spatialinformation while GIS provides geospatial data integrationas well as accurate geospatial analysis in realtime mannerZhen Geospatial Technology and InfectiousDisease SurveillanceInfectious diseases mostly adapts antimicrobial andmobility features later formed in a shape of pandemic andor epidemic Chen Cheng Lee andNishiura which forced public health authorities tounderstand not only the diseases virulence but also itsdemographic and environmentalfactors that helps inmaking spread patterns though space and time domainCroner For example the global spread of highlypathogenic avian ‚uenza HPAI H5N1 in “ withno effective vaccines led to concern among public healthdecision makers in spite of many international programsRappole and Huba´lek The reason behind theirconcern was they were lacking of disease surveillance toolin its initial stage which caused inaccessibility to populations atrisk and faced difficulties in implementingimmunization strategies at a global scale Kitler Stoto However the impact of environmentaland demographic factors also plays a major role as this caninform about the interaction between hosts and pathogensand patterns of spread in space and timeThe GIS provides dynamic maps to understand geographical distribution of diseases for analysis on frequencyof cases disease mapping spatial cluster of diseases disease association with environmentalfactors networkanalysis etc With such a visualization and analyticalJournal of the Indian Society of Remote Sensingforservice frameworkcapabilities GIS technology is holding a widespreadgrowth in public health Ahmad 2011a b Booman HanafiBojd Kolivras Martin Nykiforuk and Flaman Abdul Rasam Zhang Zhen Theseamless integration of GIS with realtime infectious diseaserelated diverse datasets through webbased mappingto the development of geospatial dashboardleadsgeospatialinfectious diseasesurveillance Dent Gao Yun Theinfectious diseaserelated data mightinclude diseasesurveillance data activeconfirmed cases and health system data hospital visits emergency services availabilitynursedoctor availabilityICUbed availability Many source geospatialstandards of GeospatialConsortium OGC are used as a Web Map ServiceWMS Web Coverage Service WCS Web ProcessingService WPS Web Feature Service WFS etc Bulatovic´ Gao to visualize accesspublish and manipulate geospatial resources Also manyother popular industrial geospatial standards are developedby ESRI Google Yahoo and MapInfo Granell to fetch locationbased data and provide infectiousdisease surveillance dashboard to monitor and control thegeographically spread of disease Zhang TheGeocoded Really Simple Syndication GeoRSS taggedXML files from GeoRSS services can also be used toprovide geocoded infectious disease news from socialmedia platform Tolentino KassHout andAlhinnawi 2013a b Kodong Historical ContextThe mapping of infectious diseases using geospatial andinformation technology to benefit public health is not a newway of tracking the diseases Ahmad Cui Hirsch Hornsby Matthew May Mujica Nicholson and Mather Noble Perl and Moalem Williams The historical disease mapping has faced manychallenges” authors rarely documented the evidencethat were used to create map after mapping had beenimplemented before the beginning of geographical information systems many errors arouse which were expandedextremely at global scales and there were no fidelityassessment of maps which resulted in inaccurate precisionBut nowadays wide range of geospatial applications areavailable in public health community with a possibilities ofvisualization analysis detection of clusters formed andcalculate diseaserelated metrics such as incidence andprevalence rate Beck Clarke Hay Jacquez Kleinschmidt Lawson and 0cJournal of the Indian Society of Remote SensingLeimich Moore and Carpenter Robinson Wilkinson The earliest mapping for visualisation ofthe linkbetween disease and place was done in on plagueepidemic in Italy Dent During cholera outbreak in the study of physician John Snow had made a novelcontribution in history of public health and epidemiologyby using cartography applications and geographic visualization in fighting cholera After years the maps wereidentified as a communication tool in understanding andtracking of infectious diseases such as the ‚uenzapandemic yellow fever and cholera Since then revolutionof webbased tools started in applied health geographyBoulos The trend of infectious disease mappingcould be seen from review of the Health GIS literature which demonstrated that research papers out of were focused on infectious disease mapping Lyseen Covid19The ongoing pandemic outbreak targeting humans™ respiratory system was recently discovered in December by the name of Coronavirus Disease Covid19 WorldHealth anization from a cluster of patients with acuterespiratory distress syndrome in Wuhan Hubei ProvinceChina Huang Lu 2020a b and spreadglobally by March This pathogenic disease is structurally related to the Coronavirus CoV which belongs tofamily Coronaviridae and the order Nidovirales Thisfamily is classified into four genera”AlphacoronavirusBetacoronavirus Gammacoronavirus and Deltacoronavirus on the basis of their phylogenetic and genomicanalysis The species of Alphacoronavirus and Betacoronavirus infect mammals causes respiratory illness inhumans and gastroenteritis in animals while species ofGammacoronaviruses and Deltacoronaviruses infect birdsbut some of them can also infect mammals Woo et alfrom Betacoronavirus The two virusgenus”Severe Acute Respiratory Syndrome SARSCoVor Middle East Respiratory Syndrome MERSCoV”hadearlier demonstrated that coronaviruses can cause significant public threat Ge The COVID19 is categorizedby World Healthanization WHO on the basis of genomic sequencinganalysis ofrespiratory tract samples which isobtained from total of nine patients Huang Lu 2020a b COVID19 has started behaving like theonceinacentury pandemic by affecting healthy adults aswell as elderly people with some health issues and byinfecting others at an exponential rate of increase thanSARS or MERSinto BetacoronavirusspecieslowerGeospatial TechnologyDuring occurrence of diseases geospatial technologies andservices could help in representing the spatiotemporalinformation and in analysing the dynamic spread of diseases As mentioned by Boulos geospatial technologies and services which performs in real time mannerare tremendously relevantto create a ˜˜spatial healthinformation infrastructure™™ In this section a review onmany geospatial technologies with enabled IT services iscarried out to understand and analyse the spread and outbreak of disease with a case study on COVID19 pandemicCitizen Scienceissues and concernsThe expansion of Citizen Science from biodiversity andecological domain Haklay MillerRushing to public health community across spatial extentsmade an urgent need to study its different forms Crowl The indepth report of EU describes taxonomyof Citizen Science in three levels European Commission described in Roy Wiggins andCrowston and Haklay Roy categorized Citizen Science by participant™s number and oftheir spread ˜˜local™™ and ˜˜mass™™ and ˜˜thoroughness™™time and resource investment or King described ˜˜for the people with the people or by the people™™ about Citizen Science activities Wiggins andCrownston classified Citizen Science projects inconservation managing natural resources action addressing localinvestigation answering scientific questions and education providingknowledge to citizens Haklay classified CitizenScience into four levels based on participant™s engagement” level is crowdsourcing in which citizens withless or no knowledge on activity perform as sensors tocomplete computing tasks level is distributed intelligence where citizens are being trained with skills forinterpretation of collected data level is participatoryscience in which citizens decide about research questionsand types of data to be collected and level is extremewhere citizens are fully involved in defining researchstrategies data collection data interpretation and performing scientific analysis Apparentlythe concept ofCitizen Science is rare in public health domain but some ofits contribution seen in some studies which not only helpsin predicting disease risks but also in combating theinfectious diseases CurtisRobles Palmer Smolinski Wilson Another approach similar to Citizen Science is ˜popularepidemiology™ in which experts and laypersons jointly 0ccollect environmental data responsible for particular healthconsequences Brown or ˜street science™ as a process in which general public communities actively engagedin defining problems framing of research questions anddecisionmaking activities about research design CorburnCrowdsourceVGI Mobile AppsDespite technological and computational developments inGeoWeb many web technologies such as jQuery andAJAX mapping APIs like Google and GPS devicesresulted in a new revolution of neogeography Turner where mapping is done by crowd and can bereached by anyone from general public members groupSuch revolution brought a trend of Volunteered GeographicInformation VGI which is first coined and explained byGoodchild 2007a According to Goodchild 2007b VGIhighlighted the human capabilities in collecting geospatialinformation by using five senses and then integrating withexternal sensors of mobile devices like GPS accelerometer camera digital compass and microphone gives valuable datasets which can neither be retrieved from satelliteimagery nor collected with any GPS receivers Anothersuccessful term in geospatial mapping using mobile technology is crowdsourcing Heipke HudsonSmith which was coined by Howe thatinvolves the collection of geospatial information or mapping of any particular activity by an undefined crowd ornetwork of people Both terms VGI and crowdsourcingslightly differ but they are usually recognized as a synonyms or even as a combined term ˜˜crowdsourcing geographic information™™ Sui Over the lastdecade VGIoriented source mobileareEpiCollect Aanensen for ecology and epidemiology NoiseTube Maisonneuve httpnoisetubenet and Noise Battle GarciaMartı´ for noise monitoring Skywatch Windoo httpwindoochfor weather monitoring Mappiness httpwwwmappinessukfor behavioural analysis MacKerron andMourato appsThe source mechanism for data collection usingAndroid devices can be performed by Data KitODK suite107 https datakit which is composed of ODK Collect and ODK Aggregate ODK Collecthttps datakitusecollect provides a customizable framework for geospatial data collection and ODKAggregate is a web application that runs on ApacheTomcat server httptomcatapache to store collecteddata through a synchronization with a databaseforexample PostgreSQL Brunette As suchsuite™s performance can be seen in various activities likeagricultural monitoring Krosing and Roybal Journal of the Indian Society of Remote Sensingmonitoring of deforestation and school attendance documentation of war crimes and health programs Anokwa Digital Contact TracingNowadays COVID19 has become the greatest threat forpublic health in last years and due to such pandemicvarious levels of lockdown are issued across the world tobreak its chain of infection transmission However this isthe first approach to invade the contagion but once itwould be lifted this pandemic would start in a new wayand might reach its highest peak by infecting more andmore population Ferguson Thereforetocombat with such a global pandemic threat anotherapproach is discovered by a group of researchers known asdigital contact tracingSmartphonebased contact tracing is known as a digitalcontact tracing which presents a sustainable solution tolimit the transmission of infectious disease by tracing theirpotential transmission routes in a population howeversuch an app presents significant concerns regarding privacy The digital contact tracing works on the principle of˜crowdsource data™ by measuring the proximity to aninfectious person In previous diseases risk surveillancethe contact tracing apps were used to pool location timestamped data to determine the exposures to risk of infectionsSacks Such data are highly personal and leadmany privacy concerns Smith but they werenot always accurate to infer the exposure risks due to noisydata Farrahi Therefore various smartphoneapps are developed in COVID19 pandemic in which someapps use location for proximity and some of them are notusing location services of mobile device subject to theprivacypreserving natureCOVID19 Contact TracingIn order to illuminate the epidemiology of COVID19 andto characterize its severity Lipsitch there is anurgent need of digital platform that captures realtimeaccurate information on COVID19 patients diseasesdiagnosis treatment and clinical reports and whom theyget interacted at which place to detect clusters and generatealerts Such information may help in understanding riskfactors of infection and in predicting the next generation ofinfectious persons FitzGerald Addressing thisunprecedented challenge many mobile apps have beendeveloped and are being used at large scale and some ofthem are as follows 0cJournal of the Indian Society of Remote Sensing¢ COVID Symptom Study COVID Symptom Tracker”This mobile app is developed in collaboration of ZoeGlobal Ltd a digital health care company and a groupof academic scientists from Massachusetts GeneralHospital and King™s College London which waslaunched in UK on March and becameavailable after days in USA This app enquires aboutage location and other diseases risks and also a selfreporting function is enabled which is associated withCOVID19 infection and exposure Drew This app retrieve updates on healthcare worker™sexperiences who are on COVID19 duty their stressand anxiety and use of personal protective equipmentPPE kits are being surveyed through this app toobserve intensity of health care workers Drew et alappimplemented¢ Aarogya Setu”This mobile app is launched on April by Government of India to aware general publicon COVID19 symptoms government advisory measures online consultation facility and dynamics ofdisease Thiscrowdsourcingapproach by which general public members enter theirdetails for selfassessment and this assessment is thenused to trace the infectious contacts or agents as adigital contact tracing concept This app uses locationservices to geolocate the users and Bluetooth tomaintain the log of contacts when one userdevicecomes in contact with another userdevice and as suchdigital contact tracing activity helps in identifying thecluster of diseases and communities which are at risk ofinfection The Aarogya Setu app was downloadedby million users within days of its launchUpadhyay and by using app™s crowdsourcedata the Indian government detected approx positive casesinformed probable users ofbeing at risk and identified potential clusters TheTimes of IndiaNumerous digital contact tracing apps are in use indifferent parts of world”TrackCOVID Yasaka TraceTogether Bay WeTrace De Carli and Google and Apple™s recently announcedjoint initiative Li and Guo COVID19 Data Visualization and ExploratoryData AnalysisWith early experiences of epidemics such as “SARSCoV Boulos and the “ MERSCoVGikonyo and other seasonal flu™s online realtime or nearrealtime mapping of diseases™ occurrencesusing geospatial technologies and web applications havealways been used as a pivotal webbased tools in trackinghealth threats and combating infectious diseases Thissection described a range of mapping dashboards based ongeospatialtechnologies for tracking and unfolding thecoronavirus disease around the world Some of the globaland national geospatial initiatives with an aim to supplyinformation faster than diseases are as summarized inTable Infectious Diseases ModellingThe intention of infectious diseases surveillance is to detectepidemics in their early stages so that the countermeasurescould be taken for preventing its wide spread Suchsurveillance tasks require many epidemiological and statistical methods with geospatial features in investigatingepidemics preferably from localized areas The reason forpreferring the local areas for investigation is because epidemics generally emerged in small areas and then spreadwidely if they are not controlled However some methodsrequire rigorous conventions in their underlying modelsand are too problematical to be applied on small areasThereforefordetecting diseases prevalence with case studies on smalldatasets which would be more useful for public healthactivitiessection discussessimple methodsthisClusteringClustering deals with the study of spatialtemporal patternsof the spread of communicable diseases and identificationof other diseaserelated aspects allied with heterogeneousgeographical distribution which might be helpful in elucidating the diseases™ spread mechanism Such study andanalysis on spacetime patterns is a kind of diseasesurveillance which involves detecting the outbreak clustersof active cases monitoring of localisation and isolation ofinfectious agents and relative risks assessment of affectedsites at early stage Clements Cromley Kulldorff This study on geographical clustering ofinfectious diseases with temporal features helps in makingstrategies that dynamically update on emergence source ofdisease outbreak to help epidemiologists and decisionmakers for identification of spread and risk zones Thusclustering helps to enable timely prevention and containment measures and timely resource allocation to mitigatethe diffusion of diseasesBased on spacetime surveillance of diseases spacetime scan statistic Kulldorff is one of the clusterdetection tools which is widely used in geographicalsurveillance of diseases during epidemic andor pandemicThe spacetime scan statistic comes with two versions”prospective and retrospective Desjardins 0cTable Summary table for geospatial dashboards for COVID19Project nameDatasetsScopePurposeJournal of the Indian Society of Remote SensingWHO Coronavirus Disease COVID19WHO™s official dataDashboard Dong httpscovid19whointGlobal Visualization of official daily counts ofconfirmed cases and deaths related toCOVID19 with time stamps using EsriArcGIS Online serviceExploratory data analysis using 3D graphto perform countrywise analysis usingpopulation confirmed cases cumulativecases deaths and cumulative deathsProvides daily aggregate case and deathcount in CSVJohns Hopkins University COVID19Aggregated data from WHO EuropeanGlobal Dashboard for visualizing realtimeDashboard Dong httpscoronavirusjhuedumaphtmlCentre for Disease Prevention andControl ECDC WorldoMeters BNONews US CDC 1Point3Arces COVIDTracking Project and DXYmapping of COVID19 with graphs onconfirmed and daily casesCritical trend analysis on new cases perday mortality and fatality analysis inpopulation timeline of outbreak etcISRO™s BHUVAN COVID19 GeospatialSolution httpsbhuvanapp3nrscgovincoronacorona_dashboarddashboarddashboardphptypecitizenCOVID19 data Source MoHFWIndiaTime series visualization of activerecovered and deceased cases fromMarch to till dateGraphical analysis on spread trend ofCOVID19 daywise and statewiseCOVID19INDIA httpswwwCM Health M handles Press Trust ofIndiaVisualization of cumulative and dailycovid19indiaIndia state press bulletins PBI and ANIreportsnumbers of confirmed active recovereddeceased and tested statewise casesthrough maps and graphsProvides daily COVID19 cases in stateand district and cases reassigned to statesthrough APIMapmyIndia COVID19 httpsmapsCOVID19 data Source MoHFWIndiaProvides API on corona dashboards tomapmyindiacomcoronadistricts_containment_zonecontainment_zone_gradientHunger Relief Centres Source MyGovHunger Night Shelter Source MyGovNDMAvisualize cases at district and state levelhotspots treatment centres testing labsquarantine centres containment zoneslockdown issues hunger relief hunterand night sheltersMonthly climate explorer for COVID19httpscdsclimatecopernicuseuappsc3sappc3smonthlyclimatecovid19explorerMonthly COVID19 cases Source JHUGlobal Visualization of COVID19 fatalities withCSSEAtmospheric composition”PM10 and NO2Source CAMS EAC4Meteorological data”humidity hPaand surface air temperature on hourly andmonthly average rate Source ERA5reanalysisclimatic and atmospheric variations onmonthly basisExploratory analysis on correlation ofpollutants and specific humidity withCOVID19 deathsExperimental COVID19 and GlobalCOVID19 cases Source JHU CSSEGlobal Visualize earthquake as a cause of increaseSeismic Risk Map httpsmaps quakemapcovid1920200520v3grm234900Global earthquake risk map Source GEMGlobal Earthquake Modelin COVID19 cases due to people™sdisplacement from damaged buildingsOwusu and difference between both is thatprospective neglects historical clusters which may havepreviously occurred before the most current time period ofanalysis with no health threat Kulldorff Thereforethe prospective version of spacetime scan statistic iscommonly used to detect statistically significant active orevolving clusters of diseases for the present time periodand when more data become available the tool can be rerun to detect new evolving clusters with update on relativerisks for each affected sites Previously the prospectivespacetime scan statistic was used in thyroid cancerKulldorff shigellosis Jones measlesYin syndromic surveillance Yih and many other diseases However cluster analysis of 0cJournal of the Indian Society of Remote Sensingdiseases can be performed through several packages andlibraries in R Go´mezRubio and Pythonsoftware Yeng The contribution of cluster detections and analysis inCOVID19 pandemic is becoming useful nowadays as itdetects active and emerging clusters of COVID19 andnotify epidemiologist decision makers and public healthcare officials which can help in eradicating infections fromaffected sites and improving interventions quarantine andisolation measures The significant applications of clustering with respect to infectious diseases modelling aredemonstrated across the world Zarikas forexample India Bhosale and Shinde USA Desjardins Hohl Brazil Martines Italy Cereda China Ji Liu 2020a b Qiu Zhang Singapore Bhosale and Shinde Pung SouthKorea Shim French Alps Danis Germany Pfefferle Sergipe Andrade etcOutlier AnalysisThe outlier is defined by Hawkins as ˜˜an observationwhich deviates so much from the other observations as toarouse suspicions thatit was generated by a differentmechanism™™ In other words when data generation processstarts behaving abnormal and reflects the abnormalities orerrors in data such abnormalities are known as outliersBansal However the outliers generally holdadvantageous information about the systems unusual characteristics and entities which impact the data generationprocess Some of the useful applications of outliers in diseases are Cleynen Dai and Bikdash Krishnan Lo Prensner Washington Wu and Krishnan Clusteringalgorithms are optimized to find clusters rather than outliersand the accuracy of outlier detection depends on how goodthe clustering algorithm captures the structure of clustersMaximum Entropy Modelling Maxent ApproachIn context of disease systems disease transmission risksdepend on distribution of pathogens species in space andtime in some complex environmental conditions Townsend and as such treatments are focused mainly on spatialdimensions therefore diseases transmission risks are purelyhandled through geographical phenomena Such geographical link with diseases leads to the challenge of spatialmapping of disease transmission which overcame throughthe branches of biodiversity science”ecology and biogeography Such approach of ecological and biogeographical modelling can be seen from various studies on diseasetransmission risks mapping for example Arboleda Deka and Morshed Ferreira Holt Mweya Nakazawa Reeves Samy Qian Zhao Zhu Following recent studies on geographical mapping ofpathogens causing disease transmission machine learningbased maximum entropy method Maxent Elith Phillips is applied on spatial records ofCOVID19 with a set of bioclimatic environmentalvariables from WorldClim Poggio Ramı´rezVillegas and Bueno Cabrera to analyse theirfavourable environmental conditions as shown in Fig and Table required in maintaining its population TheMaxent principle is to estimate the target probability distribution by applying the maximum entropy to distributionwhich is most spread or closest This study is carried out inR software Ihaka and Gentleman and a geographical dataset consists of latitude and longitude of thoseregions which were affected till March Figure depicts the habitat suitability map of virus withprobability range in colour scale to visualize the highsuitability light and dark green colour medium suitabilityyellow and dark brownlow suitability light browncolour and unsuitable grey colour Table lists thefavourable bioclimatic variables and their contribution inpercent in maintaining the suitability of virusSusceptibleInfectiousRecovered SIR ModelEpidemiology deals with the study of pattern and occurrence of diseases in space and time associated with otherfactors such as environment demography and the translation of epidemiology into mathematical equations todescribe the spread of infectious diseases is known asmathematical epidemiology Allen Rayner andBender The mathematical epidemiology model isimplemented to understand the transmission dynamics ofcommunicable diseases by categorizing population intosusceptible infectious and recovered compartments Thefirst basic model known as SusceptibleInfectiousRecovered SIR model was proposed by Kermack andMcKendrick to describe the transmission of epidemic diseases from individual to individual The SIRmodel is a set of nonlinear ordinary differential equationswhich is mathematically defined as follows¼ l N þ SðÞ 00 bSI¼ bSI 00 cI 00 lI¼ cI 00 lRdSdtdIdtdRdtð1Þð2Þð3Þ 0cJournal of the Indian Society of Remote SensingFig Predicted suitability of Betacoronavirus using data till March Table Responsible bioclimatic variables in suitability modellingHereS is the class of susceptible individuals who are not yetcontracted to diseaseI is the class of infectious people who are now infectedwith disease and become infectious to infect others¢ R is class of recovered individuals who have recoverednow and are removed from class S¢ N is a total population size N S I R and t istime in days or weeks¢ b is the contact rate of infected person with suspected¢¢¢Bioclimatic variablesPercent contributionMean temperature of coldest quarterPrecipitation of wettest monthMean diurnal rangeIsothermalityAnnual mean temperatureMax temperature of warmest monthPrecipitation of coldest quarterPrecipitation of wettest quarterAnnual precipitationPrecipitation of driest quarterMean temperature of driest quarterMean temperature of wettest quarterPrecipitation seasonalityTemperature seasonalityPrecipitation of warmest quarterMean temperature of warmest quarterTemperature annual rangePrecipitation of driest monthMin temperature of coldest monthperson per dayc is the infectious period and average infectious periodis 1c¢ l is the per capita death rate which is adjusted by birthrate lNThere are many other compartment models derived fromthe basic epidemic model SIR with more compartmentsand transitions” SusceptibleExposedInfectiousRecovered SEIR Li and Muldowney SusceptibleInfectiousExposedRecoveredDeadSEIRDPiccolomiini and Zama SusceptibleInfectiousExposedRecoveredSusceptible SEIRS Liu and Zhang SusceptibleInfectiousQuarantineRecoveredSIQR Erdem
2
acute myeloid leukemia aml is the most common type ofacute leukemia in adults caused by the clonal expansion ofundiï¬erentiated myeloid progenitor cells although mostpatients with aml can achieve complete remission by inductionchemotherapy the recurrence rate remains high and thus is themain factor aï¬ecting the outcomes of aml patients relapseoften develops from minimal residual disease in the protectivebone marrow bm microenvironment a comprehensiveunderstanding of the bm microenvironment is conducive to thediagnosis and personalized treatment of aml leukemic cellscytogenetics and molecular aberrations are the main factorsfor risk stratification in patients with aml in additionthe bm microenvironment also plays a very important role thein the homing and survival ofbm microenvironment contains various componentssuchas immune cells stromal cells endothelial progenitor cellsextracellular matrix growth factors and cytokines theinteraction between leukemic cells and bm microenvironmentaï¬ects resistance to chemotherapy in aml the modulationof bm microenvironment in aml is currently undergoingpreclinical research and early clinical trials molecular inhibitorssuch as cxcr4 inhibitors vla4 inhibitors and eselectininhibitors are currently undergoing clinical trials immunecells and stromal cells are important components of the bmmicroenvironment and are also the main ‚uencing factorsof leukemia development the estimate program isa common method to explore the microenvironment of manytumors recently it has also been used to explore theprognostic genes in the microenvironment of aml patients“ most studies have focused on diï¬erentially expressedgenes degs however the interaction and relationship betweengenes are open to investigate moreover the coding genes wereextensively explored but regions that encoded lncrnas andmirnas were less wellstudiedweighted gene coexpression network analysis wgcnais an algorithm commonly used in systems biology toexplore the correlation between gene sets and the clinic functionalization is achieved by constructing a freescalecoexpression gene network wgcna can identify highlyrelated genes and aggregate them into the same genetic modulecurrently wgcna is used in multiple fields such as cancer andnervous system or to identify potential biomarker candidates ornew therapeutic targets from genetic data “the competition endogenous rna cerna hypothesis was anew regulatory mechanism between noncoding rna ncrnaand messenger rna mrna proposed by salmena in in this theory crosstalk between cernas is achieved byabbreviations aml acute myeloid leukemia auc area under curve bmbone marrow cam cell adhesion molecules cerna competing endogenousrnas deg diï¬erentially expressed gene david the database for annotationvisualization and integrated discovery go gene ontology icam1 intercellularadhesion molecule il10ra interleukin receptor subunit alpha keggkyoto encyclopedia of genes and genomes ppi proteinprotein interactiontcga the cancer genome atlas tlr6 toll like receptor tlr8 toll likereceptor tom topological overlap matrix wgcna weighted correlationnetwork analysisimmunerelated cerna network of amlcompetitively combining shared mirnas in recent yearsthe cernas hypothesis has attracted widespread attention in thestudy of molecular and biological mechanisms of tumorigenesisand development for example previous studies have foundthat lncrnarelated cernas were involved in the biologicalprocesses of glioblastoma and breast cancer theresearch on cernas of leukemia was generally based on thediï¬erential genes screened by leukemia and normal controls but no known module based on cernas network related tomicroenvironment in leukemia has been set upinformation ofthe aml cohortin this study mrnas mirnas and lncrnas data andclinicalfrom the cancergenome atlas tcga database were used to calculate theimmune and stromal scores of these aml cases using theestimate algorithm diï¬erentially expressed mrnas andlncrnas were applied to wgcna to identify the modulesmost relevant to the aml immune microenvironment thenthe immunerelated lncrnamirnamrna cerna networkwas established to screen genes with clinical significance thesefindings will help to better understand the role oftumormicroenvironment in aml and shed light on the developmentand progression of amlmaterials and methodsdata acquisitionall data sets of aml patients were downloaded from tcgadatabase1 the data used in this study met the following criteria excluding samples combined with other malignancies samples with lncrnas and mirnas and mrnas detection datafinally all lncrnas mrnas and mirnas expression profilesof aml specimens and the corresponding clinical followupdata were downloadedidentification of differentially expressedgenesthe estimate algorithm2 was used to calculate the immunescores and stromal scores of aml samples diï¬erentiallyexpressed genes degs such as lncrnas mirnas and mrnaswere identified between high and low score groups stratified bythe median value of immune scores and stromal scores usinglimma package all q values use fdr to correct the statisticalsignificance of the multiple test lncrnas and mrnas withlog fc and fdr were regarded as diï¬erentiallyexpressed while mirnas with log fc and fdr were regarded as diï¬erentially expressed then all the degs wereentered into r version auckland nz united states forcluster analysis based on the expression value of each sample inits respective data set the results were expressed in a clustergrameach column represents a sample and each row represents theexpression level of a given gene1portalgdccancergov2sourcefenetprojectsestimateprojectfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amldavid3 wasgo and pathway enrichment analysesthe database for annotation visualization and integrateddiscoveryenrichedbiological themes of degs functions particularly go geneontology terms and kegg kyoto encyclopedia of genes andgenomes pathway enrichment p was set as thecutoï¬ criterionapplied to analyzeweighted gene coexpression networkanalysiswgcna is an algorithm for identification of gene coexpressionnetworksthrough highthroughput expression profiles ofmrnas or lncrnas with diï¬erent characteristics pairwisepearson correlation analysis was used to evaluate the weightedcoexpression relationship between all datasettopics in theadjacency matrix in this study wgcna was used to analyzemrnas and lncrnas to obtain the mrnas or lncrnas mostrelevant to aml immune microenvironmentcerna network construction andanalysisaccording to the results of wgcna we selected all mrnasand lncrnas in the most relevant module turquoise anddiï¬erentially expressed mirnas to construct a cerna networkbriefly the associated cerna network in aml was constructedfollowing three stages prediction of lncrnamirna inorder to make lncrnas and mirnas map into the interactionssuccessfully we used the miranda4 and pita5 to get targetedlncrnas that mirnas may regulate prediction of mirnamrna three online databases miranda4 targetscan6 andmirwalk7 were used simultaneously for target mrna prediction construction of lncrnamirnamrna cerna networkthe cerna network was constructed based on the negativelyregulating target relationships of mirna“mrna and mirna“lncrna correlation pairsppi network constructionthe retrieval of interacting genes string database8 wasutilized to construct a protein“protein interaction ppi networkof the degs identified in the cerna network the interactingpairs with a confidence score greater than were considered assignificant and were retained the degree represents the numberof interaction partnerssurvival analysiskaplan“meier plots weretherelationship between the overall survival of aml patientsand the expression level of mrnas lncrnas and mirnasthe statistical significance of the correlation was tested by theconstructed to illuminatelogrank test and p was considered significant theanalysis was conducted using the r package of survivalstatistical analysisgraphpad prismtm san diego ca united states or rversion auckland nz united states software was usedfor all data analyses diï¬erences across groups were comparedusing kruskal“wallis test for continuous variables diï¬erenceswere considered significant when p resultsimmune and stromal scores areassociated with aml clinicalparameterswe obtained gene expression profiles and clinical informationof aml patients from tcga database supplementarymaterial among them were male and were female with a median age range “ years oldaccording to the fab classification there were cases of m0 m1 cases m2 cases m3 cases m4 cases m5 cases m6 cases and m7 case the estimate algorithm was used tocalculate the immune scores and stromal scores of aml patientsthe median immune score was range from to and the median stromal score wasˆ’ range fromˆ’ to we analyzed the relationship between immune scores andstromal scores and clinical parameters of aml patients caseswith m4 subtype aml had the highest immune scores while caseswith m3 subtype had the lowest immune scores p figure 1a similarly m4 cases had the highest stromal scoreswhereas m0 subtypes had the lowest p figure 1baccording to cytogenetics aml patients were divided intothree groups favorable intermediatenormal and poor therewas an obvious correlation between the cytogenetic risk andthe immune scores p figure 1cfavorable vsintermediate p favorable vs poor p intermediate vs poor p but no significant correlationbetween the cytogenetic risk and the stromal scores was observedp figure 1dscoreand high immunestromalusing the median immune or stromal score as a thresholdaml patients were divided into two groups with lowimmunestromalscoresurvival analysis showed that the survival rate of aml patientswith low immune scores was significantly higher than that ofpatients with high immune scores p figure 1ehowever there was no significant diï¬erence in survival betweenpatients with low stromal scores and those with high stromalscores p figure 1f3httpdavidncifcrfgov4httpwwwmicrorna5genieweizmannacilpubsmir07mir07_exehtml6httpwwwtargetscan7http12920671508tringdbidentification of differentially expressedgenes based on immune scoresand stromal scoressetting the cutoï¬ criteria as log fc and fdr we identified mrnas figure 2a and lncrnasfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure immune and stromal scores are associated with aml clinical parameters ab distribution of immune scores a and stromal scores b for aml fabsubtypes cd the correlation between immune scores c and stromal scores d and aml cytogenetic risk ef kaplan“meier survival curve based on immunese and stromal scores f aml acute myeloid leukemiafigure heatmap of differentially expressed genes in the high and low immunestromal score groups a mrnas b lncrnas and e mirnas based onimmune scores c mrnas d lncrnas and f mirnas based on stromal scoresfigure 2b based on immune scores and mrnasfigure 2c and lncrnasfigure 2d based onstromal scores setting the cutoï¬ criteria as log fc and fdr we identified and mirnas based onimmune scores figure 2e and stromal scores figure 2frespectively the degs oflow vs high immunethefrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure top go terms in each of biological process were performed for functional enrichment clustering analysis a top25 significant go terms based onupregulated genes in immune scores b top25 significant go terms based on upregulated genes in stromal scores c top25 significant go terms based ondownregulated genes in immune scores d top25 significant go terms based on downregulated genes in stromal scores go gene ontologyscore or stromal score groups were illustrated in the heatmap figure aml and thus require further research to determine theirbiological contributionfunctional enrichment analysis of degsbased on the david the databasefor annotationvisualization and integrated discovery gene annotation tool weperformed go analyses of both upregulated and downregulateddegs the top go biological process indicated that theupregulated degs based on immune or stromal scores wereprimarily enriched in neutrophil degranulation regulationof immune response signal transduction and ‚ammatoryresponse figures 3ab while the downregulated degs basedon immunestromal scores were primarily enriched in rrnaprocessing regulation of translation regulation of transcriptionand cell diï¬erentiation figures 3cd subsequently weperformed kegg kyoto encyclopedia of genes and genomespathway enrichment and interrelation analysis kegg analysisrevealed that the upregulated degs were mainly enriched ininfection osteoclast diï¬erentiation nodlike receptor signalingpathway hematopoietic cell lineage and cell adhesion moleculescams pathways figures 4ab while the downregulateddegs were mainly enriched in ribosome metabolism pi3kaktsignaling pathway transcriptional dysregulation in cancer andmirnas in cancer figures 4cd above analyses revealedthatthese degs play a vital role in the development ofconstruction of weighted correlationnetwork analysis and identification ofkey modulesbased on the results of survival analysis degs based on immunescores were selected for subsequent analysis the best β valuein the lncrnamrna coexpression network was which wascalculated using the diï¬erential lncrnas diï¬erentialmrnas and their expression data in leukemia samples nextthe method of dynamic tree cutting was used to producecoexpression modules finally modules of lncrnamrnacoexpression networks were generated and the heat map plot oftopological overlap matrix tom was shown figure 5a eachmodule was calculated and plotted with its corresponding clinicalcharacteristics correlation analysis showed that turquoisemodule displayed the highest relationship with aml immunescores r which included mrnas and lncrnasfigure 5b these mrnas were further used to performthe gene enrichment analysis the genes were most relatedto neutrophil degranulation immune response ‚ammatoryresponse signal transduction and tolllike receptor signalingpathway figure 5c in addition genes were highly enriched ininfection osteoclast diï¬erentiation nodlike receptor signalingfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure top kegg pathway analysis were performed for functional enrichment clustering analysis a top25 significant kegg pathways based onupregulated genes in immune scores b top25 significant kegg pathways based on upregulated genes in stromal scores c top25 significant kegg pathwaysbased on downregulated genes in immune scores d top25 significant kegg pathways based on downregulated genes in stromal scores kegg kyotoencyclopedia of genes and genomespathway metabolic pathways and hematopoietic cell lineage bykegg analysis figure 5dcerna network constructionsince the turquoise module showed the highest relationshipwith aml immune scores we selected lncrnas and mrnas inthe turquoise module and diï¬erentially expressed mirnasbased on immune scores to construct a cerna network firstlybased on the pita and mircode online database that matchespotential mirnas with lncrnas a total of lncrnamirnapairs contained lncrnas and mirnas then we searchedfor the mrnas targeted by the diï¬erentially expressed mirnasusing three target gene prediction websites miranda mirwalkand targetscan using these websites we detected and target mrnas respectively based on the venn intersectionanalysis target mrnas were selected subsequently wematched the predicted target genes with the mrnas in theturquoise module then we constructed the cerna networkby integrating the mirnalncrnamrna interactions at lasta final lncrnamirnamrna cerna network was constructedwith lncrnas mirnas and mrnas figure 6aprotein“protein interaction ppinetwork analysisto further explore the interplay among the mrnas in cernawe constructed a ppi network based on the string theretrieval of interacting genes online database figure 6b inthe network tlr8 toll like receptor icam1 intercellularadhesion molecule tlr6 toll like receptor and il10rainterleukin receptor subunit alpha had higher degrees and respectively supplementary table the genes encoding these proteins have been confirmed tobe associated with immune microenvironment and leukemiaprogression “association between mrnas mirnasand lncrnas in cerna and overall amlsurvivalwe further analyzed the prognostic values of mrnas mirnasand lncrnas in the cerna network subjects were dividedinto highexpression and lowexpression cohorts accordingto the median value ofthese genes for overall survivalfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure wgcna was used to analyze genetic modules a cluster diagram of coexpression network modules based on topological overlap in mrna andlncrna b study the relationship between each module with their corresponding clinical characteristics c go analysis showed the gene symbols and geneinteractions in the turquoise module d kegg analysis was used to study the pathway enrichment in the turquoise module wgcna weighted correlation networkanalysis go gene ontology kegg kyoto encyclopedia of genes and genomesthrough the package survivalthe highexpression and lowexpression cohorts were splitfor the logrank testin rsoftware out of lncrnas ac0099485 cmahp cta331p31 fcgr2c grk6p1 linc00539 linc01272 mipepp3psmb8as1 rp11266l95 rp11320g101 rp11421f163rp11439e1910 rp11792a83 and stag3l2 out of mirnas hsamir125b5p and hsamir3383p and out of mrnas agpat3 ankrd27 cbx2 ccnd2cd300lb cyth1 erg gdf11 igf1r kiaa0513 kiaa0930larp1 lfng lpcat1 nrep nudt16 pou2f2 ppm1hptafr qsox2 rab3d ralgps2 siglec7 slc43a2 srsf6tnfaip2 tns3 trib1 zbtb5 znf70 and znrf1 wereassociated with overall survival according to the logrank testp representative genes are shown in figure age ‰¥ years vs years and risk group favorablevsintermediatenormal vs poor were also associated withoverall survival according to the logrank test p and respectively the above mentioned lncrnas mrnas and mirnas were brought into further multivariatecox proportional hazard regression analysis with age and riskgroup finally mrnas ccnd2 erg lpcat1 nudt16ralgps2 tnfaip2 and znf70 lncrnas cmahp fcgr2cpsmb8as1 rp11266l95 rp11320g101 rp11792a83 andstag3l2 and mirnas hsamir125b5p and hsamir3383p were independently associated with overall survival table discussionin recent years studies about the roles of gene mutationsand chromosomalin the occurrence anddevelopment of aml and their prognostic values have madesignificant progress however the bm microenvironmentwhich also plays an important role in the pathophysiologytranslocationsfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure cerna network construction and protein“protein interaction network a a lncrnamirnamrna cerna network was constructed by lncrnas mirnas and mrnas b a ppi network was constructed based on the string database the rectangle represents micrornas the circle represents mrnasand the triangle represents lncrnas the red represents upregulation in ishigh and the green represents downregulation in ishigh the size of the dot representsthe regulatory capacity of the mrna and larger points indicate stronger regulatory capability cerna competing endogenous rnas ppi protein“protein interactionprocess in aml are poorly understood therefore mosttreatments previously targeted only tumor cells butfewtargeted the tumor microenvironmentindepth researchon the microenvironment of leukemia will help to furtherunderstand the mechanism of leukemia development and mayfind new targets for microenvironment treatment this studyscreened microenvironmentrelated genes based on the tcgadatabase and further established microenvironmentrelatedlncrnamirnamrna cerna networks through wgcnafirst we calculated the immune scores and stromal scores ofaml patients based on the estimate algorithm and found thatthese scores were related to the fab typing of aml in additionimmune scores were significantly correlated with cytogenetic riskand overall survival estimate is a new algorithm to inferthe level of stromal and immune cells in tumor tissues andtumor purity using gene expression data high immunescores in the bm samples from patients with poor prognosisindicated that more immune cells were recruited in their bmfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure correlation between mrnas mirnas and lncrnas in cerna and overall aml survival in tcga kaplan“meier survival curves with the logrank testwere performed for the representative mrnas mirnas and lncrnas cerna competing endogenous rnas aml acute myeloid leukemia tcga the cancergenome atlasmicroenvironment this may be due to that aml cells activelyshape the bm environment and immune cells to promote diseaseprogression through cellular structural and functional changes however there was no significant correlation betweenstromal scores and cytogenetic risk or survival of aml patientssuggesting that the proportion of stromal cells were comparablein diï¬erent group possible explanation may be that stromal cellsplay an important role in solid tumors while its role inleukemia is not as strong as in solid tumors “then we identified diï¬erentially expressed mrnas mirnasstromaland lncrnas based on the immune scores orscores functional enrichment analysis indicated thatthesedegs were mainly involved in immune and ‚ammatoryresponses consistent with these results previous studies haveshown that the biology of the immune system is essentialfor the formation of a complex bm microenvironment in recent yearsthe immunologicalcharacteristics of aml has increased and the developmentof eï¬ective aml immunotherapy strategies has attractedwidespread attention the understanding ofin recent years important advances in cerna coexpressionnetwork research have developed rapidly the disruption offrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amltable multivariate cox proportional hazard regression analysis of lncrnas mrnas and mirnasgenesccnd2erglpcat1nudt16ralgps2tnfaip2znf70cmahpfcgr2cpsmb8as1rp11266l95rp11320g101rp11792a83stag3l2hsamir125b5phsamir3383phr 95ci “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “pthe cerna network balance is a major cause for tumorigenesis therefore understanding the complex interactions betweendiï¬erent cerna networks will lead to an indepth understandingof gene regulatory networks and has implications for cancertreatment in addition the lncrnamirnamrna cernanetwork can predict the prognosis of the disease for examplea lncrnamirnamrna cerna network was established basedon rnaseq data of breast cancer from tcga which consistsof mirnas lncrnas and mrnas multiplexcox regression analyses showed that four of these lncrnasadamts9as1 linc00536 al3914211 and linc00491 havesignificant prognostic value wang identified lncrnas mirnas and mrnas from a database toconstruct a lncrnamirnamrna cerna network inthe network a univariate and multivariate cox proportionalhazard regression analysis was used to establish a survivalmodel with target mrnas hoxa9 insr krit1 mybspry2 ube2v1 wee1 and znf711 where auc area undercurve is indicating the sensitivity and specificity ofprognostic prediction however the screening of diï¬erentialgenes in this study was based on leukemia patients andnormal people and did not focus on tumor microenvironmentso far there is no cerna research based on the leukemiamicroenvironment in our research the screening of diï¬erentialgenes was based on the immune score then wgcna wasused to identify the modules most relevantto the amlimmune microenvironment then using wgcna and mirnaprediction websites a lncrnamirnamrna cerna networkconsisting of lncrnas mirnas and mrnaswas constructedsubsequently we built a ppi network predicting theinteraction among the proteins encoded by the degs inthe cerna network tlr8 icam1 tlr6 and il10ra hadhigher degrees tlr8 and tlr6 are members of the tolllike receptor family which is upstream to the transcriptionittransportationthe innate immune system andfactor nfκb and part ofplays an importantin progression of aml roleicam1 is one of the cams a large class of transmembraneproteinsinvolved in the binding of cells to another cellor extracellular matrix and involved in cell proliferationdiï¬erentiation movementandtissue structure the protein encoded by il10ra is areceptor for interleukin which has been shown to mediatethe immunosuppressive signal ofinterleukin and thusinhibits the synthesis of pro‚ammatory cytokines and isreported to promote survival of progenitor myeloid cellsthrough the insulin receptor substrate2pi3kakt pathway these results indicated that this novel cerna networkwere closely associated with immune microenvironment andprogression of amlapoptosisfurthermore lncrnas mirnas and mrnas withprognostic significance were screened out which could be usedas biomarkers for prognosis among the genes with prognosticsignificance in our module of immunerelated cerna networkthere were aml related reports about cbx2 ccnd2 ergigf1r larp1 lfng nudt16 pou2f2 ptafr rab3dsiglec7 srsf6 tnfaip2 trib1 zbtb5 and znrf1 themost reported of which were erg ccnd2 and ifg1r ergtranslocation was involved in the occurrence and developmentof aml and high expression of erg was a poor prognosticfactor for patients with normal karyotype aml ccnd2mutations were more common in cbfaml and it was also afrequent mutation event in t aml ccnd2 leadedto increased phosphorylation of retinoblastoma proteins leadingto significant cell cycle changes and increased proliferation ofaml cell lines nicolas chapuis found that igf1spontaneous lesions played a key role in pi3kakt activation ofaml cells providing strong evidence for targeting igf1r as apotential new therapy for aml the functions of agpat3ankrd27 cd300lb cyth1 gdf11 kiaa0513 kiaa0930lpcat1 nrep ppm1h qsox2 ralgps2 slc43a2 tns3and znf70 in aml have not been reported we identified lncrnas with clinical significance among them only cmahpwas reported to be related to mllpositive aml andother lncrnas have not been reported in leukemia twomirnas including hsamir125b5p and hsamir3383p havebeen reported to be associated with a variety of cancers “ but no studies have been reported related to aml all theseunreported mrnas mirnas and lncrnas may be potentialnovel biomarkers or therapeutic targets for amlis importantto note that our current research haslimitations we selected the target data from the tcga publicdatabase only through the biological algorithm method weshould further verify the results of this in clinical patientsin further studyconclusionin summary a comprehensive bioinformatics analysis wasperformed on the aml dataset in tcga with an emphasison the immune microenvironment using wgcna andfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlmirna prediction programs an immunerelated lncrnamirnamrna cerna network was established and degs withprognostic value were further identified further studies of thesegenes are needed in the clinic and may provide new insights intothe pathogenesis of aml this study increases our understandingof the complex interactions between aml tumor cells and the bmmicroenvironment and may provide novel prognostic factors andtherapeutic targetsdata availability statementall data sets of aml patients were downloaded from the cancergenome atlas tcga database portalgdccancergovauthor contributionsyfl cw and zj conceptualization and design sw dataacquisition and writing “ original draft sw ly yx and dzmethodology sw and cw data analysis and interpretationyjl and yfl writing “ review and editing yfl cw and zjproject administration all authors contributed to the andapproved the submitted versionfundingthis work was supported by the national natural sciencefoundation of china grant numbers and u1804191and the henan medical science and technology research projectgrant number acknowledgmentswe thank the tcga database for the availability of the datasupplementary materialthe supplementary materialonline202001579fullsupplementarymaterialfor this can be foundwwwfrontiersins103389foncatreferences ferrara f schiï¬er ca acute myeloid leukaemia in adults lancet “ doi 101016s0140673612617279 zeng z shi yx samudio ij wang ry ling x frolova o targetingthe leukemia microenvironment by cxcr4 inhibition overcomes resistanceto kinase inhibitors and chemotherapy in aml blood “doi 101182blood200805158311 shafat ms gnaneswaran b bowles km rushworth sa the bone marrowmicroenvironment“home of the leukemic blasts blood rev “ doi 101016jblre201703004 bullinger l döhner k döhner h genomics of acute myeloid leukemiadiagnosis and pathways j clin oncol “ doi 101200jco ayala f dewar r kieran m kalluri r contribution of bonemicroenvironment to leukemogenesis and leukemia progression leukemia “ doi 101038leu2009175 uy gl rettig mp motabi ih mcfarland k trinkaus km hladnik lm a phase study of chemosensitization with the cxcr4 antagonist plerixaforin relapsed or refractory acute myeloid leukemia blood “doi 101182blood201110383406 rashidi a uy gl targeting the microenvironmentin acute myeloidleukemia curr hematol malig rep “ doi 101007s11899 austin r smyth mj lane sw harnessing the immune system in acutemyeloid leukaemia crit rev oncol hematol “ doi 101016jcritrevonc201604020 yehudairesheï¬ s attiasturgeman s sabbah r gabay t musallam rfridmandror a abnormal morphological and functional nature ofbone marrow stromal cells provides preferential support for survival of acutemyeloid leukemia cells int j cancer “ doi 101002ijc yoshihara k shahmoradgoli m martínez e vegesna r kim h torresgarciaw inferring tumour purity and stromal and immune cell admixture fromexpression data nat 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101101gr180273114 liu q jiang c xu j zhao mt van bortle k cheng x genomewide temporal profiling of transcriptome and open chromatin of earlycardiomyocyte diï¬erentiation derived from hipscs and hescs circulat res “ doi 101161circresaha116310456 salmena l poliseno l tay y kats l pandolfi pp a cerna hypothesisthe rosetta stone of a hidden rna language cell “ doi101016jcell201107014 karreth fa pandolfi pp cerna crosstalk in cancer when cebling rivalriesgo awry cancer discov “ doi 10115821598290cd13 zhang k li q kang x wang y wang s identification and functionalcharacterization of lncrnas acting as cerna involved in the malignantprogression of glioblastoma multiforme oncol rep “ doi103892or20165070 wang jd zhou hs tu xx he y liu qf liu q prediction ofcompeting endogenous rna coexpression network as prognostic markers inaml aging “ doi 1018632aging101985 ritchie me phipson b wu d hu y law cw shi w limma powersdiï¬erential expression analyses for rnasequencing and microarray studiesnucleic 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EpidemiologyEPIDEMIOLOGICAL SCIENCERisk factors for hospital admissions related to COVID19 in patients with autoimmune inflammatory rheumatic diseasesDalifer D Freites Nu±ez1 Leticia Leon Arkaitz Mucientes1 Luis Rodriguez Rodriguez Judit Font Urgelles3 Alfredo Madrid Garc­a1 Jose I Colomer1 Juan A Jover34 Benjam­n Fernandez Gutierrez3 Lydia Abasolo1Handling editor Josef S Smolen1Rheumatology Department and IDISSC La Fundacion para la Investigacion Biomedica del Hospital Clinico San Carlos Madrid Spain2Department of Health and Education Universidad Camilo Jose Cela Villafranca del Castillo Madrid Spain3Rheumatology Department Hospital Clinico San Carlos Madrid Spain4Medicine Department Universidad Complutense de Madrid Madrid Comunidad de Madrid SpainCorrespondence toDr Leticia Leon IdISSC and Rheumatology La Fundacion para la Investigacion Biomedica del Hospital Clinico San Carlos Madrid Spain lleon hcsc salud madrid Received May Revised July Accepted July Objectives To describe patients with autoimmune inflammatory rheumatic diseases AIRD who had COVID19 disease to compare patients who required hospital admission with those who did not and assess risk factors for hospital admission related to COVID19Methods An observational longitudinal study was conducted during the pandemic peak of severe acute respiratory syndrome coronavirus March to April All patients attended at the rheumatology outpatient clinic of a tertiary hospital in Madrid Spain with a medical diagnosis of AIRD and with symptomatic COVID19 were included The main outcome was hospital admission related to COVID19 The covariates were sociodemographic clinical and treatments We ran a multivariable logistic regression model to assess risk factors for the hospital admissionResults The study population included patients with AIRD and COVID19 Of these patients required hospital admission related to COVID19 The mean age on admission was years and the median time from onset of symptoms to hospital admission was “ days The median length of stay was “ days A total of patients died during admission Compared with outpatients the factors independently associated with hospital admission were older age OR p000 and autoimmune systemic condition vs chronic inflammatory arthritis OR p001 No statistically significant findings for exposure to disease modifying antirheumatic drugs were found in the final modelConclusion Our results suggest that age and having a systemic autoimmune condition increased the risk of hospital admission whereas disease modifying antirheumatic drugs were not associated with hospital admission Authors or their employers No commercial re use See rights and permissions Published by BMJTo cite Freites Nu±ez a0DD Leon a0L Mucientes a0A et a0al Ann Rheum Dis Epub ahead of print [please include Day Month Year] 101136annrheumdis2020217984INTRODUCTIONSevere acute respiratory syndrome coronavirus SARS CoV2 causes a myriad of clinical signs and symptoms together with typical laboratory abnormalities that manifest as the disease COVID191Since the confirmation of the first patient infected with SARS CoV2 in Spain in January the current COVID19 outbreak has had a considerable impact especially in the Madrid region where the highest incidence of COVID19 cases has been Key messagesWhat is already known about this subject –º The epidemiological scenario is changing daily There is little evidence for risk factors of poor outcome with COVID19 specific to autoimmune inflammatory rheumatic diseasesWhat does this study add –º Patients with an autoimmune systemic condition have a higher risk of hospital admission related to COVID19 compared with those with chronic inflammatory arthritis –º Disease modifying agents were not associated with a higher risk of hospital admission related to COVID19How might this impact on clinical practice or future developments –º Our data show that in a real world setting a high percentage of patients with autoimmune inflammatory rheumatic diseases and COVID19 required hospital admission The patients were mainly elderly with comorbidities and a systemic autoimmune conditionrecorded with more than patients admitted to the hospital until the first week of May2The incidence and severity of COVID19 disease seem to be higher in patients with risk factors such as advanced age and associated comorbidities mainly hypertension diabetes heart disease and previous respiratory diseases3 It is not clear whether patients with rheumatic diseases are more susceptible to SARS CoV2 infection or when they are infected whether they have more severe disease or a poorer outcome Previous outbreaks caused by coronaviruses did not yield overwhelming evidence that patients with rheumatic diseases are at an increased risk4 although some patients are candidates for a higher number of infections owing to their rheumatic disease predominantly systemic or the treatment they are receiving for rheumatic diseases5 Preliminary experiences in patients with COVID19 show that those with chronic arthritis treated with synthetic conventional or targeted syntheticbiologic disease modifying antirheumatic Freites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cEpidemiologydrugs DMARDs do not seem to be at a greater risk of respiratory or life threatening complications from SARS CoV2 than the general population6 The epidemiological scenario is changing and evidence on the risk factors of poor outcome with COVID19 specific to inflammatory rheumatic disease is scarce In addition there are little data on how the hospital admissions of these patients with severe COVID19 infection have evolved8The aim of our study was to describe patients with autoimmune inflammatory rheumatic diseases AIRD who had COVID19 during the pandemic peak We also explored possible risk factors associated with hospital admission related to COVID19 disease in patients with AIRD from a tertiary hospital in Madrid SpainMETHODSSetting study design and patientsThe study was performed in a public tertiary hospital Hospital Cl­nico San Carlos HCSC in Madrid Spain The catchment area is home to almost peopleWe performed a prospective observational study from March when our health area had the first hospital admission related to COVID19 to April We preselected all patients attended at the rheumatology outpatient clinic of our centre during the study period whose data were recorded in the electronic clinical history of our department HCR Penelope The inclusion criteria were age years a medical diagnosis according to International Classification of Diseases ICD10 of inflammatory rheumatic disease and symptomatic COVID19 disease assessed by medical diagnosis or confirmed with a positive SARS CoV2 PCR diagnostic testPatient data were obtained during routine clinical practice The study was conducted in accordance with the Declaration of Helsinki and the principles of Good Clinical Practice and was approved by the HCSC Ethics Committee approval number E BSVariablesThe primary outcome was admission to hospital with a medical diagnosis of COVID19 andor a positive PCR result between March and April compared with outpatients with symptomatic COVID19 diseaseThe covariables recorded were as follows sociodemographic baseline characteristics including sex age and rheumatic disease duration Type of AIRD including systemic autoimmune conditions polymyalgia rheumatica mixed connective tissue disease systemic sclerosis Sjogren™s syndrome vasculitis Raynaud phenomenon polymyositis polychondritis sarcoidosis antiphospholipid syndrome autoinflammatory syndromes and systemic lupus erythematosus and chronic inflammatory arthritis rheumatoid arthritis inflammatory polyarthritis juvenile idiopathic arthritis psoriatic arthritis axial spondyloarthritis uveitis and inflammatory bowel disease Baseline comorbid conditions including hypertension dyslipidaemia depression diabetes mellitus smoking habit kidney disease chronic liver disease respiratory diseases chronic obstructive pulmonary disease and interstitial lung disease thyroid disease heart disease valve disease arrythmias cardiomyopathy heart failure and pericarditis ischaemic vascular disease stroke cardiovascular and peripheral vascular disease venous thrombosislung embolism and cancer Treatment for inflammatory rheumatic disease a glucocorticoids b non steroidal anti inflammatory drugs NSAIDs c conventional synthetic disease modifying antirheumatic drugs csDMARDs antimalarials hydroxychloroquine and chloroquine azathioprine cyclophosphamide cyclosporine colchicine leflunomide methotrexate mycophenolate mofetilmycophenolic acid and sulfasalazine d targeted syntheticbiologic DMARDs tsbDMARDs including antitumour necrosis factor TNF alpha drugs infliximab adalimumab etanercept certolizumab and golimumab other biologics anti interleukin IL6 tocilizumab and sarilumab rituximab abatacept belimumab anti IL1723 anti IL17 ustekinumab ixekizumab and secukinumab Janus kinase JAK inhibitors tofacitinib and baricitinibTreatment had to start at least month before the beginning of the study and continue during the study period until the end of the study or hospital admission for antimalarial therapy glucocorticoids sulfasalazine NSAIDs or colchicine Regarding csDMARDs and tsbDMARDs treatment had to start at least month before the beginning of the study and continue until at least 21st March the end of the study or hospital admission In the case of rituximab the last infusion had to be at least in JanuaryData sourcesPatient sociodemographic clinical laboratory and data on treatment of rheumatic disease were obtained through HCR PenelopePatients with COVID19 were detected by warning calls to our rheumatologists or nurses or via routine telephone consultation Other infected patients were detected through their sick leave forms for COVID19 The results of SARS CoV2 PCR diagnostic assays were obtained from the microbiologyinfectious service of HCSC In addition our Hospital Central Services registered all medical admissions to HCSC This information was provided from March to AprilThe researchers carried out an exhaustive review of the clinical histories of admitted patients to identify COVID19 cases and rule out patients admitted for other reasons Once the COVID19 cases were identified we collected clinical laboratory and treatment data during admission until the end of admission either discharge or death in order to describe the progress of the disease The review was performed until 24th April in order to include follow up data from patients admitted to the hospital with COVID19Statistical analysisPatient characteristics are expressed as mean and SD or median and IQR for continuous variables categorical variables are expressed as percentages Statistical tests were performed to compare characteristics between patients admitted with COVID19 and those without hospital admissions Continuous variables were analysed using the Mann Whitney test or t test and discrete variables were analysed using the χ2 or Fisher exact test Univariable logistic regression analyses were performed to assess differences between hospital admissions related to COVID19 risk and covariates Multivariable logistic regression models adjusted for age sex and comorbidity were run in a stepwise manner to examine the possible effect of sociodemographic clinical and therapeutic factors on hospital admissions related to COVID19 The model also included csDMARDs and all other variables with a p02 from the simple regression analysis The results were expressed as the OR with its respective CIAll analyses were performed in Stata V13 statistical software Stata Corp A two tailed p value was considered to indicate statistical significanceFreites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cRESULTSA total of patients with AIRD with symptomatic COVID19 disease were included in the study table The tests were performed as an exploratory measure of the association between a variable and the outcomeMost of the patients were women with a mean age of years and a mean time since diagnosis of years The main diagnosis was rheumatoid arthritis followed by axial spondyloarthritis Many patients had at least one baseline comorbid condition the most prevalent being hypertension dyslipidaemia and lung disease Most patients were taking csDMARDs Half of the patients were taking glucocorticoids a quarter were taking NSAIDs and were taking tsbDMARDs of which adalimumab was the most frequently prescribed followed by rituximab Only one patient was taking a JAK inhibitor Interestingly of the patients taking tsbDMARDs were taking the drug in combination with a csDMARDA total of patients had to be admitted to the hospital because of COVID19 Of these were evaluated in the HCSC Emergency Department were admitted to HCSC and were transferred to the Institucion Ferial de Madrid IFEMA support hospital owing to the lack of capacity in our hospital at that time The remaining three patients were evaluated and admitted to other hospitals in the Autonomous Community of Madrid Table presents data for the patients admitted to HCSCOf the patients admitted to our hospital were women with a mean age at admission of years and median lag time from the onset of symptoms to the admission of “ days The median length of stay was “ days table At admission the median haemoglobin was “ gdL and the median total lymphocyte count was “ ngmL The median D dimer value was “ ngmL In of patients median interleukin IL6 levels were “ pgmL Patients received various antibiotics mainly azithromycin levofloxacin and third generation cephalosporinsMost patients were treated with hydroxychloroquine during admission About half received glucocorticoids Eighteen were treated with lopinavirritonavir and received the anti IL 6R antibody tocilizumab table 2FEDERA total of patients developed relevant complications during admission the most frequent being myocarditis thrombosis and kidney failure Only two patients were admitted to the intensive care unit during admission The first was a patient in 50s with mixed connective tissue disease and associated comorbidities who developed acute respiratory insufficiency and bilateral pneumonia The patient was treated with antibiotic therapy lopinavirritonavir hydroxychloroquine and βinterferon Finally the patient was extubated days later and is recovering The other was a young adult patient with systemic lupus erythematosus treated with methotrexate rituximab hydroxychloroquine and glucocorticoids who days after being diagnosed with COVID19 PCR developed an erythematous rash and generalised urticaria requiring hospitalisation in the intensive care unit owing to general clinical and laboratory worsening elevated D dimer values The patient was treated with methylprednisolone heparin and a cephalosporin A few days later the patient™s condition improved and he recovered completely at dischargeOf the patients admitted to HCSC were sent to another care centre converted hotel hospitalIFEMA support hospital when their condition improved A further patients Epidemiologywere discharged home to continue self isolation after improvement At the end of the study five patients remained in hospital A total of patients died during admission men and women with a median age of “ years Of the patients who died had relevant comorbidity diabetes mellitus pulmonary disease ischaemic vascular disease hypertension venous thrombosislung embolism lung disease and or liver disease The main diagnoses were rheumatoid arthritis followed by spondyloarthritis polymyalgia rheumatica vasculitis and Sjogren™s syndrome The results of the univariable analysis are shown in table Older age systemic autoimmune conditions vs chronic inflammatory arthritis OR CI “ p0014 hypertension diabetes mellitus lung disease heart disease and glucocorticoids were associated with statistically significant greater risk of admission to the hospital Female sex NSAIDs and anti TNF drugs vs non use were associated with a statistically significant lower risk The differences reported for the remaining variables did not reach statistical significanceThe multivariable analysis was adjusted for gender age and comorbidities related to COVID19 These comorbidities were diabetes mellitus pulmonary disease ischaemic vascular disease hypertension venous thrombosislung embolism lung disease andor liver disease table Age and systemic autoimmune conditions had more probability of hospital admissions regardless of other factors Differences in exposure to glucocorticoids were not statistically significant The type of exposed DMARDs did not reach statistical significance in the multivariate model In fact long term treatment with antimalarials OR CI “ p066 other csDMARDs including methotrexate leflunomide and azathioprine OR CI “ p09 and NSAIDs OR CI “ p05 dropped from the final model The variable tsbDMARDs was also eliminated from the final model anti TNF vs none OR CI “ p016 and non anti TNF vs none OR CI “ p03DISCUSSIONOur study aims to shed light on rheumatologists™ concerns regarding their patients We found that in a real world setting of patients with AIRD and COVID19 required hospital admission These were mainly elderly patients with more comorbidities and systemic autoimmune conditions Our data show that patients exposed to disease modifying agents do not seem to be at higher risk of hospital admission related to COVID19Of the patients included in the study with COVID19 required hospital admission Comparison of the characteristics of patients admitted to hospital because of COVID19 and those who did not require hospital admission were as follows admitted patients had a median age close to years that is more than years older than patients who were not admitted Moreover those who were admitted more frequently had baseline comorbidities and systemic autoimmune conditions As for therapy admitted patients were less frequently exposed to antimalarial and anti TNF alpha agentsThe median lag time from onset of symptoms to admission was days and almost of patients had pneumonia at admission The baseline laboratory results for admitted patients in our study are consistent with those published elsewhere9“ and are characterised by lymphopenia and elevated acute phase reactants In fact of the patients had elevated D dimers normal and elevated IL6 normal pgmL Treatment during admission varied widely as the disease proved Freites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cEpidemiologyTable Baseline demographic and clinical characteristics of patients with AIRD and with COVID19 admitted vs no admitted at the hospitalAIRD“COVID19 patientsAIRD“COVID non admitted patientsAIRD“COVID admitted patientsVariableN123N69N54P value Positive Negative Not performed Women n Age years mean SDTime since diagnosis years mean SDPCR test n Smoking habit active vs noneDiagnosis AIRD n Rheumatoid arthritis Axial spondyloarthritis Polymyalgia rheumatica Psoriatic arthritis Systemic lupus erythematosus Mixed connective tissue disease Sjogren™s syndrome Vasculitis Uveitis Systemic sclerosis Inflammatory polyarthritis Polychondritis Polymyositis Raynaud phenomenon OtherComorbidities n NSAIDs n Glucocorticoids n csDMARDs n TsbDMARDs n JAKi n Others inflammatory bowel disease antiphospholipid syndrome juvenile idiopathic arthritis autoinflammatory syndromes and sarcoidosis Heart disease arrhythmiasvalve disease cardiomyopathy and heart failure Ischaemic vascular disease stroke cardiovascular and peripheral vascular diseaseAIRD autoimmune inflammatory rheumatic disease Anti TNF tumour necrosis factor alpha COPD chronic obstructive pulmonary disease csDMARD conventional synthetic disease modifying antirheumatic drug ILD interstitial lung disease JAKi JAK inhibitor tsbDMARDs target syntheticbiologic disease modifying antirheumatic drug Hypertension Dyslipidaemia Depression Diabetes mellitus Heart disease Vascular disease Liver disease Kidney disease Lung disease ILDCOPD Cancer Venous thrombosislung embolism Thyroid disease Anti TNF alpha agent Other biologics Abatacept Tocilizumab Belimumab Rituximab Methotrexate“leflunomide“azathioprine Sulfasalazine AntimalarialsFreites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cTable Hospital admissions related to COVID19 among patients with AIRDVariableValueTable OR of hospital admission related to COVID19 in patients with AIRD univariable analysisVariable CIORPEpidemiology Haemoglobin gdL D dimer ngmL Neutrophil count —109L Lymphocyte count —109L CRP mgdL LDH UL Platelet count —109L Creatinine mgdL Ferritine ngmLAdmissions nLag time from onset of symptoms to admission days median IQRPneumonia at admission n Systemic autoimmune conditions n Laboratory data at admission median IQR COVID19 related treatments during admission n Admitted by intensive care unit during hospital admission Length of stay days median IQRDischarge reason n Azithromycin Other antibiotics Glucocorticoids Lopinavirritonavir Remdesivir Darunavircobicistat Tocilizumab Interferon HCQ Immunoglobulin Improvement home isolation Other care centre medicalised hotelIFEMA hospital Death End of study no discharge No Yes “ Data for patients patients were treated in other support centres after referral or admission in other centresCRP C reactive protein HCQ hydroxychloroquine LDH lactate dehydrogenase challenging for specialists who prescribed various combinations of drugs based on little published evidence In this sense the anti IL 6R antibody tocilizumab has proven to be beneficial in patients with COVID1912 Treatment may also be successful in the early stages of cytokine release syndrome if it can effectively block the signal transduction pathway of IL6 therefore tocilizumab and sarilumab are likely to emerge as effective drugs for patients with moderate to severe COVID1913 In our study almost of the patients were treated with tocilizumabThe patients who eventually died had a median age of years This finding is in line with data for the general population where over of deaths occurred in persons years and more than of all deaths were in people aged ‰¥ years7The multivariable regression model showed that only age increasing by per year and systemic autoimmune conditions continued to be risk factors for hospital admission related to COVID19 “ “ “ “ “ “ “ “ “ ““““““““““““ Rheumatoid arthritis Inflammatory polyarthritis Systemic lupus erythematosus Psoriatic arthritis Spondyloarthritis MTCD Sjogren syndrome Hypertension Dyslipidaemia Depression Diabetes mellitus Heart disease Vascular disease Liver disease Kidney disease Lung disease ILDCOPD Cancer Venous thrombosislung embolism Thyroid diseaseGender womenAge yearsDiagnosis AIRD one category vs the rest Disease durationSmoking habit active vs noneComorbidities yes NSAIDsGlucocorticoidscsDMARDSs TsbDMARDs JAKisOther biologics anti IL6 tocilizumab sarilumab rituximab Rtx anti IL1723 anti IL17Othercategories could not be represented polymalgia rheumatica systemicsclerosis vasculitis Raynaud phenomenon polychondritis Beh§et diseasepolymyositis uveitis inflammatory boweldisease antiphospholipid syndrome juvenile idiopathic arthritis autoinflammatorysyndromes and sarcoidosisAIRD autoimmune inflammatory rheumatic disease anti TNF tumour necrosis factor csDMARD Conventional synthetic disease modifying antirheumatic drug IL6 interleukin6 JAKi JAK inhibitors tsbDMARDs target syntheticbiologic disease modifying antirheumatic drug““““““““““““ ““ Methotrexate“leflunomide“azathioprine Sulfasalazine Antimalarial agents““““““““““ None Anti TNF agents Other biologics““As for the association between sex and risk of hospital admission we did not find a higher risk of admission in women despite the fact that rheumatic diseases are more prevalent in this group The type of diagnosis seems to play an important role in the probability of hospital admission and patients with systemic autoimmune conditions seem to have the highest risk compared with chronic inflammatory arthritisAs it has been reported elsewhere comorbidities play an important role in the risk of hospital admission15 Clinical outcomes are poorer in patients with COVID19 with a comorbid condition than in those without and a greater number of comorbidities correlates with poorer clinical outcomes16 Diabetes is a major comorbidity in COVID19 and patient™s history of diabetes is an independent risk factor for morbidity and mortality in this condition17 Diabetes has been associated with admissions to Freites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cEpidemiologyTable Multivariable analysis risk factors for hospital admission related to COVID19 in patients with AIRDVariableOR CIP value“““““Gender womenAge yearsAIRD systemic autoimmune conditions vs chronic inflammatory arthritisCOVID comorbidities yesGlucocorticoidsSystemic autoimmune conditions polymyalgia rheumatica mixed connective tissue disease systemic sclerosis Sjogren™s syndrome vasculitis Raynaud polymyositis polychondritis sarcoidosis antiphospholipid syndrome autoinflammatory syndromes and systemic lupus erythematosus vs chronic inflammatory arthritis rheumatoid arthritis inflammatory polyarthritis juvenile idiopathic arthritis psoriatic arthritis axial spondyloarthritis uvetis and inflammatory bowel disease Comorbidities including the presence of at least one of the follows hypertension heart disease vascular disease diabetes mellitus venous thrombosislung embolism chronic kidney disease liver disease and lung disease ILDCOPDAIRD autoimmune inflammatory rheumatic disease COPD chronic obstructive pulmonary disease ILD interstitial lung diseasethe intensive care unit due to COVID19 in recent series19 and has been shown to increase mortality6 Therefore we adjusted for comorbidity in the multivariable analysisTreatment with glucocorticoids lost its statistical significance in the multiple regression model However the dose was not reported in our data and in the case of these agents the risk could be dose dependent In a recent publication from a European registry the authors found that exposure to mgday was associated with a greater probability of hospitalisation21The exposure to DMARDs regardless of whether they were biological or synthetic does not seem to be associated with a higher hospital admission related to COVID19 Although we have to consider the limited number of patients in our study our results are in concordance with data reported elsewhere8 Our results should be interpreted taking into account other limitations First patients were included from a single centre Second of all the patients with COVID19 who did not require admission one third contacted the rheumatology service to report the disease and the remainder were detected through the COVID19 discharge reports sent to their primary care physician Elderly persons and homemakers who did not contact us can be considered missing Consequently there may be some selection bias between those admitted and those not admitted although this problem was addressed by adjusting for confounders in the multivariable analysis Third while it is acknowledged that clinical suspicion must be confirmed by PCR assay almost of patients admitted did not undergo PCR owing to the lack of tests or the extreme healthcare overload Nevertheless all cases included were clinically compatible and managed as COVID19The key strength of our study is that it was performed in real life conditions during then pandemic peak with access to complete sociodemographic and clinical data from our rheumatology electronic clinical history including thorough hospital admission data such as laboratory abnormalities and COVID19 treatment data from the hospital computer services Consequently this has allowed us to analyse the risk of hospital admission related to COVID19 adjusted for confounders thus avoiding possible biasAlthough we are unable to modify the factors reported here knowing them can help rheumatologists to treat and advise their patients during this new and challenging period Results provided by our study are preliminary and should be corroborated with other real life studies but we consider our findings helpful to increase the knowledge in the management of patients with AIRD and COVID19Twitter Benjam­n Fernandez Gutierrez Fergutbe2001Acknowledgements The authors would like to thank Ana M Perez for her help with data collection They would like to say a special thanks to all the rheumatologists and nurses who contributed to the care of the patients in such an innovative and conscientious wayContributors BF G LL JAJ LR R and LA conceived and designed the study DDFN JFU AMG JIC and LL collected data LA and LL performed the data analysis and interpreted the data All of the authors were involved in the drafting andor revising of the manuscriptFunding This work was supported by the Instituto de Salud Carlos III ISCIII Ministry of Health Spain CP1600916 PI1801188 and RD1600120014 and cofunded by el Fondo Europeo de Desarrollo Regional FEDER The funders had no role in study design data collection analysis manuscript preparation or decision to publishCompeting interests None declaredPatient and public involvement Patients andor the public were not involved in the design or conduct or reporting or dissemination plans of this researchPatient consent for publication Not requiredEthics approval The study was approved by the Hospital Cl­nico San Carlos institutional ethics committee approval number E BS This study was conducted according to the principles of the Declaration of HelsinkiProvenance and peer review Not commissioned externally peer reviewedData availability statement Data are available upon reasonable requestThis article is made freely available for use in accordance with BMJ™s website terms and conditions for the duration of the covid19 pandemic or until otherwise determined by BMJ You may use download and print the article for any lawful non commercial purpose including text and data mining provided that all copyright notices and trade marks are retainedORCID iDsLeticia a0Leon http orcid Luis a0Rodriguez Rodriguez http orcid REFERENCES Fernandez Gutierrez B COVID19 with pulmonary involvement An autoimmune disease of known cause Reumatol Clin “ COVID19 Situaci³n actual en La Comunidad de Madrid Informe de situaci³n del de Mayo Available httpswww comunidad madrid sites default files doc sanidad 200508_ cam_ covid19 pdf [Accessed May ] Chen N Zhou M Dong X et a0al Epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in Wuhan China a descriptive study Lancet “ Figueroa Parra G Aguirre Garcia GM Gamboa Alonso CM et a0al Are my patients with rheumatic diseases at higher risk of COVID19 Ann Rheum Dis “ Favalli EG Ingegnoli F De Lucia O et a0al COVID19 infection and rheumatoid arthritis Faraway so close Autoimmun Rev Zhou F Yu T Du R et a0al Clinical course and risk factors for mortality of adult inpatients with COVID19 in Wuhan China a retrospective cohort study Lancet “ Monti S Balduzzi S Delvino P et a0al Clinical course of COVID19 in a series of patients with chronic art
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netosis is a type of regulated cell death dependent on the formation of neutrophil extracellular traps net where netlike structures of decondensed chromatin andproteases are produced by polymorphonuclear pmn granulocytes these structuresimmobilise pathogens and restrict them with antimicrobial molecules thus preventing theirspread whilst nets possess a fundamental antimicrobial function within the innate immunesystem under physiological circumstances increasing evidence also indicates that netosisoccurs in the pathogenic process of other disease type including but not limited to atherosclerosis airway inflammation alzheimer™s and stroke here we reviewed the role ofnetosis in the development of an injury including injury to the brain lung heart kidneymusculoskeletal system gut and reproductive system whilst therapeutic agents in blockinginjuries induced by netosis in its primitive stages were also discussed this review providesnovel insights into the involvement of netosis in different an injuries and whilstpotential therapeutic measures targeting netosis remain a largely unexplored area thesewarrant further investigationkey words netosis neutrophil an injury cell death inflammation cell death is commonly segregated into necrosisand apoptosis apoptosis being programmed cell death anaesthetics pain medicine and intensive care department of surgeryand cancer faculty of medicine imperial college london chelsea andwestminster hospital fulham road london sw10 9nh uk department of anesthesiology shanghai fengxian district central hospital shanghai jiao tong university affiliated sixth people™s hospitalsouth campus fengxian district shanghai china to whom correspondence should be addressed at anaesthetics painmedicine and intensive care department of surgery and cancer faculty of medicine imperial college london chelsea and westminsterhospital fulham road london sw10 9nh uk emaildmaimperialacukfor instance during development and physiological cellular turnover whilst necrosis predominantly takesplace in an unregulated manner netosis like necrosis is a mode of cell death that involves the loss ofmembrane integrity during netosis decondensationof chromatin is thought to be initiated by peptidyl arginine deiminase pad4 its subsequent releasetogether with granule contents is vital in the innateimmune response to infection and inflammation recentstudies suggest that net formation is of central topathogenesis of an injury this review will summarise the current understanding of the molecular mechanisms of netosis and the therapeutic approaches underdevelopment targeting netinduced an injury the authors this is an open access publication 0cmolecular mechanism of netformationalthough netosis is closely associated with netformation not all net formation requires the process ofcell death to take place beforehand according to nomenclature committee on cell death the term ˜netosis™should only be used in the context of cell death and notjust based on the presence of net formation two main pathways of net formation have beendescribed and categorised according to their dependenceon the activity of nicotinamide adenine dinucleotide phosphate nadph oxidase pathway fig nadph oxidasedependent net formationthe nadph oxidasedependent molecular pathwayof net formation begins with activation of neutrophilsurface receptors by stimuli derived from pathogenic ornonpathogenic sources such as cholesterol or urate andends with cellular lysis these stimuli trigger calciumrelease from the endoplasmic reticulum er resulting inthe activation of protein kinase c pkc and the assemblyof the nadph oxidase complex generating reactive oxygen species ros following this neutrophil elastasene a protease stored in the cytoplasmic granules migrates to the nucleus in a myeloperoxidase mpodependent manner and cleaves histones to initiate chromatindecondensation this is promoted by the citrullinationof histone arginine residues by peptidylarginine deiminaseiv pad4 finally mixing of the chromatin andgranule proteins takes place as cellular membranes arebroken down interestingly there have been reports ofmitochondrial dna mtdna instead of nuclear dnabeing the source of the dna fibres in nets with observations of mtdna being released from granulocytes inresponse to disease states such as trauma and systemiclupus erythematosus sle [ ] moreover it seemsthat histone citrullination is not always required for netformation as observed by kenny and colleagues in theirstudy of neutrophils activated by the pkc agonist phorbol12myristate 13acetate pma this highlights the diversity of pathways for net formation following their induction degradation of nets can take place through severalpathways for example by dnases or endocytosed bymacrophages factors that influence net formation include phco2 and hco3ˆ’ levels which modulate neutrophil activation this explains why nets form more readily in thecahilog zhao wu alam eguchi weng and maperiphery of an inflammatory site where the ph is morealkaline this may influence treatment efficacy asnets can seal off the affected area an acidic environmenthas been hypothesised to reduce nadph oxidasedependent net formation by reducing neutrophil glycolysis nadph oxidasedependent net formation also requires neutrophils to be in the cell cycle necessitating theactivation of cyclindependent kinases cdk phosphorylating the retinoblastoma proteinnadph oxidaseindependent net formationthis mechanism of net formation is more relevant inthe context of infection as inducers of netosis here arecalcium ionophore a23187 and the potassium ionophorenigericin which are products of streptomyceschartreusensis and streptomyces hygroscopicus respectively how this pathway leads to net release ispoorly understoodnetosis and inflammationnets under physiological conditions are central topathogen clearance when there is excessive formation orsuboptimal nets are able to initiate further destructivesignalling through interaction with other tissue constituentsand the immune system moreover the antimicrobial histones and peptides within the net structure impose adirect cytotoxic effect on tissues to date there havebeen numerous accounts of netosis being present indiseases of major ans understanding of netosis inpathophysiology may offer unique opportunities for clinical managementnetosis in an injurythere is an expanding body of research describingnetosis in infectious and noninfectious an injurysummarised in fig although it is valid that in thesescenarios nuclear dna released during necrotic cell deathcan contribute to tissue injury and exacerbate the extent ofan damage here we focus on the contribution by aberrant net formation and the implication of understandingits underlying pathogenesis for therapeutic interventions 0crole of neutrophil netosis in an injuryfig type of cell death for neutrophil in an injury during solid an injury neutrophils could be prompted to undergo caspasedependent apoptosiswhich results in controlled dissolution of cell into apoptotic bodies containing cellular debris to prevent immune and inflammatory responses neutrophilextracellular traps nets form via two pathways the first is through lytic netosis a cell death pathway characterised by nuclear delobulation anddisintegration of the nuclear envelope which precedes loss of cellular polarisation chromatin decondensation and plasma membrane rupture the secondmechanism involves the nonlytic form of netosis which is not associated with cellular death but prompts expulsion of nuclear chromatin together withrelease of granule proteins through degranulation these components can assemble in the extracellular space into nets leaving behind enucleated cytoplaststhat continue to ingest microanisms in addition neutrophils could undergo unregulated necrosis that does not involve specific molecular pathwayswith uncontrolled release of cellular debris acting as dangerassociated molecular patterns damps to trigger proinflammatory responsebrainalzheimer™s disease alzheimer™s disease ad is acommon disorder of neurodegeneration characterised bygradual loss of cognitive functions in ad patients neutrophils have been observed to invade the brain parenchyma and release nets causing destruction of neural cellsand the bloodbrain barrier stroke it is well known that the adaptive immunesystem is altered after a stroke predisposing patients toinfections [“] interestingly netosis has also beendescribed as significantly impaired up until on day inthose with an ischaemic stroke though netosis inhaemorrhagic stroke patients has yet to be documented ithas been noted that the generation of ros a keyrequirement for chromatin decondensation is suppressedin these patients for up to days lungcystic fibrosis it has been well established that chronic infections in cystic fibrosis cf patients are due to thehighly viscous mucus production harbouring microbialgrowth although impaired clearance of mucus has beenprincipally named responsible there is increasing evidencethat the high viscosity is also due vast amounts of freedna found in sputum samples which was in concordance with the high concentration of neutrophil and 0ccahilog zhao wu alam eguchi weng and mafig involvement of netosis in an injury accumulating evidences now point to an important role of netosis in infectious and noninfectious solidan injury neutrophil invasion into brain parenchyma and release of neutrophil extracellular traps nets have been established in the pathophysiology ofalzheimer™s disease to cause destruction to neural cells and bloodbrain barrier abnormal netosis activity and reactive oxygen species ros response akey element to netosis initiation were observed in stroke patients the degree of neutrophil infiltration net formationcomponent eg cellfreenucleosomes and netosis have been found to correlate with the severity of a range of lung diseases including cystic fibrosis acute lung injury aliacute respiratory distress syndrome ards and lung infection netosis was also shown to be involved in allergic asthma chronic obstructive pulmonarydisease and pulmonary hypertension wherein degree of net formation correlates with disease severity during liver ischaemiareperfusion tolllikereceptordependent net release has been suggested to mediate liver inflammation and injury conversely deficiency in net release was reported indecompensated cirrhotic liver disease and could explain susceptibility to bacterial peritonitis infection in those patients net formation and netosis havefurther been implicated in atherosclerosis and myocardial infarction wherein net was found in thrombi and infarct lesion and correlate with disease severityin rheumatoid arthritis enhanced net release and netosis are observed in synovial tissue rheumatoid nodules and skin whilst proinflammatory cytokinesand autoantibodies further aggravate neutrophil infiltration and netosis neutrophils could also be potently activated by monosodiumurate msu crystals ingout joints and point to a potential role of netnetosis in gout pathogenesis moreover neutrophil activation and net deposition were also observed incolon mucosa of ulcerative colitis excessive neutrophil activation net formation and netosis could also be responsible different pregnancyrelateddisorders including preeclampsia wherein net deposition and netosis in the intervillous space may damage maternal endothelium and impair foetaloxygen exchangenets found in cf lungs the source was believed to befrom necrotic neutrophils but is now considered to besecondary to netosis additionally net productionwas found to be promoted by bacterial infection in cfairways and was defective in clearance of the bacteriapseudomonas aeruginosa nets are also named as a facilitative factor for biofilm formation there is evidencethat surfactant protein d spd responsible foropsonising pathogens and dying cells for clearance byalveolar macrophages is essential for net clearancethrough binding directly to the chromatin within the netsspd levels are decreased in cf patients and the level ofdecrease is proportional to the degree of inflammationthrough accumulation of nets 0crole of neutrophil netosis in an injurylung infection neutrophils migrated into the affectedsite and initiate the cascade of antimicrobial mechanismsincluding net generation to combat microanismsthis happens more readily in the lungs compared with inother tissues with neutrophils found to exist in higherconcentrations in pulmonary vasculature compared withsystemic blood vessels a prominent pathway leadingto net formation in infection is through the interaction oflipopolysaccharide lps with tolllike receptor tlr4found on neutrophils in patients with communityacquired pneumoniacap increased levels of cellfree nucleosomes in serumsamples used as surrogate markers of netosis werefound this was associated with prolonged hospitalisationand a greater 30day allcause mortality this findingsuggests nets could function as a novel marker of prognosis in capacute lung injury and acute respiratory distresssyndrome the degree of neutrophil influx into the lungsand net release during ali and ards positively correlates with disease progression and severity with neutrophil depletion conferring protection in a transfusionrelated ali animal model netosis seems to be akey component of ventilatorinduced lung injury vili as evidenced by detection of citrullinated histone3suggesting that this was a mode of cell death independentfrom apoptosis and necrosis the authors suggestedthat this may be due to increased levels of il1β andhmgb1 which have been both shown to be able to inducenetosisallergic asthma patients with neutrophilic asthmahave a greater severity of disease and reduced response tocorticosteroid treatment compared with the eosinophilictype the increased expression of neutrophilchemoattractant il8 in airway smooth muscle cells couldbe contributing to disease severity through inducingnetosischronic obstructive pulmonary disease netosis hasbeen documented as an integral part of chronic obstructivepulmonary disease copd pathophysiology unlike asthma where neutrophils are important in certain subtypesnetosis has been directly linked to disease severity incopd [ ] tlr4 expression one of the main potentiators for net formation is increased during copd exacerbations pulmonary hypertension nets are also able to potentiate dysregulated angiogenesis as seen in patients withchronic thromboembolic pulmonary hypertension and idiopathic pulmonary hypertension as plasma levels of dnane and mpo levels are significantly elevated moreovernets also seem to destabilise intercellular junctions andincrease endothelial cell motility through direct contactwith endothelial cells nets were found to induce theactivity of the proinflammatory transcription factor nfκbby approximately 3fold moreover nets increase thesurface expression of von willebrand factor and plateletadhesion thereby producing a prothrombotic state kidneyglomerulonephritides nets have been visualised upon immunostaining renal biopsies from patients with sleand antineutrophil cytoplasmic antibodiesassociated vasculitis aav and may be at least partially responsiblefor activating complement pathways resulting in diseaseexacerbations these autoimmune conditions alsoseem to affect the patient™s ability to degrade nets amplifying their deleterious inflammatory effects increscentic glomerulonephritis neutrophilmediated glomerular damage is worsened by addition of extracellularmpo which could have been released during netosis netosis could also contribute to the loss of immunetolerance through further externalisation of crucialautoantigens during cell death haemolytic uraemic syndrome hus plasma from affected patients exhibited a greater capacity to undergonetosis compared with healthy patients the ensuingdamage has been linked to the proinflammatory cytokinesil6 and il8 released from glomerular epithelial cellsupon stimulation by nets this potentiates microvasculature inflammation and thrombosis precipitating renal failure liverdecompensated cirrhotic liver disease a deficiency innet release has been demonstrated to play a role in theonset of endstage liver disease as neutrophils incirrhotic patients are found to have defective ros production which commonly triggers net release thismay also partially explain why these have a predispositionto recurrent bacterial infections and increased rates ofdecompensatory complications such as spontaneous bacterial peritonitis sbp this is corroborated by defectivenet release from ascitic fluid neutrophils in cirrhoticpatients compared with controls in vitro cirrhoticpatients with sbp were also found to have an increase in 0cpro and antiinflammatory cytokines in peripheral bloodand ascitic fluid ischaemiareperfusion injury ischaemiareperfusioninjury iri is an inherent consequence of liver transplantation hypovolaemia or trauma and results in the release ofdamageassociated molecular patterns damps which inturn cause net formation in a tlrdependent mannerexacerbating inflammation treatment with apeptidylargininedeiminase pad4 inhibitor ordnase has been shown to be significantly hepatoprotectivefollowing liver iri cardiovascular systematherosclerosis nets are a wellknown constituent ofatherosclerotic lesions mpo has been strongly associated with diminishing the cardioprotective effects ofhighdensity lipoprotein cholesterol hdlc through oxidation reactions and an increased enzymatic activity islinked to increased plaque rupture other proteinsfound in nets such as cathelinrelated antimicrobial peptide cramp have also been shown to contribute todisease progression moreover in vitro studies showthat hypercholesterolemia triggers net formation alargescale study in patients with suspected coronary arterydisease revealed that the markers of netosis such asextracellular dna are independently associated with disease severity coronary specimens from patients afteran acute myocardial infarction mi showed the presenceof nets in both fresh and lytic thrombi therefore suggesting netosis happens in the early stages of thrombusevolution furthermore net burden was shown tobe positively correlated with the infarct size in patientsundergoing primary percutaneous coronary interventionspostmi this is supported by increased levels of mpodna and ne in the lesion site therefore nets couldpotentially be considered as a novel biomarker in atherosclerosis diabetes mellitusinduced vasculopathy it has beenshown that neutrophils form peripheral blood of diabetesmellitus dm patients showed increased spontaneousnetosis interestingly metformin reduces the deleterious effects of netosis in a mechanism independentlyfrom glucose control one recent study showed that month treatment with metformin in predm patients reduced levels of components of nets whereas glycaemiccontrol with other medication such as insulin saw nodifference when compared with placebo controls thiscahilog zhao wu alam eguchi weng and mahas been attributed to a direct effect of metformin oninhibiting the activation of nadph oxidase musculoskeletal systemrheumatoid arthritis neutrophils from the peripheralblood and synovial fluid of patients with rheumatoid arthritisra exhibit increased netosis compared with healthy controls and patients with osteoarthritis the externalisation ofcitrullinated proteins during the process of netosis wasfound to initiate and perpetuate the aberrant immune responsein ra moreover the autoantibodies and inflammatory cytokines commonly seen in ra promote netosis resulting in avicious cycle of tissue destruction gout gout is an inflammatory disease that involvesthe deposition of monosodiumurate msu crystals injoints during acute gout there is increased movement ofneutrophils into the synovial fluid msu is a known neutrophil stimulus and it has been shown that acute gout isassociated with an increase in il1β levels a keyplayer in net formationgutulcerative colitis there is prominent neutrophil infiltration in the colon mucosa in ulcerative colitis uc and this correlates with disease severity in uc the inflammatory environment promotes neutrophil activation andil1β expression in contrast nets do not seem toplay a key role in crohn™s disease this may explain whymesalazine a known inhibitor of il1β production and thefirstline treatment for uc flareups is not therapeutic incrohn™s patients per se reproductive systempreeclampsia placentas from women diagnosed withpreeclampsia showed increased neutrophil infiltration andnetosis when compared with nonhypertensive pregnantcontrols [ ] and are probably involved in causingwidespread damage to the maternal endothelium placental and endothelial injury during pregnancy aberrantneutrophil activity during pregnancy is also associated withother severe complications including recurrent foetal loss one recent study indicated neutrophils in pregnant womenseem to have an increased propensity to undergo netosissecondary to an increase in granulocyte colonystimulating 0crole of neutrophil netosis in an injuryfactor during pregnancy progesterone has been shown toattenuate neutrophilmediated ros production whereas 17βestradiol induces intracellular ros generation in a dosedependent manner associated an injury associated with netosis fig examples of recent publications on potential therapeutic compounds targeting netosis are summarised in table netosis as a therapeutic targettargeting critical steps in net formation and degradation is critical for developing treatment strategies for netosistlr inhibitorsdexamethasone dex has been shown in vitro toreduce netosis in neutrophils that are stimulated withstaphylococcus aureus but not in those stimulated withpma the tlrs involved in s aureusinduced net formation seem to be tlr2 and tlr4 as agonists of thesereceptors rescued dex inhibition interestingly althoughfig therapeutic strategies targeting net formation stimulation of neutrophil receptors eg fc γ receptor tolllike receptor by microanisms orsterile signals leads to release of calcium ca2 from the endoplasmic reticulum er cellular ca2 overload results in activation of protein kinase c pkcassembly of the nicotinamide adenine dinucleotide phosphate nadph oxidase complex andor mitochondrial activation thus stimulating reactive oxygenspecies ros production oxidative stress promotes myeloperoxidase mpodependent migration of granular neutrophil elastase ne into the nucleus tocleave histones subsequent activation of peptidylarginine deiminase pad induces histone citrullination to cause chromatin decondensation the last stepinvolves nuclear membrane degradation and extrusion of a mixture of chromatin and granular proteins into extracellular space whereby extracellular dnaseeventually digests and removes neutrophil extracellular traps nets in this regard modulation of critical steps in net formation and degradation shown byblocking arrows might be beneficial for the treatment of inflammatory disorders figure modified and reproduced with permission fcγr fc γ receptortlr tolllike receptor 0cdex reduced net formation it did not significantly affectros production calcineurin inhibitorscalcineurin is a calciumdependent serinethreonineprotein phosphatase that is important for neutrophil activity many stages of netosis induction depend upon calcium mobilisation hence modulators of the calcineurinpathway are potential pharmacological inhibitors of netformation cyclosporine a csa an antagonist of thecalcineurin pathway has been shown to reduce the effectof key physiological activators of neutrophils this effecton netosis may in part explain csa™s efficacy in ra and steroidresistant uc patients pmainducednetosis seems to be calciumindependent as this wasnot inhibited by csa cahilog zhao wu alam eguchi weng and mapad inhibitorsusing a murine model of atherosclerosis knight andcolleagues have shown that pharmacological inhibition ofpad4 using weeks of daily clamidine injections reduced netinduced vascular damage with delayed plaqueprogression in the carotid arteries the same groupalso showed that pad inhibitors reduce disease activity inmurine models of sle by reducing endothelial dysfunction it is worth mentioning that the possibility of padinhibition as a therapeutic avenue to be pursued in netinduced an damage in glomerulonephritis has beenrecently challenged by the work of gordon and colleagueson murine models on sle with pad4 deletion theyshowed that this did not reduce endan damage asmeasured by proteinuria suggesting that mechanismsother than net formation are implicated in this complexautoimmune conditionros scavengersdnase therapythe mitochondria are a powerful source of ros ros scavengers such as nacetyl cysteine nac reducenet formation and severity of sle in patients troloxand tempol are two antioxidants which have also beenshown to prevent netosis of pmastimulated humanneutrophils in a dosedependent manner and have beenrecommended for treatment of autoimmune and inflammatory pathologies dnase therapy has been proposed to improve outcomes in cf patients through reducing mucous viscosityhowever it appears that recombinant dnase does notreduce the load of dnaprotein complexes seen innetosis one solution to this is to combine elastase withdnase in order to degrade histones and provide dnaseaccess to chromatin this combination has been shown toenhance solubilisation of sputum drug classstudymain findingstable potential therapeutic approaches targeting netosistlr inhibitorswan t et al calcineurin inhibitorsgupta ak et al dexamethasone reduced netosis in neutrophils stimulated with s aureusagonists of tlr2 and tlr4 rescued dexamethasone inhibitionros production was unaffected by dexamethasonecyclosporine a reduced the effect of key physiological stimuli that activate neutrophilssuch as il8 and suppressed netosisros scavengerspatel et al vorobjeva nv and pinegin bvnacetyl cysteine reduced net formation and severity of sle in patientsantioxidants trolox and tempol prevent netosis of in stimulated human neutrophils in apad inhibitorsknight js et al weeks of daily clamidine injections reduced netinduced vascular damage and area ofdosedependent mannerlesions in a murine model of atherosclerosispad inhibition dampens disease activity by reducing endothelial dysfunction in a murinemodel of slednase therapypapayannopoulos v staab delastase combined with dnase therapy enhances solubilisation of sputum in cystic fibrosistetrahydroisoquinolines martinez ne et al tetrahydroisoquinolines selectively target net overproduction at micromolarzychlinsky a patientsconcentrations possibly at multiple stages of net formation without compromisingnormal neutrophil function 0crole of neutrophil netosis in an injurytetrahydroisoquinolinesin contrary to the aforementioned mechanisms ofnetosis inhibitors tetrahydroisoquinolines thiqs area new class of net formation inhibitors that do not targetros formation or granular protein activity as functionalneutrophils are paramount to maintaining immune reactivity this difference offers an advantage to selectively targetnet overproduction without impairing normal functionthe exact molecular mechanisms of thiqs are yet to bedetermined however it is known that thiq inhibition ofnetosis take place at micromolar concentrations and possibly at different stages of net formation conclusionwhen regulated as part of normal physiology netsare antimicrobial and fundamental to the innate immunesystem dysregulated net formation contributes to thepathogenesis of a plethora of diseases this review hassummarised the role of netosis in pathologies of multiplebody systems as well as highlighted the stages of netosisthat has so far been investigated as emerging pharmacological targets these putative strategies seem to hold therapeutic potential and warrant further investigationauthors™ contributionsdm designed and reviewed the manuscript zc andhz wrote the first draft of the paper all authors readrevised and approved the final manuscriptcompliance with ethical standardscompeting interests the authors declare that they haveno competing interestsethics approval and consent to participate notapplicableconsent for publication not applicableopen access this is licensed under a creativecommons attribution international license whichpermits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicateif changes were made the images or other third partymaterial in this are included in the 's creativecommons licence unless indicated otherwise in a creditline to the material if material is not included in the's creative commons licence and your intended useis not permitted by statutory regulation or exceeds thepermitted use you will need to obtain permission directlyfrom the copyright holder to view a copy of this licencevisit httpcreativecommonslicensesby40references vandenabeele p l galluzzi t vanden berghe and g kroemer molecular mechanisms of necroptosis an ordered cellularexplosion nature reviews molecular cell biology “ httpsdoi101038nrm2970 lewis hd j liddle je coote sj atkinson md barker bdbax kl bicker rp bingham m campbell yh chen cwchung pd craggs rp davis d eberhard g joberty kelind k locke c maller k martinod c patten o polyakovace rise m rüdiger rj sheppard dj slade p thomas jthorpe g yao g drewes dd wagner pr thompson rkprinjha and dm wilson inhibition of pad4 activity issufficient to disrupt mouse and human net formation naturechemical biology “ httpsdoi101038nchembio1735 galluzzi lorenzo ilio vitale stuart a aaronson john m abramsdieter adam patrizia agostinis emad s alnemri et al molecular mechanisms of cell death recommendations of the nomenclature committee on cell death cell death and differentiation “ httpsdoi101038s414180170012 gupta s and mj kaplan the role of neutrophils andnetosis in autoimmune and renal diseases nature reviews nephrology “ httpsdoi101038nrneph201671 papayannopoulos v neutrophil extracellular traps in immunity and disease nature reviews immunology “httpsdoi101038nri2017105 kobayashi sd and fr deleo role of neutrophils ininnate immunity a systems biologylevel approach wiley interdisciplinary reviews systems biology and medicine “httpsdoi101002wsbm32 metzler kd c goosmann a lubojemska a zychlinsky andv papayannopoulos a myeloperoxidasecontaining complex regulates neutrophil elastase release and actin dynamics duringnetosis cell reports “ httpsdoi101016jcelrep201406044 tessarz p and t kouzarides histone core modificationsregulating nucleosome structure and dynamics nature reviewsmolecular cell biology “ httpsdoi101038nrm3890 yousefi s c mihalache e kozlowski i schmid and husimon viable neutrophils release mitochondrial dna toform neutrophil extracellular traps cell death and differentiation “ httpsdoi101038cdd200996 wang haiting ting li sheng chen gu yueying and shuang ye neutrophil extracellular trap mitochondrial dna and its 0cautoantibody in systemic lupus erythematosus and a proofofconcept trial of metformin arthritis rheumatology “ kenny ef a herzig r kruger a muth s mondal prthompson v brinkmann hv bernuth and a zychlinsky diverse stimuli engage different neutrophil extracellular trappathways elife httpsdoi107554elife24437 hakkim a bg furnrohr k amann b laube ua abed vbrinkmann m herrmann re voll and a zychlinsky impairment of neutrophil extracellular trap degradation is associatedwith lupus nephritis proceedings of the national academy of sciences of the united states of america “ httpsdoi101073pnas0909927107 farrera c and b fadeel macrophage clearance of neutrophil extracellular traps is a silent process journal of immunology “ httpsdoi104049jimmunol1300436 maueroder c a mahajan s paulus s gosswein j hahn dkienhofer mh biermann et al 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Treatment Strategy for Metastatic Spinal Tumors A Narrative ReviewSam Yeol Chang Sujung Mok Sung Cheol Park Hyoungmin Kim BongSoon ChangDepartment of Orthopedic Surgery Seoul National University Hospital Seoul Korea Metastatic spinal tumors are common and their rising incidence can be attributed to the expanding aging population and increased survival rates among cancer patients The decisionmaking process in the treatment of spinal metastasis requires a multidisciplinary approach that includes medical and radiation oncology surgery and rehabilitation Various decisionmaking systems have been proposed in the literature in order to estimate survival and suggest appropriate treatment options for patients experiencing spinal metastasis However recent advances in treatment modalities for spinal metastasis such as stereotactic radiosurgery and minimally invasive surgical techniques have reshaped clinical practices concerning patients with spinal metastasis making a demand for further improvements on current decisionmaking systems In this review recent improvements in treatment modalities and the evolution of decisionmaking systems for metastatic spinal tumors are discussedKeywords Spinal metastasis Decisionmaking system Radiosurgery Minimally invasive surgical procedures Separation surgeryIntroductionThe spine has been identified as the most common site for malignant metastasis in the musculoskeletal system and vice versa spinal metastasis is considered the most common malignant lesion in the spine [] The incidence of metastatic spinal tumors has seen an increasing trend due to the growing aging population worldwide and continued improvements in survival rates among cancer patients [] Symptomatic spinal metastasis is often the first clinical manifestation for “ of cancer patients [] whereas up to of cancer patients may experience spinal metastasis at some point during the course of their disease [] The objectives of surgical treatment in patients with metastatic spine tumors are mostly palliative Spine surgeons make an effort to maintain or improve the patient™s quality of life during the remainder of their survival by reducing pain and preserving ambulatory function via surgical treatment []Because clinical manifestations and treatment responses vary widely among cancer patients a multidisciplinary decisionmaking process that integrates medical and radiation oncology together with surgery along with assistance from pathology and diagnostic radiology deemed is essential when deciding to conduct surgical treatment for spinal metastasis [] In the literature various decisionmaking systems have been developed and introduced to date in an effort to aid in this decisionmaking process Received Jul Revised Jul Accepted Jul Corresponding author BongSoon ChangDepartment of Orthopedic Surgery Seoul National University Hospital Daehakro Jongnogu Seoul KoreaTel Fax Email bschangsnuackrCopyright ’¸ by Korean Society of Spine SurgeryThis is an Access distributed under the terms of the Creative Commons Attribution NonCommercial License httpcreativecommonslicensesbync40which permits unrestricted noncommercial use distribution and reproduction in any medium provided the original work is properly citedAsian Spine Journal ¢ pISSN eISSN ¢ wwwasianspinejournalAsian Spine JournalASJAsian Spine Journal 0cSam Yeol Chang et alAsian Spine J [] However recent advancements in the treatment of metastatic spine tumors have injected more complexity into this decisionmaking process and demanded the evolution of the decisionmaking system itself these recent advances include the development of stereotactic spine radiosurgery SRS the introduction of minimally invasive surgical techniques and the evolution of various target therapies for individual primary cancers [] In this review we discussed the development and evolution of various decisionmaking systems in spinal metastasis treatment Current concepts and recent trends in radiotherapy and surgery for spinal metastasis are also includedDecisionMaking Systems for Managing Metastatic Spinal TumorsPrognostic factors for metastatic spinal tumorsWhen attempting to choose an appropriate treatment for a patient with spinal metastasis establishing an accurate estimation of the individual™s life expectancy is the most crucial To do this one must first identify prognostic factors associated with the survival of patients with spinal metastasis As such many authors have conducted studies to try and identify prognostic factors associated with survival among spinal metastasis patients and have developed various decisionmaking systems in order to estimate survival based on these prognostic factors []In a recently published metaanalysis Luksanapruksa [] have identified independent prognostic factors associated with the survival of patients with spinal metastasis Among these factors nine factors can be classified as relating to the preoperative performance or neurological status of a patient”for example the Karnofsky Performance Score or Eastern Cooperative Oncology Group grade [] Meanwhile four factors involve the presence or the number of metastases spine bone or visceral and two factors were found to be related to primary tumors in the Tomita classification scheme [] Finally male sex and the time interval from cancer diagnosis to the start of radiotherapy are the two remaining prognostic factors independently associated with survival in spinal metastasis patients Among these primary tumor histology the presence and number of metastases and performance status are proposed as the three most important prognostic factors associated with survival in spinal metastasis patients not only in this study but also in most previous investigations []In other studies the patient™s age and comorbidities assessed using the American Society of Anesthesiologists physical status [] and Charlson Comorbidity Index [] have also been identified to be prognostic factors in spinal metastasis [] Other authors have identified laboratory abnormalities such as leukocytosis and low hemoglobulin and albumin levels as prognostic factors and included these into their decisionmaking systems [] In addition previous systemic treatment or chemotherapy has also been suggested as an independent prognostic factor by multiple authors [] Further not only preoperative chemotherapy but also the presence of available systemic treatments in the postoperative period has been hypothetically regarded as a potential prognostic factor in the literature [] In a recently published study by Chang [] the authors verified the presence of the remaining systemic treatment options to be independently associated with improved postoperative performance status and survivalClassificationbased decisionmaking systemsBased on these prognostic factors numerous decisionmaking systems or œscoring systems have been developed and introduced to estimate the life expectancy among spinal metastasis patients [] In these œclassificationbased decisionmaking systems scores for each prognostic factor identified by multivariate logistic or proportional hazards regression analyses are integrated in order to obtain a total prognostic score that reflects the estimated survival of the patient Surgeons can adopt these prognostic scores to identify patients with an estimated survival profile that is sufficient to warrant surgical treatment Although the prognostic factors included in each system vary primary tumor histology and visceral metastases are included in most systems Table In the New England Spinal Metastasis Score NESMS was introduced by Ghori [] The NESMS was developed using multicenter data and retrospectively validated in the following investigations [] The NESMS consists of modified Bauer score components and score serum albumin level and ambulatory status of the patient More recently the NESMS was validated prospectively in the Prospective Observational Study of Spinal Metastasis Treatment trial which aimed to verify the 0cTreatment of Spinal Metastasis Table Prognostic factors in decisionmaking systemsReferencesBauer [] modifiedTomita [] To kuhashi [] revisedKa tagiri [] revisedGhori [] Paulino [] Primary tumorPerformance statusNo of vertebral metastasesBone metastasisVisceral metastasisPrevious systemic treatmentOther factorsOOOOOOOOOOOOOOOOOOOOOOOOOOBr ain metastasis WBC Hb platelet albumin bilirubin Creactive protein lactate dehydrogenaseSerum albuminAge WBC Hb brain metastasisWBC white blood cell Hb hemoglobin NESMS as a reliable predictive tool in spinal metastasis patients []There have been efforts to develop novel decisionmaking systems using evolving methodologies In the Skeletal Oncology Research Group S compared multiple prognostic survival algorithms including classic nomogram and boosting algorithms using the same retrospective dataset obtained from patients [] In their study the nomogram was found intuitive and demonstrated a comparable level of performance Then in the S used machinelearning algorithms to develop a novel prognostic model for metastatic spinal disease [] which was externally validated in subsequent studies []Although these various œclassificationbased decisionmaking systems are helpful and widely used for predicting the survival of spinal metastasis patients recent studies have reported that the degree of accuracy of these classic systems eg Tomita Tokuhashi decreases over time especially in cancers with poor prognoses such as lung cancer [] This pitfall of œclassificationbased decisionmaking systems reportedly stems from the inability of these systems to reflect survival improvement due to recent evolutions in systemic treatment for primary cancers [] Another existing limitation is that these systems cannot directly guide the selection of specific treatments appropriate for patients with spinal metastasisPrinciplebased decisionmaking systemsAs an alternative to these œclassificationbased decisionmaking systems that are incapable of reflecting recent advances in oncology and guiding specific treatments several authors have proposed œprinciplebased decisionmaking systems These œprinciplebased systems do not œscore the patient and estimate survival but instead provide advice regarding which treatment is more appropriate in individual cases based on the integration of rapidly evolving treatment modalities including target therapies radiosurgery and minimally invasive surgical techniquesThe neurologic oncologic mechanical and systemic NOMS decision framework was first introduced in [] The NOMS decision framework consists of neurologic N oncologic O mechanical M and systemic S considerations integrating novel multimodal therapies including SRS and minimally invasive surgical techniques [] As a neurological N assessment approach the grading system developed by Bilsky and Smith [] was used while surgical decompression is recommended for highgrade spinal cord compressions During oncological O assessment the responsiveness of spinal metastasis to currently available treatments especially the level of tumor sensitivity to radiotherapy is evaluated Mechanically M instability of the spinal column as determined by the Spinal Instability Neoplastic Score SINS indicates the need for surgical stabilization regardless of the neurologic or oncologic status [] Finally systemic S assessment focuses on the patient™s ability in tolerating the suggested treatment Meanwhile if the general condition performance status and medical comorbidities of a patient do not allow surgery to be performed radiotherapy is instead recommendedA modification of the NOMS decision framework has also been presented in the literature Paton introAsian Spine Journal 0cSam Yeol Chang et alAsian Spine J duced the œLMNOP system as an improvement to the NOMS approach [] With this development these authors added two additional key considerations to the NOMS as follows the location and levels of metastasis L and the patient™s response to previous therapy P The P in œLMNOP stands for not only the response to prior therapy but also includes patient fitness and prognosis which was considered previously as part of the systemic S assessment in the NOMS decision framework The authors emphasized that the response of primary cancer to previous treatments including chemotherapy and radiotherapy is considered a significant factor when determining the appropriate treatment for spinal metastasis patients For instance it is anticipated that a patient diagnosed with symptomatic spinal metastasis at the initial presentation of primary cancer synchronous metastasis who would have multiple potential treatment options is more likely to experience a better prognosis than a patient diagnosed with spinal metastasis despite previous treatment metachronous metastasis Differences in the survival rates between the patients with synchronous and metachronous spinal metastasis have been confirmed in a previous research [] Therefore a more aggressive approach including surgical treatment can be considered for patients with a synchronous spinal metastatic lesion In summary œprinciplebased decisionmaking systems relative to œclassificationbased systems are able to better incorporate evolving treatment modalities and guide the selection of appropriate treatments for patients in a timely fashion Table Current trends and future directions in the development of decisionmaking systemsAdvances in cancer biology and treatment modalities are necessitating the evolution of decisionmaking systems for spinal metastasis Possible future directions to take to improve decisionmaking systems include the following the use of multicenter or multinational databases the integration of histologyspecific data the application of computational methodologies such as machinelearning algorithms and the combination of classificationbased and principlebased systems Some recent studies are already covering these trendsThe size of the study sample under assessment determines the performance and accuracy of prognostic models Although spinal metastasis is found to be relatively common data from a larger sample population beyond that of just a single institution is usually required to develop a powerful enough prognostic model For this reason recently introduced prognostic models or decisionmaking systems are generated from multicenter databases [] Future prognostic models should also have the freedom to rely on even larger databases such as multinational tumor registriesBiologic therapy including molecular target therapy and immunotherapy is believed to be an emerging gamechanger in modern cancer treatment Genetic subtype analysis of the primary cancer histology which guides selection among t hese therapies has become more essential [] In a revised prognostic system proposed by Katagiri Table The NOMS decision frameworkNeurologic NOncologic OMechanical MSystemic SDecisionLowgrade ESCCno myelopathyHighgrade ESCC±myelopathyRadiosensitiveRadiosensitiveRadioresistantRadioresistantRadiosensitiveRadiosensitiveRadioresistantRadioresistantRadioresistantRadioresistantStableUnstableStableUnstableStableUnstableStableStableUnstableUnstablecEBRTStabilization followed by cEBRTSRSStabilization followed by SRScEBRTStabilization followed by cEBRTAble to tolerate surgeryD ecompressionstabilization followed by SRSUnable to tolerate surgerycEBRTAble to tolerate surgeryDecompressionstabilization followed by SRSUnable to tolerate surgeryStabilization followed by cEBRTNOMS neurologic oncologic mechanical and systemic ESCC epidural spinal cord compression cEBRT conventional external beam radiation SRS stereotactic radiosurgery 0c [] the authors considered the availability of molecular target therapy when classifying the primary tumor For example lung cancer treated with targeted drugs was designated as an example of a moderategrowth tumor while lung cancer without targeted drugs is regarded as an example of a rapidgrowth tumor [] This application of genetic profiles to decisionmaking systems is likely to grow more specific and tailored corresponding to the evolution of molecular genetics in the futureAs previously described machinelearning algorithms have been applied in the development of prognostic models Classically prognostic models for spinal metastasis have been developed using logistic or proportional hazards regression analyses As part of its research efforts the S was able to develop prognostic models using machinelearning algorithms such as gradient boosting decision trees random forests and neural networks [] and these algorithms were externally validated elsewhere [] Like in other fields of medicine evolving computational methodologies including machinelearning algorithms should be assessed extensively in terms of their potential in the management of spinal metastasisFinally the combination of classificationbased and principlebased decisionmaking systems should be considered Classificationbased systems or prognostic models seek to estimate the patient™s remaining survival Based on this survival estimation principlebased systems then may suggest the most appropriate treatment option Until now surgeons and physicians have been employing these two separate systems in the same decisionmaking process A novel decisionmaking system could integrate these two systems together and provide survival estimations and appropriate treatment options simultaneouslyRadiotherapy for Metastatic Spinal TumorsTreatment of Spinal Metastasis single session of SRS achieved durable longterm control of spinal metastasis regardless of histology and tumor size Of note the only significant factor associated with tumor control was the radiation dose These results suggest that SRS can be effective even in cases of metastasis previously considered to be radioresistantSRS can also be a definitive treatment for the management of solitary metastasis without spinal cord compression [] Excellent local control rates of “ have allowed SRS to replace curative surgeries with high morbidities such as total en bloc spondylectomy for addressing these solitary metastases [] In patients with highgrade spinal cord compression SRS can be applied after separation surgery which will be discussed further in the following section Overall the effectiveness of SRS has been changing the role and extent of surgical treatment and in turn shifting the focus of the treatment of spinal metastasisVertebral compression fracture following stereotactic spine radiosurgeryA pitfall of SRS is an increase in vertebral compression fracture VCF following radiotherapy Risk estimates for VCF after SRS are reported to be up to as compared with just in relation to conventional radiotherapy [] The occurrence of VCF is dosedependent and caution is required if the radiation dosage exceeds Gy per fraction in highrisk patients [] Highrisk patients of older ages with lytic lesions andor with spinal malalignment can reportedly benefit from undergoing preventive stabilization surgery before SRS When determining the necessity of stabilization surgery before SRS SINS can be used to identify potentially unstable lesions [] SINS will be further covered in the following sectionStereotactic spine radiosurgery is triggering a paradigm shiftTiming of radiotherapy after surgeryEvidence in the literature supports that radiosurgery is a safe and effective modality for local tumor control with low associated complication rates in patients with spinal metastasis [] Technical improvements including intensitymodulated and imageguided radiation delivery and processing software have allowed SRS to be a gamechanger in the treatment of spinal metastasis [] A recent study by Yamada [] reported that a highdose Adequate timing of radiotherapy following surgery whose determination is related to the risk of wound complications continues to be debated among spine surgeons and radiation oncologists It is also controversial whether the interval can be shortened in patients receiving SRS Lee [] sent questionnaires to radiation oncologists and spine surgeons to gather opinions on the optimal timing of surgery and radiotherapy in spinal metastasis Based on the procured comments the auAsian Spine Journal 0cSam Yeol Chang et alAsian Spine J thors recommended that the interval be at minimum weeks regardless of radiation modalities Interestingly as compared with radiation oncologists surgeons tended to favor a shorter interval of time between surgery and radiotherapy when SRS is performed although there was no statistically significant difference in this regard []A recently published systemic review also advocated for weeks with a minimum of days between surgery and radiotherapy [] When the rates of wound complications were compared between SRS and conventional radiotherapy many studies reported reduced wound complications in SRS patients [] However due to limited highlevel evidence no definite conclusion was made regarding whether the interval could be reduced in patients undergoing SRSSurgery for Metastatic Spinal TumorsSurgical indicationsThe objective of surgical treatment in spinal metastasis was to provide pain relief support neurological improvement and in turn enhance the quality of life during the remaining survival period Clinical benefits of direct surgical decompression in patients with metastatic spinal cord compression MSCC have been well described in the literature [] The most important prerequisite for surgical treatment in spinal metastasis has been identified as the sufficient enough estimated survival time to make surgery a reasonable approach Researchers have largely recommended that a minimum of to months of remaining survival should exist when considering whether to perform surgery [] At this point a number of prognostic scoring systems previously described are being used to estimate a patient™s survival The patient should also have a good enough general condition or performance status to in order to endure surgery If these conditions are satisfied then surgery can be performed in patients with symptomatic MSCC or mechanical instabilityIn spinal metastasis the instability is assessed by the SINS [] Table The SINS is an independent and unique tool that integrates clinical and radiological components to help surgeons decide whether to conduct surgery for stabilization A score of to points suggests impending instability while that of points or more indicates existing spinal instability which requires stabilization As previously mentioned the SINS has also been incorporated into principlebased decisionmaking systems such as the NOMS and LMNOP systems [] Recent studies have reported the reliability and accuracy of the SINS system in predicting spinal adverse events including VCF especially in those patients who received radiotherapy [] although some components may still require revision []Separation surgery and minimally invasive surgeryWhen considering the surgical techniques used for spinal metastasis the literature shows a trend toward the adoption of less invasive techniques which is thought to have Table Spinal Instability Neoplastic Score systemComponentLocationJunctional occiput“C2 C7“T2 T11“L1 L5“S1Mobile spine C3“C6 L2“L4Semi rigid T3“T10Rigid S2“S5PainaYesOccasional pain but not mechanicalPainfree lesionBone lesionLyticMixed lyticblasticBlasticRadiographic spinal alignmentSubluxationtranslation presentDe novo deformity kyphosisscoliosisNormal alignmentVertebral body collapse collapse collapseNo collapse with body involvedNone of the abovePosterolateral involvement of spinal elementsbBilateralUnilateralNone of the aboveScoreaPain improvement with recumbency andor pain with movementloading of spine bFacet pedicle or costovertebral joint fracture or replacement with tumor 0cTreatment of Spinal Metastasis primarily resulted from recent advances in radiotherapy as previously described [] With the use of advanced radiation techniques surgeons can minimize surgical morbidities by avoiding extensive debulking surgery [] Fig During the separation surgery circumferential spinal cord decompression is performed only to the extent required to facilitate safe radiosurgery In a study by Laufer [] separation surgery followed by postoperative SRS resulted in a low local progression rate Other studies have also reported that this hybrid surgery“radiosurgery approach is a safe and effective treatment option for MSCC []BCADFig A 68yearold male with spinal metastasis of renal cell carcinoma at T9 The patient also had lung metastasis A Preoperative MRI shows spinal cord compression and involvement of posterior elements and left rib head at the T9 level B Following a separation surgery without spinal instrumentation MRI at postoperative 2weeks shows a decompressed spinal canal but residual metastatic tumor around the left 9th rib head C In the postoperative 1year MRI the patient showed a nearcomplete response following a single session stereotactic radiosurgery 18Gy1 fraction which was performed weeks after the separation surgery MRI magnetic resonance imaging D Planning images for the postoperative stereotatic radiosurgery following a separation surgery Asian Spine Journal 0cSam Yeol Chang et alAsian Spine J œMinimalaccess surgery is also used in treating spinal metastasis while reducing surgical morbidities In the anterior approach retractor systems and thoracoscopic assistance have been described [] Minimally invasive posterior approaches for decompression and corpectomy have also been introduced and reviewed in the literature [] Other minimally invasive techniques for spine surgery such as the intraoperative stereotactic navigation system and percutaneous pedicle screw instrumentation are being incorporated into spinal metastasis surgery as well []Studies comparing minimally invasive surgery and surgery showed that minimally invasive surgery provided equivalent or superior outcomes with reduced surgical morbidity and complications in spinal metastasis patients [] However because the quality of evidence is deemed low in the current literature no definite conclusion regarding the superiority of minimally invasive surgery over surgery can be derived and no strong recommendations have been made at this point []Role of curative surgery en bloc resectionMost surgeries for spinal metastasis are found palliative and the role of en bloc resection in spinal metastasis is decreasing even further due to improvements in radiotherapy Generally curative surgical resection of spinal metastasis has been considered in the context of a single metastasis of a slowgrowing tumor such as in renal cell thyroid and breast cancers Fig Favorable outcomes in this regard have been reported in the literature [] However some authors recently reported that curative ABECDFig A 63yearold male with spinal metastasis of thyroid carcinoma at T8 A Preoperative MRI shows pathologic fracture and spinal cord compression at the T8 level B Postoperative Xray at month shows removal T8 vertebra and reconstruction with an expandable cage following total en bloc spondylectomy C MRI at postoperative 3years shows a widely decompressed spinal canal with no tumor recurrence D Postoperative Xray at postoperative 5years shows wellmaintained instrumentation E Bone scan at postoperative 5years shows no evidence of bone metastasis MRI magnetic resonance imaging 0csurgical resection en bloc spondylectomy did not impact the oncologic outcomes of spinal metastasis patients [] Therefore a more careful and thorough decisionmaking process is required before performing curative resection surgery for spinal metastasis especially when the extended role of radiosurgery is consideredPostoperative complications and preventive measuresThe overall complication rate following surgery for spinal metastasis ranges from to in the literature [] Because surgery for spinal metastasis is performed to improve the quality of life of a patient surgeons should try to minimize all possible surgical and medical complications by implementing multidisciplinary interventions Among diverse complications those that require additional attention when found in spinal metastasis patients eg wound infection instrumentation failure and intraoperative bleeding are briefly discussed hereThe incidence of surgical site infection SSI is determined to be higher in spinal metastasis surgery reaching up to as compared with during other spine surgeries [] SSI has also been identified as the most common cause for reoperation in of reoperations following surgery for spinal metastasis [] Poor nutrition and exposure to adjuvant therapies chemotherapy and radiotherapy put spinal metastasis patients at risk for SSI [] Surgeons should provide adequate nutritional support perioperatively and secure a sufficient time interval between radiation and surgery as previously discussed to minimize SSIs Additional risk factors such as smoking obesity and medical comorbidities should also be considered []The second cause for reoperation in spinal metastasis surgery is the failure of instrumentation [] Risk factors associated with instrumentation failure include the number of operated levels prior chest wall resection and history of radiotherapy [] The necessity of additional fusion procedures while performing surgery for spinal metastasis is debated but highlevel evidence remains to be lacking at this time [] In future studies we suggest that instances of early and late failure be distinguished when assessing instrumentation failure because the mechanisms of failure between the two types seem to differ ie insufficient stability of the construct in the context of early failure versus the progression of deformity or lack of fusion in the context of late failureTreatment of Spinal Metastasis Intraoperative bleeding during spinal metastasis surgeries can be massive and can further lead to serious complications such as cardiovascular or cerebral events [] For the spinal metastasis of hypervascular tumors such as renal cell hepatocellular and thyroid cancers preoperative embolization is recommended Previous studies have verified the effectiveness of preoperative embolization in reducing intraoperative bleeding in spinal metastasis surgeries [] Surgery should be performed within hours following embolization to avoid the diminished effect of preoperative embolization []ConclusionsThe determination of appropriate treatment for a patient with spinal metastasis is a challenging task that requires multidisciplinary considerations Recent advances in radiotherapy and surgery for spinal metastasis have brought about improvements in the management of these patients Evolving decisionmaking systems are also crucial contributors to stateoftheart care of patients with metastatic spinal tumorsConflict of InterestNo potential conflict of interest relevant to this was reportedReferences White AP Kwon BK Lindskog DM Friedlaender GE Grauer JN Metastatic disease of the spine J Am Acad Orthop Surg Laufer I Sciubba DM Madera M Surgical management of metastatic spinal tumors Cancer Control Schiff D O™Neill BP Suman VJ Spinal epidural metastasis as the initial manifestation of malignancy clinical features and diagnostic approach Neurology Klimo P Jr Schmidt MH Surgical management of spinal metastases Oncologist Nathan SS Healey JH Mellano D Survival in patients operated on for pathologic fracture implications for endoflife orthopedic care J Clin Oncol Curtin M Piggott RP Murphy EP Spinal metaAsian Spine Journal 0cSam Yeol Chang et alAsian Spine J static disease a review of the role of the multidisciplinary team Orthop Surg Ahmed AK Goodwin CR Heravi A Predicting survival for metastatic spine disease a comparison of nine scoring systems Spine J Barzilai O Fisher CG Bilsky MH State of the art treatment of spinal metastatic disease Neurosurgery Tomita K Kawahara N Kobayashi T Yoshida A Murakami H Akamaru T Surgical strategy for spinal metastases Spine Phila Pa Tokuhashi Y Matsuzaki H Oda H Oshima M Ryu J A revised scoring system for preoperative evaluation of metastatic spine tumor prognosis Spine Phila Pa Bauer HC Wedin R Survival after surgery for spinal and extremity metastases prognostication in patients Acta Orthop Scand Van der Linden YM Dijkstra SP Vonk EJ Marijnen CA Leer JW Dutch Bone Metastasis Study Group Prediction of survival in patients with metastases in the spinal column results based
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environmental exposure to arsenite as3 has a strong association with the development ofhuman urothelial cancer uc and is the 5th most common cancer in men and the 12th mostcommon cancer in women muscle invasive urothelial cancer miuc are grouped into basalor luminal molecular subtypes based on their gene expression profile the basal subtype ismore aggressive and can be associated with squamous differentiation characterized byhigh expression of keratins krt1 and and epidermal growth factor receptoregfr within the tumors the luminal subtype is less aggressive and is predominatelycharacterized by elevated gene expression of peroxisome proliferatoractivated receptamma pparÎ and forkhead box protein a1 foxa1 we have previously shown thatas3transformed urothelial cells ast exhibit a basal subtype of uc expressing genesassociated with squamous differentiation we hypothesized that the molecular subtype ofthe ast cells could be altered by inducing the expression of pparÎ andor inhibiting theproliferation of the cells nontransformed and ast cells were treated with troglitazonetg pparg agonist μm pd153035 pd an egfr inhibitor μm or a combination oftg and pd for days the results obtained demonstrate that treatment of the ast cellswith tg upregulated the expression of pparÎ and foxa1 whereas treatment with pddecreased the expression of some of the basal keratins however a combined treatment oftg and pd resulted in a consistent decrease of several proteins associated with the basalsubtype of bladder cancers krt1 krt14 krt16 p63 and tfap2a our data suggeststhat activation of pparÎ while inhibiting cell proliferation facilitates the regulation of genesinvolved in maintaining the luminal subtype of uc in vivo animal studies are needed toaddress the efficacy of using pparÎ agonists andor proliferation inhibitors to reduce tumradestage of miuca1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation mehus aa bergum n knutson pshrestha s zhou xd garrett sh activation of pparÎ and inhibition of cellproliferation reduces key proteins associated withthe basal subtype of bladder cancer in as3transformed urotsa cells one e0237976 101371 pone0237976editor karl x chai university of central floridaunited statesreceived may accepted july published august copyright mehus this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the paper and its supporting informationfilesfunding seema somji und school of medicineand health sciences pilot grant das allundergraduate research and core facilities ndinbre idea program p20 gm103442 from thenational institute of general medical sciences nih one 101371 pone0237976 august one 0ccompeting interests the authors have declaredthat no competing interests existactivation of luminal pathway in basal bladder cancerintroductionbladder cancer bc is the ninth most common cancer diagnosed worldwide and in theamerican cancer society estimated that about new cases of bc would be identified inthe us and about deaths would occur from bladder cancer among bcs urothelialcell carcinomas uc are the most common being the second most diagnosed cancer of thegenitourinary tract behind prostate cancer [ ] it is the 5th most common cancer in menand the 12th most common cancer in women urothelial cancers are classified as muscle invasive miuc or nonmuscleinvasivenmiuc nonmuscleinvasive urothelial cancers have a lower tendency to progress whereasmiucs have a high rate of metastasis and a year survival rate of approximately bothmiuc and nmiuc have been subtyped into various groups with the basal and luminal subtype being the most prominent the luminal subtype of human uc includes the majority ofthe early stage noninvasive ucs and a significant number of miucs this subtype isenriched for papillary histology is less aggressive and has a more favorable patient outcome [ ] basal classified tumors have a poorer overall survival compared to luminal tumors they often exhibit squamous differentiation are aggressive and found exclusively inmiuc that metastasize and spread to distal ans about of miucs arise independent of the papillary pathway have poor outcomes and an overall year survival rate of environmental exposure to arsenite as3 has a strong association with the developmentof human uc the increased risk of uc correlates to the same endemic areas of the worldwhere populations have been identified with arsenicinduced skin cancer [“] we havedeveloped a cell culture model of arsenicinduced urothelial cancer by exposing the immortalized nontumorigenic urothelial cell line urotsa to arsenite these transformed cell lines produce tumors in athymic mice that express genes for keratin krt and asignature pattern highly similar to the basal subtype of human miuc [ ] the tumorshave a histology similar to urothelialtransitional cell carcinomas with focal areas of squamousdifferentiation [ ] which is associated with poor prognosis [ ]the molecular mechanism driving a tumor towards a basalsquamous subtype is currentlyunknown in a recent study yamashita show that transcription factor activatingprotein alpha tfap2a is expressed at high levels in basalsquamous bladder cancerenriched in areas of squamous differentiation and is associated with increase lymph nodemetastasis and distant recurrence of the disease the study also shows that increased expression of tfap2a can facilitate the expression of other transcription factors such as tumor protein p63 tp63p63 also known as p63 which is known to be associated with the basalsubtype of uc palmbos demonstrated that p63 binds to the transcriptional regulatory regions of the gene ataxiatelangiectasia group d complementing gene atdc alsoknown as trim29 and krt14 thus increasing their expression the study further showedthat both krt14 and trim29 promote the invasion of the basal subtype of uc in vitro and invivo the luminal subtype of uc is associated with the expression of the transcriptional factorsforkhead box protein a1 foxa1 gata binding protein gata3 and peroxisome proliferatoractivated receptor gamma pparÎ [ ] the activation of pparÎ with an agonistcan represses the expression of tfap2a and inhibit squamous differentiation in vitro the exact role of pparÎ signaling in carcinogenesis is somewhat unclear however theexpression of pparÎ in bladder cancers is a favorable prognostic marker both in vivoand in vitro studies indicate that pparÎ ligands such as troglitizone can promote differentiation inhibit cellular proliferation induce autophagy and enhance apoptosis in bladder cancer one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancer[“] likewise suppressing cellular proliferation with epidermal growth factor receptoregfr inhibitors has been used preclinically to reduce basallike muscle invasive bladdertumor growth although the egfr inhibitors did not have the same efficacy in nonbasalliketumors the goal of this study was to determine if the activation of pparÎ and inhibition of cellproliferation in the urotsa parent and the as3transformed urotsa isolates would repressthe expression of genes involved in maintaining the basalsquamous type of bladder cancerand induce genes that were associated with the luminaldifferentiated state of bladder cancermaterials and methodsanimalsathymic nude ncrnunu mice purchased from envigo were used in these studies themice were housed four to a cage at ˚c under a 12hour lightdark cycle food and water wasavailable ad libitum mouse heterotransplants of the urotsa transformed cell lines as3 andas4 and the rt4 cell line were produced by subcutaneous injection at a dose of x cellsin the dorsal thoracic midline of athymic nude ncrnunu mice this study adhered to allrecommendation dictated in the guide for the care and use of laboratory animals of thenih tumor sizes were assessed weekly and the animals were sacrificed when the size of thetumor was approximately “ cm or when dictated by clinical conditions euthanizationwas done by co2 asphyxiation and conformed to the american veterinary medical association guideline on euthanasia death was confirmed by ascertaining cardiac and respiratoryarrest following which the ans and tumor were harvested care of taken to ensure thatthere was no distress to the animals during the procedure the protocol was approved by theuniversity of north dakota animal care committee iacuc16122ccell culturethe urotsa parent cells and two of the as3transformed isolates as3 and as4 were cultured in in dulbeco™s modified eagle™s medium dmem supplemented with vv fetalbovine serum as described previously the cells were subcultured at a ratio usingtrypsinedta and the cultures were fed fresh growth medium every three days the urotsaparent cell line has been authenticated using short tandem repeat str analysis the as3transformed isolates used in the current study have been previously characterized for its ability to form colonies in soft agar form spheroids when grown in ultralow attachment flasksand form tumors when injected subcutaneously in immunecompromised mice [ “]the as3 can also form tumors upon intraperitoneal injection for drug treatmentsurotsa parent and the as3transformed isolates as3 and as4 were grown to confluence inserum containing medium following which the cells were incubated with a serum freemedium consisting of a mixture of dmem and hams™s f12 growth medium for h thecells were then exposed to either dimethyl sulfoxide dmso the drug vehicle troglitizonetg μm a pparÎ agonist pd153035 pd μm an epidermal growth factor receptoregfr inhibitor or a combination of tg and pd tgpd for and hours the concentrations of the drugs were chosen based on preliminary studiesvisualization of dapistained cellstoxic effects of tg and pd on the urotsa cells was determined by visualization of 406diamidino2phenylindole dapistained nuclei as described previously by this laboratory atthe indicated time points the cell monolayers were washed twice with phosphate buffered one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancersaline pbs following which the cells were fixed for min with ethanol and rehydratedwith 1ml pbs the rehydrated cells were stained with 10μl dapi 10μgml in distilled waterrna isolation and realtime pcr analysistotal rna was isolated using tri reagent molecular research center as described previously the expression of various genes was assessed with realtime reverse transcriptionpolymerase chain reaction rtpcr using primers that were purchased commercially frombiorad laboratories the genes along with the catalog number of the primers are listed insupplemental material s1 table total rna μg was transcribed to cdna using theiscript cdna synthesis kit biorad laboratories the amplification of the cdna was performed using the itaq universal sybr green supermix biorad laboratories with μlcdna and μm primers in a total volume of μl in a cfx96 realtime detection systembiorad laboratories amplification was monitored by sybr green fluorescence cyclingparameters consisted of a s hotstart followed by cycles of denaturation at ˚c for sannealing at ˚c for s and extension at ˚c for s which gave optimal amplificationefficiency the resulting levels were normalized to βactin expression assessed by the sameassay the threshold cycles cts for βactin and the resulting delta cts for the target genes arereported in s2 tablewestern blot analysiswestern blot analysis was performed as described previously the cell pellets were dissolved in 1x radioimmunoassay precipitation assay ripa lysis buffer supplemented withpmsf protease inhibitor cocktail and sodium orthovandate santa cruz biotechnology thecell suspension was sonicated and the lysate was centrifuged to remove cellular debris proteinlysates were quantified using the pierce bca protein assay kit thermoscientific pierceprior to loading samples were reduced and denatured the protein extracts were separated ontgx anykd sdspolyacrylamide gels purchased from biorad laboratories and transferred toa μm hybondp polyvinylidene difluoride membrane using semidry transfer the blotswere blocked in trisbuffered saline tbs containing tween20 tbst and wvbovine serum albumin bsa for min at room temperature the membranes were probedovernight at ˚c with the primary antibody diluted in wv bovine serum albumin allantibodies were purchased from commercial suppliers and were validated against known positive and negative expressing cell lines by western analysis prior to use in experimental protocols the source of the antibody along with their catalog numbers are reported in s3 tableafter washing times for minutes each wash in tbst the membranes were incubated withthe antimouse or antirabbit secondary antibody for min at room temperaturethe blots were visualized using the clarity„¢ western ecl blotting substrate bioradlaboratoriesimmunohistochemical stainingserial sections were cut at “ μm and immersed in preheated target retrieval solutiondako in a steamer for min the sections were allowed to cool to room temperature andimmersed into tbst for min the primary antibodies used in this study along with theirdilutions and catalogue numbers are listed in s3 table the primary antibodies were localizedusing dako peroxidase conjugated envision plus for rabbit or mouse primary antibodies atroom temperature for min liquid diaminobenzidine dako was used for visualizationcounter staining was performed for “ sec at room temperature using readytousehematoxylin dako slides were rinsed in distilled water dehydrated in graded ethanol one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancercleared in xylene and coverslipped two pathologists judged the presence and degree ofimmunereactivity in the specimensstatistical analysisall experiments were performed in triplicate and the results are expressed as the mean ± semstatistical analyses were performed using graphpad prism1 software version using oneway anova with tukey™s or dunnett™s posthoc testing for gene expression statistics wererun on the delta cycle threshold δct values that were generated from normalization to βactin levels unless otherwise stated the level of significance was p005resultseffect of troglitizone and pd153035 on the viability and morphology ofurotsa parent and ast cellsthe urotsa parent and ast cells as3 and as4 were treated with either dmso controltroglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr as seenin fig 1a“1d there was no change in the morphology of the urotsa parent cells with varioustreatments there was a change in the morphology of the as3 and as4 cells when treatedwith tg fig 1f and 1j and tgpd fig 1h and 1l the cells looked more differentiatedformed mounds and resembled the intermediate cells of the bladder there was a decrease inthe number of urotsa parent cells treated with pd and tgpd when compared to the cellstreated with tg alone or with dmso fig 1m there was also a decrease in the number ofas4 cells when treated with tg and tgpd when compared to the dmso treated cells fig1o there was no significant decrease in the number of as3 cells in any of the treatmentgroups fig 1n an examination demonstrated the lack of dead cells in the treated groups andthe decrease in cell number compared to the dmso group could be due to lack of proliferationandor increased differentiation of cellseffect of pparÎ activation and egfr inhibition on the expression ofluminal genesthe transcription factors pparÎ foxa1 and gata3 play a role in the establishment of theluminal subtype of bladder cancer [ ] studies done by varley have shown thatagonistdependent activation of pparÎ with simultaneous inhibition of egfr phosphorylation in normal human urothelial cells increases the effectiveness of the pparÎ agonist in thepresent study we investigated the effect of the pparÎ agonist tg and an egfr inhibitor pdon the expression of luminal transcriptional factors in the urotsa parent cells and the astcells expression levels for the target genes in this study are reported for a hr hr and hr timecourse for the parent as3 and as4 cells s1 s2 and s3 figs respectively for simplicity the hr gene and protein levels are reported in the main body of the manuscripttreatment with tg increased the expression of pparÎ in the urotsa parent fig 2a i iv andv cell line a similar effect was seen in as3 fig 2b i iv and v and as4 fig 2c i iv and vcell lines treatment with pd did not induce the expression of pparÎ in the urotsa parentfig 2a iv and v or as3 fig 2b iv and v cells but there was a small increase in pparÎ protein in the as4 cells fig 2c iv and v treatment with both tg and pd tgpd increasedthe expression levels of pparÎ mrna in the urotsa parent cells but there was no increase inthe protein levels there was no increase in mrna expression in the as4 cells but there was aslight increase in the protein levels fig 2c i iv and v one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig morphology and viability of urotsa parent and ast cells the urotsa parent ad and ast cells as3 eh and as4 il were treated witheither dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr the measurements were performed in triplicatesand the values reported are mean percentage of control ± sem ordinary oneway anova was performed followed by tukey™s posthoc test bars withdiffering letters indicate significant differences p 101371 pone0237976g001foxa1 gene and protein expression in the urotsa parent cells treated with tg wasreduced fig 2a ii iv and vi however the expression was increased in the as3 and as4cells fig 2b ii iv and vi and 2c ii iv and vi at the protein level treatment with pd decreasedfoxa1 protein in the parent cells but the levels were elevated in the as3 cells tgpdreduced the expression of foxa1 in the urotsa parent cells but it increased the expression offoxa1in the as4 cells at the protein leveltreatment of the urotsa parent cells with tg pd or tgpd did not increase the mrnalevels of gata3 but there was an increase in the protein levels after treatment with tg andtgpd when compared to the dmso treated group fig 2a iii iv and vii in as3 and as4cells there was an additive reduction of gata3 protein by using the combined tgpd treatment fig 2b iv and vii and 2c iv and vii one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancer one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig expression of luminal markers in urotsa parent and ast cells the urotsa parent aivii as3 bivii and as4 civii weretreated with either dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr real time rtpcranalysis was performed to verify gene expression a b ciiii western blot analysis was used to measure protein levels a b civ and theintegrated optical density iod of each protein band was calculated a b cvvii gene expression was normalized to βactin and gene andprotein are plotted as foldchange relative to the dmso control amplification was below detectable levels for pparÎ in dmso as3 so adelta cycle threshold δct value of was assigned which is higher than the highest delta ct detected for pparÎ in that cell linetriplicate measurements of gene and protein data were performed and are reported as mean ± sem ordinary oneway anova wasperformed followed by tukey™s posthoc test bars with differing letters indicate significant differences p 101371 pone0237976g002effect of tg and pd on the phosphorylation and expression levels of egfrthe effect of tg and the egfr inhibitor pd was determined on the expression and phosphorylation of egfr in the urotsa parent and ast cells only the combination of tgpdreduced the expression of egfr mrna in the urotsa parent cells however all three treatments decreased the protein levels fig 3a i ii and iii there was no basal phosphorylation ofegfr in the urotsa parent cells and none of the treatments had any effect on the phosphorylation levels fig 3a ii the expression of egfr in the ast cells varied with a decrease inas3 cells and an increase in as4 cells fig 3b i ii and iv and fig 3c i ii and iv respectivelyboth the transformed cell lines had basal phosphorylation of the egfr pegfr and treatment with pd decreased the pegfr levels in as3 fig 3b ii and iii and as4 fig 3c ii andiii which indicates that the pd treatment was effectiveeffect of the pparÎ agonist and inhibition egfr phosphorylation on theexpression of keratinsstudies performed by varley have shown that inhibition of the egfr with simultaneous activation of pparÎ signaling switches normal human urothelial cells from a squamous metaplastic phenotype to a transitional differentiated state with the repression ofkrt14 and the upregulation of krt13 and krt20 therefore we wanted to determineif the urotsa parent and the ast cells would revert more from a basal phenotype to amore transitionalintermediate phenotype when treated with tg and pd the mrnaexpression data is shown in fig and the protein expression data is shown in fig for theurotsa parent cells there was a decrease in expression of krt1 krt5 and krt14 with alltreatments fig 4a i ii and vi and fig 5a i ii and iv the protein for krt1 was undetectedin the urotsa parent cells the expression of krt6 and krt16 increased with tg butdecreased with pd and tgpd fig 4a iii iv v and vii and fig 5a iii and v the krt6antibody does not distinguish between the krt6a krt6b and krt6c isoforms and recognizes protein made by these three genes thus it is not known which isoform is beingexpressed at the protein level there was a decrease in expression of krt13 in the urotsaparent cells fig 4a viii and fig 5a i and vi in the as3 cells the expression levels of thebasal krts with various treatments was similar to the urotsa parent cells fig 4b ivii andfig 5b ivi with the exception of krt1 protein which was expressed in the as3 cells andits expression decreased with tg and tgpd treatment in the as4 cells the expression ofthe krts was similar to as3 with the exception of krt16 fig 4c iviii treatment withtg decreased the expression of krt16 the protein levels for the all the krts was similarto the mrna level with the exception of krt5 krt13 and krt16 krt5 showed anincrease in expression with pd and tgpd treatment and krt16 which showed anincrease in expression with pd fig 5c ivii in the as3 and as4 cells krt13 geneexpression was reduced however the protein levels were elevated from all three treatmentsfig 4b viii fig 4c viii and fig 5b i vii fig 5c i vii one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig expression and phosphorylation of epidermal growth factor receptor the urotsa parent aiiii as3 biiv and as4 ciiv weretreated with either dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr real time rtpcranalysis was performed to verify gene expression a b ci western blot analysis was used to measure protein levels a b cii and the integratedoptical density iod of each protein band was calculated aiii b and ciii and iv phosphorylatedegfr was not detected in the urotsa parentcell line gene expression was normalized to βactin and gene and protein are plotted as foldchange relative to the dmso control triplicatemeasurements of gene and protein data were performed and are reported as mean ± sem ordinary oneway anova was performed followed bytukey™s posthoc test bars with differing letters indicate significant differences p 101371 pone0237976g003effect of pparÎ activation and egfr inhibition on expression oftranscriptional factors p63 and tfap2a and the oncogene trim29associated with squamous differentiationrecently tfap2a has been implicated in the development of squamous differentiation inbasal cancers and activation of pparÎ is shown to represses the expression of tfap2a we therefore investigated the effects of pparÎ activation and egfr inhibition on the expression of tfap2a in urotsa parent and ast cells our results demonstrate that tg reducedtfap2a protein within the parent and as3 cells while the mrna and protein was elevatedin the as4 cells from tg exposure treatment with pd as well as tgpd decreased the one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancer one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig gene expression of keratins the urotsa parent aiviii as3 biviii and as4 civiii were treated witheither dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr real timertpcr analysis was performed to verify gene expression a b civiii gene expression was normalized to βactin andare plotted as foldchange relative to the dmso control triplicate measurements of gene levels were performed and arereported as mean ± sem ordinary oneway anova was performed followed by tukey™s posthoc test bars withdiffering letters indicate significant differences p 101371 pone0237976g004expression of tfap2a in the urotsa parent fig 6a i iv and v as3 fig 6b i iv and v andas4 cells fig 6c i iv and vtranscription factor p63 is another protein associated with human bladder cancersenriched in basalsquamous markers [ ] there was a decrease in the expression of p63 inthe urotsa parent cells with all treatments fig 6a ii iv and vi in the as3 cells the expression of p63 was low and treatment with tg increased its expression however treatment withpd and tgpd decreased its expression fig 6b ii iv and vi the expression of p63 in as4cells increased at the mrna level with tg and tgpd treatments however the protein levelswere decreased when compared to the dmso control fig 6c ii iv and vithe expression of trim29 a gene associated with the basal gene expression program was also determined in the urotsa parent and the as3transformed cells for the urotsaparent and as3 cells there was a decrease in the expression of trim29 in cells treated withpd and tgpd fig 6a and 6b iii iv and vii for as4 pd and tgpd treatments increasedtrim29 protein fig 6c iv and viiexpression of trim29 tfap2a and p63 within tumors formed fromurotsa as3 as4 and rt4 cellsurotsa as3 and as4 are considered to be of the basal molecular subtype while rt4 cellsare considered to be of the luminal molecular subtype of bladder cancer cells therefore wewanted to confirm the in vivo expression of trim29 tfap2a and p63 within the nondifferentiated basalsquamous areas of tumors originating from urotsa as3 and as4 cells incomparison to the expression in tumors originating from the luminal rt4 cells all three ofthese proteins were enriched within the nondifferentiated areas of tumors developed from theurotsa as3 and as4 cells fig 7a 7b 7d 7e 7g and 7h signs a lower expression wasobserved in the welldifferentiated areas of the urotsa as3 and as4 tumors fig 7a 7b7d 7e 7g and 7h� asterisks there was low to no staining in the rt4 cells for trim29tfap2a and p63 fig 7c 7f and 7idiscussionthe classification of uc into various subtypes has implications in the overall patient management of the disease with the basal subtype having a worse outcome when compared to theluminal subtype the molecular mechanisms involved in the development of these subtypesare not yet established however the role of some transcriptional factors is established withpparÎ playing an important role in the activation of the luminal specific genes [ ] andtfap2a playing a role in the development of the basal subtype of uc in addition studieswith normal human urothelial cells show that activation of pparÎ with an agonist along withinhibition of cellular proliferation via the egfr pathway can switch cells from a squamousmetaplastic phenotype to a more transitional differentiated phenotype combination therapiesusing egfr inhibitors and pparÎ agonists show promising results against some urothelialtumors in vivo as well as against other cancers based on these studies we sought todetermine if the urotsa parent and the as3transformed urotsa cell lines that are one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancer one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig protein expression of keratins the urotsa parent aivi as3 bivii and as4 civii were treated with either dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr western blot analysis was used to measure protein levels ai bi ci and the integratedoptical density iod of each protein band was calculated aiivi b and ciivii protein levels are plotted as foldchange relative to the dmso controltriplicate measurements of protein data were performed and are reported as mean ± sem ordinary oneway anova was performed followed by tukey™sposthoc test bars with differing letters indicate significant differences p 101371 pone0237976g005molecularly characterized as a basal subtype of bc could convert to a luminal transitionalcell type when treated with a pparÎ agonist andor an egfr inhibitor as this could affect theoverall outcome of the diseasethe urotsa parent cells when grown in serum expresses many genes that are associatedwith the basal subtype in this study treatment of these cells with the pparÎ agonist tginduced the expression of pparÎ as well as gata3 but not foxa1 in mortal human urothelial cells treatment with the pparÎ agonist shows an increase in the expression of the transcription factor foxa1 and this is contrary to what is seen in our study these differencescould be due to the cell type since we are using immortalized cells the as3transformedurotsa cells showed an induction in the expression of pparÎ and foxa1 when treated withtg however the expression of gata3 in these transformed lines was not consistent in bccell lines studies by others have shown that have shown that gata3 and foxa1 cooperatewith pparÎ activation to drive the differentiation of a basal bladder cancer subtype to a moredifferentiated luminal subtype other studies have also linked the expression of foxa1 tothe development of the luminal subtype of urothelial cancer [“] in our studies treatmentwith tg did result in the differentiation of the as3transformed cells based upon morphological changes and induced the expression of pparÎ as well as foxa1 both of which are factorsknown to drive luminal differentiation of bladder cancer cell linessignaling via the pparÎ pathway is essential for the growth arrest and terminal differentiation of adipocytes and other normal epithelial [“] and cancer cells [“] whereassignaling via the egfr receptor plays a role in cell proliferation the keratins play an essentialrole in the differentiation of epithelial cells and different keratin profiles are expressed at different stages of differentiation the normal bladder has three different stages of differentiationand these stages are marked by the expression of krt14 krt5 and krt20 krt14 isexpressed in a subset of basal cells that are thought to play a role in regeneration as well astumorigenesis whereas other basal cells and intermediate cells in the normal bladderexpress krt5 the expression of krt20 is restricted to the most differentiated cells theumbrella cells these differentiation stages of the bladder are shared by bladder cancersand malignant transformation can occur in any of the different cell types of the bladder theexpression of krt14 is seen in the least of the differentiated tumors and its expression correlates to poor prognosis krt14 whereas the expression of krt20 is restricted to differentiated bladder cancers and its expression is associated with good prognosis a study doneby varley showed that normal human urothelial cells in culture express krt14 andlack the expression of krt13 and krt20 activation of pparÎ in these cells induced theexpression of krt13 and decreased the expression of krt14 this effect was significantlyenhanced when
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" according to the who most chronic diseases including cancer can be prevented by identifyingtheir risk factors such as unhealthy diet smoking and physical inactivity this research examined the effectiveness ofa theorybased educational intervention on colorectal cancerrelated preventive nutritional behaviors among asample of anizational staffmethods in this interventional study employees of shahid beheshti university of medical sciences wererandomly divided into two groups intervention and control with cluster sampling the data gathering tool was aresearchermade questionnaire containing two parts of 10dimensional information and health belief modelconstructs the educational intervention was conducted for month and in four sessions in the form of classroomlecture pamphlet educational text messages via mobile phones and educational pamphlets through the officeautomation system two groups were evaluated in two stages pretest and posttest data were analyzed usingspss18 software analysis of covariance ancova and independent ttest intergroup comparisonsresults two groups were evaluated for variables such as age sex education level and family history of colorectalcancer and there was no significant difference between the two groups p after the months sinceintervention except for the mean score of perceived barriers which was not significant after intervention the meanscores of knowledge perceived susceptibility perceived severity perceived benefits perceived selfefficacybehavioral intention and preventive behaviors were significantly increased after the intervention in the interventiongroup compared to the control group p implementation of educational intervention based on health belief model was effective for thepersonnel and can enhance the preventative nutritional behaviors related to colorectal cancerkeywords educational intervention health belief model nutritional behavior colorectal cancer correspondence mohtashamghaffarisbmuacir1environmental and occupational hazards control research center schoolof public health and safety shahid beheshti university of medical sciencestehran iranfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0crakhshanderou bmc medical education page of nearly million new cases of colorectal cancer arediagnosed every year worldwide with nearly half of theaffected patients losing their lives due to the disease approximately of men in and of women in are diagnosed with crc during their life time the incidence of colorectal cancer in iran ranges from to per annually with a death rate of about per hundred thousand and it accounts for approximately of all gastrointestinal cancerrelated deaths according to the latest cancer record in iran colonand rectum cancer ranked third in female cancers andfifth in male cancers the global incidence of crc is predicted to increase by to more than million newcases leading to million cancer deaths by therisk of colon cancer increases with age and is higher inmen than in women various factors are involved inthe development of various types of cancerincludingcolorectal cancer which can be attributed to geneticenvironmental and dietary factors among the riskfactors of colorectal cancer nutritionalfactors areconsidered to be the most important and preventableones so that to of cases can be prevented byproper nutrition [ ] colorectal cancer is also morecommon in iran than in other asian countries [ ]therefore the need to educate people about the nutritionalbehaviors associated with colorectal cancer is becomingmore and more evident theories and models identifyfactors that influence health and behavior “ which meansthat they can be used to develop programs the most effective training programs are based on the theorydrivenapproaches which are rooted in behaviorchanging modelsalso selecting appropriate model or theory is the first stepin the process of planning a training program [ ] asone of the most widely applied theories of health behaviorthe health belief model hbm posits that six constructspredict health behavior perceived susceptibility perceivedseverity perceived benefits perceived barriers perceivedselfefficacy and cues to action fig the hbmposits that when an individual perceives a serious threatalong with a way to reduce the threat they will be morelikely to take action to reduce the threat the hbmhas been applied to predict a wide variety of healthrelatedbehaviors such as being screened for the early detection ofasymptomatic diseases the model has been applied tounderstand patients™ responses to symptoms of disease lifestyle behaviors and behaviors related to chronicillnesses which may require longterm behaviormaintenance in addition to initial behavior change the research hypotheses are an intervention based onthe hbm can significantly promote colorectal cancer preventive behaviors the score for each and every constructof the hbm eg perceived awareness and susceptibilityperceived severity perceived benefitsbarriers and perceivedselfefficacy is increased significantly after the interventionin the experimental group as compared to the controlmethodsstudy design and samplingthis interventional study was conducted at shahidbeheshti university of medical sciences tehran iranfrom october to june fig health belief model™s components and links 0crakhshanderou bmc medical education page of in thisstudy using the sample size formula ¾ z¾2δ2d2 in which δ2 α n ¼ °zˆd and with an attrition rate of finally women subjects in the experimental and in thecontrol group were considered the random samplingmethod clustering and simple random sampling wasused in this study in order to choose from four facultiesfaculties of shahid beheshti university of medicalsciences four faculties were randomly selected and fromthese four faculties two faculties were assigned as intervention group and were considered as control grouprandom sampling method was used to select samplesfrom each clusterinclusion exclusion criteriabeing under years of age having satisfaction to participate in the study and not having serious diseases including gastrointestinal diseases were the inclusion criteriaalso not willing to continue with the study not completing the questionnaire in full and not attending in morethan two educational sessions were the exclusion criteriameasuresthe researchermade questionnaire was used for datacollection in this study three sources of existed toolsliterature review and expert view were used for itemgeneration this instrument consisted of two main partsas followpart one demographic questions about age gendereducational level and economic statuspart two constructs of the health belief model whichincludes knowledge perceived susceptibility perceivedseverity perceived benefits perceived barriersperceived selfefficacy behavioral intention andbehavior table validity and reliabilityface and content validities were applied for validationphase reliability was confirmed based on methods oftestretest and internal consistency cronbach™s alphafor face validity a survey was done on “ employeesabout the difficulty in understanding the words andphrases the probability of misunderstanding the phrasesand lack of clarity in the meaning of the words somemodifications were made to the tool™s questions todetermine the content validity of the questionnaire twogastroenterologistsfive health education and healthpromotion specialists and one related expert were askedto complete the questionnaire the initial questionnairehad questions theconstructs of knowledgeperceived susceptibility perceived severity perceivedbenefits perceived barriers perceived selfefficacyintention and behavior had and questions respectively internal consistency was used todetermine the reliability of hbm structures the cronbach™s alpha coefficient was for all structures andwas statistically acceptable the retest was used to ensure the reliability of the awareness variable in this way employees completed the questionnaire twice and theicc was obtained also construct validity wasperformed by exploratory analysis method the kmovalue was and bartlett™s research showed thetable description of study instrumentconstruct knowledge refers to a theoretical or practical understanding of asubject perceived susceptibility refers to subjective assessment of risk ofdeveloping a health problem perceived severity perceived severity refers to the subjectiveassessment of severity of a health problem and its potentialconsequences perceived benefits healthrelated behaviors are also influenced bythe perceived benefits of taking an actionno of items format items truefalsedon™t know items 5point likert scalestrongly disagree to stronglyagree items5point likert scalestrongly disagree to stronglyagree items5point likert scalestrongly disagree to stronglyagreescoring range˜correct™ response ˜don™t know™response ˜incorrect™ response “strongly disagree disagree noidea agree strongly agree “strongly disagree disagree noidea agree strongly agree “strongly disagree disagree noidea agree strongly agree “ perceived barriers healthrelated behaviors are also a function ofperceived barriers to taking action perceived selfefficacy refers to an individual™s perception of his orher competence to successfully perform a behavior behavioral intention refers to a person™s perceived probability orœsubjective probability that he or she will engage in a given behavior items5 point likert scalestrongly disagree strongly agree items5 point likert scalestrongly disagree strongly agree items5point likert scalestrongly disagree to stronglyagreestrongly disagree disagree noidea agree strongly agree “strongly disagree disagree noidea agree strongly agree “strongly disagree disagree noidea agree strongly agree “ behavior refers preventative behaviors associated with colorectalcancer items5point likert scalealways to neveralways often sometimes rarely never 0crakhshanderou bmc medical education page of significant correlations among the items χ2 df p therefore the data were suitable forconducting factor analysisinterventionboth intervention and control groups were pretestedusing the questionnaire the analysis of educational needsdetermined the educational methods educational package and the number of educational sessions was obtainedby the pretestreadabilitycomprehensibility and not complexity of educational contents for participants was obtained by pretesting materialssuch as pamphlets messages etc in a sample of employees who were not included in main researchresults assurance abouteducational intervention based on educational textmassagesover the course of days ten text messages were sentto the employees in the intervention group at am mostof which had been prepared according to the educationalobjectives ofthe constructs of knowledge perceivedsusceptibility perceived benefits perceived barriers andperceived selfefficacycounseling there waseducational pamphletstwo pamphlets were given to the employees during twoseparate sessions along with simultaneous provision ofindividuala possibility ofquestioning and answering any ambiguity regarding thecontent of pamphlets the first pamphlet containedsections on the signs and symptoms of colorectal cancerand the risk factors of this cancer and the secondpamphlet contained sections on methods of preventingthis cancereducational packages in the office automation systemeducational packages were uploaded on the staff automation system for days and the employees were askedto study it during the working hoursthe intervention was conducted month and followup months after the intervention the educationalcontents were taken from the trusted sources of theministry of health complemented by what the staffneeded to know about promoting nutritional behaviorsrelated to the prevention of colorectal cancer the education varied in form across the model constructs forperceived susceptibility the facts and figures of the incident rate of colorectal cancer were presented in theclass and for perceived severityimages of colorectalcancer problems were used also for perceived barrierseducational materials were used to somehow incite theindividuals to analyze the cost of optimal behavioragainst the costs of risks time etc involved in unhealthybehavior the educational content used for perceivedbenefits intended to raise awareness on the usefulness ofhealth promoting behaviors to reduce the risk of illnessor to understand the benefits of healthy behaviors infig the research process is presented in generalethical considerationsat first a permission was obtained from the universityto conduct the study and attend the healthcare centerthe samples were assured about the confidentiality oftheir specifications and information they were also toldthat their information will only be used for the purposeof this study and the data collection the participantswere allowed to enter and leave the study at any timesuitable conditions were provided for a proper understanding of questions and responses for the subjectsafter the end of the intervention period the controlgroup was also trained using the slides that were used totrain the intervention group an informed consent wasobtained from the participants the study on whichthese data analyses are based was approved by theethical board committee of shahid beheshti universityof medical sciencesdata analysisdata were analyzed by spss software kolmogorov smirnovtest was used to check the normality of the data to assessthe effectiveness of intervention on variables of knowledgeperceived susceptibility perceived severity perceived benefits perceived barriers perceived selfefficacy behavioralintention and behavior in the intervention and controlgroups two groups were evaluated in two stages pretestand posttest data were analyzed using spss18 softwareanalysis of covariance ancova and independent ttestintergroup comparisonsthe confidence level of and the significance level of were consideredin this studyresultsthe findings of this study showed no drop out until theend of study the questionnaire was completed in bothgroups in a complete and precise manner homogenizationwas done in the two groups by controlling variables such asage sex level of education and related family history theresults showed no significant relationship within thesevariables p table effectiveness of the educationalintervention in improving knowledge perceived susceptibility perceivedseverity perceived benefits perceived selfefficacybehavioral intention and behavior once age gender andlevel of education factors were adjusted was checkedthrough ancova the results revealed that the intervention was successful in improving constructs of thehealth belief model significantly in participants table the mean score ofintention and behavior in the 0crakhshanderou bmc medical education page of fig schematic diagram of designed interventions for colorectal cancer preventionexperimental and control groups before and after theintervention is presented in fig discussionthe purpose of this study was to investigate the effectsof educationalinterventions on the promotion ofcolorectal cancer prevention nutritional behaviors thekmo and bartlett™s test p results confirmed the suitability of the model for conducting factoranalysis the kmo is in the range “ if the value ofthe inedex is near to one the data are suitable for factoranalysis kaiser at least kmo to determinestable demographic and variables in intervention and control groups before the interventionvariablegroupintervention group n n control group n n agegenderlevel of education““femalemalediplomaassociate degreeundergraduate degreeand higherhistory of specialdiet compliancefamily history of cancerchisquareyesnoyesnop “value 0crakhshanderou bmc medical education page of table comparison of intervention and control groups in terms of health belief model constructs before and after the interventionp valueconstructsgroupsbefore interventionmean ± sd ± after interventionmean ± sd ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± meandifference ± ˆ’ ± ± ± ± ˆ’ ± ± ˆ’ ± ˆ’ ± ± ± ˆ’ ± ± ˆ’ ± ± ˆ’ ± knowledgeinterventioncontrolperceived susceptibilityinterventionperceived severityperceived benefitsperceived barriersperceived self efficacybehavioral intentionbehavioranalysis of covariance ancovacontrolinterventioncontrolinterventioncontrolinterventioncontrolinterventioncontrolinterventioncontrolinterventioncontrol also bartlett test was used to confirm adequacy ofthe samples in the present study the mean score of behavioralconstruct increased after the intervention in the intervention group and there was significant differencebetween the two groups after the intervention in thisregard the results of this study are consistent with thefindings of abood hart roozitalabi alidoosti and davoodi studies behavioral intention is the thought of doing abehavior and is considered as the immediate determinant of that behavior the mean score in this construct aswell increased in the intervention group after the intervention and there was significant difference between thetwo groups after the intervention in the study of braun and gimeno the results were similar tothe results of present study selfefficacy is a keyprerequisite for behavior change there was significantdifference between mean score of perceived selfefficacyconstruct in the two groups after the intervention in thisfig mean scores of intention and behavior in the experimental and control groups before and after the intervention 0crakhshanderou bmc medical education page of regard the results of the study by braun alidoosti and hart are consistent with thisfinding perceived selfefficacy is considered as a strongmotivational source and in fact is an indicator of theability of individuals to anize themselves in pursuit ofcertain goals studies show that individuals with ahigh level of perceived selfefficacy have a greatercommitmentto engage in activities at a time ofchallenges and difficulties and spent more time andeffort on such activities such individuals are morelikely to contribute to maintaining healthy behaviors andretrieve them even after failure and they have strongerintention and motivation this not only improves thetarget adjustment but also ensures achievement andsustainability in pursuit of the goals another important factor is knowledge that can be pointed to itsrole in healthy behaviors this study showed a significant difference in the two group in terms of the meanscore of knowledge after the educationalinterventionthese results are consistent with the findings of roozitalab ho and gimeno studiesalso there was no significant difference in the controlgroup before and afterthe intervention althoughincreasing knowledge is an important step in changingattitudes and behaviors it is not a major contributor tocrc prevention achieving the intention to behave isinfluenced by individual and environmental factors so inaddition to enhancing individual aspects overcomingthe structural and environmental barriers of the healthsystem regarding the use of cancer prevention nutritional behaviors is also vital in the present study themean score of perceived susceptibility and perceived severity constructs showed a significant difference betweenthe intervention and control group after the educationalintervention studies by kolutek wang cengiz and donadiki reportedthe role of beliefs regarding public health threats perceived susceptibility and perceived severity in the healthpromotion behaviors becker believed that one™sintention to selfcare is influenced by his or her perception of vulnerability and the severity of disease outcomes therefore the need for interventions to increasethe perception of society about the irreparable complications of diseases caused by unhealthy behaviors malnutrition habits seems necessary in this study there was asignificant difference between the two groups in terms ofthe constructs of perceived benefits after the educationalintervention this result is consistent with the findings ofgrace alidoosti and abood studies also in the present study the mean score of perceived barrier construct decreased after the interventionthis was a good result but it was not statistically significant in the present study the mean score of perceivedbarrier construct decreased after the intervention which isnot consistent with the results of studies by moatari grace and gimeno the study ofrajabi identified some of the most important causes of barriers to nutrition in preventionof cancer such as the difficulty of preventativemeasuresinappropriate economic status and fear ofcancer information therefore strategies that overcome the individual and environmental barriers thataffect nutritional behaviors should be addressed byplanners and policymakerslimitationsthe limitations of this study which could have had a relative effect on its findings include the short duration ofintervention the sample size the inability to follow thelong term effect of the intervention and the selfreportingof the subjects in responding to questions however theuse of this method in such studies is inevitable and maylead to a bias of the œresearcherdesired report in thisstudy anonymous questionnaire was used to minimizethis biasthe findings of this study confirmed the effectiveness ofhealth belief modelbased education in improvement ofcolorectal cancerrelated preventive behaviors on theother hands interventions based on hbm concepts couldpromote nutritional behaviors related to colorectal cancerprevention consequently offering educational programsincluding public information campaigns workshopsvideos websites exhibitions etc should be used to informpeople about crc symptoms and risk factors alsomodelbased education will have a greater effect on nutritional behaviors improvement by focusing on perceptionsand enhancing beliefs aboutthe applicability oftheprogram and understanding the benefits and barriersabbreviationscrc colorectal cancer hbm health belief modelacknowledgementsthis is a part of an msc dissertation in health education approved by theshahid beheshti university of medical sciences the authors of this paperwould like to express their gratitude and appreciation to all the contributorswho have somehow collaborated on the design guidance andimplementation of this projectauthors™ contributionsmgh sr as and mm designed the study mm and mgh wrote the firstdraft sr and asm conducted the analyses all authors contributed towriting revising and approved the final manuscriptfundingthis study is sponsored by shahid beheshti university of medical sciences intehran the funding agencies had no role in the design of study datacollection and analysis or presentation of the resultsavailability of data and materialsthe datasets used and analyzed during the current study are available fromthe corresponding author on reasonable request 0crakhshanderou bmc medical education page of ethics approval and consent to participatethe study on which these data analyses are based was approved by theethical board committee of shahid beheshti university of medical sciencesparticipants were provided information about the study and verbalconsented by proceeding to take the survey this implied verbal consent wasapproved by the ethical board committee of shahid beheshti university ofmedical sciencesconsent for publicationnot applicablecompeting intereststhe authors have no conflict of interestsauthor details1environmental and occupational hazards control research center schoolof public health and safety shahid beheshti university of medical sciencestehran iran 2school of public health and safety shahid beheshti universityof medical sciences tehran iranreceived december accepted august screening in general practice in central england j epidemiol communityhealth “ roozitalab m moatari m gholamzadeh s saberifiroozi m zare n the effectof health belief on participation of the official administrative personnel incolorectal cancer screening programs in shiraz university of medicalsciences govaresh “ alidosti m sharifirad g hemate z delaram m najimi a tavassoli e theeffect of education based on health belief model of nutritional behaviorsassociated with gastric cancer in housewives of isfahan city daneshvarmed davodi a anoosheh m memarian r the effect of selfcare education onquality of life in patients with esophageal cancer following esophagectomyzums j “ braun kl fong m kaanoi me kamaka ml gotay cc testing a culturallyappropriate theorybased intervention to improve colorectal cancerscreening among native hawaiians prev med “ gimenogarc­a az quintero e nicol¡sp©rez d parrablanco a jim©nezsosa a impact of an educational videobased strategy on the behaviorprocess associated with colorectal cancer screening a randomizedcontrolled study cancer epidemiol ““ bandura a social cognitive theory handbook of social psychologicaltheories london sage bandura a social cognitive theory an agentic perspective annu revpsychol “luszczynska a guti©rrezdo±a b schwarzer r general selfefficacy invarious domains of human functioning evidence from five countries int jpsychol “ ho tv effects of an educational intervention on breast cancer screeningand early detection in vietnamese american women oncol nurs forumkolutek r avci ia sevig u the effects of scheduled observation at homeon health beliefs related to breast and cervical cancer screening andattitudes of married women eur j oncol nurs 201418s25 wang wl hsu sd wang jh huang lc hsu wl survey of breast cancermammography screening behaviors in eastern taiwan based on a healthbelief model kaohsiung j med sci “ cengiz b bahar z use of the health belief model in screening methodsfor colorectal cancer eur j oncol nurs 201418s27 donadiki e jim©nezgarc­a r hern¡ndezbarrera v sourtzi p carrascogarrido p de andr©s al jimeneztrujillo i velonakis e health belief modelapplied to noncompliance with hpv vaccine among female universitystudents public health “ becker mh drachman rh kirscht jp a new approach to explaining sickrole behavior in lowincome populations am j public health “ ma gx shive s tan y gao w rhee j park m kim j toubbeh jicommunitybased colorectal cancer intervention in underserved koreanamericans cancer epidemiol “ moatari m roozitalab m saber f zare m gholamzadeh s effect ofeducation on health beliefs on knowledge and participation j res med“ rajabi r sharifi a shamsi m almasi a dejam s investigating the effectof package theorybased training in the prevention of gastrointestinal cancers publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsreferencesarnold m sierra ms laversanne m soerjomataram i jemal a bray f globalpatterns and trends in colorectal cancer incidence and mortality gut american cancer society colorectal cancer facts and figures “available at httpswwwcancercontentdamcancerresearchcancerfactsandstatisticscolorectalcancerfactsandfigures 20172019pdf[accessed ]ansari r amjadi h norozbeigi n zamani f mirnasseri s khaleghnejad amalekzadeh r survival analysis of colorectal cancer in patients underwentsurgical operation in shariati and mehr hospitaltehran in a retrospectivestudy govaresh “centers for disease control and prevention cdc colorectal cancer risk byage available at httpwwwcdcgovcancercolorectalstatisticsagehtm[accessed apr ] malekzadeh r bishehsari f mahdavinia m ansari r epidemiology andmolecular genetics of colorectal cancer in iran a review kz aa saadat a jalalian hr esmaeili m epidemiology and survival analysisof colorectal cancer and its related factors trauma monthly winter239“ghaffari m mehrabi y rakhshanderou s safarimoradabadi a jafarian szeffectiveness of a health intervention based on who food safety manual iniran bmc public health “hosseini sv izadpanah a yarmohammadi h epidemiological changes incolorectal cancer in shiraz iran “ anz j surg “yazdizadeh b jarrahi a mortazavi h mohagheghi ma tahmasebi s nahvijoa time trends in the occurrence of major gi cancers in iran asian pac jcancer prev “ glanz k rimer bk viswanath k health behavior and health educationtheory research and practice john wiley sons ghaffari m rakhshanderou s safarimoradabadi a torabi s oral and dentalhealth care during pregnancy evaluating a theorydriven intervention oraldis “ becker mh the health belief model and sick role behavior health educmonogr “janz n champion v strecher vj the health belief model k glanz bk rimer“janz nk becker mh the health belief model a decade later health educ q“lp o review of translation and cultural adaptation process ofquestionnaires kellar sp kelvin ea munro's statistical methods for health care researchwolters kluwer healthlippincott williams wilkins abood da black dr feral d nutrition education worksite intervention foruniversity staff application of the health belief model j nutr educ behav“ hart ar barone tl gay sp inglis a griffin l tallon ca mayberry jf theeffect on compliance of a health education leaflet in colorectal cancer 0c"
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sarscov2 has resulted in numerous cases of coronavirus disease covid19 worldwide in addition to feverand respiratory symptoms digestive symptoms also are observed in some patients with covid19 angiotensinconverting enzyme ace2 was reported to be the receptor for sarscov2 the aim of this study was tocomprehensively investigate the digestive symptoms that occur in covid19 patients and the potential pathogenicroute of the sarscov2 infection in digestive tract ans from the oral cavity to the gastrointestinal tract weinvestigated the digestive symptoms of patients with covid19 and explored ace2 expression in digestive tractand lung cancers based on a series of bulk and singlecell rna sequencing data obtained from public databases wefound that of the patients with covid19 suffered from digestive symptoms among which pharyngalgia was the most common manifestation followed by diarrhea anorexia and nausea the bulktissue rna sequencing analysis indicated that digestive tract ans had higher ace2 expression levels compared tothe lung and the expression of ace2 in the lung increased with age singlecell rnaseq results showed that theace2positivecell ratio in digestive tract ans was significantly higher compared to the lung ace2 expression washigher in tumor cells compared to normal control nc tissues while in gastric tissues ace2 expression graduallyincreased from chronic gastritis to metaplasia to early cancer our data might provide a theoretical basis for screeningthe sarscov2 susceptible population and for the clinical classification of treatment of patients with covid19introductionthe global pandemic coronavirus disease covid19 caused by severe acute respiratory syninfection1 hasdrome coronavirus sarscov2been raging throughout the world by june covid19 has been found in countries with a totalof million confirmed cases worldwide includingover deaths2correspondence yujie liang yujie0350126com uiqing liao drliaoguiqinghotmailcom1department of oral and maxillofacial surgery hospital of stomatology sunyatsen university 56th lingyuanxi road guangzhou guangdongchina2guangdong province key laboratory of stomatology no 2nd zhongshanroad guangzhou guangdong chinafull list of author information is available at the end of the these authors contributed equally jiabin xu mei chu fan zhongedited by i amelioit has been established that sarscov2 invadeshost cells by binding to the transmembrane receptorangiotensinconverting enzyme ace23 which also isthe receptor for sarscov and hcovnl6345 ace2plays a crucial role in the cellular entry for sarscov2which means that ace2positive cells may act as targetcells and are susceptible to infection6 thus the expression of ace2 might affect the invasion path and pathogenicity of the virusthe common symptoms of covid19 include feverfatigue cough myalgia and dyspnea7“ also somepatients may suffer from digestive system symptoms suchas pharyngalgia diarrhea nausea vomiting and abdominalpain10 suggesting that the digestive tract ans also maybe targeted by the virus studies have reported that ace2was expressed in lung esophagus epithelial cells ileum11colon12 kidney bladder13 and oral mucosa14 however the authors open access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate ifchanges were made the images or other third party material in this are included in the ™s creative commons license unless indicated otherwise in a credit line to the material ifmaterial is not included in the ™s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this license visit httpcreativecommonslicensesby40official of the cell death differentiation association 0cxu cell death discovery page of table demographics and clinical characteristics ofpatients with covid19total number of cases notable ace2 expression in aerodigestive cancerscancersample type samplesize nomeanexpressionlog2 fpkm standarddeviation “lungoralnctumornctumoresophagus ncstomachcolontumornctumornctumorage yearmedianrangeage group no years‰¥ yearssex nomalefemalecomorbidity nohypertensiondiabeteschronic obstructive pulmonary disease copdchronic renal diseasescardiovascular and cerebrovascular diseaseschronic liver diseasesmalignancydigestive system diseasesigns and symptoms nofevercoughdigestive symptomspharyngalgiadiarrheaanorexianauseafatiguedyspneainformation is lacking concerning the comparative analysisof ace2 expression in the entire digestive tract from theoral cavity through the gastrointestinal tract the oropharynx and gastrointestinaltract are physiologicallyinterlinked and share similar microbial environments15they also share similarities with respect to the microenvironment associated with epithelial carcinogenesisreports show that oral bacteria play a role in the genesis ofdigestive tract tumors16“ more than of covid19cases coexist with malignant tumors10 it is unclear whether there are differences in ace2 expression betweentumor and normal cellsin this study we assessed the clinical characteristicsand investigated the digestive symptoms of patientswith covid19 in guangzhou eighth people™s hospitalofficial of the cell death differentiation associationnote data was downloaded from the cancer genome atlas tcgaace2 angiotensinconverting enzyme nc normal control ace2 angiotensinconverting enzyme nc normal controlwe explored ace2 expression in digestive tract cancersand lung cancers based on a series of bulk tissue rnasequencing data from two independent databases andsinglecelltranscriptome data from three singlecelldatabases the results showed that nearly a quarter ofcovid19 patients had gastrointestinal symptoms thatace2 expression was higher in the digestive tract thanthe lung and the ratio of ace2positive cells in tumortissues was higher than that in paracancerous normaltissuematerials and methodspatients™ involvement and data collectionfortyeight hospitalized patients admitted from january to march in guangzhou eighth people™shospital the hospital exclusively designated for covid19patients in guangzhou who were clinically diagnosed withœviral pneumonia based on their clinical symptoms feveror respiratory symptoms with typical changes in chestradiology were preliminarily included in this studypatients without or with negative test results were excludedfrom this studydemographiccharacteristicsincluding medical history comorbidities signs andsymptoms were obtained from the electronic medicalrecord system of guangzhou eighth people™s hospital andanalyzed by three independent researchers all patientsincluded in our study provided consent for the nasopharyngeal swabs and clinicalinformation collectionthis study was approved by the institutional ethics boardof guangzhou eighth people™s hospital no and complied with the declaration of helsinki concerningmedical research using human subjectsinformationclinicalnucleic acid detection of sarscov2nasopharyngeal swabs were collected and placed intoa sterile tube containing rna preservation solution 0cxu cell death discovery page of fig the expression of ace2 in different tissues a“c ace2 expression of different tissues downloaded from tcga database a ace2 expressionin all samples b ace2 expression in tumor samples c ace2 expression in nc samples black solid line median value black dotted line interquartilerange d ace2 expression in cancer cell lines downloaded from ccle database solid line mean value dotted line median value ace2 angiotensinconverting enzyme tcga the cancer genome atlas nc healthy normal control luad lung adenocarcinoma lusc lung squamous cell carcinomahnsc head and neck squamous cell carcinoma esca esophageal carcinoma stad stomach adenocarcinoma coad colon adenocarcinoma cclecancer cell line encyclopediathe swabs were sent for sarscov2 rna extractionand detection within h by a realtime reverse transcriptional polymerase chain reaction rtpcr systemby following the commercial test kit instructions da™angene cooperation cat da0930 as previously described19 briefly two pcr primer and probe sets targetingorf1ab and ncovn genes were separately added intothe same reaction tube positive and negative controlswere involved for detectionbulk tissue rna sequencing rnaseq data analysisthe rnaseq data level and clinical information forsamples were downloaded from the cancer genomeatlas tcgahttpsportalgdccancergovrepositoryusing the gdc data transfer tool the datasets used forace2 analysis included head and neck squamous cellcarcinoma hnsc esophageal carcinoma esca stomach adenocarcinoma stad colon adenocarcinomacoad lung adenocarcinoma luad and lung squamous cell carcinoma lusc gene expression waspresented as log2 fragments per kilobase per millionmapped reads fpkm the effects of age and sex onace2 expression were explored we also downloaded theboxplot for ace2 expression in human cancer cell linesfrom the cancer cell line encyclopedia ccle httpsportalsbroadinstituteccle database for verificationsinglecell rna sequencing scrnaseq data analysisthe lung cancer dataset exp0068 downloaded fromthe cancer singlecell state atlas httpbiocchrbmueducncancerseahomejsp and the lung tissue datasetsra878024 downloaded from the panglaodb httpspanglaodbseindexhtml were merged for further analysis the esophageal squamous cell carcinoma esccdataset gse81812 oral cancer dataset gse103322 gastriccancer dataset gse134520 and colorectal cancer datasetgse81861 were obtained from the gene expressionomnibushttpswwwncbinlmnihgovgeofor gse103322 cells from lymph nodes were removedthe r package seurat version was used for singlegeoofficial of the cell death differentiation association 0cxu cell death discovery page of fig the expression of ace2 in different ans grouped by age a“e the expression of ace2 in different tissues downloaded from tcgadatabase grouped by age top row the expression of ace2 in tumor samples bottom row the expression of ace2 in nc samples bold black linemedian value dotted black line range of values statistical tests mann“whitney utest ace2 angiotensinconverting enzyme tcga the cancergenome atlas nc healthy normal control luad lung adenocarcinoma lusc lung squamous cell carcinoma hnsc head and neck squamous cellcarcinoma esca esophageal carcinoma stad stomach adenocarcinoma coad colon adenocarcinomalogtransformed and scaledcell data analysis genebybarcode count matrices werenormalizedfollowed bydimension reduction using principal components analysispca uniform manifold approximation and projectionumap was used to carry out dimensionality reductionand clusteringall analyses were performed in r r version andgraphpad and the level of significance was set at p ‰¤ resultsdemographics and clinical characteristicsa total of patients diagnosed as covid19 wereincluded in the study twentyfive patients were male and were female the median patient age was years andranged from to years the majority of thepatients included in the study were under years of age of the patients had at least one underlyingcomorbidity the most common of which was hypertension three patients suffered from diabetes chronicobstructive pulmonary disease copd or chronic renaldiseasesrespectively two patients presented withcardiovascular and cerebrovascular disease one patienthad chronic liver disease malignancy or digestive systemdisease respectively the most common symptoms werefever or cough followed by digestive symptoms fatigue or dyspnea among patientswho presented with digestive system symptoms pharyngalgia was the most common manifestationfollowed by diarrhea anorexia and nausea table bulk tissue rnaseq data analysisa total of tumor samples and normal control nc samples were downloaded from the tcgadatabase table shows the expression of ace2log2fpkm in the different samples figure 1a“crespectively show ace2 expression in all tumor andnc samples from the different tissues the resultsshowed that digestive tract ans had higher ace2expression compared to the lung in digestive tractcancers ace2 expression gradually increased from theoral cavity to the esophagus stomach and the colonofficial of the cell death differentiation association 0cxu cell death discovery page of fig the expression of ace2 in different ans grouped by sex a“e the expression of ace2 in different tissues downloaded from tcgadatabase grouped by sex top row the expression of ace2 in tumor samples bottom row the expression of ace2 in nc samples bold black linemedian value dotted black line range of values statistical tests mann“whitney utest ace2 angiotensinconverting enzyme tcga the cancergenome atlas nc healthy normal control luad lung adenocarcinoma lusc lung squamous cell carcinoma hnsc head and neck squamous cellcarcinoma esca esophageal carcinoma stad stomach adenocarcinoma coad colon adenocarcinomafollowing the path of the digestive tract fig1a“c theresults were validated using the cancer cell line datafrom the ccle dataset fig 1dto analyze the effect of age or sex on ace2 expression thepatients were divided into a young group and an oldergroup ‰¥ based on age fig and the patients also wereseparated into females and males fig based on the clinicaldata in the tcga database the results revealed that for lungcancer based on either tumor samples or nc samples fig2a ace2 expression in the older group was higher comparedto the young group also for oral cancer the expression oface2 was significantly increased in the older group for thenc samples fig 2b as well as in the female group for thetumor samples fig 3bscrnaseq data analysissix different scrnaseq datasets were included in thisstudy sra878024 and exp0068 were lung normal tissueor lung cancer scrnaseq data respectively gse103322was oral cancer scrnaseq data gse81812 was scrnaseq data for the escc cell line kyse180 treated withdifferent doses of radiotherapy gse134520 includedscrnaseq data from chronic gastritis cg wild intestinal metaplasia wm severe intestinal metaplasia smand gastric cancer gc gse81861 included scrnaseqdata from colon cancer and adjacent normal tissues theresults showed that ace2positive cells accounted for thelowest proportion in lung samples with inthe nc group and in the tumor groupfig 4a the ace2positive rate in oral cancer cells was fig 4b while the ace2positive ratio inkyse180 treated with different doses of radiotherapywas for gy for gy and for gy fig 4c the ratios of ace2positive cells in the cg wm sm and gc groups were and respectively fig 4d the ace2positivecell ratio was in normal colonicmucosal cells and in colon cancer fig4e the ace2positive rate in the esophageal and oralmucosa was reported to be nearly and respectively we found that the ace2positivecell ratioofficial of the cell death differentiation association 0cxu cell death discovery page of fig see legend on next pageofficial of the cell death differentiation association 0cxu cell death discovery page of see figure on previous pagefig singlecell sequensing analysis of aerodigestive cancer cells a singlecell analysis of lung cancer cells tumor in exp0068 and nc cells insra878024 b singlecell analysis of oral cancer cells in gse103322 c singlecell analysis of escc cell line kyse180 tumor treated with different doses ofradiotherapy in gse81812 d singlecell analysis of cg wm sm and gc cells in gse134520 e singlecell analysis of colon cancer cells tumor and nc cells ingse81861 left column umap plots showing the distribution of cells colorcoded for pathology middle column umap plots showing the distribution oface2positive cell red right column stacked barplot showing the proportion of ace2positive cells red exp0068 was downloaded from the cancer singlecell state atlas sra878024 was downloaded from the panglaodb gse103322 gse81812 gse134520 and gse81861 were obtained from the geneexpression omnibus nc healthy normal control escc esophageal squamous cell carcinoma cg chronic gastritis wm wild intestinal metaplasia sm severeintestinal metaplasia gc gastric cancer umap uniform manifold approximation and projection ace2 angiotensinconverting enzyme study were younger and presented with fewer underlyingdigestive system diseases the most common digestivesymptom was pharyngalgia which was consistent withother studies2224we observed that the expression of ace2 in the lungincreased with age but was independent of sex which wasconsistent with the report by chen 25 this maypartly explain why older patients with covid19 aremore likely to develop pneumoniathis study found that ace2 was highly expressed indigestive tract tumors or paracancerous tissues compared tothe lung for both the bulk tissue analysis and the singlecellanalysis elevated ace2 expression in the digestive tract suggested that the digestive tract ans also should be consideredto be vulnerable targets for sarscov2 infection it wasreported that sarscov2 might cause a cytokine storm andmultian failure in severe pneumonia patients26 similarlysarscov2 might interact with ace2 in digestive anscausing further damage to the mucous membrane barrier andincrease inflammatory cytokine production oralrelatedsymptoms are rarely reported with covid19 infectionsbut this does not indicate that an oral infection route forsarscov2 should be excluded the oral cavity and gastrointestinal ans share similarities in the microbiomeinflammation and tumorigenesis thus we expect that theoral and gastrointestinal ans should exhibit commoninteractions in the route of sarscov2 infection furthermore recent studies have reported a high positive rate ofsarscov2 detection in saliva2728indicating that oralinfection could be an early symptom of covid19interestingly we found that the ace2positive rate intumor cells was significantly higher compared to the ncgroup moreover ace2 expression in gastric tissuesgradually increased from chronic gastritis to intestinalmetaplasia to early gastric cancer similar findings werereported for the colon29 these results suggested thatcancer patients might have a higher risk of sarscov2infection from another perspective we suspected thatincreased expression levels of ace2 affected the occurrence of digestive tract tumors ace2 hydrolyzes ang iito ang1“ which negatively regulates the active reninangiotensin system ras reports show that ang1“plays an inhibitory role in the genesis and development offig ace2 expression increases along with the path of digestivetract ace2positive cell proportion in aerodigestive cancers ace2positive cell proportion in oral mucosa reported by xu 14 ace2positive cell proportion in esophageal epithelium reported by zou 11 ace2 angiotensinconverting enzyme nc healthy normalcontrol cg chronic gastritis wm wild intestinal metaplasia sm severeintestinal metaplasia gc gastric cancerin digestive tract ans was significantly higher than inthe lung the ace2 expression also was higher in tumorcells compared to nc tissues the ace2 expression ingastric tissues gradually increased from chronic gastritisto metaplasia then cancer fig discussionthis study investigated the gastrointestinal symptoms of patients with covid19 who were admitted to theguangzhou eighth people™s hospital we explored ace2expression in digestive tract cancers and lung cancers basedon both bulk tissue rnaseq data and scrnaseq datathe median age of the patients was years whichwas similar to data reported by xu years21but was younger than the ages found in many otherreports892223 consistent with other recent reportshypertension and diabetes were the most commonunderlying comorbidities and fever and cough were themost common symptoms of covid19 infection923 inthis study of the covid19 patients exhibiteddigestive symptoms which was lower than the percentage reported by xu 21 and zhang 22 thereason for this difference may be that the patients in thisofficial of the cell death differentiation association 0cxu cell death discovery page of tumors3031 however sarscov2 could cause aninflammatory cytokine storm when the disease developsinto a longterm chronic state a potential risk mightdevelop for malignant changes to occur in the mucosaunder the effect of various cytokines therefore we suggest that when following patients with covid19 the riskof tumorigenesis should be consideredin summary the ace2positive cells in digestive tract tissuesmight provide possible routes for sarscov2 infectionace2 expression in lung tissue increased with age whichmight explain at least partially why older patients withcovid19 are more likely to develop pneumonia ace2expression was correlated to histological grading suggestingthat cancer patients might be more susceptible to sarscov future research should focus on whether the expression oface2 in digestive tract ans could affect the replication ofsarscov2 and whether sarscov2 infection could affectthe genesis or development of tumorsacknowledgementsthis work was supported by the guangdong financial fund for highcaliberhospital constructionauthor details1department of oral and maxillofacial surgery hospital of stomatology sunyatsen university 56th lingyuanxi road guangzhou guangdongchina 2guangdong province key laboratory of stomatology no 2ndzhongshan road guangzhou guangdong china 3guanghua schoolof stomatology sun yatsen university 56th lingyuanxi road guangzhou guangdong china 4guangzhou eighth people™s hospitalguangzhou medical university guangzhou guangdong china5school of stomatology wuhan university 237th luoyu road wuhanhubei chinaconflict of interestthe authors declare that they have no conflict of interestpublisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsreceived may revised july accepted july references coronaviridae study group of the international committee on taxonomy ofviruses the species severe acute respiratory syndromerelated coronavirusclassifying 2019ncov and naming it sarscov2 nat microbiol “ who coronavirus disease covid19 situation report101 httpswwwwhointemergenciesdiseasesnovelcoronavirus2019situationreportswho xu x evolution of the novel coronavirus from the ongoing wuhanoutbreak and modeling of its spike protein for risk of human transmission scichina life sci “ he l expression of elevated levels of proinflammatory cytokines insarscovinfected ace2 cells in sars patients relation to the acute lunginjury and pathogenesis of sars j pathol “ official of the cell death differentiation associationli w the s proteins of human coronavirus nl63 and severe acuterespiratory syndrome coronavirus bind overlapping regions of ace2 virology “ zhou p a pneumonia outbreak associated with a new coronavirus ofprobable bat origin nature “ moein s t smell dysfunction a biomarker for covid19int forumallergy rhinol httpsdoi101002alr22587 mao l neurological manifestations of hospitalized patients with covid in wuhan china a retrospective case series study jama neurol httpsdoi101001jamaneurol20201127 huang c clinical features of patients infected with novel coronavirus in wuhan china lancet “ fang z clinical characteristics of coronavirus pneumonia covid19 an updated systematic review medrxiv httpsdoi zou x singlecell rnaseq data analysis on the receptor ace2 expression reveals the potential risk of different human ans vulnerable to ncov infection front med “ zhang h the digestive system is a potential route of 2019ncovinfection a bioinformatics analysis based on singlecell transcriptomes biorxivhttpsdoi10110120200130927806 lin w singlecell analysis of ace2 expression in human kidneys andbladders reveals a potential route of 2019ncov infection biorxiv httpsdoi10110120200208939892 xu h high expression of ace2 receptor of 2019ncov on the epithelialcells of oral mucosa int j oral sci zhang x the oral and gut microbiomes are perturbed in rheumatoidarthritis and partly normalized after treatment nat med “ whitmore s e lamont r j oral bacteria and cancer plos pathog e1003933 chen x poor oral health is associated with an increased risk of esophageal squamous cell carcinomaa populationbased casecontrol study inchina int j cancer “ maisonneuve p amar s lowenfels a b periodontal disease edentulismand pancreatic cancer a metaanalysis ann oncol “ chen w detectable 2019ncov viral rna in blood is a strong indicatorfor the further clinical severity emerg microbes infect “ stuart t comprehensive integration of singlecell data cell “e21 xu x clinical findings in a group of patients infected with the novel coronavirus sarscov2 outside of wuhan china retrospective caseseries bmj m606 zhang j clinical characteristics of patients infected with sarscov2 in wuhan china allergy httpsdoi101111all142382020 wang c horby p w hayden f g gao g f a novel coronavirus 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Construction of a novel prognosticpredicting modelcorrelated to ovarian cancerWeichun Tang12 Jie Li3 Xinxia Chang12 Lizhou Jia1 Qi Tang12 Ying Wang4 Yanli Zheng4 Lizhou Sun5 andZhenqing Feng121National Health Commission Key Laboratory of Antibody Technique Nanjing Medical University Nanjing People™s Republic of China 2Department of Pathology Nanjing MedicalUniversity Nanjing People™s Republic of China 3Department of Nursing The Second Affiliated Hospital of Nantong University Nantong People™s Republic of China 4Department ofGynaecology and Obstetrics The Second Affiliated Hospital of Nantong University Nantong People™s Republic of China 5Department of Obstetrics and Gynecology First AffiliatedHospital of Nanjing Medical University Nanjing People™s Republic of ChinaCorrespondence Zhenqing Feng fengzhenqingnjmueducn or Lizhou Sun lizhou sun163comBackground Ovarian cancer OC is one of the most lethal gynecological cancers worldwide The pathogenesis of the disease and outcomes prediction of OC patients remainlargely unclear The present study aimed to explore the key genes and biological pathwaysin ovarian carcinoma development as well as construct a prognostic model to predict patients™ overall survival OSResults We identified upregulated and downregulated differentially expressedgenes DEGs associated with OC Gene Ontology GO term enrichment showed DEGsmainly correlated with spindle microtubes For Kyoto Encyclopedia of Genes and GenomesKEGG pathways cell cycle was mostly enriched for the DEGs The protein“protein interaction PPI network yielded nodes and edges Top three modules and ten hub geneswere further filtered and analyzed Three candidiate drugs targeting for therapy were alsoselected Thirteen OSrelated genes were selected and an eightmRNA model was presentto stratify patients into high and lowrisk groups with significantly different survivalConclusions The identified DEGs and biological pathways may provide new perspective onthe pathogenesis and treatments of OC The identified eightmRNA signature has significantclinical implication for outcome prediction and tailored therapy guidance for OC patientsBackgroundOvarian cancer OC is the most lethal malignant disease in the female reproductive system with over new cases and deaths each year worldwide [] Epithelial OC accounts for “ ofovarian malignancies listed as the most common histological type Since the ovaries locate in the deeppelvis with mere symptoms emerging at the beginning of ovarian morbid change the early detectionfor the malignancy is truly difficult Hence when OC is detected the patient is usually at an advancedstage with invasion and metastasis accompanied [] For patients in the early stage the 5year survivalrate can reach “ whereas for advancedstage patients the number is mere ˆ¼ [] Thereforeit is imperative to explore the molecular mechanisms of malignant biological behavior of OC cells andto develop more reliable molecular markers for predicting recurrence and evaluating prognosis furtherguiding clinicians for therapyAt present various highthroughput microarrays and nextgeneration sequence genomic datasetswhich were deposited in the Gene Expression Omnibus GEO [] and The Cancer Genome Atlas TCGAdatabases have been widely analyzed for identifying differentially expressed genes DEGs which couldserve as candidate diagnostic or prognostic markers further effectively improving our understanding ofthe disease from genetic perspective Whereas since the existence of tissue or sample heterogeneity inThese authors contributedequally to this workReceived April Revised July Accepted July Accepted Manuscript online July Version of Record published August The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261each independent experiment as well as the discrepancy of different data processing methods and technology platforms the DEGs identified from a singlecohort study may generate false positives Herein the Robust Rank Aggregation RRA method which analyzes the significant probability of all elements by a probabilistic model is developedto identify statistically significant genes from multiple datasets and provide more accurate and valuable informationfor clinical use far beyond one gene list [] To date a bunch of novel prognostic markers have been discovered topotentially improve the efficacy of diagnosis and prognosis of OC [] However these identified markers were onlyeffective for partial stages or grades and were difficult to apply widely Hence a prognostic model which is basedon signature gene expression level with high discriminating power to effectively assist prognosis prediction for eachpatient is required in clinical practiceIn the present study we downloaded six primary microarray datasets from the GEO database which containeda total of samples with OC samples and normal samples The geneset and relative clinical information on ovary tissues of OC patients and healthy females from TCGA and GTEx portal were also downloadedIntegrated DEGs between cancerous and normal ovarian samples were screened using the ˜limma™ R package andRRA method Gene Ontology GO and Kyoto Encyclopedia of Genes and Genomes KEGG pathways enrichmentof DEGs were performed for nextstep functional analysis The Search Tool for the Retrieval of Interacting GenesDatabase STRING and the Connectivity Map CMap online database were then used to analyze the association ofDEGs and explore the molecular mechanisms as well as drugs involved in tumorigenesis Through survival analysisprognostic mRNAs were also selected By performing Cox regression analysis we identified an eightmRNA signature model with the ability to predict the prognosis of OC patients and independent from clinical factors Our studyprovides reliable molecular markers and prognostic models for early detection and outcome prediction as well aseffective drug targets for treating OCMethodsData collectionThrough searching on the GEO Repository with ˜ovarian cancer™ we downloaded the gene expression profiles ofGSE54388 GSE40595 GSE38666 GSE27651 GSE18520 and GSE14407 and the corresponding annotation files fromthe GPL570 [HGU133 Plus ] Affymetrix Human GenomeU133 Plus Array platform GSE54388 contains ovarian tissue samples with normal ovarian surface epithelium and tumor epithelial components from highgradeserous OC patients GSE40595 includes OCassociated stroma and epithelium samples which consist of cancerassociated stroma samples and epithelial tissues from highgrade serous OC patients along with stromal component and ovarian epitheliums from the normal ovary GSE38666 comprises stroma and matchedovarian epitheliums from healthy females as well as cancer stroma and matched cancer epitheliums from OCpatients GSE27651 incorporates normal ovarian surfaces epithelial and serous borderline ovarian tumors lowgrade serous ovarian carcinomas and highgrade serous ovarian carcinomas GSE18520 covers advancedstage highgrade primary OC specimens and normal ovarian surface epithelium tissues GSE14407 involves healthy ovarian surface epithelial samples and paired serous OC epithelium Note that all samples from these GEOdatasets are classified into the cancerous or normal part to be clear the normal stromal and surface epithelium is defined as normal ovarian tissues whereas the borderline tumors as well as cancerous stromal and epithelial tissues areconsidered as malignancies Besides we also downloaded the FPKM format gene expression data and relative clinicalinformation of OC patients™ samples and normal ovarian tissues from TCGA and GTEx portal respectivelyScreening for DEGs and integration of microarray dataWe used the ˜limma™ R package [] to integrate the expression profiles from TCGA and GTEx portal standardize the data from the integrated TCGA and GTEx expression matrix as well as six GEO datasets and furtherscreen the DEGs between ovarian cancerous and normal samples The list of DEGs obtained from six GEO microarray datasets by limma analysis was further integrated by the ˜RRA™ method to get prioritized commonly upor downregulated gene list The final overlapped DEGs for subsequent biological function analysis were the combination of prioritized jointly dysregulated genes from six GEO microarrays and the results from TCGA and GTExdatabases The cutoff criteria were set as FDR and log2fold change FC GO term and KEGG pathway enrichment analysisGO classified the known genes into three main biological progress Molecular Function MF Cellular ComponentCC and Biological Process BP [] KEGG provides researchers highlayer functions of the biological system frommolecular level information [] The Enrichr online tool amppharmmssmeduEnrichr allows for GO term The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The expression heatmap of the top significantly dysregulated genes in six GEO datasetsHierarchical clustering that shows the expression profiles of mRNAs from A GSE14407 B GSE18520 C GSE27651 DGSE38666 E GSE40595 F GSE54388annotation and KEGG pathway for a cluster of genes [] We explored the biological functions of overlappedDEGs and hub modules from our protein“protein interaction PPI network using Enrichr website Pvalue was considered as significant enrichment Likewise the functional biological pathways of the top ten hub genes fromPPI network were also analyzed by the FunRich tool version [] and the top five enriched pathways of up anddownregulated genes were displayed as bar charts respectively We set the Pvalue as statistically significant The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261PPI network construction and analysisPPI networks display the relationships of various proteins according to their physical or biochemical propertiesSTRING is a database that encompasses the interaction information between known proteins and potentially interacted proteins [] In order to explore the correlations between the DEGs we used the STRING database to constructa PPI network and visualize our results by Cytoscape software [] Confidence score was set as significantMolecular Complex Detection MCODE was utilized to select hub modules of PPI networks in Cytoscape [] Weset the degree cutoff node score cutoff kscore and max Depth was set as the criterion Thenthe significant modules were performed by GO and KEGG analyses Top ten genes were defined according to thehigh degree of connectivity in STRING network [] The coexpression analysis of ten hub genes was performed bySTRING eitherValidation of the hub genesWe downloaded the raw geneset of OC patients from TCGA to explore the expression differences of hub genes inlow and highgrade tumor tissues of OC and draw the survival plot using Kaplan“Meier plotter webtool [] Thegene and protein expression level of graderelated hub genes were then confirmed by Oncomine and The HumanProtein Atlas HPA database [] Meanwhile we explored the genetic alteration information of the selected tenhub genes in OC patients by plugin cBioPortal cBio Cancer Genomics Portal tool which deposits the genomicsprofiles of various cancer types and provides analysis and visualization of the genomics datasets []Identification of candidate small molecule drugsThe CMap database was able to predict potential drugs which might reverse or induce the biological state encoded incertain gene expression signatures in OC [] The DEGs from our study were used to query the CMap databaseThe enrichment scores which represent the similarity were calculated ranging fromˆ’ to The positive connectivity score means an inducing influence on the input signature whereas drugs with negative connectivity score presentreversion impact on the characteristic in human cell lines and are considered as candidate therapeutic molecules After sorting all instances the connectivity score of various instances was filtered by Pvalue Next we investigatethe structures of these candidate molecular drugs from the Pubchem database pubchemncbinlmnihgovEstablishment and evaluation of the prognostic modelThe OC patients from the TCGA project were randomly classified into the training cohort n188 and the testingcohort n186 OSrelated genes were determined by performing univariate Cox regression analysis in the trainingcohort with the ˜Survival™ R package and further selected for the nextstep screening Least Absolute Shrinkage andSelection Operator LASSO is a parameter selection algorithm which shrinks all highdimensional regression coefficients and generates the penalty regularization parameter λ via the crossvalidation routine by ˜glmnet™ R packageTo select the optimal prognostic mRNAs we adopted LASSO regression among the selected candidate genes and further perform multivariate Cox proportional hazards regression to evaluate their independent prognostic values Theriskscore model for predicting outcomes of OC patients was the sum of each optimal prognostic mRNA expressionlevel multiplying relative regression coefficient weight calculated from the multivariate Cox regression modelRisk score patient cid2Coefficient mRNAi × Expression mRNAiiAll training cohort patients were classified into high and lowrisk groups according to the median risk score TheKaplan“Meier curves of two diverse groups were plotted using ˜survfit™ function and the receiver operating characteristic ROC curve was unfolded for OS prediction to estimate the sensitivity and specificity of the prognostic modelCox multivariate analysis was also performed to examine whether the risk score was independent of the clinical characters such as age tumor stage and grade Next we used the testing group to check the efficacy of the prognostic riskmodel Each individual in the testing cohort was also categorized as high or lowrisk case by comparing the patient™srisk score with the cutoff value calculated from the training cohort Kaplan“Meier curve analysis timedependentROC analysis and Cox multivariate analysis were performed eitherSearching tumorinfiltrating immune cells associated with patients™prognostic signaturesThe TIMER webtool allows for systematical evaluations of the relationship between the six immune cell types inthe tumor microenvironment which are B cell CD4 T cell CD8 T cell neutrophil macrophage as well as dendritic The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The volcano plot of all gene expression distribution in six GEO datasetsVolcano plot of differentially expressed mRNAs of A GSE14407 B GSE18520 C GSE27651 D GSE38666 E GSE40595 FGSE54388cell and clinical impact in various cancer types via a novel statistical method [] To further explore the prognosticsignature we used the TIMER online tool to search the most significant tumorinfiltrating immune cells according tothe TCGA OC gene data To be clear the relative gene expression levels of six types of immune cells for each patientin high and lowrisk groups from training and testing cohort were measuredResultsThe DEGs among six GEO microarray datasetsThe top significantly up and downregulated genes from each microarray dataset were displayed in the heatmapsFigure 1A“F and the distribution of all gene expression was presented in volcano plots Figure 2A“F ThroughRRA analysis of expression microarrays we identified DEGs which consisted of upregulated and downregulated genes and displayed the top dysregulated genes by ˜pheatmap™ R package in Figure Next weanalyzed the expression profiles of TCGA and GTEx getting dysregulated genes Intriguingly when these DEGs were combined with the DEGs from GEO datasets we found that genes were commonly dysregulatedin these two databases with upregulated Figure 4A and downregulated genes Figure 4BGO term and KEGG pathway enrichment analysis of DEGsTo study the potential biological function of the DEGs we performed biological pathway analysis and identifiedsignificantly enriched pathways via Enrichr web tools In GO term Figure 5A for the BP group the DEGs weremostly enriched in ˜regulation of attachment of spindle microtubes to kinetochore™ ˜cellular response to laminar fluidshear stress™ and ˜microtubule cytoskeleton anization involved in mitosis™ In MF group the dysregulated geneswere highly correlated to ˜microtubulebinding™ ˜microtubule motor activity™ and ˜tubulinbinding™ As for CC group The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure Heatmap showing the top upregulated genes and top downregulated genes according to PvalueEach row represents one gene and each column indicates one dataset Red indicates upregulation and blue represents downregulation The numbers in the heatmap indicate log FC in each dataset calculated by the ˜limma™ R package Abbreviation log FClogarithmic fold changethe DEGs were closely related to ˜condensed nuclear chromosome kinetochore™ and ˜mitotic spindle™ KEGG pathwayanalysis showed DEGs highly enriched in ˜cell cycle™ and ˜Alanine aspartate and glutamate metabolism™ Figure5B The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The intersection of up and downregulated DEGs of GEO and TCGA datasetsA upregulated intersected DEGs in both datasets B downregulated intersected DEGs in both GEO and TCGA dataset Theintersected DEGs were defined as the significant DEGsPPI network construction and modules analysisUsing the STRING database and Cytoscape software a total of DEGs were mapped into the PPI network whichincluded nodes and edges Figure 6A The PPI enrichment Pvalue was 10e16 The top three key modules Figure 6C“E within PPI network were then selected Module MCODE score Module MCODEscore Module MCODE score and the biological function of Module which consisted of nodesand edges was further analyzed GO analysis indicated that Module1 was mainly associated with ˜regulation ofattachment of spindle microtubules to kinetochore™ ˜condensed nuclear chromosome kinetochore™ and ˜microtubulemotor activity™ KEGG analysis showed that ˜cell cycle™ and ˜oocyte meiosis™ were the most highly enriched pathwaysSupplementary Figure S1The screening of Hub genes and their characteristicsThe top ten hub genes with the highest degree of connectivity were CDC45 CDK1 TOP2A CDC20 CCNB1CEP55 UBE2C HMMR FOXM1 and TPX2 Figure 6B The coexpression analysis results of the hub genes demonstrated that these genes actively interacted with each other Supplementary Figure S2 Besides we established theinteraction network of ten hub genes with their related genes and explored the biological role Supplementary Figure S2AC“F of the network by FunRich The gene alteration type and frequency as well as the most frequentlyaltered neighbor genes were also exhibited Figure Gene alteration frequency of ten hub genes among TCGAOC samples was beyond with most genes showed amplified and multiple altered Figure 7AB The top threemost frequently altered genes were FOXM1 CDC20 and CCNB1 with FOXM1 and CDC20 largely amplified whileCCNB1 deep deleted Through analysis of OC patients™ geneset from TCGA we found that CCNB1 UBE2C CDK1CEP55 as well as FOXM1 expressed significantly higher in highgrade tumors and predicted worse outcomes sincepatients overexpressed above genes owned lower overall survival OS and diseasefree survival DFS rates Figure The Oncomine database showed results from various studies were consistent to our finding Supplementary Figure S3 The HPA website also demonstrate that proteins translated by such five hub genes were overexpressed in OCtissues Supplementary Figure S4 HMMR and TPX2 were also negatively correlated to patients™ prognosis while noexpression difference was observed in diverse tumor grades and CDC20 was positively associated with tumor gradebut not correlated to patients™ outcomesRelated small molecule drugs screeningIn total DEGs were analyzed in CMap to screen small molecule drugs and the candidate molecules with top tenconnectivity score are listed in Table Five of these molecules showed a negative correlation and suggested potentialin clinical applications Among them Trichostatin A pyrvinium and 8azaguanine showed a significantly negativecorrelation and the stuctures of such candidate molecule drugs was found in the PubChem database and shown inSupplementary Figure S5 The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure GO and KEGG functional annotation for the significant DEGsA The top ten enriched BP of the DEGs B The top ten enriched CC of the DEGs C The top ten enriched MF of the DEGs DThe top ten enriched KEGG pathways of the DEGs The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The PPI network and top hub genes as well as top three modules were constructedA The PPI network of the DEGs B The hub genes of the DEGs C“E Top three hub modules were identified by Cytoscapeplugin MCODE C module1 D module2 E module3Table The top ten OCrelated small molecules with highly significant correlations in results of CMap analysisRankCMap nametrichostatin A8azaguaninepyrviniumisoflupredonequinpirolevorinostatgenisteinantimycin AheptaminolmidodrineMeanˆ’ˆ’ˆ’ˆ’ˆ’NEnrichmentP valueˆ’ˆ’ˆ’ˆ’ˆ’ The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The gene mutation overview of ten hub genes in TCGA OC patientsA Ten hub genes are altered in of queried patients B The summary of mutation type of ten hub genes in OC patientsC The network of hub genes and the most frequently altered neighbor genesTable Univariate cox regression identified DEGs correlated to patients™ OSGene IDCCND1SYNE4CCDC80TMC4MCCFOXQ1KRTCAP3CXCR4IL4I1DEFB1CSGALNACT1KLHL14MCUR1HRAbbreviation HR hazard ratioHR95LHR95HPvalueConstruction of prognostic model and evaluation of its predictive abilityUnivariate Cox regression analysis revealed that of DEGs were significantly correlated to patients™ OS in thetraining cohort Table The OSrelated genes were listed as follows CCND1 SYNE4 CCDC80 TMC4 MCC The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The clinical characteristics of CCNB1 CEP55 CDK1 FOXM1 and UBE2C in OC patientsA Five genes were overexpressed in high grade G1 and G2 compared with low grade G3 and G4 in OC BC The OS time Band DFS time C of five genes in OC patients The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure Identification of prognosisrelated mRNAs using LASSO regression modelA LASSO coefficient profiles of the mRNAs associated with the OS of OC B Plots of the crossvalidation error rates Each dotrepresents a λ value along with error bars to give a confidence interval for the crossvalidated error rateTable Multivariate Cox regression selected eight DEGs correlated to patients™ OSGene IDTMC4KLHL14CXCR4CCDC80KRTCAP3DEFB1SYNE4FOXQ1HRHR95LHR95HPvalue845E08Abbreviation HR hazard ratioFOXQ1 KRTCAP3 CXCR4 IL4I1 DEFB1 CSGALNACT1 KLHL14 and MCUR1 Through LASSO Cox regression we narrowed the number of prognosisassociated genes to according to the minimum criteria Figure Next based on the multivariate Cox model of candidate mRNAs retained their prognostic significance and werefinally selected as independent remarkable prognostic factors which were TMC4 KLHL14 CXCR4 CCDC80 KRTCAP3 DEFB1 SYNE4 and FOXQ1 Table To predict patients™ outcomes we developed an individual™s risk scoremodel as follows risk score × expression value of TMC4 × expression value of KLHL14 ˆ’ × expression value of CXCR4 × expression value of CCDC80 ˆ’ × expression value of KRTCAP3 ˆ’ × expression value of DEFB1 × expression value of SYNE4 × expression value of FOXQ1 On the basis of the median risk score patients were divided into high orlowrisk groups Kaplan“Meier curve analysis showed that the OS time of the lowrisk group was significantly longerthan the highrisk group P1147e07 Figure 10E ROC curve analysis revealed the area under the ROC curveAUC of the prognostic model was Figure 10D Meanwhile the risk scores Figure 10A of OC patients inthe training group were ranked for displaying their distribution and the survival status Figure 10B was marked onthe dot plot The expression pattern of eight prognostic mRNAs between high and lowrisk groups was also shown inthe heatmap Figure 10C Univariate and multivariate Cox regression analyses concerning the risk score and clinicalfactors showed that the prognostic model was able to serve as an independent prognostic indicator Figure 11ABROC curve analysis also showed that the AUC value of the model was much significantly higher than the tumor stage AUC grade AUC and patients™ age AUC Figure 11C Interestingly when The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure Prognostic analysis of the TCGA training modelA The risk score B survival status C expression heatmap D timedependent ROC curves and E Kaplan“Meier survival ofthe prognostic model for the TCGA OC training cohort The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The clinical independency of the risk model in training cohortUnivariate A and multivariate B regression analyses as well as timedependent ROC curve analysis CD of the prognostic valuebetween the training model and OC patients™ OS status when compared with or combined with clinical factorscombined the risk score with clinical factors the ROC curve of combination model was much higher than each aloneFigure 11DAs for the testing cohort we divided the group into highrisk and lowrisk individuals based on the trainingcohort cutoff risk score The outcomes of low and highrisk groups™ patients of the testing cohort were also measuredand the OS time of the highrisk group was significantly shorter than the lowerrisk group P1721e02 Figure12E The AUC of the prognostic model was Figure 12D The risk scores distribution Figure 12A and survivalstatus Figure 12B of OC patients as well as the eightprognostic gene expression heatmap Figure 12C in the testinggroup were also present Meanwhile the independency of the prognostic model was confirmed in testing cohort sinceunivariate and multivariate Cox regression analyses showed the model correctly predicted high or lowrisk factroups patients™ outcomes without relying on any clinical factors Figure 13AB ROC curve analysis showed that theprognostic model exhibited better sensitivity and specificity when compared with tumor stage grade and patients™age for the AUC value of the model was much higher than later Figure 13C In accordance with results from trainingcohort the combination of risk score and clinical factors showed better OS prediction capability Figure 13DThe prognostic signature correlating to immune cells infiltrationThrough TIMER webtool we analyzed the relative gene expression levels of six types of immune cells for each patientand found that genes concerning macrophage fraction were expressing significantly higher in the highrisk groupP005 compared with the lowrisk group in training cohort Figure Interestingly same result was also observed in the testing cohort Figure The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure Prognostic analysis of the TCGA testing modelA The risk score B survival status C expression heatmap D timedependent ROC curves and E Kaplan“Meier survival ofthe prognostic model for the TCGA OC testing cohort The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The clinical independency of the prognostic risk signature in testing cohortUnivariate A and multivariate B regression analyses as well as timedependent ROC curve analysis CD of the prognostic valuebetween the testing model and OC patients™ OS status when compared with or combined to clinical factorsDiscussionIn the present study we used the RRA methods to jointly analyze six GEO OC microarrays which contained OC and normal samples identifying DEGs and overlapped them with dysregulated genes of OC cohort fromTCGA and GTEx portal finally getting upregulated and downregulated genes Functional analysis showedthat DEGs were significantly enriched in the cell division cycle to be clear in the process of the mitotic spindleSpindle microtubules have been proved to play crucial role in physiological and pathological processes As for celldivision only when all chromosomes linked to spindle microtubules through kinetochores and the spindle assemblycheckpoint is satisfied this process could step to anaphase [] Suraokar et al found that the mitotic spindle assemblycheckpoint and microtubule network were significantly altered in malignant pleural mesothelioma MPM whileusing epothiloneB a nontaxane small molecule inhibitor targeting the microtubules could greatly decrease theviability of MPM cell lines [] Rogalska et al compared the antiproliferative capacity of epothilone B with paclitaxelon OC cell line SKOV3 found that this effect of Epo B was greater than latter [] The researches above wereconsistent with our study that the mitotic spindle process was dysregulated in OC progression playing importantroles in OC cell proliferation and tumor developmentPPI network construction of DEGs included nodes and edges among which we identified three keymodules Interestingly the top1 module was also highly associated with spindle microtubules and chromosome kinetochore confirming the role of cell cycle in OC pathogenesis The top ten hub genes from the PPI network were alsorecognized which were CDC45 CDK1 TOP2A CDC20 CCNB1 CEP55 UBE2C HMMR FOXM1 and TPX2 The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The expression level of immune cells related genes in high and lowrisk groups of the training cohortA Bcell fraction B dendritic fraction C CD4 Tcell fraction D CD8 Tcell fract
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" heat shock transcription factor1 hsf1 was overexpressed to promote glutaminolysis and activatemtor in colorectal cancer crc here we investigated the mechanism for cancerspecific overexpression of hsf1methods hsf1 expression was analyzed by chromatin immunoprecipitation qrtpcr immunohistochemistrystaining and immunoblotting hsf1 translation was explored by polysome profiling and nascent protein analysisbiotin pulldown and m6a rna immunoprecipitation were applied to investigate rnarna interaction and m6amodification the relevance of hsf1 to crc was analyzed in apcmin and apcmin hsf1ˆ’miceresults hsf1 expression and activity were reduced after the inhibition of wntcatenin signaling by pyrvinium orcatenin knockdown but elevated upon its activation by lithium chloride licl or catenin overexpression thereare much less upregulated genes in hsf1ko mef treated with licl when compared with licltreated wt mefhsf1 protein expression was positively correlated with catenin expression in cell lines and primary tissues aftercatenin depletion hsf1 mrna translation was impaired accompanied by the reduction of its m6a modificationand the upregulation of mir4553p which can interact with ²utr of hsf1 mrna to repress its translationinterestingly inhibition of mir4553p rescued catenin depletioninduced reduction of hsf1 m6a modificationand mettl3 interaction both the size and number of tumors were significantly reduced in apcmin mice whenhsf1 was genetically knockedout or chemically inhibiteds catenin suppresses mir4553p generation to stimulate m6a modification and subsequenttranslation of hsf1 mrna hsf1 is important for catenin to promote crc development targeting hsf1 could bea potential strategy for the intervention of catenindriven cancerskeywords colorectal cancer catenin hsf1 translation mir4553p m6a rna modification correspondence wangx118zjueducn jinhczjueducn1department of medical oncology cancer institute of zhejiang university sirrun run shaw hospital school of medicine zhejiang university hangzhouchina2labortary of cancer biology key lab of biotherapy in zhejiang sir run runshaw hospital school of medicine zhejiang university hangzhou chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0csong molecular cancer page of introductioncolorectal cancer crc is the third most common cancer with high mortality rate globally the accumulation of various genetic and epigenetic changes activatesmultiple oncogenic signaling critical for the pathogenesisof crc such as wntcatenin signaling pathway its activation will eventually initiate a transcriptiondependent oncogenic process to promote cell cycle progression and apoptosis resistance while the mechanismfor activated wntcatenin signaling to promote crcdevelopment has been wellexplored no therapeuticstargeting this pathway has been successfully developedin addition to proliferation activation and apoptosisresistance metabolism reprogramming is one of important hallmarks of cancer cells for example cancercells favor glycolysis instead of oxidative phosphorylationfor glucose metabolism even in aerobic conditionswhich was wellknown as warburg effect [ ] pyruvate the last product of glycolysis is converted into lactate rather than acetylcoa acetyl coenzyme a fortca tricarboxylic acid cycle or the krebs cycletherefore targeting enhanced glycolysis has been proposed as novel options in the prevention and treatmentof human cancers including crc however as a metabolism hub tca cycle is important in both energyproduction and biosynthesis therefore it needs to bereplenished by anaplerotic reactions such as glutaminolysis previously we reported that heat shock transcription factor hsf1 stimulated glutaminolysis toactivate mtor and promote crc development by upregulating the expression of glutaminase gls1 thecritical enzyme in glutaminolysis hsf1 expressionwas increased in crc and had a positive correlationwith shorter diseasefree survival dfs however theupstream mechanism for hsf1 overexpression in crcwas still uncleargene expression can be controlled by multiple processesincluding transcription mrna degradation translationand protein degradation while gene translation and protein degradation have been extensively investigated moreand more studies focused on mrna translation by exploring the effect of noncoding rnas such as micrornasmirnas and new modifications of mrna including n6methyladenine m6a modification [ ] mirnas canform a mirnainduced silencing complex mirisc toposttranscriptionally regulate gene expression by inhibiting capdependent initiation and stimulating mrna deadenylation [ ] on the other hand as one of the mostabundant modifications in mrna m6a modification ofmrnas usually promotes translation by recruiting initiation factors such as eif3 to the ² end of the mrna while mirnas and mrna m6a modifications play a distinct role in mrna translation the interplays betweenthem were not clarifiedin this study we found that activated wntcateninsignaling stimulated hsf1 translation to promote crcdevelopment byrepressing hsf1 mrnatargetingmir4553p to increase m6a modification of hsf1mrna therefore targeting hsf1 translation could be anew strategy for the intervention of crc and other cancers driven by activated wntcatenin signalingmaterials and methodscell antibodies and chemicalshuman crc celllines sw480 sw620 dld1 rkowere obtained from the american type culture collection atcc all cells were routinely cultured in rpmi invitrogen “ or dmem invitrogen“ supplemented with fetal bovine serumall cells were incubated at °c with co2 and humidity the following antibodies were used for western blotting and ihc hsf1 12972s cell signalingtechnology cst ab52757 abcam catenin 8480scst actin l cst flag f1804“ sigmacyclind1 ab134175 abcam cleaved parp1 9541scst mettl3 a8370 abclonal gls1 ap8809babgent pyrvinium p0027 licl cycloheximide r750107 chloroquine c6628 mg132 and pd150606 d5946 were purchased from sigmaaldrichinterfering rna sirnasirna mirna mimicsinhibitors transfectiontargeting cateninsmallmettl3 and micrornas were synthesized by genepharma shanghai china and ribobio guangzhouchina the sequence of these sirnas and mirnaswere listed in additional file table s1 sirnas andmirna mimicsinhibitors were transfected into cellsseeded overnight by lipo2000 invitrogen usa or lipofectamine rnaimax transfection reagentinvitrogenusa according to the manufacturer™s instructionsluciferase activity assaythe plasmid of catenin reporter was gifted from profximei wu zhejiang university for hsf1 activity assaya fragment containing x hse were synthesized andinserted into the pgl3basic vectors promega corporation usa the plasmid was cotransfected with prlrenilla and catenin sirna by using lipo2000 invitrogen usa or treatment with pyrvinium by xtremegene hp dna transfection reagent roche usa ²utr segment of the hsf1 was cloned by pcr andinserted into the vector pmirreporter promegathe mutation of mir455 binding sites in hsf1 ²utrwas generated by quick sitedirected mutagenesis“ stratagene usa the resultant plasmidswere cotransfected with prl renilla and mir455 mimicsby usinglipo2000 invitrogen usa h post 0csong molecular cancer page of transfection the luciferase activity was measured by thedualglo luciferase assay system promega corporation usachromatin immunoprecipitation chipchip analysis was conducted with the simplechip„¢ enzymatic chromatin ip kit cst usa antibodies usedwere antihsf1 12972s cst tcf7l2c48h11 cstand flag f1804“ sigma the primers used for thepcr analysis of precipitated dna were shown inadditional file table s2 for flagchip assay theflagcatenin vector was transiently overexpressed bytransfection after h the enrichment of flagcateninon col27a1 promoter was measured by the chip kitimmunoblotting and immunohistochemistryimmunoblotting and immunohistochemistry ihc assays were performed as previously reported for immunoblotting total proteins were extracted with ripabuffer supplemented with protease inhibitors rocheusa after heating the protein sample to “ °c for min celllysates were transferred to polyvinylidenefluoride pvdf membranes after the membranes wereblocked in milk primary antibody with gentle agitation overnight at °cfor ihc assay was performed in a tissue array containing cases of colonic tissues primary antibodies usedwere listed above the degree of immunostaining wasassessed by independent pathologists and evaluated byassigning a score of “ scores were defined as follows no staining faint staining moderate staining and strong staining final scores of and were regardedas low expression whereas scores of and consideredas high expressionapoptosis detectioncell apoptosis was measured by flow cytometry analysisand western blotting for flow cytometry analysis ofapoptosis cells were harvested and resuspended in μl x binding buffer μl fluorescein isothiocyanatefitc annexin v and propidium iodide pi bd biosciences usa were added to the cell suspensionand then incubated for min at room temperatureafter that the samples were attenuated with 400ul xbinding buffer and analyzed by acs caliburflowcytometerpuromycinlabellingto detect the change of nascent hsf1 synthesis x cells were plated in cm dish afterthe giventreatment cells were incubated with biotindcpuromycin nu925bios jena bioscience for hcells were lysed with np40 buffer mm trishclph mm nacl np40 glycerolcontaining 1x protease inhibitor cocktail after adequatecentrifugation the supernatant was incubated with 80ulstreptavidin sepharose beads ge17“ sigma byrotating at °c for h to overnight the mixture waswashed by np40 buffer for times and subjected towestern blotting using hsf1 antibodyseparatestranslatingpolysome profilingpolysome profilingor nontranslating mrnas on a sucrose gradient according tothe number of bound ribosomes as previously described in brief cells were grown to confluence before collection cells were incubated with μgml ofcycloheximide for min then cells are lysed by polysome buffer [ mmoll kcl mmoll mgcl2 triton x100 μgml cycloheximide mmoll heparin and uml rnase inhibitor takara 1x cocktail] for min on ice lysates were centrifuged rpm for min and the supernatant was layered onto a to sucrose gradient gradients were then centrifuged at rpm for min at °c and polysomebound fractions were collected using an isco densitygradient fractionation system isco lincoln ne withcontinuous monitoring based on a260nm wavelengththe rna in each fraction was extracted using trizol reagent invitrogen and analyzed by realtime rtpcrbiotin pull down assaybiotin pull down assay was performed as described previously cells were transfected with biotinylatedmir4553p probes for h and resuspended using lysisbuffer mm tris ph mm nacl mmmgcl2 uml superasein mm dtt igepal protease inhibitors lysates were incubated withprepared streptavidin beadsge healthcare yeasttrna sigma was used for blocking lysates at °c for h then washed times with binding and wash buffer mm trishcl ph mm edta m naclfinally the bound rnas were extracted and purified forqpcrrna immunoprecipitation rip assayrip assay was performed by magna riptm rnabinding protein immunoprecipitation kitmilliporeno17“ briefly × cells were lysed in μlrip lysis buffer and immunoprecipitated with antibodiesofinterest and protein g magnetic beads at °covernight followed by six times of washes in washingbuffer and protein digestion at °c total rna wasisolated and subjected to rtpcr analysis followingantibodies were used for rip n6methyladenosine synaptic mettl3 a8370 abclonal igg millipore 0csong molecular cancer page of rnasequencing2x106 wt mef and hsf1 ko mef were plated andcultured overnight following day cells were treatedwith mm licl for h cells were collected with trizol reagent the total rna was processed by nebnext®polya mrna magnetic isolation module to enrichmrna and the product rna was used for constructionlibrary via kapa stranded rnaseq library prep kitillumina sequencing libraries denatured by mnaoh to generate singlestranded dna as amplified insitu illumina cbot truseq sr cluster kit v3cboths gd4013001 illumina the ends of the generatedfragments were used to run cycles by the illuminahiseq sequencer all the experimental steps afterthe rna extraction were conducted in kangcheng biotechnology co ltd aksomics shanghai china rnasequencing was performed three timesanimal experimentsanimal care and experiments were conducted in compliance with institutional animal care and use committeeand nih guidelines the c57bl6 j mice and apcminmice were purchased from model animal research center of nanjing university marc nanjing chinahsf1 ko mice reported previously were used to generate apcmin mice hsf1ˆ’ subsequently groupsof mice wild type apcmin apcmin hsf1ˆ’ and apcmin treated with knk437 as previously reported were fed with highfat diet kcal fat for monthsthe intestine was dissected flushed with pbs and cutopen longitudinally along the main axis the number oftumors was counted and the sizes oftumors weremeasuredstatisticsall data were expressed as mean ± sd unless specifiedthe student™s ttest was performed for statistical significance analysis p value was considered as statistically significantresultscatenin activates hsf1 in crcin an effort to explore potential regulations of hsf1 wescreened chemicals generating a gene expression patternsimilar to hsf1 depletion by connective map httpportalsbroadinstitutecmapinterestingly a recently reported inhibitor of wntcateninsignaling pyrvinium had a similar effect on genomewide gene expression as hsf1 depletion fig 1aand additional file figs1ab moreover the expression signature related to wntcatenin signaling was positively correlated with the hsf1 signature fig 1b indicating a potential connection of hsf1 withwntcatenin signaling indeed pyrvinium attenuated[ ]the activity of a luciferase reporter driven by hsf1 binding sites hse heat shock response elements [ ]fig 1c and reduced the expression of wellknown transcriptional targets of hsf1 such as hsp90aa1 hspa4hspb1 and hsph1 fig 1d and additionalfile figs1c chromatin immunoprecipitation chip assayfurther confirmed the reduced interaction of hsf1 withits transcriptional targets fig 1e and additional file figs1d in consistence with pyrvinium knockdown ofcatenin by sirna also decreased hsf1 activity fig1f reduced the expression of hsf1 targets fig 1g andadditional file figs1e and attenuated the interactionof hsf1 with its targets fig 1i and additional file figs1f furthermore hsf1 targets were upregulated bythe potent gsk3 inhibitor licl in colorectal cancer cellline rko which had a low level of catenin expressionfig 1hto explore the biological relevance of hsf1 activationto catenin signaling we profiled gene expression ofwild type mouse embryonic fibroblasts wt mef andhsf1 knockout mef hsf1 ko mef before and afterlicl treatment ncbi geo gse151119 while only genes were upregulated in licl treatedhsf1 komef there were genes significantly upregulated inwt mef after licl treatment fig 1j among them genes displayed a dependence on hsf1 since theirexpression levels failed to be upregulated by licl treatment once hsf1 was depleted fig 1k in fact their expression levels had a high correlation with theexpression of a previously reported hsf1 signature fig1l furthermore genes had a hse heatshock response element hse within their promoter regions fig 1m and additional file meaning that theyare most likely bona fide targets of hsf1 indeed someof them such as tma16 dedd2 hspa9 and kif21a havebeen confirmed as the target of hsf1 by chipseqncbi geo gse57398 fig 1n and additional file figs1g taken together these results indicated that catenin can positively regulate hsf1catenin stimulates hsf1 protein translationto delineate how catenin regulates hsf1 we quantitated protein levels of hsf1 before and after inhibitingcatenin both pyrvinium and catenin depletion reduced the protein level of hsf1 fig 2a and b in contrast overexpression of exogenous catenin increasedhsf1 protein level fig 2c furthermore hsf1 proteinlevel was increased after activating wntcatenin signaling by licl treatment in both rko and mef cellsfig 2d in addition hsf1 expression correlates wellwith catenin expression in primary tissues fig 2e andf p chisquare test all of these data indicatedthat catenin upregulates hsf1 protein expression 0csong molecular cancer page of fig wntcatenin signaling activates hsf1 a chemicals influencing gene expression in a similar manner to hsf1 inhibition were screened byconnective map analysis b the correlation of wntcatenin signaling signature and hsf1 signature was detected by gepia c the effect ofpyrvinium on hsedriven promoter activity was explored by luciferase reporter assay d the effects of pyrvinium on the targets of hsf1 wereanalyzed by rtpcr e binding of hsf1 to the promoters of hsf1 targets in crc cells treated with or without pyrvinium was determined by chipf the luciferase assays of hse before and after catenin knockdown were shown as in c g and h the mrna levels of hsf1 targets with catenin knockdown or licl treatment were analyzed by rtpcr i binding of hsf1 to its targets promoter in crc cells before and after cateninknockdown was analyzed by chip j volcano plot displays differentially regulated genes in dhsf1 compared to wt parental cells with licl reddots indicate significantly regulated genes based on adjusted pvalue and logfold change logfc p log2fc k differential geneexpression analysis in wt and hsf1ˆ’ mef treated with licl were performed by rnaseq numbers of upregulated genes in two cells wereshown in venn graph l the correlation of putative hsf1dependent genes from k with reported hsf1 signature was detected by gepia mnumbers of putative hsf1dependent genes with or without hse in their promoters were summarized n representative hsf1 chipseqtracks ncbi geo gse57398 for hsecontaining genes are shown asterisks indicate p 0csong molecular cancer page of fig catenin stimulates hsf1 protein translation a the effect of pyrvinium on the protein expression of hsf1 was explored by westernblotting b the effect of catenin knockdown on hsf1 protein level was analyzed by western blotting c the protein level of hsf1 before andafter catenin overexpression was analyzed by western blotting d the effect of licl on hsf1 protein level in rko and mef was analyzed bywestern blotting e the expression of catenin and hsf1 in colorectal tissue was analyzed by immunohistochemistry staining f the correlationbetween catenin expression and hsf1 expression in colorectal tissue was analyzed by chisquare test p g the effect of catenindepletion on hsf1 with puromycin labeling was determined by western blotting h amount of hsf1 mrna in various polysome fractions wasanalyzed by rtpcrp however there were no apparent alterations in hsf1mrna level after catenin knockdown or pyrviniumtreatment additional file figs2a and s2b meanwhile the halflife of hsf1 protein was also not changedbefore and after catenin knockdown additional file figs2c inhibitors of proteasome autophagy and calpains all failed to reverse hsf1 protein downregulationinduced by catenin knockdown additionalfile 0csong molecular cancer page of figs2d all of these results implied that catenin affects hsf1 protein expression mostlikely via translationregulation therefore puromycin labeling assay wasemployed to monitor the synthesis of nascent hsf1 protein as expected the puromycin labeling of hsf1was reduced by catenin depletion fig 2g to furtherconfirm it monopolysome fractions from cytoplasmicextracts of crc cells before and after catenin depletion were collected by sucrose gradient centrifugationthe subsequent rtpcr analysis revealed that catenindepletion considerably reduced the presence of hsf1mrna in the polysome fraction but increased in nontranslating ribosome fractions fig 2h in summary catenin upregulates hsf1 expression by stimulatinghsf1 protein translationhsf1 protein translation is regulated by mir4553pas micrornas mirnas play an important role inregulating the efficiency of protein translation we wondered whether hsf1 protein translation was regulatedby micrornas based on bioinformatics screening bytargetscan mirdb and starbase some micrornas including mir4553p mir2145p mir4315p mir184mir4903p and mir375 were proposed to target ²utr of hsf1 mrna fig 3a after functional validation by western blotting mir4553p and mir2145pbut not other micrornas were capable to suppress theexpression of hsf1 protein in crc cells fig 3b andadditional file figs3a however mir2145p but notmir4553p also reduced hsf1 mrna level additionalfile figs3b what™s more an inhibitor of mir4553prather than mir2145p rescued the downregulation ofhsf1 protein by catenin knockdown fig 3c and additional file figs3c indicating that mir4553p mightbe relevant to catenininvolved regulation of hsf1protein translation indeed mir4553p inhibited the activity of luciferase driven by wild type hsf1 mrna ²utr but not its mutant unable to bind mir4553p fig3d the interaction of mir4553p with hsf1 mrnawas further confirmed by biotin pull down assay fig3e based on the analysis of tcga data httpmirtvibmssinicaedutwthe expression of mir4553p islower in colon adenocarcinoma than in normal tissuesadditional file figs3d similarly qpcr analysis revealed lower levels of mir4553p in crc tissues than inadjacent nontumor tissues fig 3f additionally wehad confirmed high expression of hsf1 in the same cohort of human crc tissues previously indeedmir4553p expression was negatively correlated with theexpression of hsf1 fig 3g on the other handmir4553p similar to hsf1 inhibition as we reportedrecently reduced the expression of hsf1 targets induced the viability inhibition and apoptosis activation ofcolorectal cancer cells fig 3hj and additional file figs3eg the seed sequence of micrornas was important for targeting mrna by basepairing indeed the seed sequence mutant of mir4553p could notdownregulate the protein level of hsf1 additional file figs4a confirming the importance of mir4553p totarget hsf1 protein expression in a word mir4553ptargets hsf1 mrna ²utr to inhibit its translationm6a modification of hsf1 mrna stimulates its proteintranslationin addition to microrna mrna modifications such asn6methyladenosine m6a play important roles in theregulation of hsf1 translation interestingly we noticedthat the matching sites of mir4553p seed sequence inhsf1 mrna ²utr contains a typical motif of m6amodification fig 4a which was supported by bioinformatic analysis httpwwwcuilabcnsramp fig 4b andadditional file figs4b moreover we have done themerip sequencing in sw620 and found that the ²utr region of hsf1 has one m6a modification site intriguingly this sequence is completely complementary tothe seed sequence of mir4553p additionalfile figs4c pcr analysis after merip m6a rna immunoprecipitation further confirmed m6a modification ofhsf1 mrna fig 4c what™s more the activity of luciferase driven by the mutant hsf1 mrna ²utr whichwas unable to bind mir4553p but retains the m6amodification site sequence drach d a g or u r a or g h a u or c [“] was higher than theactivity of luciferase driven by wild type hsf1 mrna²utr fig 3dindicating the importance of m6amodification to hsf1 expression as the main component of the methyltransferase œwriter complex [ ]mettl3 was also bound to hsf1 mrna fig 4donce its expression was depleted m6a modification ofhsf1 mrna was decreased fig 4e in consistencewith its potential roles in promoting protein translationsuch a reduction of hsf1 mrna m6a modification reduced hsf1 protein expression fig 4f and nascenthsf1 protein synthesis fig 4g furthermore mettl3depletion considerably reduced the presence of hsf1mrna in the polysome fraction but increased in nontranslating ribosome fractions fig 4h while hsf1mrna or the stability of hsf1 protein were not changed additionalfile figs4d and s4e moreoverhsf1 protein was decreased with the knockdown ofythdf1 which was the reader protein of hsf1 m6amodification fig 4i to sum up m6a modification ofhsf1 mrna was relevant to stimulate its translationcatenin suppresses mir4553p to increase hsf1 mrnam6a modificationnext we explored the interplay between mir4553p andm6a modification of hsf1 mrna both hsf1 mrna 0csong molecular cancer page of fig hsf1 protein translation is regulated by mir4553p a overlap of hsf1targeting micrornas predicted by targetscan mirdb and starbaseb the effect of mir4553p on hsf1 protein was analyzed by western blotting c the effect of mir4553p inhibitor on catenin knockdowninduced hsf1 downregulation was determined by western blotting d luciferase activity assay was used to analyze the effect of mir4553p onthe activity of ²utr with or without mir4553p binding sites p e the binding between biotinmir4553p and hsf1 mrna wasdetermined by biotin pull down assay p f expression of mir4553p in pairs of fresh crc tissues and adjacent nontumor tissues wasanalyzed by qpcr g the correlation of hsf1 protein and mir4553p in pairs of fresh crc tissues and adjacent nontumor tissues wasanalyzed h the effect of mir4553p on viability of crc cells was explored by mts assay i the effect of mir4553p on apoptosis of crc cells wasanalyzed using flow cytometry after pi and annexin vfitc double staining j apoptosis of crc cells treated with or without mir4553p wasdetermined by western blottingm6a modification and its binding to mettl3 were decreased by the overexpression of wild type mir4553pbut not its mutant unable to bind to hsf1 mrna ²utr figfigs5aand additional5afileinterestingly mettl3 depletion not only reduced m6amodification of hsf1 mrna fig 4e but also enhanced the interaction of mir4553p with hsf1 mrnafig 5b and additionalfile figs5b while the 0csong molecular cancer page of fig m6a modification of hsf1 mrna stimulates its protein translation a the sites of hsf1 ²utr binding to the seed sequence of mir4553pwas consistent with m6a rna modification elements œdrach b bioinformatic prediction of m6a modification in ²utr of hsf1 mrna c m6amodification of hsf1 mrna was analyzed by merip p d binding of mettl3 to hsf1 mrna was detected by rip p e m6amodification of hsf1 mrna with or without mettl3 depletion was analyzed by merip p f the protein level of hsf1 before and aftermettl3 depletion was detected by western blotting g the effect of mettl3 knockdown on hsf1 synthesis was determined by western blottingafter puromycin labeling h amount of hsf1 mrna in various polysome fractions was analyzed by rtpcrp i the effect of ythdf1knockdown on hsf1 protein level was analyzed by western blottingexpression of mature and primary mir4553p was notupregulated additional file figs5cd these resultsindicated that mir4553p may compete with mettl3for the m6a modification of hsf1 mrna thus inhibitinghsf1 protein translation furthermore the binding ofmir4553p to hsf1 mrna was not changed by ythdf1deletion additional file figs5e indicating that thetranslation repression of hsf1 mrna was more likely tobe mediated directly by the reduced m6a modification ofhsf1 0csong molecular cancer page of fig catenin suppresses mir4553p to increase hsf1 mrna m6a modification a m6a modification and mettl3 interaction of hsf1 mrnawith wt or mutant of mir4553p were analyzed by rip b the interaction between biotinmir4553p and hsf1 mrna with or without mettl3depletion was analyzed by biotin pull down c the effect of mir4553p inhibitor andor catenin knockdown on m6a modification of hsf1mrna was analyzed by merip d the effect of mir4553p inhibitor andor catenin knockdown on the interaction of mettl3 with hsf1 mrnawas analyzed by rip e the effect of catenin knockdown on interaction of mir4553p and hsf1 mrna was analyzed by biotin pull down f andg the levels of mature f and primary g mir4553p with catenin knockdown or licl treatment were determined by rtpcr h the correlationof col27a1 and mir455 was analyzed in linkedomics httplinkedomics i the effect of catenin depletion or licl on mrna level ofcol27a1 was analyzed by rtpcr j the interaction of catenintcf7l2 and hsf1 promoter was determined by chip k the correlation of catenin protein expression with the rna level of col27a1 was detected by linkedomics httplinkedomicsindeed mir4553pcatenindepletioninduced decrease of hsf1 mrna m6a modification fig 5c and additional file figs5f meanwhilethe interaction of mettl3 with hsf1 mrna wasinhibitorrescuedabrogated by depleting catenin fig 5d and additionalfile figs5g accompanied by the increased interactionof mir4553p to hsf1 mrna fig 5e and upregulationof mature fig 5f and additionalfile figs5h 0csong molecular cancer page of precursor additional file figs5i and primary mir4553p fig 5g and additional file figs5j in contrastwhen catenin was upregulated by overexpression or licltreatment both mature mir4553p fig 5f and additionalfile figs5h and primary mir4553p fig 5g and additional file figs5j were downregulatedprimary mir4553p was derived from a premirna hairpin encoded in intron of the collagen gene col27a1 additional file figs5k actually col27a1 expression was significantly correlated with the expression ofmir455 httplinkedomics fig 5h consistent withthis we observed col27a1 mrna levels were increasedupon catenin depletion while decreased after licl treatment fig 5i and additional file figs5l moreover catenintcf7l2 complex could interact with the promoterof col27a1 while the pair of primers negativechipprimer at a position far away from the promoter region couldnot enrich col27a1 fig 5j and additionalfile figs5m meanwhile the protein expression of cateninwas negatively correlated with rna level of col27a1httplinkedomics fig 5k these results indicatedthat the transcription of col27a1 was inhibited by wntcatenin signalingleading to decreased biogenesis ofmir4553p therefore catenin facilitates the shift frommir4553p binding to m6a modification in hsf1 mrnaby suppressing mir4553p expression eventually promoting hsf1 protein translationboth genetic and chemical inhibition of hsf1 attenuatecolorectal carcinogenesis in micein light of these in vitro findings we further exploredthe relevance of hsf1 to colorectal carcinogenesis inapcmin and apcmin hsf1ˆ’ mice since the interaction of mouse mir4553p and mouse hsf1 mrnaseems to be well conserved mouse mir4553p seed sequence cagucca the binding site in mouse hsf1mrna ²utr tggactg the expression of hsf1 andits downstream target gls1 were increased whilemir4553p expression was reduced in intestine tissuesfrom apcmin mice compared with normal c57bl6mice fig 6a and b after fed with highfat diet for months these apcmin mice developed multiple tumorsin the intestine fig 6c however both the size andnumber of tumors were significantly reduced in apcminmice treated with a chemical inhibitor of hsf1 knk437and apcmin hsf1ˆ’ mice fig 6d accompanied bythe downregulation of hsf1 targets fig 6e all of theseresults confirmed that hsf1 is a novel downstream target of wntcatenin signaling important to promotecrc developmentdiscussiongenetic changes in components of wntcatenin signaling such as deletion of the apc gene and ctnnb1mutations have been frequently detected in many typesof human cancers all of these muta
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Rapid repair of human disease‘specific single‘nucleotide variants by One‘SHOT genome editingYuji Yokouchi12 Shinichi Suzuki2 Noriko Ohtsuki12 Kei Yamamoto12 Satomi Noguchi12 Yumi Soejima3 Mizuki Goto34 Ken Ishioka5 Izumi Nakamura12 Satoru Suzuki6 Seiichi Takenoshita7 takumi era123Many human diseases ranging from cancer to hereditary disorders are caused by single‘nucleotide mutations in critical genes Repairing these mutations would significantly improve the quality of life for patients with hereditary diseases However current procedures for repairing deleterious single‘nucleotide mutations are not straightforward requiring multiple steps and taking several months to complete In the current study we aimed to repair pathogenic allele‘specific single‘nucleotide mutations using a single round of genome editing Using high‘fidelity site‘specific nuclease AsCas12aCpf1 we attempted to repair pathogenic single‘nucleotide variants SNVs in disease‘specific induced pluripotent stem cells As a result we achieved repair of the Met918Thr SNV in human oncogene RET with the inclusion of a single‘nucleotide marker followed by absolute markerless scarless repair of the RET SNV with no detected off‘target effects The markerless method was then confirmed in human type VII collagen‘encoding gene COL7A1 Thus using this One‘SHOT method we successfully reduced the number of genetic manipulations required for genome repair from two consecutive events to one resulting in allele‘specific repair that can be completed within weeks with or without a single‘nucleotide marker Our findings suggest that One‘SHOT can be used to repair other types of mutations with potential beyond human medicineThe human genome contains extensive variation including an estimated — singlenucleotide variants SNVs that determine how we look and function as well as our specific disease tendencies1“ Some SNVs are pathogenic and either directly or indirectly cause hereditary disorders4“ such as multiple endocrine neoplasia type 2B MEN2B7 and dystrophic epidermolysis bullosa DEB8 MEN2B is an autosomal dominant syndrome characterised by thyroid adrenal gland and neuronal tumours and skeletal abnormalities The majority of MEN2B cases result from a single aminoacid substitution Met918Thr in the RET protooncogene which is caused by a pathogenic SNV RET c2753TC at the second base of the codon7 DEB is an inherited disease characterised by severe recurrent skin ulcers and blistering It is caused by genetic mutations in the human type VII collagenencoding gene COL7A1 the product of which is an anchoring fibril connecting the epidermis to the dermis8 To model diseases such as these in a0vitro diseasespecific induced pluripotent stem cells iPSCs carrying pathogenic SNVs or other genetic mutations can be obtained from patients9“ Repairing these iPSCs to generate isogenic revertant cells is a promising strategy for genome repair and s up new avenues for drug 1Pluripotent Stem Cell Research Unit in Department of Thyroid and Endocrinology School of Medicine Fukushima Medical University Hikarigaoka Fukushima Japan 2Department of Thyroid and Endocrinology School of Medicine Fukushima Medical University Fukushima Japan 3Department of Cell Modulation Institute of Molecular Embryology and Genetics IMEG Kumamoto University Kumamoto Japan 4Department of Dermatology Faculty of Medicine Oita University Yufu Japan 5Department of Microbiology School of Medicine Fukushima Medical University Fukushima Japan 6Office of Thyroid Ultrasound Examination Promotion Radiation Medical Science Centre for the Fukushima Health Management Survey Fukushima Medical University Fukushima Japan 7Fukushima Medical University Fukushima Japan email yokouchyfmuacjpScientific RepoRtS 101038s41598020704017Vol0123456789wwwnaturecomscientificreports 0c discovery1314 However the repair process remains problematic and a precise and convenient genome editing procedure has not yet been developedArtificial genome repair andor modification generally starts from a targetspecific doublestrand break generated by sitespecific nucleases1516 Doublestrand breaks are then repaired by a cell™s own genome repair machineries1516 However most of the break sites are incorrectly repaired by nonhomologous end joining and can result in gene knockout through the generation of nonspecific insertions or deletions indels1718 If a repair template carrying a repair base is coadministrated however the cleavage sites can be accurately repaired via homologydirected repair HDR1516 Sitespecific nucleases such as transcription activatorlike effector nucleases or the Streptococcus pyogenes Sp Cas9 nuclease are typically used as genome editing tools for human iPSCs19“SpCas9 is a type IIA endonuclease in the class clustered regularly interspaced short palindromic repeats CRISPRCas system that has been repurposed as a programmable sitespecific nuclease for genome engineering22“ Indeed SpCas9 has become a popular genome editing tool for genetically modifying human pluripotent stem cells19 Despite its efficient cleavage activity wildtype SpCas9 has a low DNA repair rate using HDR following plasmidbased administration It recuts the repaired site because the guide RNA has a “2base mismatch tolerance during sequence recognition leading to incorrect repair by nonhomologous end joining1718 To prevent recutting a blocking mutation must be introduced into the seed sequence of the guide RNA or into the protospaceradjacent motif PAM26“However for œscarless genome editing repairs with wildtype SpCas92749 each method has its obvious strengths and weaknesses For example CORRECT which includes excellent tricks to prevent recutting of the edited target by the editing tool can be performed even if the target recognition ability of the genome editing tool is insufficient however the need for two consecutive editing steps27 In comparison MhAX has high genome editing efficiency but cannot achieve completely scarless editing because singlebase markers are required Further as with the CORRECT method MhAX editing requires two consecutive edits49 increasing cost and time requirementsAnother recentlyidentified bacterial programmable sitespecific nuclease CRISPRCas12aCpf1 is a type VA endonuclease belonging to the class CRISPRCas system32 Among identified Cas12a enzymes those from Acidaminococcus sp BV3L6 As and Lachnospiraceae bacterium ND2006 show strong cleavage activity in mammalian cells32 These Cas12a endonucleases have unique features that complement Cas9 and expand the genome editing range First Cas12a recognises a Trich PAM upstream of the protospacer whereas Cas9 recognises a Grich PAM downstream of the protospacer32 Second two PAMinteracting variants have been generated that expand the Cas12a target range3334 Third Cas12amediated cleavage generates a staggered cut on the PAMdistal region of the target sequence as opposed to the PAMproximal blunt ends generated by Cas932 Finally and perhaps most importantly the guide or CRISPR RNA crRNA exhibits highfidelity target recognition meaning that Cas12a can precisely distinguish the target sequence at a singlebase resolution3536 Consequently the resulting offtarget effects are kept to a background level These features suggest that Cas12a might be suitable for precise diseasespecific iPSC repair because its recut activity is lowCas12a has already been used to knock out pathogenic genes in cancer cells37 generate insertions or twonucleotide substitutions in iPSCs38 and to induce exonskipping in diseasespecific iPSCs39 Thus we investigated whether Cas12a could be used to carry out allelespecific singlenucleotide repair of iPSCs carrying the pathogenic SNVs found in MEN2B and DEB patients in a single round of genome editing To accomplish this we used an AsCas12a PAM variant and singlenucleotide mismatch detection polymerase chain reaction SNMDPCR analysis in diseasespecific iPSCs to develop a precise convenient genomeediting procedure we have called OneSHOT One allelespecific single HDR and singlestranded oligodeoxynucleotide ssODN transient drug selection with SNMDPCR screening OneSHOT provides scarless singlenucleotide substitution of a pathogenic SNV in diseasespecific iPSCs within a0weeks The final modification rate is within a practical range for handpicking cloning Our findings suggest that this simple low cost procedure could be used for genome editing in a single step drastically reducing the time currently needed for scarless SNV repairResultsPrinciples of One‘SHOT repair of single‘nucleotide mutations AsCas12a is a highfidelity RNAguided sitespecific nuclease that binds to the target genomic DNA site via a 20nt guide sequence in the crRNA allowing it to discriminate the target sequence at the single nucleotide level Fig a0 Following the addition of a crRNA designed for a specific target sequence containing a singlenucleotide mutation AsCas12a selectively binds to the target sequence on the mutant allele and induces a doublestrand break leaving the wildtype sequence on the alternative allele unaffected Fig a0 In the presence of a ssODN wildtype sequence template the mutant nucleotide in the target sequence can be œrepaired to the wildtype sequence via the cellular HDR machinery To mark the repaired allele we labelled the ssODN with a singlenucleotide marker in the vicinity of the mutant nucleotide This label allowed us to easily identify the generepaired clones by allelespecific amplification40“42SNMDPCR detection of the singlenucleotide marker Fig a0 A complete outline of the OneSHOT workflow for SNV repair is provided in the Supplementary Information and in Supplementary Fig a0S1Allele‘specific single‘nucleotide substitution in MEN2B‘specific iPSCs Before carrying out allelespecific singlenucleotide repair of the pathogenic RET mutation we assessed whether the OneSHOT approach could be used to accomplish allelespecific singlenucleotide substitution of the wildtype alleleWe established FB414 human iPSCs from a patient with MEN2B using a Sendai viral vector protocol43 We then confirmed that the FB414 cells exhibited an embryonic stem celllike morphology and expressed pluripotent gene markers indicating that they were authentic iPSCs Supplementary Fig a0S2 and X7 To identify Scientific RepoRtS 101038s41598020704017Vol1234567890wwwnaturecomscientificreports 0cguide seqcrRNAHigh Fidelity SiteSpecific Nuclease SSN AsCas12aCpf1target alleleDSBTYCValleleTYCVHDRTYCVTYCVby SNMDPCR ntssODN w markerFigure a0 OneSHOT principles AsCas12a pale yellow and crRNA orange and grey lines selectively bind to a target sequence containing a pathogenic SNV red triangle on the target allele Binding leads to a doublestrand break in the target sequence on the target allele left but not in the corresponding wildtype sequence containing the wildtype nucleotide blue triangle on the alternative nontarget wildtype allele right When the ssODN repair template bluegreen line with the wildtype nucleotide blue triangle and a singlenucleotide marker a silent mutation for SNMDPCR screening green triangle is cotransfected with AsCas12a into the cells the target site on the pathogenic allele is repaired using the template by the endogenous HDR machinery In this case the intended geneedited clones are easily identified by positive screening for the singlenucleotide marker because the repaired expathogenic allele now carries the singlenucleotide markerpossible target sites for AsCas12a around the SNV of interest we first searched for PAMs recognised by wildtype AsCas12a or the RR and RVR variants which recognise TYCV and TATV PAMs respectively3334 We identified two PAM sites for the RR variant TYCV Y CT V ACG TTCC located 12bp upstream of the target nucleotide on the sense strand and TTCA located 7bp upstream of the target nucleotide on the antisense strand Fig a02a magenta lines Based on this information we designed two pairs of crRNAs crRNA_RET1 and crRNA_RET1 a0m and crRNA_RET2 and crRNA_RET2 a0m which contain guide sequences that specifically recognise wildtype and mutant target sequences respectively Fig a02aTo test the cleavage activity and targetrecognition specificity of AsCas12a_RR using these crRNAs we performed a T7E1 assay using 409B2 human iPSCs carrying the wildtype RET sequence in the target site Fig a02a middle The crRNAs for the wildtype sequence crRNA_RET1 and crRNA_RET2 each exhibited significant cleavage activity towards the wildtype target sequence Fig a02b P and P respectively By contrast the crRNAs for the mutant sequence crRNA_RET1 a0m and crRNA_RET2 a0m showed extremely weak activity Fig a02b P for both A more accurate ICE analysis showed no significant activity of the crRNAs on the WT allele Supplementary Fig a0X2a These results indicate that the crRNAs for the mutant sequence do not have significant if any activity on the WT alleleHowever the observed cleavage activity of AsCas12a_RR in conjunction with crRNA_RET1 was significantly higher than that with crRNA_RET2 Fig a02b P Supplementary Fig a0X2a Puromycin treatment further promoted the cleavage activity of AsCas12a_RR with crRNA_RET1 Fig a02b P To test the applicability of the method to carry out allelespecific singlenucleotide substitution in human iPSCs we attempted to replace the wildtype nucleotide RET c2753T at the Met918 site in the wildtype allele in FB414 MEN2BiPSCs Fig a02c Following electroporation of the pY211puro vector which expresses AsCas12a_RR and crRNA_RET1 Fig a02c blue line and a ssODN modification template ssODN_RET_M918T_I913silentC carrying both a variant nucleotide at Met918 and a singlenucleotide marker at Ile913 Fig a02c red C and light green C respectively into FB414 cells we conducted SNMDPCR screening Overall clones were positive for the substitution Fig a02c d and GE1 in Table a0 Direct sequencing of the target sequence revealed that clones contained the wildtype allelespecific introduction of the mutant nucleotide at the target site T C substitution resulting in the Met918Thr substitution Fig a02e red arrow along with the singlenucleotide marker T C substitution leading to a silent mutation at Ile913 Fig a02e blue arrow The HDR efficiency was Table a0We then searched for offtarget sequences corresponding to the target sequence using the web tool CHOPCHOP v244 and detected no indels in either of the predicted two offtarget sites by Sanger sequencing Table a0 GE1 and by AmpliSeq Supplementary Table a0X4 These results indicated that the OneSHOT method could be used to replace a single nucleotide in an allelespecific manner while minimising offtarget effects As in the preliminary experiment direct sequencing analysis around the target sites revealed no duplication events in the unintended geneedited clones suggesting that most of the intended geneedited clones had clonally proliferated Supplementary Fig a0S3 GE1Scientific RepoRtS 101038s41598020704017Vol0123456789wwwnaturecomscientificreports 0c–¸Figure a0 Singlenucleotide substitution of the RET wildtype sequence in MEN2B iPSCs a Human RET locus containing the MEN2B mutation and crRNA of AsCas12a_RR for the mutation Top exon of the RET locus Middle the wildtype WT allele sequence Blue letters indicate the wildtype nucleotide at Met918 underlined Bottom the mutant allele sequence Red letters indicate the single missense mutation caused by a TC substitution producing a Met918Thr substitution underlined Coloured lines indicate the guide sequence template for the crRNA The pink line indicates the AsCas12a_RR PAM Coloured dashed lines indicate the sites cleaved by AsCas12a_RR with the corresponding crRNA b T7E1 assay using human wildtype iPSCs 409B2 electroporated with AsCas12a_RR and the different crRNAs crRNA_RET1 crRNA_RET1 a0m crRNA_RET2 or crRNA_RET2 a0m targeting exon Left the cropped gel images Arrowheads indicate cleaved bands The fulllength gels are presented in Supplementary Figure a0S7 Right statistical analysis of the cleavage activity and specificity of AsCas12a_RR with the crRNAs following selection with different concentrations of puromycin c HDRmediated editing for generating artificial homozygous MEN2B using AsCas12a_RR with crRNA_RET1 selectively targeting RET_Met918 in the wildtype allele d SNMDPCR analysis of the first round of screening The cropped gel image is shown here The arrowhead indicates positive PCR amplicon a0bp The fulllength gel is presented in Supplementary Figure a0S8 e Sequencing of the original and modified MEN2B iPSCs FB414 Top original sequence of RET exon with a T C substitution in the MEN2B mutant allele Bottom the modified RET sequence The T C substitution resulting in a homozygous Met Thr substitution Red and blue arrows indicate the positions of the pathogenic SNV and the singlenucleotide marker respectively Underlining indicates the codons affected by the editing A more detailed explanation is provided in the œExtended Figure Legends in the Supplementary InformationAllele‘specific single‘nucleotide repair of a pathogenic RET variant To repair the pathogenic SNV RET c2753TC in the mutant allele in FB414 cells we first tested the cleavage activity and target recognition specificity of AsCas12a_RR using crRNA_RET1 a0m and crRNA_RET2 a0m Fig a02a in a homozygous MEN2B iPSC line with mutations in RET exon in both alleles GE19 genotype RETMet918ThrMet918Thr RETIle913 silentC Fig a02e bottom The T7E1 assay confirmed that the MEN2B target sequence was selectively cleaved by AsCas12a_RR with either crRNA_RET1 a0m or crRNA_RET2 a0m but not with crRNA_RET1 or crRNA_RET2 Fig a03a The ICE analysis revealed that only the AsCas12a_RR with crRNA_RET1 a0m exhibited strong cleavage activity on the target sequence Supplementary Fig a0X2b therefore we selected the crRNA_RET1 a0m for use in subsequent experimentsWe then carried out OneSHOT repair in FB414 cells using AsCas12a_RR with crRNA1m and a ssODN repair template containing a repair nucleotide at Met918 and a singlenucleotide marker at Ile913 Fig a03b T in blue and C in light green respectively Subsequent SNMDPCR screening showed that clones were positive Fig a03c GE2 in Table a0 while direct sequencing confirmed that of the positive clones contained the introduced wildtype nucleotide at the target site C T substitution leading to a Thr918Met substitution repair Fig a03d red arrow These clones also contained the singlenucleotide marker T C substitution leading to a silent mutation at Ile913 Fig a03d blue arrow The overall HDR efficiency was Table a0 GE2 and we detected no offtarget effects by Sanger sequencing Table a0 GE2 and by AmpliSeq Supplementary Table a0X4 As in the preliminary experiment direct sequencing analysis around the target sites revealed no duplication events in the unintended geneedited clones suggesting that most of the intended geneedited clones had clonally proliferated Supplementary Fig a0S3 GE2Allele‘specific single nucleotide repair of a pathogenic variants in RET and COL7A1 without a single‘nucleotide marker We next investigated whether the OneSHOT method could be used to repair the pathogenic SNV in RET without including the singlenucleotide marker which would achieve true scarless repair We therefore performed OneSHOT repair in the FB414 cells using AsCas12a_RR crRNA_RET1 a0m and the ssODN repair template with only a wildtype nucleotide at Met918 In the subsequent SNMDPCR screening for the pathogenic SNV no amplicons were obtained from repaired clones because the pathogenic SNV was lost from the mutant allele Fig a04a Overall we identified negative clones by SNMDPCR screening for the pathogenic SNV and direct sequencing revealed that carried only the wildtype nucleotide at Met918 Fig a04cd and GE4 in Table a0 In this experiment the overall HDR efficiency was Table a0 GE4 and no indels were detected in the two predicted offtarget sites by Sanger sequencing Table a0 GE4 and by AmpliSeq Supplementary Table a0X4We next attempted to perform scarless repair of a pathogenic SNV in iPSCs derived from a patient with DEB to confirm the applicability of the approach for other hereditary diseases We generated iPSCs from a patient with DEB autosomal recessive compound mutation COL7A1pGly2138Ter COL7A1c3591del13insGG and aimed to substitute the pathogenic SNV c6412G T pGly2138Ter in exon Supplementary Fig a0S4ab Scarless OneSHOT using AsCas12a_RR with crRNA_COL7A11 a0m plus the repair template scarlessly repaired the pathogenic SNV in the mutant allele Supplementary Fig a0S4cde and GE5 in Table a0 with a substitution rate of No indels were detected in the seven predicted offtarget sites Supplementary Table a0S2 Supplementary Table a0X4 Unlike the scarless OneSHOT for RET_Met918Thr in FB414 cells Fig a0 GE4 in Table a0 identical sequences within the target site were observed among the unintended geneedited clones suggesting that these clones were likely duplicated Supplementary Fig a0S3 GE5Scientific RepoRtS 101038s41598020704017Vol1234567890wwwnaturecomscientificreports 0cabcRET locus on Chr 10q11exon M918PAM crRNA_ RET1 bpWT allelecrRNA_RET2 PAMPAM M918TcrRNA_RET1mMutant allelecrRNA_RET2m PAM M NCpuro crRNA_RET 1m 2m T7E1 assay in WT iPSC 409B2 nsns xedni ledniNCcr2mpuro cr2mpuro cr2mpuro cr2puro cr2puro cr1puro cr1puro cr1mpuro cr1mpuro cr2puro cr1puro cr1mpuro crRNApuro treatmentRETI913 C T PAMRET M918 C TcrRNA_RET1WT allele sequencessODN_RET_M918T_I913silentCModified WT allele sequence Mutant allele sequencede1KM M1KIle913 A T T A T TCMet918 A TC G A C GIleIleMetThrThrScientific RepoRtS 101038s41598020704017Vol0123456789wwwnaturecomscientificreports 0cGene editing CellGE1GE2GE3GE4GE5FB414FB414FB414FB414B1173Genotype phenotypeOriginal †’ DestinationRETM918T †’ RETM918TM918T I913_silentCMEN2Ba †’ MEN2B homo with SN MarkerRETM918T †’ RET I913_silentCMEN2Ba †’ MEN2B revertant with SN MarkerRETM918T †’ RET I920_silentCMEN2Ba †’ MEN2B revertant with SN markerRETM918T †’ RETMEN2Ba †’ MEN2B scarless revertantCOL7A1G2138X del13 ins GG †’ COL7A1 del13 ins GGDEBb †’ DEB scarless revertantNo of total picked clones TCNo of 1st screening passed clones SNMD PCRNo of 2nd screening passed clonessequencing1stTC 2nd1st 2ndTC Table OneSHOT and scarless OneSHOT gene editing GE experiments After electroporation of the AsCas12a_RR expression vector and the ssODN template into the cells the crude DNA samples from the singlecell derived colonies that expanded on the master plates were subjected to SNMDPCR in the first screening round For positive screening colonies with amplifiable “200bp fragments from the SNMDPCR primer pair were the intendedclone candidates GE13 For negative screening colonies lacking PCR amplification were the intendedclone candidates GE4 and In the second screening round we directly read the sequences around the target site of the DNA fragments amplified by Tks Gflex DNA polymerase in each sample silentC a silent mutation generated by replacement with a cytidine for SN marker a Multiple endocrine neoplasia type 2B B Dystrophic epidermolysis bullosa Positive screening results Negative screening resultsSampleSiteGenomic location No of mis matchesOriginalRET exon target1chr10 GE1GE1GE2GE2GE3GE3GE4GE4Offtarget Offtarget Offtarget Offtarget Offtarget Offtarget Offtarget Offtarget chr15 chr4 chr15 chr4 chr15 chr4 chr15 chr4 Sequencea including mismatchesTTCC AGT TAA ATG GAT GGC AAT TGTTCC cGTTAAtTGGtTGG CAA TTG TTCC AcTTA AAT GcATG GCA tTTG TTCC cGTTAAtTGGtTGG CAA TTG TTCC AcTTA AAT GcATG GCA tTTG TTCC cGTTAAtTGGtTGG CAA TTG TTCC AcTTA AAT GcATG GCA tTTG TTCC cGTTAAtTGGtTGG CAA TTG TTCC AcTTA AAT GcATG GCA tTTG Indel ratio b Table Offtarget effects of AsCas12a_RR in gene editing experiments “ GE1GE4 After amplifying the offtarget candidates predicted by CHOPCHOP v2 from the intended geneedited iPSC clones we directly read the sequences around the candidate sites after Sanger sequencing with specific primers a Underline indicates the PAM of the AsCas12a_RR variant Lower letters indicate mismatched bases in the offtarget candidates as compared with the original target sequence b Number of indel clones relative to the number of analysed clonesDiscussionMany hereditary human diseases are caused by singlenucleotide mutations These singlebase alterations have the potential to drastically alter protein structure and function Although most singlenucleotide mutations are completely harmless silent repair of pathogenic SNVs would significantly improve the quality of life and life expectancy of patients with hereditary diseases Thus in the present study we investigated whether we could achieve scarless repair of pathogenic SNVs in pluripotent stem cells from patients with two different types of hereditary disease MEN2B and DEB More importantly we aimed to carry out the repairs in a single stepUsing the OneSHOT approach developed in this study we successfully repaired a RET gene SNV in MEN2B iPSCs with the addition of a singlenucleotide selective marker in a single step We then confirmed that the same technique could be used to carry out scarless repair in MEN2B and DEBspecific iPSCs without the need for the singlenucleotide marker Scarless repair where no trace of gene editing is left around the target sequence is the goal of any gene editing technique because it safely repairs mutations in noncoding genomic regions without any secondary effects In contrast the inclusion of marker sequences during gene editing can have downstream effects Such secondary effects include the introduction of noncoding SNVs to cryptic splice sites Scientific RepoRtS 101038s41598020704017Vol1234567890wwwnaturecomscientificreports 0ccausing abnormal RNA splicing4546 and mutations that introduce a premature termination codon resulting in unstable mRNA46 Noncoding mutations affecting regulatory elements can also interfere with gene regulation through loss of function resulting in reduced gene expression or gain of function resulting in gene mis or overexpression4748 Therefore scarless repair is crucial for maintaining genome integrity and preventing unknown secondary effects in the target geneSeveral other methods of pathogenic SNV repair have been developed including CORRECT2627 and MhAX49 However all currently available methods have inherent obstacles to achieving scarless SNV repair in a fast and errorfree manner To overcome some of these obstacles we used the AsCas12a nuclease which has highfidelity targetrecognition3536 circumventing the need for a blocking base to inhibit recutting as is required in other methods2627 We also performed SNMDPCRbased negative screening for the pathogenic SNV which easily detects candidate clones containing the intended alteration As a result of these modifications we achieved absolute scarless editing of the RET and COL7A1 SNVs see Fig a0 Supplementary Fig a0S4 and GE4 and GE5 in Table a0 Another advantage of the AsCas12a nuclease was the ability to carry out SNV repair in a single step because only one round of HDR is required for gene editing Fig a0 The OneSHOT method was used to repair the SNVs in RET and COL7A1 within a 3week period with sufficient efficiency for handpicking In contrast other methods can take up to “ a0months to generate the intended geneedited clone because two rounds of HDRMMEJ may be required262749 However similar to our approach the CORRECT method can achieve scarless singlenucleotide substitution thus ensuring high sequence fidelity around the target site in geneedited cells Fig a0 and Supplementary Fig a0S4 Conversely MhAX leaves a silent single nucleotide mutation around the target site for use in screening49 Another difference is that the dsDNA template in MhAX can be randomly integrated into the genome outside of the target site by nonhomologous end joining50 whereas the ssODN templates used for OneSHOTscarlessOneSHOT and CORRECT approaches are not randomly integrated51 Thus the OneSHOT method developed for SNV repair in the current study appears to have several advantages over currently available methods see Supplementary Table a0S3In the CORRECT procedure the cuttomutation distance the distance between the CRISPRSpCas9 cleavage site and the blocking mutation is a crucial factor for HDR efficiency and zygosity determination2627 We therefore searched for more appropriate sites for the singlenucleotide markers by first comparing the efficacies of three singlenucleotide markers set in different positions around the target site using a PCRrestriction fragment length polymorphism RFLP assay52 We found that two of the markers showed similar HDRspecific cleavage activity while no cleavage activity was detected for the third marker Supplementary Information and Supplementary Fig a0S5 suggesting that Ile920 could be used as an alternative singlenucleotide marker Testing of HDR efficiency in FB414 cells following OneSHOT repair using the alternative marker again confirmed that the singlenucleotide substitutions in the geneedited clones were effectively detected by positive screening using SNMDPCR for a singlenucleotide marker Supplementary Information and Supplementary Fig a0S5 We do note however that the efficiency of identification might depend on the position of the singlenucleotide marker and the primers used for SNMDPCRDespite our success in repairing the pathogenic SNVs in a single step the study has several limitations The OneSHOT method only requires one PCR run thereby reducing the time and cost compared with standard PCR“RFLP screeningbased methods which require up to three steps5253 However we found that falsepositive clones are included in the population after the first SNMDPCR screen Supplementary Fig a0X8 Therefore we are currently designing a simple way to discriminate false clones from authentic clones using a PCRbased procedure We also noted that the geneedited cell lines generated by OneSHOT are not always clonal This situation arises because high cell densities occur in the culture during puromycin selection a0days and in the recovery culture “ a0days prior to clonal expansion However assessment of our data suggests that a 1day recovery culture and sufficient singlecell suspension at the reseeding stage can prevent duplication and ensure clonal establishment of the geneedited cells Using the current protocol we estimate that the HDR substitution rate is “ While this is sufficient to permit a handpicking cloning protocol it is lower than that achieved by Cas12a in fertilised eggs from model animals3954 We hoped to improve this rate by combining OneSHOT with other procedures based on alternative principles such as introducing a blocking base into the repair template2627 andor using HDRNHEJ modification compounds3863“ We have examined whether the modification compounds can promote HDR however the compounds examined in this study had no HDRpromoting effects in our experimental system Supplementary Fig a0X4It is important to emphasise though that the procedure depends on highfidelity target recognition by the sitespecific nuclease Thus the only enzymes appropriate for the OneSHOT procedure include highfidelity variants of engineered SpCas955“ or naturally highfidelity Cas9 orthologues59“ Finally while we confirmed the expression of pluripotency markers in the geneedited clones data not shown we next aim to carry out functional analyses to confirm the differentiation potential of the repaired cells Therefore further work is needed to finetune the protocol and to confirm differentiation potential and functionality of the proteins in the corrected cell populationsTo increase the reliability of the OneSHOT method it is important to show the robustness of OneSHOT and the fidelity of the repair In order to demonstrate these issues we performed targeted NGSbased deep AmpliSeq analysis of the target sequence With regard to repair fidelity the AmpliSeq analysis showed that accurate singlenucleotide substitutions were achieved by HDR that were faithful to the ssODN template and occurred at sufficient frequency Supplementary Fig a0X3ac “ These results suggest that the method has good repair fidelity With regard to the robustne
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bousmalis leveraged cgan with the contentsimilarity loss to generate realistic target images and jointly trained the gan discriminator with the task network unidirectional translation has been applied to remove dataset variations for example bentaieb et al designed a stain normalization approach using a task conditional gan to translate he images to a reference stain madani et al proposed a semisupervised approach for cardiac abnormality classification using using gan discriminator for abnormality classification in minimally labeled xray images and showed that the adversarial loss could reduce domain overfitting mahmood translated real endoscopy images to graphicallyrendered synthetic colon images with groundtruth annotations for depthestimation during surgical navigation unidirectional translation has also been applied to crossmodality scenario for instance zhao proposed a modified unet to translate paired brain ct to mri bidirectional translationbidirectional image translation also known as reconstructionbased dt leverages two gans constraining the mapping space by enforcing semanticconsistency between the original and reconstructed images cyclegan by zhu is one of the most popular architectures for bidirectional translation cyclegan utilizes cycleconsistency to constrain the translation mapping and improve the quality of generated images cyclegan has been expanded to handle larger domain shifts with semanticconsistency loss functions cycada multidomain translation stargan and translation between two domains with multimodal conditional distributions munit in supervised learning bidirectional translation expands the training data to make the segmentation task model imia yearbook of medical informatics 2020choudhary 0crobust the translation and segmentation network can be trained either independently two stages or jointly zhang et al presented a onestage framework with an additional shapeconsistency loss in cyclegan to achieve better segmentation masks and lower failures chartsias used a twostage framework to segment mri images using ct images cai combined segmentation loss on generated images as an additional shape constraint for 3d translation and leveraged mri for pancreas segmentation in ct images in the unsupervised setting image translation is used to create labeled data for the target domain huo proposed a joint optimization approach for the synthesis and segmentation of ct images using labeled mri their framework achieved comparable performance in comparison to the fully labeled case there are a few observations about gans a cyclegan does not guarantee consistent translation of minor anatomical structures and boundaries and thus needs additional constraints like gradient and shape consistency for instance jiang incorporated tumorshape and featurebased losses to preserve tumors while translating ct data to mri data b attention networks can account for varying transferability of different image regions for instance liu proposed a novel attentionbased unet as a gan generator to translate hardtogenerate textured regions from mri to ct for alternate scenarios such as 3d2d paired images or semisupervised dadt zhang segmented xray images by using synthetic xray images created from accessible 3d ct annotations nguyen et al used semisupervised da with paired ct images to constrain cyclegan to generate more realistic images pan leveraged mri to generate missing pet images for patients for alzheimer™s disease diagnosis chen proposed stateoftheart unsupervised segmentation method using bidirectional dadt between mri and ct combining cyclegan with shared feature encoder layers between domains their method resembled cycada and showed the efficacy of combining dt with featurebased alignment c dadt can be used for singlemodality medical imaging chen leveraged a cyclegan with semanticaware adversarial loss to perform lung segmentation across different chest xray datasets latent feature space transformation in domain adaptation unlike the imagetoimage translation in dtda the lfstda transforms the source domain and target domain images to a shared latent feature space to learn a domaininvariant feature representation the goal is to minimize domainspecific information while preserving the taskrelated information the lfstda can be trained in an unsupervised fashion to obtain a domaininvariant representation or in a concurrent manner where the representation network and the task network eg image classification network are trained simultaneously to improve the performance lfstda is used in three basic implementations divergence minimization adversarial training and crossdomain reconstruction compared to dtda lsftda is more computationally efficient because it focuses on translating relevant information only instead of the complete image also featurebased domain alignment outperforms dtda by preserving taskcritical features divergence minimizationa simple approach to learn domaininvariant features and remove distributionshift is to minimize some divergence criterion between source and target data distributions common choices include maximum mean discrepancy mmd correlation alignment coral [ ] contrastive domain discrepancy cdd and wasserstein distance mmd coral and wasserstein distances are classagnostic divergence metrics and do not discriminate class labels when aligning samples cddbased da aligns samples based on their labels by minimizing the intraclass discrepancy and maximizing the interclass discrepancy mmd and coral are two of the most utilized divergence metrics that match the firstorder moment mean and the secondorder moment covariance of distributions however the represented hidden features can be complicated in the real world and may not be fully characterized by mean and covariance wasserstein distance aligns feature distributions between domains via optimal transport theory compared to the adversarialbased approaches divergencebased da has not been as widely explored in medical imaging for crossmodality da zhu utilized maximum mean discrepancy to map mr and pet images to a common space to mitigate missing data several works have used samemodality da to mitigate dataset variations in xray retinal fundus and electron microscopy images adversarial traininginstead of minimizing a divergence metric adversarial methods train a discriminator typically a separate network in an adversarial fashion against the feature encoder network the goal of the feature network is to learn a latent representation such that the discriminator is unable to identify the input sample domain from the representation for medical imaging featurebased adversarial domain adaptation has been widely utilized for various applications for example in crossmodality adaptation zhang applied a domain discriminator to adapt models trained for pathology images to microscopy images lsftda is also used for singlemodality adaptation to overcome dataset variations in pathology images mr images and ultrasound images for example lafarge have utilized a domain discriminator to mitigate the color variations of histopathology images for mitosis detection in breast cancer kamnitsas have applied a domain discriminator to mr images from different scanners and imaging protocols to improve the brain lesion segmentation performance reconstructionbased adaptationthe reconstructionbased adaptation maximizes the interdomain similarity by encoding images from each domain to reconstruct images in the other domain the reconstruction network decoder performs feature alignment by recreating the feature extractor™s input while the feature extractor encoder transforms input image into latent representation ghifary proposed imia yearbook of medical informatics 2020advancing medical imaging informatics by deep learningbased domain adaptation 0cdrcn for object detection using only target domain data reconstruction while bousmalis et al proposed a domain separation network that extracts image representations in two subspaces the private domain features and the shareddomain features the latter being used to reconstruct input image for medical imaging reconstructionbased methods are less developed and are usually combined with adversarial learning for samemodality adaptation oliveira et al have combined imagetoimage translation with a featurebased discriminator to mitigate the variations in xray images and improve segmentation performance for crossmodality adaptation ouyang combined variational autoencoder vae with adversarial training to adapt mr to ct scans challenges and opportunities domain selection and direction of domain adaptationselecting related domains for effective knowledge transfer is an openresearch area in ml in medical imaging domains are often selected based on the type of imaging technique eg radiology anatomy availability of labeled data and whether the modalities are complementary for the underlying task regarding whether da could be performed symmetrically across domains the potential information loss in a particular direction is critical for assessing task performance for example for unsupervised da from ct to mri reverse da may sometimes be needed to preserve tumors for supervised da between multiple he stained images tellez showed that mitosisdetection and cancer tissue classification in a particular color space leads to higher accuracy typically to assess domain relationship and da direction it is necessary to use a largescale empirical studies such as exploring bidirectional da across multiple datasets b a representationshift metric to roughly quantify the risk of applying learnedrepresentations from a particular domain to a new domain or c multisource da which automatically explores latent source domains in multisource datasets and quantifies the membership of each target sample however such experimentation requires extensive benchmarking studies that are lacking in medical imaging transferability of individual samplesmost da studies for medical imaging assume that all samples are equally transferable across two domains thus they focus on globally aligning domain distributions however the ability to transfer or align varies across clinical samples because of a intradomain variations eg in multimodal da between mri and ct each modality can have contrast variations b noisy annotations due to human subjectivity c target label space being a subset of source label space and d varying transferability among different image regions eg tumors are difficult to translate and could be missed during ct to mri imagetranslation some samples in the source domain may be less useful and can lead to negative transferring which adversely impacts da selecting relevant samples or reducing the impact of outlier samples using transferability frameworks is a potential solution some strategies include weighting samples based on classifier discrepancy downweighting outlier classes using the classification probability for target data leveraging openset based optimization and leveraging an attention mechanism to focus on hardtotransfer samples or using a noise coadaption layer recent medical imaging studies have explored sample selection and transferability assessment using reverse classification accuracy attentionbased unet and transferable semantic representations limitations of domain adaptation in medical imagingfor medical imaging most dlbased da uses adversarial methods primarily gan for unsupervised da adversarial methods are prone to errors because the discriminator can be confused and there is no guarantee that the domain distributions are sufficiently similar moreover the generator in gan is prone to œhallucinating content to convince the discriminator that data belongs to the target distribution as such cyclegan could be trained to synthesize tumors in images of healthy patients beyond applying consistency constraints during image translation artifacts which are not directly visible in synthesized images are also important for consideration for example cyclegans incorporate highfrequency information in the intermediate representation used by the second generator to translate the image back to the source domain this high frequency information can interfere with downstream tasks dtda approaches require translating the entire image increasing the complexity of the models for largesized medical images like whole slide images few studies [ ] have compared adversarial da methods for mrict translation however a comprehensive comparison of various featurebased da approaches is lacking future studies could explore combining dtda and lfstda approaches moreover current frameworks typically focus on sourcetarget domain pair while many tasks such as stain normalization in histopathology images can be multidomain leveraging synthetic datada for medical imaging can be applied in relatively underexplored applications such as singleview 3d reconstruction or temporal disease analysis this could benefit imageguided surgery in which training data is very scarce and difficult to obtain one way is to leverage synthetic data with ground truth information adapting it to the real data this approach has been successfully applied in natural images reverse domain adaptation ie translating real data to synthetic data is also a promising solution mahmood generated synthetic endoscopy data with known depth information by using an anatomical colon model and a virtual endoscope this simulated data was used for 3d reconstruction of real endoscopic images pan translated mr data to generate synthetic pet images to infer missing patient scans for temporal analysis of alzheimer™s disease another area that could benefit from synthetic data is skin lesion detection imia yearbook of medical informatics 2020choudhary 0ctable summary of da studies in medical imaging categorized by da methodology task modality anatomy and learning scenarios s segmentation c classification 3dr 3d reconstructionda method framework task source target domains anatomy learning scenario publications single modality dt lfst unidirectional reconstruction s c s c c s s s s s s s divergence s reconstruction s adversarial crossmodality radiology xray different demographics xray different demographics synthetic data †’ mri xray different demographics ultrasound different sources xray different disease states ultrasound different sources mri different flair sequences mri moderate to severe tbi mri different disease types demographics mri different vendors mri crossinstitutional xray different disease types demographics contrast lung xray different datasets breast lung lung brain lung fetal head lung heart brain brain brain brain brain spine reconstruction ct †’ mri s ct †’ xray s mri †” ct s mri †’ ct s s ct †” mri s 3d ct †” mri s mri †’ ct mri †’ ct mri t1 †’ t2 s ct †’ mri s mri †’ pet c mri †’ ct s 3dr ct †’ 3d rendered depthmap mri †’ pet c ct†’ mri s s mri †’ ct reconstruction s 3d mri †’ ct unidirectional divergence adversarial dt lfst single modality dt lfst adversarial unidirectional c c c s s divergence wsi different he stains wsi different stains wsi crossinstitutional wsi different stains microscopy different specimens crossmodality dt lfst reconstruction s s unidirectional adversarial c wsi ck †’ pdl1 wsi he †” if wsi †’ microscopy lung lung heart heart heart heart breast pancreas abdomen abdomen brain hip thigh pelvis brain brain colon brain abdomen heart heart pathology breast colon ovary breast prostate colon brain lung breast bladder lung colon opthalmology single modality dt unidirectional adversarial divergence lfst s s s s retinal fundus multiple datasets retinal fundus multiple datasets retinal fundus multiple datasets retinal fundus multiple datasets retina retina retina retina unsupervised semisupervised unsupervised semisupervised unsupervised chen tang gholami et a madani yang dong degel orbesarteaga kamnitsas novosad yan shanis venkataramani oliveira unsupervised supervised unsupervised supervised unsupervised unsupervised supervised unsupervised unsupervised unsupervised jiang zhang chen chartsias zhang cai huo liu hiasa pan zhao mahmood zhu yang dou ouyang bentaieb lafarge ren hou chacon kapil brieu zhang zhao javanmardi wang zhuang imia yearbook of medical informatics 2020advancing medical imaging informatics by deep learningbased domain adaptation 0c conclusions and future directionsdeep learning has been widely applied to medical imaging data analysis however the lack of wellannotated images and the heterogeneity of multicenter medical imaging datasets are two key challenges for dl performance da has emerged as an effective approach for minimizing domainshift and leveraging labeled data from distinct but related domains fstda and dtda are two popular approaches to minimize the distribution divergence in multiple medical imaging studies exploring samemodality or crossmodality scenarios they have proven to achieve good performance particularly in unsupervised da settings and organ segmentation tasks current approaches are primarily adversarial with domains being selected based on certain heuristics and underlying tasks extensive benchmarking studies are needed to quantify the domain relationship for different imaging modalities and to compare adversarial and nonadversarial approaches varying sample transferability and multimodal domains for medical imaging are two other major issues one strategy is to explore downweighting or attentionbased networks also alternative multimodal frameworks such as munit can be explored finally for certain application areas in medical imaging such as 3d reconstruction and temporal disease analysis where da is relatively unexplored synthetic data can be usedacknowledgmentsthe work was supported in part by grants from the national science foundation eager award nsf1651360 children™s healthcare of atlanta and georgia tech partnership grant giglio breast cancer research fund petit institute faculty fellow award and carol ann and david d flanagan faculty fellow research fund for professor may d wang this work was also supported in part by the scholarship from china scholarship council csc under the grant csc no the content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the nihreferences mendelson ds rubin dl imaging informatics essential tools for the delivery of imaging services acad radiol oct20101195“ litjens g kooi t bejnordi be setio aaa ciompi f ghafoorian m a survey on deep learning in medical image analysis med image anal dec4260“ adlermilstein j jha ak sharing clinical data electronically a critical challenge for fixing the health care system jama apr sharma p shamout fe clifton da preserving patient privacy while training a predictive model of inhospital mortality arxiv preprint arxiv191200354 dec zhang y wei y zhao p niu s wu q tan m huang j collaborative unsupervised 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the diagnostic platform utilizing the detection of biomarkers in various body fluids called œliquidbiopsy can revolutionize precision medicine precision medicine is aimed at attaining betterpersonalized care by the development of the latest diagnostic and prognostic methods thatconsider individual variability kaur liquid biopsy is being utilized for noninvasiveabbreviations 5hmc 5hydroxy methyl cytosine ccfdnas circulating cellfree deoxyribonucleic acids ccffetalnascirculating cellfree fetal nucleic acids ccfmirnas circulating cellfree mirnas ccfnas circulating cellfree nucleic acidsccfrnas circulating cellfree ribonucleic acids mtdna mitochondrial dnaedited byrui henriqueportuguese oncology instituteportugalreviewed bynaoko hattorinational cancer center researchinstitute japanigor kovalchukuniversity of lethbridge canadacorrespondencejyotdeep kaurjyotdeep2001yahoocoinspecialty sectionthis was submitted toepigenomics and epigeneticsa section of the frontiers in geneticsreceived february accepted july published august citationrahat b ali t sapehia dmahajan a and kaur j circulating cellfree nucleic acids asepigenetic biomarkers in precisionmedicine front genet 103389fgene202000844frontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkersprognostic and predictive purposes efficient and reliable markerswithin the body fluids can help in personalized treatmentdecisions for monitoring disease and survival ccfnas haveemerged as such markers for screening diagnosis prognosismanagement and treatment of various cancers autoimmuneneurological and mitochondrial diseases prenatal diagnosisdiagnosis of pregnancyrelated complications pos diabetes ‚ammation rheumatoid arthritis stroke and traumaswarup and rajeswari an increased amount of ccfnasis observed in these disorders making liquid biopsies moresensitive rapid accurate and preferable alternatives for variousinvasive diagnostic methods pos ccfnas present in blood circulation include cellfree genomicdnas ccfdnas and cellfree mtdna kohler thierry and cellfree rnas ccfrnas includingproteincoding messenger rna mrna regulatory noncodingrnas like micrornas mirnaslong noncoding rnaslncrnas circular rnas and rnas involved in translationlike transfer rnas trnas and ribosomal rnas rrnaspos the ccfnas dnas and rnas are generally released into theblood circulation either by apoptosis necrosis or active secretionin healthy persons the origin of ccfnas is mainly attributed tolymphoid and myeloid tissues snyder while in thecase of various clinical conditions the associated or the aï¬ectedtissues would release the extra amount of ccfnas into bloodswarup and rajeswari devonshire in a patternspecific to the pathophysiological condition hunter noferesti various genetic as well as epigenetic biomarkers havebeen explored for ccfnabased liquid biopsy as geneticbiomarkers are less consistent and provide more variabilityacross studies epigenetic markers which are more generalizedbetween samples present as a promising alternative for earlydiagnosis and monitoring of the diseases these epigeneticmarks are tissue specific and reflect the pattern of diseaseprogression zeng furthermore epigeneticbiomarkers are dynamic with most techniques required foranalysis ofthese biomarkers that are already available inclinical laboratoriescare thein future precise patientthe use of epigenetic marks has revolutionized the fieldof noninvasive molecular diagnosisreplacing traditionalscreening and treatment methods these assays have greatpotentialepigeneticmarks for ccfnas reflect the pattern specific for the tissuecontributing to these ccfnas therefore the use of epigeneticmarkers can help in the diagnosis of various diseases evenbefore the onset of actual symptoms and hence help inbetter management ofthe disease precision medicine hasimproved health care by theidentification of diï¬erentstagessubsets of diseases precise diagnosis and treatmentfurthermore the development of advanced analytical softwaretechniques like machine learning and artificialintelligencecan enhance precision medicine ahlquist beltrangarcia these are used in combination withnextgeneration sequencing to identify novel ccfnabasedepigenetic markersepigenetic biomarkers in ccfnasreliable markers are required to guide personalized treatmentdecisions for monitoring disease progression and survivalthe presence of epigenetic marks on ccfnas specific toa particular clinical condition is widely being explored toadvance personalized medicine a perfect epigenetic markerfor precision medicine should be able to detect the diseasewith high sensitivity predict the risk of disease developmentand its progression and monitor the therapeutic responseofbeltrangarcia ccfdnas areassociated with various epigenetic marks schwarzenbach like dna methylation hydroxymethylcytosine 5hmcand posttranslational modifications of histones in additionnucleosome positioning and occupancy on ccfdnas haveexhibited high sensitivity and specificity in liquid biopsybasedmethods for disease detection and classificationthe patientthe5methylcytosine5mc modificationat cpgdinucleotides is the most abundant form of dna methylationit plays an important role in the regulation of gene expressionand is widely used as an epigenetic biomarker for ccfdnabasedassays dna methylation has replaced many genetic mutationor proteinbased markers these 5mc biomarkers are alsovaluable in identifying tissuespecific methylation to estimatetumor burden and tissue of origin in ccfdnas in additionto 5mc 5hydroxymethylcytosine 5hmc is also used as anepigenetic mark on ccfdnas zeng 5hmc is createdby the oxidation of 5mc by “ translocation tet proteinsalthough 5hmc is far less abundant compared to 5mc it ismore distinctly distributed among diï¬erent transcriptionallyactive regions which emphasizes its potential as a diagnosticmarker genomewide analysis of 5hmc pattern can providemore information about the potential of this epigenetic markerfor ccfdnas zeng nucleosome positioning has emerged as a recent biomarkerto distinguish the tissue of origin of ccfdna based onderived nucleosome maps snyder performed deepsequencing on ccfdnas and observed a distinct pattern ofnucleosome positioning between healthy persons and cancerpatients correlating with the tissues of origin snyder this emphasizes the use of nucleosome maps whichconsist of occupancy of transcription factor and nucleosomeas the epigenetic marks to distinguish normal versus cancerccfdnas hence nucleosome positioning can also be used toidentify various cancers that generally require invasive biopsiesfor definitive diagnosis moreover genomewide nucleosomepositioning of ccfdnas is utilized to infer pathological statesof multiple disease types a comprehensive public databasecalled cellfree epigenome atlas cfea provides the epigenomeprofile of ccfdnas from various human diseases and canhelp in a better understanding of collected data yu ccfdna are generally associated with nucleosomesand histone proteins histone proteins are posttranslationallymodified at amino acid residues located on their n andcterminal tails these modifications act as epigenetic marksthat can specifically distinguish diseaserelated ccfdnas in bloodsamples various types of histone modifications are associatedfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkersarewith the development and pathogenesis of human diseaseszhao and shilatifard in addition to dna markers rna markers like mrnasmirnas lncrnas and circrnas are also getting attention in thefocus of clinical research pos most of the currently available diagnostic tests based onccfnas use either dna methylation markers or the diï¬erentialexpression of mirnas these biomarkersrelativelyeasily detected and estimated using accessible techniqueslike methylight methylspecific pcr methylationsensitivehighresolution melting and pyrosequencing garc­agimnez dna methylation specific to fetal and tumor dnahas been reported in pregnant women and cancer patientsrespectively wong poon the pattern ofthe methylation in these ccfdnas has been traced back to theirtissue of origin lun sun diï¬erentiallymethylated markers have been reported in ccfdnas like inspromoter in diabetes and reg1a and cux2 genes in pancreaticcancer lehmannwerman promoter methylationof serpinb5 rassf1a and stat5a act as epigenetic fetalmarkers in maternal blood chim chan rahat 2016atumor dna depending on the copy number mostly targetedmethylation sequencing is carried out in such cases which hasa greater potential for the detection of lower levels of ccfna inpatients with earlystage diseasechromatinbased chipseq experiments are revolutionizingour understanding of the complexes associated with chromatindynamics ongoing advances such as nanochipseq allow chipseq to be analyzed from far fewer cells necessary for embryologyand development studies nakato and shirahige theemergence of chipexo that digests the ends of dna fragmentsnot bound to protein is quite promising furey howeverthe application of these techniques to identify biomarkers islimited due to the expertise and cost associatedcriticalchipseq also providesinformation on otherchromatin modifiers such as histone marks and the enzymesthat modify these marks in diseases such as cancer chipseq has identified the role of aberrant h3k79 methylationby the methyltransferase dot1l in mixed lineage leukemiamllrearranged leukemias bernt in additionto chipseq diï¬erentlike chippcr elisabased assays or mass spectrometry are used to detect andquantify histone modifications on ccfnas in serum or plasmaadli and bernstein techniquesdiagnostic approach forepigenetic modifications in ccfnathe various diagnostic approaches to study the epigeneticmodifications in the nucleic acids include methylated cpgisland recovery assay mira and methylcap that rely onmethylcpgbinding domains mbd to capture methylateddna after dna fractionation either by restriction digestion orsonication mitchell these methods can also becombined with microarray or ngs technologies methylcapseq to identify biomarkers for cancer diagnosis and dnamethylation maps of cancer genomes simmer reduced representation bisulfite sequencing rrbs meissner is an efficient method for absolute quantification ofthe methylation status of more than one million cpg sites atsingle basepair resolution covering regions of moderate to highcpg density lee new techniques such as wholegenome bisulfite sequencing wgbs allows for an unbiasedassessment of dna methylation at singlebase resolution withfull coverage of more than million cpg sites in the humangenome and by using this technique in the clinical settingsrelevant biomarkers were identified in colorectal and breastcancers and certain types of leukemia berman some of the techniques are used in clinical settingslikeparallel shotgun sequencing and targeted sequencing norwitzand levy for noninvasive prenatal testing wgs for fetalgene detection lo and cancer personalized profilingby deep sequencing cappseq to quantify circulating tumordna newman despite the advancement of the techniques to study epigeneticmodifications the use of epigenetic biomarkers present onccfnas is limited due to their lower levels in the blood circulationin the case of cancer wgs is applied to only “ of cellfreeccfnas as epigenetic biomarkersin various diseasesthe detection and quantification of ccfnas viz rna dnafetal dna fetal rna mtdna and mitochondrial rna andmirna levels in body fluids are of clinical significance theseccfnas have the potential to act as biomarkers for diagnosisas well as prognosis of various diseases fleischhacker andschmidt breitbach such as diï¬erent cancersobstetric autoimmune neurological and mitochondrial diseasesas well as prenatal diagnosis etc kandel shaw although the most studied area of epigenetics is dnamethylation yet in the clinical setting there are only a fewmethylation markers various blood or tissuebased cohortwellpowered studies have recently shown that changes in thedna methylation are not only observed frequently in cancersbut also in other broad range of complex diseases includingneurodegenerative metabolic autoimmune and ‚ammatorydiseases although at a lower frequency tost dna methylation analysis of ccfdna might provide avaluable option in some cases when the blood“brain barrieris temporarily disrupted it was recently demonstrated by thedetection of unmethylated fragments of mbp3 and wm1 specificfor oligodendrocytes in about of patients with relapsingmultiple sclerosis zachariah cfrnas are also presentin the patient™s serumplasma in addition to ccfdnas higherlevels of circulatory rnases were observed in cancers and variousdiseases like cerebral attack preeclampsia etc and surprisinglyrna found in the circulation was found to be stable umu changes in the expression of intracellular mirnahave been causally linked with many diseases that include canceresquelakerscher and slack cardiovascular diseasesfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkersnavickas neurodegenerative diseases gupta etc such changes in expression of mirna are eithersimilar or distinct in the serum of a particular set of patientsthus enabling mirna detection in serum as biomarkers ofhuman diseases backes therefore ccfnas playa prominent role in the pathogenesis and diagnosis of variousdiseases further research is required in this field to ensure thewidespread application of these markers in clinical settingsccfnas in cancerevery year about million new cases of cancer are reportedexcluding skin cancer other than melanoma that cause about million deaths accounting for of deaths in a yearferlay an estimated number of more than million new cancer cases are likely to be diagnosed and cancer deaths are expected in the united states in whichdeciphers almost deaths per day siegel thesix major hallmarks of cancer hanahan and weinberg are uncontrolled cell growth and division programmed cell deathavoidance invasion metastasis and angiogenesis the diagnosisof cancer usually occurs following the appearance of signs orsymptoms or through screening and investigations like xraysblood tests endoscopy ct scans etc biopsy tissue examinationindicates the type of proliferating cells genetic abnormalitiesand histological grade and other characteristics thereforeadvanced measures such as estimating prognosis risk assessmentfor early diagnosis biomarkers and observing the response totherapy can lead to successful treatment positive outcomes andimprovement of the quality of life for patients the tissue biopsymatched ccfdna is considered as surrogate marker due to itsrelease from the tumor sites de mattosarruda it is proven to be a noninvasive rapid and sensitive markerfor diagnosis prognosis and therapy response monitoring indiï¬erent cancers volik in addition the integrityof ccfdna extent of ccfdna fragmentation may be utilizedas a promising biomarker for diagnosis and prognosis of cancermadhavan ccfnas as diagnostic and prognosticbiomarkers for cancerserum or plasma ccfna serves as a œliquid biopsy which is usefulfor various applications in diagnostics and avoids the necessityfor biopsy of tumor tissue the levels of ccfna in blood andlymphatic circulation are aï¬ected by degradation clearance andvarious other physiological events liver and kidney clear nucleicacids from the blood and they have a halflife of diï¬erent timeintervals in the circulation that varies from min to severalhours fleischhacker and schmidt mirnas appear to beextremely stable but their rate of clearance from the blood is notwell studied in cancer patients thus owing to the uniqueness ofthis research areaccfdnas in cancerccfdnas consists of both genomic dna gdna as well asmtdna there is a production of longer uneven fragments ofdna by necrosis in cancer patients and shorter dna fragmentsfrom apoptosis hence increased levels of longer dna fragmentsin the bloodstream have been targeted as a potential marker forthe presence of malignant tumor dna arkoboham tumor cells are the origin of ccfdna in the blood of cancerpatients stroun aberrations specific to tumors likeoncogene and tumor suppressor gene mutations wang methylation of dna fujiwara and instabilityof microsatellite dna shaw were recognized inccfdna tumorigenesis and its progression are monitored bythe change in various epigenetic modifications patients withdiï¬erent types of malignancies have methylated dna in theirserum or plasma one of the most important methods foranalyzing malignancy is by detecting the presence of methylatedccfdna in cancer patientsfor early diagnosis of colorectal cancer crc analysisof promoter hypermethylation in blood and fecal dna hasthe potential to be used as a noninvasive test and eï¬ortsare made for clinical application of these molecular markersvarious studies have observed mgmt rassf2a wif1 ngfrand sept9 as aberrantly methylated genes used as diagnosticbiomarkers in patients with crc lee powrozek several potential methylation biomarkers have beenfound that diï¬erentiate plasma from breast cancer patients andthat from control subjects hoque remarkablytwo independent studies recognized cst6 as being methylateddiï¬erentially between breast cancer and control plasma samplesradpour chimonidou for lungcancer an early focus was to search methylated cdkn2a as aplasma diagnostic biomarker studies observed hypermethylationof cdkn2a in the plasma of patients with lung cancer ascompared to cancerfree controls zhang shox2was identified as a potential biomarker in a retrospective studydone by researchers from the theracode a diagnostic firmkneip a recent study by a group as part ofthe australian pancreatic cancer genome initiative apgihas observed elevated levels of aberrant methylation in theimportant cell signaling pathways thus suggesting its possibilityas a disease biomarker they worked on a group of sixcandidate genes nptx2 sarp2 uchl1 ppenk cdkn2aand rassf1a and observed diï¬erential methylation in thepromoters of all the genes in pancreatic cancer and healthycontrols except in cdkn2a promoter which was methylateddiï¬erentially between pancreatic cancer patients and thosehaving chronic pancreatitis park epigenetic eventsin the progression of cancer include the promoter regionthe genes piclass gstp1 and apchypermethylation ofwhich are the most common somatic genome abnormalities incolorectal and prostate cancer ellinger rassf1ararb sept9 esr1 and cdkn2a are the important methylatedgenes that have shown utility in prognosis using ccfdnaassays in many patients methylation of histones is an activeprocess with vital roles in diï¬erentiation and developmenttumorigenesis also occurs due to aberrant levels of histonemethylation the promoters associated with h3k4 are primarilytrimethylated by set1a and set1b set1a plays a vitalrole in oncogenic function in breast cancer metastasis lungfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkerscancer and colorectal cancer zhao and shilatifard table presents the frequently hypermethylated genes invarious cancer typesccfmirnas in cancerin various cancers mirna expression dysregulation has beenobserved that suggests its role in many processes necessaryfor the progression of cancer like proliferation cell deathmetastasis and resistance to treatmentiorio and croce during the development of the liver mirna expressionchanges dynamically mir500 is one such oncofetal mirnathat is important for the diagnosis of hepatocellular carcinomayamamoto lately in nonsmall cell lung cancernsclc mir1246 and mir1290 were recognized as tumorinitiating and cellspecific mirnas zhang mir was found to be a significant prognostic factor for osccpatients based on cox regression analysis in addition mir could serve as a valuable biomarker in oscc patientsto predict the clinical response to chemoradiotherapy lin a study by alhasan showed a serumsignature of 5mirnas mir135a mir106a mir200c mir and mir433 predicted a very highrisk prostate canceralhasan expression levels of mir21 mir23bmir200c and mir200b were upregulated in metastatic breastcancer when compared to early breast cancer patients thereforesupporting the notion that ccfmirnas presents a tool with thecrucial diagnostic and prognostic implication in breast cancerpapadaki furthermore a study discovered thatincreased mir122 expression was significantly associated witha reduction in the overall survival as well as progressionfreesurvival in breast cancer patients saleh elevationin the levels of serum mir29 mir122 mir155 and mir was observed in cholangiocarcinoma although mirnaslevels before surgery were inappropriate as survival prognosticmarker however postsurgery decrease in the serum mir122levels was significantly linked with better patient prognosisloosen ccfnas in treatment and cancerprogressionccfdna analysis is a noninvasive process that allows day to daypatient followup and monitoring of response toward treatmentges both genetic and epigenetic changes areexhibited by ccfdna stroun the study of thesechanges might provide valuable information to mold the choiceof treatment by clinicians given the limitations of the noveltargeted therapiesabnormal hypermethylation at cpg islands occurs rarely innonmalignant and normally diï¬erentiated cells so the releaseof dna from tumor cells can be found with a prominentextent of sensitivity even when the excess of dna is releasedfrom normal cells and this characterizes its potential clinicalapplication wong in this context promoter regionhypermethylation of ink4a occurs very early in the progressionof hepatocellular carcinoma hcc and henceit serves asa valuable biomarker for noninvasive diagnosis as well asprediction of response to therapy huang isatherapyimmunotherapyidentification ofrapidly developingsignificant mirnasinmany cancers because of various advantages over standardchemotherapythatprovides a foresight of response in cancer immunotherapy wouldenable better patient selection and enhancement of therapeuticefficacy and provide a novel target antonia chen mirna21 is a cellfree oncogenic mirna whichhas been known as a potential regulator of stat3 and thusit could be detected in various tumors ji thuscirculating mirna21 can act as a biomarker for response incancer immunotherapy wu in the mycnamplified neuroblastoma progression mycnis detected in circulating dna this phenomenon was found tobe associated strongly with the quick progression of tumors andpoor outcomes combaret loss of heterozygosityloh and abnormal methylation at the promoter region ofmycn were detected using ccfdna which showed elevatedlevels in patients of highgrade glioma detection of promoterregion hypermethylation of myod1 in serum may serve asa potential prognostic marker for discriminating patients ofcervical cancer at high risk for lymph node metastasis or relapsewidschwendter moreover the investigation of circulating mirnas presentsgreat potentialin revealing new insights into their role intherapy and diagnosis mirna serum signatures mir345 5pmir330 3p and mir9 3p were found to be significantlyupregulated in patients of prostate cancer pca when comparedto healthy individuals the role of mir3455p to act as anoncomir through cdkn1a targeting makes it a potential targetfor pca therapeutically tinay ccfdnasin glioma wereassociated with diï¬erentialmethylation levels of mgmt cyclindependent kinase inhibitor2a multiple tumor suppressor p16ink4a p73 and retinoicacid receptor beta rarb balana weaver wakabayashi all these studies propose acrucial role of epigenetic marks in ccfnas in cancertargetedtherapy as well as pathogenesisccfnas in cancer precision medicineprecision oncology is an approach that includes the molecularprofiling of tumors to identify eï¬ective therapeutic strategiesa clinical research program initiated by the englander institutefor precision medicine eipm in uses wholeexomesequencing of metastatic and primary tumors to identifyindividualized therapeutic options and to help guide clinicaldecision making by prospective followup of patients rennert oncology is the obvious choice for heightening theimpact of precision medicine several targeted therapies havebeen developed that have shown profound benefits recentlynovel immunological approaches produced insightful responsessnyder in addition the identification of epigenetic biomarkers leadsto more precise disease prognosis especially in therapeutic areasthat are linked with a high degree of variability concerningsurvival van neste research carried out in severalcancers like glioblastoma reveals that levels of 5hmc are criticalin the regulation of genes having a crucial role in disease andfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkerstable frequently hypermethylated genes in various cancer typesgenecancer typereferencesitih5 dkk3 brca1 erbeta apc gstp1 esrbrassf1ap16arf bax bcl2 cdh1 dapk ednrb eomes faddpcdh17 pou4f2sept9 hltf nell1 cea tac1vhlrbtmeff2 prdm13ost2 mgmtapc gstp1st6galnac3 znf660brca1 rassf1a rassf2ahtertp16ink4a timp3 thbs1breast cancerprostate canceresophageal liver and pancreasbladder cancerkloten cheuk vu liu house abern wang colorectal cancerkidney tumorsretinoblastomalung cancerrenal cell carcinomaprostateovarian cancerleptomeningeal carcinomatosis in csfgliomatham semaan ma ohtanifujita palmisano lee su hauser haldrup giannopoulou lonning bougel liu show that global reduction in 5hmc over the genome leads topoor clinical outcomes in these patients johnson epigenetic changes introduced common genetic mutations inan in vitro model of lung cancer vaz epigeneticbased diagnostics can detect early disease signals and thuscan provide possibilities for clinicalintervention before theprogression of symptomsthe detection of ccfnas could be exploited by targetedtherapies approved lately and eventually benefit the patientsscrutinizing cancers by analyzing ccfna dynamics in blood orserum is an innovative and emerging research area as far as theexisting research advancement and the growth of the medicalindustry are concerned we consider that ccfna assays may beemployed for realtime personalized treatments in the future forcancer patients based on their ccfnas or ccfdna methylationlevels for diagnosis and prognosis nevertheless there is muchscope for improvement before the application of this technologyin clinical settingsuse of ccffetalnas in prenataldiagnosis and pregnancyrelateddisordersthe apoptosisnecrosis ofduring pregnancytrophoblastsarising from syncytiotrophoblast is the prime source of therelease of ccffetalnasinto the maternal blood litton the presence of ccffetalnas has pavedthe way for noninvasive prenatal diagnosisand earlylo prediction of pregnancyrelated complications the use of ccffetalnas has gradually replacedinvasive techniques like amniocentesis or chorionic villussampling serr ccffetaldna comprises “ ofthe maternal ccfdna wang andcan be efficiently detected atthe fifth week of gestationguibert the amount of ccffetaldna inmaternal blood increases progressively throughout pregnancybirch ccfnas in prenatal diagnosisprenatal diagnosis is an established practice for the managementof pregnancy as well as avoidance of prenatalneonatal deathsthe leading causes for such deaths are genetic disorderbirth defects congenital malformations and chromosomalabnormalities like trisomy down™s syndrome edward™ssyndrome and patau syndrome and sex chromosomeaneuploidies like monosomy x turner syndrome carlson andvora therefore successful management of pregnancydemands efficient and timely prenatal diagnosis to determine theoutcome of pregnancy timely detection of neural tube defectsis already providing early prenatal treatment resulting in betterneonatal outcomes adzick ccffetaldna is clinically used for the detection of fetal sexand multiple anomalies based on paternally inherited mutationsbianchi recent studies have discovered many fetalepigenetic biomarkers for ccffetalnabased liquid biopsies inclinical samples that have demonstrated high clinical potentialin disease diagnosis prognosis and pregnancy managementthese epigenetic modifications are specific to the fetus and helpto distinguish fetal nucleic acids from maternal nucleic acidsjones and takai clinical testing of recently developedfetal epigenetic markers can help in the proper managementof personalized care the first reported use of fetalderivedepigenetic marker in maternal body fluids had come frompoon who utilized an imprinted h19igf2 locusbased on parentoforiginspecific methylation and the maternaland the paternal copies of the gene were distinguished inmaternal blood poon based on the placental originof ccffetaldna having placentaspecific dna methylationpattern the genomic regions that show diï¬erential methylationbetween the placenta and the maternal blood cells can actas a marker for fetal dna in maternal blood the promoterregion of maspin serpinb5 is the first such reported universalfetal dna marker with detectable hypomethylationin thebackground of hypermethylated maternal sequences the fetalorigin of these hypomethylated maspin has been confirmedby the clearance of these sequences within h of deliveryfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkerschim the clinical use of hypomethylated maspinis limited by the required bisulfite treatment of ccffetaldna as this treatment can degrade around of the dnagrunau thus decreasing the amount of alreadylow levels of fetal dna in maternal blood such limitationwas overcome by the detection of fetalderived hypermethylatedrassf1a in maternal blood for prenatal diagnosis chan hyland tounta 2011b the maternalhypomethylated rassf1a ccfdna can be removed by treatmentwith methylationsensitive restriction enzyme digestion leavingbehind fetal hypermethylated rassf1a ccffetaldna chan various other fetalderived diï¬erentially methylatedsequences have also shown a similar potential to act as fetal dnaepigenetic markers in maternal blood table ccffetaldna methylation markers have the potential ofbeing used as both quantitative as well as qualitative markersin prenatal diagnosis as qualitative markers these are used toestimate the false positives during the determination of fetalgender rh status and paternally inherited polymorphisms chan while as quantitative markers these can estimate thelevels of ccffetaldna in maternal plasma such an applicationof ccffetaldna finds its use in the detection of chromosomalaneuploidies lun based on the location of themaspin gene on chromosome hypomethylated fetal maspinhas been used to calculate the allelic ratio to diagnose trisomy with sensitivity tong fetal trisomy was detected by analyzing chromosomal dosage via targetingof fetal hypermethylated hlcs sequences in the combinationof microfluidics digital pcr rassf1a on chromosome and zfy on the y chromosome were used as referencestong fragmentation pattern of ccffetaldnain maternal plasma has been successfully used for enrichmentmethod in size separation manner on agarose gel electrophoresisramezanzadeh various nextgeneration sequencing and highthroughputtechniques have catalyzed the identification of newer and novelfetal epigenetic markers further advancing noninvasive prenataldiagnosis the microarraybased approach has identified manyfetal epigenetic markers with diï¬erential methylation betweenchorionic villus samples and maternal blood on chromosomes and for aneuploidy detection chu combining highresolution tiling oligonucleotide array withmethylated dna immunoprecipitation medip has helped ina genomewide screen for detecting the diï¬erential methylatedsites between placental tissue and maternal blood cells it hasdetected various new fetal epigenetic markers on chromosomes and
0
" IBDFecal calprotectinEndoscopic activityIBD noninvasive managementThe term IBD is usually used for referring to a group of ‚ammatory gastrointestinal diseases mainly Crohn'sdisease and ulcerative colitis Accordingly IBD arises as a result of inappropriate immune response to intestinalcommensal anisms among genetically susceptible individuals Performing colonoscopy and histopathologicevaluation on an ‚amed bowel biopsy specimen are currently considered as gold standards for diagnosis andmanagement of IBD Correspondingly these techniques are known to be invasive and costly In recent decadesfecal calprotectin as a biomarker has received much attention for the diagnosis and noninvasive managementof IBD Up to now many studies have investigated the efficacy of fecal calprotectin in the areas of IBD diï¬erentiation from IBS prediction of endoscopic and histologic activities of IBD and prediction of disease recurrenceAlthough some of these studies have reported promising results some others have shown significant limitationsTherefore in this paper we reviewed the most interesting ones of these studies after a brief discussion of thelaboratory measurement of fecal calprotectin Moreover we attempted to provide an answer for the question ofwhether fecalcalprotectin could be considered as a potential surrogate marker for colonoscopy IntroductionInflammatory bowel disease IBD is a long life disease with remission and relapse periods IBD arises as a result of inappropriateimmune response to intestinal commensal anisms in individualswith genetic predisposition and consequently causes ‚ammation andintestinal ulcers [] In addition IBD has a complex pathogenesis andmany factors such as dysbiosis oxidative stress and epigenetics thatmay also be involved in disease pathogenesis [“] Ulcerative colitisUC and Crohn's diseases CD are known as two main forms of IBDAccordingly these diseases cause intestinal ulcers and some annoyingsymptoms such as diarrhea abdominal pain and rectal bleeding Occasionally the severity of these symptoms is very high which can leadpatients to be hospitalized In this regard therapeutic approaches totreat these diseases mainly focus on prolonging remission and are almost similar however diï¬erential diagnosis can also help to treat thedisease in a more eï¬ective way For example 5ASA which is acommon drug in the treatment of IBD is less eï¬ective on maintainingremission in CD patients On the other hand antibiotic therapy is notrecommended for the treatment UC but it can be eï¬ective on CD patients [][] Diï¬erential diagnosis is a serious challenge because CDand UC have significant similarities in terms of their clinical endoscopic and histological features However there are some diï¬erencesbetween UC and CD which are summarized in Table1 In addition tointestinal complications UC and CD also have significant extraintestinal manifestations For example it was shown that UC is significantly associated with Primary sclerosing cholangitis and CD is alsoassociated with cholelithiasis especially in cases that the ileum is involved [] Furthermore CD can cause fistulas to the urinary systemwhich leads intestinal bacteria to enter the urethra and recurrent urinary tract infections [] Both CD and UC can cause several disorderssuch as arthritis Erythema nodosum pyoderma gangrenosum andanemia which are known as the most important extraintestinal manifestations of IBD [][] The latest statistics showed that the globalŽ Corresponding author at Department of Clinical Biochemistry and Laboratory Medicine Faculty of Medicine Tabriz University of Medical Sciences DaneshgahStreet PO Box Tabriz IranEmail address vagharimtbzmedacir M VaghariTabari101016jcca202008025Received July Received in revised form August Accepted August Available online August Elsevier BV All rights reserved 0cF KhakiKhatibi et alTable1Clinical endoscopic and histological features of CD and UCClinical FeaturesFeaturesRectal bleedingAbdominal painFeverMucus defectionIntestinal obstructionPerineal diseasePostoperative recurrenceASCA positiveANCA positiveEndoscopic FeaturesCDOccasionallyFrequentlyFrequentlyOccasionallyYESYESYESFrequentlyNot commonUCFrequentlyOccasionallyNot commonFrequentlyNONONONot commonFrequentlyFeaturesCDUCLocationMucosal involvementDepth of ulcerationfistulaCobblestone appearanceAphthous ulcerationMucosal friabilityHistological featuresFeaturesGranulomasCrypt abscessesPatchinessAny part of GI tractDiscontinuousDeepYesYESFrequentlyNot commonCDFrequentlyNot commonFrequentlyColon and rectumContinuoussuperficialNONOOccasionallyFrequentlyUCRareFrequentlyNot commonprevalence of IBD currently is on the rise and it is not an exaggerationif we consider it as a global serious health problem [] According to areport published in IBD has the highest prevalence rate inEurope and its prevalence in the newly industrialized countries of AsiaAfrica and South America also appears to be increased over the pastthree decades []Unfortunately the peak of the disease is at the young age of“ years old [] therefore in addition to the suï¬ering from ‚icts on the patients it also has many negative eï¬ects on societyMoreover many financial burdens are annually imposed on countriesfor controlling and treating this chronic disease The invasive diagnosticand therapeutic measures are currently undertaken to diagnose andmanage IBD which are unpleasant for patients as well as having thehigh associated costs Now the gold standard method for diagnosingIBD and monitoring patient status is performing colonoscopy examination and histopathologic evaluation which are invasive measures[] Therefore in recent years many studies have been conducted tofind a suitable laboratory marker with sufficient sensitivity and specificity for the purpose of diagnosing and noninvasive management ofIBD A high proportion of these studies have investigated the efficacy offecal calprotectin in diagnosing and monitoring patients Althoughsome of these studies reported auspicious results there are still somedoubts on the eï¬ectiveness of fecal calprotectin on diagnosing andmonitoring IBD patients So in this review we addressed the advantages and limitations of fecal calprotectin for the diagnosis andmanagement of IBD The role of fecal calprotectin in diagnosis and management ofIBDThe efficacy of fecal calprotectin as an laboratory marker in various areas of IBD diagnosis and management have been studied including IBD diï¬erentiation from irritable bowel syndrome IBS evaluation of endoscopic activity of the disease evaluation of histologicalactivity of the disease and prediction of disease recurrence andClinica Chimica Acta “response to treatment In following after a brief introduction andmentioning the important points regarding laboratory measurement offecal calprotectin we reviewed the most interesting findings in all ofthe abovementioned areas Calprotectin A clinically valuable proteinCalprotectin is an antimicrobial protein mainly secreted by neutrophils This protein competes with bacteria over zinc thus kills thebacteria However this is not the only contribution that it has to antimicrobial activity Moreover this protein has many potential clinicalapplications such as the elevated serum levels that have been observedunder various immunological and immunopathological conditionsSerum calprotectin levels rapidly increase in response to bacterial infections in the kidney and heart or during transplant rejection At theearly stages of ‚ammation of the lung serum calprotectin can also beconsidered as a reliable marker besides plasma levels of calprotectinappear to be useful in reflecting disease activity in ‚ammation of thejoints [] In addition it has been demonstrated that serum calprotectin levels are increased in patients with bacterial sepsis so it can beconsidered as a reliable biomarker [] In Neonatal Sepsis the serumlevel of calprotectin increases as well as a sensitivity of and aspecificity of that have been reported for serum calprotectin indiagnosis of Neonatal Sepsis [] It has been recently shown thatserum calprotectin levels increase in patients with aneurysmal subarachnoid hemorrhage and higher levels in the first of onset areassociated with a poor prognosis at the first three months [] Serumcalprotectin levels also increase in patients with rheumatoid arthritisand even in patients with a moderate to high disease activity who havenormal or low CRP levels so they appear to be more efficient at reflecting disease activity []Some studies have also investigated the efficacy of serum calprotectin in the diagnosis of cancers Correspondingly in one of thesestudies it was shown that serum calprotectin levels significantly increased in patients with laryngeal carcinoma compared with healthyindividuals and those with benign laryngeal pathologies Moreover inthis study a direct relationship was also observed between serum levelsof calprotectin and stage of cancer [] Another study showed that theserum level of calprotectin increased in patients with papillary thyroidcarcinoma but it significantly decreased after operation [] Alsoregarding the efficacy of serum and saliva calprotectin for the diagnosisof IBD impressive results have been reported [][] A study onpatients with IBD both UC and CD have shown that serum calprotectinlevels were directly correlated with fecal calprotectin levels and weremore potent in IBD diagnosis compared to CRP and albumin This studyalso indicated that the combination of serum calprotectin with CRP oralbumin can be helpfulin the prediction of treatment escalationespecially in patients with CD [] However no significant correlationwas observed between serum calprotectin and fecal calprotectin levelsin patients with CD and UC as well as a slight correlation betweenserum calprotectin level and CRP that was observed only in patientswith UC [] Another study showed that the serum level of calprotectin was significantly higher in patients with CD compared to healthyindividuals In addition although a significant correlation was observedwith the clinical activity of the disease no significant correlation wasfound between the level serum calprotectin and endoscopic activity ofthe disease [] The efficacy of salivary calprotectin in the diagnosisof IBD has also been studied which showed that salivary calprotectinsignificantly increased in patients with IBD compared to healthy individuals In this study AUC values for unstimulated saliva and stimulated saliva to distinguish IBD patients from healthy individualswere reported to be and respectively [] However thepopularity of calprotectin is mainly due to the use of fecal calprotectinin the diagnosis and management of IBD that is discussed in the following 0cF KhakiKhatibi et alClinica Chimica Acta “ Laboratory measurement and reference intervalFecal calprotectin is a stable protein that remains stable for “ daysat room temperature [] This property is an excellent advantage for alaboratory marker Also it seems that keeping the specimen at refrigerated temperature °C can increase the stability of fecal calprotectin [] However evidence has been obtained regarding thatthe stability of this protein decreases after staying for three days atroom temperature On the other hand it is not also recommended tokeep samples in the refrigerator for more than days [] It seemsthat fecal calprotectin remains stable up to one year at ˆ’ °C []Measurement of fecal calprotectin can be done both qualitatively andquantitatively Accordingly in the qualitative measurement monoclonal antibodies are used to detect fecal calprotectin and the positiveresults are characterized by the appearance of colored lines on the testcassette However in the qualitative one only positive or negative results are reported and despite of sensitivity test specificity in theevaluation of disease activity was reported to be only It seems thatthe main application of this test is to diï¬erentiate healthy individualsfrom IBD patients rapidly however some studies have shown that it isnot accurate enough in this case as well [][] Nevertheless asignificant concordance has been reported between home test resultsIBDoc and fecal calprotectin laboratory measurement results whenQuantum Blue calprotectin ELISA kit was used Notably the agreements between results were and depending on the selectedcutoï¬s [] Several commercial kits are also available for fecal calprotectin qualitative test known as rapid calprotectin These tests reportpositive results ranged from to µgg There are also severalcommercial kits that can be used for the quantitative measurement offecal calprotectin These kits are usually designed in terms of the ELISAmethod and some have a measurement range between and µgg Moreover the chemiluminescence immunoassays CLIAmethod can also detect values between and µgg Fluoro enzyme immunoassays FEIA and particle enhanced turbidimetric immunoassays PETIA can also be used for the measurement of fecalcalprotectin In this regard one of the most serious challenges to thelaboratory evaluation of fecal calprotectin is the determination of theupper limit in healthy individuals Among healthy adults there is asignificant agreement on µgg as an upper limit One study suggested values up to µgg in people over years old and up to µgg in children aged between and years old as referenceranges of fecal calprotectin in healthy individuals []Fecal calprotectin levels appear to be higher in healthy infants andchildren under four years old than in adults and further studies areneeded in this regard to determine the acceptable upper limit for diagnosis of pediatric IBD [] Table lists the median levels of fecalcalprotectin in healthy individuals with diï¬erent ages reported in somestudies According to these reports age can aï¬ect fecal calprotectinlevels Fecal calprotectin and IBD diagnosisOnly a small percentage of patients complaining of abdominal painand diarrhea have IBD In many cases IBS as a functional gastrointestinal disorder is known as the cause of such clinical symptomsPatients with IBS have normal colonoscopy results while IBD patientsindicate abnormal colonoscopy results and have intestinal ulcersUnfortunately the significant prevalence of IBS and the overlap between clinical symptoms and IBD can increase the colonoscopy rateTherefore a noninvasive diagnostic marker can be very helpful in thisregard Notably the first evidence of the efficacy of fecal calprotectin inthe diagnosis of IBD was obtained in the 1990s Røseth et al in proposed a method for measuring Calprotectin in stool specimens []One of the first and most interesting studies regarding fecal calprotectinutility in IBD diagnosis was the study by Røseth et al published in In this study patients with ulcerative colitis were studied and according to their results fecal calprotectin levels are higher in patientswith ulcerative colitis compared to healthy controls This study havealso shown that even patients with low disease activities had higherlevels of fecal calprotectin compared to healthy individuals []Subsequent studies somehow confirmed and complemented the findings of this study In another study published in AUC values of CI “ were reported for fecal calprotectin in thediagnosis of colorectal ‚ammation [] Moreover in a study onchildren with IBD it was shown that the level of fecal calprotectin washigher in these patients compared to healthy children so it can beconcluded that it is also directly correlated with ESR levels [] In astudy published in Kolho et al reported AUC values of CI “ for fecal calprotectin in the diagnosis of pediatric IBD [] In a study on patients with Crohn disease a sensitivity of and a specificity of at cutoï¬ of μgg have been reportedfor fecal calprotectin in diagnosis of the disease [] The results of ourrecent study along with other studies showed that fecal calprotectin ispreferred over traditional ‚ammatory biomarkers such as CRP andESR in the diagnosis of IBD [][] Diamanti et al reported a sensitivity of and a specificity of for fecal calprotectin at a cutoï¬ of μgg in IBD diagnosis [] In our recent study a sensitivityof and a specificity of at a cutoï¬ of μgg were observed for fecal calprotectin in the diagnosis of IBD however oursample size was and the majority of patients were in the active phaseof the disease []In another study conducted on patients with ulcerative colitis asensitivity of and a specificity of at cutoï¬ of μgg havebeen reported in this regard [] In one study it was shown that fecalcalprotectin in cutoï¬ of μgg is able to distinguish patients withIBD from patients without IBD patients with diseases other than IBDpatients with IBS and healthy persons with sensitivity and specificity [] Caviglia et al in their study reported a sensitivity of and a specificity of at a cutoï¬ of μgg for fecalcalprotectin in diï¬erentiating between IBS and IBD [] Howeversome studies have reported significantly lower values Accordingly in astudy on patients with ulcerative colitis Kalantari et al reported asensitivity of and a specificity of at a cutoï¬ of μgg []Besides there is a considerable agreement between fecal calprotectinand capsule endoscopy findings in patients with Crohn's disease Asensitivity of and a specificity of have also been reported at acutoï¬ of mgkg for fecal calprotectin in predicting CE findings anddiagnosis of Crohn's disease [] In another study lower sensitivityand specificity rates sensitivity specificity were reportedfor fecal calprotectin in this regard [] Furthermore in one studythat examined the efficacy of fecal calprotectin in predicting wirelesscapsule endoscopy findings a sensitivity of and a specificity ofTable Reported median levels of fecal calprotectin in healthy individuals of diï¬erent agesAgesMedian levels of fecal calprotectin range µggNumber of subjectsUsed kitUp to monthChildren “ yearsChildren “ yearsAdultsOver years “ “ “ “ “Bühlmann ELISABühlmann ELISACALPRO® Calprotectin ELISA Test ALPPhiCalPhicalReference[][][][][] 0cF KhakiKhatibi et al were reported for this biomarker at μgg in the diagnosis ofsmall bowel ‚ammation in Crohn's disease [] Given these findings it seems that fecal calprotectin has no ideal sensitivity and specificity for the diagnosis of IBD where the small intestine is involvedBesides there are some preanalytical limitations which are explainedin the next sections Therefore optimistically speaking fecal calprotectin measurement can eliminate the need for colonoscopy Howeverin a metaanalysis performed to evaluate the efficacy of fecal calprotectin and some other ‚ammatory markers to diï¬erentiate betweenIBD and IBS the probability of IBD was less than at fecal calprotectin values lower than µgg or CRP values lower than mgdL[] Therefore it seems that fecal calprotectin can be helpful at leastin ruling out the possibility of IBD in patients with IBSlike symptoms aswell as reducing the rate of colonoscopy Moreover it should be notedthat although a systematic review has reported pooled sensitivity andspecificity above for fecal calprotectin to diï¬erentiate between IBDand IBS it emphasized more on the possibility of falsepositive resultsin low cutoï¬ points [] Hence performing extensive studies indiï¬erent countries on the healthy population and the IBD patient is beneeded to determine a suitable cutoï¬ with maximum sensitivity andspecificity and minimum falsepositive resultsTable summarizes the results of various clinical investigationsregarding fecal calprotectin utility in the diï¬erential diagnosis of IBDfrom IBS and Table4 summarizes some metaanalysis results in thisregard As shown in Table the most important limitation of the majority of clinical studies conducted to date is the small sample size Alarge global study may be helpful in providing a more precise evaluation of fecal calprotectin clinical value in discrimination between IBDand nonIBD diseases Fecal calprotectin and endoscopic and histologic activity evaluationUndoubtedly one of the most serious challenges in the managementof IBD is evaluating the endoscopic and histologic activities of thedisease Nowadays colonoscopy and histopathologic examinations arethe routine tools for the assessment of mucosal healing in patients withIBD As noted earlier several scoring systems have been devised toscore disease activity based on the findings of colonoscopy and histopathologic examinations In recent years many promising results havebeen reported regarding the correlation between these scores and fecalcalprotectin levels In addition many studies have been performed inthe last decade all of which cannot be reviewed in this article The firstevidence of a link between fecal calprotectin and disease endoscopicactivity was obtained in the late 1990s In one of the first studiesRoseth et al found a significant correlation between fecal calprotectinlevels and endoscopic and histologic activities in patients with ulcerative colitis [] Furthermore in another study they observed that IBDpatients who were in remission clinically and had normal fecal calprotectin levels less than mgL had normal colonoscopy results[] These interesting findings indicate that fecal calprotectin can beconsidered as a biomarker in the evaluation of endoscopic activity andClinica Chimica Acta “Table4summarized results of some metaanalysis regarding the utility of fecal calprotectin in discrimination between patients with IBD and without IBDSample sizePooled SensitivityPooled SpecificityReferences[][][][][]mucosal healing in IBD patients Also these studies were the startingpoint of extensive studies that have been conducted up to now In astudy conducted on patients with Crohn's disease Sipponen et alinvestigated the sensitivity and specificity of fecal calprotectin in predicting endoscopic activity of Crohn's disease [] Correspondinglythe researchers used the Crohn's Disease Endoscopic Index of SeverityCDEIS scoring system in their study to evaluate the endoscopic activity of Crohn's disease As a result they found that there was a significant correlation between the endoscopic activity of the disease andthe level of fecal calprotectin Besides the findings of this study demonstrated that fecal calprotectin at µgg cutoï¬ can predict theendoscopic activity of Crohn's disease with sensitivity and specificity In another study CDEIS and Mayo Disease Activity IndexMDAI were used to evaluate the endoscopic activity of Crohn's diseaseand ulcerative colitis respectively According to the results of thatstudy on IBD patients there was a significant correlation between fecalcalprotectin levels and disease endoscopic activity [] Another studyshowed that fecal calprotectin is more strongly correlated with theendoscopic activity of the disease in ulcerative colitis compared to theRachmilewitz clinical activity index In addition in this study theoverall accuracy of fecal calprotectin for endoscopically active diseaseidentification was obtained as []Some studies have also shown the superiority of fecal calprotectinover traditional ‚ammatory markers like CRP Besides one studyfound that fecal calprotectin was more strongly correlated with theSimple Endoscopic Score for Crohn's disease SESCD compared to theCRP and even Crohn's disease activity index CDAI [] The modifiedBaron Index was also used in another study to evaluate the endoscopicactivity of ulcerative colitis As a result it was shown that calprotectinis more strongly correlated with the endoscopic activity of ulcerativecolitis compared to CRP and clinical activity of the disease [] In thisregard similar results were also observed in our recent study in whichthe Ulcerative Colitis Endoscopic Index of Severity UCEIS and SESCDwere used [] Therefore fecal calprotectin appears to be superior totraditional ‚ammatory markers in the prediction of IBD endoscopicactivity The high values of sensitivity and specificity that were mentioned earlier have raised the hope that using fecal calprotectin canreduce colonoscopy rate for patients™ monitoring However severalrecent studies have reported some significantly lower values Accordingly in a recent study in which Mayo Endoscopic Score [MES] wasused to evaluate the endoscopic activity of ulcerative colitis aTable Summary of the results of some studies regarding the utility of fecal calprotectin in discrimination between patients with IBD and without IBDNumber of IBD patientsAge groupLocationCut oï¬SensitivitySpecificity CD and UC CD and UC CD and UC and unclassified68CD and UC CD and UC and unclassified CD and UC CD and UC UC CD UCAdultsAdultsAdultsBoth adult and pediatricpediatricAdultspediatricAdultsAdultsBoth adult and pediatricTaiwanChinaItalySpainFinlandIranItalyIranDenmarkIndia48µgg µgg150µgg150µgg595µgg784µgg160µgg164µgg150µgg188µggAUCReferences[][][]SPSrefidbib60[][][][][][][] 0cF KhakiKhatibi et alClinica Chimica Acta “Table Summary of the results of some studies regarding the correlation of fecal calprotectin with endoscopic activity in IBD patientsAge groupStudylocationUsedendoscopicactivity indexCorrelationcoefficientrReferenceNumberof IBDpatients CD UC UC CD UCAdultsAdultsAdultsAdultsAdultsFinlandIranSwitzerlandSwitzerlandSwitzerland ModifiedCDEISUCEISRachmilewitzSESCD UC CDAdultsAdults UC CD UC CD CD UC UCAdultsAdultsAdultsAdultsAdultsAdultsAdultsBaron ScoreRachmilewitzSESCDGermanyUSA andCanadaJapanItalyItalyBrazilFranceFranceSouth Korea UCEISMattsSESCDMayo scoreSESCDCDEISMayo score[][][][][][][][][][][][][][]sensitivity of and a specificity of were reported for fecalcalprotectin at µgg to diï¬erentiate active endoscopic from inactiveMES or from MES or [] In another study the sensitivityand specificity of fecal calprotectin at a cutoï¬ of µgg for diï¬erentiating MES ‰¤ in patients with ulcerative colitis were and respectively [] Overall as presented in Table several studiesperformed in diï¬erent countries reported the correlation between fecalcalprotectin and IBD endoscopic activity Although some of these studies reported a strong correlation some others reported a relativelyweak correlation As noted earlier there are significant diï¬erencesbetween the reports on the sensitivity and specificity of fecal calprotectin to predict the endoscopic activity of IBD Undoubtedly a widerange of factors from sample size and the inclusionexclusion criteriato preanalysis variables and indexes used to evaluate the endoscopicactivity may also contribute to these diï¬erences However fecal calprotectin does not appear to be a very reliable marker for the predictionof IBD endoscopic activity so currently it seems a bit optimistic toconsider fecal calprotectin as a reliable alternative for colonoscopy Inthis regard further studies are still needed However under some certain circumstances such as pregnancy or pandemics the use of fecalcalprotectin to evaluate IBD endoscopic activity can be helpfulPregnant patients with IBD have serious limitations for colonoscopyexamination and it has been recommended that colonoscopy should beonly performed in the second trimester of pregnancy and where there isa strong indication [] Therefore noninvasive markers such as fecalcalprotectin can be helpful during pregnancy In one study physicianglobal assessment [PGA] which is a clinical symptombased criterionwas used to evaluate IBD activity and subsequently the associationbetween fecal calprotectin and this criterion was investigated in pregnant women with IBD The results of this study showed a significantcorrelation between fecal calprotectin and PGA levels at prepregnancyduring pregnancy and postpartum stages [] In another study asignificant association was reported between fecal calprotectin levelsand clinical activity of IBD in pregnant women Moreover it was shownthat stool calprotectin at a cutoï¬ of mgkg had a sensitivity between and as well as a specificity between and in the assessment of IBD clinical activity at diï¬erent stages ofpregnancy [] A recently published systematic review has also confirmed the conclusions obtained from these studies [] According tothese results it seems that fecal calprotectin is not aï¬ected by physiological changes during pregnancy however it is significantly correlatedwith IBD clinical activity during pregnancy Therefore from the viewpoint of relatively acceptable sensitivity and specificity in predictingthe endoscopic activity of IBD fecal calprotectin may be considered as anoninvasive biomarker for the evaluation of IBD endoscopic activity inpregnant women In addition under pandemic conditions fecal calprotectin can be very helpful Following the COVID19 pandemicwhich began in late and is still ongoing healthcare systems indiï¬erent countries were forced to impose significant limitations oncolonoscopy Therefore noninvasive IBD management and fecal calprotectin as a noninvasive laboratory marker have become moreimportant than before The combination between disease clinical activity and fecal calprotectin has been recommended as a noninvasiveapproach that can help in making decisions on treatment duringCOVID19 pandemic [] Therefore it seems that fecal calprotectincan be considered as an alternative for colonoscopy used for IBD endoscopic activity evaluation during pandemic Fecal calprotectin appears to be associated with IBD histologic activity as well Given thedifficulty in the evaluation of the histologic activity of Crohn's disease[] some studies have been focused on the ulcerative colitis andmany scoring systems have been devised so far Correspondingly thesesystems score the disease's histologic activity based on histologic observationsTherefore for this purpose a biopsy of the intestinal tissue is required which can be prepared by colonoscopy and then sent to thelaboratory In this regard one of these histologic scoring systems isRobert™s score that was used in one of our recent studies where weobserved a significant correlation between the level of fecal calprotectinand the histologic activity of ulcerative colitis which was calculatedbased on the Robert™s scoring system [] Theede et al also used themodified Harpaz Index and performed some interesting studies in thisregard In one of their studies fecal calprotectin was found to be significantly associated with the histologic activity of the ulcerative colitisand it was shown that it could predict histological mucosal healingAUC CI95 “ Sensitivity Specificity andCutoï¬ mgkg [] In another study on patients with endoscopically inactive ulcerative colitis Mayo endoscopic score the researchers showed that patients with ulcerative colitis who were inendoscopic remission but had histologically active disease had higherlevels of fecal calprotectin compared to patients with no histologicallyactive disease versus mgkg P Also despite thehigh specificity the sensitivity of fecal calprotectin in theprediction of score of histological activity was achieved as at mgkg [] In a recent study the Geboes
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"Biochemistry Biomarkers Medicine and Health Sciences Diagnostic Medicine Gastroenterology and Hepatology Gastrointestinal Cancers Oncology Basic Cancer Research Metastasis Cancers and Neoplasms Carcinomas Adenocarcinomas Colon Adenocarcinoma Gastrointestinal Tumors Pathology and Laboratory Medicine Anatomical Pathology Surgical Pathology Molecular Pathology The Clinical Implication of Cancer-Associated Microvasculature and Fibroblast in Advanced Colorectal Cancer Patients with Synchronous or Metachronous Metastases Cancer-Associated Microenvironment in Advanced Colorectal Cancer Kwak Yoonjin 1 2 Lee Hee Eun 1 Kim Woo Ho 1 2 Kim Duck-Woo 3 Kang Sung-Bum 3 Lee Hye Seung 4 * 1 Department of Pathology Seoul National University Hospital Seoul South Korea 2 Department of Pathology Seoul National University College of Medicine Seoul South Korea 3 Department of Surgery Seoul National University Bundang Hospital Seongnam-si Gyeonggi-do South Korea 4 Department of Pathology Seoul National University Bundang Hospital Seongnam-si Gyeonggi-do South Korea Lo Anthony W. I. Editor The Chinese University of Hong Kong Hong Kong * E-mail: hye2snu.ac.kr. Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: HEL WHK HSL. Performed the experiments: HEL WHK HSL. Analyzed the data: YK HEL HSL. Contributed reagents/materials/analysis tools: HEL WHK DWK SBK HSL. Wrote the paper: YK HEL WHK DWK SBK HSL. 2014 18 3 2014 9 3 e91811 11 1 2014 14 2 2014 2014 Kwak et al This is an open-access distributed under the terms of the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original author and source are credited. Background We aimed to evaluate the clinical significance of microvessel density (MVD) lymphatic vessel density (LVD) and cancer-associated fibroblasts (CAFs) in relation to tumor location in advanced colorectal cancer (CRC). Methods Using immunohistochemistry we examined 181 advanced CRC patients for CD31 and D2-40 to measure MVD and LVD respectively ?-smooth muscle actin (SMA) and desmin to identify CAFs and PTEN to examine genetic changes of CAFs. To evaluate the regional heterogeneity of these properties we examined tissue from four sites (the center and periphery of the primary cancer a distant metastasis and a lymph node metastasis) in each patient. Results MVD LVD and CAFs showed significant heterogeneity with respect to the tumor location. LVD was the greatest in the center of the primary cancers and the amount of CAFs was the lowest in distant metastases. In distant metastases those from the lung had higher LVD and MVD but fewer CAFs than those from the liver peritoneum or ovary. Patients with low MVD and LVD in the center of the primary cancer had worse outcomes and patients with few CAFs in distant metastases and in the primary tumor had a lower survival rate. PTEN expression in CAFs in distant metastases was lost in 11 of 181 CRC patients (6.1%) which was associated with a worse prognosis. Conclusions The microenvironment including cancer-associated microvasculature and fibroblasts is heterogeneous with respect to the tumor location in CRC patients. Therefore heterogeneity of microenvironments should be taken into account when managing CRC patients. This study was supported by grant number 03-2011-012 from the Seoul National University Bundang Hospital Research Fund. The funder had no role in study design data collection and analysis decision to publish or preparation of the manuscript. Introduction Although the mortality rates of colorectal cancer (CRC) patients have decreased in most western countries and in several developing countries in Asia advanced CRC patients who initially present with stage IV disease or those who develop distant metastases several months after diagnosis still have a lower five-year survival rate [1] [2]._ENREF_4 Recently the range of systemic chemotherapy has expanded and targeted therapy including epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) inhibitor therapies have been used in advanced CRC patients increasing patient survival [3]. However some CRC patients respond poorly to targeted therapy despite presenting positive results in targeted therapy-specific mutation studies [4]. One possible explanation for this therapeutic failure is tumor heterogeneity; several studies have reported that CRCs possess a heterogenic genotype or phenotype including KRAS p53 and BRAF [5]“[7]. Therefore the differing characteristics of the primary tumor site and the corresponding metastatic an need to be clarified to improve the management of CRC patients with metastatic diseases. Furthermore understanding the clinicopathological characteristics of advanced CRC is important for the development and improvement of systemic therapies. Since Paget et al. first described the cancer microenvironment by the œseed and soil theory [8] there has been growing evidence that cancer-associated stroma might affect the cancer cells themselves and contribute to cancer progression [9]. The main components of the cancer microenvironment are microvasculature (microvessels and lymphatic vessels) inflammatory cells and cancer-associated fibroblasts (CAFs) [10]“[12]. The current method of verifying angiogenetic and lymphangiogenetic activity in cancer tissue is to assess microvessel density (MVD) and lymphatic vessel density (LVD) respectively. MVD has been proposed as a surrogate marker of cancer-associated angiogenesis to identify patients with a high risk of recurrence or those with poor prognoses for various cancers including CRC [13] [14]; however the prognostic correlation of angiogenesis in CRC is still controversial [15] [16]. Similar to angiogenesis LVD has received interest as a means of lymphatic metastasis and survival [17] [18] but its role in tumor progression is still unclear [19]. The other prominent component of stroma CAFs are consistently activated and affect many aspects of tumor initiation invasion and progression [9]. While some studies have suggested that CAFs may inhibit tumor progression [20] [21] other studies have proposed that CAFs may promote progression in prostate breast and skin cancers [22]“[24]. In the context of CRC Tsujino et al. have suggested that ?-smooth muscle actin (SMA)-expressing CAFs might be a useful indicator of poor prognosis. However these results were restricted to stage II and III CRCs [25]. In addition to cancer cells genetic alterations in CAFs have demonstrated including the loss of heterozygosity microsatellite instability and genetic mutations [26] [27]. Recently genetic inactivation of PTEN in CAFs was reported in breast cancer patients [28]. Trimboli et al. identified that PTEN loss in stromal fibroblasts resulted in extensive extracellular matrix remodeling and angiogenesis which characteristic of tumor progression [28]. However expression loss of PTEN and its clinical significance have not been investigated in colorectal cancer patients. The aim of this study was to investigate the characteristics of microenvironments including microvasculatures and CAFs in advanced CRC patients. Additionally we assessed the intratumoral heterogeneity in the primary tumor and the discordance between primary tumor and distant metastasis microenvironments. "
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Electronic health records EHRs contain rich documentation regarding disease symptomsand progression but EHR data is challenging to use for diagnosis prediction due to its highdimensionality relative scarcity and substantial level of noise We investigated how to bestrepresent EHR data for predicting cervical cancer a serious disease where early detectionis beneficial for the outcome of treatment A case group of patients with cervical cancerwere matched to ten times as many controls and for both groups several types of eventswere extracted from their EHRs These events included clinical codes lab results and contents of free text notes retrieved using a LSTM neural network Clinical events are describedwith great variation in EHR texts leading to a very large feature space Therefore an eventhierarchy inferred from the textual events was created to represent the clinical texts Overallthe events extracted from free text notes contributed the most to the final prediction and thehierarchy of textual events further improved performance Four classifiers were evaluatedfor predicting a future cancer diagnosis where Random Forest achieved the best resultswith an AUC of from a year before diagnosis up to one day before diagnosis Weconclude that our approach is sound and had excellent discrimination at diagnosis but onlymodest discrimination capacity before this point Since our study objective was earlier disease prediction than such we propose further work should consider extending patient histories through eg the integration of primary health records preceding referral to hospitalIntroductionInformation on disease progression documented in electronic health records EHRs is apotential source of valuable new knowledge which could lead to improved health care [“]Since EHR information is derived directly from health care there is a great interest on how tobest use this source for reallife applications by way of advanced medical informatics Applications that can benefit from EHR mining include clinical decision support adverse eventa1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Weegar R Sundstro¨m K Usingmachine learning for predicting cervical cancerfrom Swedish electronic health records by mininghierarchical representations e0237911 101371journalpone0237911Editor Sreeram V Ramagopalan University ofOxford UNITED KINGDOMReceived January Accepted August Published August Copyright Weegar Sundstro¨m This is an access distributed under the terms ofthe Creative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement The data set consistsof second party data and is part of Health Bank Swedish Health Record Research Bank which ismanaged by the Department of Computer andSystems Sciences at Stockholm University dsvsusehealthbank The ethical permissionsassociated with this data set does not allow for access to the data For interestedresearchers inquiries can be made to the directorof Health Bank herculesdsvsuse or to thecorresponding author to access the data in thesame way as the authors ie on site at DepartmentPLOS ONE 101371journalpone0237911 August PLOS ONE 0cof Computer and Systems Sciences at StockholmUniversityFunding Both authors RW and KS were fundedby the Nordic Information for Action eScienceCenter of Excellence in HealthRelated eSciencesNIASC project number wwwnordicehealthse The funders had no role in studydesign data collection and analysis decision topublish or preparation of the manuscriptCompeting interests The authors have declaredthat no competing interests existUsing machine learning for predicting cervical cancer from Swedish electronic health recordsdetection and risk prediction [] One important potential of mining of EHRs is to generatenew clinical hypotheses and since EHRs document large populations observed over time theyallow for investigations regarding the relationship between clinical events and outcomes []Compared to cohort studies where specific information about individuals is typically collected at predefined time intervals the use of EHR data has several distinct strengths but alsolimitations Some of the advantages of using EHR data are that data are collected continuouslythat a richer set of information types is included and that less resources may be required toacquire the data However since EHRs are not primarily used to collect data for research purposes EHR data are typically sparse and can contain a high level of noise making this type ofdata challenging to analyze []In this work the focus is prediction of a major human cancer ie cervical cancer usingEHRs as input Globally cervical cancer is one of the dominating cancer forms in womenwith half a million cases each year In Sweden due to prevention through anized cervicalscreening the disease is rarer but there are still about cases of cervical cancer per year andthe incidence has risen in the past two years The median age of women getting the diagnosisis years and thus it is a disease which strikes relatively early in life [] Improved predictionof risk could lead to interventions at an earlier stage where the illness may still exist as a precancerous lesions amenable to surgical removal For cancer diseases earlier diagnosis couldalso lead to improvements in the outcomes of treatment and quality of life as well as for survival [] and for cervical cancer relative survival is higher when the cancer is detected at anearly stage [] The latter improvement is termed downstaging and would be of value especially in a disease such as cervical cancer where higher stages are associated with very highmortality Machine learning models created from EHRs could if sufficiently accurate potentially lead to an earlier diagnosis and increased knowledge regarding the events preceding adiagnosis In this work the aim is therefore to apply machine learning to EHRs and to explorehow well classifiers can identify future cervical cancer cases To this end both the informativeness of different event types found in EHRs diagnosis codes drug codes free text proceduresand lab results was evaluated together with the issue of how to best represent such events fordiagnosis predictionMachine learning methods have been applied to EHRs for predicting a number of differentoutcomes both for specific diseases and also for the risk of mortality and hospitalization andthe number of studies using EHR data for creating risk prediction models is increasing []Zhao and Weng [] used variables known to be related to pancreatic cancer andweighted them using PubMed s The variables included symptoms labresults andcomorbidities and were extracted from EHRs for cases and controls Each selected variable was assigned a weight according to if the association to pancreatic cancer mined from thes was positive or negative and a Bayesian Network Inference model using the assignedweights gave a better predictive performance compared to not using the weights derived fromPubMedIn their study on using machine learning to develop risk prediction models from healthrecord data Mani [] aimed to identify patients at risk of type diabetes They extracted variables representing demographic information clinical findings and laboratory valuesfor over patients where of were patients with a diabetes type diagnosis and theremaining were controls Three data sets representing different time intervals were createdIn the first one all data up to the diagnosis date were included while in the second and thirdones data were included from over days and over days before the diagnosis A numberof classifiers were evaluated for predicting which patients would develop type diabetes andan AUC of was achieved for each of the three time intervalsPLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordsHuang [] aimed to predict future cases of depression the severity of the depressionand the response to treatment In brief patients with a future depression diagnosis andat least years of data before the diagnosis were matched to six times as many controlsDiagnosis codes medication codes demographics and free text from EHRs belonging to thecases and controls were used as input and their model could predict depression diagnoses months in advance with an AUC area under the curve of and when including all available data up to the diagnosis data the corresponding result was an AUC of Kop [] used structured data from EHRs to predict future cases of colorectal cancerCRC Six months of data for over patients whereof CRC cases were extractedfrom primary care health records The data included ICPC codes International Classificationof Primary Care ATC codes Anatomical Therapeutic Chemical Classification System andlaboratory values Temporal and cooccurrence patterns were mined from the data set andused as input for three different classifiers CART decision trees logistic regression and Random Forest An AUC of was achieved and the input features were ranked according tothe importance factor provided by the logistic regression model Most of the features with highranks corresponded to events known to be linked to colorectal cancer providing validation oftheir modelFurther promising examples of studies have used neural network models such denoisingautoencoders in combination with Random Forest convolutional networks and recurrentnetworks for risk prediction of subsequent clinically relevant diseases through the use of EPRdata [“]Materials and methodsEthics statementThe use of health record data in this project was approved by the Regional Ethical ReviewBoard in Stockholm Sweden which determined that informed consent from the study participants was not required The data was anonymized before being accessed by the researchersEthical permission number Feature extractionWhen health records are mined a first necessary step is feature extraction during which dataof interest are selected and extracted from the records There are two possible ways of selectingwhich features to include either a topdown approach is used where domain or expert knowledge guides the feature selection or a bottomup approach is applied with an datadriven feature selection [] A benefit of using the bottom up approach is that it allows fordetecting new previously unknown links between events and outcomes since no a prioriassumptions are made regarding which features are relevant to include in the model []EHRs contain several information types requiring different levels of preprocessing beforethey can be included in a machine learning model Often a distinction is made between structured and unstructured information where diagnosis codes drug codes and demographicinformation such as age or gender is considered as structured and therefore easier to representfor machine learning purposes Free text on the other hand is regarded as unstructured information as it is not possible to directly determine the value or meaning of an EHR free textnote This free text makes up a substantial part of the documentation in EHRs and since itdescribes a patient™s health status symptoms and treatments it is potentially valuable toinclude free text in risk prediction models Free text notes require a higher level of preprocessing and therefore Natural Language Processing methods may be applied to structure the freetext and extract relevant information from it []PLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordsThe level or degree of structure can vary also for the structured data for example even ifthe type of a lab test is coded the result of the test might be a in free text with different unitsused for the same test Such information can be regarded as semistructured and thereforeadditional preprocessing is required also for these types of eventsDataThe health records collected for this study comes from Karolinska University Hospital inStockholm from the years [] These records contain coded information such asdiagnosis codes drug codes and procedural codes semistructured information such as labtest results and free text From this data set the patients with an ICD10 diagnosis coderepresenting cervical cancer ie a code starting with C53 were selected as the case groupNext a control group was created where each patient in the C53 case group was matched onage to ten other control women who did not have any C53 diagnosis on record in the dataSince one aim of this work was to investigate the events leading up to a first diagnosis of cervical cancer only cases with EHR data before the diagnosis date of cancer were included andall patients with the code Z854C previous cervical cancer were also filtered out leaving cases and controls Five basic event types were extracted from the ERHs for both casesand controls clinical entities found in the free text notes diagnosis codes drug codes labresults and procedure codes The extracted data were then divided into intervals for the firstof which all data up to the day before diagnosis were included For the next interval only dataregistered at least days before diagnosis were included and so on in intervals of days upto a year before diagnosis The available data was divided into two parts a development setcontaining of the data and a test set with the remaining The development set was setaside for hyperparameter tuning of the classifiers and for feature selection The test set wasused to evaluate the selected classifiers through a process of 10fold crossvalidation wherebyten rounds of training and testing was performed with of the data used for training and for testing for each round The development set was not included in the final evaluationof the classifiersICD codesThe health records contain diagnosis codes from the Swedish version of the ICD10 codesInternational Statistical Classification of Diseases and Related Health Problems”Tenth Revision ICD10 codes are hierarchically arranged with chapters at the highest level thesechapters are further divided into sections subsections and full codes This makes it possible toinclude both very finegrained information in form of the full diagnosis codes as well as higherlevel information such as chapters and subsections Table gives an example of the ICD10Table Example from the ICD hierarchyLevelChapterSectionExampleC00D48 NeoplasmsC51C58 Malignant neoplasms of female genital ansSubsectionC53 Malignant neoplasm of cervix uteriCodeC530 Malignant neoplasm endocervixSizeIn dataThe top of the hierarchy consists of different chapters where chapter II comprises codes for neoplasms Furtherdown in the hierarchy more detailed information is included in the codes The column titled Size gives the numberof different codes for each level in the complete code hierarchy and the last column In data shows the number ofdifferent codes included in the current data sets101371journalpone0237911t001PLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordsFig ICD chapters and sections for the case group and the control group The left part corresponds to the case group and the right part the control group The innercircle represents the ICD chapters and the outer circle the ICD sections included in the chapter the labels show the sections with the most frequent codes101371journalpone0237911g001hierarchy for the code C530 Fig provides a visualization of the distribution of ICD10 codesfor the control group and the C53 case group Comparing cases and controls it can be notedthat many of the patients in the case groups have experienced other types of cancers ofcases and of controls had a diagnosis code from chapter II of ICD10 which is the chaptercontaining codes for tumorsneoplasms Additionally a large part of these codes came fromthe section representing codes for malignant neoplasms of the female genital ans C51C58other than cervical cancerATC codesThe next feature type are the ATC codes from the Anatomical Therapeutic Chemical Classification System representing drugs As with ICDcodes these codes are hierarchical and therefore it was possible to represent them with different levels of detail Table shows an exampleof the included ATC levelsClinical entities extracted from textMost of the data extracted from the EHRs were in the form of free text notes and differentapproaches have previously been used to create representations of the free text in EHRs OneTable Example from the ATC code hierarchyLevelMain group3rd level subgroup4th level subgroupFull codeExampleN Nervous systemN02B Other analgesics and antipyreticsN02BE AnilidesN02BE01 ParacetamolIn dataThe last column of the table shows how many different codes appear in the current data set for each code level101371journalpone0237911t002PLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordsFig Example of patient record text This example contains one multiword Disorder œsquamous cell carcinoma insitu101371journalpone0237911g002possibility is to map the text to some existing ontology or terminology Roque []matched free text to ICD codes to enrich coded information extracted from EHRs This hasthe advantage of getting a coded representation practical for interpretation and machinelearning purposes however mapping directly from clinical text to standard terminologies canlead to low recall [] as the language used in clinical text differs from the standardised language in terminologies Tools for matching text to terminologies such as MetaMap [] arenot available for Swedish Another approach which also reduces the size of the feature spacewas applied by Miotto [] They included free text in their model by extracting entitiesfrom clinical notes using the Biomedical Annotator and topic modeling this approachwas effective for predicting diagnoses but has the drawback of reduced interpretabilityHere named entity recognition NER was applied to the free text notes belonging to casesand controls Using NER allowed for extracting the most relevant parts of the text and compared to including the full texts in a bagofwords model named entities makes it possible torepresent multiword expressions such as diabetes typ diabetes type and cancer in situ Toextract these entities from the free text a bidirectional long shortterm memory biLSTMnetwork was trained on notes annotated by two medical experts The notes used as trainingdata for the network were annotated for the entity types Body part annotations Disorder annotations and Finding annotations For this work findings and disorderswere included as events and following the SNOMED definitions a œFinding represent bothnormal and abnormal observations regarding a patient while a œDisorder always is the resultof an underlying pathological process see Fig A large corpus of clinical texts was used to generate word2vec [] embeddings of the textsas input representation for the network This corpus contained GB of text from SwedishEHRs with a complete vocabulary of about words The properties of the LSTM network are described in detail in Weegar [] This network was applied to all clinical notesfor the cases and the controls to extract all mentions of findings and disorders from these textswhich contained on average tokens for each patient Next negation detection was used toidentify negated entities using a rulebased module NegEx adapted specifically to the domainof Swedish clinical text Swedish [] This was an important step because clinical notes oftencontain documentation of discussions and reasoning around a potential condition in text andit is thus particularly common that findings are negated Indeed in this material up to ten percent of findings and six percent of the extracted disorders were actually in the negated formSimilarly to mitigate the risk of including information regarding individuals other than theactual patient to whom the record belonged deriving from eg discussions on family historyof disease clinical notes with headings related to family members were excluded The use ofNER for event extraction resulted in events for the controls on average For the cases onlyevents occurring before the C53 diagnosis were included on average eventsThe extracted events were lemmatized which reduced the scarcity of the free text data aslemmatization maps inflected versions of a word to the same basic representation By usinglemmatization words such as hudirritationskin irritation singular and hudirritationerskinirritations plural will be considered as the same event Similarly the event hostadecoughedPLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordspast tense will be joined with hostarcoughs present tense But even after lemmatizationthe number of different events extracted from the texts is very large There were about different events appearing at least two times This is partially due to the possibility of describing the same event in many different ways in writing for example œfractured patella andœpatella fracture refers to the same type of event but will be considered as two different eventsas their surface forms are different Therefore to further reduce the feature space two additional preprocessing steps were applied to the textual events Firstly a spelling normalizationmodule was used to group different spelling variations of the same concept This grouping wasachieved through calculating the edit distance between each pair of events A Levenshtein editdistance of two strings is a measurement of how different the two strings are and is calculatedby counting the number of insertions deletions and substitutions of characters that is requiredto make two strings equal [] Groups of events were formed where each member in thegroup had at most an edit distance of one from some other member of the group and the samefirst letter as all other members in the group as it was found that allowing larger distancesintroduced errors For multiword expressions the distance was calculated per word and itwas also required that each included word had the same first letter The spelling differences inthe text data were mainly the result of misspellings but also of inflections or instances of writing the same concept as a either a compound word or a as two separate words After groupingthe events using edit distance all variations were exchanged with the most frequent surfaceform One example of such a group is the spelling variations for thyroid cancerthyreoideacancer tyreoideacancer thyroidecancer thyreoidacancer thyreoidcancer tyroideacancer tyreoidecancer thyroidea cancer thyroideacancer thyreoidecancerwhere each variation was mapped to the most frequently occurring form œthyreoideacancerThis normalization mapped about different surface forms of events into about different groupsNext a hierarchical representation of the events extracted from the free texts was createdThe hierarchy was constructed by first sorting the events extracted from the texts according totheir length and placing oneword entities at the top of the hierarchy Next for each level anyevent that contained all words of an event on the higher level was added as a child node of thatevent In this hierarchy the oneword event œleukaemia at the top level had the child nodesœacute leukaemia and œchronic leukaemia where œchronic leukaemia in turn had the childnodes œchronic myeloid leukaemia and œchronic lymphocytic leukaemia Events furtherdown in the hierarchy became gradually more detailed as they often contained some type ofmodifier to its parent node such as œnormal œserious or œmalignant capturing many relevant relationships between eventsA hierarchy of four levels was created with oneword entities at level one and events containing four or more words at the fourth level It is worth noting that when representing thetextual events for a single patient the longer lowlevel events had influence over higher levelrepresentations in the same way as for the ICD or ATC code hierarchies Using the ICD codehierarchy a diagnosis can be represented by the corresponding full lowlevel code or by thesection or chapter it belongs to In a similar way the lower level event œchronic myeloid leukaemia can be represented by the higher level event œleukaemiaThis hierarchy of textual events was however different from the ICD and ATC code hierarchies in two ways Firstly each child node could have more than one parent node an œinfectedwound had the parent œwound and the parent œinfected The second difference was thatwhile the top levels are the smallest in the code hierarchies the opposite is true for the textualevents There was a higher number of short textual events and the short events also make up alarger part of the total set of textual events The top level of the hierarchy contained PLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordsdifferent events followed by events at level two events at level three and events at level fourLab resultsThe included data for lab results consists of the type of test and the result of the test Includingthe value of test result can be too finegrained for the task of prediction since many tests arerare [] Therefore the result is represented as being either inside or outside the referencerange for a test value For example the result of a Hemoglobin test can either be in the normalrange for Hemoglobin above the range or below it Therefore each lab result in the includedhealth records will be represented by one of three different features depending on the outcomeof the testProcedure codesProcedure codes classify procedures including surgical procedures medical investigationspreventive measures and treatments for example the code AK044 corresponding to an ultrasound of the kidneys The codes are used in health records for statistical and administrativepurposes and have been included as an event type in this workA number of the events that were extracted from the EHRs were unique meaning that theyonly occurred for a single patient Since such events cannot contribute to the diagnosis prediction but only increase the scarcity of the data they were removed at this stage leaving in total different events in the data set Each case had on average different events and events in total before the diagnosis For the controls the corresponding numbers were on average different events and an average total of The reason for the larger number of eventsfor the control group is that only data up to a cervical cancer diagnosis is included for the casegroup and all events after the diagnosis were discarded For the controls on the other hand alldocumented events were kept even if they occurred after the time of their matched case™s diagnosis Since the objective of our study was correct classification based on EHR events withpotentially low expected contrast between cases and controls we opted for this choice to maximize the control information for the classifiers to be trained on minimize the risk ofmisclassification of disease status by ensuring to the best of our knowledge that no hospitalbased female controls were diagnosed with cervical cancer later during the study period and to increase generalizability to a reallife clinical situation where EHRs are available but diseasestatus of the individuals is not already known Fig gives an overview of the available data forthe different time intervals However some events will appear outside of the study periodleading to data censoring [ ] Important and informative events could have occurred beforethe start of data collection and this also means as in most casecontrol or other studies thatwe do not know whether some members of the control group develop cervical cancer after thestudy end point when no more EHR data was available to us However this is a wellknownfact resulting from this type of study design and does not invalidate the comparison made during the actual study period definedClassification experimentsFour different classifiers were used to evaluate the appropriateness of the different featuretypes for classification of future C53 cases These classifiers were Random Forest ComplementNaive Bayes Bernoulli Naive Bayes and Support Vector Machines all implemented in Scikitlearn [] The first classifier Random Forest is an ensemble classifier robust to noise andlarge feature spaces [] Complement Naive Bayes [] is capable in cases of data sets withclass imbalance Bernoulli Naive Bayes additionally takes absence of features into account forPLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordsFig Available data for the case group The xaxis denotes the number of days before a diagnosis and events only appearing onetime in the data set have been excluded At days before a diagnosis events were available for patients101371journalpone0237911g003classification [] and Support Vector Machines SVM is a classifier well suited for highdimensional spaces []This classification task can be understood as a text classification problem where each eventextracted from a patient record corresponds to a word and each patient is represented by a vector with the same dimension as the complete vocabulary of events For Random Forest andComplement Naive Bayes the input vectors consisted of the raw counts of events For Bernoulli Naive Bayes binary input vectors were used and for the SVM classifier normalized vector counts were used as input Binary vectors correspond to if a patient ever experienced anevent and count vectors also represent how many times each event occurred Another possibility is to use tfidf term frequencyinverse document frequency weights The idea behindtfidf is to give more weight to the events that are representative for individual patients However using tfidf did not improve classification resultsResultsEvent typesEach type of event was evaluated individually for its ability to correctly classify the patients Fig shows the average AUC using the four classifiers over time for each event type ICD codesATC codes Procedure codes Clinical entities and Lab results The average AUC was calculated as the sum of the scores AUCi for the individual classifiers divided by the number of classifiers AUCavg ¼ the classifiers were the clinical entities extracted from the text as the highest AUC scores wereachieved using only the text entities ðAUC1 þ AUC2 þ AUC3 þ AUC4Þ The most informative event type forPLOS ONE 101371journalpone0237911 August PLOS ONE 0cUsing machine learning for predicting cervical cancer from Swedish electronic health recordsFig Average AUC for the different event types over time101371journalpone0237911g004Event levelsSince the data in EHRs typically are noisy high dimensional and sparse it is necessary toinvestigate data representation how the information included in the records should be presented to the model [] In this work both which types of feature to include and also the detailwith which those features should be represented was evaluatedIt has previously been found that including features derived from several levels of hierarchical clinical codes improves the performance for predicting adverse drug events from EHR data[] and as the input events can be represented with different levels of detail the next set ofexperiments aimed to evaluate the most suitable representation of the events in this regardUsing the most detailed levels such as a full ICD10 code gives very detailed information buta very sparse data set By including the higherlevel representations the sparsity of the data canbe reduced Fig shows the average AUC o
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"Methylnaltrexone was developed at the University of Chicago and licensed to Progenics Pharmaceuticals subsequently sub-licensed to Salix Pharmaceuticals. Dr. Moss was a paid consultant for Progenics Pharmaceuticals and currently is a paid consultant for Salix Pharmaceuticals. He receives royalties through the University of Chicago. There are no patent(s) or patent applications relating to material pertinent to this . This does not alter the authors' adherence to all PLOS ONE policies on sharing data and materials. Conceived and designed the experiments: FEL JM RS PAS. Performed the experiments: TM BM VAP FEL. Analyzed the data: JM PAS. Contributed reagents/materials/analysis tools: PAS JM. Wrote the paper: PAS. 2014 24 3 2014 9 3 e91577 16 9 2013 13 2 2014 2014 Lennon et al This is an open-access distributed under the terms of the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original author and source are credited. Recent epidemiologic studies implying differences in cancer recurrence based on anesthetic regimens raise the possibility that the mu opioid receptor (MOR) can influence cancer progression. Based on our previous observations that overexpression of MOR in human non-small cell lung cancer (NSCLC) cells increased tumor growth and metastasis this study examined whether MOR regulates growth factor receptor signaling and epithelial mesenchymal transition (EMT) in human NSCLC cells. We utilized specific siRNA shRNA chemical inhibitors and overexpression vectors in human H358 NSCLC cells that were either untreated or treated with various concentrations of DAMGO morphine fentanyl EGF or IGF. Cell function assays immunoblot and immunoprecipitation assays were then performed. Our results indicate MOR regulates opioid and growth factor-induced EGF receptor signaling (Src Gab-1 PI3K Akt and STAT3 activation) which is crucial for consequent human NSCLC cell proliferation and migration. In addition human NSCLC cells treated with opioids growth factors or MOR overexpression exhibited an increase in snail slug and vimentin and decrease ZO-1 and claudin-1 protein levels results consistent with an EMT phenotype. Further these effects were reversed with silencing (shRNA) or chemical inhibition of MOR Src Gab-1 PI3K Akt and STAT3 (p<0.05). Our data suggest a possible direct effect of MOR on opioid and growth factor-signaling and consequent proliferation migration and EMT transition during lung cancer progression. Such an effect provides a plausible explanation for the epidemiologic findings. Support was provided from institutional and/or departmental sources and National Institutes of Health grant CTSA UL1 TR000430. The funders had no role in the study design data collection and analysis decision to publish or preparation of the manuscript. Introduction The role of anesthesia and analgesia in the recurrence and metastatic rate of malignancies has recently received considerable attention [1] [2] [3] [4]. Retrospective studies have demonstrated a diminished incidence of cancer recurrence following regional anesthesia with lower doses of opioids following surgery for breast prostate colon cancer and melanoma although other studies have failed to detect significant differences [5] [6] [7] [8]. Some hypotheses to explain these differences in recurrence rates include immune suppressive effects and direct effects on tumor cell growth [9] [10] [11]. Our research has focused on the mu opioid receptor (MOR) and its role in directly regulating cellular changes leading to tumor growth and metastasis [4] [12] [13]. Effective therapeutic strategies for lung cancer the leading cause of cancer-associated mortality worldwide are extremely limited exemplifying the need for early diagnosis and novel therapeutic interventions [14] [15]. We have previously reported that the MOR is upregulated in several types of human non-small cell lung cancer (NSCLC) [12]. Further we have shown that overexpression of MOR in human NSCLC increases primary tumor growth and metastasis in xenograft models [13]. However the exact cellular changes regulated by MOR in NSCLC are incompletely defined [4]. For cancer cells to grow and metastasize there needs to be a loss of cell-cell adhesion (characterized by a reduction of epithelial cell adhesion proteins including the tight junction proteins ZO-1 and claudin-1) followed by acquisition of mesenchymal characteristics including a loss of baso-apical polarization cytoskeletal remodeling and increased cell motility (characterized by increases in specific cytoskeletal proteins (i.e. vimentin) and transcription factors (i.e. Slug and Snail) [16] [17] [18] [19]. This orchestrated oncogenic process is referred to as epithelial mesenchymal transition (EMT) [16] [17] [18] [19] [20] [21] [22]. Growth factor receptors including the epidermal growth factor receptor (EGFR) are often overexpressed and/or mutated in NSCLC and regulate oncogenic processes including tumor cell proliferation migration and EMT transition [23] [24] [25] [26] [27]. Several therapies targeting the EGFR in NSCLC exist including tyrosine kinase inhibitors (gefitinib erlotinib) and monoclonal antibodies (cetuximab)[28] [29] [30] [31]. However the overall survival rate for NSCLC remains low [32] [33] [34]. Recently Fujioka et al. have demonstrated that morphine can stimulate EGFR signaling pathways including the serine/threonine kinases Akt and MAP kinase in NSCLC suggesting a role for MOR inhibition as a potential therapeutic strategy for NSCLC [35]. Based on the recent interest of the effects of anesthesia and analgesia regimens on the recurrence and metastatic potential of various cancers [1] [2] [3] [4] our previous published data indicating the MOR is upregulated in lung tissue from patients with NSCLC [12] overexpression of MOR promotes tumor growth and metastasis in human NSCLC xenograft models [13] as well as data from Fujioka et al. demonstrating MOR regulation of EGF-induced signaling events in NSCLC [35] this study investigated the functional effects of MOR in the fundamental oncogenic processes of opioid and growth factor-induced human lung cell migration proliferation and epithelial mesenchymal transition (EMT)[16] [17] [18] [19] [20]. Since there is currently very little information on opioid and/or MOR regulation of EMT and the molecular mechanisms integrating cancer cell proliferation migration and EMT this study investigated the detailed molecular mechanisms for these events which can have potential clinical utility. Methods Cell Culture and Reagents The human NSCLC cell H358 was obtained from ATCC (Walkersville MD) and cultured in Roswell Park Memorial Institute complete medium (Cambrex East Rutherford NJ) at 37°C in a humidified atmosphere of 5% CO2 95% air with passages 6“10 used for experimentation. Unless otherwise specified reagents were obtained from Sigma (St. Louis MO). Reagents for SDS-PAGE electrophoresis were purchased from Bio-Rad (Richmond CA) and Immobilon-P transfer membrane was purchased from Millipore "
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colorectal carcinoma crc as one of the most commongastrointestinal malignancies has developed the world™sfourth most deadly cancer with a high rate of incidence andmortality [ ] liver metastasis which is the most commonform for crc metastasis is the leading cause of high mortality for this severe malignancy however few clinical prevention and treatment measures could be available for tumormetastasis therefore it is really urgent to develop new biomarkers and explore the underlying mechanism for crcmetastasis and eventually develop new therapeutic strategiesfor crc patientsduring cancer progression metabolic reprograming withincreasing glucose utilization is termed as warburg aï¬ectwhich is accompanied by altered pyruvate and mitochondrialmetabolism the fate of pyruvate is the core manifestationto distinguish normal cells and tumor cells through metabolism in normal cells pyruvate was used for efficient atpproduction directly into mitochondria however pyruvatewas converted into lactate in cytosol despite of normoxicand hypoxic conditions in cancer cells this may be dueto the impaired process of pyruvate from the cytosol intothe mitochondrial matrix which is a critical metabolic steplinking glycolysis and mitochondrial oxidative phosphorylation the mitochondrial pyruvate carriermpc a 0c of immunology researchmultimeric complex containing two distinct proteins mpc1and mpc2 which is located in the inner mitochondrialmembrane is responsible for efficient mitochondrial pyruvate uptake loss of mpc expression or activity blockspyruvate entry into the tca cycle which results in a metabolism switch to increase glycolysis and the compensatoryusage of glutamine [ ]existed studies have reported that mpc1 was related withimmunoregulation stemness metabolism cellular morphology etc [ “] currently the important role of mpc1was uncovered in several tumors in crc and esophagealsquamous cell carcinoma decreased mpc1 results in accelerated aerobic glycolysis and malignant progression [ ] inlung adenocarcinoma mpc1 deficiency accelerates lung adenocarcinoma progression through the stat3 pathway in prostate cancer mpc1 was reported to be involved instemness and metabolism which regulated by couptfii[ ] in renal cell carcinoma hypoxiainduced loss ofmpc1 enhanced the expression of mmp7 and mmp9 to promote cell invasion collectively these data suggestedthat mpc1 maybe serves as a suppressor to disrupt tumormalignancy however whether mpc1 is involved in crcmetastasis and the underlying mechanisms remain to beillustratedin the present study we figured out the relationshipbetween the mpc1 expression and crc liver metastasiswe identified that decreased mpc1 was closely correlatedwith patient™s metastasis as well as led to poor prognosisfunctionally mpc1 overexpression could attenuate themigration and invasion capacities of crc cells both in vitroand in vivo mechanically mpc1 suppressed crc metastasisthrough mediating the wntcatenin signaling thus ourfinding firstly revealed a critical role of mpc1 in crc livermetastasis materials and methods data mining seven geo datasets gse21510 gse5206gse20916 gse9348 gse89393 gse67675 and gse4183and tcga were used to analyze the mpc1 expression pattern in crc the primary data for tcga datasets weredownloaded wwwcancergov the primary data feo datasets were downloaded at wwwncbinlmnihgovgeo the oncolnc database httpwwwoncolnc was used to detect the prognostic value of mpc1 inthe tcga cohort patients and clinical specimens in our study a total of patients containing paired crc tissues and adjacentnontumor tissues were enrolled from the department of gastrointestinal surgery renji hospital school of medicineshanghai jiao tong university among them cases ofliver metastases were collected and only cases wereavailable with complete followup data for survival analysisinformed consents were signed by all patients the researchwas approved by the research ethics committee of renjihospital and carried out in accordance with ethical standardsas formulated in the helsinki declarationtable sequences of primers used for realtime pcrprimermmp7 forwardmmp7 reverseecadherin forwardecadherin reversesnail1 forwardsnail1 reversemyc forwardmyc reversegapdh forwardgapdh reversesequence ²²gagtgagctacagtgggaacactatgacgcgggagtttaacatatgagtgtcccccggtatcttcacgagcagagaatcataaggcggtatccagagctgtttggaaacattttcctcccaggccatcacagccctcactcacacagattccacaaggtgcctgggctacactgagcaccaagtggtcgttgagggcaatg cell culture and cell transduction human crc celllines hct116 ht29 sw620 rko sw480 and lovoand mouse crc cell line mc38 were gained from the cellbank of the chinese academy of sciences shanghai chinaall cells were cultured in dmem medium supplemented°with fetal bovine serum and antibiotics at c in ahumidified incubator with co2mc38 cells were transfected with lentivirus containing aluciferase reporter plasmid stable transfection cells werescreened with μgml blastisidin for days which termedmc38luc in addition one short hairpin rna shrnasequence against mpc1 was packaged as lentivirus andtransfected into mc38luc cells lovo and sw480 were transfected with lentivirus containing fulllength human mpc1cdna or empty vehicle control stable transfection cells werescreened under μgml puromycin for days and verifiedby western blot in those assays all lentiviral transfectionswere performed in the presence of μgml polybrene immunohistochemical ihc the protocol of this assayand quantify the mpc1 protein expression level were performed according to previously reported primary antibodies used as follows mmp7 yt2663 immunoway ecadherin cst snail1 ab53519 abcam and mycyp0861 immunoway cellular immunofluorescence assays were performedaccording to the previous description briefly cells wereincubated with antibodies against catenin ab32572abcam and incubated with alexa 488conjugated secondaryantibody the nuclei were stained with ²6diamidino2phenylindole dapi western blotting wholecell lysates or nuclear proteinwas extracted using a protein extraction buï¬er beyotimeshanghai china or nucleoprotein extraction kit sangonbiotech c500009 respectively proteins were resolved bysdspage and transferred onto nitrocellulose nc membranes using standard methods primary antibodies used asfollows mpc1 ab74871 abcam catenin ab32572abcamlamin ac ab8984 abcam and gapdhab9485 abcam speciesspecific secondary antibodies used 0c of immunology researchtable related enzyme and carrier of pyruvate analyzed in this studygenepdha1pdhbpdk4pdhxmpc2pdk2pdk1pdk3mpc1pcpdp1pdprpdha2pdp2pklrdescriptionpyruvate dehydrogenase lipoamide alpha pyruvate dehydrogenase lipoamide betapyruvate dehydrogenase kinase isozyme pyruvate dehydrogenase complex component xmitochondrial pyruvate carrier pyruvate dehydrogenase kinase isozyme pyruvate dehydrogenase kinase isozyme pyruvate dehydrogenase kinase isozyme mitochondrial pyruvate carrier pyruvate carboxylasepyruvate dehydrogenase phosphatase catalytic subunit pyruvate dehydrogenase phosphatase regulatory subunitpyruvate dehydrogenase lipoamide alpha pyruvate dehydrogenase phosphatase catalytic subunit pyruvate kinase liver and rbclocationmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion inner membranemitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion inner membranemitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrionas follows irdye goat antimouse igg licor andirdye goat antirabbit igg licorttest was used for comparison between groups p wasconsidered as statistically significantsynthesized using the primescript realtime pcr total rna was extracted using trizoltakara according to the manufacturer™sinstructionscdna wasreversetranscriptionpolymerasechain reaction rtpcr kittakara the qpcr was performed using sybr green masˆ’–ct method was used to analyze theter mix roche the data and gapdh was used as a loading control the primersequences are listed in table liver metastasis model this study was performed inaccordance with the recommendations in the guide for thecare and use of laboratory animals and relevant chineselaws and regulations the protocol was approved by theinstitutional animal care and use committee iacucof shanghai jiao tong university the mc38 cells transplanting luciferaseexpressing were injected into the spleensof c57bl6n mice n with a concentration of cellsmouse two weeks later the mice were killed andthe liver metastasis tissues were harvested luciferase reporter assay the protocols of this assaywere operated in accordance with previous reported ng top reporter plasmid wntcatenin signalingor ng fop reporter plasmid negative control ofwntcatenin signaling and ng renilla were mixedand transfected into crc cells using lipofectamine dualluciferase reporter assay system promega was usedto detect the firefly and renilla luciferase activities statistical analysesdata are shown as means ± sd spss chicago il usaand graphpad prism software were used to manipulate statistical analyses kaplanmeier method was used to calculatecumulative survival time the chisquare test or student™s results mpc1 expression was aberrantly decreased in crcpyruvate is a pivotal intermediate in the process of cellmetabolism which connects glycolysis and the tca cycleto determine the potential maladjustment genes involvedin pyruvate metabolism which is located in mitochondriaas shown in table we analyzed the tcga dataset containing crc and their normal counterparts the resultsshowed that multiple genes are significantly upregulated ordownregulated in crc t tissues compared to normal colonn notably mpc1 had a log2 fold change less than figures 1a and 1b as known to us pyruvate translocation from the cytoplasm to the mitochondria is the first stepinto the tca cycle which needs mpc1mpc2 heterodimerin the analysis no significant change was found in mpc2therefore mpc1 was selected for further study the expression pattern of mpc1 was further analyzed in five independent geo datasetsgse21510 gse5206 gse20916gse9348 and gse4183 consistently we found thatmpc1 was downregulated with statistical diï¬erence in crctissues figure 1c and inflammatory tissue supplementary figure in comparison to their normal counterpartsmeanwhile we found decreased mpc1 expression inhuman crc tissues compared to their normal counterpartsfigure 1d in addition a similar phenomenon wasrevealed in aomdss induced mouse crc modelsfigure 1e then we evaluated the protein level of mpc1used a tissue microarray containing matching cancerand corresponding adjacent nontumortissues whichsubjected ihc staining the expression of mpc1 wasscored as œ  based on the staining area andintensity figure 1f we found that more lower mpc1expression score as œ and œ was presented in crc 0c of immunology researcheulavp gol“tcga t nmpc1“mpc2“log2 fold changenoisserpxe cpm evitalertcgaŽŽŽn normaltumornoisserpxe cpm evitalergeo datasetsŽŽŽŽŽŽŽŽŽŽŽŽgse21510 gse5206 gse20916 gse9348normaltumorabhuman samplecrcnormalaom dssnormalccrcesacesac“esacesacdempc1 lower expressionmpc1 higher expressionfŽŽ egatnecrepromutlamron“gfigure expression pattern of mpc1 in crc a volcano plot showed fold changes xaxis and corresponding p values log10 yaxis ofpyruvate metabolismrelated genes located in mitochondria analyzed in the tcga dataset between paired normal and crc samplesstudent™s ttest b the comparison of mpc1 expression in tumor and matched normal tissues using the tcga dataset student™s ttest ˆ—ˆ—ˆ—p c expression analysis of mpc1 in tumors and corresponding normal tissue using four independent geo datasetsgse21510 gse5206 gse20916 and gse9348 student™s ttest ˆ—ˆ—ˆ—p d ihc staining of mpc1 expression in matched crctumor and nontumor tissues scale bar μm e ihc staining of mpc1 expression in the aomdss induced crc mouse models andcontrol animal scale bar μm f representative ihc staining of mpc1 expression in tissue microarray from the ren ji cohortwhich contained crc patients and paired adjacent normal tissue n the tumor tissues were divided into two groups based on theexpression level which scored as œ  scale bar μm g the percentage of tissue displaying diï¬erent expression level ofmpc1 in crc tumor and adjacent nontumor tissues fisher™s exact test ˆ—ˆ—p tissues figure 1g overall these results revealed thatmpc1 was disrupted during crc decreased mpc1 enhanced tumor metastasis capabilityand predicated poor prognosis in crc it is well known thatcrc is one of the most malignant tumors given its strongmetastasis ability so we tried to figure out whether thempc1 expression was correlated with metastasis we foundthat lower mpc1 expression was closely correlated withmetastasis p lymph node invasion p and tnm stage p which revealed by the analysisbetween the clinical significance and mpc1 expression incrc table next to illuminate the expression patternof mpc1 in diï¬erent process of crc data mining was carried out by two independent geo datasets gse21510 andgse89393 which contained normal tissue primary crcand metastatic lesion in the liver mcrc as shown infigures 2a and 2b mpc1 expression was gradually downregulated in patients with an increase in metastasis ability asimilar result was found in mice cells ct26 with high livermetastasis hmct26 or poor liver metastasis pmct26figure 2c which isolated by in vivo selection in an orthotopic mouse model of colon cancer metastasis to the liverfurther analysis showed that mpc1 protein expression was 0c of immunology researchtable clinicopathological correlation of mpc1 expression in theren ji crc cohortclinicopathological featureexpression ofmpc1lowhighp value χ2testage years‰¥gendermalefemalemetastasisyesnolymph node invasionyesnotumor size cm‰¥ cmtnm stageiiiiiiivkras mutationmutationno mutationgradually downregulated in normal tissue primary crc andliver metastasis crc crlm tissues figure 2d furthermore survival analysis showed that patients with lowermpc1 expression had a worse outcome compared to thepatients with higher mpc1 expression using the tcgacohort figure 2e and ren ji cohort figure 2f additionally among patients with metastasis worse prognosiswas emerged in the mpc1 low cases figure 2g while nosignificant association was observed in patients withoutmetastasis figure 2h mpc1 overexpression impaired crc cells motility bothin vitro and in vivo to evaluate the role of mpc1 on themotility of crc cells the transwell assay was performedfirstly we examined low mpc1 protein expression in humanand high mpc1 protein expression in mouse mc38 crccells by western blot figure 3a mpc1overexpressingstable cell lines were established using a lentivirus carrying the mpc1 gene in lovo and sw480 cells and theoverexpression efficiency was confirmed by immunoblotsfigure 3b mpc1 knockdown in lucmc38 cells wereestablished and verified by wb figure 3c mpc1 overexpression exhibited significantly weaker migration and invasion ability than the control cells in both lovo figure 3dand sw480 figure 3e cells following the liver metastasismodel of crc was established by spleen orthotopicallyinjecting mc38 cells transplanting luciferaseexpressingwhich would simulate mc38 metastasis to the liver throughsplenic veinportal vein the results revealed that the mpc1knockdown promoted mc38 cells metastasis to the liverthrough detecting the luminescence intensity monitoredby bioluminescence imaging figure 3f notablythenumber of metastatic liver nodules in the mpc1 silencinggroup wasin the control groupfigure 3g histological examination also proved thatmpc1 knockdown decreased the metastatic potential ofcrc in vivo figure 3gthan thatsmaller decreased mpc1 activated the wntcatenin pathwayby promoting nuclear translocation of catenin to furtherexplore the underlying mechanism of mpc1mediated inhibition of crc metastasis the tcga database was used toperform gsea analysis the results indicated that mpc1was involved in the wntcatenin signaling when set themrna expression median as a cutoï¬ figure 4a anddualluciferase reporter gene assay revealed that mpc1 overexpression obviously inhibited the activity of wntcateninpathway figure 4b which confirmed the result abovewe then tested the distribution changes of catenin innuclear and cytoplasmic which was a crucial step inwntcatenin pathway as shown in figure 4c no significant diï¬erence was emerged in the total amount ofcatenin between mpc1 overexpression cells and the control cells figure 4c however obviously decreased distribution of nuclear catenin was presented in mpc1overexpression cells compared to that in the control cellsas revealed by the stronger gray corresponding to cateninfigures 4c and 4d consistently immunofluorescenceif staining showed that mpc1 overexpression weakenednuclear catenin localization in both lovo and sw480 cellswhen compared to control cells figures 4e and 4f asimilar phenomenon was observed in mouse liver metastasistissues by ihc figure 4g following qpcr analyses displayed some downstream target genes of catenin such asmmp7 ecadherin snail1 and myc obviously increasedin ecadherin and decreased in mmp7 snail1 and mycwere observed after the mpc1 overexpression in both lovoand sw480 cells compared to that in the control cellsfigures 4h and 4i meanwhilethe opposite trendwas observed in mc38 cell after mpc1 knockdownfigure 4j and similar results were observed in mouseliver tissue detected by ihc figures 4k“4n takentogether the data above indicated that mpc1 mediatedcrc cell metastasis through the wntcatenin pathway discussionaccumulating evidences have shown that reprogrammedenergy metabolism conduces to the tumor malignant propertiesincluding enhanced crc liver metastatic capacity[“] mitochondria as the primary site of energy production regulate the pyruvate metabolism under both physiologic and pathologic conditions the first step of the tcacycle is mediated by mpc which transports pyruvate into 0cnoisserpxe cpm evitalergse21510ŽŽŽnormal crc mcrcnoisserpxe cpm evitalernormalacrccrlmesacesac of immunology researchgse67675ŽŽŽpmct26 hmct26ctcgan n p hr time daysgse89393ŽŽnormal crcmcrcbnoisserpxe cpm evitaler““Ž egatnecreplavivrus llarevolamroncrcmlrc“lavivrus llarevoren ji cohortn p hr n time monthsfdlavivrus llarevolavivrus llarevocrc with metastasis n n p hr n time monthsmpc1 higher expressionmpc1 lower expressiongecrc without metastasis n n n p hr time monthshfigure decreased mpc1 enhances tumor metastasis and predicts poor prognosis in crc a b data mining showed mpc1 expressionwas gradually decreased in normal tissue primary crc and liver lesion of metastatic crc mcrc from two independent geo datasetsoneway anova a gse21510 ˆ—ˆ—ˆ—p b gse89393 ˆ—ˆ—p c data from gse67675 revealed that mpc1 expressionwas lower in high liver metastasis ct26 cells hmct26 than that in poor liver metastasis ct26 cells pmct26 student™s ttestˆ—ˆ—ˆ—p d gradually decreased mpc1 expression was presented in normal tissue primary crc and liver metastasis crccrlm tissue scale bar μm n fisher™s exact test ˆ—p e kaplanmeier overall survival os curves in the tcgadataset of crc patients according to the mrna expression of mpc1 the lower quartile value of expression was utilized as a cutoï¬logrank test p f kaplanmeier os curve for the mpc1 expression in the ren ji cohort logrank test p gkaplanmeier os curve for the mpc1 expression in patients with metastasis logrank test p h kaplanmeier os curve forthe mpc1 expression in patients without metastasis logrank test p mitochondrial from cytoplasm in the beginning tcgawas used to analyze the relative genes involved in pyruvatemetabolism which is located in mitochondria the resultsrevealed that mpc1 expression was significantly downregulated in crc tissues meanwhile the geo datasets analysisas well as ihc staining on crc patients™ tissue and mousemodels confirmed this trend these phenomena may indicate that loss of mpc activity enhanced tumorigenic glucoseutilization by blocking mitochondrial pyruvate uptake andoxidation interestingly in the course of data analysis wefound that the expression of mpc1 was decreased at thestage of intestinal inflammation which was not diï¬erentfrom that in tumor tissue mpc1 has been reported to beinvolved in immune regulation of peripheral t cell homeostasis through metabolic regulation and a decrease inmpc1 was found at the earliest stages of crc hence 0c of immunology researchhumanmouselovosw480lovosw480mpc1gapdhmpc1““mpc1gapdhmpc1““mpc1gapdhwsthwsovolokrcmtchablovoŽŽrotcevitnelcpmitnelrotcevitnelcpmitneldlefi rep sllec edavnidlefi rep sllec edavnirotcevitnelcpmitneldsw480rotcevitnelcpmitnelcncpmŽcncpmdlefi rep sllec detargimdlefi rep sllec detargimcŽŽŽcncpmŽŽcncpmmc38shnc shmpc1¨¯ŽŽ sp xufl latotcnhscpmhscnhscpmhsradiancepseccm2srfemc38ŽŽsuledon sisatsatemcnhscpmhsgfigure mpc1 overexpression inhibits the motility of crc cells in vitro and in vivo a mpc1 expression in human and mouse crc celllines examined by western blot gapdh serves as loading control b mpc1 overexpression in lovo and sw480 cells c mpc1 silencing by shrnampc1 in mouse mc38 cells d e transwell assays showed that upregulated mpc1 suppressed the invasion and migration ability of lovod and sw480 e cells quantification of invaded and migrated cells was performed for five randomly selected fields values are means ± sd frepresentative bioluminescence photograph of mice spleen implanted with luciferaseexpressing mc38 cells treated with shmpc1 or controlvector total flux was quantified by the ivis system to verify the ability of liver metastasis g representative image of liver metastases andquantified by the nodules in mice inoculated with mc38 cells treated with shmpc1 or control vector as well as representative images ofhe staining of the liver metastatic lesions scale bar μm student™s ttest ˆ—p ˆ—ˆ—p ˆ—ˆ—ˆ—p 0c of immunology researchse erocs tnemhcirnewnt signaling pathwaynes “p mpc1 highmpc1 lowoitar pof pot evitaler𝛽cateninlamin ac𝛽cateningapdhmpc1alovosw480““csuelcunlatot icanmalnnetac𝛽iŽŽŽŽlovoncmpc1bŽsw480Žlovosw480ncmpc1d𝛽catenindapimerge𝛽catenindapimerge𝛽catenincpmcnovolnoisserpxe evitaleregnahc doflelovoŽŽŽŽlianscymŽpmmncmpc1nirehdacehcpmcnwsfsw480ŽŽŽlianscymŽŽpmmncmpc1nirehdaceifigure continuedcmcnhscpmhsmc38gŽŽlianscymŽŽpmmŽnirehdaceshncshmpc1jnoisserpxe evitaleregnahc doflnoisserpxe evitaleregnahc dofl 0c of immunology researchmmp7ecadherinsnail1myccmcnhscpmhscmcnhscpmhscmcnhscpmhscmcnhscpmhsklmnfigure decreased mpc1 activates the wntcatenin pathway by promoting nuclear translocation of catenin a gsea analysis ofmpc1 expression in crc using the tcga dataset nes normalized enrichment score b luciferase reporter gene assay of crc cellstreated with mpc1 overexpression or not c the expression of total catenin and nuclear catenin was detected in control and mpc1overexpression crc cells respectively gapdh and lamin ac were used as the loading control of total and nuclear protein respectivelyd the gray value analysis of nuclear catenin in mpc1overexpression cells and control cells e f mpc1 overexpression could inhibitthe nuclear translocation of catenin in crc cells scale bar μm g ihc staining of catenin in mouse liver metastatic lesionsinoculated with mc38 cells treatment with shmpc1 or control vector scale bar μm h i relative mrna expression level of catenin target genes in crc cells with mpc1 overexpression or control vector j relative mrna expression level of catenin targetgenes in mc38 cells with shmpc1 or control vector k“n relative protein expression level of catenin target genes in mouse livertissue detected by ihc scale bar μm student™s ttest ˆ—p ˆ—ˆ—p we suspect that mpc1 is involved in bowel inflammation totumorigenesis and more studies need to be devised to illustrate this processfollowing in the analysis between the clinical significance and mpc1 expression in crc we found that mpc1expression was especially correlated with metastasis inspiredby this we detected the mpc1 expression pattern in normaltissue primary crc and metastasis crc by geo datasetsand patients™ tissue all results revealed that gradually downregulated mpc1 was in patients with an increase in metastasis ability survival analysis indicated that worse outcome waspresented in patients with lower mpc1 expression especiallyin patients with metastasis additionally function assays verified that mpc1 overexpression could attenuate the migration and invasion capacities of crc cells in vitro andmpc1 knockdown could enhance the metastasis capacityin vivo and existing studies have revealed that the mpc1was participated in metastatic dissemination of pgc1αtransduced cholangiocarcinoma through elevating reactiveoxygen species ros production besides mpc inhibitor uk5099 treatment could trigger strong invasive capacitythrough blocking pyruvate translocation into the mitochondria so as to attenuate mitochondrial oxidative phosphorylation and trigger aerobic glycolysis these phenomenatogether with our results indicate that mpc1 could act as atumor suppressor through inhibiting tumor metastasis andexisting studies have shown that mpc1 could alter the maintenance and fate of stem cells through regulating cancermetabolism in crc however the relationship betweenthe metabolism and tumor metastasis regulated by mpc1was not being mentioned thus further evidence should bereceived to confirm thissubsequently the underlying mechanism of mpc1 inregulating metastasis was explored for this purpose gseaanalysis was performed the results indicated that mpc1was involved in the wntcatenin signaling the next seriesof experiments also confirmed that mpc1 could mediate thewntcatenin pathway by redistribution of catenin previous reports showed that cytoplasmic catenin phosphorylated in nterminally localized to sites of cellcell contact isassociated with ecadherin and was required for intact cellcell adhesions without any change detected in the levels oftotal catenin [“] simultaneously cell“cell adhesionbased on cadherin binding with catenin limited wnt signals in addition catenin was reported to interact withusp9x to inhibit the degradation of catenin through thedeubiquitination of catenin in breast cancer a constitutive irs1 and catenin protein interaction activated mycexpression in acute lymphoblastic leukemia cells inhcc catenin was reported to interact with yap1 to leadto rapid tumorigenesis hence it is reasonable to guessthat accumulated cytoplasmic catenin maybe crosstalkwith other genes or involved in other biological processeswhat is more some downstream target genes of cateninsuch as mmp7 ecadherin snail1 and myc were changedin expression as known to us mmp7 is a member of theproteolytic enzyme family which promotes the invasionand metastasis of tumor cells by degrading the basementmembrane and extracellular matrix and previous studies had evidenced for involvement of mmp7 activation incolorectal cancer liver metastases [ ] ecadherin andsnail1 were considered as the epithelialmesenchymal transition emt marker which was involved in metastasis ofmalignant tumor moreover ecadherin was reportedto be involved in cellcell junction to regulate cancer invasionand metastasis [ ] and the gsea analysis also revealedthat mpc1 could aï¬ect the cellcell contacts data notshown as described previously mmp7 could facilitatemorphological transition by cleaving ecadherin thecommunication between the cells is disrupted when ecadherin was shredded leading to destructed cell adhesionand induction of emt followed by increased cell migration inspired by this we assumed that mpc1 could mediatetumor cell motility through aï¬ecting mmp7 activity cellcell 0c of immunology researchcontacts and emt however further studies need to be performed to clarify the detailed underlying mechanisms conclusionsin conclusion we firstly demonstrated that decreased mpc1was closely correlated with patient™s metastasis as well as ledto poor outcome moreover mpc1driven nuclear translocation of catenin contributed to crc cell motility thismeans that mpc1 has the potential to be a diagnostic biomarker and therapeutic target for metastasis patientsabbreviationscrc colorectal carcinomampc mitochondrial pyruvate carrieremt epithelialmesenchymal transitiongsea gene set enrichment analysisdata availabilitythe data used to support the findings of this study are available from the corresponding author upon requestconflicts of interestall authors declare no conflicts of interestauthors™ contributionsyahui wang and guangang tian conceived and designedthe study chunjie xu and kaixia zhou obtained and anized the data guangang tian analyzed the data zhigangzhang and jianren gu contributed reagentsmaterialsanalysis tools xueli zhang and guangang tian wrote themanuscript guangang tian chunjie xu and kaixiazhou contributed equally to this workacknowledgmentsthis work was supported by the national natural sciencefoundation of china no to zhigang zhangand the natural science foundation of shanghai no18zr1436900 to xuelili zhangsupplementary materialsœsupplementary figure expression analysis of mpc1 innormal ibd and tumor tissues using the gse4183 datasetoneway anova was used to analyze the statistical diï¬erences between ibd adenoma and crc tissues ns no significance student™s ttest ˆ—ˆ—ˆ—p ˆ—ˆ—p ˆ—p supplementary materialsreferences h brody œcolorectal cancer nature vol no r l siegel k d miller s a fedewa et al œcolorectal cancerstatistics  ca a cancer for clinicians vol no pp “ a j rauckhorst and e b taylor œmitochondrial pyruvatecarrier function and cancer metabolism current opinion ingenetics development vol pp “ m g vander heiden l c cantley and c b thompsonœunderstanding the warburg eï¬ect the metabolic requirements of cell proliferation science vol no pp “ s herzig e raemy s montessuit et al œidentification andfunctional expression of the mitochondrial pyruvate carrierscience vol no pp “ c yang b ko c t hensley et al œglutamine oxidationmaintains the tca cycle and cell survival during impairedmitochondrial pyruvate transport molecular cell vol no pp “ t bender g pena and j c martinou œregulation of mitochondrial pyruvate uptake by alternative pyruvate carrier complexes the embo vol no pp “ a g ramstead j a wallace sh lee et al œmitochondrialpyruvate carrier promotes peripheral t cell homeostasisthrough metabolic regulation of thymic development cellreports vol no pp “2899e6 x zhou z j xiong s m xiao et al œoverexpression ofmpc1 inhibits the proliferation migration invasion and stemcelllike properties of gastric cancer cells oncotargets andtherapy vol pp “ d li c wang p ma et al œpgc1α promotes cholangiocarcinoma metastasis by upregulating pdha1 and mpc1 expression to reverse the warburg eï¬ect cell death diseasevol no j c schell k a olson l jiang et al œa role for the mitochondrial pyruvate carrier as a repressor of the warburg eï¬ectand colon cancer cell growth molecular cell vol no pp “ y li x li q kan et al œmitochondrial pyruvate carrierfunction is negatively linked to warburg phenotype in vitroand malignant features in esophageal squamous cell carcinomas oncotarget vol no pp “ h zou q chen a zhang et al œmpc1 deficiency accelerateslung adenocarcinoma progression through the stat3 pathway cell death disease vol no l wang m xu j qin et al œmpc1 a key gene in cancermetabolism is regulated by couptfii in human prostatecancer oncotarget vol no pp “ y zhong x li d yu et al œapplication of mitochondrialpyruvate carrier blocker uk5099 creates metabolic reprogramand greater stemlike properties in lncap prostate cancer cellsin vitro oncotarget vol no pp “ x p tang q chen y li et al œmitochondrial pyruvatecarrier functions as a tumor suppressor and predicts theprognosis of human renal cell carcinoma laboratory investigation vol no pp “ g a tian c c zhu x x zhang et al œccbe1 promotesgist developmentthrough enhancing angiogenesis andmediating resistance to imatinib scientific reports vol no article c xu g tian c jiang et al œnptx2 promotes colorectalcancer growth and live
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" oral administration is the most common way to deliver drugs to the systemic circulation or targetans orally administered drugs are absorbed in the intestine and metabolized in the intestine and liver in theearly stages of drug development it is important to predict firstpass metabolism accurately to select candidatedrugs with high bioavailability the caco2 cell line derived from colorectal cancer is widely used as an intestinalmodel to assess drug membrane permeability however because the expression of major drugmetabolizingenzymes such as cytochrome p450 cyp is extremely low in caco2 cells it is difficult to predict intestinalmetabolism which is a significant factor in predicting oral drug bioavailability previously we constructed a mouseartificial chromosome vector carrying the cyp cyp2c9 cyp2c19 cyp2d6 and cyp3a4 and p450 oxidoreductasepor 4cypsmac genes and increased cyp expression and metabolic activity in hepg2 cells via transfer of thisvectorresults in the current study to improve the caco2 cell assay model by taking metabolism into account weattempted to increase cyp expression by transferring the 4cypsmac into caco2 cells the caco2 cells carryingthe 4cypsmac showed higher cyp mrna expression and activity in addition high metabolic activity availabilityfor permeation test and the potential to assess drug“drug interactions were confirmeds the established caco2 cells with the 4cypsmac are expected to enable more accurate prediction ofthe absorption and metabolism in the human intestine than parental caco2 cells the mammalian artificialchromosome vector system would provide useful models for drug developmentkeywords mammalian artificial chromosome chromosome transfer cytochrome p450 intestinal metabolismcaco2 cell correspondence kazukitottoriuacjp1division of genome and cellular functions department of molecular andcellular biology school of life science faculty of medicine tottoriuniversity nishicho yonago tottori japan2chromosome engineering research center cerc tottori university nishicho yonago tottori japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cohta bmc biotechnology page of bioavailability is an important area of concern in drugdevelopment poor oral bioavailability has led to drugwithdrawal oral drug bioavailability is often limited bymetabolizing enzymes and efflux transporters in the gut the caco2 cell line derived from human colon carcinoma is a commonly used model for estimating the intestinal absorption of new drug candidates althoughcaco2 cells express a variety of efflux and uptake transporters they have an absence or low levels of cytochrome p450 cyp isoforms such as cyp3a4 andcyp2c that are typically expressed in the human intestinal epithelium therefore caco2 cells are of limited use in evaluating the role of metabolism inintestinal absorption after oral administration to predict the intestinal absorption of drugs more accurately itis necessary to modify caco2 cells to increase their expression of cyp isoformssome studies reported that cyp3amediated metabolism in caco2 cells was enhanced by transfection withboth cyp3a4 and cyp oxidoreductase por [ ] treatment with 1α25dihydroxyvitamin d3 [ ] or the combination of transfection with cyp3a4 and treatment withboth sodium butyrate and 12otetradecanoylphorbol13acetate in contrast few studies aimed at enhancingmultiple cyp isoforms in caco2 cells have been performed honkakoski and his collaborators created caco2cell lines expressing nuclear receptors pregnane x receptor and constitutive androstane receptor [“] thesenuclear receptors upregulated the expression of somecyp isoforms in caco2 cells but cyp activities remainedvery low in the absence of 1α25dihydroxyvitamin d3therefore a new approach is needed to introduce multiple cyp isoforms in caco2 cellsmammalian artificial chromosome ac vectors derived from native chromosomes have several advantagesover conventional vectors acs segregate freelyfrom host chromosomes through a set of cell divisionsand are adapted to carry multiple target genes with a desired copy number and mbsized genomic regions withendogenous regulatory elements furthermore acs carrying genes of interest can be transferred into varioustarget celllines via microcellmediated chromosometransfer mmct considering these advantages acshave been used to generate several model cells for pharmacokinetic and toxicokinetic studies previously a lack of cyp3a4 expression in the caco2cell line was addressed through the introduction of exogenous cyp3a4 and por which is a coenzyme ofcyps via a human artificial chromosome hac vectorderived from human chromosome [ ] the haccarrying cyp3a4 and por genes conferred sufficientcyp3a activity to parental caco2 cells to be useful forpredicting the intestinal extraction ratio in humansrecently a mouse artificial chromosome mac vectorconstructed from native mouse chromosome wasused to increase the activity of multiple cyps in hepg2cells which are a liver cancer cell line typically exhibiting low cyp activity in this study four cyp genescyp3a4 cyp2c9 cyp2c19 cyp2d6 and a pene were loaded on the mac 4cypsmac and transferred to hepg2 cells tchepg2 to make the cellsmore suitable as a model to evaluate drug“drug interactions ddis and hepatotoxicity in the initial screeningof candidate drugs the expression and activity ofcyps in tchepg2 were comparable to those in humanhepatocytes and this expression was sustained after along culture period because of the stability of the macin human cells regarding the assessment of ddisthe activity of cyps in tchepg2 was reduced in a concentration and timedependent manner by specific inhibitors which reflects the conditions in primary humanhepatocytes furthermore metabolic toxicity of aflatoxinb1 which is converted to its active metabolite viacyp3a4 and exerts hepatotoxicity through dna damage was clearly recapitulated in tchepg2 cells rather than parental hepg2 cells this study suggestedthat tchepg2 can provide a useful model to assess notonly hepatic metabolism but also cypmediated hepatotoxicity during the early stages of drug development andthe system using the mac can improve the existingcellbased modelin the current study we aimed to utilize previouslyconstructed 4cypsmac to generate a novel caco2 cellline with increased activity of multiple major cyps the4cypsmac was transferred to caco2 cells via mmctto establish caco2 cells carrying the 4cypsmac andthe caco2 4cypsmac cells were examined to determine whether they exhibited sufficient cyp activity foruse in initial drug screeningresultsmmct and analyses of acquired clonescho cells carrying a mac vector with cyp2c9cyp2c19 cyp2d6 cyp3a4 por and gfp genes wereprepared 4cypsmac fig 1a using cho cells asdonor cells and caco2 cells as recipient cells weattempted to generate caco2 cells carrying the 4cypsmac via mmct fig 1a after selection four drugresistant gfppositive clones were obtained caco24cypsmac fig 1b to examine whether the cypand por genes were introduced into the obtainedclones genomic pcr analyses were performed chocells with the 4cypsmac and caco2 cells were usedas positive and negative controls respectively consequently a band of the desired size was observed for eachprimer set in the candidate clones fig 1c next achromosome specimen was prepared from the acquired 0cohta bmc biotechnology page of fig introduction of the 4cypsmac into caco2 cells a transfer of the 4cypsmac into caco2 cells the structure of the mac carrying fourcyps and por is shown at the top a cag promoter was placed upstream of each gene a schematic view of the transfer of the 4cypsmac tocaco2 cells is shown at the bottom the 4cypsmac was transferred from cho cells to caco2 cells through the mmct method b an image ofgfp fluorescence in parental caco2 cells and caco2 4cypsmac cells the gfp fluorescence indicates the presence of the 4cypsmac in thecaco2 cells the white bars indicate a distance of μm c results of genomic pcr analyses to detect four cyp and por transgenes on the macin caco2 cells donor cho cells and caco2 cells are positive and negative controls respectively d images of fish analyses of caco2 cellscarrying the 4cypsmac red and green signals indicate the mac and transgenes respectively the arrow shows the 4cypsmac and the insetshows an enlarged image of the 4cypsmacclones and fish analysis was performed to check thekaryotype fish analysis revealed that a single copy of the4cypsmac existed in the candidate clones fig 1drtqpcr analysis was performed to examine themrna expression level of the introduced cyps and porin the obtained clones compared with that in parentalcaco2 cells the gene expression level was particularlyhigh in the caco2 4cypsmac and caco2 4cypsmac clones fig among the introduced genespor expression was only slightly enhanced in theseclones basal expression of por in parental caco2 cells ishigh as observed in a previous study caco2 4cypsmac showed high expression of the majority of genescompared with caco2 4cypsmac the caco2 cellline consists of a heterogeneous population of cells therefore difference of the gene expression levels between obtained clones may partly depend on the population into which the 4cypsmac has been introducedalthough the other two clones obtained by mmct stillshowed higher expression levels than the parental caco2cells the level of increase was moderate therefore we selected caco2 4cypsmac and caco2 4cypsmac clones for further analyses to evaluate the availability asan improved model system these results suggest that wesuccessfully transferred the 4cypsmac to caco2 cellsand the genes on the mac were highly expressedmonolayer formation of caco2 4cypsmac cellswe seeded caco2 4cypsmac and caco2 4cypsmac cells at a concentration of × cellswell 0cohta bmc biotechnology page of fig gene expression analyses of caco2 cells with the 4cypsmac the expression levels of the four cyps and por in caco2 cells with the4cypsmac the relative expression levels of the four cyps and por genes of the parental caco2 cells and acquired clones were analyzedthrough rtqpcr gapdh was used for normalization mean ± se n in a millicell 24well cell culture insert plate the caco 4cypsmac cells spread across the entire membrane and formed a cell layer while the caco2 4cypsmac cells did not spread and there were gaps in thecell layer fig 3a caco2 4cypsmac appeared toaggregate and form multiple layers rather than spreadand form a single layer it was reported that multilayeredareas appeared in the cell population for late passagecells the teer value was measured using amillicellers fig 3b the teer value is an index oftight junction formation and when the value is almostconstant it is considered that a cell layer has formedwith the exception of the caco2 4cypsmac clonethe caco2 cells and caco2 4cypsmac cellsshowed an increase in teer value untilit plateauedafter d the caco2 cells and caco2 4cypsmac showed almost equivalent teer values because monolayer formation is essential for the permeation test thesubsequenttests were performed using the caco24cypmac cellsculture timedependent change in gene expressiontotal rna was extracted from caco2 cells andcaco2 4cypsmac cells on the 4th 11th and22nd days after seeding we compared the expressionlevel of each gene on each day and confirmed thatthe expression levels of the four cyps and por increased in both the caco2 cells and caco2 4cypsmac cells fig 3c the expression levels of allgenes analyzed were significantly higher in the caco24cypsmac cells on the 22nd day than those inparental caco2 cells the gene expression levelincreased depending on the culture time and the geneexpression levels of the caco2 4cypsmac cellsestablished in the current study were higher thanthose of parental caco2 cells with the exceptions ofcyp3a4 and cyp2d6 the expression levels in parental caco2 cells were higher in all cases until day the parental caco2 cell line appears to have higherpotential to enhance the expression of cyp2c9 andcyp2c19 during differentiation 0cohta bmc biotechnology page of fig monolayer formation assay a images of bright and gfp fluorescence from cells seeded on the membrane of a millicell24 plate in caco24cypsmac cells did not spread throughout the membrane and did not form a cell layer but in caco2 4cypsmac there were no gapsbetween cells and they formed a cell layer the white bars indicate a distance of μm b transepithelial electrical resistance teer values ofcaco2 cells caco2 4cypsmac and caco2 4cypsmac cells c culture timedependent change in gene expression the relative expressionlevel was evaluated in caco2 and caco2 4cypsmac mean ± se n the expression levels in caco2 and caco2 4cypsmac at day were compared with those in humanadult intestine additional file figure s1 in caco24cypsmac the expression levels of cyp2c9 andcyp2c19 were comparable and that of cyp2d6 washigher than in human adult intestine although cyp3a4expression was significantly enhanced in caco2 4cypsmac compared with that in parental caco2 cellsthe expression was stilllower than in human adultintestinecyp metabolic activity measurementa p450glo assay with each specific substrate wasemployed to measure the metabolic activity of cyps inthe caco2 4cypsmac cells which had high geneexpression levels as confirmed through rtqpcr analysis the activities of all four cyps were higher in thecaco2 4cypsmac clone than in caco2 cellsfig 4a the resultsthe introducedindicate that4cypsmac expressed functional cyps and increasedthe total activity of each cyp in the caco2 cells theenhancements in the rates of metabolic activity ofcyp2c9 cyp2c19 and cyp2d6 were generally correlated with those of mrna expression however therewas a gap between the enhancement of the rate ofcyp3a4 mrna expression and that of metabolic activity this may have been because the parental caco2 originally had extremely low expression of cyp3a4mdz permeability testa permeation test was conducted using midazolammdz a cyp3a substrate to examine whether the cellsreflected the behavior of small intestinal epithelial cellsin terms of mdz permeation the penetration test wasperformed on day after cell seeding when the teervalue plateaued we measured the amount of ²ohmdz in each of the donor side apical recipient sidebasal and intracellularly the amounts of ²oh mdz 0cohta bmc biotechnology page of fig activity of each cyp in the caco2 4cypsmac cells a the metabolic activity of each cyp in caco2 4cypsmac cells the relative activityfor each cyp was measured by comparing the parental caco2 cells and the caco2 4cypsmac mean ± se n b permeability test usingmdz the permeability test was performed d after seeding caco2 cells and caco2 4cypsmac whereby μm mdz was added to theapical side and after min the apical intracellular and basal supernatants were collected the ²oh mdz in the supernatant was measuredthrough lcmsms c cyp3a4 inhibition test ketoconazole an inhibitor of cyp3a4 was added to the caco2 4cypsmac and incubated for h a luminescent substrate was measured to detect cyp3a4 activity with different concentrations of ketoconazolein alllayers of the caco2 4cypsmac cells werehigher than in those of caco2 cells fig 4b moreoverer was calculated using eq and the results were and for caco2 and caco2 4cypsmac respectively er indicates the rate of metabolism during cellpermeation cyp3a4 was scarcely expressed in parentalcaco2 cells so the er value was extremely low howevercaco2 4cypsmac cells showed an er of which was higher than in the caco2 cells and mdz wasmetabolized by cyp3a4 when passing through the cellsinhibition testto determine the availability of the established clone forthe assessment of ddis we added ketoconazole an inhibitor of cyp3a4 to the caco2 4cypsmac cells andexamined whether the metabolic activity was reduced ketoconazole at and μm was addedand cells were incubated at °c for h followed by themeasurement of metabolic activity the metabolic activityof cyp3a4 decreased as the inhibitor concentration increased fig 4c the activity of cyp3a4 in caco2 4cypsmac appeared to be sufficient for the inhibition test compared with that in parental caco2 cells in addition to thepermeation test for cyp3a4 inhibition of cyp3a4™s function by ketoconazole in caco2 4cypsmac cells was alsoconfirmed this suggests that the established cells could beused for ddi testing of drugs that are substrates ofcyp3a4 therefore the caco2 4cypsmac cells moreaccurately reflect the behavior of cyp3a4 substrates in human epithelial cells than parental caco2 cellsdiscussionin the current study we introduced four cyps and porinto caco2 cells to increase their drug metabolic 0cohta bmc biotechnology page of abilities which are typically low this study was intendedto establish a better human cell model to more preciselyevaluate the behavior of drugs in the small intestine the4cypsmac was successfully introduced into the caco2cells and successfully increased cyp activityin contrastin this studythe gene expression and activity of cyps in tchepg2 carrying the 4cypsmac are either comparableto or higher than those in primary human hepatocytes to the other cypscyp3a4 mrna expression was still low in caco2 carrying the 4cypsmac compared with the level in human adult intestine despite the significant enhancementof mrna expression regarding the genes on the4cypsmac each is present as a single copy becausethe nature of gene regulation is supposed to differ between hepg2 and caco2 changing copy number of thecyp3a4 gene may further optimize the expression profile of caco2 cells to that of human intestinethe established caco2 4cypsmac cells with particularly high gene expression showed high activity in all cypsin the future we will conduct metabolic tests inhibitiontests and permeation tests using drugs that are substratesfor other cyps and investigate whether the caco2 4cypsmac cells reflect the behavior of drugs in small intestinal epithelial cells the cyp expression level in the humansmall intestine is reported to be approximately forcyp3a4 approximately for cyp2c9 approximately for cyp2c19 and approximately for cyp2d6 it will be necessary to evaluate whether the proportion ofcyp expression in the caco2 4cypsmac is close tothat of the human small intestineif cyp metabolic capacity is guaranteed in the established clones the established cell line can be used asnew human small intestine model cells in recent yearssmall intestine model cells prepared from induced pluripotent stem cells have been reported but such cells areconsidered difficult to use for screening large quantitiesof drug candidate compounds however caco2cells are easy to handle therefore it is possible to usecypmodified caco2 cells to test large numbers of candidate compounds as a first screeningwako osaka japan supplemented with fetal bovine serum fbs and μgml g418 parental caco2cells atcc® htb37„¢ atcc manassas va usawere maintained in dulbecco™s modified eagle™s mediumdmem wako supplemented with fbs memnonessential amino acids gibco thermo fisher scientific waltham ma usa m hepes gibco mmsodium pyruvate gibco mm glutamax gibcoand penicillinstreptomycin wako caco2 cells withthe 4cypsmac were maintained in the above mediumsupplemented with μgml g418 these cells werecultured at °c in co2microcellmediated chromosome transfertransfer of 4cypsmac from cho cells to caco2 cellswas performed using a standard procedure brieflydonor cho cells were cultured in f12 medium supplemented with fbs and μgml colcemid after h microcells were isolated through centrifugation withdmem containing cytochalasin b and filtration thenmicrocells suspended in phytohemagglutinin p phapdmem were poured onto caco2 cells in a 6cm dishand incubated for min the cells were treated withpolyethylene glycol peg solution g of peg1000 ml of dmem ml of dimethyl sulfoxide for minfollowed by washing with dmem after h of recoveryculture cells were seeded in a 24well collagencoatedplate corning ny usa and maintained with selectionmedium h after seeding thereafter the medium waschanged twice a week to obtain drugresistant clonesbecause the mac carries a gfp gene gfppositiveclones were selected from the drugresistant clonesgenomic pcr analyseswe extracted genomic dna from cell lines using a genomic dna extraction kit with dnasefree rnase gentra systems minneapolis mn usa the primers forthe genomic pcr are listed in additional file tables1 they amplified each gene region on the 4cypsmac we used kod fx takara otsu japan in accordance with the manufacturer™s instructionsthe mammalian artificial chromosome vector systemwould provide useful models for drug development theestablished caco2 cells with the 4cypsmac are expected to more accurately predict absorption and metabolism in the human intestine than parental caco2 cellsmethodscell culturechinese hamster ovary cho cells jcrb0218 jcrbcell bank nibiohn osaka japan carrying the 4cypsmac were maintained in ham™s f12 nutrient mixturefish analysestrypsinized cells were incubated for min in m kcland fixed with methanol and acetic acid and thenslides were prepared using standard methods fish analyses were performed using the fixed metaphase of each cellhybrid using digoxigeninlabeled roche germany dna[mouse cot1 dna invitrogen carlsbad ca usa] andbiotinlabeled dna [pac 4cypspor] essentially as described previously chromosomal dna was counterstained using dapi sigmaaldrich st louis mo usaimages were captured using an axioimagerz2 fluorescencemicroscope carl zeiss germany 0cohta bmc biotechnology page of rtqpcrwe extracted mrna using the rneasy mini kit qiagen germany and synthesized firststrand cdna usingthe high capacity cdna reverse transcription kit applied biosystems foster city ca usa the primersare listed in additional file table s1 for rtqpcranalysis tb green premix ex taq takara was usedand relative mrna expression was evaluated throughthe δδct method gapdh was used for normalizationculture timedependent expression level change of fourcypscaco2 cells and caco2 4cypsmac were seededin a 6cm dish at a concentration of × cellswell cells were lysed using trizol invitrogen causa on days and after seeding and rnawas extracted and purified using an rneasy mini kitqiagen thereafter cdna synthesis was performedusing the highcapacity cdna reverse transcriptionkit applied biosystemsactivity test of the four cypswe tested the metabolic activity ofthe four cypsusing the p450glo„¢ assay promega madison wiusa the luminogenic substrates used for the testwere luciferinipa cyp3a4 luciferinme egecyp2d6 luciferinh cyp2c9 and luciferinhege cyp2c19 cells wereseeded in 48wellcollagencoated plates corning at a density of × cellswell after h the medium was changedand h later we added transport medium tm containing substrate tm was prepared using hanks™ balanced salt solution hbss with mm nahco3 mm glucose and mm hepes which was adjusted to ph after incubation we added detectionreagent and measured the luminescence using an infiniteandcyp2c19 required h of incubation while cyp2d6and cyp3a4 required h after the measurementthe cells were washed with pbs dissolved in lysisbuffer and diluted fivefold the same amount of celltiter glo promega was added to μl of the lysateand luminescence measurement was performed tonormalize data to the number of viable cellswako cyp2c9f500 platereadermidazolam mdz permeability testthe obtained clones were assessed in an mdz permeation test each cell was seeded on a 24well cell cultureinsert plate millipore billerica ma usa at a concentration of × cellswell the medium was changedonce a week after seeding and every d after the secondweek transepithelial electrical resistance teer wasmeasured using millicellers millipore before mediumexchange the test was performed d after seedingfor the test tm ph prepared by adding mmnahco3 mm glucose and mm hepes tohbss at ph was used the donor side solutionwas prepared by dissolving μm mdz in tm ph the acceptor side solution was prepared by adding fbs to tm ph on the test day themedium was removed from the culture insert seededwith the cells and the cells were rinsed twice withtm ph tm ph and tm ph wereadded to the apical and basal chambers respectivelyand cells were incubated at °c for min the testwas started by adding the donor side solution to thedonor side chamber and the acceptor side solution tothe acceptor side chamber thirty minutes after thestart of the test the solution in the donor side andacceptor side chambers was collected moreover tomeasure the amount of mdz and ²hydroxy mdz²oh mdz in the cells after the test the cultureinsert was quickly rinsed three times with icecoldtm ph the membrane was cut using a cutterand μl of icecold tm ph was added cellswere detached from the membrane through sonicationand a cell suspension was used as a sample thesesamples were deproteinized and stored at ˆ’ °cuntil measurementlcmsms was used for the measurement of mdzand ²oh mdz in the samples qtrap5500 sciexframingham ma usa and a prominence uflc system shimadzu kyoto japan were combined for measurement the hplc conditions and msms conditionsare shown in additional file table s2 quantitativeanalysis was performed in multiple reaction monitoringmode mass transitions mz were †’ formdz †’ for ²oh mdz and †’ for ²oh mdz d4 data were analyzed usinganalyst software sciexequation formula to calculate extraction ratio erer ¼metabolite donorþreceiverþintracellularpþparent receiverþintracellularððþ þ pmetabolite donorþreceiverþintracellularðþtokyo chemicalinhibition testketoconazoleindustry tokyojapan was used as an inhibitor against cyp3a4 andchanges in metabolic activity were measured using ap450glo assay with luciferinipa cells were seededin a 48well collagencoated plate at × cellswell and the medium was changed after d thenext daythe medium was collected cells werewashed twice with pbs and then μl of tm ph containing ketoconazole tokyo chemical industry at and μm was added toeach set of three wells tm was adjusted to ph byadding mm nahco3 mm glucose and mm 0cohta bmc biotechnology page of received april accepted august referencesbenet l wu c hebert m wacher v intestinal drug metabolism andantitransport processes a potential paradigm shift in oral drug delivery jcontrol release “xie f ding x zhang qy an update on the role of intestinal cytochromep450 enzymes in drug disposition acta pharm sin b “takenaka t kazuki k harada n kuze j chiba m iwao t matsunaga t abes oshimura m kazuki y development of caco2 cells coexpressingcyp3a4 and nadphcytochrome p450 reductase using a human artificialchromosome for the prediction of intestinal extraction ratio of cyp3a4substrates drug metab pharmacokinet “hu m li y davitt cm huang sm thummel k penman bw crespi cltransport and metabolic characterization of caco2 cells expressing cyp3a4and cyp3a4 plus oxidoreductase pharm res “schmiedlinren p thummel ke fisher jm paine mf lown ks watkins pbexpression of enzymatically active cyp3a4 by caco2 cells grown onextracellular matrixcoated permeable supports in the presence of1alpha25dihydroxyvitamin d3 mol pharmacol “fan j liu s du y morrison j shipman r pang ks upregulation oftransporters and enzymes by the vitamin d receptor ligands 1alpha25dihydroxyvitamin d3 and vitamin d analogs in the caco2 cell monolayer jpharmacol exp ther “cummins cl mangravite lm benet lz characterizing the expression ofcyp3a4 and efflux transporters pgp mrp1 and mrp2 in cyp3a4transfected caco2 cells after induction with sodium butyrate and thephorbol ester 12otetradecanoylphorbol13acetate pharm res “korjamo t honkakoski p toppinen mr niva s reinisalo m palmgren jjmonkkonen j absorption properties and pglycoprotein activity of modifiedcaco2 cell lines eur j pharm sci “korjamo t monkkonen j uusitalo j turpeinen m pelkonen o honkakoskip metabolic and efflux properties of caco2 cells stably transfected withnuclear receptors pharm res “kublbeck j hakkarainen jj petsalo a vellonen ks tolonen a reponen pforsberg mm honkakoski p genetically modified caco2 cells withimproved cytochrome p450 metabolic capacity j pharm sci “mes to hbss the cells were preincubated for h at °c and 1000fold diluted cyp3a4 substrate wasadded one hour later μl of the supernatant wascollected from the well mixed with μl of detectionreagent and the luminescence value was measuredusing an infinite f500 plate reader wakosupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12896020006378additional file figure s1 comparison of gene expression betweenhuman small intestine and day culture of caco2 and caco2 4cypsmac table s1 primer sequences for genomic pcr and rtqpcrtable s2 lcmsms analysis conditions mdz ²oh mdzabbreviationscyp cytochrome p450 ac artificial chromosome mmct microcellmediated chromosome transfer por p450 oxidoreductase hac humanartificial chromosome mac mouse artificial chromosome ddi drug“druginteraction cho chinese hamster ovary mdz midazolamteer transepithelial electrical resistance er extraction ratioacknowledgmentswe thank satoru iwado at tottori university for technical assistance with theexperiments we also thank dr hiroyuki kugoh dr masaharu hiratsuka drhiroyuki satofuka and dr takahito ohira at tottori university for criticaldiscussions this research was partly performed at the tottori bio frontiermanaged by tottori prefecture we thank edanz group wwwedanzeditingcomac for editing a draft of this manuscriptauthors™ contributionsall authors conceived and designed the experiments yo and kka performedthe experiments yo sa kko and yk wrote the paper mo and yksupervised the study all authors read and approved the final manuscriptfundingthis work was supported in part by the basis for supporting innovative drugdiscovery and life science research binds from the japan agency formedical research and development amed under grant numberjp18am0301009 ykavailability of data and materialsthe data and materials used andor analyzed during the current study areavailable from the corresponding author on reasonable requestethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting interestsdr mitsuo oshimura is ceo and a shareholder of trans chromosomics incdr satoshi abe is a member of trans chromosomics inc and the otherauthors declare no conflict of interestauthor details1division of genome and cellular functions department of molecular andcellular biology school of life science faculty of medicine tottoriuniversity nishicho yonago tottori japan 2chromosomeengineering research center cerc tottori university nishicho yonagotottori japan 3trans chromosomics inc nishicho yonagotottori japan 4laboratory of biopharmaceutics meijipharmaceutical university noshio kiyose tokyo japan oshimura m uno n kazuki y katoh m inoue t a pathway fromchromosome transfer to engineering resulting in human and mouseartificial chromosomes for a variety of applications to biomedicalchallenges chromosom res “satoh d abe s kobayashi k nakajima y oshimura m kazuki y human andmouse artificial chromosome technologies for studies of pharmacokineticsand toxicokinetics drug metab pharmacokinet “kazuki y hoshiya h takiguchi m abe s iida y osaki m katoh m hiratsukam shirayoshi y hiramatsu k ueno e kajitani n yoshino t kazuki k ishiharac takehara s tsuji s ejima f toyoda a saka
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Costello syndrome is an autosomal dominant disorder that is caused by germline HRAS mutations Patients withCostello syndrome present craniofacial abnormalities cardiac defects and cancer predisposition as well as skinabnormalities including papillomas keratosis pilaris and eczematous dermatitis However the mechanisms underlyingthe dermatological abnormalities remain unclear Here we demonstrated that knockin mice expressing an Hras G12Smutation HrasG12S mice are susceptible to develop atopic dermatitis ADlike skin lesions including eczemapruritus elevated serum IgE levels acanthosis and the ltration of mast cells basophils and type2 innate lymphoidcells in the dermis after stimulation with house dust mite allergens Dermatophagoides farinae Dfb Reduced skinbarrier function increased proliferation of phosphorylated ERK pERKpositive epidermal cells and increased Th2typecytokines as well as epithelial cellderived cytokines including IL33 were observed in the skin tissue of HrasG12Smice compared with Hras mice Cultured HrasG12S keratinocytes exhibited increased IL33 expression after Dfbstimulation PD0325901 an MEK inhibitor ameliorated ADlike symptoms in HrasG12S mice showing decreasedproliferation of pERKpositive epidermal cells and decreased expression of IL33 Our findings indicate that theepidermis of HrasG12S mice stimulated by Dfb strongly induced IL33 expression and type2 innate lymphoid cellsresulting in ADlike skin lesions These results suggest that the epidermis of HrasG12S mice are prone to developmentof eczematous dermatitis stimulated with house dust mite allergensIntroductionThe skin is a stratified epithelium consisting of severallayers of cells in various stages of differentiation In orderto maintain normal skin homeostasis the proliferationdifferentiation and response of epidermal cells to externalstimuli must be tightly regulated1 The RASMAPK signaling pathway plays a crucial role in cell proliferationdifferentiation and apoptosis23 A strong activation of theRASMAPK pathway in skin is known to resultinCorrespondence Yoko Aoki aokiymedtohokuacjp1Department of Medical Genetics Tohoku University Graduate School ofMedicine Sendai Japan2Department of Pediatrics Tohoku University Graduate School of MedicineSendai JapanFull list of author information is available at the end of the Edited by E Candiepithelial cancers and melanoma45 Pigmented lesionshyperkeratosis pruritus curly hair and hyperplasia havealso been observed in vemurafenib a BRAF inhibitortreated patients6 The balance of the RASMAPK signaling pathway could be particularly important for epidermalhomeostasisCFCNoonan syndrome Costello syndrome and cardiofaciocutaneoussyndrome are phenotypicallyoverlapping genetic disorders characterized by craniofacial dysmorphia congenital heart defects and psychomotor retardation7 These syndromes are commonlycaused by germline mutations in components of the RASMAPK pathwaytermed RASopathies which constitutively activate the RASMAPK pathway89 Of thesesyndromes Costello syndrome is characterized by short The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate ifchanges were made The images or other third party material in this are included in the ™s Creative Commons license unless indicated otherwise in a credit line to the material Ifmaterial is not included in the ™s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this license visit httpcreativecommonslicensesby40Official journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of stature craniofacial abnormalities congenital heart diseases hypertrophic cardiomyopathy and intellectual disability10 Approximately patients with Costellosyndrome develop malignant tumors including rhabdomyosarcoma and bladder carcinoma In we identified germline HRAS mutations in patients with Costellosyndrome11 A nucleotide change that cause the substitution of glycine at codon to serine pG12S in oneallele of HRAS has been observed in of Costellosyndrome patients The G12S mutation which has beenidentified in somatic cancer is an oncogenic mutationthat activates the downstream pathway Patients withCostello syndrome develop a variety of skin abnormalitiesincluding palmoplantar keratoderma acanthosis nigricans eczema loose skin cutis laxa darker skin colorand papillomata around nose and anus However thepathogenesis of dermatological abnormalities remainsunclearWe have recently generated a strain of knockin miceexpressing an Hras G12S mutation the most frequentmutation in Costello syndrome12 which exhibited facialdysmorphia cardiomyocyte hypertrophy and kidneyanomalies Impaired mitochondrial fatty acid oxidationwas observed in HrasG12S mice fed a highfat diet13Skin abnormalities including papillomas have not beenobserved in young HrasG12S mice weeks old underspecific pathogenfree conditions In contrast HrasG12Smice over weeks of age or highfat diet fedHrasG12Smice had cutaneous lesions due to scratching Supplementary Fig 1a under the same pathogenicfree condition Although we have not analyzed the histology of skinof HrasG12S mice over weeks of age gross appearances of the skin lesions and scratching behavior suggestthat they are atopic dermatitislike In the current studywe tested to generate experimentally induced dermatitisin HrasG12S mice and found that HrasG12S micedeveloped more severe atopic dermatitis ADlike lesionsthan Hras mice after treatment with house dust miteallergens Dermatophagoides farinae Dfb Furthermorethese ADlike skin lesions in HrasG12S mice werereversedbyan MEK inhibitorPD0325901treatment withResultsDfb ointment induces ADlike skin lesions in HrasG12SmiceWe first tested the effect of picryl chloride whichinduce contact dermatitis and imiquimod which inducepsoriasis on the skin of Hras and HrasG12S miceSupplementary Fig 1b c but no difference in skinlesions was observed between them Supplementary Fig1d In contrast the treatment with Dfb ointment developed severe dermatitisincluding severe erythemaOfficial journal of the Cell Death Differentiation Associationhemorrhage scarring and eczema in the dorsal skin ofHrasG12S mice but not in Hras mice Fig 1a andSupplementary Fig 2a The ears of HrasG12S micebecame thick with edema erosion and excoriationFig 1b The dermatitis score was significantly higher inDfbtreated HrasG12S mice than in any other group ofmice SDStreated control Hras mice DfbtreatedHras mice and SDStreated control HrasG12Smice on day Fig 1c and Supplementary Table Other dermatitis parameters including the ear swellingFig 1d and the scratching behavior Fig 1e increasedsignificantly in Dfbtreated HrasG12S mice comparedwith Dfbtreated Hras mice Serum IgE levels weresignificantly higher in HrasG12S mice compared toHras in nontreated baseline ± ngmL vs ± ngmL P Supplementary Fig Although the difference was not statistically significant in SDS treatment groups IgE levels were higher inHrasG12S mice compared to Hras ± ngmLvs ± ngmL P Fig 1f as well as in theDfb treatment groups ± ngmL vs ± ngmL P Fig 1f These symptoms werealso seen in Hras mice but skin lesions are moresevere in HrasG12 mice In both groups of mice the IgEelevations were triggered by Dfb ointmentWe next examined if Dfbinduced dermatitisinHrasG12S mice is caused by the same pathology as inHras miceHistological analysis revealed hyperkeratosis and epidermal hyperplasia in the dorsal skin of DfbtreatedHrasG12S mice Fig 2a The epidermis of HrasG12Smice became thicker than that of Hras mice althoughDfb treatment increased the epidermal thickness in bothHras and HrasG12S mice Supplementary Fig 2b Inthe ADlike skin lesions Dfbtreated HrasG12S micedisplayed increased number of mast cells toluidine blueand tryptase 1 a marked increase in the numbersof T cells CD4 and dendritic cells MHC class IIFig 2a b and Supplementary Fig 2c e Western blottinganalysis revealed that the levels of CD4 protein weresignificantly increased in Dfbtreated mice compared withcontrol mice Supplementary Fig 2d In line with theacanthosis of Dfbtreated HrasG12S mice an increasednumber of phosphohistone H3positivecells wereobserved in the suprabasal epidermis layers of HrasG12Smice Fig 2b Although phosphorylated ERK pERKpositive cells were also increased in the epidermis of Dfbtreated Hras and HrasG12S mice the immunostainedarea in HrasG12S mice was significantly larger than thatin Hras mice Fig 2b and Supplementary Fig 2f g Inaddition we examined the expression of filaggrin andclaudin1 as epidermal barrier markers in AD14“ Adecreased expression of claudin1 was observed in Dfb 0cKatata Cell Death and Disease Page of Fig Dfb treatment induces atopic dermatitislike skin lesions in HrasG12S mice a b Skin and ear features of mice on day after treatmentof Dfb ointment HrasG12S mice showed dermatitis by repeated application of Dfb a The severity of ear swelling responses to Dfb was strongerin HrasG12S than Hras mice b c Dermatitis scores of only SDStreated control Hras and HrasG12S and Dfbtreated Hras andHrasG12S mice n per group d Time course of ear thickness from Hras and HrasG12S mice after treatment with SDS or Dfb n pergroup e The number of scratching bouts per min assessed by the video n per group f Serum IgE levels in Hras and HrasG12S mice aftertreatment with SDS or Dfb n per group Data are presented as mean ± SD Significance in c d and f was analyzed by oneway ANOVAand the Tukeyˆ’Kramer method P P P and P HrasG12S Dfb vs Hras Dfb P P P and P HrasG12S Dfb vs HrasG12S SDS  P   P    P and     P Hras Dfb vs Hras SDSSignificance in e was analyzed by twotailed Student™s t test P treated HrasG12S mice compared with control HrasG12Smice Fig 2c d Together these results indicate that Dfbapplied HrasG12S mice exhibited more severe ADlikeskin lesions than Hras mice including acanthosis withhyperproliferation of pERKpositive cells in the epidermisas well as increased ‚ammatory cells and reducedclaudin1 expressionThe skin of Dfbapplied HrasG12S mice shows an increaseof itchassociated factors and ‚ammatory cytokinesTo further characterize the ADlike skin lesions weevaluated the levels of the itchassociated factors and‚ammatory cytokinesin the skin of DfbtreatedHrasG12S mice Itchrelated neuronal markers including skin Tac1 Klk7 and Klk14 mRNA levels or PAR2 andOfficial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of Fig See legend on next pageOfficial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of see figure on previous pageFig Histological analysis reveals acanthosis with hyperproliferation of pERKpositive epidermal cells increased ‚ammatory cells andreduced claudin1 expression in the dorsal skin of Dfbtreated HrasG12S mice a Skin tissue stained with HE and TB b c Immunohistochemistryof CD4 tryptase 1 pERK pHH3 and claudin1 in the skin a“c Scale bars μm d Lysates from the skin were immunoblotted with antiClaudin1antibody Band intensities were quantified and compared among the four groups The expression levels were normalized to GAPDH n in eachgroup Data are presented as mean ± SD Significance was analyzed by oneway ANOVA and the Tukeyˆ’Kramer method P P twotailedStudent™s t testIl1 pro‚ammatory cytokineEndothelin proteins17“ were significantly higher thanthose in control HrasG12S and Dfbtreated Hras miceFig 3a“c Regarding ‚ammatory cytokines the skinIl4mRNA levels ofTh2related cytokine and epidermalderived cytokinesIl33 and thymic stromal lymphopoietin Tslp were significantly elevated in Dfbtreated HrasG12S mice compared with control HrasG12S and Dfbtreated Hrasmice Fig 3d IL33 leads to the activation of type2innate lymphoid cells ILC2s through ST2 IL33 receptor21 In Dfbtreated HrasG12S mice the skin mRNAlevels of St2 were also significantly increased Fig 3dImmunohistochemistry analysis revealed that DfbtreatedHrasG12S mice showed enhanced expression levels ofIL33 and TSLP in the basal epidermal layers and thesurface of epidermis respectively Fig 3e Likewise theIL33 protein levels were significantly higher in the skin ofDfbtreated HrasG12S mice than in Dfbtreated Hrasmice Fig 3fIncreased numbers of ILC2 and increased IL33 expressionwere observed in the epidermis of HrasG12S miceTo investigate when and how ‚ammatory cytokinesare induced in HrasG12S mice we performed flowcytometry on skin and ear samples days after Dfbapplication that is once the ADlike skin lesions hadbegun to appear Fig 1c The accumulation of basophilsand ILC2s was observed on skin samples of DfbtreatedHrasG12S mice days after Dfb application Fig 4a Asignificant increase in mast cells eosinophils basophilsand ILC2s was also observed in the ears of HrasG12Smice Fig 4b On the other hand these immune cellswere hardly detected in Hras and HrasG12S mice days after SDS application Supplementary Fig The mRNA levels of ‚ammatory cytokines in the skinfrom HrasG12S mice days after Dfb applicationshowed that Il1 and Il33 were significantly elevated inthe skin of Dfbtreated HrasG12S mice compared withDfbtreated Hras mice Fig 5aAtopic dermatitis is characterized by increased serumIgE acanthosis loss of skin barrier function and ltration of immune cells including Th2 cells dendriticcells eosinophils basophils and mast cells2223 On thataccount we next examined the response of immune cellsin these mice HRAS is known to be highly expressed inOfficial journal of the Cell Death Differentiation Associationthe epidermal cells but not in immune cells2425 Indeedno significant difference was found in the population ofimmune cells from the spleen and lymph node LN Theproliferation of naive CD4 T cells and Th2 immuneresponse was comparable between Hras and HrasG12Smice at weeks of age suggesting that ADlike dermatitismay not be caused by different response of immune cellsFig 6a b Then we examined whether an increasedproduction of IL33 and TSLP was observed in primaryepidermal keratinocytes in response to Dfb Six hoursafter Dfb stimulation the mRNA level of Il1 not Tslpwas significantly elevated in cultured epidermal keratinocytes of Hras and HrasG12S mice Fig 5b NotaIl33 in the Dfbstimulatedblykeratinocytes of HrasG12S mice were significantlyincreased compared with those of nonstimulatedHrasG12S and Dfbstimulated Hras keratinocytessuggesting that epidermal keratinocytes with Hras G12Smutation have increased IL33 expression after stimulation with Dfb Fig 5bthe mRNA levels ofPD0325901 reduces skin lesions in DfbstimulatedHrasG12S miceamelioratesTreatment with MEK inhibitorstheabnormalities observed in RASopathyrelated modelmice26“ We investigated the effects of an MEK inhibitor PD0325901 on skin lesionsin DfbtreatedHrasG12S mice Supplementary Fig 5a Ten days oftreatment with PD0325901 or saline resulted in a significant improvement of the dermatitis score and earswelling in HrasG12S mice Fig 7a b PD0325901treatment resulted in a marked reduction of epidermalthickness mast cell numbers and pERKpositive epidermal cells as well as recovered expression levels ofclaudin1 Fig 7c d and Supplementary Fig 5b c ThemRNA levels of Il1 Il4 Il33 St2 and Klk14 were significantly lowerin the skin of PD0325901treatedHrasG12S mice than that of vehicle salinetreatedHrasG12S mice Fig 7e and Supplementary Fig 5dThese results suggest that ERK inhibition partially ameliorates Dfbinduced skin lesions in HrasG12S miceDiscussionHere we demonstrated that mice expressing a germlineHras G12S mutation but not Hras mice developed 0cKatata Cell Death and Disease Page of Fig See legend on next pageOfficial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of see figure on previous pageFig Expression of itchassociated factors and ‚ammatory cytokines is enhanced in Dfbinduced skin lesions in HrasG12S mice a c dRelative mRNA expression related to neuronal factors a Tac1 and Ngf skin proteases c Klk5 Klk7 and Klk14 and ‚ammation d Il1 Il4 Tslp Il33and St2 in the dorsal skin mRNA levels were normalized to those of 18s rRNA n per group b f Protein extracts from dorsal skin wereimmunoblotted with antiPGP95 antiPAR2 antiEndothelin and antiIL33 antibody n in each group GAPDH same data as in Fig 2d f Bandintensities were quantified and compared among the four groups Expression levels were normalized to GAPDH e Immunohistochemistry of IL33and TSLP Scale bars μm Data are presented as mean ± SD Significance was analyzed by oneway ANOVA and the Tukeyˆ’Kramer method P P P and P P twotailed Student™s t testADlike skin lesions under conditions of Dfb exposureThe levels of IL1 and epithelial cellderived cytokinesIL33 and TSLP were also increased in DfbtreatedHrasG12S mice In addition an increased production ofIL1 IL4 and IL33 as well as ‚ammatory cellsbasophils and ILC2s was observed in the dorsal skin ofHrasG12S mice before the development of ADlike skinlesions Analysis of the underlying mechanism revealedthat Dfbstimulated keratinocytes in HrasG12S miceinduced IL33 production while in naive CD4 T cellsfrom the spleen the Th2 immune response was comparable between Hras and HrasG12S mice Finallythe inhibition of ERK activation by PD0325901 treatmentameliorated the ADlike skin lesions and IL33 production Together these data indicate that germline HrasG12S activating mutation causes ADlike skin lesions viathe ERKIL33 axis Fig cellsepidermalIn the present study after repeated stimulation withDfb HrasG12S mice showed hyperproliferation of pERKpositiveand increased IL33expression in the dorsal skin Increased IL33 expressionwas also observed in primary epidermal keratinocytesfrom HrasG12S mice after Dfb stimulation which issimilar to the reports that the activation of RASMAPKsignaling was associated with increased IL33 expressionin cancer cells2930 Recently IL33 was found to inducethe Th2 ‚ammatory response in allergic diseasesespecially AD31 Excess IL33 is also associated with skinbarrier dysfunction and ILC2 functions which is partiallyregulated by the RASMAPK signaling pathway3233Epithelialspecific IL33 transgenic mice have been foundto develop ADlike dermatitisincluding acanthosispruritus increased IgE serum levels reduced claudin1expression and increased production of eosinophils mastcells and ILCs3334 Of note IL33 and its receptor ST2are highly expressed in the skinderived ILC2s of ADpatients and lung ILC2s of patients with allergic airwaydiseases respectively3536 Importantly consistent with thephenotype of IL33 transgenic mice and AD patients Dfbtreated HrasG12S mice showed ADlike skin lesionsreduced claudin1 expression increased IL33 expressionhyperproliferation of pERKpositive epidermal cells andincreased ILC2 production Collectively excess IL33could lead to ERK activation resulting in an increase ofILC2s and impaired skin barrier Fig Official journal of the Cell Death Differentiation AssociationThe pathogenesis of skin lesions observed in Costellosyndrome includes dermal connective tissue abnormalities cutis laxa and deep palmer and plantar creaseshyperproliferative skin disease palmoplantar keratoderma cutaneous papilloma and acanthosis nigricansand ‚ammatory skin abnormalities sensitive skineczema and pruritus A previous study showed that inthe skin fibroblasts of Costello syndrome elastic fiberswere not assembled due to a functional deficiency of theelastinbinding protein as a result of an unusual accumulation ofchondroitin sulfatebearing proteoglycans3738 Regarding the hyperproliferative skin lesions ithas been reported that the root cause of papillomashyperkeratosis and epidermal hyperplasia such as psoriasis is the activation of the RASMAPK pathway39“However the pathophysiological mechanism of ‚ammatory skin abnormalities in Costello syndrome remainsunclear In the present study Dfbtreated HrasG12S micedisplayed pruritus and eczema Recently mice withepidermisspecific BRAFRAF1 deficient also showed ADlike dermatitis which is characterized by increased serumIgE levels and a Th2 response43 The elevated IgE levelshave not been systematically examined or reported inCostello syndrome patients However it is possible thatthe allergic reaction stimulated by house dust mites couldbe involved in the development of ‚ammatory skinabnormalities in Costello syndrome patientsincludingsensitive skin pruritus and eczemaimprove HRASdrivenAt present acitretin has been reported to treat palmoplantar keratosis in patients with Costello and CFC syndrome4445 Several reports have demonstrated that MEKtumorigenesis46inhibitorsimpaired enamel formation in the teeth27 and longtermdepression in the hippocampus in Hras G12V knockinmice28 as well as hyperkeratosis and hyperplasia in theforestomach of BrafQ241R mice26 In the present studyPD0325901 treatment of the ADlike skin lesions inHrasG12S mice was found to reverse these lesions byreducing hyperproliferation of pERKpositive epidermalcells and the production of ‚ammatory cells and cytokines including IL1 IL4 and IL33 Treatment withU0126 an MEK inhibitor in human keratinocytes hasalso been found to restore the reduced expression levels ofclaudin1 and filaggrin and increase ERK activationthrough excess IL333347Indeed reduced claudin1 0cKatata Cell Death and Disease Page of Fig An increase in ILC2s and basophils observed in the skin and ear of Dfbtreated HrasG12S mice a b Flow cytometric analysis of skin aand ear b cells from Hras and HrasG12S mice collected days after Dfb application Eosinophils CD45 SSChi siglecF Basophils CD45siglecFˆ’ FcεRIα DX5 Mast cells CD45 siglecFˆ’ FcεRIα DX5ˆ’ ILC2 CD45 Linˆ’ CD3ε CD4 CD8a CD11c FcεRIα NK11 CD19 Ter119 CD5 F4 Gr1 Sca1 GATA3 n in each group Data are presented as mean ± SD Significance was analyzed by twotailed Student™s t test P P expression was improved in Dfbtreated HrasG12S miceafter PD0325901 treatment Recently hypertrophic cardiomyopathy in Noonan syndrome patients were treatedby MEK inhibitor48 So MEK inhibitors could be effectivein patients with RASopathies The most common sideeffect of trametinib MEK inhibitor in human patients isOfficial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of Fig Expression of ‚ammatory cytokines is enhanced in the skin of Dfbtreated HrasG12S mice in the early stage of dermatitisa Relative mRNA expression related to ‚ammation including Il1 Il4 Il13 Tslp Il33 and St2 in the dorsal skin of Hras and HrasG12S mice on day after treatment of Dfb mRNA levels were normalized to those of 18s rRNA n per group b Relative mRNA expression of Il1 Tslp and Il33 inDfbstimulated ngμl h or vehicletreated PBS h keratinocyte from Hras and HrasG12S mice mRNA levels were normalized to those ofGapdh Results represent five independent experiments Data are presented as mean ± SD Significance was analyzed by oneway ANOVA and theTukeyˆ’Kramer method P P and P skin rush and common toxicity associated with vemurafenib BRAF inhibitor is cutaneous abnormalities suchas keratoacanthoma and squamous cell carcinoma by themechanism of paradoxical MAPK pathway activation49Therefore the balance of RASMAPK signaling plays animportant role in the emersion of skin abnormalitiesAdjusting dosage of MEK inhibitors may be effective onskin lesions of patients with Costello syndromeHere Dfbtreated HrasG12S mice exhibited increasedIL33 expression through hyperproliferation of pERKpositive epidermal cells Additionally we show thatPD0325901 treatment ameliorated the ADlike skinlesions in HrasG12S mice under conditions of exposureto Dfb Thus it will be interesting to investigate whethertreatment with IL33 antibody reduces the ADlike skinlesions in HrasG12S mice Our findings provide additional perspective that HrasG12S mice will serve as avaluable model to study pathophysiology and potentialtherapeutic approaches in ADMaterials and methodsMiceHrasG12S mice on a C57BL6J background have beendescribed previously13 Male mice were analyzed inthis studyGenotypingThe genomic DNA was extracted from the tail tissueusing a Maxwell Mouse Tail DNA Purification KitPromega Madison WI USA or the alkaline lysismethod For the alkaline lysis method a small piece ofeach tail mm was incubated in mM NaOH for min at °C After the addition of M TrisHClpH the extracts were used for PCR Genotyping ofOfficial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of Fig No significant difference was found in Th2 cytokine responses and the proliferation of naive CD4 T cells between Hras andHrasG12S mice a Naive CD4 T cells were isolated from the spleen of Hras and HrasG12S mice at weeks of age After that the cells werestained with CSFE and cultured with mouse antiCD3 monoclonal antiCD28 monoclonal recombinant mouse IL2 recombinant mouse IIL4 andantiIFNγ antibodies for days After incubation the cells were fixed permeabilized and stained with IL5 and IL13 antibodies Flow cytometricanalysis was performed n in each group b After days culture of purified naive T cells from spleen of Hras and HrasG12S mice theproliferation index was measured by flow cytometry n in each group Data are presented as mean ± SD Significance was analyzed by twotailedStudent™s t testHras and HrasG12S mice was performed by PCRusing KOD FX Neo TOYOBO Osaka Japan The primers used for PCR have been described previously1350Induction of dermatitisAtopic dermatitislike skin lesions were induced in malemice at weeks of age according to the manufacturer™sinstructions The mice were anesthetized with isofluraneand their dorsal hair was removed using an electric clipper2000AD Natsume Seisakusho Tokyo Japan For sensitization mg of Biostir AD Biostir Inc Kobe Japanan ointment of Dfb extract was applied to the shaveddorsal skin and ears Three or four days later hair growthwas removed with an electric clipper and μl of SDS SigmaAldrich St Louis MO USA was applied tothe shaved dorsal skin and ears to disrupt the skin barrierAfter h mg of Biostir AD was applied to theirshaved dorsal skin and ears to induce ADlike skin lesionsThese procedures were repeated twice a week for daysThe mice were sacrificed on day to collect skin and earsamples Supplementary Fig 2a To assess the effect ofDfb ointment to Hras and HrasG12S mice they wererandomly divided into four groups SDStreatedHras SDStreated HrasG12S Dfbtreated Hrasand Dfbtreated HrasG12S using single blinding testEvaluation of dermatitis and ear thicknessThe dermatitis scores were evaluated twice a weekaccording to the development of four symptoms erythemahemorrhage of dorsal skin scarringdryness ofdorsal skin edema of ear and excoriationerosion ofear51 The total dermatitis score maximum score wasdefined as the sum of individual scores none mild moderate severe for each symptom SupplementaryTable Ear thickness was measured with a digimaticmicrometer CLM115QM Mitutoyo Kanagawa JapanMeasurement of scratching behaviorOn day scratching behavior was monitored by videoGZHM890 JVC Kanagawa Japan for min TheOfficial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of Fig See legend on next pageOfficial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of see figure on previous pageFig MEK inhibitor reduces the clinical severity of dermatitis in Dfbstimulated HrasG12S mice a b Gross appearances dermatitis score andear thickness in vehicle saline or PD0325901treated Hras and HrasG12S mice after days of daily injections n per group All of the micein these figures were treated with Dfb The circle and square show vehicle group without MEKi with same amount of saline c Skin tissuestained with HE and TB in Hras and HrasG12S mice with PD0325901 or vehicle treatment n per group d Immunohistochemistry of pERKand claudin1 in skin c d Scale bars μm e Relative mRNA expression of Il1 Il4 and Il33 in dorsal skin mRNA levels were normalized to those of18s rRNA vehicle group n PD0325901 group n Data are presented as mean ± SD Significance was analyzed by oneway ANOVA and theTukeyˆ’Kramer method P P and P HrasG12S PD0325901 vs HrasG12S vehicle P HrasG12S vehicle vs Hrasvehicle  P onetailed Student™s t testFig Germline HrasG12S mutation causes ADlike skin lesions via ERKIL33 axis Exposure to Dfb allergen induces ADlike skin lesionsincluding eczema acanthosis and pruritus in HrasG12S mice Dfbtreated HrasG12S keratinocytes show increased IL33 expression throughhyperproliferation of pERKpositive epidermal cells Excess IL33 activates basophil and ILC2containing ST2 receptors These activated immune cellsinduce the production of type2 ‚ammatory cytokines such as IL4 Furthermore excess IL33 can activate ERK signaling resulting in reducedclaudin1 expression and skin barrier dysfunction DC dendritic cellsnumber of scratching bouts was assessed by replaying thevideo Each incidence of scratching behavior was definedas rubbing of ears and dorsal skin with forepaws hindpaws and mouthHistology and immunohistochemistryThe dorsal skins were fixed in neutral bufferedformalin for paraffinembedded specimen and paraformaldehyde for frozen specimen Embedded tissueswere sectioned at μm paraffinembedded specimensor μm frozen specimens Paraffinembedded specimens were stained with hematoxylin and eosin HEand toluidine blue TB Epidermalthickness wasmeasured in five randomly selected areas × μmof each HEstained sample Mast cells were counted inten randomly selected areas × μm of each TBstained sample For immunohistochemistry the paraffinembedded sections were deparaffinized using xylene andrehydrated with ethanol Frozen specimens were driedsufficiently with a dryer Antigens were activated using aHistofine simple stain kit Nichirei Bio Sciences TokyoJapan The antibodies used are described in Supplementary Table Signals were visualized with a DABSubstrate KitNichirei Bio Sciences Sections werecounterstained with hematoxylin pERK immunostainedareaepidermis was measured in five randomlyOfficial journal of the Cell Death Differentiation Association 0cKatata Cell Death and Disease Page of selected areas × μm of each pERKstainedsampleSpleen and LN immune cell preparation and flowcytometryQuantitative reverse transcriptionPCRTotal RNA extraction and purification of keratinocyteswas performed according to the standard proceduresusing an RNeasy Mini Kit Qiagen Hilden Germany andQIAshredder Qiagen The extraction and purification ofthe total RNA from the dorsal skin and cDNA synthesiswere performed as previously described26 Quantitativerealtime PCR was performed using THUNDERBIRDSYBR qPCR MIX TOYOBO in a StepOnePlus ThermoFisher Scientific Waltham MA USA The amplificationprimers are described in Supplementary Table Eachsample was run in duplicateWestern blottingSkin tissue was homogenized in lysis buffer mM TrisHCl pH and SDS containing phosphatase and protease inhibitor P5726 and P8340 SigmaAldrich Supernatants were collected after centrifugation and the proteinconcentration was determined using a BioRad ProteinAssay BioRad Laboratories Hercules CA Western blotanalyses were performed as previously described26 Brieflythe proteins were transferred onto nitrocellulose membranesand blocked with nonfat milk in Trisbuffered salinewith Tween20 mmolL TrisHCl pH mmolLNaCl and Tween20 for h at room temperature Themembranes were incubated overnight at °C with theantibodies described in Supplementary Table All membranes were visualized using the Western Lightning ECLPlus Kit PerkinElmer Waltham MA USA The bandintensities were quantified using NIH ImageJ softwareELISASerum IgE was determined using LBIS Mouse IgEELISA Kit FUJIFILM Wako Shiba
2
"tea is the second most popular beverage consumed in theworld next to water green tea is a kind of nonfermentedtea produced from the plant camellia sinensis it is favoredby people for its fresh flavor and health benefits and consumed worldwide especially in east asian countriesgreen tea contains caï¬eine and polyphenolic compoundsknown as catechins catechins are the primary bioactivesubstances and present significant biological propertiestea leaves™ drycatechins constitute up to ofweight among that egcg is the main and the most abundant catechin [ ] egcg has been traditionally regardedas beneficial mainly ascribed to its antioxidant action the antioxidant eï¬ects of egcg are manifested in scavenging free radicals in the body and inhibiting the formation ofros the results of earlier studies suggested that egcgcould decrease the risk of several human disorders associatedwith oxidative stress on the other hand egcg also displays significantprooxidant eï¬ects usually under highdose conditions theprooxidant actions of egcg play a dual role being both beneficial and harmful while achieving desired outcomes inchronic disease prevention and treatment reports about thetoxicity of egcg are also emerging a growing body ofevidence continues to demonstrate a variety of harmfuleï¬ects from excessive consumption of green tea or oraladministration of highdose egcg supplement highdoses of egcg not only cause cytotoxicity in vitro but alsoresult in living body hepatotoxicity nephrotoxicity andgastrointestinal disorders vomiting and diarrhea the oral bioavailability of egcg is not so profound inhealthy humans as it was only of the total ingestion most of the ingested egcg is absorbed in the smallintestine and substantially degraded in the large intestine bymicrobiota action the eï¬ective dosage of egcg mightbe close to or higher than the toxic dosage in practical applications considering its low bioavailability therefore it is 0coxidative medicine and cellular longevitynecessary to understand the potential toxicity doses andusage of egcg in this review the prooxidant eï¬ects ofegcg in health benefits and adverse eï¬ects were discussedespecially concerning their underlying mechanisms involvedand doses used this review is aimed at harnessing the prooxidant eï¬ects of egcg for human health maintenance whileavoiding toxicity thereby better guiding the safety consumption of green tea and egcg chemical structure andautooxidation of egcgbasic catechins contain two or more aromatic ringshydroxyl group on carbon three position andor the higherdegree of hydroxylation of the b ring would be primarilyresponsible for the potent antioxidant activities of catechinsfigure 1a previous structureactivity relationshipstudies of catechins have demonstrated the importance ofthe number and location of the phenolic hydroxyl groupson antioxidative capacity egcg has the remarkablepotential to scavenge radicals and chelate metal ion theseabilities could be ascribed to the presence of dihydroxyand trihydroxy groups in a ring b ring and d ringfigure 1b the catechol structure of egcg makes it susceptible todegradation via autooxidation figure under normal°physiological conditions ph c egcg is autooxidized and converted to oquinone through nonenzymaticaldehydrogenation of phenolic hydroxyl groups at b ring when the cell culture medium is exposed in the airegcg could be oxidized by oxygen and yields superoxide andanion radicals o2‹…egcg are essential intermediate products in egcg autoox and oxygen could function as oxidants for furidation o2ther oxidation of egcg finally resulting in the formationof oquinone accompanying the generation of hydrogen could also form substantial amountsperoxide h2o2 o2of h2o2 via disproportionation reaction one egcgmolecule could produce more than two h2o2 molecules inphosphate buï¬er at neutral ph and egcg radicals ‹…egcg o2autooxidation of egcg generates substantial ros theros comprises singlet oxygen hydroxyl radicals superoxideperoxides and h2o2 h2o2 is in a dominant position andusually is regarded as a toxic agent when the ros levelexceeds cellular antioxidant capacity oxidative stress willoccur in other words this is the result of an imbalancebetween prooxidant and antioxidant eï¬ects inclusion ofantioxidant defense enzymes such as catalase cat andsuperoxide dismutase sod could minimize h2o2 levelwhich is essential to maintain the redox balancethe concentration of egcg in the cell environmentseems to be a primary factor in explaining its prooxidanteï¬ects for example egcg treatment alone diminisheddna strand breakage of human blood lymphocytes at lowconcentrations μm while it induced dna strandbreakage at higher concentration μm thusegcg acts as an eï¬ective antioxidant at low doses withinthe range of high nanomolar and low micromolar levelswhile egcg represents a prooxidant at high doses howeverthis blurred boundary might vary depending on the type ofradical stimulants cellular environment and duration ofexposure to egcg health benefitsuntil now egcg has been a major research subject withinthe field of healthpromoting eï¬ects the potential role ofthe prooxidant eï¬ects of egcg in cancer and obesity prevention and treatment as well as the antibacterial actionsachieved demonstrable results in previous studies prooxidant eï¬ects and anticancer activity of egcgcancer is one of the most common and lifethreateningdiseases occurring among humankind egcg as a naturalproduct has drawn a great deal of attention from boththe scientific community and the general public indeedegcg has shown both prophylactic and therapeutic efficacy in multiple human cancers several mechanisms havebeen proposed to accountfor the inhibitory action ofegcg against cancer formation and growth the prooxidant eï¬ects of egcg were thought to be potential mechanisms for anticancer action the anticancer mechanismsvaried depending on the cell type dose andor time oftreatment table [“]apoptosis is the bestdescribed form of programmed celldeath the induction of apoptosis represents a universal andideal therapeutic strategy for cancer control cell apoptosiscould be triggered by either the intrinsic mitochondrial pathway or the external death receptor pathway the mitochondrial pathway could be induced by intracellularstresses such as oxidative stressthe apoptosistriggering eï¬ects of ros have beennoted in vitro table egcg inhibited cell growth ina dosedependent manner and the decrease in the numberof viable cells was mainly due to apoptosis caused by theegcginduced intracellular ros as early as the last century scientists found that egcg induced h2o2 formationin human lung cancer celllines h661 and 21bes andexogenously added cat completely prevented egcginduced cell apoptosis which suggested that h2o2 isinvolved in the apoptosis process provoked by egcg similar actions were also found in various cancersand tumor cells table thioredoxin trx and thioredoxin reductase trxr are pivotal regulators of cellularredox homeostasis decreased trxtrxr activity mightcontribute to the increased ros level high concentrationof egcg inactivated trxtrxr via the formation of egcgtrx1 and egcgtrxr conjugates which was linked to theelevation of ros level in hela cells and further promotedcancer cell death moreover one of the biochemical hallmarks of apoptosis is genomic dna fragmentation chen performed the dna fragmentation assay in theskov3 cells indicating that egcg induced apoptosis bycausing dna damage this result was consistent withother studies in ovarian and cervical cancer cells [ ]in terms of molecular mechanisms intrinsic apoptosisleads to the release of mitochondrial cytochrome c afterbeing released into the cytoplasm cytochrome c stimulates 0coxidative medicine and cellular longevityohbohohohhoohbohohohohdohocoaohobhoocaohafigure a basic structure of catechins b chemical structure of egcgohbegcgohohautooxidationph75 °cohboautooxidation·egcgoh“o2h2o2ohboooquinonefigure superoxidemediated chain reaction the formation of oquinoneapoptosome formation followed by activation of caspasecascades egcgmediated mitochondrial ros couldpromote cytochrome c release to the cytosol the antiproliferative action of egcg on prostate cancer and breast cancer is mediated through apoptosis as evident from caspase9[ ] the cells susceptible to egcginduced apoptosisalso showed activation of caspase3 moreover theincreased ros level was observed to result in the stimulationof mitogenactivated protein kinase mapk themapk signaling pathwayincluding extracellular signalregulated kinase erk jun nterminal kinase jnks andp38 plays a vital contribution in cell proliferation diï¬erentiation apoptosis and stress response egcg induced oxidative stress via generation of ros and thereafter activatedthe jnk pathway leading to changes in mitochondrial membrane potential and release of cytochrome c in ht29 humancolon adenocarcinoma cells and mia paca2 pancreaticcancer cells [ ] together these results suggest thategcginduced apoptosis is mediated through ros generation and might subsequently activate the cell intrinsic pathway in the presence of transition metals such as copper andiron h2o2 could convert to a potent oxidant hydroxyl radical via the fenton reaction nakagawa found that egcg μm produced h2o2 and triggered fenton reactionto form highly toxic hydroxyl radicals which resulted in lymphoblastic leukemia jurkat cell death in the presence offeiii and cuii egcg μm induced dna damagein hl60 cells as 8oxo78dihydro2²deoxyguanosine oxodg content increased which was a characteristic ofoxidative dna damage nevertheless no significantincrease in 8oxodg was observed in h2o2resistant colonhp100 cells suggesting that h2o2 was involved in cellulardna damage egcg could inhibit cell proliferation andinduce apoptosis through cellular dna breakage in diï¬erentcancer cell lines such dna breakage involved the mobilization of endogenous copper ions and the generation ofros moreover the observation of site specificity of dnadamage by egcg is valuable cuiimediated dna damageby egcg occurred most frequently at t and g residues egcg was able to mobilize endogenous copper ions andgenerate hydroxyl radicals in situ hydroxyl radicalsserved as the proximal dna cleaving agentleading todna breakage in the nuclei this result was possibly due tothe interaction of egcg with chromatinbound copper ionsand then the nondiï¬usible hydroxyl radicals were formed atthe binding site hence hydroxyl radical generated nearbydna was well established as the cause of strand scissionbecause the concentration of copper is significantly very highin various malignancies egcg could induce cancer celldeath through the metal iondependent pathway thispathway was independent of mitochondriamediated programmed cell deaths such action involved in metal ionmediated dna cleavage would be an important mechanismof anticancer properties of egcgin addition to being transported into the cell egcgcould also function on the cell membrane fraction to regulatethe surface growth factor receptor earlier studies foundthat autooxidation of egcg led to epidermal growth factorreceptor egfr inactivation in human esophageal cancer 0ccell linesbladder cancernbtiibreast cancermcf7mcf7cervical cancerhelahelacolon canceroxidative medicine and cellular longevitytable role of prooxidant eï¬ects in the anticancer activity of egcg based on cell culture studiesegcgconcentrationtimebiological eï¬ectsreferences μm hinduced early apoptosis through dna damage μgml hinduced cell growth inhibition and apoptosis by downregulating survivinexpression via suppressing the akt pathway and activating caspase9 μm hinduced apoptosis at low doses via activation of jnk caspase9 and caspase3while inducing necrosis at high doses which is related to diï¬erences in rosgeneration and atp levels μm μm and h hincreased cell death through dna damageinduced cell death through generation of ros and inactivation of trxtrxrhct116 μm hinduced apoptosis through induction of ros and epigenetic modulation ofapoptosisrelated gene expressionht29 μm hendometrial carcinomaishikawa μm hinduced apoptotic cell death via activating the jnk pathway accompanyingmitochondrial transmembrane potential transition and cytochrome c releaseic50 was μminduced apoptosis via ros generation and p38 map kinase activationic50 was μmesophageal cancerkyse lung cancer μm hinactivated egfr by superoxide generated from autooxidation of egcg μm μm h h h μm hdisplayed strong growth inhibitory eï¬ects against lung tumor cell linesinhibited cell growth through induction of ros ic50 was μmic50 was μminduced apoptosis via h2o2 production and hydroxyl radical formationinduced apoptosis by modifying the redox systemh661 and h1299 μmh1299lymphoblastic leukemiajurkatmyelomaim9 rpmi8226and u266oral cancerscc25 andscc9ovarian cancer μm hreduced cell viability by inducing mitochondrialocalized ros and decreasingsirt3 expressionskov3 μgml dpancreatic cancerpanc1 μm hmia paca2 μm hinhibited cell proliferation and induced apoptosis by inhibiting cell cycle arrest andinducing dna damageinduced apoptosis through generation of ros as well as caspase3 andcaspase9 activationinduced stress signals by damaging mitochondria and rosmediatedjnk activationprimary eï¬usionlymphomabcbl1 and bcprostate cancer μgml hinduced apoptosis and autophagy through ros generationpc3 and μm hreduced cell survival and increased apoptosis caused a significant alteration incaspase9 alternative splicing 0coxidative medicine and cellular longevitycell line kyse one possible explanation is thath2o2 produced from egcg autooxidation in the cell culturemedium could attack and inactivate egfr leading to theinhibition of egfr phosphorylationand preferentialit is worth considering whether high amounts of egcgcould cause damage to normal cells egcgmediated rosproduction was particularly observed in cancer cells compared with normal cells the selectivity of egcginducedapoptosis in cancer cells might be due to the diï¬erentialinducibility of rosexpression ofapoptosisrelated genes moreover tao found thategcg induced diï¬erential mitochondrial dysfunction andoxidative stress in normal and oral cancer cells these eï¬ectswere related to the diï¬erential modulation of sirtuin sirt3 and its downstream targets including glutathionegsh and sod considering the cytotoxicity of egcgin normal cells the ic50 value in normal cells was checkedand showed to be more than μm while that for thecorresponding cancer cells was μm these resultssuggested that cancer cells are more sensitive to egcg thannormal cells and ros might be selectively toxic to cancercellsin addition to being used as preventive agents individually egcg could also be used as adjuvant therapies generally cooperative interaction of two or more agents couldtarget more signaling pathways thus eï¬ectively improvingagent chemosensitivity reducing untoward eï¬ects of treatment expanding the scope of action and showing highertherapeutic outcomes drug resistance is a dauntingchallenge in cancers prooxidant activities of egcg wereproposed to contribute to overcoming drug resistancehighlighted by the fact that h2o2 production induced byegcg increased the potency of cisplatin in ovarian cancercells by three to sixfold in contrast cisplatin alone washighly resistant to the treatment in some cancer cell linescopper transporter ctr1 is a critical determinant toincrease cisplatin uptake egcg could upregulate ctr1expression through the stimulation of ros simultaneous treatment of arsenic trioxide ato with egcgshowed oxidativemediated induction of apoptosis in leukemia cancer cells egcg acted as a prooxidant andincreased intracellular h2o2 and atoinduced hemeoxygenase1 ho1 provided ferrous iron to increase thefenton reaction in both cases cellular oxidative damageeventually occurredin general under typical cell culture conditions egcghas been known to generate i extracellular ros via autooxidative reaction in the cell culture medium ii ros in cellular mitochondria and iii intracellular ros through thefenton reaction upon cell entry figure these three pathways contribute diï¬erently to cancer cells but eventuallycause cell damage and death cancer initiation and progression are generally divided into several stages when egcgacts as an antioxidant it might more eï¬ectively enhance antioxidant capacity at the cancer prevention stage whereaswhen egcg acts as a prooxidant it might be more criticalat suppressing tumor growth stage one possible suppositionis that tumor cells may be more susceptible to oxidativestress because their increased growth rate and metabolismcause a heightened basal ros level the degree of cell proliferation and diï¬erentiation seems to be one factor aï¬ectingthe ros production ability of egcg future research willbe required to determine if egcg is a much more potentros inducer in diï¬erentiated than in undiï¬erentiated cancercells although a limited amount of data has shown that theseprooxidant eï¬ects can occur in vivo it is essential to understand when and to what extent the antioxidant or prooxidanteï¬ects of egcg are working in diï¬erent stages of cancers inanimal models prooxidant and antiobesity eï¬ects of egcg obesity is ametabolic disease characterized by abnormal or excessive fataccumulation it is generally associated with an increased riskof chronic diseases including diabetes hypertension anddyslipidemia a large and growing body of studies hasinvestigated the antiobesity eï¬ects of egcg in cellular andanimal experiments and the underlying mechanismsthe clinical manifestations of obesity are adipocytehyperplasia and hypertrophy in vitro studies have well demonstrated that egcg could inhibit adipocyte growth andinduce adipocyte death through its prooxidant eï¬ects hung reported that high concentrations of egcg μm reduced the cell viability of preadipocytes by induced the appearance of dna fragmentation andincreased the activity of the apoptotic enzyme caspase3 egcg was demonstrated to raise ros level anddescend gsh level in preadipocytes and adipocytes whichinduced oxidative stress thus resulting in decreased cell number ²ampregulated protein kinase ampk represents ametabolitesensing protein kinase hwang foundthat the release of ros by egcg stimulation could furtheractivate ampk rapidly in 3t3l1 adipocytes a recent studyalso proved that ampk was activated by exogenous h2o2and this activation was not through direct redox signalingto ampk but was a secondary consequence of redox eï¬ectson other processes egcg activates ampk via the generation of ros subsequently blocks anabolic pathways and promotes the catabolicpathway and suppresses gluconeogenesis and adipogenesisconsequently leading to body weight reduction and metabolic syndrome alleviation figure the activation ofampk modulates the expression of genes and proteinsinvolved in lipid metabolism downregulates the expressionof fat synthesis proteins and upregulates lipid catabolismproteins it was shown that egcg inhibited the expressions of glucose 6phosphatase g6pase for gluconeogenesis phosphoenolpyruvate carboxykinaseforgluconeogenesis fatty acid synthase fas for fatty acid synthesis acetylcoa carboxylase acc for fatty acid synthesis hydroxymethylglutarylcoa reductase hmgrforregulatory elementbinding proteinscholesterolsrebpsfor sterol synthesis peroxisome proliferatoractivated receptor gamma pparγ for lipid synthesis andstorage and ccaatenhancerbinding protein alphacebpα for adipogenesis as well as enhanced the expression of acylcoa oxidase aco for fatty acid oxidationperoxisome proliferatoractivated receptor alpha pparαpepcksterol 0coxidative medicine and cellular longevityautooxidationrosegcgcellrosfe2cu2fentonreaction·ohegfrcytochrome ccell damagecell deathcaspase9caspase3cell culture mediumfigure prooxidant eï¬ects of egcg in cell cultureegcggeneraterosactivateampkmodulateg6pase pepck fasacc hmgr srebpsppar𝛾 cebp𝛼aco ppar𝛼 cpt1acad pgc1𝛼ucps atglfat synthesislipid catabolismantiobesityfigure eï¬ects of egcg on lipid metabolism via ros and ampkfor fatty acid oxidation carnitine palmitoyltransferase1cpt1 for fatty acid oxidation acylcoa dehydrogenaseacad for fatty acid oxidation peroxisome proliferatoractivated receptor gamma coactivator1α pgc1α for fattyacid oxidation uncoupling proteins ucps for thermogenesis and adipose triglyceride lipase atgl for triglyceridehydrolysis [“]accordingly the prooxidant eï¬ects of egcg play avital role in preventing the initiation and progression ofobesity egcg could cause oxidative stress thus damagingadipocyte directly and activating ampk and then aï¬ectingrelative genes and protein expression and signal transduction in various tissues indirectly however the increase ofoxidative stress in fat accumulation might be an importantpathogenic mechanism of obesityrelated metabolic syndrome such as diabetes firm conclusions as to whetherprooxidant eï¬ects of egcg could perform on body weightbody fat and adipose weight in humans will require morethorough clinical studies prooxidant and antibacterial eï¬ects of egcg egcgexhibits a broad spectrum of bactericidal activity against various bacteria its bactericidal eï¬ects include damage to thebacterial cell membrane and inhibition of fatty acid synthesisand enzymatic activity h2o2 which is generated byegcg appears to play an indispensable role in the bactericidal actions of egcg the bactericidal action of egcgwas related to h2o2 level as bactericidal action was inhibitedby the increase of cat concentration egcg was foundto have bactericidal activity at higher concentrations in thesalmonella assay highly correlated with h2o2 production egcg showed a dosedependent “ μm inhibition on escherichia coli e coli op50 strain growth this inhibitory action was associated with a profoundincrease in intracellular oxidative stress caused by egcghence the use of egcg as a prooxidant is well supportedby these studiesegcg was shown to have broad antibacterial spectrumeï¬ects on both grampositive and gramnegative bacterianevertheless egcg might function through diï¬erent mechanisms against grampositive and gramnegative bacteriaintracellular ros level was determined by flow cytometrythe results indicated that damage on gramnegative e colicell walls was induced by egcg depending on h2o2 release 0coxidative medicine and cellular longevity in contrast the damages on grampositive staphylococcus aureus resulted from a combination between egcg andpeptidoglycan layer because the outer membrane ofgramnegative bacteria was mainly composed of negativelycharged lipopolysaccharides which could resist the destruction of egcg they are less susceptible to egcg thangrampositive bacteria bacterial cell membrane damage not only prevents thebinding of bacteria to host cells but also inhibits the abilityof the bacteria to combine with each other and form biofilms egcg was known to attack the lipid bilayer of bacterialcell membranes leading to physical disruption of the membrane as for the cell walls results from atomic forcemicroscopy suggested that the subminimum inhibitory concentrations of egcg treatment mgl to e colio157h7 strains could lead to temporary changes in the cellwalls cui such changes were due to the damagecaused by h2o2 generated from egcg moreover egcgcaused cell membrane damage via increased intracellularros level and led to potassium leakage these are potentiallyconducive to the antibiofilm efficacy of egcg against vibriomimicus which is a foodborne pathogen in seafood andwater in addition egcg also regulates the expression of oxidative stressrelated genes oxyr and soxrs systems are activated upon exposure to oxidative stress oxyr induces katgand soxrs induce soda strongly when cells are stressed byexogenous h2o2 egcg treatment upregulated katgand soda expression in e coli these results verified the roleof ros in egcgmediated bacterial inhibition the cpxsystem is thought to control protein homeostasis in the cellenvelope when e coli was exposed to egcg apoptosis happened because of ros formation by the cpx system rpos is a general stress regulator in response to oxidativestress egcg could cause a reduction in the expression forrpos indicating that egcg induced oxidative stress in bacterium models the potential prooxidant properties of egcg could beattributedin part to its suppressive eï¬ects on bacteriamore broadly research is also needed to determine relativesignaling pathways and proteomic factors egcg is superexcellent natural products it could increase the efficacy ofbactericidal eï¬ect when it aids other fungicides morerecent attention has been focused on the impact of greentea and green tea polyphenols on the intestinal microflorawhether egcg intervention would change the diversity ofmicrobiota and lead to microbiota death is also in need offurther investigation adverse effectsin recent years egcg has become one of the most aggressively promoted food supplement products in daily lifeegcg entered the market and its safety has raised queriesthe prooxidant eï¬ect of egcg is not necessarily advantageous they might have implications regarding potential toxicity hence it is necessary to systematically explore theharmful eï¬ects of egcg and the mechanisms prooxidant and hepatotoxicity eï¬ects of egcg a considerable amount of literature has been published on hepatotoxicity of green teaderived products it is noteworthythat the hepatotoxicity of green tea and its derived productswas initially found in some diet products in after beingthe cause of liver injury in subjects france and spain governments have suspended the marketing of exolise whichwas a weightloss phytotherapeutical drug in the pasttwo decades reports on liver disorders caused by green teaingestion with overdose of egcg content have graduallyemerged the liver is a major drug metabolic organ in the bodythe bioavailability of egcg in rats was determined after min of oral administration mgkg by detecting theconcentration of egcg in plasma and diï¬erent tissuesincluding the liver the results showed that the concentrationof egcg in the liver μmolkg was four times higherthan in that in the blood plasma μmolkg moreover utilizing anatomy egcg could trigger liver damagewhereas no visible abnormalities were found in other tissuesand organs [ ] hence it could be preliminarily concluded that the liver is the toxic target organ of egcgat the cellular level egcg demonstrated cytotoxic eï¬ectin cultured rat hepatocytes it was shown that μm egcgtreatment on freshly isolated rat hepatocytes caused timedependent cytotoxicity the hepatocyte was incubatedwith egcg for h resulting in liver cell function reduceddose dependently the decrease of lactate dehydrogenase ldh a marker of cell membrane damage wasobserved in rat hepatocytes egcg also caused damageto the outer mitochondrial membrane by the fact that mitochondrial membrane potential collapsed in animal experiments table the severity of egcginduced toxicity is relevant with dose route of administration and period of treatment [ “] biochemicaland histopathological analysis showed that liver samples ofmice displayed diï¬erent degrees of liver injury liver functionindexes of plasma alanine aminotransferase alt andaspartate aminotransferase ast activity increased in adosedependent mannermalondialdehyde mda and 4hydroxynonenal hne are final products of lipid peroxidation present biochemical markers of oxidative stress metallothionein mtand γhistone 2ax γh2ax are molecular markers of oxidative stress oral high dose of egcg mgkgd to cf mice for two days significantly enhanced the formation ofmda in the liver and elevated the expression of hepaticmt and γh2ax protein and increased positive staining for4hne in liver samples intraperitoneal administrationof egcg or mgkgd for five days raised serum hne level and western blot analysis showed that hepaticγh2ax was markedly increased all these biomarkersillustrated that egcgtriggered hepatotoxicity in vivo wasinduced by oxidative stressprevious pharmacological studies have shown that undernormal physiological conditions egcg is metabolizedthrough methylation sulfation and glucuronidation andthen excreted in urine subsequently whereas at toxicdoses these pathways might be saturated and the excessive 0canimal typefemale swissalbino micemalekunmingmiceegcgdosagemgkgd andmalekunmingmice and and male nd4micemale cf1micewistar rats ofboth sexesmale cd1micemicefemaleswisswebster miceoxidative medicine and cellular longevitytable hepatotoxicity of egcg based on animal modelsroute ofadministrationdurationresultsreferenceip and po dip treatment increased serum bilirubin markers po treatment didnot show any dosedependent changes except alt marker dtolerable dose of egcg was mgkg for ip and mgkg foripipigigpo d dserum alt ast 4hne il2 il6 and il10 and hepatic γh2axwere raised hepatic nrf2target gene expression was increasedthe fatality rate was single doseserum alt ast 4hne il6 and il10 and hepatic γh2ax wereraised hepatic nuclear and cytosolic nrf2 proteins were suppressed d dmouse growth was not aï¬ected the dosage was considered asmaximum tolerable dosehepatotoxicity occurred major hepatic antioxidant enzymes weresuppressed nrf2mediated rescue response was inducedsingle dosemice died in a dosedependent manner andthe nrf2 pathway was not activated nrf2 and its target genes were h dsuppressedalt was slightly increased histopathology of the liver showedcongestion of sinusoids and central and portal veinssingle dosealt was markedly increased histopathology of the liver showeddegenerative hepatocytes and a small number of vacuoles d dmouse survival was reduced by mouse survival was reduced by hepatic mda mt and γh2axwere increasedsingle dosealt was increased by 108fold mouse survival was reduced by egcg2²cysteine and egcg2³cysteine were detected in theurineposingle dosemice were lethargic and their respiration was labored and andipipipsingle doseplasma alt was increased mice died within h h degcg thiol conjugates egcg2²cysteinyl and egcg2³cysteinyl were detected in the urine of mice died plasma alt activity was elevated severe hepaticnecrosis occurredamount of egcg would be oxidized to form oquinonewhich could react with gsh to form egcg thiol conjugates therefore it could be inferred that high dose of egcgresults in the accumulation of oquinones and the metabolites of oquinones are biomarkers of oxidative stress twoegcg thiol conjugates egcg2²cysteinyl and egcg2²²cysteinyl were detected in the pooled h urine of micetreated with a dose of or mgkg intraperitonealip injection of egcg however egcg thiol conjugateswere not found when the dose was or mgkg bwip when cf1 mice were treated with a single doseof mgkg intragastric ig administration of egcgboth egcg2²cysteinecysteine weredetected in the pooled h urine gsh conjugate ofand egcg2²²egcg was also detected in hepatocytes incubated withegcg these findings indicated that the formation ofdetectable amounts of egcg thiol conjugates appears toresult from the administration of toxic doses of egcgnuclear factor erythroidrelated factor nrf2 an essential antioxidant transcription factor regulates the expressionof many antioxidant and phase ii detoxifying enzyme genessuch as ho1 and nadphquinone oxidoreductase1nqo1 through antioxidant response element are undernormal metabolic and physiologic states nrf2 is repressed inthe cytoplasm by kelchlike echassociated protein1keap1 while under oxidative stress conditions nrf2 dissociates from keap1 and translocates to the nucleus to bind toare the activation of the nrf2are signaling pathway 0coxidative medicine and cellular longevityrepresenting a major cellular defense against oxidative stresscould stimulate the expression of downstream antioxidantenzymes a previous study revealed that toxic doses ofegcg and mgkg ip inhibited hepatic antioxidantenzymes sod cat and glutathione peroxidase and exacerbated oxidative damage in hepatocytes after treatmentwith egcg the expression of nrf2 decreased in the cytosoland increased in the nucleus indicative of nrf2 activationas a result mrna expression of ho1 nqo1 and otherhepatic nrf2target genes was induced in a dosedependentmanner accordingly a conclusion could be made that themolecular mechanisms underlying highdose egcg potentialtoxicity involve activation of the nrf2are signaling pathwayand suppression o
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Millions of people are suffering from cancers but accurate early diagnosis and effectivetreatment are stilllong noncoding RNAslncRNAs have been proven to play an important role in diseases especially cancersThese lncRNAs execute their functions by regulating gene expression Thereforeidentifying lncRNAs which are related to cancers could help researchers gain a deeperunderstanding of cancer mechanisms and help them find treatment options A largenumber of relationships between lncRNAs and cancers have been verified by biologicalexperiments which give us a chance to use computational methods to identifycancerrelated lncRNAs In this paper we applied the convolutional neural network CNNto identify cancerrelated lncRNAs by lncRNA™s target genes and their tissue expressionspecificity Since lncRNA regulates target gene expression and it has been reportedto have tissue expression specificity their target genes and expression in differenttissues were used as features of lncRNAs Then the deep belief network DBN wasused to unsupervised encode features of lncRNAs Finally CNN was used to predictcancerrelated lncRNAs based on known relationships between lncRNAs and cancersFor each type of cancer we built a CNN model to predict its related lncRNAs Weidentified more related lncRNAs for kinds of cancers Tencross validation has beenused to prove the performance of our method The results showed that our method isbetter than several previous methods with area under the curve AUC and areaunder the precision“recall curve AUPR To verify the accuracy of our results casestudies have been doneKeywords long noncoding RNA lncRNA cancer convolutional neural network CNN deep belief network DBNmachine learningINTRODUCTIONFour to nine percent of the sequences™ transcription are long noncoding RNAs lncRNAs inmammalian genomes Canzio Ji lncRNA was regarded as the noise ofgenome transcription and did not have biological functions at first However an increasing numberof studies have reported that lncRNA is widely Robinson involved in chromosomeEdited byLei DengCentral South University ChinaReviewed byHao LinUniversity of Electronic Science andTechnology of China ChinaInner Mongolia University ChinaJuan WangCorrespondenceNan Dudunan05aliyuncomGanfeng Xiexiegfaliyuncom These authors share first authorshipSpecialty sectionThis was submitted toMolecular Medicinea section of the journalFrontiers in Cell and DevelopmentalBiologyReceived June Accepted June Published August CitationLiu Z Zhang Y Han X Li C Yang XGao J Xie G and Du N Identifying CancerRelated lncRNAsBased on a Convolutional NeuralNetwork Front Cell Dev Biol 103389fcell202000637Frontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cLiu et alA Method to Identify CancerRelated lncRNAsgenomicimprintingchromatin modificationsilencingtranscriptional activationinterference andnuclear transport Cheng 2018a Recently it has beenproven to be associated with many kinds of cancerstranscriptionalThe secondary structure spliced form and subcellularlocalization of most lncRNAs are conserved Karner which is very important for lncRNA to execute functionsHowever compared to the functions of microRNAs miRNAsand proteins the function oflncRNA is more difficult todetermine According to the position of lncRNA in the genomerelative to proteincoding genes it can be divided into five typessense antisense bidirectional intronic and intergenicMany researchers have found lncRNAs play an important rolein cancers Avgeris Cheng 2018b Zhao and neurodegenerative diseases Peng and Zhao as other biological molecules Zhang T Bai Cheng 2019a Liang Although manyresearchers have verified many associations between lncRNAsand cancers by biological experiments compared with ourknowledge about diseaserelated genes we still do not knowenough about diseaserelated lncRNAs Considering the timeand money cost of finding diseaserelated lncRNAs more andmore researchers tend to use computational methods to identifydiseaserelated lncRNAs These methods could be divided intothree categories machine learning methods network methodsand other methodsMachine learning methods build models based on thesimilarities of diseases orlncRNAs and their biologicalcharacteristics Cheng Cheng 2019b Zeng Zou Lan developed thelncRNA“disease association prediction LDAP which is amethod based on bagging support vector machine SVM toidentify lncRNA“disease associations They used similarities oflncRNAs and diseases as the features Yu developedcollaborative filtering naive Bayesian classifier CFNBC based onnaive Bayesian They integrated miRNA“lncRNA associationsmiRNA“disease associations and lncRNA“disease associationsto infer more lncRNA“disease associations Considering thediscriminative contributions of the similarity association andinteraction relationships among lncRNAs disease and miRNAsXuan 2019a developed a dual convolutional neuralnetwork CNN with attention mechanisms to predict diseaserelated lncRNAsNetwork methods are the most common way to identifyassociations between diseases and lncRNAs nowadays Gu Yu Zhang J Kuang Wang L Liu Thiskind of method would build one or multiple networks toinfer new information Wang L built a lncRNA“miRNA“disease interactive network and used their novel methodœLDLMD to predict associations between lncRNAs and diseasesSumathipala used a multilevel network topologywhich includes lncRNA“protein protein“protein interactionprotein“disease relationship to use network diï¬usion algorithmto predict diseaserelated lncRNAs The graph convolutionalnetwork GCN and CNN were used on a lncRNA“miRNA“disease network by Xuan 2019b Deng builtlncRNA similarity network disease similarity network miRNAsimilarity network and their associations Then they calculatedthe metapath and feature vector for each lncRNA“disease pair inthe heterogeneous information networkOther methods may borrow the feature extraction methodor similarity conjecture of network methods but the core ofthis method is matrix decomposition or matrix completionLu developed the geometric matrix completionlncRNA“disease association GMCLDA which is a methodbased on geometric matrix completion They calculated diseasesimilarity based on Disease Ontology DO and calculatedthe Gaussian interaction profile kernel similarity for lncRNAsThen they inferred diseaserelated lncRNAs based on theassociation patterns among functionally similar lncRNAs andsimilar diseases Wang Y proposed a weightedmatrix factorization to capture the interintraassociationsbetween diï¬erent types of nodes Then they approximated thelncRNA“disease association matrix using the optimized matricesand weights to predict diseaserelated lncRNAs Localityconstrained linear coding label propagation Latent DirichletAllocation LLCLPLDA was developed by Xie Firstly localconstraint features of lncRNAs and diseases wereextracted by localityconstrained linear coding LLC Thenthey predicted diseaserelated lncRNAs by label propagationLP strategyHowever previous methods did not consider the regulatingtarget gene expression of lncRNA which is an important functionof lncRNA and plays an important role in associations betweenlncRNAs and diseases In addition deep learning methods arean important tool and have shown their power in bioinformaticsChen Lv Wei Wu Zhao 2019abc Therefore in this paper we used thisinformation as features of lncRNA In addition the expressionof lncRNA in diï¬erent tissues were also used as the featuresof lncRNA Then the deep belief network DBN was used toencode and the CNN was used to classifyMETHODSFeature ExtractionTissue Expression Specificity of Long NoncodingRNACompared with proteincoding geneslncRNA shows strongtissue specificity The specificity of lncRNAs in diï¬erent kindsof tissues and cell types has been proven by many biologicalexperiments The diï¬erent expression also plays an importantrole in essential cellular processes Sasaki testedthe expression of lncRNAs in diï¬erent tissues and found lncRNAs exhibited tissuespecific expression and oflncRNAs were only expressed in one discrete tissue Thereforethe expression of lncRNAs in diï¬erent tissues were used asthe featuresWe obtained the expression of lncRNAs in diï¬erenttissues which included adipose adrenal breast colon heartkidney liver lung lymph node ovary placenta prostate testisand thyroidTherefore the dimension of each lncRNA™s expression featureis ˆ— Frontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cLiu et alA Method to Identify CancerRelated lncRNAsTherefore the dimension of each lncRNA™s target gene featureis ˆ— Deep Belief NetworkThe DBN can eï¬ectively learn complex dependencies betweenvariables Zhao 2019d The DBN contains many layers ofhidden variables which can eï¬ectively learn the internal featurerepresentation of the data and can also be used as an eï¬ectivenonlinear dimensionality reduction methodWhen the observable variables are known the joint posteriorprobabilities of the hidden variables are no longer independentof each other so it is difficult to accurately estimate the posteriorprobabilities of all hidden variables The posterior probability ofearly DBN is generally approximated by Monte Carlo methodbut its efficiency is relatively low which makes its parameterlearning difficult In order to eï¬ectively train the DBN weconvert the sigmoid belief network of each layer to a restrictedBoltzmann machine RBM The advantage of this is that theposterior probabilities of the hidden variables are independentof each other which makes it easy to sample In this way theDBN can be regarded as being stacked from top to bottom bymultiple RBMs and the hidden layer of the Lth RBM is used asthe observable layer of the L 1th RBM Further the DBN canbe trained quickly by layerbylayer training that is starting fromthe bottom layer and training only one layer at a time until thelast layer The specific layerbylayer training process is to trainthe RBM of each layer in turn from bottom to top Assuming wehave trained the RBM in the first L1 layer we can calculate theconditional probability of the bottomup hidden variablesphihiˆ’ σ bi Wihiˆ’where bi is the bias of ith layer of RBM Wi is the connectionweight hi is the ith layer of RBMThe process of training DBN is as followsFIGURE The number of target genes for each long noncoding RNAlncRNAFIGURE The distribution of the number of target genes lncRNA longnoncoding RNAreverseTarget Gene of Long Noncoding RNAQuantitativechainreaction qRTPCR and Western blot were used to testthe diï¬erentexpression genes after knocking down oroverexpressing lncRNAstranscriptasepolymeraseWe obtained target genes of lncRNA from LncRNA2TargetInput train dataset ˆvn learning rate λJiang As we can see in Figure there are kinds of lncRNAsOne lncRNA has more than target genes Then we drawthe distribution of the number of target genes correspondingto lncRNAAsshown in Figure most ofthe target genes arecorresponding to less than five lncRNAs Therefore if we usedthem to be the features of lncRNAs the features would be sparseTherefore we only select the most common target genes to bethe features The genes which are corresponding to more thanfive lncRNAs were selected as the features of lncRNAs There are kinds of genes Then we need to encode these genesF [G1 G2 · · · G45]where G1 denotes the first gene of these genes and F denotesthe feature of lncRNA For each lncRNA if G1 is the target geneof it then G1 otherwise G1 Output weight matrix Wl bias al and blFor l 1LInitialization Wi al bi Sample from train dataset ˆh0For i lˆ’Sample hi based on phi ˆhiˆ’EndSet hi1as the train sample to train lth layer ofRBMEndSince the dimension of expression feature and target genefeature are diï¬erent we should reduce the dimension of targetgene feature and make it the same as the expression feature™sTherefore in this paper two layers of RBM were used to builda DBN modelThe number of nodes oftheand respectively Sigmoid function was used astwo layers was theFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cLiu et alactivation functionσ x eˆ’xTherefore the dimension of final features is ˆ— F cid20 G1 G2 · · · G13E1 E2 · · · E13 cid21A Method to Identify CancerRelated lncRNAsConvolutional Neural NetworkThe power of CNN in dealing with bioinformatic problems hasbeen proven by many researchers We selected CNN as theclassifier based on two reasons The dimension of features is ˆ— which can be regarded as an image The outstandingperformance of CNN in image classificationThere are five layers in our CNN model The structure of CNNis shown as Table where G1 G2 · · · G13 denotes target gene feature after DBNand E1 E2 · · · E13 denotes the expression of lncRNAs in diï¬erent tissuesTABLE The structure of convolutional neural network CNNLayersParameterConvolutional layerPooling layerConvolutional layerPooling layerFully connected layerOutputFilter kernel size Activation function tanhpool size Activation function tanhFilter kernel size Activation function tanhpool size Activation function tanhUnits Activation function tanhUnits Activation function sigmoidWork FrameFigure shows the work frame of our method œDBN“CNNThere are three steps of our methods Firstly we should extractfeatures of lncRNAs There are two parts of features expressionfeature and target gene feature Then DBN was used to encodethe target gene feature After encoding the two kinds of featureswere combined together Finally CNN was used to classifyRESULTSData DescriptionThe known associations between lncRNA and diseases wereobtained from LncRNADisease database Bao Wetotally obtained kinds of cancerrelated lncRNAs The numberof their corresponding lncRNAs is shown as Figure As shown in Figure People™s understanding of cancerrelated lncRNAs varies widely We have known more than lncRNAs for some cancers but few lncRNAs are known for somecancers To better build our model we only selected cancerswhich have more than related lncRNAs Therefore kindsof cancers were selectedFIGURE Work frame of deep belief network DBN“convolutional neural network CNN lncRNA long noncoding RNAFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cLiu et alA Method to Identify CancerRelated lncRNAsFIGURE The number of long noncoding RNAs lncRNAs for each cancerTABLE The performance of deep belief network DBN“convolutional neuralnetwork CNN in cancersCancerArea undercurve AUCArea under precisioncurve AUPRCervical cancerBreast cancerColorectal cancerStomach cancerUrinary bladder cancerLung cancerOvarian cancerThyroid cancerProstate cancerLiver cancerPancreatic cancerOvarian epithelial cancerGallbladder cancerEndometrial cancerColon cancerEsophageal cancerThetargetgenes oflncRNAs were obtained fromLncRNA2Target database We have discussed about this insection Target Gene of Long Noncoding RNAFIGURE The receiver operating characteristic ROC curves of the threemethods DBN deep belief network CNN convolutional neural network PCAprincipal component analysisFIGURE The area under the precision“recall curve AUPR of the threemethods DBN deep belief network CNN convolutional neural network PCAprincipal component analysisThe expression oftissues wasobtained from NONCODEV5 Zhao We only usedhuman datalncRNAs in diï¬erentThe Performance of Deep BeliefNetwork“Convolutional Neural NetworkWe did 10cross validation on each cancer Area under the curveAUC Cheng Dao Zhang and areaunder the precision“recall curve AUPR were used to evaluatethe performance of DBN“CNN The results are shown in Table As we can see in Table the performance of DBN“CNN isquite diï¬erent in diï¬erent cancers This may be caused by thediï¬erent sample sizes The average AUC is and AUPR is Comparison ExperimentsTo verify the superior of DBN“CNN we compared it with similarmethods Since the main function of DBN is to reduce dimensionprincipal component analysis PCA has the same functionTherefore instead of using DBN to encode we used PCA thistime and CNN was used to classify the features after PCA We callthis method PCA“CNN In addition we also used the deep neuralnetwork DNN to replace CNN so this comparison method wascalled DBN“DNNWe used these three methods to test on cancers andsummarized the results to get a final AUC and AUPR for eachmethod The receiver operating characteristic ROC curves areshown in Figure As shown in Figure the blue curve denotes the results ofDBN“CNN The red and black curves denote PCA“CNN andDBN“DNN respectively As we can see DBN“CNN performedbest among these three methods The AUC of DBN“CNN is which is better than and for PCA“CNN andDBN“DNN respectivelyFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cLiu et alA Method to Identify CancerRelated lncRNAsAs shown in Figure the AUPR of DBN“CNN is the highestwith the least standard errorCase StudyLiu found down syndrome cell adhesion molecule antisense RNA DSCAMAS1 is associated with breast cancerby constructing two suppression subtracted cDNA librariesMartensUzunova reported the associationbetween H19 and bladder cancer They also pointed out that H19could be the biomarker of bladder cancerShi measured the expression level of lncRNAsLoc554202 in breast cancer tissues and found that Loc554202was significantly increased compared with normal control andassociated with advanced pathologic stage and tumor sizeCONCLUSIONSIncreasing evidence has shown the relationship between lncRNAsand cancers lncRNAs could be the biomarkers to help diagnosecancer and also help researchers understand the mechanismof cancers Compared with people™s knowledge of diseaserelated protein coding genes we knew few about diseaserelated lncRNAs However the biological experiments for findingdiseaserelated lncRNAs are timeconsuming and expensiveTherefore in this paper we proposed a novel method foridentifying cancerrelated lncRNAs We called this methodœDBN“CNN which is a fusion of DBN and CNN Two kindsof features were used based on the biological background SincelncRNAs have tissuespecific expression and the expression ofcancer tissues is diï¬erent from normal tissues the expressionoftissues could provide importantin diï¬erentlncRNAsREFERENCESAvgeris M Tsilimantou A Levis P K Tokas T Sideris D C StravodimosK Loss of GAS5 tumour suppressor lncRNA an independentmolecular cancer biomarker for shortterm relapse and progression in bladdercancer patients Br J Cancer “ 101038s4141601803206Bai Y Dai X Ye T Zhang P Yan X Gong X PlncRNADBa repository of plant lncRNAs and lncRNARBP protein interactions CurrBioinform “ Bao Z Yang Z Huang Z Zhou Y Cui Q and Dong D LncRNADisease an updated database of long noncoding RNAassociateddiseases Nucleic Acids Res D1034“D1037 101093nargky905Canzio D Nwakeze C L Horta A Rajkumar S M Coï¬ey E L Duï¬y EE Antisense lncRNA transcription mediates DNA demethylationto drive stochastic protocadherin α promoter choice Cell “653e15 101016jcell201903008Chen X Shi W and Deng L Prediction of disease comorbidity usinghetesim 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and Developmental Biology wwwfrontiersinAugust Volume 0cLiu et alA Method to Identify CancerRelated lncRNAsKuang L Zhao H Wang L Xuan Z and Pei T A novel approachbased on point cut set to predict associations of diseases and LncRNAs CurrBioinform “ Lan W Li M Zhao K Liu J Wu FX Pan Y LDAP a webserver for lncRNAdisease association prediction Bioinformatics “ 101093bioinformaticsbtw639Liang C Changlu Q He Z Tongze F and Xue Z gutMDisorder acomprehensive database for dysbiosis of the gut microbiota in disorders andinterventions Nucleic Acids Res Liu D Rudland P Sibson D and Barraclough R Identification ofmRNAs diï¬erentiallyexpressed between benign and malignant breast tumourcells Br J Cancer “ 101038sjbjc6600456Liu X Hong Z Liu J Lin Y Alfonso RP Zou Q Computational methods for identifying the critical nodes in biologicalnetworks Brief Bioinform “ 101093bibbbz011Lu C Yang M Li M Li Y Wu F and Wang J Predicting humanlncRNAdisease associations based on geometric matrix completion IEEE 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cancer cellsBiochem Biophys Res Commun “ 101016jbbrc201402144Network diï¬usion approach to predictlncRNA disease associationsusing multitype biological networks LION Front Physiol 103389fphys201900888Wang L Xuan Z Zhou S Kuang L and Pei T A novel modelassociations based on the LncRNA“for predicting LncRNAdiseaseMiRNAdisease interactive network Curr BioinformWang Y Yu G Wang J Fu G Guo M and Domeniconi C Weightedmatrix factorization on multirelational data for LncRNAdisease associationprediction Methods “ 101016jymeth201906015Wei L Su R Wang B Li X Zou Q and Gao X Integrationof deep feature representations and handcrafted featuresto improvethe prediction of N 6methyladenosine sites Neurocomputing “ 101016jneucom201804082Wu B Zhang H Lin L Wang H Gao Y Zhao L A similarity searching system for biological phenotype images using deepconvolutional encoderdecoder architecture Curr Bioinform “ Xie G Huang S Luo Y Ma L Lin Z and Sun Y LLCLPLDA a novelmodel for predicting lncRNA“disease associations Mol Genet Genomics “ 101007s00438019015908Xuan P Cao Y Zhang T Kong R and Zhang Z2019a Dualconvolutional neural networks with attention mechanisms based methodfor predicting diseaserelated lncRNA genes Front Genet 103389fgene201900416Xuan P Pan S Zhang T Liu Y and Sun H 2019b Graph convolutionalnetwork and convolutional neural network based method for predictinglncRNAdisease associations Cells 103390cells8091012Yu G Fu G Lu C Ren Y and Wang J BRWLDA birandomwalks for predicting lncRNAdisease associations Oncotarget “ 1018632oncotarget19588Yu J Xuan Z Feng X Zou Q and Wang L A novel collaborativefiltering model for LncRNAdisease association prediction based on the NaïveBayesian classifier BMC Bioinform 101186s1285901929850Zeng X X Wang W Deng G S Bing J X and Zou Q Prediction ofpotential diseaseassociated microRNAs by using neural networks Mol TherNucleic Acids “ 101016jomtn201904010Zhangand Deng LJ Zhang Z Chen ZIntegratinglncRNAdisease associationIEEEACM Transac Comput Biol Bioinform “multiple heterogeneous networks for novelinference 101109TCBB20172701379Zhang T Tan P Wang L Jin N Li Y Zhang L RNALocate aresource for RNA subcellular localizations Nucleic Acids Res D135“D138 101093nargkw728Zhang Z M Tan J X Wang F Dao F Y Zhang Z Y and LinH Early diagnosis of hepatocellular carcinoma using machinelearning method Front Bioeng Biotechnol 103389fbioe2020Zhao T Cheng L Zang T and Hu Y 2019a Peptidemajor histocompatibilitycomplex class I binding prediction based on deep learning with novel featureFront Genet 103389fgene201901191and Cheng LIdentifyingAlzheimer™s diseaserelated proteins by LRRGD BMC Bioinform 101186s1285901931247Zhao T Hu Y Zang T2019bZhao T Hu Y Zang T and Cheng L MRTFB regulates the expressionof NOMO1 in colon Proc Natl Acad Sci USA 101073pnas2000499117Zhao T Hu Y Zang T and Wang Y 2019c Integrate GWAS eQTLand mQTL Data to Identify Alzheimer™s diseaserelated genes Front Genet 103389fgene201901021Zhao T Wang D Hu Y Zhang N Zang T and Wang Y 2019d IdentifyingAlzheimer™s diseaserelated miRNA based on semiclustering Curr Gene Ther “ Zhao Y Li H Fang S Kang Y Wu W Hao Y NONCODE an informative and valuable data source of long noncoding RNAs NucleicAcids Res D203“D208 101093nargkv1252Zou Q Xing P Wei L and Liu B Gene2vec gene subsequenceembedding for prediction of mammalian N6methyladenosine sites frommRNA RNA “ 101261rna069112118Conflict of Interest The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestCopyright Liu Zhang Han Li Yang Gao Xie and Du This is an openaccess distributed under the terms of the Creative Commons Attribution License CCBY The use distribution or reproduction in other forums is permitted providedthe original authors and the copyright owners are credited and that the originalpublication in this journal is cited in accordance with accepted academic practiceNo use distribution or reproduction is permitted which does not comply with thesetermsFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0c'
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ShortTerm Consequences ofPediatric Anticancer TreatmentRegarding Blood Pressure MotorPerformance Physical Activity andReintegration Into Sports StructuresTina Keiser Dominik Gaser Christiane Peters Renate OberhofferFritz Sabine Kesting   and Irene von Luettichau  Edited byKirsten K NessSt Jude Children™s Research Hospital Department of Sports Medicine and Exercise JustusLiebig University GieŸen GieŸen Germany Department of Sport andHealth Sciences Institute of Preventive Pediatrics Technical University of Munich Munich Germany Department ofPediatrics and Children™s Cancer Research Center Kinderklinik M¼nchen Schwabing TUM School of Medicine TechnicalUnited StatesReviewed bySeth E KarolSt Jude Children™s Research HospitalUnited StatesJacques GrillInstitut Gustave Roussy FranceCorrespondenceSabine KestingsabinekestingtumdeIrene von LuettichauIreneTeichertvonLuettichaumritumde These authors sharesenior authorshipSpecialty sectionThis was submitted toPediatric Oncologya section of the journalFrontiers in PediatricsReceived January Accepted July Published August CitationKeiser T Gaser D Peters COberhofferFritz R Kesting S and vonLuettichau I ShortTermConsequences of PediatricAnticancer Treatment RegardingBlood Pressure Motor PerformancePhysical Activity and ReintegrationInto Sports StructuresFront Pediatr 103389fped202000463University of Munich Munich GermanyBackground Cardiovascular diseases in childhood cancer survivors are knownlate sequelae following treatment Arterial stiffness pulse wave velocity PWV andcentral systolic blood pressure cSBP are potential predictors to assess the statusof cardiovascular health Frequent inpatient stays and reduced physical activity PAduring treatment can lead to noticeable impairments regarding motor skills and physicalperformance The present study examined parameters of cardiovascular health motorperformance and the status of integration into sports structures shortly after cessationof treatmentMethods A crosssectional monocentric study was conducted from April to June Participants “ yrs mixed cancer entities during maintenance therapy andfollowup care were recruited Peripheral and central systolicdiastolic blood pressurepSBP pDBP cSBP and PWV were assessed using the MobilOGraph® The MOONtest MOtor performance in pediatric ONcology was used to scale motor performancePA levels and status ofintegration into sports structures were assessed with aquestionnaire referring to the KiGGS study All measured data were compared topublished reference valuesResults Forty participants ± years female were recruited ± years posttreatment PSBP zscore ± p pDBP ± p and cSBP ‰¥ years ± p were significantly increasedcompared to reference values PWV was also elevated but not significantly Motorperformance was reduced in almost all motor abilities Thirtysix percent of the examinedgroup did not participate in physical education at school to the full extent Only reported hour of daily moderatetovigorous PA as recommended for children andadolescents by the World Health anization Half of the participants were active sportsclub members before treatment but one third did not resume their former membershipFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerConclusionIncreased cardiovascular parameters and impaired motor performanceshortly after cessation of treatment physical inactivity and low rates of integration intoregular sports programs highlight the support needed Young cancer patients shouldreceive early support in coping with physical limitations preferably soon after diagnosisMotor deficits could be reduced by applying targeted interventions Furthermorea regular sports therapy program during in and outpatient care could increaseengagement in PA to possibly counteract risk factors and improve cardiovascular healthKeywords childhood cancer cardiovascular health motor performance physical activity sports reintegrationblood pressure arterial stiffnessINTRODUCTIONExtensive research and optimized treatment regimens resulted inan increase of the 5year survival rate to in the USA and of the 15year survival rate to for patients under theage of in Germany However childhood cancer is a raredisease It contributes only around to all malignant diseasesin developed countries Worldwide children underthe age of and adolescents aged between and are diagnosed with cancer every year As a consequence ofthe success in the treatment of childhood cancer the importanceof survival quality and prevention of late sequelae have receivedmore attention during the last yearsKnown negativelongterm consequences ofintensivetreatment for childhood and adolescent cancer patients ofteninclude adverse eï¬ects on the cardiovascular system Cardiovascular diseases are the most frequently reported causesof death in childhood cancer survivors following secondarytumors Arterial stiï¬ness pulse wave velocity PWV andcentral systolic blood pressure cSBP are potential predictorsfor cardiovascular diseases frequently investigated in medicalresearch to evaluate the status of a patient™s cardiovascularhealth PWV describes the velocity of the pressure wave in the aortawhich spreads from the left ventricle through the arterial vascularsystem during systole Noninvasive investigation of the PWVvia ultrasound or oscillometric methods provides informationon the elasticity ofthe vascular system and enables earlyrecognition of damages in the vessels Thus in order to detectpotential structural modifications in the vascular system andindicators for arterial stiï¬ness at an early stage this subclinicalparameter should be surveyed continually Previous data indicatea positive correlation of PWV with arterial vascular stiï¬ness Moreover elevated PWV reflecting subclinical vasculardamage was shown in pediatric patients after hematopoieticstem cell transplantation On the contrary another studyinvestigated elevated blood pressure levels but no statisticallyAbbreviations PWV Pulse Wave Velocity WHO World Health anizationcSBP central systolic blood pressure pSBP peripheral systolic blood pressurepDBP peripheral diastolic blood pressure BMI Body mass index MOONMotor performance in pediatric oncology KiGGS German Health Interviews andExamination Survey for Children and Adolescentssignificant variation for PWV in pediatric cancer survivorscompared to healthy children and adolescents In addition to the abovementioned late sequelae severalproblems already arise during treatment and often persistthroughout survivorship For instancelongterminpatient stays and reduced physical activity during treatmentcan lead to noticeably reduced physical performance ofsurvivors and reintegration into sportschildhood cancerstructures mightberehabilitationprocess throughoutfrequentaï¬ectedIn healthy populations reduced physical activity leadsto negative consequencesfor cardiovascular health Additionally the necessary use of anthracyclines in almost ofapplied therapy regimens in childhood cancer increases the riskof cardiovascular morbidity and mortality eightfold comparedto agematched patients not receiving anthracyclines indicatingthe importance of reducing such longterm consequences Due to a poor state of health and impaired immune functionsports options such as physical education at school engagementin sports clubs or recreational sports are no longer feasibleduring therapy Consequently reintegration after cessation oftreatment is associated with even more barriers due to diseaseand treatmentrelated impairments Circumstances ofanticancer treatment can lead to inactivity resulting in deficitsof fine and gross motor skills reduced muscle strength andpoor physical fitness following treatment Especiallymotor performance in pediatric bone tumor patients oftenremains reduced until at least years after cessation of treatment Impairments of physical performance have been shown topersist throughout survivorship which may complicate thesurvivors™ reintegration into both social and sports structures aswell as the development of a longterm active lifestyle According to a questionnairebased study childhood cancersurvivors™ reintegration rate into physical education at school isvery low especially after treatment for bone tumors The lackof comprehensive oï¬ers of physical activity promotion and motordevelopment might exacerbate motor impairments and problemsof reintegration into sports structures Von Korn et al examined motor performance usingthe Fitnessgram Rcid13 as well as peripheral blood pressure centralblood pressure and PWV using the MobilOGraph Rcid13 in childrenafter treatment for childhood cancer n aged ± years ± years postdiagnosis Their results show reducedFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood Cancermotor performance of childhood cancer survivors compared toreference values of healthy children However no correlationcould be drawn regarding cardiovascular parameters and motorperformance The present crosssectional study aimed atinvestigatingvarious parameters of cardiovascular health motor performanceand status of physical activity in children and adolescentsshortly after cessation of anticancer treatment or during ongoingoral maintenance therapy The collection of such data isof considerable importance for the early detection of healthimplications related to both disease and treatment Moreoverthe findings will help to support the development of preventivestrategies regarding the health of children and adolescents treatedfor cancer Appropriate strategies during primary and secondaryprevention and following cancer treatment tertiary preventionneed improvementsMATERIALS AND METHODSDesignThe crosssectional monocentric study was performed over aperiod of months April“June at our institution Theassessment of cardiovascular parameters using the MobilOGraph Rcid13 was followed by the MOON test MOtor performance inpediatric ONcology to evaluate motor performance Finally theparticipants completed a standardized questionnaire referring tothe KiGGS study German Health Interview and ExaminationSurvey for Children and Adolescents to collect data regardingtheir current level of physical activity and status of integrationinto sports structures The Ethics Committee of the School ofMedicine of the Technical University of Munich approved thestudy project number SSR Participation was voluntaryand informed written consent was signed by each participant aswell as by his or her legal guardian All data was collected encodedpseudonym and in accordance with privacy policy standardsParticipantsPrior to addressing the participants all eligible children andadolescents were screened using the electronic patient recordSAP Rcid13 ERP Patients were recruited during routine followupvisits The following inclusion criteria were applied childrenand adolescents during maintenance therapy and followupcare of a pediatric oncological disease and currently agedbetween and years No restriction was applied regardingthe period posttreatment Exclusion criteria were medicalcontraindications such as fever acute infection orthopedicrestrictions and mental retardation insufficient knowledgeof the German language and absence of written informedconsent The attending physician confirmed participation forall recruited children and adolescents Following these inclusioncriteria children and adolescents were initially found eligibleFigure Outcome MeasuresAnamnestic and Anthropometric DataAnamnestic and clinical data ie type of cancer treatmentregime end of therapy was obtained from the electronic patientrecord SAP Rcid13 ERP The nursing staï¬ assessed anthropometricdata height and weight during routine medical examinationseca electronic column scaleseca mechanicalmeasuring rod Body mass index BMI was calculated as aratio of body weight kg per square body height m2 By usingthe reference values of a healthy German cohort BMI wasconverted into percentiles and classified in underweight 10thpercentile normal weight 10thˆ’90th percentile and overweight90th percentile Cardiovascular ParametersPrior to the measurement the participants had to rest for atleast min in a supine position PWV central blood pressureand peripheral blood pressure were assessed using the MobilOGraph Rcid13 IEM GmbH Stolberg Germany and HMS ClientServer Version an oscillometric noninvasive methodMeasurements were performed on the left upper arm The cuï¬was inflated twice with a rest of s in between Cuï¬ sizewas chosen according to the circumference of the participant™sleft upper arm An ARCSolver Algorithm calculated the cSBPindirectly as well as the PWV from recorded brachial pulseRaw data was transformed into zscores and compared by usingzscores of a healthy reference cohort PSBP and pDBPwere compared to references from the national cohort of children and adolescents KiGGS study For the assessmentof PWV and cSBP values references from Elmenhorst et al of healthy children and young adults were used To evaluatethe results of the parameters cSBP and PWV the examinedparticipants were separated into two groups years and‰¥ years According to the age distribution of the referencevalues participants years were compared to heightmatchedreference values and participants ‰¥ years were compared toagematched referencesThe measurement using the MobilOGraph Rcid13 was previouslyused several times in pediatric patients “ and was alsosuccessfully applied in childhood cancer survivors Motor PerformanceTo quantify motor performancethe MOON test MOtorperformance in pediatric ONcology was applied Thetest assesses motor abilities coordination speed flexibility andstrength and consists of eight test items eyehand coordinationinserting pins static balance static stand upper extremitycoordination throwing at a targetspeed reaction testmuscular endurance sittostand flexibility stand and reachhand grip strength handheld dynamometry and muscularexplosive strength medicine ball shot The test lasts min onaverage Data of each item was compared to published age andsexmatched reference values of a healthy population within anage range of to years Data of participants older than years was compared to reference values of healthy 17yearoldssince reference values of healthy 18yearolds are not availableCalculation of a total score is not possible within this toolInstead each item was analyzed individually and the percentagedeviation to reference values was computedFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerFIGURE Flow chart of recruitment Out of participants eligible could not be addressed due to several medical examinations in different departments of thehospital and resulting in missing time slots Longer periods posttreatment are associated with fewer appointments for followups and more medical examinationstake place in day This does not necessarily mean that the children who could not be included in the study due to missing time slots are medically more complexTen participants refused participation Thus the sample included participantsPhysical Activity and Reintegration Into SportsStructures After Acute TreatmentPhysical activity levels and status of integration into sportsstructures were assessed with a standardized selfreportingreferring to the KiGGS study Thequestionnairequestionnaire wassupplemented by several disease andtreatmentrelated aspects in accordance with the study ofKesting et al to investigate potential barriers regardingreintegration and participation in sports activities eg barrierswith respect to exemption from physical education at school ornonparticipation in sports clubs sports therapy oï¬ers duringtreatment The KiGGS study oï¬ers the reference values ofhealthy children and adolescents n for comparison ofour dataData AnalysisCardiovascular parameters were analyzed and compared to thehealthy reference population with the one sample ttest Motorperformance was analyzed using the Wilcoxon signedranktest in comparison to age and sexmatched reference valuesPearson correlation was applied to calculate possible associationsbetween motor performance and cardiovascular parametersBMI and the period posttreatment The MannWhitneyUtestwas performed to evaluate anthracyclinemediated eï¬ects oncardiovascular health as well as diï¬erences within subgroupsregarding diï¬erent entities and levels of physical activity motorperformance physical education at school and achievement ofphysical activity recommendationsExplorative twosided statistical tests were conducted andp ‰¤ was considered statistically significant No adjustmentfor multiple comparison was conducted Correlations coefficientρ were classified according to Cohen Descriptive statistics were calculated with Microsoft Excelversion for demographic characteristics and medicaldata GraphPad Prism version was used to perform allfurther statistical analyses Data analysis was performed inconsultation with the Institute of Medical Informatics Statisticsand Epidemiology of the Technical University of MunichRESULTSParticipantsOut of eligible children and adolescents who met the inclusioncriteria a total of participants female with variouscancer entities were recruited and examined Table Performed AssessmentsFor various reasons not alltests could be realized withall participants In participants cardiovascularparameters could not be evaluated due to missing time slotsFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerTABLE Anthropometric and medical characteristics of the participants n CharacteristicsN Mean ± SD MedianRangeAge at diagnosis years ± Age at assessment years ± ““ years‰¥ years Period postdiagnosis years ± Period posttreatment years ± ““ year“ years yearsLeukemiaLymphomaBone tumorBrain tumorOther solid tumors Body mass index¢ kgm2 ± “UnderweightNormal weightOverweightChemotherapyAnthracycline applicationCumulative dose mgm2RadiotherapyChestdirected radiation Anthracycline chest radiation Surgical tumor resectionRelapse ± “FIGURE Cardiovascular parameters shown in zscores and compared topublished reference values pSBP peripheral systolic blood pressurepDBP peripheral diastolic blood pressure PWV pulse wave velocity cSBPcentral systolic blood pressure Significant values p ‰¤ Results are given in Mean ± SD M median RangeOther solid tumors alveolar rhabdomyosarcoma n carcinoid tumor of the appendixn nephroblastoma n focal nodular hyperplasia liver n mature cysticteratoma ovary n thoracic ganglioneuroma n papillary thyroid carcinoman neuroblastoma n ¢BMI was converted into percentiles and classified in underweight 10th percentilenormal weight 10thˆ’90th percentile and overweight 90th percentile Thirteen participants received a combination of anthracyclines mainly the combination ofDoxorubicin and Daunorubicinduring routine appointment and a lack of willingness to prolongthe outpatient visit Six of the participants could not performthe MOON test due to medical limitations current orthopedicrestrictions n examinationrelated limitations ie a draintube in the crook of the arm for followup MRT n andabandonment due to lack of time n The central venousdevice has already been explanted in all participants prior toour studyOf the remaining participants not everyone performedevery test item Two participants could not perform the testitem speed due to a drain tube in the crook of the armFour participants could not accomplish the test item muscularexplosive strength due to orthopedic restrictions as well asexaminationrelated limitations Three participants did notperform the item muscular endurance of the legs n hadcrutches n severe muscular deficit in the legs n lackof time Two participants could not perform the test item handgrip strength with both hands due to lack of time n andinfusion needle in the crook of the arm n The test itemupper extremity coordination was measured in n participantsbecause reference values are provided for children between and years onlyCardiovascular HealthIn participants all parameters were assessed Based on theunderlying reference values for cSBP and PWV of Elmenhorstet al the group was separated into participants aged years heightmatched reference values and ‰¥ years agematched reference values PSBP zscore ± p pDBP zscore ± p as well as cSBP values‰¥ years zscore ± p were significantlyincreased compared to reference values of healthy children andadolescents Figure PWV was elevated but not significantly years zscore ± p ‰¥ years zscore ± p Comparison of cardiovascular parameters of participantswho received anthracyclines during intense therapy with acumulative dose of ± mgm2 and subjects whodid not receive cardiotoxic agents did not show statisticallysignificant diï¬erencesMotor PerformanceThe participants™ n motor performance wasreduced in almost all motor abilities compared to the referencevalues of healthy children and adolescents Table Significantimpairments became obvious in the following dimensionsmuscular explosive strength p upper extremitycoordination p muscular endurance of the legs p Frontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerTABLE Results of the MOONtest compared to reference values n Motor abilityTest itemEyehand coordinationStatic balanceφSpeedInserting pins timeStatic stand contactsReaction test timeUpper extremity coordinationθFlexibilityThrowing at a target pointsStand and reach cmMuscular explosive strengthMedicine ball shot meterMuscular endurance legsSittostand secHand grip strengthHandheld dynamometry kgRightLeftNMean ± SD of differenceMedian pvalueto reference values ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’All results were compared to the reference values of each single test item Speed could only be measured in participants muscular explosive strength in participants andhand grip strength in right and left participants due to lack of time orthopedic restrictions or drain tube in the crook of the armFor the test items static balance and flexibility the absolute differences were used as the measured values would have fluctuated around zero and would have givenoversized percentagesφ Static balance was assessed counting the contacts with a foot to the ground while balancing on a rail in this context a negative difference to reference values represents fewercontacts and therefore better resultsθ Although all participants completed this test item reference values are provided for children between and years onlyM median abs indicates absoluteBold numbers indicate significant values p ‰¤ TABLE Results of the MOONtest comparing participants treated for leukemialymphoma and participants treated for brain tumors compared to reference valuesLeukemiaLymphoma n Brain tumor n Motor abilityEyehand coordinationStatic balanceφSpeedFlexibilityMuscular explosive strengthMuscular endurance legsHand grip strength rightHand grip strength leftNMean ± SD Median ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’NMean ± SD Median pvalueˆ’ ± ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ All results were compared to the reference values of each single test item φ Static balance was assessed counting the contacts with a foot to the ground while balancing on a rail in this context a negative difference to reference values represents fewercontacts and therefore better resultsFor the test items static balance and flexibility the absolute differences were used as the measured values would have fluctuated around zero and would have givenoversized percentagesDue to insufficient number of participants the comparison regarding the test item upper extremity coordination was disregarded reference values only provided for children aged and yearsM median abs indicates absoluteBold numbers indicate significant values p ‰¤ and hand grip strength on the right hand p The performance of eyehand coordination speed flexibility andhand grip strength on the left hand were also reduced but notsignificantly In the test item static stand the study participantsperformed slightly better compared to the reference populationFor upper extremity coordination throwing at a target onlyreference values from children aged “ years are availableTherefore comparison of the collected data was only possiblewith the same age group n Data for olderparticipants was not collectedComparing motor performance of participants diagnosedwith leukemialymphoma n and participantsn diagnosed with brain tumorsrevealedsome diï¬erences Table Children and adolescents treatedfor brain tumors performed significantly worse in eyehandcoordination than participants treated for leukemialymphomap Moreover the performances in all other testedmotoric dimensions of participants diagnosed with braintumor were deteriorated compared to participants treated forleukemialymphomas but not significantlyTo determine influencing factors on motor performance thecorrelation between motor performance results and BMI as wellas the period posttreatment was performed With increasingBMI values of static balance deteriorated significantly ρ Frontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerFIGURE Pearson correlation between static balance and BMI kgm2¢ leukemialymphoma —¦ bone tumor –³ brain tumor cid7 other solid tumorsHorizontal line indicates the reference values The absolute difference fromreference values is given number of contacts to the ground A negativedifference means fewer ground contacts and therefore better performanceThirty four participants performed the testp which corresponds to a moderate to high correlationFigure A negative diï¬erence to reference values means fewercontacts to the ground and therefore better performance in staticbalance Furthermore some nonsignificant correlations werefound Deteriorated eyehand coordination ρ ˆ’ p and flexibility ρ ˆ’ p were also associatedwith a higher BMI However superior values in upper extremitycoordination ρ p muscular explosive strengthρ p hand grip strength right ρ p left ρ p and muscle endurance ofthe legs ρ p were associated with increasedBMI The test item speed showed no association with BMIρ p A longer period posttreatment was significantly associatedwith decreased eyehand coordination ρ ˆ’ p corresponding to a moderate correlation Figure Especially participants treated for a brain tumor with a longerperiod posttreatment showed deteriorated values in eyehandcoordination Speed performance was deteriorated in participantswith longer posttreatment period ρ ˆ’ p Physical Activity and Reintegration IntoSports StructuresAccording to the selfreported questionnaire n did not participate in physical education at school to full extend n were not admitted to school sports activitiesand n were partly excluded Table Neitherof the two participants treated for bone tumor was takingpart in physical education at school n whereaschildren with other tumors participated to a notably higherrate Treatmentrelated muscular deficits n andosteonecrosis n were the most common reasons forparticipants not taking part in physical education at schoolFIGURE Pearson correlation between eyehand coordination and periodposttreatment months ¢ leukemialymphoma —¦ bone tumor –³ brain tumorcid7 other solid tumors Horizontal line indicates the reference values Thirtyfour participants performed the test itemTABLE Participation in physical education at school subdivided into entitiesn Participation Partial exemption Full exemptionN N N Entire group n LeukemiaLymphoma n Bone tumor n Brain tumor n Other solid tumors n Abs N number Four out of children were still attending kindergarten and could notanswer the question regarding participation in physical education at schoolOnly n reported moderatetovigorous physicalactivity for min daily as generally recommended by the WHOfor healthy children and adolescents Table This percentageis comparable to the achievements in the healthy referencepopulation n Every second participant questioned was an active memberin a sports club whereas n did not return to asports club following cancer treatment Almost onethird n has never been a sports club member Reasons fornot engaging in sports club activities of participants wereno interestfun n physical weakness n no time n anxiety n andphysicianbased prohibition due to clear medical reasons n Nearly all participants n were active inrecreational sportsFurther analyses pointed toward diï¬erences in physicalactivity and sports club participation especially betweenparticipants with brain tumors and leukemialymphomas Thenumber of participants in recreational sports was reported highin both groups leukemialymphoma and brain tumor In contrast a diï¬erence was found in sports club activitySixtysix percent of leukemialymphoma patients were membersFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerTABLE Physical activity and engagement in sports club and recreationalsportsEntire groupReference populationn n Physical activity guidelinesWHO mindayDaily physical activityDaily walking distance kmDaily walking distance “ kmDaily walking distance kmSports club activity currentlyFormer membershipRecreational sports activityAbs N number Reference values derived from the national cohort of healthy childrenand adolescents in the KiGGS study German Health Interviews and Examination Surveyfor Children and Adolescents of a sports club whereas only of participants with a braintumor were active in a sports club On the other hand almosthalf of the children treated for brain tumor and of thechildren treated for leukemialymphoma were former membersConcerning possible correlations between motor abilities andphysical education at school participation in sports clubs orrecreational sports defined as activeinactive as well as meetingthe physical activity recommendations no significant associationscould be determined Likewise the comparison of motor abilitiesof participants receiving sports therapy during treatment didnot show any correlation Almost half of the participants n took part in a sports therapy programme duringtreatment which was mainly oï¬ered as care and varied greatlyin terms of training interventions without any standardizationDISCUSSIONtreatmentThe results of our study clearly present evidence for deterioratedcardiovascular function in children and adolescents shortlyafter cessation of cancerIncreased pSBP andincreased pDBP are risk factors for cardiovascular diseasesregarding guidelines for arterial hypertension Potentialcardiovascular consequences such as stroke sudden deathheart failure and peripheral artery disease due to elevatedblood pressure values are described in the aforementionedguidelines as well as in the literature Accordinglychildhood cancer survivors with elevated blood pressure are atrisk to experience such cardiovascular late eï¬ects Regarding10years survivors of childhood cancer a higher prevalenceof hypertension is assumed and cardiovascular diseaserelated deaths are eighttimes more likely in childhoodcancer survivors compared to the general population Our findings support previous study results which depictcomplications such as increased blood pressure prehypertensionand hypertension in children and adolescents treated for cancer This study aimed at investigating specific parameters thatcould serve as early predictors for potential damage to thecardiovascular system Recent evidence of cSBP as a suitableparameter to determine the elasticity of blood vessels suggeststhat cSBP is more closely related to cardiovascular events in thefuture than brachial blood pressure Increased cSBP inparticipants in our study may result from early changes in arterialwall stiï¬ness As a further parameter to detect early impairmentsin elasticity of the vascular system PWV was investigated Incontrast to prior studies no decisive change was observedin PWV While anthracycl
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"and vimentin was positive in p120ctn cytoplasmic-positive lung cancer cells. .0088064.t001 Correlation between E-cadherin vimentin and lymph node metastasis and p120ctn. p120ctn N membrane cytolymph/nucleolus X2 p E-cadherin negative 56 9 47 30.166 <0.01 positive 22 18 4 Vimentin negative 53 23 30 5.633 0.022 positive 25 4 21 Lymph node metastasis No 41 19 22 5.251 0.032 Yes 37 8 29 Localization of p120ctn is consistent with E-cadherin in lung cancer cells We examined the protein expression levels of p120ctn and E-cadherin in normal HBE cells and nine lung cancer cell lines by Western blot and found that they all expressed mainly isoforms 1A (120 kDa) and 3A (100 kDa) of p120ctn (A). Although the protein expression levels of p120ctn were not related to E-cadherin the localization (membrane or cytoplasm) of p120ctn was always consistent with that of E-cadherin. We then screened cells expressing high levels of p120ctn and E-cadherin in the membrane (H460 cells) or cytoplasm (SPC cells) as well as those expressing low levels of p120ctn and E-cadherin in the membrane (H4299 cells) or cytoplasm (LK2 cells) for further study (B). .0088064.g002 Expression and localization of p120ctn and E-cadherin in H460 SPC H1299 and LK2 cells. (A) Western blot analyses showed expression of p120ctn and E-cadherin in nine lung cancer cell lines and HBE. (B) By immunofluorescence analysis the expression of E-cadherin and p120ctn were observed restricted to the cell membrane at cell-cell adherens junctions in H460 and H1299 cells whereas they both were confined to the cytoplasm in SPC and LK2 cells. Different functions of p120ctn isoform 1A in EMT are dependent on E-cadherin subcellular localization Knockdown of endogenous p120ctn isoform 1A by siRNA-p120ctn-1A resulted in decreased E-cadherin expression and increased N-cadherin snail and vimentin expression in H460 cells (A). However knockdown of endogenous p120ctn-1A by siRNA-p120ctn-1A showed opposite results in SPC cells where we found increased E-cadherin expression and decreased N-cadherin snail and vimentin expression (B). In comparison with the control the ablation of p120ctn isoform 1A also enhanced the H460 cells invasiveness (17.33±1.25 vs. 36.33±1.70 P<0.01) (C) whereas reduced the SPC cells invasiveness (23.0±0.82 vs. 13.0±0.82 P<0.01) (D). These results revealed that the p120ctn isoform 1A plays a different role in EMT and cell invasiveness in different E-cadherin subcellular locations. .0088064.g003 p120ctn isoform 1A plays a different role in regulating EMT in H460 and SPC cells. (A) Ablation of p120ctn isoform 1A decreased E-cadherin expression and increased N-cadherin snail and vimentin expression in H460 cells. (B) SPC cells were treated as in (A) and the opposite results were obtained. (C) Ablation of p120ctn isoform 1A enhanced the invasiveness of H460 cells (**P<0.01). (D) Ablation of p120ctn isoform 1A decreased the invasiveness of SPC cells (**P<0.01). Inhibitory function of p120ctn isoform 3A on EMT is not affected by differences in E-cadherin subcellular localization To verify whether p120ctn isoforms 1A and 3A play different roles in regulating EMT their expression plasmids were transiently transfected into lung cancer cells with low expression of p120ctn (H1299 with membrane E-cadherin expression and LK2 with cytoplasmic E-cadherin expression). The western-blot analysis demonstrated that overexpression of the p120ctn isoform 1A led to increased E-cadherin expression and decreased N-cadherin vimentin and snail expression (A); on the contrary the decreased E-cadherin expression and increased N-cadherin vimentin and snail expression were observed in LK2 cells (B). Overexpression of the p120ctn isoform 1A also reduced the H1299 cell invasiveness (52.0±2.65 vs. 33.33±2.64 P<0.01) (C) while enhanced the LK2 cell invasiveness (18.0±0.82 vs. 39.66±2.05 P<0.01) (D).. Overexpression of p120ctn isoform 3A led to increased E-cadherin expression decreased N-cadherin vimentin and snail expression (A 4B) and reduced cell invasiveness (52.0±2.65 vs. 29.66±1.53 P<0.01; 18.0±0.82 vs. 8.33±expression 0.47 P<0.01) (C 4D) in both of these cell lines. These results further confirmed that the p120ctn isoform 1A had a different effect on EMT depending on the subcellular localization of E-cadherin. They also revealed that the p120ctn isoform 3A maintained an inhibitory role in the EMT of lung cancer cells whether E-cadherin was localized to the membrane or the cytoplasm. .0088064.g004 p120ctn isoform 3A maintains the role of inhibitiing EMT independently of E-cadherin localization. (A B) Both H1299 (E-cadherin membrane localization) and LK2 cells (E-cadherin cytoplasmic localization) transiently transfected with the p120ctn isoform 3A plasmid showed increased E-cadherin expression and decreased N-cadherin vimentin and snail expression. (C D) Transient transfection of p120ctn isoform 3A plasmids into H1299 and LK2 cells resulted in decreased cell invasiveness (**P<0.01). (E) E-cadherin remained localized on the membrane in H1299 cells and in the cytoplasm of LK2 cells after transfection of the p120ctn isoform 3A plasmid. Discussion The phenomenon of EMT in tumor cells often leads to decreased cell adhesion and increased mobility and this transition is accompanied by decreased E-cadherin expression and increased expression of N-cadherin vimentin and other mesenchymal biomarkers [3] [4] [5] [6] [7]. As an important factor for stabilizing E-cadherin p120ctn plays a role in inhibiting or promoting tumor cell proliferation and invasion that is dependent on whether E-cadherin is expressed or not [16] [17]. Furthermore p120ctn isoforms 1A and 3A have shown different effects on E-cadherin expression and tumor cell invasiveness which are based on differences in the localization of E-cadherin [18]. These results strongly suggest that p120ctn most likely regulates the EMT of tumor cells by affecting E-cadherin expression and that p120ctn isoforms 1A and 3A play different roles in EMT expressing E-cadherin in different subcellular locations. We first found that the p120ctn membrane expression was positively correlated with E-cadherin expression and negatively correlated with vimentin expression and lymph node metastasis while the cytoplasmic expression of p120ctn was negatively correlated with E-cadherin expression and positively correlated with vimentin expression and lymph node metastasis by immunohistochemistry. Although these results were consistent with previous studies [13] [14] they further suggested that p120ctn likely affects the EMT by influencing the expression of E-cadherin and vimentin and thereby the cell invasion and metastasis in non-small cell lung cancer (NSCLC). To confirm the different impacts of p120ctn isoforms 1A and 3A on EMT in cells expressing E-cadherin in different locations we selected H460 and H1299 cells with E-cadherin membrane expression and SPC and LK2 cells with E-cadherin cytoplasmic expression for further analysis. Plasmids expressing the p120ctn isoforms 1A and 3A were constructed and the full-length p120ctn siRNA was synthesized for these experiments. Since the sequence beyond amino acids 1“101 of p120ctn isoform 1A is similar to that of p120ctn isoform 3A [24] [25] we could not design an interference sequence specifically for p120ctn isoform 3A. Therefore we had to further study the impact of the two isoforms on EMT and cell invasiveness in lung cancer cells with different E-cadherin locations specifically by knocking down p120ctn isoform 1A in H460 and SPC cells with high p120ctn expression and transfecting cDNA plasmids for exogenous p120ctn isoforms 1A and 3A into H1299 and LK2 cells with low expression of p120ctn. Knockdown of p120ctn isoform 1A in H460 cells destroyed the epithelial cell adhesion complexes. E-cadherin expression was also downregulated due to the loss of its important stabilizing factor p120ctn isoform 1A which was consistent with previous studies [20] [26]. Decreased E-cadherin expression and disrupted cell-cell adhesion may induce EMT [27] [28] [43] [44] which results in increased N-cadherin vimentin and snail expression and enhanced cell invasiveness. On the other hand overexpressed p120ctn isoforms 1A and 3A was shown to bind E-cadherin located on the membrane proactively in tumor cells [29] and then inhibit the degradation of E-cadherin and stabilize its expression contributing to the formation of effective epithelial cell adhesion complexes [30] [31] [32]. As these series of processes maintained the normal cell-cell adhesion connection and inhibited EMT there was increased E-cadherin expression and decreased N-cadherin vimentin and snail expression as well as inhibited cell invasiveness in H1299 cells. Previous studies have shown that although p120ctn isoform 1A could bind E-cadherin in the cytoplasm they could not form effective adhesion complexes on the membrane between epithelial cells [33]. Furthermore the cytoplasmic E-cadherin is likely not to be the full-length E-cadherin but instead cleaved E-cadherin fragments such as E-cad/sE-cad (80 kDa) and E-cad/CTF2 (33 kDa) [34]. The E-cad/CTF2 fragment can bind to p120 in the cytoplasm and then translocate into the nucleus and bind the transcriptional repressor of Kaiso to activate the Wnt/b-catenin pathway [35] [36] finally promoting the EMT of tumor cells and enhancing cell invasion and metastasis [37]. Moreover others have shown that p120ctn-1A is related to abnormal expression of E-cadherin and poor prognosis [38]. These studies illustrated that the cytoplasmic p120ctn isoform 1A can play a role in promoting tumor cell EMT invasion and metastasis. Based on the above we observed on the one hand that the effect of p120ctn isoform 1A to promote tumor cell EMT invasion and metastasis would be lifted by its ablation resulting in increased E-cadherin expression decreased N-cadherin vimentin and snail expression and inhibited invasiveness in SPC cells. On the other hand transfection of the p120ctn isoform 1A plasmid into LK2 cells expressing cytoplasmic E-cadherin resulted in decreased E-cadherin expression increased N-cadherin vimentin and snail expression and enhanced cell invasiveness. Although the precise role of p120ctn during EMT induction is still unclarified previous studies suggested that knockdown of all isoforms of p120ctn could induce EMT indirectly [27] [28] [43] [44]. All inductions were based on decreased E-cadherin expression and intercellular adhesion in previous studies which were also confirmed by our study in H460 cells with E-cadherin membrane localization. Unlike the H460 cells knockdown of the p120ctn isoform 1A in SPC cells with E-cadherin cytoplasmic expression could not decrease E-cadherin expression and intercellular adhesion. Instead we found increased E-cadherin expression and decreased cell invasiveness indicating that the EMT could not be induced by this pathway in SPC cells. It was worth noting that the same result was observed in LK2 and H1299 cells transfected with the p120ctn isoform 3A plasmid both showing increased E-cadherin expression decreased N-cadherin vimentin and snail expression and inhibited cell invasiveness. These results suggested that p120ctn isoform 3A has the function of inhibiting EMT of lung cancer cells and this function is independent of the cellular E-cadherin localization. Past research had also confirmed a shift from p120ctn isoform 3A to p120ctn isoform 1A expression after the induction of EMT [9] [10] [11] which indirectly indicates that p120ctn isoform 3A may inhibit EMT while p120ctn isoform 1A promotes EMT. In addition we also noticed that p120ctn and E-cadherin protein expression levels were significantly increased after transfection of the p120ctn-3A plasmid into LK2 and H1299 cells but p120ctn and E-cadherin were still mainly restricted to the cell membrane at cell-cell adherens junctions in H1299 cells. By contrast E-cadherin and p120ctn were almost exclusively located in the cytoplasm in LK2 cells (E). As a cell adhesion molecule E-cadherin is known to be only located on the cell membrane with the potential to inhibit EMT while in the cytoplasm it is often cleaved into fragments and therefore functions differently from the molecules located on the cell membrane [34]. Thus the cytoplasmic E-cadherin would theoretically not play a role in inhibiting EMT. Based on the above analysis we speculated that there may be some interaction between p120ctn isoform 3A and snail which plays a role in suppressing EMT in lung cancer cells expressing cytoplasmic E-cadherin but this hypothesis requires further study. Importantly we also found that knockdown of p120ctn-1A in SPC cells with cytoplasmic E-cadherin resulted in decreased twist expression (B). Meanwhile transfection of LK2 cells which also showed cytoplasmic localization of E-cadherin with the p120ctn isoform 1A plasmid resulted in increased twist expression (B). However no changes in twist expression were observed in the rest of the experiments (A 4A). As a transcription factor and master gene regulator of EMT [39] [40] twist can downregulate E-cadherin expression [41] and upregulate N-cadherin and other mesenchymal biomarkers [42]. Increased twist expression in LK2 cells transfected with the p120ctn isoform 1A plasmid indicated that transcriptional activation took place and further suggested that the p120ctn isoform 1A may have translocated into the nucleus upon binding of E-cad/CTF2 in the cytoplasm consequently activating the Wnt signaling pathway to promote EMT. Decreased twist expression in SPC cells transfected with p120ctn-1A-siRNA indicated that transcriptional activity was downregulated and suggested that ablation of p120ctn isoform 1A resulted in the inhibtion of EMT by removing the stimulatory effect of the Wnt signaling activity by p120ctn isoform 1A. In the H460 and H1299 cells with E-cadherin localized in the membrane the unchanged twist expression confirmed that p120ctn isoforms 1A and 3A could bind to E-cadherin and maintain effective cell-cell adhesion in order to suppress EMT instead of affecting the Wnt/twist pathway. Intriguingly overexpression of p120ctn isoform 3A did not change twist expression in LK2 cells expressing cytoplasmic E-cadherin indicating that p120ctn isoform 3A did not activate transcription. Therefore we firmly believe in the above hypothesis that p120ctn isoform 3A may interact with snail in some manner to influence E-cadherin expression and suppress EMT in lung cancer cells carrying cytoplasmic E-cadherin. Previous studies have observed that p120ctn-1A restored the cytoplasmic expression of E-cadherin whereas p120ctn-3A could not [20] which seems to be contradictory with the results of this study. However the method in previous studies of knocking down p120ctn expression and then transfecting p120ctn isoforms 1A and 3A plasmids into cells is different from that in the current study in which cells were only transiently transfected with p120ctn isoforms 1A and 3A plasmids. Therefore the different research methods may have led to different effects on E-cadherin. We also noted that in previous studies decreased and almost undetectable levels of E-cadherin by ablation of p120ctn resulted in the failure of exogenous p120ctn-1A to translocate into the nucleus to activate the Wnt/b-catenin pathway and decrease E-cadherin expression due to the deletion of the binding partner E-cad/CTF2. However the LK2 and H1299 cell lines used in these experiments expressed E-cadherin in the present study. E-cadherin binds primarily to unphosphorylated p120ctn isoform 3A whereas tyrosine-phosphorylated p120ctn isoform 1A interacts exclusively with N-cadherin [23]. In the previous studies exogenous p120ctn isoform 3A was prevented from binding and stabilizing E-cadherin after its ablation while in the present study the exogenous p120ctn isoform 3A could stabilize E-cadherin expression directly on the membrane or indirectly by increasing its cytoplasmic expression via regulation of snail expression. Of course all of these findings will need to be further investigated. In conclusion we for the first time found that p120ctn isoforms 1A and 3A to have different functions in EMT of lung cancer cells with E-cadherin expressed in different subcellular locations. When E-cadherin was localized on the cell membrane p120ctn isoforms 1A and 3A both could inhibit EMT and reduce the cell invasiveness phenotype."
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edited byfang zhoucas lamvac biotech co ltd chinareviewed bybart evertsleiden university medicalcenter netherlandsmaria rosa bonouniversity of chile chileandrew john staggqueen mary university of londonunited kingdomcorrespondencedipayan rudradipayanrudragmailcomrudradpostechackrsinhyeog imiimshpostechackr present addresshyunja kokobiolabs inc seoul south koreasungwook hongdepartment of microbiology andimmunology centre for immunologyuniversity of minnesota medicalschool minneapolis mnunited states¡these authors have contributedequally to this work§deceasedspecialty sectionthis was submitted toimmunological tolerance andregulationa section of the frontiers in immunologyreceived april accepted july published august citationko hj hong sw verma r jung jlee m kim n kim d surh cdkim ks rudra d and im sh dietary glucose consumptionpromotes raldh activity in smallintestinal cd103cd11b dendriticcells front immunol 103389fimmu202001897intestinal dendritic cells dcs are critical for the initiation and regulation of innate and adaptiveimmunity by delivering self or foreign antigens to t cells “ the intestine is spontaneouslyexposed to innumerable antigens comprising of intestinal microbes as well as dietarycomponents to maintain immune homeostasis intestinal dcs regulate the balance betweenthe tolerogenic immune response by inducing cd4foxp3 regulatory t cells treg cells “and the protective immune responses by inducing eï¬ector t cells dysregulation of thisbalance by harmful pathogens or dietary intake results in ‚ammatory disorders such as‚ammatory bowel disease ibd celiac disease and food allergy frontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityintestinal dcs are located in the peyer™s patches ppsmesenteric lymph nodes mlns and lamina propria lp andcomprise cellular subsets that have diï¬erent origins and functions “ among these dc subtypes intestinal cd103 dcshave the unique function that metabolizes vitamin a to retinoicacid ra through the activation of aldehyde dehydrogenase member a2 [aldh1a2 also called retinaldehyde dehydrogenaseraldh2] enzyme the ra produced by intestinaldcs play an important role in orchestrating immune responsesimprinting guthoming specificity on t cells b cells andinnate lymphoid cells ilcs inducing igaproducing b cellspromoting tgfdependent diï¬erentiation of induced tregcells suppressing the diï¬erentiation of th17 cells enhancing il production by Îδ t cells and ilcs as well as inducing eï¬ectorfunctions in t cells “while vitamin a derived from dietary intake can induceraldh enzymatic activity the ra produced from intestinalepithelial cells iecs by raldh1 and stroma cells in lp andmln by raldh2 in a trans activating mechanism is alsocapable of inducing raldh expression in intestinal dcs “ furthermore recent data suggest that ra is also involvedin the development of a gut homing precursor for intestinaldcs in the bone marrow as well as is required for theirtranscriptional programming and maturation severalendogenous factors that regulate raldh expression in lpdcs are also reported cytokines such as il4 and granulocytemacrophage colonystimulating factor gmcsf induce orenhance the expression of raldh enzymes in lpdcs whileprostaglandin e2 pge2 negatively regulates raldh activitythrough the induction of inducible cyclic amp early repressoricer “ despite these findings whether additionalcomponents in diet can induce raldh activity in the intestineand promote immune tolerance remains unknown in this studywe uncover a hitherto unknown role of dietary glucose in shapingup intestinal immunological tolerance by facilitating raldhexpression specifically in intestinal lpdcsresultsmice administered antigen free diet havedefects in development and raldhactivity in cd103cd11b silpdcsto investigate the ‚uence of commensal microbiota and foodcomponents on intestinal immunity we utilized the previouslyestablished œantigen free af mice model where germfreegf mice are raised on welldefined elemental diet [termedœantigen free diet afd] devoid of macromolecules such asproteins and starches when dcs in small intestine wereassessed we observed comparable frequencies of cd11cmhcii silpdcs in specific pathogen free spf gf and af micefigures 1ab however indepth analyses revealed alterationin the frequencies of tolerogenic dc subtypes the frequencyof cd103cd11b silpdcs a subset known to be a majortolerogenic dc population was slightly but significantly lowerin af when compared to spf and gf mice figure 1c acompensatory increase on the other hand was observed inthe cd103cd11bˆ’ silpdc compartment more interestinglywhile the expression of the characteristic dc surface markerslargely remained comparable in all three groups figure s1athe expression of all three representative genes tested namelyaldh1a2 indoleaminepyrrole 23dioxygenase ido1 andtransforming growth factor beta tgfb1 that are functionallyimplicated in tolerogenic phenotype of cd103cd11b dcswere dramatically reduced in af mice figure 1d interestinglythe expression of aldh1a2 was found to be specifically reduced inmice raised under afd a phenomenon that was not observedin gf mice figure 1d left panel on the other hand theabsence of gut microbiota appeared to partially ‚uence theexpression of ido1 and tgfb1 which was further enhanced byafd figure 1d middle and right panel these results indicatedthat certain dietary components otherwise absent or underrepresented in afd have most specific and the largest ‚uenceon the expression of aldh1a2 for this study we therefore focusedon the ‚uence of normal diet on raldh activity in silpdcsraldh is an enzyme that irreversibly metabolizes vitamin ato ra which in turn acts as a key modulator of mucosal immuneresponses “ to determine whether in concert to itsreduced expression the function of raldh in lpdcs fromaf mice was also negatively aï¬ected we next examined raldhenzyme activity in lpdcs from spf gf and af mice usingthe aldefluor assay in this assay which has been previouslyemployed in the context of cd103 lpdcs and mlndcs the raldh enzyme activity is measured in individual cellsby flow cytometry with a fluorescent substrate based assay system in agreement with the results obtained by realtime pcranalysis cd103 silpdcs both cd11b and cd11bˆ’ subsetsfrom af mice displayed significantly lower enzyme activity whencompared with spf and gf mice figures 1ef figure s1b ofnote the characteristic frequencies of the aforementioned silpdc subtypes in spf gf and af mice remained unaltered evenafter performing this assay suggesting that this enzyme assay didnot interfere with the phenotype of silpdcs figure s1cin order to further understand the role of dietary componentson raldh activity we next analyzed silpdc raldh activityin mice at diï¬erent stages of their lives after subjecting them tospecific dietary conditions we observed that raldh activityin preweaned gf mice weeks of age was significantlylower than in adult gf mice and comparable to af mice thiswas dramatically restored to the level equivalent to adult gfmice within a week after weaning figure 2a furthermorewhen mice raised in af condition were fed with normal chowdiet ncd raldh activity in cd103cd11b silpdcs waspromptly recovered within a week figure 2b mirroring thisan opposite phenomenon was observed when ncd in gf micewas replaced with afd figure 2c these results suggested thatdietary components in ncd absent in afd is required as theinitial trigger for raldh gene expression after mice are weanedthereby promoting enzyme activity as well as homeostasis ofcd103cd11b silpdcsthe above results also implied that supplementing af micewith ra the final product of the enzymatic reaction and a knownfeedback inducer of raldh activity would be sufficient indriving raldh activity in these mice indeed when adult affrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure dietary intake affects differentiation and raldh activity in cd103 silpdcs cell suspensions were prepared from silp harvested from agematchedadult ˆ¼12weekold spf gf and af mice and phenotypic and raldh activity analyses of silpdcs were carried out a representative fluorescenceactivatedcontinuedfrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure cell sorting facs plots of silpdc subpopulations gated on linˆ’ cd3ˆ’b220ˆ’ cells b statistical quantification of percentage left and total numbersright of cd11cmhcii cells in mice from indicated experimental groups c graph displays percentage of silpdc subpopulations in cd11cmhcii cells bcdata are combined from four independent experiments d realtime analyses of mrna expression of indicated gene products normalized against hprt mrna levelsef representative facs plots left and quantification right of relative mean fluorescence intensity mfi of aldefluor in cd103cd11b e and cd103cd11bˆ’f silpdc subpopulations from indicated groups deab is a raldh inhibitor 01mfi is calculated by subtracting background deab mfi from aldefluor mfi relative 01mfi indicates ratio of 01mfi in experimental samples vs control data are combined from six independent experiments mean ± sem are indicated statisticalsignificance was determined by oneway anova bef and twoway anova c with turkey™s multiple comparison tests p ns not statisticallysignificantmice were supplemented in their diet with ra it resulted incomplete recovery of raldh activity figure 2d interestinglyin all the cases changes in raldh activity also correlatedwith the frequencies of cd103cd11b silpdcs suggestingits role in diï¬erentiation as well as function of these cells ofnote while the above results were obtained in a gf setting thebasic findings from these experiments could also be recapitulatedin mice raised in spf conditions thereby confirming thatmice with normal repertoire of gut flora are equally aï¬ectedby dietary components with respect to raldh activity insilpdcs figures s2a“craldh activities in different intestinalraproducing cells are differentiallyaffected by dietwe next sorted to understand whether the ‚uence of dieton raldh activity is an lpdc specific phenomenon orwhether other raldh expressing cells are also aï¬ected itis wellestablished that within the gut associated lymphoidtissues besides lpdcs ra converting enzymes are alsoexpressed in lp associated stroma cells lpscs small intestineepithelial cells iecs as well as mlndcs the ra producedfrom these cellsis known as a localsource of ra for inducing the raldh expression in cd103silpdcs “in particular iecsthe nonhematopoietic scs in secondary lymph nodescomprise three diï¬erent cell types based on the expressionof surface markers lymphatic stroma cells [lscs also calledfibroblast reticular cells frc]lymphatic endothelial cellslecs and blood endothelial cells becs amonglpscs cd45ˆ’epcamˆ’ in smallintestine the lscs thatexpressed podoplanin pdpn and are cd31ˆ’ were foundto be capable of activating raldh enzymes figure 3a aspreviously reported interestingly unlike lpdcstheraldh activity in lpscs remained comparable between gfand af mice figure 3b in contrast when iecs were analyzedthe expression of aldh1a1 raldh1 the major gene encodingfor raldh enzyme in these cells was found to be reducedin af mice figure 3c of note while the iecs are wellestablished to have raldh activity “ the baseline ofthis activity in these cells is known to be significantly lower thanlpdcs therefore our attempt to measure raldhactivity in iecs was unsuccessful due to lower sensitivity ofthe aldefluor assay however albeit comparatively lowerraldh activity on a per cell basis the cumulative contributionof iecs in ra production is understandably of larger significancesince numerically there are many more iecs than the other celltypes in the intestinefinally when mlndcs were analyzed the raldh activityin particular within the cd103cd11b dc population in afmice was found to be significantly albeit to a lesser extentlower than that of gf mice figure 3d taken together theseresults suggested that dietary components diï¬erentially ‚uenceraldh activity in diï¬erentregulatory dc populationswhereas mlndcs and iec are aï¬ected lpscs appeared toremain unaï¬ected from dietary contributions these results alsoimplied that the overall reduction of ra synthesis cumulativelyamong these cell types eventually contributed to the reducedraldh activity in the lpdcs in af miceproteins starches and minerals in diet donot ‚uence raldh activity incd103cd11b silpdcssince for this study we focused on raldh activity insilpdcs we next wished to define which dietary factorswere required to trigger the initial raldh activity in thesecells while in our initial findings we observed both thesubtypes cd103cd11b and cd103cd11bˆ’ silpdcs tohave reduced raldh activity in af mice figures 1ef thecell recovery of cd103cd11bˆ’ silpdcs from spf and gfmice were low and the level of enzyme activity in this celltype showed variability among individual mice figures 1cftherefore henceforth in this study we focused on the raldhactivity in cd103cd11b silpdcswe first confirmed that the reduction in raldh activityin cd103cd11b silpdcs was not a consequence of lowvitamin a contentin af diet thereby compromising theprecursor for the assayed reaction based on information offood compositions from the suppliers and from our perviousreport final consumption of vitamin a per day by gf andaf mice were comparable [table s1 and ] nonethelessthere remained a possibility that albeit equal consumptionthe absorption of vitamin a into the small intestine may becompromised in af mice however when gf mice were weanedon af diet supplemented with times more vitamin a inthe usual form of œoil mix or were administrated additionalœoil mix by oral gavage failed to recover the reduction inraldh activity in cd103cd11b silpdcs figure s3we thus concluded that mere unavailability of the precursorvitamin a was not a cause of reduced raldh activity inthese cellsto this end we modified the compositions of purifieddiet by removing individual food components table s2 withfrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure dietary components in normal chow readily trigger and maintain raldh activity in silpdcs in mice after weaning cell suspensions prepared from silpwere subjected to aldefluor assays and raldh activity in cd103cd11b silpdcs and percentage of silpdc subpopulations in cd11cmhcii cells wereanalyzed by flow cytometry a gf mice 3weeks old before weaning preweaned gf mice weaned onto ncd for days and adult gf mice were analyzed braldh activity and frequencies of silpdc subpopulations in cd11cmhcii cells in adult af mice ˆ¼12weekold after feeding ncd for and days c raldh activity and frequencies of silpdc subpopulations in cd11cmhcii cells in adult gf mice ˆ¼12weekold after feeding afd for and weeks d raldh activity and frequencies of silpdc subpopulations in cd11cmhcii cells in adult gf af or af mice that were administered intraperitoneal injection of alltrans ra µg per mouse in soybean oil every other day for days data are combined from two to three independent experimentsmean ± sem are indicated bc statistical significance was determined by oneway anova with turkey™s multiple comparison test p p p ns not statistically significantthe presumption that taking out or adding back individualcomponents in otherwise welldefined diet may lead us towardidentifying the dietary component required to trigger raldhactivity to obtain relatively accurate results under in vivosettings the modified diets were designed to contain similaramount of vitamin a as in ncd tables s1 s2 and thefrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure raldh activity in intestinal raproducing cells is differentially regulated by diet a“c cell suspensions from mln and silp harvested from agematchedadult ˆ¼12weekold gf and af mice were subjected to aldefluor assays and raldh activity of lpscs and mlndcs were analyzed by flow cytometry arepresentative facs plot of lpsc distinguished by cd31 and pdpn from cd45epcam cells red dots in facs plot indicate aldefluor positive cells histogramsdepict the mfi of aldefluor in lpsc subtypes b graph displays the level of raldh activity in lscs data is combined from two independent experiments c iecswere isolated from small intestine si by stripping with edta and analyzed for expression of aldh1a1 raldh1 by realtime pcr upon normalization to hprt mrnalevels fold change indicates ratio target gene in experimentcontrol data are combined from four independent experiments d representative facs plots ofmlndc subpopulations distinguished by cd103 and cd11b from cd11cmhciilinˆ’ cells and the raldh activity in cd103cd11b mlndcs andcd103cd11bˆ’ mlndcs data are combined from four independent experiments mean ± sem are indicated statistical significance was determined bytwotailed unpaired ttest p p ns not statistically significantexperiments were performed primarily in gf condition in orderto eliminate the ‚uence of microbiota in addition to avoidany ‚uence from ncd during the preweaned period neonateaf mice were utilized and these mice were weaned onto eachmodified diet for “ weeksas a starting point we took advantage of two commerciallyavailable diets with welldefined dietary compositions in the socalled amino acid defined diet aadˆ— like the afd employed sofar proteins were replaced with amino acids however there wereseveral diï¬erences between their compositions table s2 whilefrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure depletion of macromolecules from purified diet do not alter the raldh activity in cd103cd11b silpdcs af mice ˆ¼4weekold were weaned ontospecific diets for ˆ¼ weeks following which the indicated analyses were carried on a a cartoon depicting experimental scheme left panel aad is a sterilized formcontinuedfrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure of amino aciddefined diet aad that contains three times more vitamin a than aad where protein macromolecules are replaced with amino acidsaad_stf indicates aad from which cornstarch and maltodextrin are removed the level of raldh activity in cd103cd11b silpdcs in the indicated groups arepresented by relative aldefluor 01mfi right panel b representative facs plots left panel frequencies middle panel and absolute numbers right panel of totalcd4foxp3treg cell and cd4foxp3nrp1lo peripheral treg ptreg cell populations in silp of mice subjected to the indicated diet regimes c experimentalscheme left panel and relative aldefluor 01mfi of silpdcs in the indicated experimental groups right panel min_mix afd indicates afd mixing with the mineral mixpowder td94049 data are combined from four independent experiments mean ± sem are indicated statistical significance was determined by oneway anovawith turkey™s multiple comparison test p p p ns not statistically significantafd is a liquid diet where the fibers are provided as cellulosebedding aadˆ— diet are edible solid pellets with cellulose mixedwith the food components unlike afd aadˆ— diet containedstarches in the form of maltodextrin and corn starch and whilethe source of sugar in afd was glucose that in aadˆ— was sucrose furthermore in terms of mineral compositionthere are substantial diï¬erences between the groups the secondcommercially available diet is aadˆ—_stf which was largelysimilar to aadˆ— but was devoid of starches note the œˆ— inaadˆ— indicates a sterilizable form of the diet which is otherwisesimilar to its traditional form aad but with three times morevitamin a to account for presumed losses during sterilization byirradiation surprisingly the mice groups weaned in both aadˆ—and aadˆ—_stf showed a complete recovery of raldh activityin cd103cd11b silpdcs to an extent similar to the controlncd fed group figure 4a these results were independentof microbiota since the characteristic drop in raldh activityof cd103cd11b silpdcs in mice raised in spf conditionscould also be recovered by aad figure s2d taken togetherthese findings led to two important conclusions first starchesare not involved second antigens in the form of peptidesderived from proteins are also dispensable as far as raldhactivity in cd103cd11b silpdcs is concerned to this endwe also considered a possibility that an artifact arising fromunaccounted protein contamination in the amino acid defineddiets may be responsible for the observed recovery of raldhactivity we therefore quantified the generation of peripheralregulatory t ptreg also referred to as itreg when inducedin vitro cell population in the silp of these mice ptreg cells area type of treg cells that are extrathymically generated primarilyat mucosal sites and are distinguished from their thymic ttregcounterparts by the lack of expression of the membrane boundcoreceptor neuropilin1 nrp1 notably in a previousreport we have demonstrated that diet derived proteins are theprimary cause for the generation of cd4foxp3nrp1ˆ’ ptregcells in the small intestine and af mice display dramaticallyreduced ptreg population in silp indeed we found that whiletotal foxp3 treg populations comprising of ttreg and ptregcells remained comparable the frequencies and numbers offoxp3nrp1ˆ’ ptreg cells among total treg population couldonly be recovered in mice fed with ncd and not aadˆ—and aadˆ—_stf figure 4b therefore the recovery of raldhactivity in aadˆ— and aadˆ—_stf groups were not due to anyprotein contamination in the aadˆ— dietwe next wished to exclude the possibility that diï¬erencesin minor food components such as minerals between afdand purified diet table s2 was responsible for diï¬erences inraldh activity for that we prepared afd by supplementingwith mineral mix powder td94049 derived from aadˆ—min_mixafd as shown in figure 4c min_mixafd failedto induce raldh activity taken togetherthese resultsconcluded that proteins starches and minerals in diet werenot responsible for the induction of raldh activity incd103cd11b silpdcsoptimum glucose level in diet is requiredto induce raldh activity incd103cd11b silpdcsif the macromolecules and micromolecules in diet were notinvolved lack of which factors in afd compromise raldhactivity in cd103cd11b silpdcs to investigate furtherwe compared the food compositions between aadˆ—_stf andafd table s2 which were the closest among the four typesof diets tested in case of aadˆ—_stf this diet does not containproteins and starches as in afd but does contain unknowndietary factors responsible for the induction of raldh activityin silpdcs there are few diï¬erences between the compositionof these two diets the diet forms pellet vs liquid thecarbohydrate sources sucrose vs glucose the amount ofcarbohydrate sucrose ˆ¼ vs glucose ˆ¼ based on thisobservation as well as in order to minimize the diï¬erencesin diet forms we first generated a liquid form of afd with of sucrose afd_s500 or glucose afd_g500germ free diet gfd or aadˆ—_stf were used as positivecontrols afd with usual glucose designated as afd_g220here was used as negative control the neonate af micewere weaned onto each diet for “ weeks and were analyzedfor raldh activity in cd103cd11b silpdcs figure 5aleft panel indeed afd supplemented with sucroseresulted in significantly enhanced raldh activity in these cellsmore interestingly we observed a similar increase in raldhactivity in cd103cd11b silpdcs even when the source ofcarbohydrate was changed to glucose instead of sucrosefigure 5a middle panel these eï¬ects were found to be specificfor cd103cd11b silpdcs since raldh1 expression iniecs remained unaltered upon carbohydrate supplementationfigure 5a right panel these results suggested that regardlessofits optimum concentrationis important and glucose being a monosaccharide unit ofcarbohydrate is sufficient for the initial triggering of raldhactivity in silpdcsthe source of carbohydratein order to further understand whether the positive eï¬ectof dietary carbohydrate supplementation is specific for raldhactivity or if it can also aï¬ect diï¬erentiation of other immunecells we determined the frequencies of th1 th2 and ptregfrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure supplementing glucose in af diet restores raldh activity in cd103cd11b silpdcs a cartoon depicting experimental scheme left panelneonate af mice were weaned onto specific diets for ˆ¼ weeks afd contains of glucose afd_g220 afd_g500 indicates of glucose in afd andafd_s500 indicates of sucrose in afd aad_stf was utilized as a positive control of raldh activity in lpdcs aldefluor assays were performed on cellsuspensions from silp and raldh activity of silpdc was analyzed by flow cytometry the level of raldh activity in cd103cd11b silpdcs is represented asrelative 01mfi of aldefluor middle panel aldh1a1 expression was also determined in sorted iecs relative to hprt control right panel data are combined from twoindependent experiments b“d graphs display the percentage of tbet in cd4 t cells th1 b gata3 in cd4 t cells th2 c and nrp1lo populations amongin cd4foxp3 treg cells ptreg d data are combined from at least two independent experiments mean ± sem are indicated statistical significance wasdetermined by oneway anova with turkey™s multiple comparison test p p p ns not statistically significantcells in these mice supplementation of afd with additionalsucrose or glucose did not aï¬ect tbet th1 or gata3 th2 cellsfigures 5bc neither recovered the characteristically reducedptreg population in silp figure 5dwe next directly investigated the eï¬ect of glucose on raldhactivity by employing in vitro culture conditions spldcsmlndcs and cd11c silpdcs were magnetically purifiedand cultured either without glucose in commercially availableglucosefree media without glucose but in the presence of rawith glucose or in the presence of glucose and ra for hand then measured for raldh activity in terms of baselineraldh activity as expected spldcs displayed the least whichwas significantly increased in the presence of ra glucose onthe other hand had minimal eï¬ect figure 6a although mlndcs had the highest baseline raldh activity among the threegroups tested neither ra nor glucose had an impact figure 6bin contrast silpdcs derived from 2weeks old neonatal spfmice which had low basal raldh activity at steady state wassignificantly increased in the presence of glucose alone while raitself induced slight increase of this enzyme activity the highestactivity was observed when both ra and glucose was presentfigure 6c moreover this raldh activity promoting eï¬ect ofglucose was also observed when silp mixed lymphocytes derivedfrom 8weeks old adult mice were cultured with glucose for hunder the culture conditions tested ra by itself was found tohave minimal eï¬ect on the already high raldh activity whereasthe addition of glucose in the media was able to further boost thisactivity figure 6dfinally in order to ascertain functional relevance of thesefindings we sorted to determine whether supplementationfrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure glucose induces raldh activity specifically in silpdcs mlndcs and spleen dcs spldc from adult spf mice or silpdcs from 2weeks oldneonatal spf mice were either purified a“c or total single cell suspensions were isolated from adult spf mice si d cells were cultured for h in glucosefreecontinuedfrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure media with or without treatments following which aldefluor assay was performed fluorescence intensities of aldefluor in cd8ˆ’cd11b spldcsa cd103 mlndcs b and cd103cd11b silpdcs cd were analyzed by flow cytometry raldh activity depicted in overlaid histograms and bar graphsare from the cells cultured with glucose mm or ra nm or both the line graphs indicate raldh activity from the cells cultured with different concentrations ofglucose and mm in the presence or absence of ra nm data shown is representative of at least three independent experiments mean ± sem areindicated statistical significance was determined by oneway anova with turkey™s multiple comparison test p p p ns notstatistically significantof glucose presumably through the generation of ra canfacilitate the generation of itreg cells for this we performedan in vitro itreg induction assay with magnetically purifiedcd11chi dcs with œhigh cd11c expression that were pretreated with glucose and incubated with naïve t cells undersuboptimal itreg inducing condition we observed significantincrease in itreg induction when silpdcs were pretreatedwith glucose compared to mock this eï¬ect of glucose pretreatment was specific for silpdcs and was not observedwhen mlndcs were used in the assay figures 7ab andfigure s4 these results in accordance to the results presentedin figure 6b suggested that the silp dcs are particularlymore susceptible to glucose treatment and thereby presumablythrough enhanced raldh activity acquire superior itreg cellinduction capacity compared to mln dcs it is to be notedhowever that while the purified cd11chimhcii dcs usedin this assay are presentthere aresome site specific diï¬erences with regard to the expressionof mhcii and cd11c in mln and silp compared to silpmln has significantly higher proportion of cd11cˆ’mhciiand cd11cintmchiiˆ’ with œintermediate cd11c expressionpopulations figure s4 left panels furthermore the expressionof mhcii in purified mln dcs is lower than that of silpdcs figure s4 right panels these observations raised theformal possibility that enhanced mhcii expression in silprather than glucose mediated enhanced raldh activity maybe responsible for increased itreg conversion however pretreatment with ra either alone or in the presence of glucoseresulted in equally efficient itreg induction irrespective of thesource of the dcs suggesting that the benefit of glucose pretreatment is indeed primarily due to enhanced ra productionfigures 7ab lastly when itreg induction was carried outin vitro in a dc independent manner supplementation ofthe media with excess glucose had little eï¬ect thereby furthersubstantiating the role of dc derived raldh in this processfigure s5in similar frequenciesdiscussionthere is accumulating evidence suggesting that vitamin a and itsmetabolites play a pivotal role in maintaining various biologicalprocesses “ dietary supplementation of ra in thecontext of cutaneous t cell lymphoma and acute promyelocyticleukemia have been shown to have beneficial outcome “ furthermore many studies highlight anti‚ammatoryactivities of ra at mucosal sites and tissues such as intestinalmucosa airways lung central nervous system and skin “ in addition to the eï¬ect of ra on cancer and ‚ammatorydiseases vitamin a or its metabolites play an important roleto suppress dietinduced obesity and insulin resistance “therefore a better understanding of the cellular and molecularparameters responsible for vitamin a metabolism is of greatbiological relevance in this study by identifying the role of dietand the importance of glucose consumption for establishingraldh activity early in life in small intestine dcs we makesubstantial contribution to our knowledge related to the role ofnutritional components in establishing immunological toleranceat mucosal sitesby feeding af diet to mice raised under germ free conditionwe found that small intestine cd103cd11b lpdcs require adietary component as an initial trigger for raldh activity sincevitamin a in diet is known to be essential to activate raldhand generate ra in lpdcs lpscs mlndcs mlnscs andiecs one explanation of this observation could bethe possibility that at steady state af mice consume less vitamina compared to gf mice raised on ncd however our furtherexperiments in conjunction with a previous report stronglyindicate that the availability of vitamin a and the way it is feddoes not account for the low raldh activity in af m
0
"preadipocytes diï¬erentiate into adipocytes During this process the preadipocytes ceaseto proliferate begin to accumulate lipid droplets and develop morphologic and biochemical characteristics of mature adipocytesMesenchymal stem cells MSCs are a type of adult stem cells known for their high plasticity and capacity to generate mesodermaland nonmesodermal tissues Many mature cell types can be generated from MSCs including adipocyte osteocyte and chondrocyteThe diï¬erentiation of stem cells into multiple mature phenotypes is at the basis for tissue regeneration and repair Cancer stem cellsCSCs play a very important role in tumor development and have the potential to diï¬erentiate into multiple cell lineagesAccumulating evidence has shown that cancer cells can be induced to diï¬erentiate into various benign cells such as adipocytesfibrocytes osteoblast by a variety of small molecular compounds which may provide new strategies for cancer treatmentRecent studies have reported that tumor cells undergoing epithelialtomesenchymal transition can be induced to diï¬erentiateinto adipocytes In this review molecular mechanisms signal pathways and the roles of various biological processes in adiposediï¬erentiation are summarized Understanding the molecular mechanism of adipogenesis and adipose diï¬erentiation of cancercells may contribute to cancer treatments that involve inducing diï¬erentiation into benign cells IntroductionAdipogenesis is the process through which mesenchymalstem cells MSCs commit to the adipose lineage and diï¬erentiate into adipocytes During this process preadipocytescease to proliferate begin to accumulate lipid droplets anddevelop morphologic and biochemical characteristics ofmature adipocytes such as hormoneresponsive lipogenesisand lipolytic programs Currently there are mainly twomodels of benign adipocyte diï¬erentiation in vitro One isfibroid pluripotent stem cells which can diï¬erentiate intonot only adipocytes but also muscle cartilage and othercells There are two kinds of fibroid pluripotent stem cellsbone marrow and adipose mesenchymal stem cells Anothergroup is fibroblastic preadipocytes which have a single direction of diï¬erentiation namely lipid diï¬erentiation including3T3L1 and 3T3F422A cells [] Cancer cells with tumorinitiation ability designated as cancer stem cells CSCshave the characteristics of tumorigenesis and the expressionof specific stem cell markers as well as the longterm selfrenewal proliferation capacity and adipose diï¬erentiationpotential [] In addition to CSCs [] cancer cells undergoing epithelialmesenchymaltransformation EMT havebeen reported to be induced to diï¬erentiate into adipocytes[“] Lung cancer NCIH446 cells can be induced to differentiate into neurons adipocytes and bone cells in vitro[] The adipogenesis diï¬erentiation treatment is promisingin the p53 gene deletion type of fibroblastderived cancer[] Cancer cells with homologous recombination defectssuch as ovarian and breast cancer cells with breast cancersusceptibility genes BRCA mutations can be inducedto diï¬erentiate by poly ADPribose polymerase PARP 0cStem Cells Internationalinhibitors [] The nuclear receptor peroxisome proliferatoractivated receptor Î PPARÎ agonist antidiabetic thiazolidinedione drug can induce growth arrest and adipogenicdiï¬erentiation in human mouse and dog osteosarcoma cells[] Thyroid cancer cells expressing the PPARÎ fusion proteinPPFP can be induced to diï¬erentiate into adipocytes bypioglitazone [] Adipogenesis can be induced in welldiï¬erentiated liposarcoma WDLPS and dediï¬erentiatedliposarcoma DDLPS cells by dexamethasone indomethacininsulin and 3isobutyl1methyl xanthine IBMX []In this review we highlight some of the crucial transcription factors that induce adipogenesis both in MSCs and inincluding the wellstudied PPARÎ and CCAATCSCsenhancerbinding proteins CEBPs [] as well as othercell factors that have been recently shown to have an important role in adipocyte diï¬erentiation We focus on understanding the complex regulatory mechanism of adipocytediï¬erentiation that can contribute to the clinical treatmentof human diseases including those caused by obesity andadipocytes dysfunction especially for the malignant tumorwhich can be transdiï¬erentiated into mature adipocytes Adipocyte DifferentiationCell proliferation and diï¬erentiation are two opposingprocesses and there is a transition between these two processes in the early stages of adipocyte diï¬erentiation Theinteraction of cell cycle regulators and diï¬erentiation factors produces a cascade of events which ultimately resultsin the expression of adipocyte phenotype [] Adipogenesishas diï¬erent stages Each stage has a specific gene expression pattern [] In general adipocyte diï¬erentiation ofpluripotent stem cells is divided into two phases The firstphase known as determination involves the commitment ofpluripotent stem cells to preadipocytes The preadipocytescannot be distinguished morphologically from their precursor cells but also have lost the potential to diï¬erentiate intoother cell types In the second phase which is known asterminal diï¬erentiation the preadipocytes gradually acquirethe characteristics of mature adipocytes and acquire physiological functions including lipid transport and synthesisinsulin sensitivity and the secretion of adipocytespecificproteins []The diï¬erentiation of precursor adipocytes is also dividedinto four stages proliferation mitotic cloning early diï¬erentiation and terminal diï¬erentiation [] After the precursors are inoculated into the cell culture plates the cellsgrow exponentially until they converge After reaching contact inhibition the growth rate slows and gradually stagnatesand the proliferation of precursor adipocytes stops which isvery necessary for initiating the diï¬erentiation of precursoradipocytes Adipocyte precursors exhibit transient mitosiscalled œclonal expansion a process that relies on the actionof induced diï¬erentiation factors Some preadipocyte cellsmouse cell lines 3T3L1 3T3F442A undergo one or tworounds of cell division prior to diï¬erentiation [] whereasother cell lines mouse C3H10T12 diï¬erentiating into adipocyte do not undergo mitosis clonal expansion []Whether œmitotic clonal expansion is required for adiposediï¬erentiation remains controversial However it is certainthat some of the checkpoint proteins for mitosis regulateaspects of adipogenesis [ ] When cells enter the terminaldiï¬erentiation stage the de novo synthesis of fatty acidsincreases significantly the transcription factors and adipocyterelated genes work cooperatively to maintain precursor adipocyte diï¬erentiation into mature adipocytes containing largelipid droplets [] Regulatory Pathways inPreadipocytes CommitmentAdipocyte diï¬erentiation is a complex process in which geneexpression is finely regulated The most basic regulatory network of adipose diï¬erentiation has not been updated inrecent years but some factors and signaling pathways thatdo aï¬ect adipose diï¬erentiation have been continuouslyreported Adipocyte diï¬erentiation is the result of the geneexpression that determines the phenotype of adipocyteswhich is a complex and delicate regulatory process Figure Wnt Signal Pathway in Adipogenesis Wnt signaling isimportant for adipocytes proliferation and diï¬erentiationboth in vitro and in vivo [] The Wnt family of secretedglycoproteins functions through paracrine and autocrinemechanisms to ‚uence cell fate and development Wntprotein binding to frizzled receptors initiates signalingthrough catenindependent and independent pathways[] Wnt signaling inhibits adipocyte diï¬erentiation in vitroby blocking the expression of PPARÎ and CEBPα [] Constitutive Wnt10b expression inhibits adipogenesis Wnt10b isexpressed in preadipocytes and stromal vascular cells butnot in adipocytes In vivo transgenic expression of Wnt10bin adipocytes results in a reduction in white adipose tissuemass and absent brown adipose tissue development []Wnt10a and Wnt6 have also been identified as determinantsof brown adipocyte development [ ] Wnt5b is transiently induced during adipogenesis and promotes diï¬erentiation [] indicating that preadipocytes integrate inputs fromseveral competing Wnt signals The Hedgehog HH Signaling Pathway MechanismThree vertebrate HH ligands including sonic hedgehogSHHIndian hedgehog IHH and desert hedgehogDHH have been identified and initiated a signaling cascademediated by patched Ptch1 and Ptch2 receptors [ ]HH signaling had an inhibitory eï¬ect on adipogenesis inmurine cells such as C3H10T12 KS483 calvaria MSCslines and mouse adiposederived stromal cells [] Thesecells were visualized by decreased cytoplasmic fat accumulation and the expression of adipocyte marker genes after HHsignaling was inhibited [] Although it is generally agreedthat HH expression has an inhibitory eï¬ect on preadipocytediï¬erentiation the mechanisms linking HH signaling andadipogenesis remain poorly defined [] ERKMAPKPPAR Signal Pathway Extracellularregulated protein kinase ERK is required in the proliferativephase of diï¬erentiation ERK activity blockade in 3T3L1 0cStem Cells InternationalDEX insulin DEMXWNT 10band othersSHHPBC SMOTGF𝛽P SMAD3 SMAD3Testosterone𝛽catentinARIRSPI3KAKTCREBPKAPCREBFOXO1A2TCFLEF GATA23CEBP𝛽MAPKG3K3𝛽P2CEBP𝛽CEBPαPPARá¿«BMPsSMAD1SREBPAdipocytegenesFigure Regulation pathways in preadipocytes commitment BMP and Wnt families are mediators of MSCs commitment to producepreadipocytes Exposure of growtharrested preadipocytes to diï¬erentiation inducers IGF1 glucocorticoid and cAMP triggers DNAreplication leading to adipocyte gene expression due to a transcription factor cascade The dotted line indicates an uncertain molecularregulatory mechanismcells and embryonic stem cells can inhibit adipogenesis Inthe terminal diï¬erentiation phase ERK1 activity leads toPPARÎ phosphorylation which inhibits adipocyte diï¬erentiation This implies that ERK1 activity must be reduced afteradipocyte proliferation so that diï¬erentiation can proceedThis reduction is mediated in part by mitogenactivatedprotein kinase MAPK phosphatase1 MKP1 [ ]These extracellular and intracellular regulation factors causeadipocytespecific gene expression and eventually lead toadipocyte formation Adipocyte DifferentiationRegulatory Proteins PPARÎ and Adipocyte Diï¬erentiation PPARÎ is a member of the nuclearreceptor superfamily and is both necessaryand sufficient for adipogenesis [] Forced expression ofPPARÎ is sufficient to induce adipocyte diï¬erentiation broblasts [] Indeedthe proadipogenic CEBPs andKrüppellike factors KLFs have all been shown to induceat least one of the two PPARÎ promoters In contrast antiadipogenic transcription factor GATA functioned in part byrepressing PPARÎ expression [] PPARÎ itself has twoisomers The relative roles of PPARÎ1 and PPARÎ2 in adipogenesis remain an question PPARÎ2 is mainlyexpressed in adipose tissue while PPARÎ1 is expressed inmany other tissues Although both can promote adipocytediï¬erentiation PPARÎ2 could do so eï¬ectively at very lowligand concentration compared with PPARÎ1 [] The twoprotein isoforms are generated by alternative splicing andpromoter usage and both are induced during adipogenesisPPARÎ1 can also be expressed in cell types other than adipocytes Ren [] used engineered zincfinger proteins tothe expression ofthe endogenous PPARÎ1 andinhibitPPARÎ2 promoters in 3T3L1 cells Ectopic expression ofPPARÎ2 promotes adipogenesis whereas that of PPARÎ1does not Zhang reported that PPARÎ2 deficiencyimpairs the development of adipose tissue and insulin sensitivity []There are transcriptional cascades between adipocytesgenes including PPARÎ and CEBPα which are the coreadipocyte diï¬erentiation regulators In the early stage of adipocyte diï¬erentiation the expression of CEBP and CEBPδincrease which upregulates CEBPα expressionfurtheractivate PPARÎ PPARÎ activating CEBPα in turn resultsin a positive feedback PPARÎ binding with retinoic acid Xreceptor RXR forms diï¬erent heterodimers The variousdimmers can combine with the PPARÎ response elementPPRE and initiate the transcription of downstream genesfor diï¬erentiation into adipocytes []CEBPs participate in adipogenesis and several CEBPfamily members are expressed in adipocytesincludingCEBPα CEBP CEBPÎ CEBPδ and CEBPhomologous protein CHOP The temporal expression of thesefactors during adipocyte diï¬erentiation triggers a cascadewhereby early induction of CEBP and CEBPδ leads toCEBPα expression This notion is further supported by thesequential binding of these transcription factors to severaladipocyte promoters duringadipocyte diï¬erentiationCEBP is crucial for adipogenesis in immortalized preadipocyte lines CEBP and CEBPδ promote adipogenesis atleast in part by inducing CEBPα and PPARÎ CEBPαinduces many adipocyte genes directly and plays an important role in adipose tissue development Once CEBPα isexpressed its expression is maintained through autoactivation [] Despite the importance of CEBPs in adipogenesis 0cStem Cells Internationalthese transcription factors clearly cannot function efficientlyin the absence of PPARÎ CEBP cannot induce CEBPαexpression in the absence of PPARÎ which is required torelease histone deacetylase1 HDAC1 from the CEBPαpromoter [] Furthermore ectopic CEBPα expressioncannot induce adipogenesis in PPARΓ“ï¬broblasts []However CEBPα also plays an important role in diï¬erentiated adipocytes Overexpression of exogenous PPARÎ inCEBPαdeficient cells showed that although CEBPα isnot required for lipid accumulation and the expression ofmany adipocyte genes it is necessary for the acquisition ofinsulin sensitivity [ ] Figure Human fibroblasts withthe ability to diï¬erentiate into adipocytes also do not undergomitotic cloning amplification However PPARÎ exogenousligands need to be added to promote adipocyte diï¬erentiation Therefore it can be inferred that mitotic cloning expansion can produce endogenous ligands of PPARÎ [] BMP and Transforming Growth Factor TGF inAdipocyte Diï¬erentiation A variety of extracellular factorsaï¬ect the preadipocyte commitment of stem cells includingbone morphogenetic protein BMP []transforminggrowth factor TGF [] insulininsulinlike growthfactor IGF1 [] tumor necrosis factor α and interleukin [] matrix metalloproteinase [] fibroblast growthfactor FGF and FGF2 [] BMP and TGF have variedeï¬ects on the diï¬erentiation fate of mesenchymal cells []The TGF superfamily members BMPs and myostatinregulate the diï¬erentiation of many cell types includingadipocytes [] TGF inhibitor can promote adipose diï¬erentiation of cancer cells with a mesenchymal phenotypein vitro and transgenic overexpression of TGF impairsadipocyte development [] Inhibition of adipogenesis couldbe obtained through blocking of endogenous TGF with adominantnegative TGF receptor or drosophila mothersagainst decapentaplegic protein SMAD inhibitionSMAD3 binds to CEBPs and inhibits their transcriptionalactivity including their ability to transactivate the PPARÎ2promoter [ ] Exposure of multipotent mesenchymalcells to BMP4 commits these cells to the adipocyte lineageallowing them to undergo adipose conversion [] Theeï¬ects of BMP2 are more complex and depend on the presence of other signaling molecules BMP2 alone has little eï¬ecton adipogenesis and it interacts with other factors such asTGF and insulin to stimulate adipogenesis of embryonicstem cells [] BMP2 stimulates adipogenesis of multipotentC3H10T12 cells at low concentrations and can contribute tochondrocyte and osteoblast development at higher concentrations [] KLFs in Adipocyte Diï¬erentiation During adipocyte differentiation some KLF family members are overexpressedsuch as KLF4 KLF5 KLF9 and KLF15 while KLF16 expression is reduced [ ] KLF15 is the first KLF family members which were identified to be involved in adipocytediï¬erentiation Its expression increased significantly on thesixth day of 3T3L1 adipocyte diï¬erentiation and peakedon the second day of adipocyte induction in MSCs andmouse embryonic fibroblasts Inhibition of KLF15 by siRNAor mutation led to a decrease in PPARÎ CEBPα fatty acidbinding protein FABP4 and glucose transporter GLUT4 However overexpression of KLF15 in NIH3T3cells was found to be associated with lipid accumulation aswell as increases in PPARÎ and FABP4 [] Mice with complete absence of KLF5 showed embryonal lethality and micewith singlechromosome KLF5 knockout showed a significant reduction in white fat in adulthood suggesting thatKLF5 plays an important role in adipocyte diï¬erentiationKLF5 can be activated by CEBP or CEBPδ which isinvolved in early adipocyte diï¬erentiation KLF5 can beactivated by CEBP or CEBPδ which is involved in earlyadipocyte diï¬erentiation Direct binding of KLF5 to thePPARÎ2 promoter in combination with CEBPs inducesPPARÎ2 expression [] Transfection of KLF5 dominantnegative mutants in 3T3L1 cells reduced lipid droplet accumulation and inhibited PPARÎ and CEBPα expressionwhereas overexpression of wild KLF5 significantly promotedadipocyte diï¬erentiation even without exogenous hormonestimulation Similar to KLF5 KLF9 knockdown can inhibitthe expression of a series of adipocyte diï¬erentiation genessuch as PPARÎ CEBPα and FABP4 hence inhibitingadipocyte diï¬erentiation However KLF9 overexpressiondid not upregulate the expression of PPARÎ and CEBPα[] In addition KLF4 can transactivate CEBP by bindingto the region of KB upstream of the CEBP promoter and promote lipid diï¬erentiation [] KLF6 can forma complex with histone deacetylase3 HDAC3 inhibitingpreadipocyte factor1 Pref1 expression and promotinglipid diï¬erentiation [] KLF2 is highly expressed in adiposeprogenitors and its expression decreases during the processof lipid diï¬erentiation Overexpressed KLF2 can bind to theCACCC region of PPARÎ2 proximal promoter and inhibitlipid diï¬erentiation as well as the expression of PPARÎCEBPα and sterolregulated elementbinding proteinsSREBP by inhibiting the promoter activity [] RNAsequence analysisshowed that KLFl6 expression wasdecreased on the first day of adipocyte diï¬erentiation of3T3L1 cells Adipocyte diï¬erentiation was promoted byKLF16 knockdown but was inhibited by KLF16 overexpression via inhibition of PPARÎ promoter activity [] In addition KLF3 and KLF7 were also found to play a negativeregulatory role in adipocyte diï¬erentiation [ ] Signal Transducers and Activators of TranscriptionSTATs and Adipocyte Diï¬erentiation The activated STATprotein enters the nucleus as a dimer and binds to the targetgene to regulate gene transcription In the adipocyte diï¬erentiation of mouse 3T3L1 cells the expression of STAT1 andSTAT5 was significantly increased while that of STAT3and STAT6 was not significantly changed [] In the adipocyte diï¬erentiation of human subcutaneous adipose precursor cells STAT1 expression was significantly decreased[] while the expression of STAT3 and STAT5 wasincreased and STAT6 expression was unchanged [] Therole of STAT1 in adipocyte diï¬erentiation is not clearbecause its expression trend in humans and mice diï¬ersduring the adipocyte diï¬erentiation process Early adipocytediï¬erentiation of 3T3L1 cells was inhibited by STAT1 0cStem Cells InternationalKLF5SREBP1cKLF15KLF2CHOPCEBPá¿«KROX20LigandCEBP𝛽CEBP𝛿GATA23PPARá¿«CEBP𝛼ProadipogenicAntiadipogenicGenes of terminaladipocytedifferentiationFigure A cascade of transcription factors that regulate adipogenesis PPARÎ is one of the key transcription factors in adipogenesis and thecore of the transcriptional cascade that regulates adipogenesis PPARÎ expression is regulated by several proadipogenic blue andantiadipogenic red factors CEBPα is regulated through a series of inhibitory protein“protein interactions Some transcription factorfamilies include several members that participate in adipogenesis such as the KLFs Black lines indicate eï¬ects on gene expression violetlines represent eï¬ects on protein activityagonist interferon Î Loss of STAT1 in 3T3L1 cells can rescue the inhibition of adipocyte diï¬erentiation caused byprostaglandin factor 2α [] Other studies have found thatSTAT1 is required for adipose diï¬erentiation and STAT1overexpression in C3H10T12 cells can prevent the inhibition of lipid diï¬erentiation caused by Bcell lymphoma6knockdown [] There was no abnormal adipose tissuein STAT1 knockout mice [] STAT3 not only aï¬ectsthe proliferation of 3T3L1 cells but also coregulates theiradipocyte diï¬erentiation with high mobility group protein [] The FABP4 promoter was used to specificallyknock out STAT3 in the adipose tissue of mice and theresults showed that mice weight significantly increasedand the adipocyte quantity increased compared with thewildtype mice [] STAT5A and STAT5B have diï¬erenteï¬ects on adipocyte diï¬erentiation Abnormal adipose tissuewas found in the mice with STAT5A or STAT5B knockout ordouble knockout and the amount of adipose tissue was onlyonefifth of the original adipose tissue in mice withoutknockdown [] Histone Modification in Adipocyte Diï¬erentiation Histone deacetylase sirtuin SIRT plays an important rolein biological processes such as stress tolerance energymetabolism and cell diï¬erentiation [] During the adipocyte diï¬erentiation of C3H1012 cells SIRT1 expressiondecreased [] Overexpression of SIRT1 activated theWnt signal which caused the deacetylation of cateninThe accumulation of catenin in the nucleus could inhibitadipocyte diï¬erentiation SIRT1 knockdown resulted inincreased acetylation of the histones H3K9 and H4K16 inthe secreted frizzledrelated protein sFRP and sFRP2 promoters thereby promoting transcription of these genes andpromoting lipid diï¬erentiation [] Forkhead box proteinO FOXO is a member of the transcription factor FOXOfamily It can recruit cyclic AMP response elementbindingprotein CBPhistone acetyltransferase p300 to initiate anacetylation The acetylated FOXO1 can be phosphorylatedby phosphorylated protein kinase B PKBAKT The phosphorylation of FOXO1 by AKT inhibits the transcriptionalactivation of FOXO1 The acetylation of FOXO1 lost the ability of DNAbinding affinity and promoted its shuttling fromnuclei to cytoplasm [] SIRT1 and SIRT2 can deacetylateand active FOXO1 Activated FOXO1 nonphosphorylatednuclear FOXO1 in the nucleus binds to the promoters of target genes encoding p21 p27 and PPARÎ and initiates subsequent transcriptions [] SIRT2 inhibits the acetylation andphosphorylation of FOXO1 thereby induces the accumulation of activated FOXO1 in the nucleus Activated FOXO1could inhibit adipogenesis via PPARÎ [“] Lysinespecific histone demethylase LSD1 expression increasedduring the adipocyte diï¬erentiation of 3T3L1 cells LSD1could reduce the dimethylation levels of histone H3K9 andH3K4 in the CEBPα promoter region thereby promotingadipocyte diï¬erentiation [] SET domaincontaining SETD8 catalyzed the monomethylation of H4K20 andpromoted PPARÎ expression The activation of PPARÎ transcriptional activity leads to the induction of monomethylatedH4K20 and modification of PPARÎ and its targets therebypromoting adipogenesis [] Enhancer of zeste homolog EZH2 is a methyltransferase and can bind methyl groupsto histone H3K27 which is also necessary for lipid diï¬erentiation The absence of EZH2 in brown fat precursors results inreduced levels of the Wnt promoter histone H3K27me3which is also saved by the ectopic EZH2 expression or theuse of a Wntcatenin signal inhibitor [] In addition histone demethylases such as lysinespecific histone demethylase LSDKDM KDM6 and histone lysine demethylasePHF2 are also involved in adipose diï¬erentiation andKDM2B inhibits transcription factor activator protein 2αpromoter via H3K4me3 and H3K36me2 [] Role of microRNA and Long NoncodingRNA in AdipogenesismicroRNA miR can bind and cut target genes or inhibittarget gene translation Endogenous siRNA can be producedby the action of Dicer enzyme and bind to a specific proteinto change its cellular location [] Many kinds of miRsare involved in regulating adipocyte diï¬erentiation The 0cStem Cells Internationalexpression of miR143 increased during the diï¬erentiationof adipose progenitor cells Overexpression of miR143promoted gene expression involved in adipose diï¬erentiationand triglyceride accumulation Inhibition of miR143 prevented the adipose diï¬erentiation of human fat progenitorcells [ ] Additionally miR8 promotes adipocyte diï¬erentiation by inhibiting Wnt signaling [] Moreover miR miR103 miR21 miR519d miR210 miR30miR204211 and miR375 also play a certain role in promoting adipocyte diï¬erentiation while miR130 miR448and let7y inhibit lipid diï¬erentiation [ ] In additionto miRs long noncoding RNA LncRNA is a type of noncoding RNA and is important during epigenetic regulationand can form a doublestranded RNA complex with mRNAcauses protein transcription Lncu90926 inhibits adipocytediï¬erentiation by inhibiting the transactivation of PPARÎ2[] As a novel LncRNA HOXAAS3 expression increasedduring the adipose diï¬erentiation of MSCs and HOXAAS3 silencing reduced the marker gene of adipose diï¬erentiation and inhibited the adipose diï¬erentiation [] Zhu et al[] reported that HOXAAS3 interacted with EZH2 toregulate lineage commitment of MSCs HOXA AS3 canregulate the trimethylation level of H3K27 in the Runx2promoter region by binding to EZH2 Therefore HOXAAS3 is considered to be an epigenetic switch regulating MSCslineage specificity [] Adipocyte diï¬erentiationassociatedLncRNA can act as a competitive endogenous RNA of miR in the process of lipid diï¬erentiation thereby promotingthe expression of SIRT1 the target gene of miR204 and thusinhibiting lipid diï¬erentiation [] The LncRNA NEAT1can also regulate adipocyte diï¬erentiation under the ‚uence of miRNA140 [] Other LncRNA including LncRNABlnc1 and Plnc are also involved in regulating adipocytediï¬erentiation [ ] Other Biochemical Response Involved inAdipocyte Differentiation Unfolded Protein Responses in Adipocyte Diï¬erentiationIn the endoplasmic reticulum of eukaryotes unfolded protein response involves three proteinsinositolrequiringenzyme 1α doublestranded RNAdependent proteinkinaselike ER kinase and activating transcription factorATF 6α [] Knockdown of ATF6α aï¬ects the expressionof adipocytes genes and inhibits C3H10T12 adipocyte differentiation [] The inhibitory eï¬ect of berberine on adipocyte diï¬erentiation of 3T3L1 cells is also due to inducedCHOP and decorin expressions and this inhibitory eï¬ectis ameliorated by CHOP knockout [] In the adipocytediï¬erentiation process of 3T3L1 cells increases in PPARÎand CEBPα as markers of adipocyte diï¬erentiation wereaccompanied by an increase in the corresponding proteinexpressions of phosphorylated Eukaryotic translation initiaEIF 2α phosphorylated endoribonucleasetion factorIRE1α ATF4 CHOP and other unfolded protein responsesEndoplasmic reticulum stress inducer or hypoxic endoplasmic reticulum stress can inhibit adipocyte diï¬erentiationAdditionally EIF2α mutation results in continuous activation or overexpression of CHOP which also inhibits adipocyte diï¬erentiation [] After the initiation of adiposediï¬erentiation numerous diï¬erentiationassociated proteinsare synthesized Exogenous endoplasmic reticulum stressinducers can lead to excessive endoplasmic reticulumresponse which in turn aï¬ects the synthesis of proteinsrelated to diï¬erentiation and inhibits adipocyte formationFigure Role of Oxidative Stress in Adipogenesis During thedirectional diï¬erentiation of MSCs mitochondrial complexI and III and NADPH oxidase NOX4 are the main sourcesof oxygen species ROS production Currently it is believedthat ROS aï¬ects not only the cell cycle and apoptosis but alsodiï¬erentiation through ‚uencing the signaling pathwaysincluding the Wnt HH and FOXO signaling cascade duringMSCs diï¬erentiation [] The diï¬erentiation ability ofstem cells is determined by the arrangement of perinuclearmitochondria which specifically manifests as low ATPcellcontents and a high rate of oxygen consumption The lackof these characteristics indicates stem cell diï¬erentiation[] Adipocyte diï¬erentiation is a highly dependent ROSactivation factor related to mitosis and cell maturation[] Schroder found that exogenous H2O2 could stimulate adipocyte diï¬erentiation of mouse 3T3L1 cells andhuman adipocyte progenitor cells in the absence of insulinH2O2 regulates adipocyte diï¬erentiation of 3T3L1 cells ina dosedependent manner High doses of H2O2 and μM promote adipocyte diï¬erentiation [ ] Tormos found that ROS synthesis increased in humanMSCs at the early stage of adipose diï¬erentiation and targeted antioxidants could inhibit lipid diï¬erentiation Byknocking down Rieske ironsulfur protein and ubiquinonebinding protein ROS produced by mitochondrial complexIII was found to be necessary in initiating adipose diï¬erentiation [] However other studies have shown that theexpression levels of adiponectin and PPARÎ were decreasedby using H2O2 “ mM in 3T3L1 cells [] Free radical nitric oxide NO also promotes lipid diï¬erentiationbecause treatment with NO inducer hydroxylamine or NOsynthase NOS substrate arginine can significantly induceadipose diï¬erentiation of rat adipose progenitor cells NOSinduced adipose diï¬erentiation mainly via eNOS rather thaniNOS [] ROS can induce adipose diï¬erentiation primarily by inhibiting Wnt FOXO and HH signaling pathwaysthat inhibit lipid diï¬erentiation Autophagy in Adipocyte Diï¬erentiation The increase inautophagosomes during lipid diï¬erentiation indicates thatautophagy may play an important role in lipid diï¬erentiation[] Baerga confirmed that the adipocyte diï¬erentiation efficiency was significantly inhibited in mouse embryonic fibroblasts lacking autophagyrelated gene Atg agene encoding an essential protein required for autophagy[] Knockdown of Atg5 in 3T3L1 cells promotesproteasomedependent degradation of PPARÎ2therebyinhibiting adipocyte diï¬erentiation [] Zhang reportedthat autophagyrelated gene 7Atg7 is also crucial for adipose development Atg7deficient mice were slim and onlyhad of white fat compared to wildtype mice and the 0cStem Cells InternationalCEBP𝛽 geneEBF1 geneKLF4EGR2CEBP𝛽CytosolCEBP𝛿 geneCEBP𝛿KLF5genePPARá¿« geneKLF5NR2F2NFKB11433RELASREBF1A2RXRAPPARá¿«PPARá¿«RXRA heterodimerPPARá¿«RXRAcorepressor complexFABP4Ligands of PPARá¿«FAM120BTHRAP3EP300NCOA2NCOA3HELZ2NCOA1CREBBPEBF1ADIPOQ geneAIDRFCEBP𝛼 geneZNF638ZNF467CEBP𝛼NCOR1HDAC3NCOR2 SLC2A4 geneGLUT4 geneLEP geneFABP4 geneCDK4CCND3PLIN1 genePCK1 geneFABP4CD36 genePPARARXRAcoactivator complexPPARá¿«fatty acidRXRAmediatorcoactivator complexANGPTLgenePPARGC1AMediator complex consensusLPL geneNucleoplasmProteins bind to gene promotersTranscription of genes into proteinsActing on proteins compoundingTGF𝛽1WNT1WNT10BTNF77233ADIPOQGLUT4SLC2A4 tetramerLEPFABP4lipid dropletPLIN1PCK1PaPa Pa4xPalmCCD36PaANGPTL4LPLFigure Regulation of adipocyte diï¬erentiation A regulatory loop exists between PPARÎ and CEBP activation Transcription factor CoeEBF activates CEBPα CEBPα activates EBF1 and EBF1 activates PPARÎ CEBP and CEBPδ act directly on the PPARÎ gene bybinding its promoter and activating transcription CEBPα CEBP and CEBPδ can activate the EBF1 gene and KLF5 The EBF1 and KLF5proteins in turn bind the promoter of PPARÎ which becomes activated Other hormones such as insulin can aï¬ect the expression ofPPARÎ and other transcription factors such as SREBP1c PPARÎ can form a heterodimer with the RXRα In the absence of activatingligands the PPARÎRXRα complex recruits transcription repressors such as nuclear receptor corepressor NCoR NCoR1 andHDAC3 Upon binding with activating ligands PPARÎ causes a rearrangement of adjacent factors Corepressors such as NCoR2 are lostand coactivators such as Transcription intermediary factor TIF2 CBP and p300 are recruited which can result in the expression of CyclicAMPresponsive elementbinding protein CREB followed by PPA
2
" microwave ablation mwa is widely used to treat unresectable primary and secondary malignanciesof the liver and a limited number of studies indicate that ablation can cause not only necrosis at the in situ site butalso an immunoreaction of the whole body this study aimed to investigate the effects of mwa on cytokines inpatients who underwent mwa for a hepatic malignancymethods patients admitted to the oncology department in the first affiliated hospital of soochow universitybetween june and february were selected peripheral blood was collected from patients with a hepaticmalignancy treated with mwa the levels of cytokines il2 ifnÎ tnfα il12 p40 il12 p70 il4 il6 il8 il10and vascular endothelial growth factor vegf were detected with a milliplex® map kit the comparison times wereas follows before ablation h after ablation days after ablation and days after ablation data were analyzedusing a paired sample ttests and spearman™s correlation analysisresults a total of patients with hepatic malignancies were assessed there were significant differences in il2il12 p40 il12 p70 il1 il8 and tnfα at h after mwa significant increases 2fold vs before ablation wereobserved in il2 il1 il6 il8 il10 and tnfα after mwa elevated il2 and il6 levels after ablation werepositively correlated with energy output during the mwa procedures wa treatment for hepatic malignancies can alter the serum levels of several cytokines such as il2 and il6keywords microwave ablation hepatic malignancy cytokines il2 il6 immunoregulation primary and secondary malignancies of the liver have asubstantial impact on morbidity and mortality worldwidein china hepatocellular carcinoma hcc has the secondhighest mortality rate of malignancies the treatmentof primary and secondary hepatic malignancies via correspondence lengbengsudaeducn jing zhao qiang li and merlin muktiali contributed equally to this work2department of oncology the first affiliated hospital of soochow universitysuzhou china5division of neurosurgery city of hope beckman research institute duartecalifornia usafull list of author information is available at the end of the interventional imaging therapy is undertaken by investigators in the field of interventional radiology and possibly bya smaller group of practitioners known as interventionaloncologists whose major focus is cancer care via minimally invasive approaches [ ] recently percutaneous ablation therapy has been widely accepted as a radicaltreatment method for hcc and its fiveyear survival rateis similar to that of resection microwave ablationmwa is widely used to treat unresectable hcc and recurrent hcc and has the advantages of minimal invasiona good curative effect and no side effects due to radiationor chemotherapy immune checkpoint inhibitors icis the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhao bmc cancer page of such as pd1pdl1 and ctla4 antibodies have beenwidely applied in several cancers and studies have indicated that ici treatment could enhance the effect of ablation evidence hasindicated that hyperthermicdestruction causes the release of a large population of heterogeneous tumor antigens and inflammatory cytokinesmay play crucial roles in this process cytokines aremediators that regulate a broad range of processes involved in the pathogenesis of cancer several cytokineswhich can arise from either tumor cells or immunocytes such as tumor necrosis factor tnfα interleukinil1 il6 il8 il10 and vascular endothelial growthfactor vegf have been linked with cancers and can either promote or inhibit tumor development the serumlevels of cytokines differ during cancer development although cytokines have been found to be altered after anticancer treatment such as chemotherapy and radiotherapy[ ] few investigations have focused on cytokines beforeand after mwa it is still unknown whether the above cytokines changed before andor after mwa in patientswith hepatic malignancies in this study we investigatedthe effects of mwa on the serum levels of cytokines inpatients with hepatic malignanciesmethodspatients and samplesthe patient population examined in this study was derivedfrom the first affiliated hospital of soochow universitypatients were admitted to the oncology department between june and february the total number ofpatients was with liver metastases and primaryliver cancers the inclusion criterion was a tumor locatedat a hepatic site either primary or metastases all patients with metastatic hepatic malignances should be givensystematic treatments chemotherapy or target therapyand get at least stable disease sd or partial responsepr for more than days informed consent for blooddraw and the relevant therapy was obtained from all patients the protocol was approved by the human ethicscommittee of the first affiliated hospital of soochowuniversity and was conducted in accordance with thedeclaration of helsinki all written informed consent wasobtained from all participants and clearly stated wholeblood ml was drawn into edta anticoagulant tubeson days ˆ’ to before and h days and days afterablation mostly on the last day of the course for cytometry and cytokine analysesablation procedurethe ablation procedure used in this research was mwathe puncture site and pathway were determined underthe guidance of a computed tomography ct scanlocal infiltration anesthesia was achieved by using lidocaine the placement of microwave ablation probeswas guided by a ct scan or ultrasonic device and allprobes were placed at the maximum diameter layerdouble probes were employed when the maximumdiameter of the tumor was up to cm the power andtime of ablation were designed for each patient in therange of w and min respectively basedon the size number and position of the tumor theboundaries of ablation zones were designed as extended cm upon the tumor sitecytokine detectiona milliplex map kit with human cytokinechemokinepanels that measured ifnÎ il2 il6 il8 il10 il12p40 il12 p70 il1 tnfα and vegf was utilized according to the manufacturer™s instructions briefly chemically dyed antibodybound beads were mixed withstandard or sample incubated overnight at °c washedand then incubated with a biotinylated detection antibodyafter the beads were washed they were incubated with astreptavidin phycoerythrin complex and the mean fluorescent intensities were quantified on a luminex analyzer luminex corporation all samples were measured in duplicate standard curves of known concentrations of recombinant human cytokineschemokines wereused to convert fluorescence units to cytokine concentration units pgml the minimum detectable concentrations were as follows ifnÎ pgml il2 pgmlil12 p40 pgml il12 p70 pgml il1 pgml il6 pgml il8 pgml il10 pgml tnfα pgml and vegf pgml all resultsbelow the minimum concentrations were processed as theminimum concentrationsstatistical analysisibm spss statistics software was used for the statistical analysis along with graphpad prism for figurecreations normally distributed numerical data areexpressed as the mean ± standard deviation and nonnormally distributed numerical data are expressed as themedian and confidence interval ci cytokinesat different times were compared using a onetailedpaired ttest spearman™s correlation analysis was executed to determine the correlation between clinical indexes and cytokine levels p indicates a significantdifferenceresultsclinical characteristics of the enrolled patientsas shown in table a total of patients with tumorslocated on the liver liver metastases primary livercancers were analyzed the patients™ cytokine levelswere compared according to time before treatment h after treatment days after treatment and daysafter treatment 0czhao bmc cancer page of table clinical characteristics of the patients enrolled n characteristicsexmalefemaleagepathogenesisprimarysecondaryprimary site for metastatic hepatic malignancescolon rectalpancreasstomachebreastothersmaximum tumor length mmablation probe usedablation time minaverage power per probe w ± ± ± ± average energy time × power time × power–¼–¼ time and power indicate the time and power respectively ofdifferent probes used during the operation ± ifnÎ il12 p40 and il12 p70 were slightly increasedafter mwa treatmentas shown in table and fig the median level ofifnÎ before the mwa treatment was pgml ci “ pgml at days and days after themwa treatment there was a slight increase comparedto that premwa with median levels of pgml ci “ pgml and pgml ci“ pgml respectively the median level of il p40 before the mwa treatment was pgml ci “ pgml there was a slight increase to pgml ci “ pgml days postmwathe median il12 p70 level before the mwa treatmentwas pgml ci “ pgml and increasedto pgml ci “ pgml days afterthe mwa treatment and to pgml ci “ pgml days postmwa no significant alteration in the vegf median level was detected after themwa treatmentil2 il1 il6 il8 and il10 were elevated over 2foldafter the mwa treatmentas shown in table fig and fig the median levelof il2 before the mwa treatment was pgml ci “ pgml there was a significant increase at h postmwa with a median level of pgml ci “ pgml the median level ofil1 before the mwa treatment was pgml ci “ pgml and a significantincrease wasnoted days after the mwa treatment pgml ci “ pgml the median level of il6before the mwa treatment was pgml ci“ pgml and significantly increased daysafter the mwa treatment pgml ci “ pgml the median level ofil8 before themwa treatment was pgml ci “ pgml and increased significantly to pgml ci“ pgml days after the mwa treatmentthe median level of il10 before the mwa treatmentwas pgml ci “ pgml and increasedsignificantly days after the mwa treatment pgml ci “ pgml the median level oftnfα before the mwa treatment was pgml ci “ pgml and increased significantlyto pgml ci “ pgml days afterthe mwa treatmentlevelselevated il2 and il6 levels after ablation were positivelycorrelated with energy output during mwato further evaluate the relationship between the increased cytokineand mwa treatment weemployed the concept of œenergy time × power time × power time and power indicated thetime and power of different probes used in the operation to reflect total hyperthermic damage to hepatictissues during the mwa procedure as shown in table and fig the il2 levels at h postmwa and the il levels at days postmwa illustrated significant correlations with energy the relative indexes were and respectivelydiscussionas technology continues to develop other types of localtherapy such as radiotherapy chemical ablation andhyperthermal ablation for primary and metastatic livercancer are increasingly being used mwa for liver malignances is reserved for patients who cannot undergosurgical removal or for whom other treatments havefailed a consensus guideline was recently developed to address indications for mwa in these patientsthermal ablation is a process that heats the target tissueto a temperature that causes immediate coagulative necrosis usually over °c terminal treatment requiresthat a necrotic area surrounds the target site with anadditional “10mm margins however in the liverhigh tissue perfusion and large blood vessels can cause aœheat sink effect around the ablation zone making itdifficult to achieve terminal ablation the heat sink 0czhao bmc cancer page of table median levels of cytokines before and after mwacytokineifnÎil2premwa pgml ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ h postmwa pgml ci “ ci “ –¼ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “il12 p40il12 p70il1il6il8il10tnfαvegf p vs premwa –¼ 2fold vs premwa days postmwa pgml ci “ ci “ ci “ ci “ ci “ –¼ ci “ –¼ ci “ –¼ ci “ –¼ ci “ –¼ ci “ days postmwa pgml ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “effect can lead to sublethal temperatures and the retention of malignant cells thereby increasing the likelihoodof local tumor progression ltp however an incompletely ablated zone containing immune cells andcancer cells as well as functional vessels could establisha serious inflammatory site that may provide tumorspecific antigens cytokines and activated immune cellsin our study significant increases in the secretion ofchemokines il8 proinflammatory cytokines il1il12 ifnÎ and tnfα and antiinflammatory cytokines il10 were observed after mwa il8 is mainlyproduced by macrophages the classical biological activity of il8 is to attract and activate neutrophils whichcan lead to a local inflammatory response however recent studies have indicated that il8 both macrophageand cancer cellderived can recruit myeloidderivedsuppressor cells mdscs into the tumor microenvironment eventually inhibiting antitumor immunity andpromoting cancer progression [ ] il1 is mainlyproduced by macrophages b cells and nk cells couldproduce il1 under certain circumstances generallycells can only synthesize and secrete il1 after beingstimulated by foreign antigens or mitogens il1 couldpromote the th1 response promoting the activation ofdendritic cells dcs and cytotoxic t lymphocytesctls il12 is mainly produced by b cells and macrophages human il12 is a heterodimer with two subunits p40 kd and p35 kd which areinactivated in isolated form in general il12 functionsas a combination of two subunits il12 p70 while p40alone possesses partial functions of il12 p70 it™s mentionable that il12 p40 and p35 are not expressed inequal proportions so the amounts of il12 p40 and il p70 are different in one cell il12 can stimulate theproliferation of activated t cells and promote the differentiation of th0 cells into th1 cells moreover il12could induce the cytotoxic activity of ctls and nk cellsand promote the secretion of several cytokines such asifnÎ and tnfα previous research indicatedthat tnfα may play a crucial role in mwa in combination with immunotherapy notably our data illustrated that the il12 results were consistent with thoseof ifnÎ after the ablation operation but not with thoseof tnfα this result indicated that upregulation ofifnÎ may be a major effect of the il12 increase aftermwa on the other handan antiinflammatory and immunosuppressive cytokine wasevaluated after mwa il10 is a multicellularderivedmultifunctional cytokine that regulates cell growth anddifferentiation and could participate in inflammatoryand immune responses il10 was reported to increaseafter thermal ablation in the literature [ ] strategiesto inhibit il10induced immunosuppression after thermal ablation treatment would be of interestil10asablation therapy can mediate antitumor immunity astumor tissue necrosis caused by ablation may release various antigens that eventually form a kind of œin situ vaccination moreover ablative therapy can not onlydirectly kill cancer cells in situ but also regulate immunecells and promote the immune function of patients withliver cancer [ ] many immunoregulatory cytokineswere released or expressed after thermal ablation notablythe cytokines released after thermal ablation can regulatethe positive and negative aspects of the cancer immunecycle previously researchers demonstrated that proinflammatory cytokines such as il1 il6 il8 il18 andtnfα were increased several hours or days after thermalablation [ ] to our knowledge terminal tumorthermal ablation may not only cause local heat injury intissues surrounding the tumor site but also induce a systemic reaction this systemic reaction would becaused by different mechanisms first interventional operation may result in trauma to the liver although this procedure is very minimally invasive the healing process maycause alteration of some cytokines second heat injurycould cause acute thermal necrosis in liver and tumor 0czhao bmc cancer page of fig levels of cytokines before and after mwa treatment slightly increased ifnÎ il12 p40 and il12 p70 levels after mwa treatment over fold enhancement of il2 h postmwa and of il1 il6 il8 il10 and tnfα d postmwa p 0czhao bmc cancer page of fig trends in cytokines significantly altered after mwa treatment the levels of il2 at h postmwa il1 at d postmwa il6 at dpostmwa il8 at d postmwa and il10 at d postmwa were elevated over 2fold compared to the levels premwatable correlation between the ablation energy and significantly elevated cytokinesenergyvsil2 h postmwaenergyvsil1 d postmwaˆ’energyvsil6 d postmwaenergyvsil8 d postmwaenergyvsil10 d postmwaenergyvstnfα d postmwaspearman™s rp value onetailed p 0czhao bmc cancer page of fig correlation between the ablation energy and the serum levels of il2 and il6 the serum levels of il2 at h postmwa and il6 at dpostmwa were positively correlated with energy output during the mwa procedureand nonspecifictissues and release of necrotic tissue fragments into bloodcould cause immunological reactions including nonspecific and specific reactions generally cytokines affectedby wound healingimmunologicalreactions do not last longer than those affected by specificimmunologicalreactions ablation treatmentinducedspecific immunological reactions are more complicatedand could affect more immunocytes [ ] which wouldmake this process last longer than other reactions theseexplanations may be the reason why the cytokine changeslasted different durations moreover cytokines affected bythe second manner would be positively correlated withthe ablation scale which is why we employed the œenergyindex in our ablation operation design to receive a terminal ablation larger tumor would cost higher energy including higher power and longer duration time terminaltumorthermal ablation would release tumorrelatedneoantigen to blood circulation eventually induce a systemic reaction this reaction is dependent on the scale ofthermal injury and the local immunological microenvironment of the tumor our findings indicated that il2 andil6 were significantly altered after the ablation procedureand positively correlated with mwa energy il2 is commonly derived from activated t cells primarily th1 cellsil2 can stimulate t cells to proliferate and differentiateactivate natural killer nk cells and macrophages and enhance the functions of cytotoxic t lymphocytes ctls our data illustrated that il2 is significantly increased at h after mwa indicating that il2 may induce a nonspecific immune response after mwa but il decreased after h postmwa in our study suggesting that the il2induced immune response may not belong lasting mentionable many cytokines detected il8il1 il12 were mainly derived from macrophagewhich was a widely distributed antigen presenting cellthis result support the theory that mwa could releasefragment of cancer cells into blood as neoantigen macrophages could response to this proceed and cause a systemic immunoreaction additional cytokines alterationsuch as il6 after ablation may be no anspecific inliver evidences indicate that increase of il6 was not onlyoccurred in liver ablation researches focus on lung cancerincluding primary lung cancer and pulmonary metastasesdemonstrated that serum il8 il1 il6 il10 il12and tnfα were significantly raised after radiofrequencythermal ablation moreover joseph found that imageguided thermal ablation of tumors located in lung liver orsoft tissues increases plasma levels of il6 and il10 another question remain unveiled was if our result wasœcancerspecific we checked literature about cytokinemodulation after thermal ablation in benign diseases andonly got limit evidences based on benign thyroid nodules and adenomyosis according to these literatureil6 levels did not show any significant difference aftertreatment compared with pretreatment values indicatingthat elevation of il6 may be caused by tumour antigenreleased by ablation treatment however the ablationenergy used in thyroid nodules was much lower thanliver and lung which would lead to a false negativein cytokine detection to the research about adenomyosis on the other hand experiment design was determined to followup the il6 at months afterhifu ablation as our data demonstrated mostly cytokines were return to premwa level after monthdetection after months may miss the modulation ofil6 overall few evidences support that some of thecytokines were altered in a œcancerspecific mannerwhile no solid results could confirm that further animal experiments were required to make a clarifieddata and answer this question 0czhao bmc cancer page of thetumorassociated immunein recent years ablationinduced systemic effects suchasresponse haveattracted increased attention de baere t first reported two cases of spontaneous regression of multiplepulmonary metastases occurring after radiofrequencyablation of a single lung metastasis although growing evidence suggests that thermal ablation can inducespontaneous regression of the socalled œabscopal effecton distant tumors the incidence rate of such an effect israre probably due to uncontested immunological activation caused by one ablation treatment and the lack ofimmuneamplification management in it was described that in situ tumor destruction can provide a useful antigen source forthe induction of antitumorimmunity however clinical studies could not sufficiently utilize such an effect until the development ofimmune checkpoint inhibitors icis [ ] icis suchas pd1pdl1 and ctla4 antibodies are widely applied in several cancers and studies have indicated thatici treatment could enhance the effect of ablation evidence indicates that hyperthermic destruction causesthe release of a large population of heterogeneous tumorantigens and inflammatory cytokines may play crucialroles in this process however opposite evidence indicated that incomplete radiofrequency ablation couldinduce inflammation which may accelerates tumor progression and hinders pd1 immunotherapy suggesting that ablation treatment may promote tumorprogression our data demonstrated that il6 was significantly increased after mwa treatment il6 is derived from monocytes macrophages dcs th2 cells andsometimes cancer cells and it plays a key role in t cellproliferation and survival the role of il6 appearsto be rather complex korn classified il6 as œdifferentiation factor which could involve in differentiation ofth17 cells however il6 does not direct the commitment to the th1 or th2 cell lineage but controls theproliferation and survival of immunocytes cooperatingwith other cytokines such as tgf tnf or il21 for instance il6 activated stat3 pathway in naivecd4 t cells in the presence of the morphogen tgfbpromotes the population expansion of th17 cells recent evidence indicates that il6 plays an indispensable role in t cellinfiltration to the tumor sitewhich could benefit immunomodulatory therapy incontrast il6 can increase mdscs inhibit the development and maturation of dendritic cells dcs and inhibit the polarization of th1 cells eventuallyresulting in negative immunomodulatory effects according to muneeb ahmed™s work the adjuvant uses ofa nanop smallinterfering rna sirna can besuccessfully used to target the il6mediated locoregional and systemic effects of thermal ablation il6 knockout via a nanop antiil6 sirna in mice coulddecrease the local vegf level at the ablation site therefore how to utilize the positive effect of il6 whileavoiding the negative effect after mwa needs further investigation preclinical research indicated that il6 andpdl1 blockade combination therapy reduced tumorprogression in animal models [ ] thus an antiil strategy after ablation should be considered whencombined with ici therapy previous studies and ourshave demonstrated that most cytokine levels returned topretreatment levels days after ablation this resultsuggests that h to days after ablation may be optimal timing for additional immunomodulatory therapysour results reported here support the evidence for terminal tumor thermal ablation could cause heat injury totissues surrounding the tumor site and release neoantigento blood circulation eventually induce a systemic reactionthis reaction could lead to a detectable alteration of cytokine levels further investigation is required to revealwhether the cytokines altered by mwa treatment couldaffect cancer progression whether positive or negativeabbreviationsmwa microwave ablation hcc hepatocellular carcinoma icis immunecheckpoint inhibitors tnf tumor necrosis factor il interleukinvegf vascular endothelial growth factor sd stable disease pr partialresponse ct computed tomography ci confidence interval ltp likelihoodof local tumor progression mdscs myeloidderived suppressor cellsctls cytotoxic t lymphocytes nk natural killer sirna small interfering rnaacknowledgementsnot applicableauthors™ contributionsjz conceptualization data curation writing“original draft and writing“review and editing ql conceptualization and writing“review and editingmm conceptualization and writing“review and editing brconceptualization and writing“review and editing and collect samples yhexecute milliplex assay and collect data dpl patient enrollment executemwa ablation and collect samples zl execute mwa ablation and collectsamples dml patient enrollment execute mwa ablation and collectsamples yx execute milliplex assay and collect data mt conceptualizationand writing“review and editing rl conceptualization data curation formalanalysis visualization writing“original draft and writing“review and editingall authors have read and approved the manuscriptfundingthis work was supported by the national natural science foundation ofchina the natural science foundation ofjiangsu province of china bk20140295 the jiangsu governmentscholarship for oversea studies js2018179 and the œsix one projects forhighlevel health personnel in jiangsu province lgy2018077availability of data and materialsthe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethe protocol was approved by the human ethics committee of the firstaffiliated hospital of soochow university and was conducted in accordancewith the declaration of helsinki patients were informed that the bloodsamples were stored by the hospital and potentially used for scientific 0czhao bmc cancer page of research and that their privacy would be maintained all written informedconsent was obtained from all participants and clearly statedconsent for publicationnot applicablecompeting intereststhere is no financial or personal relationship with other people oranizations that could inappropriately influence bias this workauthor details1department of radiation oncology the first affiliated hospital of soochowuniversity suzhou china 2department of oncology the first affiliatedhospital of soochow university suzhou china 3department of lymphatichematologic oncology jiangxi cancer hospital nanchang china4department of interventional radiology the first affiliated hospital ofsoochow university suzhou china 5division of neurosurgery city of hopebeckman research institute duarte california usareceived january accepted august referencesfu j wang h precision diagnosis and treatment of liver cancer in chinacancer lett “bruix j han kh gores g llovet jm mazzaferro v 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expression level in nasopharyngealcarcinoma patients after treatment with definitive intensitymodulatedradiotherapy imrt plos one 2017122e0172264kim mj jang jw oh bs kwon jh chung kw jung hs jekarl dw lee schange in inflammatory cytokine profiles after transarterial chemotherapy inpatients with hepatocellular carcinoma cytokine “ gillams a goldberg n ahmed m bale r breen d callstrom m chen mhchoi bi de baere t dupuy d thermal ablation of colorectal livermetastases a position paper by an international panel of ablation expertsthe interventional oncology sans frontieres meeting eur radiol “ ahmed m solbiati l brace cl breen dj callstrom mr charboneau jwchen mh choi bi de baere t dodd gd 3rd imageguided tumorablation standardization of terminology and reporting criteriaa 10yearupdate radiology “ chiang j hynes k brace cl flowdependent vascular heat transfer duringmicrowave thermal ablation conf proc ieee eng med biol soc “ huang hw influence of blood vessel on the thermal lesion formationduring radiofrequency ablation for liver tumors med phys najjar yg rayman p jia x pavicic pg jr rini bi tannenbaum c ko jhaywood s cohen p hamilton t myeloidderived suppressor cellsubset accumulation in renal cell carcinoma parenchyma is associated withintratumoral expression of il1beta il8 cxcl5 and mip1alpha clin cancerres “ alfaro c teijeira a onate c perez g sanmamed mf andueza mp alignanid labiano s azpilikueta a rodriguezpaulete a tumorproducedinterleukin8 attracts human myeloidderived suppressor cells and elicitsextrusion of neutrophil extracellular traps nets clin cancer res “kundu m roy a pahan k selective neutralization of il12 p40 monomerinduces death in prostate cancer cells via il12ifngamma proc natl acadsci u s a “ onishi h kuroki h matsumoto k baba e sasaki n kuga h tanaka mkatano m morisaki t monocytederived dendritic cells that capture deadtumor cells secrete il12 and tnfalpha through il12tnfalphanfkappabautocrine loop cancer immunol immunother “ yu z geng j zhang m zhou y fan q chen j treatment of osteosarcomawith microwave thermal ablation to induce immunogenic cell deathoncotarget “ yang w wang w liu b zhu b li j xu d ni y bai l liu gimmunomodulation characteristics by thermal ablation therapy in cancerpatients asia pac j clin oncol 2018145e490“erinjeri jp thomas ct samoilia a fleisher m gonen m sofocleous ctthornton rh siegelbaum rh covey am brody la imageguidedthermal ablation of tumors increases the plasma level of interleukin6 andinterleukin10 j vasc interv radiol “ den brok mh sutmuller rp van der voort r bennink ej figdor cg ruerstj adema gj in situ tumor ablation creates an antigen source for thegeneration of antitumor immunity cancer res “ zerbini a pilli m laccabue d pelosi g molinari a negri e cerioni sfagnoni f soliani p ferrari c radiofrequency thermal ablation forhepatocellular carcinoma stimulates autologous nkcell responsegastroenterology “ zhang h hou x cai h zhuang x effects of microwave ablation on tcellsubsets and 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Millions of people are suffering from cancers but accurate early diagnosis and effectivetreatment are stilllong noncoding RNAslncRNAs have been proven to play an important role in diseases especially cancersThese lncRNAs execute their functions by regulating gene expression Thereforeidentifying lncRNAs which are related to cancers could help researchers gain a deeperunderstanding of cancer mechanisms and help them find treatment options A largenumber of relationships between lncRNAs and cancers have been verified by biologicalexperiments which give us a chance to use computational methods to identifycancerrelated lncRNAs In this paper we applied the convolutional neural network CNNto identify cancerrelated lncRNAs by lncRNA™s target genes and their tissue expressionspecificity Since lncRNA regulates target gene expression and it has been reportedto have tissue expression specificity their target genes and expression in differenttissues were used as features of lncRNAs Then the deep belief network DBN wasused to unsupervised encode features of lncRNAs Finally CNN was used to predictcancerrelated lncRNAs based on known relationships between lncRNAs and cancersFor each type of cancer we built a CNN model to predict its related lncRNAs Weidentified more related lncRNAs for kinds of cancers Tencross validation has beenused to prove the performance of our method The results showed that our method isbetter than several previous methods with area under the curve AUC and areaunder the precision“recall curve AUPR To verify the accuracy of our results casestudies have been doneKeywords long noncoding RNA lncRNA cancer convolutional neural network CNN deep belief network DBNmachine learningINTRODUCTIONFour to nine percent of the sequences™ transcription are long noncoding RNAs lncRNAs inmammalian genomes Canzio Ji lncRNA was regarded as the noise ofgenome transcription and did not have biological functions at first However an increasing numberof studies have reported that lncRNA is widely Robinson involved in chromosomeEdited byLei DengCentral South University ChinaReviewed byHao LinUniversity of Electronic Science andTechnology of China ChinaInner Mongolia University ChinaJuan WangCorrespondenceNan Dudunan05aliyuncomGanfeng Xiexiegfaliyuncom These authors share first authorshipSpecialty sectionThis was submitted toMolecular Medicinea section of the journalFrontiers in Cell and DevelopmentalBiologyReceived June Accepted June Published August CitationLiu Z Zhang Y Han X Li C Yang XGao J Xie G and Du N Identifying CancerRelated lncRNAsBased on a Convolutional NeuralNetwork Front Cell Dev Biol 103389fcell202000637Frontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cLiu et alA Method to Identify CancerRelated lncRNAsgenomicimprintingchromatin modificationsilencingtranscriptional activationinterference andnuclear transport Cheng 2018a Recently it has beenproven to be associated with many kinds of cancerstranscriptionalThe secondary structure spliced form and subcellularlocalization of most lncRNAs are conserved Karner which is very important for lncRNA to execute functionsHowever compared to the functions of microRNAs miRNAsand proteins the function oflncRNA is more difficult todetermine According to the position of lncRNA in the genomerelative to proteincoding genes it can be divided into five typessense antisense bidirectional intronic and intergenicMany researchers have found lncRNAs play an important rolein cancers Avgeris Cheng 2018b Zhao and neurodegenerative diseases Peng and Zhao as other biological molecules Zhang T Bai Cheng 2019a Liang Although manyresearchers have verified many associations between lncRNAsand cancers by biological experiments compared with ourknowledge about diseaserelated genes we still do not knowenough about diseaserelated lncRNAs Considering the timeand money cost of finding diseaserelated lncRNAs more andmore researchers tend to use computational methods to identifydiseaserelated lncRNAs These methods could be divided intothree categories machine learning methods network methodsand other methodsMachine learning methods build models based on thesimilarities of diseases orlncRNAs and their biologicalcharacteristics Cheng Cheng 2019b Zeng Zou Lan developed thelncRNA“disease association prediction LDAP which is amethod based on bagging support vector machine SVM toidentify lncRNA“disease associations They used similarities oflncRNAs and diseases as the features Yu developedcollaborative filtering naive Bayesian classifier CFNBC based onnaive Bayesian They integrated miRNA“lncRNA associationsmiRNA“disease associations and lncRNA“disease associationsto infer more lncRNA“disease associations Considering thediscriminative contributions of the similarity association andinteraction relationships among lncRNAs disease and miRNAsXuan 2019a developed a dual convolutional neuralnetwork CNN with attention mechanisms to predict diseaserelated lncRNAsNetwork methods are the most common way to identifyassociations between diseases and lncRNAs nowadays Gu Yu Zhang J Kuang Wang L Liu Thiskind of method would build one or multiple networks toinfer new information Wang L built a lncRNA“miRNA“disease interactive network and used their novel methodœLDLMD to predict associations between lncRNAs and diseasesSumathipala used a multilevel network topologywhich includes lncRNA“protein protein“protein interactionprotein“disease relationship to use network diï¬usion algorithmto predict diseaserelated lncRNAs The graph convolutionalnetwork GCN and CNN were used on a lncRNA“miRNA“disease network by Xuan 2019b Deng builtlncRNA similarity network disease similarity network miRNAsimilarity network and their associations Then they calculatedthe metapath and feature vector for each lncRNA“disease pair inthe heterogeneous information networkOther methods may borrow the feature extraction methodor similarity conjecture of network methods but the core ofthis method is matrix decomposition or matrix completionLu developed the geometric matrix completionlncRNA“disease association GMCLDA which is a methodbased on geometric matrix completion They calculated diseasesimilarity based on Disease Ontology DO and calculatedthe Gaussian interaction profile kernel similarity for lncRNAsThen they inferred diseaserelated lncRNAs based on theassociation patterns among functionally similar lncRNAs andsimilar diseases Wang Y proposed a weightedmatrix factorization to capture the interintraassociationsbetween diï¬erent types of nodes Then they approximated thelncRNA“disease association matrix using the optimized matricesand weights to predict diseaserelated lncRNAs Localityconstrained linear coding label propagation Latent DirichletAllocation LLCLPLDA was developed by Xie Firstly localconstraint features of lncRNAs and diseases wereextracted by localityconstrained linear coding LLC Thenthey predicted diseaserelated lncRNAs by label propagationLP strategyHowever previous methods did not consider the regulatingtarget gene expression of lncRNA which is an important functionof lncRNA and plays an important role in associations betweenlncRNAs and diseases In addition deep learning methods arean important tool and have shown their power in bioinformaticsChen Lv Wei Wu Zhao 2019abc Therefore in this paper we used thisinformation as features of lncRNA In addition the expressionof lncRNA in diï¬erent tissues were also used as the featuresof lncRNA Then the deep belief network DBN was used toencode and the CNN was used to classifyMETHODSFeature ExtractionTissue Expression Specificity of Long NoncodingRNACompared with proteincoding geneslncRNA shows strongtissue specificity The specificity of lncRNAs in diï¬erent kindsof tissues and cell types has been proven by many biologicalexperiments The diï¬erent expression also plays an importantrole in essential cellular processes Sasaki testedthe expression of lncRNAs in diï¬erent tissues and found lncRNAs exhibited tissuespecific expression and oflncRNAs were only expressed in one discrete tissue Thereforethe expression of lncRNAs in diï¬erent tissues were used asthe featuresWe obtained the expression of lncRNAs in diï¬erenttissues which included adipose adrenal breast colon heartkidney liver lung lymph node ovary placenta prostate testisand thyroidTherefore the dimension of each lncRNA™s expression featureis ˆ— Frontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cLiu et alA Method to Identify CancerRelated lncRNAsTherefore the dimension of each lncRNA™s target gene featureis ˆ— Deep Belief NetworkThe DBN can eï¬ectively learn complex dependencies betweenvariables Zhao 2019d The DBN contains many layers ofhidden variables which can eï¬ectively learn the internal featurerepresentation of the data and can also be used as an eï¬ectivenonlinear dimensionality reduction methodWhen the observable variables are known the joint posteriorprobabilities of the hidden variables are no longer independentof each other so it is difficult to accurately estimate the posteriorprobabilities of all hidden variables The posterior probability ofearly DBN is generally approximated by Monte Carlo methodbut its efficiency is relatively low which makes its parameterlearning difficult In order to eï¬ectively train the DBN weconvert the sigmoid belief network of each layer to a restrictedBoltzmann machine RBM The advantage of this is that theposterior probabilities of the hidden variables are independentof each other which makes it easy to sample In this way theDBN can be regarded as being stacked from top to bottom bymultiple RBMs and the hidden layer of the Lth RBM is used asthe observable layer of the L 1th RBM Further the DBN canbe trained quickly by layerbylayer training that is starting fromthe bottom layer and training only one layer at a time until thelast layer The specific layerbylayer training process is to trainthe RBM of each layer in turn from bottom to top Assuming wehave trained the RBM in the first L1 layer we can calculate theconditional probability of the bottomup hidden variablesphihiˆ’ σ bi Wihiˆ’where bi is the bias of ith layer of RBM Wi is the connectionweight hi is the ith layer of RBMThe process of training DBN is as followsFIGURE The number of target genes for each long noncoding RNAlncRNAFIGURE The distribution of the number of target genes lncRNA longnoncoding RNAreverseTarget Gene of Long Noncoding RNAQuantitativechainreaction qRTPCR and Western blot were used to testthe diï¬erentexpression genes after knocking down oroverexpressing lncRNAstranscriptasepolymeraseWe obtained target genes of lncRNA from LncRNA2TargetInput train dataset ˆvn learning rate λJiang As we can see in Figure there are kinds of lncRNAsOne lncRNA has more than target genes Then we drawthe distribution of the number of target genes correspondingto lncRNAAsshown in Figure most ofthe target genes arecorresponding to less than five lncRNAs Therefore if we usedthem to be the features of lncRNAs the features would be sparseTherefore we only select the most common target genes to bethe features The genes which are corresponding to more thanfive lncRNAs were selected as the features of lncRNAs There are kinds of genes Then we need to encode these genesF [G1 G2 · · · G45]where G1 denotes the first gene of these genes and F denotesthe feature of lncRNA For each lncRNA if G1 is the target geneof it then G1 otherwise G1 Output weight matrix Wl bias al and blFor l 1LInitialization Wi al bi Sample from train dataset ˆh0For i lˆ’Sample hi based on phi ˆhiˆ’EndSet hi1as the train sample to train lth layer ofRBMEndSince the dimension of expression feature and target genefeature are diï¬erent we should reduce the dimension of targetgene feature and make it the same as the expression feature™sTherefore in this paper two layers of RBM were used to builda DBN modelThe number of nodes oftheand respectively Sigmoid function was used astwo layers was theFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cLiu et alactivation functionσ x eˆ’xTherefore the dimension of final features is ˆ— F cid20 G1 G2 · · · G13E1 E2 · · · E13 cid21A Method to Identify CancerRelated lncRNAsConvolutional Neural NetworkThe power of CNN in dealing with bioinformatic problems hasbeen proven by many researchers We selected CNN as theclassifier based on two reasons The dimension of features is ˆ— which can be regarded as an image The outstandingperformance of CNN in image classificationThere are five layers in our CNN model The structure of CNNis shown as Table where G1 G2 · · · G13 denotes target gene feature after DBNand E1 E2 · · · E13 denotes the expression of lncRNAs in diï¬erent tissuesTABLE The structure of convolutional neural network CNNLayersParameterConvolutional layerPooling layerConvolutional layerPooling layerFully connected layerOutputFilter kernel size Activation function tanhpool size Activation function tanhFilter kernel size Activation function tanhpool size Activation function tanhUnits Activation function tanhUnits Activation function sigmoidWork FrameFigure shows the work frame of our method œDBN“CNNThere are three steps of our methods Firstly we should extractfeatures of lncRNAs There are two parts of features expressionfeature and target gene feature Then DBN was used to encodethe target gene feature After encoding the two kinds of featureswere combined together Finally CNN was used to classifyRESULTSData DescriptionThe known associations between lncRNA and diseases wereobtained from LncRNADisease database Bao Wetotally obtained kinds of cancerrelated lncRNAs The numberof their corresponding lncRNAs is shown as Figure As shown in Figure People™s understanding of cancerrelated lncRNAs varies widely We have known more than lncRNAs for some cancers but few lncRNAs are known for somecancers To better build our model we only selected cancerswhich have more than related lncRNAs Therefore kindsof cancers were selectedFIGURE Work frame of deep belief network DBN“convolutional neural network CNN lncRNA long noncoding RNAFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cLiu et alA Method to Identify CancerRelated lncRNAsFIGURE The number of long noncoding RNAs lncRNAs for each cancerTABLE The performance of deep belief network DBN“convolutional neuralnetwork CNN in cancersCancerArea undercurve AUCArea under precisioncurve AUPRCervical cancerBreast cancerColorectal cancerStomach cancerUrinary bladder cancerLung cancerOvarian cancerThyroid cancerProstate cancerLiver cancerPancreatic cancerOvarian epithelial cancerGallbladder cancerEndometrial cancerColon cancerEsophageal cancerThetargetgenes oflncRNAs were obtained fromLncRNA2Target database We have discussed about this insection Target Gene of Long Noncoding RNAFIGURE The receiver operating characteristic ROC curves of the threemethods DBN deep belief network CNN convolutional neural network PCAprincipal component analysisFIGURE The area under the precision“recall curve AUPR of the threemethods DBN deep belief network CNN convolutional neural network PCAprincipal component analysisThe expression oftissues wasobtained from NONCODEV5 Zhao We only usedhuman datalncRNAs in diï¬erentThe Performance of Deep BeliefNetwork“Convolutional Neural NetworkWe did 10cross validation on each cancer Area under the curveAUC Cheng Dao Zhang and areaunder the precision“recall curve AUPR were used to evaluatethe performance of DBN“CNN The results are shown in Table As we can see in Table the performance of DBN“CNN isquite diï¬erent in diï¬erent cancers This may be caused by thediï¬erent sample sizes The average AUC is and AUPR is Comparison ExperimentsTo verify the superior of DBN“CNN we compared it with similarmethods Since the main function of DBN is to reduce dimensionprincipal component analysis PCA has the same functionTherefore instead of using DBN to encode we used PCA thistime and CNN was used to classify the features after PCA We callthis method PCA“CNN In addition we also used the deep neuralnetwork DNN to replace CNN so this comparison method wascalled DBN“DNNWe used these three methods to test on cancers andsummarized the results to get a final AUC and AUPR for eachmethod The receiver operating characteristic ROC curves areshown in Figure As shown in Figure the blue curve denotes the results ofDBN“CNN The red and black curves denote PCA“CNN andDBN“DNN respectively As we can see DBN“CNN performedbest among these three methods The AUC of DBN“CNN is which is better than and for PCA“CNN andDBN“DNN respectivelyFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cLiu et alA Method to Identify CancerRelated lncRNAsAs shown in Figure the AUPR of DBN“CNN is the highestwith the least standard errorCase StudyLiu found down syndrome cell adhesion molecule antisense RNA DSCAMAS1 is associated with breast cancerby constructing two suppression subtracted cDNA librariesMartensUzunova reported the associationbetween H19 and bladder cancer They also pointed out that H19could be the biomarker of bladder cancerShi measured the expression level of lncRNAsLoc554202 in breast cancer tissues and found that Loc554202was significantly increased compared with normal control andassociated with advanced pathologic stage and tumor sizeCONCLUSIONSIncreasing evidence has shown the relationship between lncRNAsand cancers lncRNAs could be the biomarkers to help diagnosecancer and also help researchers understand the mechanismof cancers Compared with people™s knowledge of diseaserelated protein coding genes we knew few about diseaserelated lncRNAs However the biological experiments for findingdiseaserelated lncRNAs are timeconsuming and expensiveTherefore in this paper we proposed a novel method foridentifying cancerrelated lncRNAs We called this methodœDBN“CNN which is a fusion of DBN and CNN Two kindsof features were used based on the biological background 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performedbest Finally case studies have been done to verify the accuracy ofour results We found potential lncRNAs for kinds of cancerswhich can be a kind of guidance for researchers finding novelcancerrelated lncRNAsDATA AVAILABILITY STATEMENTThe datasets presented in this study can be found in onlinerepositoryrepositoriesrepositories Theandnumbersbethesupplementary materialaccessionnamesfoundcantheofinAUTHOR CONTRIBUTIONSND and GX designed the research ZL performed the researchand wrote the manuscript YZ and XH acquired the dataand reviewed and edited the manuscript CL XY and JGanalyzed the data All authors reviewed the manuscript andprovided commentsinformation flow by a random walk BMC Genomics 19Suppl 101186s1286401743386Cheng L Yang H Zhao H Pei X Shi H Sun J 2019a MetSigDisa manually curated resource for the metabolic signatures of diseases BriefBioinform “ 101093bibbbx103Cheng L Zhao H Wang P Zhou W Luo M Li T 2019bComputational Methods for identifying similar diseases molecular 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Biol Bioinform “multiple heterogeneous networks for novelinference 101109TCBB20172701379Zhang T Tan P Wang L Jin N Li Y Zhang L RNALocate aresource for RNA subcellular localizations Nucleic Acids Res D135“D138 101093nargkw728Zhang Z M Tan J X Wang F Dao F Y Zhang Z Y and LinH Early diagnosis of hepatocellular carcinoma using machinelearning method Front Bioeng Biotechnol 103389fbioe2020Zhao T Cheng L Zang T and Hu Y 2019a Peptidemajor histocompatibilitycomplex class I binding prediction based on deep learning with novel featureFront Genet 103389fgene201901191and Cheng LIdentifyingAlzheimer™s diseaserelated proteins by LRRGD BMC Bioinform 101186s1285901931247Zhao T Hu Y Zang T2019bZhao T Hu Y Zang T and Cheng L MRTFB regulates the expressionof NOMO1 in colon Proc Natl Acad Sci USA 101073pnas2000499117Zhao T Hu Y Zang T and Wang Y 2019c Integrate GWAS eQTLand mQTL Data to Identify Alzheimer™s diseaserelated genes Front Genet 103389fgene201901021Zhao T Wang D Hu Y Zhang N Zang T and Wang Y 2019d IdentifyingAlzheimer™s diseaserelated miRNA based on semiclustering Curr Gene Ther “ Zhao Y Li H Fang S Kang Y Wu W Hao Y NONCODE an informative and valuable data source of long noncoding RNAs NucleicAcids Res D203“D208 101093nargkv1252Zou Q Xing P Wei L and Liu B Gene2vec gene subsequenceembedding for prediction of mammalian N6methyladenosine sites frommRNA RNA “ 101261rna069112118Conflict of Interest The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestCopyright Liu Zhang Han Li Yang Gao Xie and Du This is an openaccess distributed under the terms of the Creative Commons Attribution License CCBY The use distribution or reproduction in other forums is permitted providedthe original authors and the copyright owners are credited and that the originalpublication in this journal is cited in accordance with accepted academic practiceNo use distribution or reproduction is permitted which does not comply with thesetermsFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0c'
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" microwave ablation mwa is widely used to treat unresectable primary and secondary malignanciesof the liver and a limited number of studies indicate that ablation can cause not only necrosis at the in situ site butalso an immunoreaction of the whole body this study aimed to investigate the effects of mwa on cytokines inpatients who underwent mwa for a hepatic malignancymethods patients admitted to the oncology department in the first affiliated hospital of soochow universitybetween june and february were selected peripheral blood was collected from patients with a hepaticmalignancy treated with mwa the levels of cytokines il2 ifnÎ tnfα il12 p40 il12 p70 il4 il6 il8 il10and vascular endothelial growth factor vegf were detected with a milliplex® map kit the comparison times wereas follows before ablation h after ablation days after ablation and days after ablation data were analyzedusing a paired sample ttests and spearman™s correlation analysisresults a total of patients with hepatic malignancies were assessed there were significant differences in il2il12 p40 il12 p70 il1 il8 and tnfα at h after mwa significant increases 2fold vs before ablation wereobserved in il2 il1 il6 il8 il10 and tnfα after mwa elevated il2 and il6 levels after ablation werepositively correlated with energy output during the mwa procedures wa treatment for hepatic malignancies can alter the serum levels of several cytokines such as il2 and il6keywords microwave ablation hepatic malignancy cytokines il2 il6 immunoregulation primary and secondary malignancies of the liver have asubstantial impact on morbidity and mortality worldwidein china hepatocellular carcinoma hcc has the secondhighest mortality rate of malignancies the treatmentof primary and secondary hepatic malignancies via correspondence lengbengsudaeducn jing zhao qiang li and merlin muktiali contributed equally to this work2department of oncology the first affiliated hospital of soochow universitysuzhou china5division of neurosurgery city of hope beckman research institute duartecalifornia usafull list of author information is available at the end of the interventional imaging therapy is undertaken by investigators in the field of interventional radiology and possibly bya smaller group of practitioners known as interventionaloncologists whose major focus is cancer care via minimally invasive approaches [ ] recently percutaneous ablation therapy has been widely accepted as a radicaltreatment method for hcc and its fiveyear survival rateis similar to that of resection microwave ablationmwa is widely used to treat unresectable hcc and recurrent hcc and has the advantages of minimal invasiona good curative effect and no side effects due to radiationor chemotherapy immune checkpoint inhibitors icis the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhao bmc cancer page of such as pd1pdl1 and ctla4 antibodies have beenwidely applied in several cancers and studies have indicated that ici treatment could enhance the effect of ablation evidence hasindicated that hyperthermicdestruction causes the release of a large population of heterogeneous tumor antigens and inflammatory cytokinesmay play crucial roles in this process cytokines aremediators that regulate a broad range of processes involved in the pathogenesis of cancer several cytokineswhich can arise from either tumor cells or immunocytes such as tumor necrosis factor tnfα interleukinil1 il6 il8 il10 and vascular endothelial growthfactor vegf have been linked with cancers and can either promote or inhibit tumor development the serumlevels of cytokines differ during cancer development although cytokines have been found to be altered after anticancer treatment such as chemotherapy and radiotherapy[ ] few investigations have focused on cytokines beforeand after mwa it is still unknown whether the above cytokines changed before andor after mwa in patientswith hepatic malignancies in this study we investigatedthe effects of mwa on the serum levels of cytokines inpatients with hepatic malignanciesmethodspatients and samplesthe patient population examined in this study was derivedfrom the first affiliated hospital of soochow universitypatients were admitted to the oncology department between june and february the total number ofpatients was with liver metastases and primaryliver cancers the inclusion criterion was a tumor locatedat a hepatic site either primary or metastases all patients with metastatic hepatic malignances should be givensystematic treatments chemotherapy or target therapyand get at least stable disease sd or partial responsepr for more than days informed consent for blooddraw and the relevant therapy was obtained from all patients the protocol was approved by the human ethicscommittee of the first affiliated hospital of soochowuniversity and was conducted in accordance with thedeclaration of helsinki all written informed consent wasobtained from all participants and clearly stated wholeblood ml was drawn into edta anticoagulant tubeson days ˆ’ to before and h days and days afterablation mostly on the last day of the course for cytometry and cytokine analysesablation procedurethe ablation procedure used in this research was mwathe puncture site and pathway were determined underthe guidance of a computed tomography ct scanlocal infiltration anesthesia was achieved by using lidocaine the placement of microwave ablation probeswas guided by a ct scan or ultrasonic device and allprobes were placed at the maximum diameter layerdouble probes were employed when the maximumdiameter of the tumor was up to cm the power andtime of ablation were designed for each patient in therange of w and min respectively basedon the size number and position of the tumor theboundaries of ablation zones were designed as extended cm upon the tumor sitecytokine detectiona milliplex map kit with human cytokinechemokinepanels that measured ifnÎ il2 il6 il8 il10 il12p40 il12 p70 il1 tnfα and vegf was utilized according to the manufacturer™s instructions briefly chemically dyed antibodybound beads were mixed withstandard or sample incubated overnight at °c washedand then incubated with a biotinylated detection antibodyafter the beads were washed they were incubated with astreptavidin phycoerythrin complex and the mean fluorescent intensities were quantified on a luminex analyzer luminex corporation all samples were measured in duplicate standard curves of known concentrations of recombinant human cytokineschemokines wereused to convert fluorescence units to cytokine concentration units pgml the minimum detectable concentrations were as follows ifnÎ pgml il2 pgmlil12 p40 pgml il12 p70 pgml il1 pgml il6 pgml il8 pgml il10 pgml tnfα pgml and vegf pgml all resultsbelow the minimum concentrations were processed as theminimum concentrationsstatistical analysisibm spss statistics software was used for the statistical analysis along with graphpad prism for figurecreations normally distributed numerical data areexpressed as the mean ± standard deviation and nonnormally distributed numerical data are expressed as themedian and confidence interval ci cytokinesat different times were compared using a onetailedpaired ttest spearman™s correlation analysis was executed to determine the correlation between clinical indexes and cytokine levels p indicates a significantdifferenceresultsclinical characteristics of the enrolled patientsas shown in table a total of patients with tumorslocated on the liver liver metastases primary livercancers were analyzed the patients™ cytokine levelswere compared according to time before treatment h after treatment days after treatment and daysafter treatment 0czhao bmc cancer page of table clinical characteristics of the patients enrolled n characteristicsexmalefemaleagepathogenesisprimarysecondaryprimary site for metastatic hepatic malignancescolon rectalpancreasstomachebreastothersmaximum tumor length mmablation probe usedablation time minaverage power per probe w ± ± ± ± average energy time × power time × power–¼–¼ time and power indicate the time and power respectively ofdifferent probes used during the operation ± ifnÎ il12 p40 and il12 p70 were slightly increasedafter mwa treatmentas shown in table and fig the median level ofifnÎ before the mwa treatment was pgml ci “ pgml at days and days after themwa treatment there was a slight increase comparedto that premwa with median levels of pgml ci “ pgml and pgml ci“ pgml respectively the median level of il p40 before the mwa treatment was pgml ci “ pgml there was a slight increase to pgml ci “ pgml days postmwathe median il12 p70 level before the mwa treatmentwas pgml ci “ pgml and increasedto pgml ci “ pgml days afterthe mwa treatment and to pgml ci “ pgml days postmwa no significant alteration in the vegf median level was detected after themwa treatmentil2 il1 il6 il8 and il10 were elevated over 2foldafter the mwa treatmentas shown in table fig and fig the median levelof il2 before the mwa treatment was pgml ci “ pgml there was a significant increase at h postmwa with a median level of pgml ci “ pgml the median level ofil1 before the mwa treatment was pgml ci “ pgml and a significantincrease wasnoted days after the mwa treatment pgml ci “ pgml the median level of il6before the mwa treatment was pgml ci“ pgml and significantly increased daysafter the mwa treatment pgml ci “ pgml the median level ofil8 before themwa treatment was pgml ci “ pgml and increased significantly to pgml ci“ pgml days after the mwa treatmentthe median level of il10 before the mwa treatmentwas pgml ci “ pgml and increasedsignificantly days after the mwa treatment pgml ci “ pgml the median level oftnfα before the mwa treatment was pgml ci “ pgml and increased significantlyto pgml ci “ pgml days afterthe mwa treatmentlevelselevated il2 and il6 levels after ablation were positivelycorrelated with energy output during mwato further evaluate the relationship between the increased cytokineand mwa treatment weemployed the concept of œenergy time × power time × power time and power indicated thetime and power of different probes used in the operation to reflect total hyperthermic damage to hepatictissues during the mwa procedure as shown in table and fig the il2 levels at h postmwa and the il levels at days postmwa illustrated significant correlations with energy the relative indexes were and respectivelydiscussionas technology continues to develop other types of localtherapy such as radiotherapy chemical ablation andhyperthermal ablation for primary and metastatic livercancer are increasingly being used mwa for liver malignances is reserved for patients who cannot undergosurgical removal or for whom other treatments havefailed a consensus guideline was recently developed to address indications for mwa in these patientsthermal ablation is a process that heats the target tissueto a temperature that causes immediate coagulative necrosis usually over °c terminal treatment requiresthat a necrotic area surrounds the target site with anadditional “10mm margins however in the liverhigh tissue perfusion and large blood vessels can cause aœheat sink effect around the ablation zone making itdifficult to achieve terminal ablation the heat sink 0czhao bmc cancer page of table median levels of cytokines before and after mwacytokineifnÎil2premwa pgml ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ h postmwa pgml ci “ ci “ –¼ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “il12 p40il12 p70il1il6il8il10tnfαvegf p vs premwa –¼ 2fold vs premwa days postmwa pgml ci “ ci “ ci “ ci “ ci “ –¼ ci “ –¼ ci “ –¼ ci “ –¼ ci “ –¼ ci “ days postmwa pgml ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “effect can lead to sublethal temperatures and the retention of malignant cells thereby increasing the likelihoodof local tumor progression ltp however an incompletely ablated zone containing immune cells andcancer cells as well as functional vessels could establisha serious inflammatory site that may provide tumorspecific antigens cytokines and activated immune cellsin our study significant increases in the secretion ofchemokines il8 proinflammatory cytokines il1il12 ifnÎ and tnfα and antiinflammatory cytokines il10 were observed after mwa il8 is mainlyproduced by macrophages the classical biological activity of il8 is to attract and activate neutrophils whichcan lead to a local inflammatory response however recent studies have indicated that il8 both macrophageand cancer cellderived can recruit myeloidderivedsuppressor cells mdscs into the tumor microenvironment eventually inhibiting antitumor immunity andpromoting cancer progression [ ] il1 is mainlyproduced by macrophages b cells and nk cells couldproduce il1 under certain circumstances generallycells can only synthesize and secrete il1 after beingstimulated by foreign antigens or mitogens il1 couldpromote the th1 response promoting the activation ofdendritic cells dcs and cytotoxic t lymphocytesctls il12 is mainly produced by b cells and macrophages human il12 is a heterodimer with two subunits p40 kd and p35 kd which areinactivated in isolated form in general il12 functionsas a combination of two subunits il12 p70 while p40alone possesses partial functions of il12 p70 it™s mentionable that il12 p40 and p35 are not expressed inequal proportions so the amounts of il12 p40 and il p70 are different in one cell il12 can stimulate theproliferation of activated t cells and promote the differentiation of th0 cells into th1 cells moreover il12could induce the cytotoxic activity of ctls and nk cellsand promote the secretion of several cytokines such asifnÎ and tnfα previous research indicatedthat tnfα may play a crucial role in mwa in combination with immunotherapy notably our data illustrated that the il12 results were consistent with thoseof ifnÎ after the ablation operation but not with thoseof tnfα this result indicated that upregulation ofifnÎ may be a major effect of the il12 increase aftermwa on the other handan antiinflammatory and immunosuppressive cytokine wasevaluated after mwa il10 is a multicellularderivedmultifunctional cytokine that regulates cell growth anddifferentiation and could participate in inflammatoryand immune responses il10 was reported to increaseafter thermal ablation in the literature [ ] strategiesto inhibit il10induced immunosuppression after thermal ablation treatment would be of interestil10asablation therapy can mediate antitumor immunity astumor tissue necrosis caused by ablation may release various antigens that eventually form a kind of œin situ vaccination moreover ablative therapy can not onlydirectly kill cancer cells in situ but also regulate immunecells and promote the immune function of patients withliver cancer [ ] many immunoregulatory cytokineswere released or expressed after thermal ablation notablythe cytokines released after thermal ablation can regulatethe positive and negative aspects of the cancer immunecycle previously researchers demonstrated that proinflammatory cytokines such as il1 il6 il8 il18 andtnfα were increased several hours or days after thermalablation [ ] to our knowledge terminal tumorthermal ablation may not only cause local heat injury intissues surrounding the tumor site but also induce a systemic reaction this systemic reaction would becaused by different mechanisms first interventional operation may result in trauma to the liver although this procedure is very minimally invasive the healing process maycause alteration of some cytokines second heat injurycould cause acute thermal necrosis in liver and tumor 0czhao bmc cancer page of fig levels of cytokines before and after mwa treatment slightly increased ifnÎ il12 p40 and il12 p70 levels after mwa treatment over fold enhancement of il2 h postmwa and of il1 il6 il8 il10 and tnfα d postmwa p 0czhao bmc cancer page of fig trends in cytokines significantly altered after mwa treatment the levels of il2 at h postmwa il1 at d postmwa il6 at dpostmwa il8 at d postmwa and il10 at d postmwa were elevated over 2fold compared to the levels premwatable correlation between the ablation energy and significantly elevated cytokinesenergyvsil2 h postmwaenergyvsil1 d postmwaˆ’energyvsil6 d postmwaenergyvsil8 d postmwaenergyvsil10 d postmwaenergyvstnfα d postmwaspearman™s rp value onetailed p 0czhao bmc cancer page of fig correlation between the ablation energy and the serum levels of il2 and il6 the serum levels of il2 at h postmwa and il6 at dpostmwa were positively correlated with energy output during the mwa procedureand nonspecifictissues and release of necrotic tissue fragments into bloodcould cause immunological reactions including nonspecific and specific reactions generally cytokines affectedby wound healingimmunologicalreactions do not last longer than those affected by specificimmunologicalreactions ablation treatmentinducedspecific immunological reactions are more complicatedand could affect more immunocytes [ ] which wouldmake this process last longer than other reactions theseexplanations may be the reason why the cytokine changeslasted different durations moreover cytokines affected bythe second manner would be positively correlated withthe ablation scale which is why we employed the œenergyindex in our ablation operation design to receive a terminal ablation larger tumor would cost higher energy including higher power and longer duration time terminaltumorthermal ablation would release tumorrelatedneoantigen to blood circulation eventually induce a systemic reaction this reaction is dependent on the scale ofthermal injury and the local immunological microenvironment of the tumor our findings indicated that il2 andil6 were significantly altered after the ablation procedureand positively correlated with mwa energy il2 is commonly derived from activated t cells primarily th1 cellsil2 can stimulate t cells to proliferate and differentiateactivate natural killer nk cells and macrophages and enhance the functions of cytotoxic t lymphocytes ctls our data illustrated that il2 is significantly increased at h after mwa indicating that il2 may induce a nonspecific immune response after mwa but il decreased after h postmwa in our study suggesting that the il2induced immune response may not belong lasting mentionable many cytokines detected il8il1 il12 were mainly derived from macrophagewhich was a widely distributed antigen presenting cellthis result support the theory that mwa could releasefragment of cancer cells into blood as neoantigen macrophages could response to this proceed and cause a systemic immunoreaction additional cytokines alterationsuch as il6 after ablation may be no anspecific inliver evidences indicate that increase of il6 was not onlyoccurred in liver ablation researches focus on lung cancerincluding primary lung cancer and pulmonary metastasesdemonstrated that serum il8 il1 il6 il10 il12and tnfα were significantly raised after radiofrequencythermal ablation moreover joseph found that imageguided thermal ablation of tumors located in lung liver orsoft tissues increases plasma levels of il6 and il10 another question remain unveiled was if our result wasœcancerspecific we checked literature about cytokinemodulation after thermal ablation in benign diseases andonly got limit evidences based on benign thyroid nodules and adenomyosis according to these literatureil6 levels did not show any significant difference aftertreatment compared with pretreatment values indicatingthat elevation of il6 may be caused by tumour antigenreleased by ablation treatment however the ablationenergy used in thyroid nodules was much lower thanliver and lung which would lead to a false negativein cytokine detection to the research about adenomyosis on the other hand experiment design was determined to followup the il6 at months afterhifu ablation as our data demonstrated mostly cytokines were return to premwa level after monthdetection after months may miss the modulation ofil6 overall few evidences support that some of thecytokines were altered in a œcancerspecific mannerwhile no solid results could confirm that further animal experiments were required to make a clarifieddata and answer this question 0czhao bmc cancer page of thetumorassociated immunein recent years ablationinduced systemic effects suchasresponse haveattracted increased attention de baere t first reported two cases of spontaneous regression of multiplepulmonary metastases occurring after radiofrequencyablation of a single lung metastasis although growing evidence suggests that thermal ablation can inducespontaneous regression of the socalled œabscopal effecton distant tumors the incidence rate of such an effect israre probably due to uncontested immunological activation caused by one ablation treatment and the lack ofimmuneamplification management in it was described that in situ tumor destruction can provide a useful antigen source forthe induction of antitumorimmunity however clinical studies could not sufficiently utilize such an effect until the development ofimmune checkpoint inhibitors icis [ ] icis suchas pd1pdl1 and ctla4 antibodies are widely applied in several cancers and studies have indicated thatici treatment could enhance the effect of ablation evidence indicates that hyperthermic destruction causesthe release of a large population of heterogeneous tumorantigens and inflammatory cytokines may play crucialroles in this process however opposite evidence indicated that incomplete radiofrequency ablation couldinduce inflammation which may accelerates tumor progression and hinders pd1 immunotherapy suggesting that ablation treatment may promote tumorprogression our data demonstrated that il6 was significantly increased after mwa treatment il6 is derived from monocytes macrophages dcs th2 cells andsometimes cancer cells and it plays a key role in t cellproliferation and survival the role of il6 appearsto be rather complex korn classified il6 as œdifferentiation factor which could involve in differentiation ofth17 cells however il6 does not direct the commitment to the th1 or th2 cell lineage but controls theproliferation and survival of immunocytes cooperatingwith other cytokines such as tgf tnf or il21 for instance il6 activated stat3 pathway in naivecd4 t cells in the presence of the morphogen tgfbpromotes the population expansion of th17 cells recent evidence indicates that il6 plays an indispensable role in t cellinfiltration to the tumor sitewhich could benefit immunomodulatory therapy incontrast il6 can increase mdscs inhibit the development and maturation of dendritic cells dcs and inhibit the polarization of th1 cells eventuallyresulting in negative immunomodulatory effects according to muneeb ahmed™s work the adjuvant uses ofa nanop smallinterfering rna sirna can besuccessfully used to target the il6mediated locoregional and systemic effects of thermal ablation il6 knockout via a nanop antiil6 sirna in mice coulddecrease the local vegf level at the ablation site therefore how to utilize the positive effect of il6 whileavoiding the negative effect after mwa needs further investigation preclinical research indicated that il6 andpdl1 blockade combination therapy reduced tumorprogression in animal models [ ] thus an antiil strategy after ablation should be considered whencombined with ici therapy previous studies and ourshave demonstrated that most cytokine levels returned topretreatment levels days after ablation this resultsuggests that h to days after ablation may be optimal timing for additional immunomodulatory therapysour results reported here support the evidence for terminal tumor thermal ablation could cause heat injury totissues surrounding the tumor site and release neoantigento blood circulation eventually induce a systemic reactionthis reaction could lead to a detectable alteration of cytokine levels further investigation is required to revealwhether the cytokines altered by mwa treatment couldaffect cancer progression whether positive or negativeabbreviationsmwa microwave ablation hcc hepatocellular carcinoma icis immunecheckpoint inhibitors tnf tumor necrosis factor il interleukinvegf vascular endothelial growth factor sd stable disease pr partialresponse ct computed tomography ci confidence interval ltp likelihoodof local tumor progression mdscs myeloidderived suppressor cellsctls cytotoxic t lymphocytes nk natural killer sirna small interfering rnaacknowledgementsnot applicableauthors™ contributionsjz conceptualization data curation writing“original draft and writing“review and editing ql conceptualization and writing“review and editingmm conceptualization and writing“review and editing brconceptualization and writing“review and editing and collect samples yhexecute milliplex assay and collect data dpl patient enrollment executemwa ablation and collect samples zl execute mwa ablation and collectsamples dml patient enrollment execute mwa ablation and collectsamples yx execute milliplex assay and collect data mt conceptualizationand writing“review and editing rl conceptualization data curation formalanalysis visualization writing“original draft and writing“review and editingall authors have read and approved the manuscriptfundingthis work was supported by the national natural science foundation ofchina the natural science foundation ofjiangsu province of china bk20140295 the jiangsu governmentscholarship for oversea studies js2018179 and the œsix one projects forhighlevel health personnel in jiangsu province lgy2018077availability of data and materialsthe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethe protocol was approved by the human ethics committee of the firstaffiliated hospital of soochow university and was conducted in accordancewith the declaration of helsinki patients were informed that the bloodsamples were stored by the hospital and potentially used for scientific 0czhao bmc cancer page of research and that their privacy would be maintained all written informedconsent was obtained from all participants and clearly statedconsent for publicationnot applicablecompeting intereststhere is no financial or personal relationship with other people oranizations that could inappropriately influence bias this workauthor details1department of radiation oncology the first affiliated hospital of soochowuniversity suzhou china 2department of oncology the first affiliatedhospital of soochow university suzhou china 3department of lymphatichematologic oncology jiangxi cancer hospital nanchang china4department of interventional radiology the first affiliated hospital ofsoochow university suzhou china 5division of neurosurgery city of hopebeckman research institute duarte california usareceived january accepted august referencesfu j wang h precision diagnosis and treatment of liver cancer in chinacancer lett “bruix j han kh gores g llovet jm mazzaferro v liver cancer approachinga personalized care j hepatol suppls144“rognoni c ciani o sommariva s bargellini i bhoori s cioni r facciorussoa golfieri r gramenzi a mazzaferro v transarterial radioembolizationfor intermediateadvanced hepatocellular carcinoma a budget impactanalysis bmc cancer nault jc sutter o nahon p gannecarrie n seror o percutaneoustreatment of hepatocellular carcinoma state of the art and innovations jhepatol “yin j dong j gao w wang y a case report of remarkable response toassociation of radiofrequency ablation with subsequent atezolizumab instage iv nonsmall cell lung cancer medicine baltimore 20189744e13112shi l chen l wu c zhu y xu b zheng x sun m wen w dai x yang m pd1 blockade boosts radiofrequency ablationelicited adaptiveimmune responses against tumor clin cancer res “lippitz be cytokine patterns in patients with cancer a systematic reviewlancet oncol 2013146e218“jin yb zhang gy lin kr chen xp cui jh wang yj luo w changes ofplasma cytokines and chemokines expression level in nasopharyngealcarcinoma patients after treatment with definitive intensitymodulatedradiotherapy imrt plos one 2017122e0172264kim mj jang jw oh bs kwon jh chung kw jung hs jekarl dw lee schange in inflammatory cytokine profiles after transarterial chemotherapy inpatients with hepatocellular carcinoma cytokine “ gillams a goldberg n ahmed m bale r breen d callstrom m chen mhchoi bi de baere t dupuy d thermal ablation of colorectal livermetastases a position paper by an international panel of ablation expertsthe interventional oncology sans frontieres meeting eur radiol “ ahmed m solbiati l brace cl breen dj callstrom mr charboneau jwchen mh choi bi de baere t dodd gd 3rd imageguided tumorablation standardization of terminology and reporting criteriaa 10yearupdate radiology “ chiang j hynes k brace cl flowdependent vascular heat transfer duringmicrowave thermal ablation conf proc ieee eng med biol soc “ huang hw influence of blood vessel on the thermal lesion formationduring radiofrequency ablation for liver tumors med phys najjar yg rayman p jia x pavicic pg jr rini bi tannenbaum c ko jhaywood s cohen p hamilton t myeloidderived suppressor cellsubset accumulation in renal cell carcinoma parenchyma is associated withintratumoral expression of il1beta il8 cxcl5 and mip1alpha clin cancerres “ alfaro c teijeira a onate c perez g sanmamed mf andueza mp alignanid labiano s azpilikueta a rodriguezpaulete a tumorproducedinterleukin8 attracts human myeloidderived suppressor cells and elicitsextrusion of neutrophil extracellular traps nets clin cancer res “kundu m roy a pahan k selective neutralization of il12 p40 monomerinduces death in prostate cancer cells via il12ifngamma proc natl acadsci u s a “ onishi h kuroki h matsumoto k baba e sasaki n kuga h tanaka mkatano m morisaki t monocytederived dendritic cells that capture deadtumor cells secrete il12 and tnfalpha through il12tnfalphanfkappabautocrine loop cancer immunol immunother “ yu z geng j zhang m zhou y fan q chen j treatment of osteosarcomawith microwave thermal ablation to induce immunogenic cell deathoncotarget “ yang w wang w liu b zhu b li j xu d ni y bai l liu gimmunomodulation characteristics by thermal ablation therapy in cancerpatients asia pac j clin oncol 2018145e490“erinjeri jp thomas ct samoilia a fleisher m gonen m sofocleous ctthornton rh siegelbaum rh covey am brody la imageguidedthermal ablation of tumors increases the plasma level of interleukin6 andinterleukin10 j vasc interv radiol “ den brok mh sutmuller rp van der voort r bennink ej figdor cg ruerstj adema gj in situ tumor ablation creates an antigen source for thegeneration of antitumor immunity cancer res “ zerbini a pilli m laccabue d pelosi g molinari a negri e cerioni sfagnoni f soliani p ferrari c radiofrequency thermal ablation forhepatocellular carcinoma stimulates autologous nkcell responsegastroenterology “ zhang h hou x cai h zhuang x effects of microwave ablation on tcellsubsets and cytokines of patients with hepatocellular carcinoma minim
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Purpose Pancreatic ductal adenocarcinomas exhibit a high degree of desmoplasia due to extensive extracellular matrix deposition Encasement of mesenteric vessels by stroma in locally advanced pancreatic cancer LAPC prevents surgical resection This study sought to determine if the addition of a monoclonal antibody to connective tissue growth factor pamrevlumab to neoadjuvant chemotherapy would be safe and lead to improved resectability in this surgically adverse patient populationMethods In this phase III trial patients with LAPC were randomised to gemcitabinenab paclitaxel plus Arm A n24 or minus Arm B n13 pamrevlumab Those who completed six cycles of treatment were assessed for surgical eligibility by protocol defined criteria Resection rates progression free and overall survival were evaluatedResults Eighteen patients in Arm A and seven in Arm B completed six cycles of therapy with similar toxicity patterns In Arms A and B carbohydrate antigen “ response as defined by ‰¥ decline from baseline occurred in and respectively Sixteen per cent of patients were radiographically downstaged by National Comprehensive Cancer Network criteria in Arm A and in Arm B Positron emission tomography normalised in vs of patients in Arm A vs Arm B respectively and correlated with surgical exploration Eligibility for surgical exploration was vs p00019 and resection was achieved in vs of patients in Arm A vs Arm B p01193 respectively Postoperative complication rates were not different between armsConclusions Neoadjuvant chemotherapy with pamrevlumab holds promise for enhancing resection rates in patients with LAPC without added toxicity This combination merits evaluation in a larger patient cohortIntRoduCtIonPancreatic cancer is currently the third leading cause of cancer death in the USA1 and by it will likely become the second leading cause of cancer related death after Key questionsWhat is already known about this subject –º Pamrevlumab is anti CTGF1 inhibitor highly safe when given to patients with pancreatic cancer and potentially adds to the activity of gemcitabine and erlotinib when given in metastatic diseaseWhat does this study add –º This study examines a the safety and activity of pamrevlumab when added to gemcitabine and nab paclitaxel in locally advanced pancreatic cancer b the impact of this regimen and surgical resection and postoperative safety c explores the utility of a novel study design for locally advanced pancreatic cancerHow might this impact clinical practice –º This study has the potential to lead to practice changing activity in locally advanced pancreatic cancer via the eventual approval of pamrevlumab for use in this situation the promulgation of a new study design for locally advanced pancreatic cancer and increased potential for surgical resection and thus prolonged OS curability in locally advanced pancreatic cancerlung cancer surpassing breast and colon cancer2 Surgical resection is generally necessary for treatment with curative intent or to extend life expectancy3 However only of patients have disease amenable to upfront curative resection at the time of diagnosis4 Approximately “ of patients are diagnosed with locally advanced disease5 determined surgically unresectable per National Comprehensive Cancer Network NCCN guidelines6 Patients with locally advanced pancreatic cancer LAPC have a prognosis similar to those with metastatic disease with a historical median overall survival OS of Picozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c access“ months with recent trials demonstrating median OS of months7 Recent single institution retrospective studies have reported the potential for resection of LAPC with neoadjuvant therapy irrespective of imaging findings with promising results8 However these are limited by significant selection bias lack of strict radiographic classification and chemotherapy standardisation Current prospective trials have documented resection rates of LAPC in the range of to therefore novel approaches are needed to improve patient outcomesThe tumour biology inherent to pancreatic ductal adenocarcinoma PDAC significantly contributes to the poor outcomes seen in this disease Notably PDAC exhibits a high degree of desmoplasia often in association with elevated connective tissue growth factor CTGF expression12 CTGF appears to play a central role in the biology of pancreatic cancer affecting both its cellular biology and its extracellular matrix composition This leads to many simultaneous biological effects that promote pancreatic cancer growth including increased cellular proliferation and differentiation increased cellular adherence and migration antiapoptosis vascular permeability angiogenesis and suppression of tumour immunological responses13 This stroma may also contribute to the radiographic imaging findings of mesenteric vessel involvement or encasement that is used to determine resectability of pancreatic tumours Executing a pharmacological intervention on the pancreatic cancer stromal environment is therefore a major goal of the development of novel pancreatic cancer therapeuticsPamrevlumab is a human monoclonal antibody that targets CTGF Preclinical studies showed that CTGF overexpression is associated with both desmoplasia and gemcitabine resistance in the KPC pancreatic cancer mouse model14 When pamrevlumab was used in combination with gemcitabine sensitivity to gemcitabine was enhanced which correlated with inhibition of XIAP an antiapoptotic protein15 When tested in patients with advanced pancreatic cancer Stage IV and locally advanced Stage III treated with gemcitabine and erlotinib in a phase III study n75 pamrevlumab displayed multiple favourable outcomes16We hypothesised that through inhibition of the downstream effects of CTGF overexpression on tissue adhesion and other mechanisms pamrevlumab may influence resectability of PDAC tumours With this in mind this novel phase III randomised multicentre trial was designed to explore the safety and efficacy of pamrevlumab in combination with gemcitabinenab paclitaxel in LAPC with special emphasis on surgical eligibility and safetyMetHodsstudy designThis was a phase III randomised trial of safety and efficacy in patients with LAPC who received gemcitabine and nab paclitaxel with or without pamrevlumab as neoadjuvant therapy The randomisation was preplanned and blinded to the investigator The study was approved by individual institutional review boards at nine US institutions and conducted according to the Declaration of Helsinki The trial was registered at clinicaltrials gov as NCT eligibilityKey protocol eligibility requirements included biopsy proven diagnosis of PDAC radiographic staging consistent with locally advanced unresectable disease as defined NCCN guidelines V2 clinical stage confirmed by diagnostic laparoscopy radiographically measurable disease per Response Evaluation Criteria in Solid Tumors RECIST V11 Eastern Cooperative Oncology Group ECOG performance status of or adequate haematological renal and hepatic function no prior therapy for PDAC and no concomitant cancer diagnosis within the past yearsstudy schemaEligible patients were randomised to Arm A or Arm B to receive a total of six treatment cycles “ weeks of therapy figure Patients in Arm A received pamrevlumab mgkg by intravenous infusion on Days and of each day cycle with an additional dose given on Day in the first cycle Patients in both Arms A and B received gemcitabine mgm2 by intravenous infusion on Days and of each day treatment cycle nab paclitaxel mgm2 by intravenous infusion on Days and of each day cycle Doses for gemcitabine and nab paclitaxel were modified for haematological and non haematological toxicity as per standard of care SOC15 Patients remained on therapy for six treatment cycles “ weeks unless they had disease progression an intolerable adverse event AE or toxicity withdrew consent or were withdrawn at the investigator™s discretion All patients were followed for drug toxicity until days after the last drug dose Patients undergoing surgery were followed for days following hospital discharge for surgical complications CTGF levels were obtained prior to treatment from all patients Plasma samples for pamrevlumab level determination were obtained from all patients receiving this drug After all protocol specified therapy was completed patients were followed for disease progression survival and additional oncological therapy Postoperative complications including day readmissions and day mortality were notedResponse assessmentPatients were evaluated for response by the following measures carbohydrate antigen CA “ measured at baseline first day of each cycle and end of treatment EOT RECIST V11 read based on full body CT imaging high resolution dual phase fine cut CT imaging at baseline and every weeks thereafter fluorodeoxyglucose FDG positron emission tomography PET imaging and NCCN V2 resectability criteria at baseline and EOTPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c accessFigure Patient flow and surgery outcomes In Arm A four of the eligible subjects had their surgeries cancelled 1portal vein thrombosis 3medical issues precluding surgery In Arm A four eligible subjects underwent surgery but resection was not achieved 3metastatic disease discovered 1extensive SMA encasement In Arm B one eligible subject underwent surgery but resection was not achieved 1extensive vascular encasement SMA superior mesenteric arterysurgical assessmentSubjects who finished six cycles of combination chemotherapy were evaluated for eligibility for surgical exploration per protocol PP defined criteria Given that patients included in the trial were determined to be initially unresectable by radiographic imaging and NCCN criteria objective criteria were developed to standardise attempts at surgical resectionPatients were deemed eligible for surgery if one or more of the following criteria were met reduction in plasma CA “ level by ‰¥ at EOT compared with baseline reduction in FDG PET maximum standardised uptake value SUVmax by ‰¥ at EOT compared with baseline radiological tumour response per RECIST of partial response PR or complete response CR at EOT or met the definition of resectable or borderline resectable per NCCN guidelines Subjects were classified as ineligible for surgical exploration if any of the following occurred development of distant metastases or local progression on CT scan tumour anatomy precluding vascular reconstruction unreconstructible local complications preventing surgery eg portal vein PVsplenic vein thrombosis pancreatitis or decline in performance status to a Karnofsky score ‰¤ or Picozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668absolute contraindication to surgery from comorbidity eg recovery from myocardial infarction or uncontrolled diabetes The final decision regarding whether resection was to be performed was made by the treating surgeonendpointsSafety endpoints included serious adverse events SAE during neoadjuvant therapy and surgical complications postresection The efficacy endpoints included surgical eligibility R0 resection R0R1 resection median OS progression free survival PFS and year survival rate All patients were followed and data analysis was stratified by PP population and intention to treat ITT cohortstatistical considerationsThe comparison between selected clinical characteristics toxicity profiles and eligibility for surgical exploration or completed surgical resection was performed using the χ² test Exact CIs for the point estimates as well as the treatment difference were obtained from the SAS PROC FREQ procedure with the EXACT option The two treatment arms were compared using the Cochran Mantel Haentzel test controlling for baseline factors TNM stage ECOG CA “ PET SUVmax 0c accesssuperior mesenteric artery SMA involvement coeliac abutment and so on as prespecified in the protocol All cause mortality was used in determining OS which was analysed by the Kaplan Meier method Survival status was updated within month before the data cut off date Data from patients who were alive at the cut off date were censored for survival analysis All statistical tests were performed at the significance level of α005 using two sided testsResultsPatient characteristics and dispositionThirty seven patients were randomised to study treatment to Arm A pamrevlumabgemcitabinenab paclitaxel and to Arm B gemcitabinenab paclitaxel alone Patient characteristics at baseline are summarised in table All patients enrolled were unresectable by NCCN criteria patients had tumour arterial involvement SMA encasement ° coeliac abutment Table Patient characteristicsBaseline demographics “ years “ years ‰¥ years Median Male FemaleAge group Sex BMI kgm2 Mean SD Median Min maxECOG Grade Grade TNM stage T3 N0 M0 T3 N1 M0 T4 N0 M0 T4 N1 M0 T4 NX M0Location of the tumour in the pancreas Non resectability per NCCN criterion Head Body Tail Median tumour size mm ° SMA encasement Any coeliac abutment Inferior vena cava invasion or encasement Unreconstructible SMVportal occlusion Aortic invasion and encasementArm AGNPPN24 Arm BGNPN13 TotalN37 to to to · OK as isNot mutually exclusiveBMI body mass index ECOG Eastern Cooperative Oncology Group G gemcitabine n number of subjects NCCN National Comprehensive Cancer Network NP nab paclitaxel P pamrevlumab PV portal vein SMA superior mesenteric artery SMV superior mesenteric veinPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c accesswithout gastroduodenal artery involvement or aortic involvement inferior vena cava invasion and unreconstructible PVsuperior mesenteric vein SMV occlusion A higher percentage of patients with SMA encasement ° were randomised to Arm A vs Arm B Patient disposition is summarised in figure Twenty four patients in Arm A received gemcitabinenab paclitaxel and pamrevlumab patients completed six treatment cycles Six patients discontinued treatment early due to progressive disease three patients AEs two patients or physician decision one patient Thirteen patients in Arm B received gemcitabinenab paclitaxel patients completed six treatment cycles Six patients discontinued treatment early due to progressive disease two patients AEs two patients or patientphysician decision two patientssafetySAEs are summarised in table Forty one per cent of patients had a treatment emergent SAE Arm A Arm B No individual toxicity category occurred with frequency except systemic infection patients There was no demonstrable increase in any toxicity with the addition of pamrevlumab to gemcitabinenab paclitaxel chemotherapyTable Summary of treatment emergent serious adverse eventsSystem organ classpreferred term Ascites Nausea Pancreatitis Vomiting Device occlusion Drug withdrawal syndrome FeverNo of patients with any treatment emergent SAEBlood and lymphatic disorders Haemolytic uremic syndrome LymphadenopathyCardiac disorders Cardiac failure Supraventricular tachycardiaGastrointestinal disorders General disorders and administrative site conditions Hepatobiliary disorders Infections Sepsis Cellulitis Urinary tract infectionInjury poisoning and procedural complications Respiratory thoracic and mediastinal disorders Skin and subcutaneous disorders Cholangitis Hyperbilirubinaemia Craniocerebral injury Pneumonitis Pulmonary embolism RashArm An24n Arm Bn13n Overalln37n · OK as isPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c accessResponse to therapyIn Arm A had ‰¥ CA “ decline at EOT response by RECIST PR ‰¥ decline in PET SUVmax and were radiographically downstaged by NCCN criteria During the treatment period the median CA “ decline was patients were non secretors Seven out of patients had best objective RECIST response CRPR Some patients had ˜exceptional™ responses defined as normalisation or ‰¥ decline of CA “ patients or normalisation PET SUVmax in In Arm B had ‰¥ CA “ decline at EOT response by RECIST PR ‰¥ decline in PET SUVmax and were radiographically downstaged by NCCN criteria Four out of patients had best objective RECIST response CR PR In Arm B of patients had an œexceptional CA “ response and had an ˜exceptional™ PET response as defined by either ‰¥ normalized Ca response normalized SUV max andorradiographic downstaging post therapy completion surgical evaluationOverall of the total study patients were eligible for surgical exploration using protocol defined criteria Arm A Arm B p00019 Resection was completed in of the patients Arm A Arm B p01193 Details of the nine resected patients are shown in table In Arm A of the patients were eligible for surgical exploration in the ITT population and of the patients were eligible in the PP population patients who completed six cycles of treatment In Arm A out of eligible patients ultimately underwent surgical exploration for resection five operations were cancelled four due to drug toxicitymedical comorbidity sepsis gallbladder perforation declined performance status and fever and one patient declined Eight out of patients in Arm A were resected R0 R1 The remaining four patients who were explored were not resected due to progression or unresectable disease intraoperatively In Arm B of the patients were eligible for surgical exploration in the ITT population and were eligible in the PP population Of the two subjects found to be surgically eligible only one was resected as the other patient had local progressionPredictors of resectionHigh CA “ response ‰¥ decline andor normalisation was contributive to surgical eligibility vs p03 Normalisation versus non normalisation of PET SUVmax was predictive of surgical eligibility vs p0013 and successful resection vs p0002 Combining these two criteria was highly predictive for surgical eligibility vs p0003 and completed vs p0002 resection All nine successful resections were identified by one or both of these criteria Table Summary of resected patientsSitesubject IDTreatmentarmResponse to treatmentNCCNbaselineNCCNend of treatmentResection status“““““““““AAAAAAAAB UnresectablecoeliacUnresectableSMA SMVUnresectablecoeliacUnresectablecoeliacUnresectableSMVUnresectableSMAUnresectableSMA SMV coeliacUnresectableSMAUnresectablecoeliacUnresectablecoeliacUnresectableSMA SMVUnresectablecoeliacBorderline resectableUnresectableSMVUnresectableSMAUnresectablecoeliacUnresectableSMAUnresectablecoeliacR0R1R0R0R1R1R1R0R0Protocol defined criteria CA “ decrease FDG PET SUVmax decrease ‰¥ RECIST V11 response PR or CR NCCN resectable or borderline resectable criteriaCA carbohydrate antigen CR complete response FDG fluorodeoxyglucose NCCN National Comprehensive Cancer Network PET positron emission tomography PR partial response RECIST Response Evaluation Criteria in Solid Tumors SMA superior mesenteric artery SMV superior mesenteric vein SUVmax maximum standardised uptake valuePicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0cConversely radiographic features of response did not correlate with operative potential Neither RECIST response nor radiographic downstaging per NCCN criteria statistically correlated with completed resectionsurgical complicationsPostoperative complications were summarised according to the Clavien Dindo classification posthoc analysis Ischaemic gastritis and ulceration and right lower lung lobe collapse were reported for one patient in Arm A Grade II There was one episode of clinically significant pancreatic leak in each arm Grade IIIA no reoperations and no day or day surgical mortality were noted One patient in Arm B had a delayed gastric perforation following a distal pancreatectomy with coeliac axis resection likely due to thermal injury and was treated non operatively Grade IIIB No wound complications or superficial site infections were noted in either group Four out of patients and out of patients in Arm A and B respectively were readmitted within days and the difference between treatment arms was not clinically or statistically significantsurvivalAs of the data cut off date of evaluable patients were known to be alive with a median length of follow up at approximately months PFS was months CI to and months CI to in Arm A and Arm B respectively One year survival and median OS were and months CI accessto in Arm A and and months CI NR in Arm B The median OS for all patients who were eligible for surgical exploration Arm A Arm B vs ineligible Arm A Arm B was months CI NR vs months CI to p00766 The median OS for resected Arm A Arm B vs non resected patients Arm A Arm B was not reached CI NR vs months CI to p00141 figure dIsCussIonThe treatment of LAPC with neoadjuvant therapy remains challenging and there is no established SOC Several high volume centres have reported their single centre experiences with varying neoadjuvant chemotherapy and chemoradiation strategies8 The combination of more active regimens delivered over an extended period and surgeons™ comfort with resecting and reconstructing major mesenteric vessels has led to an increase in resection rates A meta analysis of studies using FOLFIRINOX has demonstrated resection rates ranging from to in LAPC17 One of the larger studies including patients with LAPC reported a resection rate of that was more dependent on duration of therapy months than chemotherapy regimen FOLFIRINOX or gemcitabine based18 Recently a single institution and single arm prospective study of neoadjuvant FOLFIRINOX and losartan with selective use of radiation in patients with LAPC reported an R0 resection rate of Figure Overall survival Resected vs Non resected patientsPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c access However the lack of randomisation makes it difficult to determine what aspect of therapy was responsible for this effect and only of resected patients needed any type of vascular reconstruction perhaps suggesting more favourable outcome than usually seen in locally advanced disease These retrospective studies contain significant interpretative challenges including selection bias treatment variables including radiation and the use of different definitions of LAPCthe anti CTGF mechanism of action With respect to gemcitabine based therapy a recent large scale prospective trial of patients with LAPC treated with induction gemcitabine with or without erlotinib followed by radiation only patients were able to undergo resection10 Furthermore the addition of erlotinib to gemcitabine did not improve any downstaging capacity and there was no difference in survival with the addition of radiation More recently the LA PACT trial examining the role of induction gemcitabine nab paclitaxel found that only out of patients with LAPC were able to undergo surgery following neoadjuvant therapy with combination gemcitabine and nab paclitaxel for six cycles by investigator™s choice11 Last although FOLFIRINOX has been the most studied induction combination chemotherapy regimen in this population recent randomised data from European patients who received neoadjuvant FOLFIRINOX versus gemcitabinenab paclitaxel prior to resection20 showed no clear difference with respect to R0R1 to resection rate vs p0135 or OS vs months p0268Given for pamrevlumab its use in the neoadjuvant setting has the potential to impact tumour regression and modulate the desmoplastic niche and possibly affect tumour margins allowing for improved resection rates Previous studies have looked at the ability of gemcitabinenab paclitaxel to reduce cancer associated fibroblasts resulting in a ˜softening™ of tumours by endoscopic ultrasound elastography21 This stromal depletion also translated into a decrease of SUV uptake on PET22 In the study reported herein we combined gemcitabine and nab paclitaxel with pamrevlumab to explore its effect in terms of therapeutic response the impact on eligibility for surgical exploration and improved resection rates in locally advanced patientsThe protocol specified therapeutic response criteria CA “ PET SUVmax RECIST and NCCN criteria were used as criteria to determine eligibility for surgical exploration in LAPC This is a novel approach specific to the protocol and allows participating patients to be explored for resection when otherwise they may not qualify by current treatment standards NCCN criteria For example by NCCN conversion alone ie converted from unresectable to borderline resectable only of patients in Arm A would have been eligible for surgical exploration However by protocol criteria of patients in Arm A were eligible for surgical exploration A higher percentage of patients were eligible for surgical exploration by the above criteria in Arm A vs Arm B vs respectivelyOverall the rate of successful resections in the pamrevlumab treated group was higher than in the control group but this did not reach statistical significance most probably due to small sample size Of the nine subjects that were successfully resected in this trial only one was converted by NCCN criteria to borderline resectable prior to surgical exploration Despite this phenomenon the data support the hypothesis that pamrevlumab a human monoclonal antibody with anti CTGF mechanism of action could alter tumour characteristics allowing resection in otherwise unresectable patients This hypothesis needs to be confirmed and patients should be stratified by coeliac andor SMA involvementThe most common predictive factors for eligibility for surgical exploration and resection were CA “ decline and PET SUV max response which are indicators of tumour response to treatment The combination of these two factors proved to be a highly sensitive objective readout for prediction of potential surgical success Both the ability of CA “ response and the inability of radiographic response RECIST and NCCN criteria of resect ability to predict surgical outcome has been observed by others3 and these observations deserve further examination in subsequent clinical trials In the MPACT study both CA “ and PET response correlated to improved survival in metastatic patients treated with gemcitabine and nab paclitaxel23 Recent surgical series of patients with borderline resectable and LAPC have also corroborated their impact in the localised setting25 Correlation of clinical response with plasma levels of endogenous CTGF and pamrevlumab exposure as shown in the prior study by Picozzi et al16 may provide added prognostic and predictive insightWith regard to safety no major incremental toxicity in any category was noted with the addition of pamrevlumab to gemcitabinenab paclitaxel In addition a higher number of patients were able to complete six cycles of the three drug combination including pamrevlumab when compared with gemcitabinenab paclitaxel alone Pamrevlumab is well tolerated and considered safe compared with the SOC drugs for patients with PDAC These observations represent a very favourable attribute when considering potential neoadjuvant chemotherapy in a patient population with the frequency of medical problems typically seen in LAPC In addition there were no signals of increased surgical morbidity or wound healing problems with CTGF blockade by pamrevlumab In fact there were only two clinically significant pancreatic leaks one in each arm which is comparable to national outcome data from high volume pancreatic surgery centres Similarly readmissions following resection were comparable between arms and reflected the complexity of this challenging patient populationFinally while survival data are not yet mature both patients who were eligible for surgery and those that Picozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0cwere ultimately resected had longer PFS and OS highlighting the importance of surgical resection of the tumour Therefore more investigation into newer agents targeting LAPC and increased consideration of candidacy for surgery in those patients who do not progress on therapy or suffer toxicity should be of utmost importance to improve outcomes in this diseaseIn conclusion this is the first prospective randomised multicentre trial examining the role of neoadjuvant therapy in LAPC with prespecified criteria for surgical exploration The use of pamrevlumab in combination with gemcitabine and nab paclitaxel showed a potential to enhance tumour response and increase resection rates Further evaluation of this drug combination in the neoadjuvant treatment setting for LAPC is warranted and a larger phase III trial with resection and survival endpoints is ongoingContributors FibroGen Inc was the study sponsor that designed the study in consultation with the Principal Investigator VP and surgical co investigator FGR All authors except those of the sponsor contributed patients to the study FibroGen was responsible for data collection and analysis All authors reviewed the manuscript and signed off on its accuracyFunding The study was funded by FibroGen Inc San Francisco CAdisclaimer The corresponding author has the right to grant on behalf of all authors and does grant on behalf of all authors an exclusive licence or non exclusive for government employees on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article if accepted to be published in ESMO editions and any other BMJPGL products to exploit all subsidiary rights as set out in our licenceCompeting interests MC MZ SP EK and EC are employees of FibroGen and hold stock andor stock options
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pulmonary disease COPD is due to structural changes and narrowing of small airways and parenchymaldestruction loss of the alveolar attachment as a result of pulmonary emphysema which all lead to airflow limitation Extracorporeal shock waves ESW increase cell proliferation and diï¬erentiation of connective tissue fibroblasts To date no studiesare available on ESW treatment of human bronchial fibroblasts and epithelial cells from COPD and control subjects We obtainedprimary bronchial fibroblasts from bronchial biopsies of patients with mildmoderate COPD and control smokers with normallung function 16HBE cells were also studied Cells were treated with a piezoelectric shock wave generator at low energy mJmm2 pulses After treatment viability was evaluated and cells were recultured and followed up for and h Cellgrowth WST1 test was assessed and proliferation markers were analyzed by qRTPCR in cell lysates and by ELISA tests in cellsupernatants and cell lysates After ESW treatment we observed a significant increase of cell proliferation in all cell types CKitCD117 mRNA was significantly increased in 16HBE cells at h Protein levels were significantly increased for cKit CD117 at h in 16HBE p and at h in COPDfibroblasts p � for PCNA at h in 16HBE p � for y1 CD90 at and h in CSfibroblasts p � and p � for TGF1 at h in CSfibroblasts p � for procollagen1 at h inCOPDfibroblasts p � and for NFκBp65 at and h in 16HBE p � and p � In the peripheral lung tissueof a representative COPD patient alveolar type II epithelial cells TTF1 coexpressing cKit CD117 and PCNA were occasionally observed ese data show an increase of cell proliferation induced by a low dosage of extracorporeal shock waves in16HBE cells and primary bronchial fibroblasts of COPD and control smoking subjects Backgrounde progressive chronic airflow limitation in chronic obstructive pulmonary disease COPD is due to two majorpathological processes i remodeling and narrowing ofsmall airways and ii destruction of the lung parenchymawith loss of the alveolar attachments as a result of pulmonaryemphysema [] Chronic ‚ammation in the lung plays a 0cCanadian Respiratory Journaltherapycentral role in both the small airway remodeling and thepulmonary emphysema [“] Lung volume reductionsurgery and lung transplantation while possible in endstageCOPD are restricted to just a few selected patients []httpwwwgoldcopdcom RegenerativeforCOPD includes mesenchymal stromal cell MSC or tissueengineering therapies But while bone marrow MSC oradipose tissue MSC treatments showed promising results inmice with induced emphysema [] clinical trials performedin COPD patients have been discouraging [ ] ere are alarge number of animal studies in which lung regenerationhas been successfully stimulated For instance in a rat modelof elastaseinduced emphysema administration of alltransRA ATRA stimulated alveolar regeneration [] keratinocyte growth factor KGF FGF7 administered afterpneumonectomy augmented alveolarization [] administration of HGF stimulated alveolar regeneration enhancedlung vascularization and improved exercise tolerance andgas exchange [] intratracheal administration of bFGF torats and dogs with elastaseinduced emphysema improvedalveolar dimensions and lung microvessel density [] andVEGF administration enhanced postpneumonectomy alveolar growth in mice [] But again the attempts tostimulate lung regeneration in COPD patients with emphysema with orally administered ATRA yielded no differences in computed tomography CT lung function orquality of life scores between treatment groups [ ] andRARc selective agonist administration also showed nodiï¬erences in CT scores or lung function in treated vsnontreated COPD patients [ ] However the therapeutic potential of regenerative pharmacology is still at thebeginning of its development And many authors haveshown that the human lung also in adulthood retains asignificant regenerative potential from the large to the smallairways and in terminal and respiratory bronchioles [] andthat tissue regeneration is achieved in two ways by proliferation of common diï¬erentiated cells andor by deployment of specialized stemprogenitor cells [ ]Extracorporeal shock wave therapy ESWT is applied inmany musculoskeletal diseases and in regenerative medicinebased on its capability to induce neoangiogenesis osteogenesis regeneration and remodeling through stem cellstimulation [] ESW in combination with tenogenicmedium improved the diï¬erentiation of human adiposederived stem cells hASCs into tenoblastlike cells []ESW combined with osteogenic medium increased the osteogenic diï¬erentiation of treated hASCs [] while stemcell diï¬erentiation into myofibroblasts was partially reducedby ESW treatment [] But to our knowledge no data areavailable on ESW treatment of primary bronchial fibroblastsof patients with COPD and control healthy smokers orbronchial epithelial cells 16HBEMarkers of cell proliferation include CD117 cKit orSCFR a receptor tyrosine kinase protein that binds to stemcell factor SCF expressed on hematopoietic stem cells Itcan also be expressed by mast cells melanocytes in the skininterstitial cells of Cajal in the digestive and urogenital tract[] cardiac pericytes [] amniotic fluid stem cells []stemprogenitor cells in conducting airway epithelium ofporcine lung [] and dendritic cells in the lung []Another marker of cell proliferation is proliferating cellnuclear antigen PCNA It is expressed in the nuclei of cellsand is involved in DNA replication DNA repair andchromatin remodeling [ ] In the lung of COPD patients alveolar type II epithelial cells and endothelial cells[] and small airway bronchiolar epithelium [] expressdecreased PCNA levels compared with related nonCOPDcontrol groups A third marker of cell proliferation is CD90y1thymocyte diï¬erentiation antigen1 a glycophosphatidylinositol cell surface protein expressed by thymocytes CD34 cells mesenchymal stem cells endothelialcells and cardiac fibroblasts It is also considered a marker ofmultipotent mesenchymal stem cells when expressed inassociation with other markers CD29 CD44 CD73CD105 [ ]We aimed in this study to analyze the proliferative eï¬ectof shock waves when applied as an external challenge toprimary bronchial fibroblasts of COPD patients and controlsmokers and to immortalized bronchial epithelial cells16HBE To this end we investigated cell markers expression related to this proliferative stimulus Methods Ethics Statement Collection and processing of bronchialbiopsies at the Institute of Veruno NO and collection andprocessing of the peripheral lung tissues at the UniversityHospital of Orbassano during lung resection for a solitaryperipheral neoplasm were approved by the ethics andtechnical committees ofthe Istituti Clinici ScientificiMaugeri CTS p102 and San Luigi Hospital OrbassanoTO CE N Italy the study complied withthe Declaration of Helsinki and written informed consentwas obtained from each participant Cell Culture and Treatments We used the SV40 large Tantigentransformed 16HBE cellline which retains thediï¬erentiated morphology and function of normal humanbronchial epithelial cells NHBE [] and primary humanbronchial fibroblasts obtained from bronchial biopsies ofpatients with COPD n � and control smoking subjectsn � with normal lung function Primary bronchial fibroblasts were obtained from bronchial biopsies obtainedfor diï¬erent protocol studies [] Bronchial biopsies weretreated with type II collagenase min at °C InvitrogenGIBCA and cultured in DMEM until confluentprimary fibroblasts were obtained 16HBE cells and primarybronchial fibroblasts were maintained in Dulbecco™s modified minimum essential medium DMEM supplementedwith vv fetal bovine serum FBS IUmL penicillin μgmL streptomycin 1x nonessential amino acids mMsodium pyruvate and mM glutamine °C CO2 []When cells were “ confluent the complete mediumwas replaced with DMEM with FBS for starvation time h e shockwave generator utilized for the in vitroexperiments was a piezoelectric device Piezoson Richard Wolf Knittlingen Germany designed for clinical 0cCanadian Respiratory Journaluse in orthopedics and traumatology Aliquots of mL ofcell suspension adjusted to × cellsmL were placed in mm polypropylene tubes completely filled with culturemedium e shock wave unit was kept in contact with thecellcontaining tube by means of a waterfilled cushionCommon ultrasound gel was used as a contact mediumbetween the cushion and tube ESW treatment was as follows energy flux density EFD � mJmm2 pulsesfrequency � shockss is EFD is a mediumhigh energywe already used for previous in vitro diï¬erentiation studiesin tendons [] After treatment cell viability was evaluatedby trypan blue exclusion and primary fibroblasts werepassaged in DMEM complete for hours 16HBEcells were cultured for and h because of their lowerresistance to starvation T0 corresponds to hours post ESWtreatment for all experiments reported Nontreated fibroblasts or 16HBE cells were used as controls Cell growth wasevaluated by the colorimetric test WST1 All experimentswere performed in quadruplicate ie four independentexperiments for each type of treatment ESW or noESWand each time exposure Extraction and Quantification of RNA and qRTPCRfrom Primary Bronchial Fibroblasts and 16HBE Total RNAfrom treated and nontreated cells was purified and isolatedusing an RNAspin Mini RNA Isolation kit GE HealthcareLife Sciences Pittsburgh USA following the manufacturer™sinstructions Total RNA was resuspended in μL nucleasefree water RNA concentration was determined using aUVvisible spectrophotometer λ260280 nm EppendorfBioPhotometer plus and stored at ˆ’°CQiagenQT00000728e expression of genes of interest was measured usingSYBR Green Qiagen UK for qPCR in a Corbett RotorGene Corbett Cambridge UK system Onestep realtime PCR was carried out by amplifying mRNA using theQuantiFast„¢ SYBR Green RTPCR kitITaccording to the manufacturer™s instructions and the genespecific primers Qiagen IT We detected the expression ofcKit or SCFR CD117 Cat QT01844549 Qiagen PCNACat QT00024633 y1 CD90Cat QT00023569TGF1CatProcollagenIQT01005725 and NFκBp65 Cat QT01007370 Weperformed independent experiments and quantitative PCRmeasurements in quadruplicate for each type of treatmentESW or noESW and each time exposure Briefly the PCRreaction mix prepared in a total volume of μL was run onthe Rotor Gene Q Qiagen IT and the following PCR runprotocol was used °C for min reverse transcription°C for min PCR initial activation step amplificationcycles of °C for s denaturation and °C for scombined annealingextension followed by melting curveanalysis to ensure the specificity of PCR amplificationGlyceraldehyde phosphate dehydrogenase GAPDHQT01192646 Qiagen was used as the reference gene forevery target gene per sample and the data were normalizedagainst the respective GAPDH signaling Cycle thresholdCT values were determined using the Rotor Gene Qsoftware RotorGene Q Series Software eCatexpression levels of all genes studied were normalized toGAPDH levels in each sample to determine the expressionbetween treated and nontreated cells using the ˆ’ΔCt method[] for primary bronchial fibroblasts and the ˆ’ΔΔCt for16HBE cells [] ELISA Tests in the Supernatants or Cell Lysates of ESWTreated and Nontreated Cells Protein extraction andquantification in the supernatants or cell lysates of ESWtreated and nontreated cells were performed as reported inTable Suppliers Cat Numbers dilution conditions anddetection limits of the ELISA kits used are also reported eELISA kits WST1 cell proliferation kit and MPERmammalian protein extraction kit were used according tothe manufacturer™s instructions Table CKit CD117PCNA and NFκBp65 were quantified in cell lysates CD90TGF1 and procollagen1 were quantified in the cellsupernatants Immunohistochemistry of the Lung Parenchyma of Patients with COPD Samples were frozen in liquid nitrogenprecooled is tane after embedding in OCT and used forcryostat sectioning and immunostaining of some cellproliferationrelated molecules Single immunostainings offrozen sections were performed with mouse anti“thyroidtranscription factor1 TTF1 sc53136 Santa Cruz rabbitanticKit CD117 ARG51826 ARGBIO and rabbit antiPCNA PAS27214 ermo Fisher primary antibodiesAntibody binding was demonstrated with secondary antibodies antimouse Vector BA and antirabbitVector BA followed by ABC kit AP AK5000VECTASTAIN and FastRed Substrate red color Doublestainings were performed using also ABC kit Elite PK6100VECTASTAIN and diaminobenzidine substrate browncolor for identification of TTF1 positive alveolar type IIepithelial cells [] coexpressing cKit CD117 or PCNAantigens Slides were included in each staining run usinghuman tonsil nasal polyp or breast cancer as positivecontrols For the negative control slides normal nonspecificmouse or rabbit immunoglobulins Santa Cruz Biotechnology Santa Cruz CA USA were used at the same proteinconcentration as the primary antibodiesmean± standardthe unpaired ttest Probability values of p were Statistical Analysis Group data were expressed asorinterquartile range IQR for morphologichistologic dataDiï¬erences between treatment groups were analyzed usingor mediandeviationrangeconsidered significant Data analysis was performed usingthe Stat View SE Graphics program Abacus Concepts IncBerkeley CA USA Results ESW Eï¬ects on Cell Proliferation ESW treatment at adosage of mJmm2 pulses frequency � shockssof primary bronchial fibroblasts from COPD patients n � 0cCanadian Respiratory JournalPackyearsExsmokercurrent smokerTable Clinical characteristics of chronic obstructive pulmonary disease COPD patients and control smokers who provided bronchialfibroblasts for œin vitro experimentsSubjectsCOPD1COPD2COPD3± Mean± SD± Mean± SDIndividual and mean± standard deviation SD data M male F female FEV1 forced expiratory volume in s BD bronchodilator FVC forced vitalAge years MFMMM”MMM”± ± ± ± ± ± ± CurrentCurrentCurrentFEV1 postBDFEV1 preBDCurrentCurrentNDNDND”FEV1FVCCS1CS2CS3Ex””capacity ND not determined Patients were classified according to the Global Initiative for Chronic Obstructive Lung disease httpgoldcopdorg levels ofseverity for COPD For COPD patients FEV1FVC are postbronchodilator values ANOVA test FEV1 p � FEV1FVC p � No significantdiï¬erences were observed for age p � and packyears p � smokedTable List of ELISA tests cell proliferation and protein extraction kits used For ELISA tests dilution of the supernatants or cell lysatesamples used and detection limits are also reportedTargetcKit or SCFR CD117PCNAy1 CD90TGF1Procollagen1NFκBp65WST1 cell proliferationMPER mammalian protein extraction ermo Scientific ngmL “ ngmL ngmL “ ngmL pgmL “ pgmL pgmL “ pgmL pgmL “ pgmL17pgmL “ pgmLCloudClone CorpCloudClone CorpCloudClone CorpCloudClone CorpCloudClone CorpSEA121 HuSEA591MiSEB404 HuSEA124 HuSEA957 HuKHO0371KA1384Dilution PBS PBSDetection limit range diluent buï¬erInvitrogenAbnovaNo dilNo dilNo dilSupplierCat ashowed a significantly increased proliferation index at and h after treatment compared with nontreated bronchial fibroblasts Figure 1a ESWtreated primary bronchial fibroblasts from control smokers with normal lungfunction n � also showed a significant increase of theproliferation index at and h aftertreatmentFigure 1b Treated bronchial epithelial cells 16HBEshowed significantly increased proliferation index values at and h after treatment when compared with nontreated16HBE cells Figure 1c ESW Eï¬ects on mRNA and Protein Levels of Cell Proliferation and Cell Remodeling Markers Primary bronchialfibroblasts from COPD patients n � control smokersn � and human bronchial epithelial cells 16HBE werestimulated with extracorporeal shock waves at a dosage of mJmm2 pulses and compared with paired nonstimulated primary bronchial fibroblasts and 16HBE cellsCKit mRNA was significantly increased in ESWtreated16HBE cells at h p � and decreased in CSfibroblasts at h p � compared with nontreated cellsFigures 2b and 2c Furthermore a tendency to increased cKit mRNA levels was observed after ESW treatment for COPDfibroblasts Figure 2a CKit protein wassignificantly increased in the cell lysates at h after ESWtreatment in primary bronchial fibroblasts of COPD patientsp � Figure 2d and in 16HBE cells p at h after ESW treatment Figure 2f No significantchanges were observed for cKit protein in ESWtreatedprimary bronchial fibroblasts from control smokers CSbronchialfibroblastswith normal lung function Figure 2e PCNA mRNAlevels were not significantly changed in ESWtreated fibroblasts and 16HBE cells when compared with nontreatedcells Figures 3a“3c PCNA protein in the cell lysatesshowed a tendency to be increased in primary bronchialfibroblasts of CS p � at h after ESW treatmentFigure 3e and a significant increase was observed at hT0 in 16HBE cells p � after ESW treatmentFigure 3f No significant changes were observed inprimaryof COPD patientsFigure 3d y1 CD90 mRNA levels were not significantly diï¬erent in ESWtreated fibroblasts and 16HBE cellscompared with nontreated cells Figures 4a“4c y1CD90 protein in the cell supernatants was significantlyincreased in primary bronchial fibroblasts of CS at hp � after ESW treatment Figure 4e No significant changes were observed in primary bronchial fibroblastsof COPD patients or in 16HBE cells Figures 4d and 4fTGF1 mRNA levels were not significantly changed in ESWtreated fibroblasts and 16HBE cells when compared withnontreated cells Figures 5a“5c TGF1 protein in thecell supernatants was significantly increased in primarybronchial fibroblasts of CS at h p � after ESWtreatment Figure 5e No significant changes were observed in primary bronchial fibroblasts of COPD patients orin 16HBE cells Figures 5d and 5f Procollagen1 mRNAlevels were not significantly diï¬erent in ESWtreated fibroblasts and 16HBE cells compared with nontreated cellsFigures 6a“6c Procollagen1 protein in the cellsupernatants wasincreased in primarysignificantly 0cCanadian Respiratory JournalabIncreased cell proliferation was observed in all cellular types studied after challenge with ESW Ttestˆ—ˆ—p andˆ—ˆ—ˆ—p Figure WST1 test for evaluation of cell proliferation after extracorporeal shock wave ESW stimulation of primary bronchial fibroblastsof COPD patients n � a primary bronchial fibroblasts of control smokers n � b and bronchial epithelial cells 16HBE ccbronchial fibroblasts of COPD patients at h p � after ESW treatment Figure 6d No significant changeswere observed in primary bronchial fibroblasts of CS or in16HBE cells Figures 6e and 6f NFκBp65 mRNAlevels were not significantly changed in ESWtreated fibroblasts and 16HBE cells when compared with nontreatedcells Figures 7a“7c NFκBp65 protein in the cell lysates was decreased in primary bronchial fibroblasts ofCOPD patients at h p � after ESW treatmentFigure 7d and increased in 16HBE cells at h p � and h p � after ESW treatment Figure 7f Nosignificant changes were observed in primary bronchial fibroblasts of CS Figure 7e Immunohistochemistry in the Lung Parenchyma of COPDPatients of Alveolar Type II Epithelial Cells Expressing cKitand PCNA In the lung parenchyma of COPD patientsalveolar type II epithelial cells were identified by the use ofanti“thyroid transcription factor1 TTF1antibodyImmunopositivity for cKit CD117 and PCNA was alsooccasionally observed in alveolar septa Figure Doublestaining used for identification of TTF1 cells coexpressingcKitFigures 9a and 9b and PCNAFigures 9c and 9d showed that alveolar type II epithelial cells coexpressing cKit and PCNA were present eventhough rarely observedCD117 Discussionis study shows that extracorporeal shock waves induce cellproliferation of bronchial epithelial cells 16HBE and primary bronchial fibroblasts of COPD patients and controlsmokers As far as markers of cell proliferation are concerned cKit CD117 was increased in bronchial epitheliumat both mRNA and protein levels h after ESW treatmentand it was also increased in primary bronchial fibroblasts at h after ESW challenge Other markers indicative of cellproliferation were also increased PCNA protein increased inCOPDWST1NO ESWESWT24T48T72T0012345ˆ—ˆ—ˆ—ˆ—ˆ—ˆ—ˆ—ˆ—ˆ—CST0T24T48T72NO ESWESWWST1012345ˆ—ˆ—ˆ—ˆ—ˆ—16HBET0T24T48NO ESWESWWST1012345ˆ—ˆ—ˆ—ˆ—ˆ—ˆ— 0cCanadian Respiratory JournaladbecfFigure CKit CD117 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial fibroblasts of COPDpatients a d primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In bronchial epithelium 16HBEcKit increased at mRNA c and protein f levels In primary bronchial fibroblasts of COPD patients cKit increased at protein level d Ttest was used for comparative purposes and p values are reported in the graphsadbecfFigure Proliferating cell nuclear antigen PCNA mRNA a b c and protein d e f expression after ESW treatment in primary bronchialfibroblasts of COPD patients a d primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In bronchialepithelium 16HBE PCNA increased at protein f level Ttest was used for comparative purposes and p values are reported in the graphsT0T24T48T7201234102030COPD CNCOPD ESWcKit CD117 “ˆ† Ct0180021103960767CS CNCS ESWcKit CD117 “ˆ† CtT0T24T48T7202461020304050002016HBE CN16HBE ESWcKit CD117 “ˆ†ˆ† CtT0T24T480246204060800435041800320010020030006839038680037305624COPD CNCOPD ESWcKit CD117 ngmLT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SWCS CNCS ESW000500100015002000250006727038680877507636cKit CD117 ngmLT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW16HBE CN16HBE ESW020406080P 000010791501364cKit CD117 ngmLT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT0T24T48T72PCNA “ˆ† Ct0005101520COPD CNCOPD ESWT0T24T48T7205101520PCNA “ˆ† CtCS CNCS ESWT0T24T48000510152025PCNA “ˆ†ˆ† Ct16HBE CN16HBE ESW01450027520139305288COPD CNCOPD ESWT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW00102030PCNA ngmL00002004006008010000512084270367304489PCNA ngmLCS CNCS ESWT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW0123004620190109820PCNA ngmL16HBE CN16HBE ESWT0 CNT0 SWT24 CNT24 SWT48 CNT48 SW 0cCanadian Respiratory JournalacebdfFigure y1 CD90 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial fibroblasts of COPDpatients a d primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In primary bronchial fibroblasts ofcontrol smokers y1 increased at protein level at and h e Ttest was used for comparative purposes and p values are reported in thegraphsbronchial epithelial cells at h after ESW challenge y1CD90 protein increased in CS“primary bronchial fibroblasts at and h after ESW treatment molecules morerelated to remodeling such as TGF1 protein were increased in CS“primary bronchial fibroblasts at h afterESW treatment and procollagen1 protein increased at hfollowed by a decrease at h in COPD“primary bronchialfibroblasts after ESW treatment A marker of ‚ammationtranscription factor NFκBp65 protein was decreased inCOPD“primary bronchial fibroblasts at h after ESWtreatment but it was increased in CS“primary bronchialfibroblasts and in bronchial epithelial cells after ESWtreatment Markers of cell proliferation such as cKit andPCNA were observed in the peripherallung of COPDT0T24T48T72COPD CNCOPD ESWThy1 CD90 “ˆ† Ct0005101520CS CNCS ESWThy1 CD90 “ˆ† CtT0T24T48T72012316HBE CN16HBE ESWThy1 CD90 “ˆ† ˆ†CtT0T24T4801020304009523087570853209221COPD CNCOPD ESWT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW00200040006000800010000Thy1 CD90 pgmLCS CNCS ESW01500003150239300410T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW000200004000060000Thy1 CD90 pgmL16HBE CN16HBE ESW035960811001447T0 CNT0 SWT24 CNT24 SWT48 CNT48 SW010203040Thy1 CD90 pgmL 0cCanadian Respiratory JournaladbecfFigure TGF1 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial fibroblasts of COPD patients ad primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In primary bronchial fibroblasts of controlsmokers TGF1 increased at protein level at h e Ttest was used for comparative purposes and p values are reported in the graphsadbecfFigure Procollagen1 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial fibroblasts of COPDpatients a d primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In primary bronchial fibroblasts ofCOPD patients procollagen1 increased at protein level d at h T0 followed by a decrease at h panel d Ttest was used forcomparative purposes and p values are reported in the graphsT0T24T48T72TGF 1 “ˆ† Ct0005101520COPD CNCOPD ESWT0T24T48T72TGF 1 “ˆ† Ct00051015CS CNCS ESWT0T24T48TGF 1 “ˆ† Ct0005101516HBE CN16HBE ESW07196046450373903445T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SWCOPD CNCOPD ESWTGF 1 pgmL005010015004487044930863500385T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SWCS CNCS ESWTGF 1 pgmL0005001000150004757089490102101199T0 CNT0 SWT24 CNT24 SWT48 CNT48 SW16HBE CN16HBE ESWTGF 1 pgmL010203040T0T24T48T72Procollanen1 “ˆ† Ct000510152025COPD CNCOPD ESWT0T24T48T72Procollagen1 “ˆ† Ct000510152025CS CNCS ESWT0T24T48Procollagen1 “ˆ†ˆ† Ct00051015202516HBE CN16HBE ESW00220057350024202359T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SWCOPD CNCOPD ESW00100020003000Procollagen1 pgmL00541053750944605958T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW000100002000030000Procollagen1 pgmLCS CNCS ESW010340898407490T0 CNT0 SWT24 CNT24 SWT48 CNT48 SW050100150200Procollagen1 pgmL16HBE CN16HBE ESW 0cCanadian Respiratory JournaladbecfFigure NFκBp65 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial fibroblasts of COPD patientsa d primary bronchial fibroblasts of control smokers b e and bronchial epithelial cells c f In bronchial epithelium 16HBE NFκBp65 increased at protein panel f level at and h of exposure In primary bronchial fibroblasts of COPD patients NFκBp65 decreased atprotein level d at h In primary bronchial fibroblasts of control smokers NFκBp65 increased at protein level e at h Ttest wasused for comparative purposes and p values are reported in the graphspatients and both these markers were occasionally coexpressed by alveolar epithelial type II cells TTF1 in thesepatientsExtracorporeal shock wave therapy is applied in regenerative medicine since it is capable of inducing neoangiogenesis osteogenesis and remodeling through stemcell stimulation [] On the other hand while regenerativetherapy applied to mice with induced emphysema has shownpromising results [] clinical trials performed in COPDpatients were discouraging [ ] Since the human lung alsoin adulthood maintains a significant regenerative potential[“] due to proliferation of diï¬erentiated of stemprogenitor cells andor by their stimulation [ ] we hereinvestigated the proliferative action of ESW at low dosage inbronchial epithelial cells and in primary bronchial fibroblasts of control smokers CS and patients with COPD Ourdata show that all the cell types studied significantly increased their proliferation index WST1 test after ESWtreatment in agreement with data previously obtained formuscle cells or tendon fibroblasts [] Interestingly thecKit CD117 receptor tyrosine kinase protein and mRNAwere increased in 16HBE cells and cKit protein also increased in primary bronchial fibroblasts of COPD patientsafter ESW stimulation It is not clear however if this cellresponse represents an intermediate dediï¬erentiation step ora simple proproliferative stimulus for stimulated 16HBEcells and COPD“primary bronchial fibroblasts Since weexposed welldiï¬erentiated cells we believe that this transitory increment may be interpreted as a proproliferativestimulus induced by ESW exposureIn bronchial epithelial cells 16HBE proliferating cellnuclear antigen PCNA considered a marker of cellproliferation was increased after ESW stimulation confirming again the proproliferative role of ESW exposurefor these lung structure cells is finding in view of thedecreased PCNA levels reported in the lung of COPDpatients [ ] compared with control subjects is particularly relevant since ESW stimulation may contrastthese lower PCNA levels characterizing the damaged lungof these patientse increased y1 CD90 protein level shown afterESW exposure in CS“primary bronchial fibroblasts was notobserved in ESWtreated COPD“primary bronchial fibroblasts or in 16HBE treated cells PCNA protein alsotended to be higher in CS“primary bronchial fibroblastsafter ESW treatment but not in COPD“primary bronchialfibroblasts ese diï¬erences in the response to ESWchallenge of COPD“ and CS“primary bronchial fibroblastsmay in part be due to the reduced proliferation capacity ofthese cells derived from COPD lungs as previously reported [ ] In our welldiï¬erentiated ESWexposedfibroblasts we interpret the increment of y1 protein NFκBp65 “ˆ† Ct012345T24T48T72T0COPD CNCOPD ESW NFκBp65 “ˆ† Ct01234T24T48T72T0CS CNCS ESW NFκBp65 “ˆ†ˆ† Ct000510152025T24T48T016HBE CN16HBE ESW00805026820020907045NFκBp65 pgmL0050000100000150000T72 CNT72 SWT24 SWT48 CNT48 SWT0 SWT24 CNT0 CNCOPD CNCOPD ESWNFκBp65 pgmL036510427702233003830020000400006000080000100000T0 SWT24 CNT24 SWT48 CNT48 SWT0 CNT72 SWT72 CNCS CNCS ESWNFκBp65 pgmL0015500002062865000100001500002004006008001000T0 SWT24 CNT24 SWT48 CNT48 SWT0 CN16HBE CN16HBE ESW 0cCanadian Respiratory JournalFigure Photomicrographs showing thyroid transcription factor1 TTF1 expression panels a b cKit CD117 c d and proliferatingcell nuclear antigen PCNA e f in the peripheral lung tissue of a representative patient with chronic obstructive pulmonary diseaseCOPD Arrows indicate positively stained cells mainly located in the alveolar septa Bars � micronsafter ESW treatment”like that of cKit”as a proproliferative stimulus induced by the treatmentWe found increased levels of secreted TGF1 inCS“primary bronchial fibroblasts h after ESW stimulation TGF signaling pathways are involved in the regulationof many cell functions and in the maintenance of cellularhomeostasis [] We recently reported a decrease of TGF1and TGF3 in bronchiolar epithelium and alveolar macrophages of COPD patients compared with CS [] and thisdecrease may favor the increase of autoimmunity responsesin these patients [] We speculate that the inductionthrough ESW challenge of an increase of TGF in bronchialfibroblasts may play a role in the TGF repositioning andgain in homeostatic function of this important protein in thelungs of COPD patientsTGF induced extracellular matrix and procollagen1production has been reported in pulmonary fibroblasts[] even though it was also reported that the increase ofprofibrotic markersincluding procollagen1 in humanlung fibroblasts may be NLRP3 ‚ammasome dependentand TGF independent [] and associated with increased‚ammation ofthe lung [] We here observed aTTF a0Lung COPDCD a0Lung COPDPCNA a0Lung COPDabcdef 0cCanadian Respiratory JournalFigure Photomicrographs showing alveolar type II epithelial cells TTF1 cells red color coexpressing cKit CD117 brown color ab and PCNA brown color c d in the peripheral lung tissue of a representative patient with COPD Positive doublestained cells can berecognized in the alveolar septa even though their presence was only rarely observed Arrows indicate positively stained cells located in thealveolar septa Bars � micronstransitory increase of procollagen1 protein in COPD“primary bronchial fibroblasts at h after ESW
2
previous studies have shown a strong coexistence of colorectal neoplasia crn and cardiovascular diseases cvd this study was aimed to summarize the available evidence on association of cvd risk with early crn detection in asymptomatic populations pubmed web of science and embase were systematically searched for eligible studies published until dec studies exploring the associations of recommended cvd risk assessment methods eg risk scores carotid artery plaque and coronary artery calcium score [cacs] with risk of crn were included metaanalyses were conducted to determine the overall association of cvd risk with the crn a total of studies were finally included the association of carotid artery plaque with the risk of colorectal adenoma ad was weakest pooled odds ratio [or] confidence interval [ci ] participants with cacs100 had about 2fold increased risk of ad than those with cacs0 the pooled ors were ci and ci for the risk of advanced colorectal neoplasia an and ad respectively in participants with framingham risk score frs20 when compared to participants at low risk frs10 frs might help identify subgroups at increased risk for an but further studies are needed keywords cardiovascular disease risk assessment colorectal neoplasiaintroductionboth colorectal cancer crc and cardiovascular diseases cvd are the leading causes of mortality and morbidity worldwide12 previous studies have shown a strong coexistence of colorectal neoplasia crn and cvd probably due to the shared risk factors eg smoking obesity and metabolic syndrome and pathophysiological mechanisms eg chronic inflammation and oxidative stress3“current guidelines8“ recommend assessing the cvd risk in healthy people using risk estimation scores such as framingham risk score frs1112 procam13 and the pooled cohort equation14 which are based on individuals™ medical history and easily available laboratory data in addition assessment of subclinical atherosclerosis by imaging modalities could be added as risk modifiers to help make clinical decisions for borderline or intermediaterisk adults8“ routine use of imaging modalities is not recommended for cvd risk assessment in clinical practice due to the medical costs or invasiveness but incorporation of imaging data such as the anklebrachial index abi coronary artery calcium score cacs and carotid artery plaques cap could improve the prediction of cvd risk15“clinical epidemiology “ chen this work is published and licensed by dove medical press limited the full terms of this license are available at wwwdovepresscomtermsphp and incorporate the creative commons attribution “ non commercial unported v30 license httpcreativecommonslicensesbync30 by accessing the work you hereby accept the terms noncommercial uses of the work are permitted without any further permission from dove medical press limited provided the work is properly attributed for permission for commercial use of this work please see paragraphs and of our terms wwwdovepresscomtermsphp 0cchen dovepressvarious risk scores have also been developed for predicting advanced colorectal neoplasia an18“ although several studies2526 have reported that elevated blood lipids the well documented cvd risk factor and history of cvd were associated with increased risk of crc the majority of risk scores developed for an did not include them into the models27 recent studies have reported the associations between cvd risk assessment and risk of1 crn higher frs estimating the 10year risk of developing coronary heart disease chd1112 was significantly associated with the higher risk of an frs vs frs10 odds ratio [or] confidence interval [ci] “ abi was associated with 13fold increased risk of an in a recent study29 cap and cacs were also found to be positively related to the increased risk of adenoma ad and an in several studies30“given a number of shared risk factors and mechanisms between cvd and crc and the emerging epidemiological evidence of association between cvd risk and crc there is a possibility that cvd risk assessment could help trigger crc screening therefore the aim of this review was to provide an overview of the cvd risk assessment methods and their associations with the risk of crn fully understanding of the current knowledge and existing gap might promote better prevention and treatment for cvd and crc circulating and urinary biomarkers have either no or only limited value when added to cvd risk estimation score systems834 thus only score models and imaging methods recommended as risk modifiers abi cacs and cap in the guidelines8“ were included in this reviewmaterials and methodsthis systematic review was conducted following the procedure recommended by the cochrane collaboration35 and was reported according to the preferred reporting items for systematic reviews prisma checklist36 ethical approval and patient informed consent were not necessary since all the data included in the current study were obtained from previously published studiesand metaanalyses remaining publications and reference lists were scrutinized studies that fulfilled the predefined criteria were includedinclusion and exclusion criteriawe required that included studies meet the following criteria published as an original research in a peer reviewed cardiovascular risk has been assessed using either score models or imaging methods recommended as risk modifiers abi cacs and cap in the guidelines3 only included participants who were considered asymptomatic reported the association of cvd risk assessment results with the risk of crn studies were excluded if they were published as conference proceedings dissertations or s only or were not published in english pico eligibility criteria for this review were presented in the supplementary table s1data extractiontwo authors yc and xc independently performed data extraction of all included studies the following information was ed author publication year study period number of participants age number of males outcome ad an and so on data source medical records questionnaires or both cvd risk assessment and association indexdiscriminatory accuracy or hazard ratio [hr] specificity sensitivity or area under the receiver operator characteristic curve values] in case of any disagreement consensus was obtained by discussionquality assessment in eligible studiesrisk of bias and applicability were assessed according to quality assessment of diagnostic accuracy studies2 quadas237 quadas2 evaluates the risk level of bias composed of four basic components patient selection index test reference standard flow and timing clinical applicability is also assessed for the first three components the risk of bias and concerns regarding applicability for each study was then rated as œhigh œlow or œunclearliterature search strategiespubmed embase and web of science were searched up to december to identify the relevant papers the searched items were presented in the appendix which mainly covers expressions for cvd risk score models recommended imaging modalities crn and discriminatory accuracy or strength of association after removal of duplicates titles and s of records were screened according to the inclusion and exclusion criteria full texts of the statistical analysiswe pooled ors for the same cvd risk assessment index using r statistical software version and the r œmeta package version for frs and cacs ors were pooled separately for different levels of scores using the lowest level as reference two kinds of outcomes ad and an were reported in the studies using frs for cvd risk assessment and thus ors were pooled separately for different outcomes heterogeneity across studies was evaluated submit your manuscript wwwdovepresscom dovepress clinical epidemiology 0cdovepress chen using cochrane™s q statistic with p value and the i2 statistic if significant heterogeneity was observed i2 or pqstatistics a randomeffects model was used to calculate pooled estimates otherwise a fixedeffects model was used35 twosided p values of or lower were considered to be statistically significantresultsliterature search resultsa total of records were obtained in the initial search including citations from pubmed citations from embase and citations from web of science after removal of duplicates n1609 and exclusion due to our predefined criteria n5727 records were qualified for fulltext assessment fortyfour records were excluded due to the inclusion and exclusion criteria finally a total of studies28““ including one study which was identified through crossreferences were included the detailed information of the selection process was presented in figure study characteristicstable summarized the basic characteristics of the included studies published between and of the included studies nine were from korea and the other three studies were from japan austria and turkey respectively the study periods stretched from to with sample sizes ranging from to only one was designed as a prospective study41 and the others were crosssectional studies most studies included participants aged older than years and only one study enrolled subjects aged years32 in addition most studies were predominantly in men with proportions of males among participants ranging from to four cvd risk assessment methods abi cap cacs and frs were used in the included studies all studies explored the role of cvd risk assessment method on the detection of ad and some of risk adenoma3032 and an2829384243focused on colorectal high them also figure flowchart of inclusions of studies about relation of cvd risk to crn note adapted from moher d liberati a tetzlaff j preferred reporting items for systematic reviews and metaanalyses the prisma statement plos med creative commons license and disclaimer available from httpcreativecommonslicensesby40legalcode36 abbreviations cvd cardiovascular disease crn colorectal neoplasiaclinical epidemiology submit your manuscript wwwdovepresscom dovepress 0cchen dovepresstable basic characteristics of included studies about relation of cvd risk to colorectal neoplasiastudycountrystudy periodnumber of participantsyamaji y kim j kim h cha jm yun ke choi sh yang mh kim hb lee yj 2019a41lee jy niederseer d basyigit s japankoreakoreakoreakoreakoreakoreakoreakoreakoreaaustriaturkey““““““““““age years mean±sd ± ± 530b median± ± ± ± 526b± ± ±male n outcomec data sourcececum intubation ratedcvd risk assessment ad anad hraadad anad hraadadadadad anad anad anmrqmrqmrqmrmrqmrqmrmrmrqmrqmrqmrqnrnrnrnr‰¥nrabicapcapcapcacscacscacscacscacsfrsfrsfrsnotes ait is a retrospective followup study and all the other studies are crosssectional bsd was not reported cdetected by colonoscopies in all included studies d100 cecum intubation rate participants with failure of cecum intubation were excluded nr not reported studies mentioned that colonoscopies were extended to cecum in the methods section but did not reported the success rate of cecum intubation abbreviations abi anklebrachial index ad colorectal adenoma an advanced colorectal neoplasia cacs coronary artery calcium score cap carotid artery plaque cvd cardiovascular disease frs framingham risk score hra high risk adenoma mr medical records nr not reported q questionnaires sd standard deviationquality assessment of studiesthe results for the quality of included studies using the quadas2 tool are presented in table regarding patient selection one study by kim did not provide detailed information about patient selection31 thus the risk of bias and applicability concerns were rated unclear for this domain in this study otherwise no major risk of bias or applicability concerns were identifiedassociation of cvd risk assessed by different methods with crc risktable described the details of the cvd risk assessment methods in the included studies abi was associated with 13fold ci increased risk of an29 three studies reported the weak association between cap and risk of ad303138 one of them also showed an increased risk of an in the participants with cap but the results were not statistically significant or table risk of bias and applicability judgements in quadas2studyrisk of biasapplicability concernstotalpatient selectionindex testreference standardflow and timingpatient selectionindex testreference standardyamaji y kim j kim h cha jm yun ke choi sh yang mh kim hb lee yj lee jy niederseer d basyigit s totalˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšnotes œ_ high risk œˆš low risk œ unclear riskˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšsubmit your manuscript wwwdovepresscom dovepress clinical epidemiology 0cdovepress chen table details of the cvd risk assessment methods in the included studies about relation of cvd risk to colorectal neoplasiastudycategoriesboutcome or[ ci]yamaji y kim j abnormal abiabnormal abicap yescap yeskim h cap yescha jm yun ke choi sh cap yescap yescacs cacs “cacs cacs cacs “cacs cacs cacs “cacs yang mh201339cacs kim hb cacs “cacs “cacs ‰¥lee yj 2019a41cacs lee jy niederseer d basyigit s frs intermediatefrs highfrs intermediatefrs highfrs intermediatefrs highfrs intermediatefrs highfrs intermediatefrs highfrs intermediatefrs highadanadhraadadanadadadhrahrahraadadadadadadadadadadananadadananadadanan[ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ]hr [ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ]notes ain participants without adenoma cacs0 at baseline compared to cacs0 increased the risk of colorectal adenoma at followup colonoscopy hr ci bthe lowest level was defined as reference abbreviations abi anklebrachial index ad colorectal adenoma an advanced colorectal neoplasia cacs coronary artery calcium score cap carotid artery plaque frs framingham risk score hra high risk adenoma hr hazard ratio or odds ratio ci confidence interval ci in addition the presence of cap was associated with increased risk of colorectal high risk adenoma or ci four studies reported ors for different levels of cacs with cacs0 as reference32333940 highest cacs levels seemed to be associated with the increased risk of ad with or ranging from to the 10year chd risk estimated by frs was categorized as low risk intermediate risk “ and high risk ‰¥ participants with high risk of 10year chd had increased risk of either ad or an in the study by basyigit participants at high chd risk had about 4fold or ci increased risk of an28metaanalyses of available ors for different cvd risk assessment methodsmetaanalyses were performed in the studies that provided ors and their cis for the same cvd risk assessment index the association of cap with the risk of ad was weakest the pooled or ci a medium level of cacs cacs “ was associated with 134fold increased risk of ad when compared to the lowest category of cacs cacs0 participants with cacs100 had an increased risk of ad and the pooled or was ci the pooled ors were ci and ci for the risk of an and ad respectively in participants with high chd risk frs20 when compared to participants at low chd risk frs10 further details were presented in table and in the supplementary figures s1“discussionthis systematic review summarized the associations of recommended cvd risk assessment methods with risk of crn in asymptomatic populations a total of studies including four different methods were identified among these methods frs was most strongly associated with risk of both an and ad participants with frs20 have about 34fold and 23fold increased risk of an and ad respectively when compared to participants at low chd risk frs10 only one study29 reported that abnormal abi greatly increased the risk of an thus it was not included in the metaanalysisboth crc and cvd are thought to develop via a process of insulin resistance inflammation and oxidative clinical epidemiology submit your manuscript wwwdovepresscom dovepress 0cchen dovepresstable metaanalysis of odds ratios for different cvd risk assessment toolsstudycvd risk assessmentcategoriesaoutcomeor cicapcacscacscacsfrsfrsfrsfrsyes vs nocacs vs cacs0cacs “ vs cacs0cacs vs cacs0intermediate vs low riskhigh vs low riskintermediate vs low riskhigh vs low riskadadadadadadanan note athe lowest level was defined as reference abbreviations ad colorectal adenoma an advanced colorectal neoplasia cacs coronary artery calcium score cap carotid artery plaque cvd cardiovascular disease frs framingham risk score or odds ratio ci confidence intervalstress74547 which might partially explain why they share a number of risk factors eg alcohol consumption tobacco use physical activity use of antiinflammatory agents obesity and diabetes mellitus4548 in addition several cellular metabolismrelated pathways eg ampk and pparγ signaling pathways eg wnt signaling pathway and genetic pathways eg lrp6 mutation and tcf7l2 polymorphism are not only associated with accelerated atherosclerosis and an increased risk of cvd but also linked to cancer development and progression7 better understanding of these overlaps might promote shared management of prevention and treatment for both disordersrisk of an in this review the strength of associations between identified cvd risk assessment methods and the risk of crn was generally weak except frs which was modestly associated with frs20 vs frs10 frs was calculated based on age total cholesterol highdensity lipoprotein cholesterol smoking status systolic blood pressure and treatment of blood pressure which are typically available in the medical records44 compared to the more sophisticated risk calculators232449 for predicting an which need variables such as physical activity red meat intake and vegetable consumption frs has relatively higher generalizability and lower recall bias a recent study has recommended the combined preventive screening and research efforts in the prevention of both cvd and cancer50 if participants with highrisk of cvd predicted by frs could be recommended to have a screening for crn which will help increase compliance and uptake of crc screening as persons who are aware of their increased risk are more likely recommendations furthermore it also maximizes the medical values of the comply with to expert information participants obtain from a clinical examination or risk assessment and thus reduces the time and costs for health carehowever there are some issues that merit our attention firstly the included studies are all crosssectional which limits the comparisons between frs and the previously developed risk prediction models for crc secondly frs has its own limitations frs only estimates 10year chd risk for all individuals years or older but not the overall cvd risk in addition it is developed based on the american population while most of study participants are asians in the included studies studies have shown that frs overestimated cvd risk in the asian cohorts51“ at last the included studies tended to yield results with wide ci probably due to the limited number of participants the wider the ci the less the precision in summary higher cvd risk might trigger concurrent crc screening which should be further validated on largescale studies and future studies could consider about using the overall cvd risk score models developed from data of local cohorts to predict the risk of crcas for imaging data the association of cap or cacs with risk of ad is not strong enough that imaging index alone might not be useful for informing early detection of crn similarly routine screening with imaging modalities to predict future cardiovascular events is generally not recommended in clinical practice but use of these imaging techniques has been shown to improve cvd risk assessment and serve as a guide for initiating preventive therapies8“ a high cacs can help modify the predicted risk obtained from frs alone especially among patients in the intermediaterisk category16 up to now only one risk score developed in the multiethnic study of atherosclerosis mesa study used both cacs and submit your manuscript wwwdovepresscom dovepress clinical epidemiology 0cdovepress chen traditional risk factors to predict the 10year chd risk55 inclusion of cacs in the mesa risk score offered significant improvements in risk prediction cstatistic vs p factors in the risk models like smoking behaviors and blood lipids are closely related to the incidence and progression of cvd but they are not direct markers of current status of atherosclerosis this might help explain why the performance of risk models is improved by adding markers with anatomical delineation through imaging technology accounting for the higher performance of the combined use of risk scores and imaging tools on cvd risk assessment further studies could consider about exploring the association of combined form of them with the risk of crcwe also observed that less than half of included studies reported the associations of cvd risk with both risk of an and ad2829384243 colonoscopy is considered to as a valid primary screening tool for crc and is able to detect both ad and an the lower prevalence of an and the limited number of participants in several included studies might limit the power to explore the relation of an with cvd risk which could partly explain why most of studies did not include an as outcome therefore the findings should be carefully interpreted and further validated on largescale studiesour study has some strengths comprehensive search strategies along with welldefined eligibility criteria were used to help identify relevant s in addition two reviewers independently extracted data and assessed the risk of bias in the included studies however several limitations should also be addressed firstly the current meta analysis was based on observational studies there were the possibilities of potential effects of unknown or residual confounding factors on our results secondly as we only considered about established cvd risk models and recommended imaging modalities the potential of other cvd risk assessment index on the detection of crn was not summarized and compared in this study however it is also reasonable to just include these methods since their feasibility and performance for cvd risk prediction have been well approved in the clinical practice thirdly cut off values and group comparisons for the same cvd risk assessment method varied in the included studies which limits the synthesis of results for example the cut off values for cacs are the tertiles of cacs in the study by kim 40 however cacs was categorized into three groups with cut off values at and in the other studies3233 therefore less studies were included in the metaanalysis which might influence the accuracy of the pooled results lastly most of studies were conducted in asian populations which is an inherent limitation of the included studies thus our findings might not be applicable to other populations and needs to be externally validated in racially diverse populationsconclusionsto our knowledge this is the first review that applies metaanalyses to determining the overall association of recommended cv risk assessment methods with the risk of crn in the asymptomatic population frs calculated based on shared risk factors of cvd and crc shows potential to help identify subgroups at increased risk for an whether the combination of frs and imaging index is useful for the optimal evaluation of crn risk remains to be solved in the future studies cvd risk might inform crc screening which needs more research in the future to validate its feasibility and effectivenessabbreviationsabi anklebrachial index ad colorectal adenoma an advanced colorectal neoplasia cacs coronary artery calcium score cap carotid artery plaque chd coronary heart disease ci confidence interval crc colorectal cancer crn colorectal neoplasia cvd cardiovascular disease frs framingham risk score hr hazard ratio hra high risk adenoma mr medical records nr not reported or odds ratio prisma preferred reporting items for systematic reviews and metaanalyses quadas2 quality assessment of diagnostic accuracy studies2 q questionnaires sd standard deviationfundingthis research was funded by national natural science foundation of china grant number disclosurethe authors report no conflicts of interest in this workreferences bray f ferlay j soerjomataram i siegel rl torre la jemal a global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries ca cancer j clin “ doidoi103322caac21492 joseph p leong d mckee m et al reducing the global burden of cardiovascular disease part the epidemiology and risk factors circ res “ doidoi101161circresaha117308903 chan aoo man hj kwok fl et al prevalence of colorectal neoplasm among patients with newly diagnosed coronary artery disease j am med assoc doidoi101001jama29812 clinical epidemiology submit your manuscript wwwdovepresscom dovepress 0cchen dovepress chan aoo lam kf tong t coexistence between colorectal canceradenoma and coronary artery disease results from patients aliment pharmacol ther “ doi doi101111j13652036200602958x wang sc schulmanmarcus j fantauzzi j et al colon cancer laterality is associated with atherosclerosis and coronary artery disease j gastrointest oncol doidoi1021037jgo20180918 kahr pc hammerl s huberschönauer u et al atrial fibrillation a new indicator for advanced colorectal neoplasia in screening colonoscopy j clin med doidoi103390jcm8071083 masoudkabir f sarrafzadegan n krahn a et al cardiovascular disease and cancer evidence for shared disease pathways and pharmacologic prevention cardiovascular disease and cancer evidence for shared disease pathways and pharmacologic prevention hhs public access atherosclerosis “ doidoi101016 jatherosclerosis201706001 piepoli mf hoes aw agewall s european guidelines on cardiovascular disease prevention in clinical practice the sixth joint task force of the european society of cardiology and other societies on cardiovascular disease prevention in clinical practice constituted by representatives of societies and by invited experts developed with the special contribution of the european association for cardiovascular prevention rehabilitation eacpr atherosclerosis “ doidoi101016jatherosclerosis201605037 arnett dk blumenthal rs albert ma et al accaha guideline on the primary prevention of cardiovascular disease a report of the american college of cardiologyamerican heart association task force on clinical practice guidelines circulation 201914011e596“e646 doidoi101161cir0000000000000678 mach f baigent c catapano al esceas guidelines for the management of dyslipidaemias lipid modification to reduce risk eur heart j “ doi cardiovascular doi101093eurheartjehz455 grundy sm becker d clark lt et al detection evaluation and treatment of high blood cholesterol in adults adult treatment panel iii circulation “ doidoi101161circ106 cleeman ji executive summary of the third report of the national cholesterol education program ncep expert panel on detection evaluation and treatment of high blood cholesterol in adults adult treatment panel iii j am med assoc “ doi doi101001jama285192486 assmann g cullen p schulte h simple scoring scheme for calculating the risk of acute coronary events based on the 10year follow up of the prospectiv
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increasing relevancy of geospatial technologies such as geographic information system GIS inthe public health domain particularly for the infectious disease surveillance and modelling strategies Traditionally thedisease mapping tasks have faced many challenges” authors rarely documented the evidence that were used to createmap before evolution of GIS many errors aroused in mapping tasks which were expanded extremely at global scalesand there were no fidelity assessment of maps which resulted in inaccurate precision This study on infectious diseasesgeosurveillance is divided into four broad sections with emphasis on handling geographical and temporal issues to help inpublic health decisionmaking and planning policies geospatial mapping of diseases using its spatial and temporalinformation to understand their behaviour across geography the citizen™s involvement as volunteers in giving healthand disease data to assess the critical situation for disease™s spread and prevention in neighbourhood effect scientificanalysis of healthrelated behaviour using mathematical epidemiological and geostatistical approaches with capacitybuilding program To illustrate each theme recent case studies are cited and case studies are performed on COVID19 todemonstrate selected modelsKeywords Geospatial technology 01 Citizen Science 01 Public health 01 COVID19 01 Mathematical epidemiologyIntroductionThe public health sector™s increasing demand for mappinganalytics and visualization had started a date back in thelast years which has resulted in a growing informationage technology for communicable disease surveillance andepidemiology Baker Bos and Blobel Friede Friede Khan Reeder Yu and Edberg This continuous publichealth burden with advances in information technology Sameer SaransameeriirsgovinPriyanka SinghPriyankaiirsgovinVishal KumarVishalkumariirsgovinPrakash ChauhanprakashiirsgovinIndian Institute of Remote Sensing Indian Space Researchanisation Kalidas Road Dehradun Indiacombined with spatial data led to the development ofvarious tools and systems that provides visualization ofdisease data in space and time Dredger Kothari Robertson and Nelson Schriml et alThe first integral definition of public health was given byWinslow as ˜˜science and art of preventing diseaseprolonging life and promoting health through the anized efforts and informed choices of society anizations public and private communities and individuals™™The American Public Health Association APHA mentioned public health as a practice of preventing the spreadof disease and an aim of promoting good health from smallcommunities to across the world Turnock Advances in information technology and spatial features resultedin geospatialtechnology which is acute for mappingsurveillance predicting outbreaks detecting clustering andanalysing spread patterns of infectious diseases with epidemic or pandemic potential in communities and acrossterritories AvRuskin Carpenter Castronovo Dominkovics Gao 0c Heymann and Brilliant Hills Klompas Reis Geospatial technology has provided visualization and analytical tools topublic health professionals and decision makers to executediseases control programs in affected andor suspectedregions and make analysis and predictions possible thatwas once technologically out of reachGeospatial technology includes geographical information systems GIS global positioning systems GPS andsatellitebased technologies such as remote sensing RSGIS is known for geographic data capture input updatemanipulation transformation analysis query modellingand visualization of all forms of geographically referencedinformation through the set of computer programs BonhamCarter GPS provides positioning navigationand timing PNT services by capturing data from satellitesand providing it to users Eldredge and RS isan earth observation instrumentthat delivers regionalinformation on climatic factors and landscape featuresTherefore GPS and RS provide regional and spatialinformation while GIS provides geospatial data integrationas well as accurate geospatial analysis in realtime mannerZhen Geospatial Technology and InfectiousDisease SurveillanceInfectious diseases mostly adapts antimicrobial andmobility features later formed in a shape of pandemic andor epidemic Chen Cheng Lee andNishiura which forced public health authorities tounderstand not only the diseases virulence but also itsdemographic and environmentalfactors that helps inmaking spread patterns though space and time domainCroner For example the global spread of highlypathogenic avian ‚uenza HPAI H5N1 in “ withno effective vaccines led to concern among public healthdecision makers in spite of many international programsRappole and Huba´lek The reason behind theirconcern was they were lacking of disease surveillance toolin its initial stage which caused inaccessibility to populations atrisk and faced difficulties in implementingimmunization strategies at a global scale Kitler Stoto However the impact of environmentaland demographic factors also plays a major role as this caninform about the interaction between hosts and pathogensand patterns of spread in space and timeThe GIS provides dynamic maps to understand geographical distribution of diseases for analysis on frequencyof cases disease mapping spatial cluster of diseases disease association with environmentalfactors networkanalysis etc With such a visualization and analyticalJournal of the Indian Society of Remote Sensingforservice frameworkcapabilities GIS technology is holding a widespreadgrowth in public health Ahmad 2011a b Booman HanafiBojd Kolivras Martin Nykiforuk and Flaman Abdul Rasam Zhang Zhen Theseamless integration of GIS with realtime infectious diseaserelated diverse datasets through webbased mappingto the development of geospatial dashboardleadsgeospatialinfectious diseasesurveillance Dent Gao Yun Theinfectious diseaserelated data mightinclude diseasesurveillance data activeconfirmed cases and health system data hospital visits emergency services availabilitynursedoctor availabilityICUbed availability Many source geospatialstandards of GeospatialConsortium OGC are used as a Web Map ServiceWMS Web Coverage Service WCS Web ProcessingService WPS Web Feature Service WFS etc Bulatovic´ Gao to visualize accesspublish and manipulate geospatial resources Also manyother popular industrial geospatial standards are developedby ESRI Google Yahoo and MapInfo Granell to fetch locationbased data and provide infectiousdisease surveillance dashboard to monitor and control thegeographically spread of disease Zhang TheGeocoded Really Simple Syndication GeoRSS taggedXML files from GeoRSS services can also be used toprovide geocoded infectious disease news from socialmedia platform Tolentino KassHout andAlhinnawi 2013a b Kodong Historical ContextThe mapping of infectious diseases using geospatial andinformation technology to benefit public health is not a newway of tracking the diseases Ahmad Cui Hirsch Hornsby Matthew May Mujica Nicholson and Mather Noble Perl and Moalem Williams The historical disease mapping has faced manychallenges” authors rarely documented the evidencethat were used to create map after mapping had beenimplemented before the beginning of geographical information systems many errors arouse which were expandedextremely at global scales and there were no fidelityassessment of maps which resulted in inaccurate precisionBut nowadays wide range of geospatial applications areavailable in public health community with a possibilities ofvisualization analysis detection of clusters formed andcalculate diseaserelated metrics such as incidence andprevalence rate Beck Clarke Hay Jacquez Kleinschmidt Lawson and 0cJournal of the Indian Society of Remote SensingLeimich Moore and Carpenter Robinson Wilkinson The earliest mapping for visualisation ofthe linkbetween disease and place was done in on plagueepidemic in Italy Dent During cholera outbreak in the study of physician John Snow had made a novelcontribution in history of public health and epidemiologyby using cartography applications and geographic visualization in fighting cholera After years the maps wereidentified as a communication tool in understanding andtracking of infectious diseases such as the ‚uenzapandemic yellow fever and cholera Since then revolutionof webbased tools started in applied health geographyBoulos The trend of infectious disease mappingcould be seen from review of the Health GIS literature which demonstrated that research papers out of were focused on infectious disease mapping Lyseen Covid19The ongoing pandemic outbreak targeting humans™ respiratory system was recently discovered in December by the name of Coronavirus Disease Covid19 WorldHealth anization from a cluster of patients with acuterespiratory distress syndrome in Wuhan Hubei ProvinceChina Huang Lu 2020a b and spreadglobally by March This pathogenic disease is structurally related to the Coronavirus CoV which belongs tofamily Coronaviridae and the order Nidovirales Thisfamily is classified into four genera”AlphacoronavirusBetacoronavirus Gammacoronavirus and Deltacoronavirus on the basis of their phylogenetic and genomicanalysis The species of Alphacoronavirus and Betacoronavirus infect mammals causes respiratory illness inhumans and gastroenteritis in animals while species ofGammacoronaviruses and Deltacoronaviruses infect birdsbut some of them can also infect mammals Woo et alfrom Betacoronavirus The two virusgenus”Severe Acute Respiratory Syndrome SARSCoVor Middle East Respiratory Syndrome MERSCoV”hadearlier demonstrated that coronaviruses can cause significant public threat Ge The COVID19 is categorizedby World Healthanization WHO on the basis of genomic sequencinganalysis ofrespiratory tract samples which isobtained from total of nine patients Huang Lu 2020a b COVID19 has started behaving like theonceinacentury pandemic by affecting healthy adults aswell as elderly people with some health issues and byinfecting others at an exponential rate of increase thanSARS or MERSinto BetacoronavirusspecieslowerGeospatial TechnologyDuring occurrence of diseases geospatial technologies andservices could help in representing the spatiotemporalinformation and in analysing the dynamic spread of diseases As mentioned by Boulos geospatial technologies and services which performs in real time mannerare tremendously relevantto create a ˜˜spatial healthinformation infrastructure™™ In this section a review onmany geospatial technologies with enabled IT services iscarried out to understand and analyse the spread and outbreak of disease with a case study on COVID19 pandemicCitizen Scienceissues and concernsThe expansion of Citizen Science from biodiversity andecological domain Haklay MillerRushing to public health community across spatial extentsmade an urgent need to study its different forms Crowl The indepth report of EU describes taxonomyof Citizen Science in three levels European Commission described in Roy Wiggins andCrowston and Haklay Roy categorized Citizen Science by participant™s number and oftheir spread ˜˜local™™ and ˜˜mass™™ and ˜˜thoroughness™™time and resource investment or King described ˜˜for the people with the people or by the people™™ about Citizen Science activities Wiggins andCrownston classified Citizen Science projects inconservation managing natural resources action addressing localinvestigation answering scientific questions and education providingknowledge to citizens Haklay classified CitizenScience into four levels based on participant™s engagement” level is crowdsourcing in which citizens withless or no knowledge on activity perform as sensors tocomplete computing tasks level is distributed intelligence where citizens are being trained with skills forinterpretation of collected data level is participatoryscience in which citizens decide about research questionsand types of data to be collected and level is extremewhere citizens are fully involved in defining researchstrategies data collection data interpretation and performing scientific analysis Apparentlythe concept ofCitizen Science is rare in public health domain but some ofits contribution seen in some studies which not only helpsin predicting disease risks but also in combating theinfectious diseases CurtisRobles Palmer Smolinski Wilson Another approach similar to Citizen Science is ˜popularepidemiology™ in which experts and laypersons jointly 0ccollect environmental data responsible for particular healthconsequences Brown or ˜street science™ as a process in which general public communities actively engagedin defining problems framing of research questions anddecisionmaking activities about research design CorburnCrowdsourceVGI Mobile AppsDespite technological and computational developments inGeoWeb many web technologies such as jQuery andAJAX mapping APIs like Google and GPS devicesresulted in a new revolution of neogeography Turner where mapping is done by crowd and can bereached by anyone from general public members groupSuch revolution brought a trend of Volunteered GeographicInformation VGI which is first coined and explained byGoodchild 2007a According to Goodchild 2007b VGIhighlighted the human capabilities in collecting geospatialinformation by using five senses and then integrating withexternal sensors of mobile devices like GPS accelerometer camera digital compass and microphone gives valuable datasets which can neither be retrieved from satelliteimagery nor collected with any GPS receivers Anothersuccessful term in geospatial mapping using mobile technology is crowdsourcing Heipke HudsonSmith which was coined by Howe thatinvolves the collection of geospatial information or mapping of any particular activity by an undefined crowd ornetwork of people Both terms VGI and crowdsourcingslightly differ but they are usually recognized as a synonyms or even as a combined term ˜˜crowdsourcing geographic information™™ Sui Over the lastdecade VGIoriented source mobileareEpiCollect Aanensen for ecology and epidemiology NoiseTube Maisonneuve httpnoisetubenet and Noise Battle GarciaMartı´ for noise monitoring Skywatch Windoo httpwindoochfor weather monitoring Mappiness httpwwwmappinessukfor behavioural analysis MacKerron andMourato appsThe source mechanism for data collection usingAndroid devices can be performed by Data KitODK suite107 https datakit which is composed of ODK Collect and ODK Aggregate ODK Collecthttps datakitusecollect provides a customizable framework for geospatial data collection and ODKAggregate is a web application that runs on ApacheTomcat server httptomcatapache to store collecteddata through a synchronization with a databaseforexample PostgreSQL Brunette As suchsuite™s performance can be seen in various activities likeagricultural monitoring Krosing and Roybal Journal of the Indian Society of Remote Sensingmonitoring of deforestation and school attendance documentation of war crimes and health programs Anokwa Digital Contact TracingNowadays COVID19 has become the greatest threat forpublic health in last years and due to such pandemicvarious levels of lockdown are issued across the world tobreak its chain of infection transmission However this isthe first approach to invade the contagion but once itwould be lifted this pandemic would start in a new wayand might reach its highest peak by infecting more andmore population Ferguson Thereforetocombat with such a global pandemic threat anotherapproach is discovered by a group of researchers known asdigital contact tracingSmartphonebased contact tracing is known as a digitalcontact tracing which presents a sustainable solution tolimit the transmission of infectious disease by tracing theirpotential transmission routes in a population howeversuch an app presents significant concerns regarding privacy The digital contact tracing works on the principle of˜crowdsource data™ by measuring the proximity to aninfectious person In previous diseases risk surveillancethe contact tracing apps were used to pool location timestamped data to determine the exposures to risk of infectionsSacks Such data are highly personal and leadmany privacy concerns Smith but they werenot always accurate to infer the exposure risks due to noisydata Farrahi Therefore various smartphoneapps are developed in COVID19 pandemic in which someapps use location for proximity and some of them are notusing location services of mobile device subject to theprivacypreserving natureCOVID19 Contact TracingIn order to illuminate the epidemiology of COVID19 andto characterize its severity Lipsitch there is anurgent need of digital platform that captures realtimeaccurate information on COVID19 patients diseasesdiagnosis treatment and clinical reports and whom theyget interacted at which place to detect clusters and generatealerts Such information may help in understanding riskfactors of infection and in predicting the next generation ofinfectious persons FitzGerald Addressing thisunprecedented challenge many mobile apps have beendeveloped and are being used at large scale and some ofthem are as follows 0cJournal of the Indian Society of Remote Sensing¢ COVID Symptom Study COVID Symptom Tracker”This mobile app is developed in collaboration of ZoeGlobal Ltd a digital health care company and a groupof academic scientists from Massachusetts GeneralHospital and King™s College London which waslaunched in UK on March and becameavailable after days in USA This app enquires aboutage location and other diseases risks and also a selfreporting function is enabled which is associated withCOVID19 infection and exposure Drew This app retrieve updates on healthcare worker™sexperiences who are on COVID19 duty their stressand anxiety and use of personal protective equipmentPPE kits are being surveyed through this app toobserve intensity of health care workers Drew et alappimplemented¢ Aarogya Setu”This mobile app is launched on April by Government of India to aware general publicon COVID19 symptoms government advisory measures online consultation facility and dynamics ofdisease Thiscrowdsourcingapproach by which general public members enter theirdetails for selfassessment and this assessment is thenused to trace the infectious contacts or agents as adigital contact tracing concept This app uses locationservices to geolocate the users and Bluetooth tomaintain the log of contacts when one userdevicecomes in contact with another userdevice and as suchdigital contact tracing activity helps in identifying thecluster of diseases and communities which are at risk ofinfection The Aarogya Setu app was downloadedby million users within days of its launchUpadhyay and by using app™s crowdsourcedata the Indian government detected approx positive casesinformed probable users ofbeing at risk and identified potential clusters TheTimes of IndiaNumerous digital contact tracing apps are in use indifferent parts of world”TrackCOVID Yasaka TraceTogether Bay WeTrace De Carli and Google and Apple™s recently announcedjoint initiative Li and Guo COVID19 Data Visualization and ExploratoryData AnalysisWith early experiences of epidemics such as “SARSCoV Boulos and the “ MERSCoVGikonyo and other seasonal flu™s online realtime or nearrealtime mapping of diseases™ occurrencesusing geospatial technologies and web applications havealways been used as a pivotal webbased tools in trackinghealth threats and combating infectious diseases Thissection described a range of mapping dashboards based ongeospatialtechnologies for tracking and unfolding thecoronavirus disease around the world Some of the globaland national geospatial initiatives with an aim to supplyinformation faster than diseases are as summarized inTable Infectious Diseases ModellingThe intention of infectious diseases surveillance is to detectepidemics in their early stages so that the countermeasurescould be taken for preventing its wide spread Suchsurveillance tasks require many epidemiological and statistical methods with geospatial features in investigatingepidemics preferably from localized areas The reason forpreferring the local areas for investigation is because epidemics generally emerged in small areas and then spreadwidely if they are not controlled However some methodsrequire rigorous conventions in their underlying modelsand are too problematical to be applied on small areasThereforefordetecting diseases prevalence with case studies on smalldatasets which would be more useful for public healthactivitiessection discussessimple methodsthisClusteringClustering deals with the study of spatialtemporal patternsof the spread of communicable diseases and identificationof other diseaserelated aspects allied with heterogeneousgeographical distribution which might be helpful in elucidating the diseases™ spread mechanism Such study andanalysis on spacetime patterns is a kind of diseasesurveillance which involves detecting the outbreak clustersof active cases monitoring of localisation and isolation ofinfectious agents and relative risks assessment of affectedsites at early stage Clements Cromley Kulldorff This study on geographical clustering ofinfectious diseases with temporal features helps in makingstrategies that dynamically update on emergence source ofdisease outbreak to help epidemiologists and decisionmakers for identification of spread and risk zones Thusclustering helps to enable timely prevention and containment measures and timely resource allocation to mitigatethe diffusion of diseasesBased on spacetime surveillance of diseases spacetime scan statistic Kulldorff is one of the clusterdetection tools which is widely used in geographicalsurveillance of diseases during epidemic andor pandemicThe spacetime scan statistic comes with two versions”prospective and retrospective Desjardins 0cTable Summary table for geospatial dashboards for COVID19Project nameDatasetsScopePurposeJournal of the Indian Society of Remote SensingWHO Coronavirus Disease COVID19WHO™s official dataDashboard Dong httpscovid19whointGlobal Visualization of official daily counts ofconfirmed cases and deaths related toCOVID19 with time stamps using EsriArcGIS Online serviceExploratory data analysis using 3D graphto perform countrywise analysis usingpopulation confirmed cases cumulativecases deaths and cumulative deathsProvides daily aggregate case and deathcount in CSVJohns Hopkins University COVID19Aggregated data from WHO EuropeanGlobal Dashboard for visualizing realtimeDashboard Dong httpscoronavirusjhuedumaphtmlCentre for Disease Prevention andControl ECDC WorldoMeters BNONews US CDC 1Point3Arces COVIDTracking Project and DXYmapping of COVID19 with graphs onconfirmed and daily casesCritical trend analysis on new cases perday mortality and fatality analysis inpopulation timeline of outbreak etcISRO™s BHUVAN COVID19 GeospatialSolution httpsbhuvanapp3nrscgovincoronacorona_dashboarddashboarddashboardphptypecitizenCOVID19 data Source MoHFWIndiaTime series visualization of activerecovered and deceased cases fromMarch to till dateGraphical analysis on spread trend ofCOVID19 daywise and statewiseCOVID19INDIA httpswwwCM Health M handles Press Trust ofIndiaVisualization of cumulative and dailycovid19indiaIndia state press bulletins PBI and ANIreportsnumbers of confirmed active recovereddeceased and tested statewise casesthrough maps and graphsProvides daily COVID19 cases in stateand district and cases reassigned to statesthrough APIMapmyIndia COVID19 httpsmapsCOVID19 data Source MoHFWIndiaProvides API on corona dashboards tomapmyindiacomcoronadistricts_containment_zonecontainment_zone_gradientHunger Relief Centres Source MyGovHunger Night Shelter Source MyGovNDMAvisualize cases at district and state levelhotspots treatment centres testing labsquarantine centres containment zoneslockdown issues hunger relief hunterand night sheltersMonthly climate explorer for COVID19httpscdsclimatecopernicuseuappsc3sappc3smonthlyclimatecovid19explorerMonthly COVID19 cases Source JHUGlobal Visualization of COVID19 fatalities withCSSEAtmospheric composition”PM10 and NO2Source CAMS EAC4Meteorological data”humidity hPaand surface air temperature on hourly andmonthly average rate Source ERA5reanalysisclimatic and atmospheric variations onmonthly basisExploratory analysis on correlation ofpollutants and specific humidity withCOVID19 deathsExperimental COVID19 and GlobalCOVID19 cases Source JHU CSSEGlobal Visualize earthquake as a cause of increaseSeismic Risk Map httpsmaps quakemapcovid1920200520v3grm234900Global earthquake risk map Source GEMGlobal Earthquake Modelin COVID19 cases due to people™sdisplacement from damaged buildingsOwusu and difference between both is thatprospective neglects historical clusters which may havepreviously occurred before the most current time period ofanalysis with no health threat Kulldorff Thereforethe prospective version of spacetime scan statistic iscommonly used to detect statistically significant active orevolving clusters of diseases for the present time periodand when more data become available the tool can be rerun to detect new evolving clusters with update on relativerisks for each affected sites Previously the prospectivespacetime scan statistic was used in thyroid cancerKulldorff shigellosis Jones measlesYin syndromic surveillance Yih and many other diseases However cluster analysis of 0cJournal of the Indian Society of Remote Sensingdiseases can be performed through several packages andlibraries in R Go´mezRubio and Pythonsoftware Yeng The contribution of cluster detections and analysis inCOVID19 pandemic is becoming useful nowadays as itdetects active and emerging clusters of COVID19 andnotify epidemiologist decision makers and public healthcare officials which can help in eradicating infections fromaffected sites and improving interventions quarantine andisolation measures The significant applications of clustering with respect to infectious diseases modelling aredemonstrated across the world Zarikas forexample India Bhosale and Shinde USA Desjardins Hohl Brazil Martines Italy Cereda China Ji Liu 2020a b Qiu Zhang Singapore Bhosale and Shinde Pung SouthKorea Shim French Alps Danis Germany Pfefferle Sergipe Andrade etcOutlier AnalysisThe outlier is defined by Hawkins as ˜˜an observationwhich deviates so much from the other observations as toarouse suspicions thatit was generated by a differentmechanism™™ In other words when data generation processstarts behaving abnormal and reflects the abnormalities orerrors in data such abnormalities are known as outliersBansal However the outliers generally holdadvantageous information about the systems unusual characteristics and entities which impact the data generationprocess Some of the useful applications of outliers in diseases are Cleynen Dai and Bikdash Krishnan Lo Prensner Washington Wu and Krishnan Clusteringalgorithms are optimized to find clusters rather than outliersand the accuracy of outlier detection depends on how goodthe clustering algorithm captures the structure of clustersMaximum Entropy Modelling Maxent ApproachIn context of disease systems disease transmission risksdepend on distribution of pathogens species in space andtime in some complex environmental conditions Townsend and as such treatments are focused mainly on spatialdimensions therefore diseases transmission risks are purelyhandled through geographical phenomena Such geographical link with diseases leads to the challenge of spatialmapping of disease transmission which overcame throughthe branches of biodiversity science”ecology and biogeography Such approach of ecological and biogeographical modelling can be seen from various studies on diseasetransmission risks mapping for example Arboleda Deka and Morshed Ferreira Holt Mweya Nakazawa Reeves Samy Qian Zhao Zhu Following recent studies on geographical mapping ofpathogens causing disease transmission machine learningbased maximum entropy method Maxent Elith Phillips is applied on spatial records ofCOVID19 with a set of bioclimatic environmentalvariables from WorldClim Poggio Ramı´rezVillegas and Bueno Cabrera to analyse theirfavourable environmental conditions as shown in Fig and Table required in maintaining its population TheMaxent principle is to estimate the target probability distribution by applying the maximum entropy to distributionwhich is most spread or closest This study is carried out inR software Ihaka and Gentleman and a geographical dataset consists of latitude and longitude of thoseregions which were affected till March Figure depicts the habitat suitability map of virus withprobability range in colour scale to visualize the highsuitability light and dark green colour medium suitabilityyellow and dark brownlow suitability light browncolour and unsuitable grey colour Table lists thefavourable bioclimatic variables and their contribution inpercent in maintaining the suitability of virusSusceptibleInfectiousRecovered SIR ModelEpidemiology deals with the study of pattern and occurrence of diseases in space and time associated with otherfactors such as environment demography and the translation of epidemiology into mathematical equations todescribe the spread of infectious diseases is known asmathematical epidemiology Allen Rayner andBender The mathematical epidemiology model isimplemented to understand the transmission dynamics ofcommunicable diseases by categorizing population intosusceptible infectious and recovered compartments Thefirst basic model known as SusceptibleInfectiousRecovered SIR model was proposed by Kermack andMcKendrick to describe the transmission of epidemic diseases from individual to individual The SIRmodel is a set of nonlinear ordinary differential equationswhich is mathematically defined as follows¼ l N þ SðÞ 00 bSI¼ bSI 00 cI 00 lI¼ cI 00 lRdSdtdIdtdRdtð1Þð2Þð3Þ 0cJournal of the Indian Society of Remote SensingFig Predicted suitability of Betacoronavirus using data till March Table Responsible bioclimatic variables in suitability modellingHereS is the class of susceptible individuals who are not yetcontracted to diseaseI is the class of infectious people who are now infectedwith disease and become infectious to infect others¢ R is class of recovered individuals who have recoverednow and are removed from class S¢ N is a total population size N S I R and t istime in days or weeks¢ b is the contact rate of infected person with suspected¢¢¢Bioclimatic variablesPercent contributionMean temperature of coldest quarterPrecipitation of wettest monthMean diurnal rangeIsothermalityAnnual mean temperatureMax temperature of warmest monthPrecipitation of coldest quarterPrecipitation of wettest quarterAnnual precipitationPrecipitation of driest quarterMean temperature of driest quarterMean temperature of wettest quarterPrecipitation seasonalityTemperature seasonalityPrecipitation of warmest quarterMean temperature of warmest quarterTemperature annual rangePrecipitation of driest monthMin temperature of coldest monthperson per dayc is the infectious period and average infectious periodis 1c¢ l is the per capita death rate which is adjusted by birthrate lNThere are many other compartment models derived fromthe basic epidemic model SIR with more compartmentsand transitions” SusceptibleExposedInfectiousRecovered SEIR Li and Muldowney SusceptibleInfectiousExposedRecoveredDeadSEIRDPiccolomiini and Zama SusceptibleInfectiousExposedRecoveredSusceptible SEIRS Liu and Zhang SusceptibleInfectiousQuarantineRecoveredSIQR Erdem
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"purpose squamous cell carcinomas and adenocarcinomas are the most common types of cervical cancercompared to squamous cell carcinomas adenocarcinomas are more common in younger women and have apoorer prognosis yet so far no useful biomarkers have been developed for these two types of cancer in thefollowing study we examined the combination of cytokeratin p63 p40 and muc5ac for distinguishingsquamous cell carcinoma scc from adenocarcinoma of the cervix aecmaterials and methods a total of scc and aec were collected immunohistochemical analyses wereconducted to determine the expression of ck56 p63 p40 ck7 and muc5ac one pathologist who was blinded tothe patient™s clinical and pathological data interpreted the staining resultsresults muc5ac and ck7 were detected in and of aec cases compared to and of scccases p the specificity of muc5ac was higher than that of ck7 in aec p the sensitivity of muc5accombined with p40 or p63 was similar to that of ck7 but the specificity was slightly higher than that of ck7 inaec moreover the expression of muc5ac was correlated with the degree of tumor differentiation inadenocarcinomas p and was not related to the prognosis of cervical adenocarcinoma and subtypess muc5ac may be useful as a biomarker for differential diagnoses between squamous carcinoma andadenocarcinoma of the cervixkeywords cervical adenocarcinoma cervical squamous cell carcinoma muc5ac ck7 correspondence xiaofangzhangsdueducn hailing li and xiaotong jing contributed equally to this work2department of pathology school of basic medical science shandonguniversity jinan shandong p r china5department of pathology school of basic medical science shandonguniversity jinan shandong p r chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cli diagnostic pathology page of thelastintroductioncervical cancer is the fourth most common carcinomain women responsible for “ of cancerrelateddeaths worldwide [ ] squamous carcinoma is themost common type of cervical carcinoma followed byadenocarcinoma nevertheless overthreedecades a significant increase in adenocarcinoma caseshas been observed in many developed countries especiallyin younger women papsmear screening also knownas pap test is still considered the main screening methodfor cervical cancer especially for squamous carcinoma compared to squamous carcinoma the adenocarcinomaof the cervix is more common in younger women and hasa poorer prognosis therapeutic approaches includechemoradiotherapy ccrt which has been proven tobe effective for squamous carcinoma of the cervix but notfor adenocarcinoma of the cervix due to its highchemo and radioresistance therefore differentiatingadenocarcinoma from squamous carcinoma is importantin order to provide patients with most suitable therapyp63 p40 and cytokeratin 56ck56 are the mostcommon panel ofimmunochemical markers for thediagnosis of squamous carcinoma p63 and ck56are traditional markers that indicate squamous differentiation in primary lung neoplasms most squamouscarcinomas and large cell carcinomas are positive forck56 warth found that the probability of acorrect sqcc diagnosis using ck56 is p63 a transcriptional regulator has a crucial role in thedevelopment and differentiation of stratified squamousepithelium it is usually strongly expressed in the basalkeratinocytes [“] vosmik analyzed patientswith cervical squamous cell carcinoma and found that had positive expression of p63 p40 is a new specific marker for distinguishing squamouscarcinomas from adenocarcinoma whose specificity isabout in lung carcinomas however the positiveexpression of ck56 p63 and p40 are only found in a fewadenocarcinomas [ ] kriegsmann suggested theuse of either ck56 or p40 over p63 in the routine diagnostic setting ck7 is expressed in many ductal andglandular epithelial cells mainly gallbladder hepatic ductsand pancreatic ducts in tissues of the female genital tractovary endometrium fallopian tube and cervix and in thebreast lung and urinary tract tissues in the normalcervical tissue and adenocarcinoma ck7 staining wasobserved in the columnar cells of endocervical glandshashiguchi found the different rates of ck7 in patientswith cervical intraepithelial neoplasia and those with invasivecarcinomas vs [ ] thus far no efficientmarkers have been developed for distinguishing squamouscell carcinoma and adenocarcinoma in the endocervixmucins are a family of large glycoproteins expressedon the epithelial cell surfaces including ducts of lacrimalglands in the eye salivary glands the lining of the respiratory gastrointestinal urothelial and reproductive tracts muc5ac belongs to gelforming mucins multiple histological studies have highlighted that muc5acis expressed in the conjunctiva middle ear nasopharynxlungs gallbladder and stomach under normal conditionswhere it provides protection to corresponding epithelialsurfaces from different factors some research hasshown that muc5ac may be a potential biomarker inpancreatic cancer tissues dimaio found thatanterior gradient homolog and muc5ac are usefulpositive markers of adenocarcinoma in the setting ofabsent or diminished p63 and cytokeratin staining inesophageal carcinoma it is also expressed in theendocervix yamanoi found that muc5ac waslargely expressed in typical legh atypical legh gasmda and gasnonmda thus we speculated thatmuc5ac could be expressed in other adenocarcinomasand might be used for the differential diagnosis of adenocarcinoma and squamous carcinoma the aim of thisstudy was to examine the combination of cytokeratin p63 p40 and muc5ac for distinguishing squamous cellcarcinoma scc from the adenocarcinoma in the cervixaecmaterials and methodstissue sampleswe analyzed poorly to moderately differentiated cervical squamous carcinoma scc and adenocarcinomasof endocervix aec all tissues were collected from thedepartment of human pathology of qilu hospitalshandong university china from to specimenswere retrieved from the pathology files of the departmentof pathology at the same hospital after collection all specimens were fixed in buffered formalin hematoxylin eosin he stains were available for review paraffin blockswere used for immunohistochemical staining all the slideswere reviewed by two experienced pathologistshistopathological and clinical variables including agetumor size differentiationinfiltrate depth and lymphnode metastasis were summarized in table followupinformation was available in aec with the followuptime ranging from to months mean monthsimmunohistochemistryfour to five micronthick paraffin sections of the cases were dewaxed rehydrated in graded alcohols andprocessed using the pv9000 detection kit zsbio commerce store beijing china briefly antigen retrieval wasperformed in a microwave oven for min in mm trisedta buffer mm tris base mm edta solution tween ph endogenous peroxidase activitywas blocked with a h2o2methanol solution for min slides were then incubated in normal goat 0cli diagnostic pathology page of table comparison of clinicopathological features between cervical squamous cell carcinoma and cervical adenocarcinomasquamous cell carcinomasn adenocarcinoman χ p valueage‰¤ size cm ‰¥ unknowndifferentiationpoormoderatewellunknown infiltrate depth of mesenchyme‰¤ unknownlymph node metastasisnoyesunknown serum for min to prevent nonspecific binding sampleswere then incubated overnight at °c with a primary antibody phosphate buffered saline pbs was used instead ofthe first antibody as a negative control consequentlysamples were incubated with reagent atroomtemperatureroomfor min and reagent attemperature for min finally the tissues were stainedwith diaminobenzidine dab the antibodies used in thisstudy are listed in table scoring methodstaining results were interpreted by one pathologist whowas blinded to the patient™s clinical and pathologicaldata for ck56 ck7 and muc5ac more than oftumor cells with a membrane or cytoplasmic brownyellow granules were considered positive for p63 andp40 the positive standard was that more than oftumor cells have brownyellow granules in the nucleusstatistical analysisstatistical analysis was performed with spss softwareversion spss inc chicago ii usa chisquareor fisher™s exacttests were used when comparingfrequencies between two groups probability values lessthan were considered statistically significantresultsthe expression of ck56 p63 p40 ck7 and muc5ac inscc and aecihc for the five proteins was performed on humanprimary cervical cancersincluding scc and aec as shown in fig and fig muc5ac ck56and ck7 were mainly expressed in the cell membranetable immunohistochemical antibodiesantibodymuc5acnozm0395ck56ck7p40p63zm0313zm0071zm0472zm0406vendorzsbio commerce store beijing chinazsbio commerce store beijing chinazsbio commerce store beijing chinazsbio commerce store beijing chinazsbio commerce store beijing chinadiluationready to useready to useready to useready to useready to use 0cli diagnostic pathology page of fig the expression of ck56 p63 p40 ck7 and muc5ac in a case of poordifferentiated squamous cell carcinoma by ihc a he b ck56positive staining c p63 positive staining d p40 positive staining e ck7 positive staining f muc5ac negative staining ×fig the expression of ck56 p63 p40 ck7 and muc5ac in case of poordifferentiated adenocarcinoma invasive stratified mucinproducingcarcinoma ismile by ihc a he b ck56 negative staining c p63 negative staining d p40 negative staining e ck7 negative staining f muc5acpositive staining × 0cli diagnostic pathology page of and cytoplasm while p40 and p63 were mainly locatedin the nucleussignificant effect on the prognosis of cervical adenocarcinoma patients p as shown in fig tumorin thecells ofwe found that muc5ac exhibited prominent immucervical aecnoreactivitymuc5ac and ck7 were detected in and of aec cases compared to and of scc casesbesides for aec the specificity of muc5ac was muchhigher than that of ck7 p moreover the sensitivity of ck56 p40 and p63 was and respectively and the specificity was and respectively in aec table through the combined detection of p40 or p63 wecompared muc5ac and ck7 again we found that thesensitivity and specificity of muc5ac in aec combinedwith p40 or p63 were and respectively and respectively the sensitivity and specificity of ck7 combined with p40 or p63 were and and respectively table thesensitivity of muc5ac combined with p40 or p63 wassimilar to that of ck7 while the specificity was slightlyhigher than that of ck7expression of muc5ac and ck7 in cervicaladenocarcinoma subtypeswe further detected the expression of muc5ac insubtypes of aec table among cases of usualtype cervical adenocarcinoma cases were muc5acpositive and cases were ck7 positive and there wasno statistical difference p in cases of mucinous adenocarcinoma nosthe expression rate ofmuc5ac and ck7 were both moreover out of cases of gastric mucinous adenocarcinomaexpressed muc5ac and of them were ck7 positivep the positive rate of the muc5ac in mucinouscarcinoma intestinal type villous tubular adenocarcinomaendometrioid adenocarcinoma clear cell carcinoma serouscarcinoma adenosquamous carcinoma and invasive stratified mucinproducing carcinoma ismile was and respectively the expressionrate of muc5ac had no statistical difference among thesesubtypes all p correlation between muc5ac expression andclinicopathological characteristics in cervicaladenocarcinomathis study further analyzed the relationship between theexpression of muc5ac and clinicopathological featuresin cervical adenocarcinoma table the expression oftumormuc5ac was correlated with the degree ofdifferentiation p a lower degree oftumordifferentiation was associated with a lower expressionrate of muc5ac there was no significant correlationbetween the expression of muc5ac protein and agetumor size depth of myometrialinvasion and lymphnode metastasis all p kaplan meier analysisrevealed that the expression of muc5ac protein had nodiscussion and sidentification of previously unutilized sensitive biomarkers is still a priority for improved differential diagnosis of cervical aec and scc at present ck56 p63p40 and ck7 are the main biomarkers for differentiatingcervical adenocarcinoma from squamous cell carcinomack56 is a kind of high molecular weight basal cellkeratin 58kda and 56kda which is mainly expressed inthe basal cells of squamous epithelium and ductal epithelium and some squamous epithelial germinal layercells myoepithelial cells and mesothelial cells butpoorly expressed in glandular epithelial cells someresearch results showed that ck56 has high sensitivityand specificity in the diagnosis ofsquamous celltable sensitivity and specificity of muc5acãck56ãck7ãp40ãp63 in cervical squamous cell carcinoma and adenocarcinomamarkerssensitivityspecificitysquamous cell carcinomasn adenocarcinoman muc5acck7ck56p40p63ck56and p40ck56and p63muc5acand p40ˆ’muc5acand p63ˆ’ck7and p40ˆ’ck7and p63ˆ’ 0cli diagnostic pathology page of table the correlation of muc5ac and the clinical variants inthe cervical adenocarcinomathe expression of muc5acpositivenegativeχ valuep valueage‰¤ v size cm ‰¥ differentiationpoorwellmoderateinfiltrate depthof mesenchyme‰¤ lymph nodemetastasisnoyescarcinoma [“] in contrast other studies showedhigh sensitivity but low specificity when diagnosing thistype of tumor p63 is a member of the p53 family a classical tumorsuppressor gene family it is located on chromosome3q27“ filho showed good sensitivity whendetecting squamous cell carcinoma with a positive rateof contrary kaufmann suggested thatp63 could also be expressed in a small number of adenocarcinoma basal cell carcinoma and transitional epithelial carcinoma moreover p63 can also be used as amarker of myoepithelial cells and prostate basal cellstherefore p63 lacks absolute specificity for squamousdifferentiationp40 is a subtype of p63 protein expressed in squamousepithelial cells including epidermis and hair folliclesurothelial cells myoepithelial cells ofthe mammarygland sweat gland and salivary gland and basal cells ofthe prostate which are highly specific in labeling squamous epithelium bishop showed that in cases of squamous cell carcinoma of the lung and cases of adenocarcinoma of the lung the sensitivity andspecificity of p63 were and respectivelythe sensitivity and specificity of p40 in the diagnosis ofsquamous cell carcinoma of the lung were and respectively therefore p40 is considered as ahighly specific and sensitive tumor biomarker of squamous epithelial origin in this study we used immunohistochemistry to detectck56 p63 and p40 in cervical squamous cell carcinomaand adenocarcinoma the sensitivity of ck56 p40 andp63 was and respectively and thespecificity was and respectivelymoreover the specificity of ck56 is slightly lower thanthat of p40 and p63 we also found that a combinationof ck56 with p40 or p63 slightly decreased the sensitivity and and increased the specificity and which in turn increased the accuracy of diagnosing squamous cell carcinomack7 is a kind of low molecular weight keratin mainlyexpressed in glandular epithelium and transitional epithelial cells of most normal tissues many studieshave found that ck7 is not only expressed in adenocarcinoma but also in squamous intraepithelial neoplasiacervical squamous cell carcinoma lung squamous cellcarcinoma and esophageal squamous cell carcinomalee found a positive expression of ck7 in fig survival analysis of muc5ac expression in cervical adenocarcinoma 0cli diagnostic pathology page of table expression of muc5ac and ck7 in differentadenocarcinoma subtypessubtypesmuc5acusual typeχ valueck7p valuepositivenegativemucinous adenocarcinoma nospositivenegativegastric typepositivenegativeintestinal typepositivenegativevillous tubular adenocarcinomapositivenegativeendometrioid adenocarcinomapositivenegativeclear cell carcinomapositivenegativeserous carcinomapositivenegativeismilepositivenegativeadenosquamous carcinomapositivenegative““““““““““ismile invasive stratified mucinproducing carcinoma cases with scc and cases withciniii furthermore yamada found that ck7expression in esophageal squamous cell carcinoma butalso in iiiaiib stage esophageal squamous cell carcinoma suggest poor tumor differentiation and thus canbe used as an independent prognostic factor ourstudy showed that the positive rate of ck7 was incervical poorly differentiated squamous cell carcinomawhich further suggested that ck7 is not an ideal markerfor differentiation between squamous cell carcinoma andadenocarcinomamucin is a high molecular weight glycosylated proteinsecreted by epithelial cells in the respiratory tractgastrointestinal tract and urogenital tract which has animportant role in the protection of epithelium cell adhesion signal transduction immune activation and inhibition at present at least mucins have been found inthe female reproductive system riethdorf and albarracin used immunohistochemistrymethods to detect the expression of muc5ac in different female reproductive system malignant tumors theyfound that muc5ac was highly expressed in cervicaladenocarcinoma and poorly expressed inendometrial adenocarcinoma all of themwere expressed in the primary ovarian mucinous tumor but not in colon adenocarcinoma therefore they concluded that muc5ac could beused as an effective marker to distinguish the origin ofpelvic tumors and distinguish primary ovarian tumorsand colorectal metastasis as well as endometrial adenocarcinoma from cervical metastasis [ ] in thisstudy we found positive expression of muc5ac in cases of cervical adenocarcinoma and in cases of squamous carcinoma which wasconsistent with riethdorf™s study the sensitivity ofmuc5ac and ck7 to cervical adenocarcinoma was and respectively but the specificity ofmuc5ac was much higher than that of ck7 through the joint detection of p40 or p63 wecompared muc5ac and ck7 again and found that thesensitivity and specificity of muc5ac combined withp40 or p63 were and respectively and respectively the sensitivity and specificity ofck7 combined with p40 or p63 were and and respectively these results showed thatthe sensitivity of muc5ac combined with p40 or p63was similar to that of ck7 butthe specificity wasslightly higher than that of ck7 therefore muc5ac issuperior to ck7 in the diagnosis of cervical adenocarcinoma and squamous cell carcinomabesides we preliminarily detected the expression ofmuc5ac in different types of cervical adenocarcinomaand found no significant difference these data suggestedthat muc5ac has no diagnostic significance in the classification of cervical adenocarcinoma at the same time weanalyzed the relationship between the expression ofmuc5ac and the prognosis of cervical adenocarcinomaand the result revealed that muc5ac was not related tothe prognosis of cervical adenocarcinomaoverall our observations strongly suggest that muc5acmay be useful as a biomarker for differential diagnosesbetween squamous carcinoma and adenocarcinomaabbreviationsscc squamous cell carcinoma aec adenocarcinoma of the cervixck cytokeratin he hematoxylin eosin pbs phosphate buffered salinedab diaminobenzidine ismile invasive stratified mucinproducingcarcinoma 0cli diagnostic pathology page of authors™ contributionsxiaofang zhang designed the study and drafted the manuscript hailing liand xiaotong jing analyzed the data and carried out theimmunohistochemistry jie yu and tingguo zhang read the pathologicalsections jinan liu collected the clinical data and carried our followupshiming chen made the slides the authors read and approved the finalmanuscript downey p cummins r moran m gulmann c if it's not ck56 positive ttf negative it's not a squamous cell carcinoma of lung apmis “ warth a muley t herpel e meister m herth fj schirmacher p weichert whoffmann h schnabel pa largescale comparative analyses ofimmunomarkers for diagnostic subtyping of nonsmallcell lung cancerbiopsies histopathology “fundingthis work was supported by the national natural science foundation ofchina no and technology development foundation of yantaino ws017availability of data and materialsnot applicableethics approval and consent to participateall tissue samples from patients were collected and protocols wereperformed according to the procedures approved by the research ethicscommittee of shandong medical university all patients provided informedconsentcompeting intereststhe authors declare that they have no competing interestsauthor details1department of pathology weifang traditional chinese hospital weifangshandong p r china 2department of pathology school of basic medicalscience shandong university jinan shandong p r china 3department ofpathology the fourth hospital of jinan the third affiliated hospital ofshandong first medical university jinan shandong p r china 4departmentof oncology yuhuangding hospital yantai shandong p r china5department of pathology school of basic medical science shandonguniversity jinan shandong p r chinareceived july accepted august referenceskurman rj carcangiu ml herrington cs who classification of tumours offemale reproductive ans4th ed lyon iarc press takeuchi s biology and treatment of cervical adenocarcinoma chin jcancer res “young rh clement pb endocervical adenocarcinoma and its variants theirmorphology and differential diagnosis histopathology “forouzanfar mh foreman kj delossantos am lozano r lopez ad murraycj naghavi m breast and cervical cancer in countries between and a systematic analysis lancet “galic v herzog tj lewin sn neugut ai burke wm lu ys hershman dlwright jd prognostic significance of adenocarcinoma histology in womenwith cervical cancer gynecol oncol “favero g pierobon j genta ml araujo mp miglino g del cpdm deandrade ch fukushima jt baracat ec carvalho jp laparoscopicextrafascial hysterectomy completion surgery after primary chemoradiationin patients with locally advanced cervical cancer technical aspects andoperative outcomes int j gynecol cancer “rose pg java jj whitney cw stehman fb lanciano r thomas gm locallyadvanced adenocarcinoma and adenosquamous carcinomas of the cervixcompared to squamous cell carcinomas of the cervix in gynecologiconcology group trials of cisplatinbased chemoradiation gynecol oncol“ ma y fan m dai l kang x liu y sun y xiong h liang z yan w chen kexpression of p63 and ck56 in earlystage lung squamous cell carcinoma isnot only an early diagnostic indicator but also correlates with a goodprognosis thorac cancer “kaufmann o fietze e mengs j dietel m value of p63 and cytokeratin as immunohistochemical markers for the differential diagnosis of poorlydifferentiated and undifferentiated carcinomas am j clin pathol “ barbieri ce pietenpol ja p63 and epithelial biology exp cell res “senoo m pinto f crum cp mckeon f p63 is essential for the proliferativepotential of stem cells in stratified epithelia cell “ pozzi s zambelli f merico d pavesi g robert a maltere p gidrol xmantovani r vigano ma transcriptional network of p63 in humankeratinocytes plos one 200943e5008 vosmik m laco j sirak i beranek m hovorkova e vosmikova h drastikovam hodek m zoul z odrazka k prognostic significance of humanpapillomavirus hpv status and expression of selected markers her2neuegfr vegf cd34 p63 p53 and ki67mib1 on outcome after chemoradiotherapy in patients with squamous cell carcinoma of uterine cervixpathol oncol res “ nobre ar albergaria a schmitt f p40 a p63 isoform useful for lung cancerdiagnosis a review of the physiological and pathological role of p63 actacytol “stolnicu s hoang l hankobauer o barsan i terinte c pesci a avielronens kiyokawa t alvaradocabrero i oliva e and others cervicaladenosquamous carcinoma detailed analysis of morphologyimmunohistochemical profile and clinical outcomes in cases modpathol “toyoshima m momono y makino h kudo t oka n sakurada j suzuki hkodama h yoshinaga k cytokeratin 7positivecytokeratin 20negative cecaladenocarcinoma metastatic to the uterine cervix a case report world jsurg oncol hashiguchi m masuda m kai k nakao y kawaguchi a yokoyama maishima s decreased cytokeratin expression correlates with theprogression of cervical squamous cell carcinoma and poor patientoutcomes j obstet gynaecol res “lee h lee h cho yk cytokeratin7 and cytokeratin19 expression in highgrade cervical intraepithelial neoplasm and squamous cell carcinoma andtheir possible association in cervical carcinogenesis diagn pathol krishn sr ganguly k kaur s batra sk ramifications of secreted mucinmuc5ac in malignant journey a holistic view carcinogenesis “thornton dj rousseau k mcguckin ma structure and function ofthe polymeric mucins in airways mucus annu rev physiol “ rose mc voynow ja respiratory tract mucin genes and mucinglycoproteins in health and disease physiol rev “ balmaña m duran a gomes c llop e lópezmartos r ortiz mr barrabés sreis ca peracaula r analysis of sialyllewis x on muc5ac and muc1mucins in pancreatic cancer tissues int j biol macromol “ dimaio ma kwok s montgomery kd lowe aw pai rkimmunohistochemical panel for distinguishing esophageal adenocarcinomafrom squamous cell carcinoma a combination of p63 cytokeratin muc5ac and anterior gradient homolog allows optimal subtyping humpathol “ yamanoi k ishii k tsukamoto m asaka s nakayama j gastric gland mucinspecific oglycan expression decreases as tumor cells progress from lobularendocervical gland hyperplasia to cervical mucinous carcinoma gastrictype virchows arch “ reisfilho js simpson pt martins a preto a gartner f schmitt fcdistribution of p63 cytokeratins and cytokeratin in normal and neoplastic human tissue samples using tarp4 multitumor tissuemicroarray virchows arch “ yamada a sasaki h aoyagi k sano m fujii s daiko h nishimura myoshida t chiba t ochiai a expression of cytokeratin predicts survival instage iiiaiib squamous cell carcinoma of the esophagus oncol rep “ baker ac eltoum i curry ro stockard cr manne u grizzle we chhieng dmucinous expression in benign and neoplastic glandular lesions of theuterine cervix arch pathol lab med “ 0cli diagnostic pathology page of riethdorf l o'connell jt riethdorf s cviko a crum cp differentialexpression of muc2 and muc5ac in benign and malignant glandularlesions of the cervix uteri virchows arch “ albarracin ct jafri j montag ag hart j kuan sf differential expression ofmuc2 and muc5ac mucin genes in primary ovarian and metastatic coloniccarcinoma hum pathol “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"
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coutard b valle c de lamballerie x canard b seidah ng and decroly e the spike glycoprotein of the new coronavirus 2019ncovcontains a furinlike cleavage site absent in cov of the same clade antiviral res 101016jantiviral2020104742 cao z wu y faucon e and sabatier jm sarscov2 covid19 keyroles of the ˜reninangiotensin™ systemvitamin d impacting drugand vaccine developments infect disord drug targets “ 1021741871526520999200505174704 zhang z chen l zhong j gao p and oudit gy ace2ang17 signaling and vascular remodeling sci china life sci “s1142701446933 paz ocaranza m riquelme ja garcia l jalil je chiong m santos ras counterregulatory reninangiotensin system incardiovascular disease nat rev cardiol “ 101038s4156901902448 ziegler cgk allon sj nyquist sk mbano im miao vn tzouanas cn sarscov2 receptor ace2 is an interferonstimulatedgene in human airway epithelial cells and is detected in specific cell subsets across tissues cell “101016jcell202004035 marrero mb schieffer b paxton wg heerdtt l 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"elucidate the molecular mechanism underlying the involvement of abnormal DNA methylation in the development of glioma and identify potential newtargets for glioma therapyMethods The GSE79122 chip achieved from the Gene Expression Omnibus GEOdatabase containing glioma samples and normal samples was analyzed Methylationspecific polymerase chain reaction MSPCR or MSP reverse transcriptionPCR andWestern blot analysis were used to confirm the methylation level and expression level ofthe interleukin receptorassociated kinase IRAK3 gene in glioma cells glioma samplesand the corresponding normal samples In vitro the proliferation apoptosis rate migrationand invasion abilities of glioma cells were detected by Cell Counting Kit8 assay Transwellassay enzymelinked immunosorbent assay and flow cytometry respectively Besides thexenograft assay of nude mice was used to confirm the effect of the IRAK3 on glioma in vivoResults Microarray analysis showed that the IRAK3 was one of the most hypermethylated genesin glioma and the related mitogenactivated protein kinase MAPK signaling pathway wasactivated More experiments supported the higher methylation level and lower expression levelof the IRAK3 in glioma tissues and cell lines The viability migration and invasion ability ofglioma cells significantly reduced and the apoptosis rate increased with the overexpression anddemethylation of the IRAK3 in vitro Besides treatment with the MAPK signaling pathwayinhibitor PD325901 alone or the overexpression or demethylation of the IRAK3 had a similareffect as the overexpression or demethylation of the IRAK3 alone in glioma cells In vivoxenotransplantation experiments in nude mice confirmed that the overexpression and demethylation of the IRAK3 and suppression of the MAPK signaling pathway inhibited the development ofgliomaConclusion IRAK3 inhibited the development of glioma progression through the MAPKsignaling pathwayKeywords glioma IRAK3 MAPK signaling pathway methylationIntroductionGlioma also known as glioblastoma is one of the most common primary malignant braintumors The average incidence rate of glioma is in individuals1 Despiteimprovements in neurosurgery and radiotherapychemotherapy most patients die within months of diagnosis1 and less than patients survive for years or even more2Recently the molecular mechanisms of glioma have gained increasing attention so as tofind some better methods to defeat this disease Previous studies evaluated that longtermsurvivors of glioma often displayed some favorable molecular characteristics such as thesubmit your manuscript wwwdovepresscomDovePresshttp102147CMARS252772Cancer Management and Research “ Wu This work is published and licensed by Dove Medical Press Limited The full terms of this license are available at wwwdovepresscomtermsphpand incorporate the Creative Commons Attribution “ Non Commercial unported v30 License httpcreativecommonslicensesbync30 By accessing the workyou hereby accept the Terms Noncommercial uses of the work are permitted without any further permission from Dove Medical Press Limited provided the work is properly attributed Forpermission for commercial use of this work please see paragraphs and of our Terms wwwdovepresscomtermsphp 0cWu et alDovepresshypermethylation of O6methylguanine DNA methyltransferase MGMT promoter3 which is known as a meaningfulpredictive biomarker for the favorable prognosis of the chemotherapeutic drugs4 Therefore this study proposed that epigenetic regulation might play a key role in the development ofglioma which deserves further research for understanding thiscancerDNA methylation isan epigenetic modificationinvolved in many biological processes especially geneexpression regulation5 The DNA methylation patterns ofnormal cells are controlled well by a balance betweenmethylation and demethylation However this balance isalways disturbed in cancer cells through ectopic deficientor excessive methylation leading to abnormal biologicalactivities6 Hypermethylation of CpG islands on specificpromoters inhibiting the transcription of downstreamtumor suppressor genes has been discovered in manycancers which may provide clinicians a new strategy forpatients with cancer7 For instance the promoter region ofSEPT9 is hypermethylated in colorectal cancer Hence theSEPT9 gene methylation assay might become a potentialoption for the early detection and screening of colorectalcancer8 CpG islands also display aberrant hypermethylation at a large number of loci and define the subgroup ofglioma910 However the underlying molecular events ofDNA methylation and glioma development remain poorlyunderstoodRecently technical advances in genomewide expression analysis have enabled an improved understanding ofthe diagnosis and prognosis of many types of tumors11Genomewide DNA methylation analysis allows comprehensive DNA methylation profiling of the whole genomehelping in the effective identification of novel genes thathave a potential value in clinical practice12 Previous studies have reported many specific methylation signaturegenes in differenttypes of cancers such as thyroidcancer13 lung squamous cell carcinoma14 hepatocellularcarcinoma15 prostate cancer16 and so on using DNAmethylation analysisThis study aimed to examine the genomewide DNAmethylation profiling of glioma tissuesrevealing thehypermethylation of several genes in glioma Then oneof these hypermethylated genes IRAK3 was selected toconductSubsequentlya relationship between IRAK3 and MAPK signaling pathway was demonstrated by using DNA methyltransferaseinhibitor overexpression of IRAK3 and MAPK signalingpathway inhibitor which can disrupt the development ofcomprehensiveaanalysisgliomas in vitro and in vivo The findings might providesome clues for the regulatory role of DNA methylationand the underlying application of targeted treatment ingliomaMethodsTissue SamplesGlioma tissues and adjacent normal tissues were collectedfrom patients with primary glioma n admitted to theZhangye People™s Hospital Affiliated to Hexi UniversitySample collection and use was performed according to theapproval of the ethics committee of the Affiliated Hospitalof Shandong University Written informed consent wasprovided by every patient with glioma All samples werefrozen in liquid nitrogen and stored at “°C All humanspecimens were obtained with the approval by theInstitutional Ethics Committee of Zhangye People™sHospital Affiliated to Hexi UniversityCell CultureHuman gliocyte cell line HEB and glioma cell linesSHG44 U251 GOS3 HS683 and SF539 wereobtained from Bena Culture Collection Beijing ChinaHEB U251 HS683 and SF539 cells were grown in highsugar Dulbecco™s modified Eagle™s medium DMEMwith fetal bovine serum FBS SHG44 cells weregrown in RPMI1640 medium containing NaHCO3 gL glucose gL and sodium pyruvate gLwith FBS The GOS3 cells were grown with minimum essential medium with Earle™s Balanced Saltswith FBS and mML glucose All cells were cultured at °C under a humidified atmosphere with CO2Cell TransfectionpcDNA31“interleukin receptor“associated kinase pcDNA31IRAK3 was obtained from GenePharma ShanghaiChina and 5azadC 5aza2ʹdeoxycytidine 5azadCand signaling pathway inhibitor PD325901 were obtainedfrom SigmaAldrich MO USA U251 cells were seeded insixwell plates at — cellswell and cultured at °C andthe cell confluence reached “ CO2 untilTransfections were executed using Lipofectamine Invitrogen CA USA following the manufacturer™s protocols The medium was changed with the complete mediumafter h of transfectionsubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressTable qRTPCR PrimersWu et alAccession NumberSequence 5ʹ3ʹForwardReverseForwardReverseForwardReverseForwardReverseACCCAAACTAACTGATTTTGCCAAGAGAAATTCCGAGGGCAGGCGACCTCCCATGGCAATTTTAACAGAGACAGGCATGGGAAGCCATCTCGACCAGTCCGTCTAGTTGGTCTGTCTCCGCTAAATACGGACTGCAGCCCTGAGGTCAATGAAGGGGTCGTGeneIRAK3MEK1CfosNM_007199NM_002755NM_005252GAPDHNM_008084GenomeWide DNA MethylationAnalysisFor DNA methylation profiling Infinium HumanMethylation450K BeadChip illuminaga_rnaseqv2100 was obtainedfrom the Gene Expression Omnibus GEO database TheDNA methylation data of chip number GSE79122 whichcontained glioma tissues glioblastomas diffuseastrocytomas and anaplastic astrocytomas and controlbrain tissues were analyzed The Infinium MethylationAbbiotec CA USA and Illumina BeadStudio softwareGenetech Biotech Taipei Taiwan were used to measurethe methylation profiles of modified DNA and loci CpGThe methylated signal intensity at particular CpG loci and450K BeadChip assay were presented as β values and percentage respectivelyQuantitative RTPCRTotal cellular RNA was extracted using PureLink Invitrogenfollowing the manufacturer™s protocol RT was performed on µg total cellular RNA using a HighCapacity cDNA ReverseTranscription Kit with RNase Inhibitor Applied Biosystemspurchased from Thermo Fisher Scientific MA USASubsequently quantitative reverse transcriptionpolymerasechain reaction RTPCR was performed using ArcturusParadise Plus qRTPCR Kit Applied Biosystems ThermoFisher Scientific Comparative expression values were calculated by the “ΔΔCt method GAPDH was used for internalreference All primer sequences involved are listed in Table converted into uracil without changing the state of methylated cytosine The IRAK3 methylation level in glioma wasidentified using methylationspecific PCR MSP The MSPprimers are listed in Table EnzymeLinked Immunosorbent AssayThe human interleukin IL6 enzymelinked immunosorbent assay kit Sangon Biotech Shanghai China was usedto test the IL6 level in the glioma cell culture medium Theglioma cell culture medium was centrifuged at rpm for min to remove cells and polymers The supernatant fluidstored at “°C was preserved in the supernatant fluid at “°C A normal glioma cell culture medium was used as thecontrolWestern Blot AnalysisLysis buffer RIPA Thermo Fisher Scientific and NPERThermo Fisher Scientific were used to extract proteinsfrom glioma cells and tissues respectively Then μg totalprotein per sample was separated using SDSPAGE andtransferred to polyvinylidene fluoride membranes ThermoFisher Scientific The membranes were probed with primaryantiIRAK3 antibody ab8116 Abcam MA USA antiMEK1 phospho S298 antibody [EPR3338] ab96379 antiERK1 ERK2 antibody [ERK7D8] ab54230 anticFosphospho T232 antibody ab17933 and antiGAPDH antibody [6C5] ab8245 Abcam as control The number ofTable MethylationSpecific PrimersDNA Methylation AssayGenomic DNA in glioma tissues and cells was extracted andtreated with bisulfite using CpGenome DNA ModificationKit Chemicon CA USA following the manufacturer™sprotocol All unmethylated cytosine residues in DNA wereGeneIRAK3 forwardIRAK3 reverseIRAK3 forwardIRAK3 reverseSequence 5ʹ3ʹ5ʹTCGGGATAGTGGTTAATATTTC3ʹ5ʹTTTTTTTCGTTTTTCGTAAAA3ʹ5ʹ AGTTTGGGATAGTGGTTAATATTTT 3ʹ5ʹ TTTTTTTCATTTTTCATAAAAAAA 3ʹCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressFigure Genomewide methylation data were obtained from the GEO database for available glioma9 adjacent mucosa A Density of methylated DNA intensity byeach sample B Type I and Type II assays showed variant β value distributions The differences between probe types were regulated by normalization procedures whichshowed that represented unmethylated sites while represented fully methylated sites C Multidimensional scaling plot showing differential clustering of control versustumor tissues D Dendrogram produced for probes in normal and tumor tissues E Heatmap of top differentially methylated imprinted CpG sitesAbbreviation MG malignant gliomasubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alFigure Distribution of top differentially methylated imprinted CpG sites A Distribution of top differentially methylated imprinted CpG sites according toCpG islands island sea shelf and shore B Distribution of top differentially methylated imprinted CpG sites according to the position relative to genes 1stexon ² UTRs or ² UTRs body IGR TSS1500 and TSS200 C Combined genetic and epigenetic annotation information revealed the distribution of the top differentially methylated imprinted CpG sitesbinding proteins was measured using AlphaEaseFC softwareGenetic Technologies FL USACell Counting Kit8 AssayU251 cells were seeded in 96well plates and allowed toadhere for “ h at °C Then these cells were transfectedwith pcDNA31IRAK3 and treated with 5azadC orPD325901 After “ h Cell Counting Kit8 DojindoKumamoto Japan was used to test the cell viability at respective time points To summarize mL of the triazoliumsubstrate was added to each well and coincubated with cellsat °C for h The absorbance was measured at nm andCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressFigure DNA methylation analysis of tumor tissues and normal tissues and analysis of methylation degree for seven CpG sites of IRAK3 A The expression of the top candidate genes was analyzed IRAK3 was hypermethylated significantly in tumor tissues compared with normal tissues B The number of IRAK3“methylated CpG islands ineach isosite C“I Seven CpG sites for IRAK3 which are presented in the boxplot displayed a decreased methylation in the tumor group The boxplot for cg Ccg D cg E cg F cg G cg H and cg IAbbreviation MG malignant gliomasubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alFigure Analysis of the IRAK3related signaling pathway A The top signaling pathways with the highest and lowest correlation with glioma B The top signalingpathway“related genes enriched in the glioma the MAPK signaling pathway was highly expressed C The MAPK signaling pathway was activated in glioma D The status ofthe top enriched signaling pathways in gliomaAbbreviation MG malignant gliomaCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressthe results were normalized to untreated cells at respectivetime pointsCell Migration and Invasion AssayBoth cell migration and invasion were performed using theTranswell assay For cell migration assay — U251cells were seeded in a serumfree medium in the upperchamber which contained a polyethylene terephthalatemembrane with mm in diameter and μm in poresize The bottom chamber was prepared with FBS asa chemoattractant After incubating at °C for hnonmigrated cells were scraped from the upper surfaceof the membrane with a cotton swab and the cells migrating to the bottom chamber were fixed with paraformaldehyde and stained with crystal violet Finally the stainedcells were counted under a microscope at — magnification in five randomly selected fields for quantificationinvasion assay — U251 cells weresuspended in mL of serumfree DMEM and thentreated using the same procedure as for the migrationassay following the manufacturer™s protocol but the chambers mm BD Biosciences San Jose CA USA wereplated with BD BioCoat MatrigelFor cellFlow CytometryEach treatment of U251 cells was washed with phosphatebuffered saline and resuspended in μL of AnnexinV binding buffer After staining with Alexa Fluor Annexin V and 7AAD Viability Staining Solution for min in the dark at room temperature these cells were analyzed using multicolor flow cytometer Data were analyzedusing Kaluza softwarethe mice were keptXenograft StudiesMale BALBc nude mice weeks were maintained underpathogenfree conditions Allina temperaturecontrolled airconditioned conventional animal house with a h light“dark cycle and given free accessto food and water Besides animal health and behaviour weremonitored every two days All experiments were approvedby the Ethics Committee of Zhangye People™s Hospital toguarantee that all studies involving experimental animalswere performed in full compliance with National Institutesof Health Guidelines for the Care and Use of LaboratoryAnimals The U251 — cells100 μL cells were transfected with pcDNA31IRAK3 and then transferred to micen12 Normal U251 cells were transferred to mice in the5azadC and PD325901 groups and then 5azadC andPD325901 were subcutaneously injected respectively intothe posterior flank of nude mice After and days™culture the mice were sacrificed and the tumor size wasmeasured The tumor volume was measured using a caliperfollowing the formula length — width22Statistical AnalysisAll data were collected from three independent experiments and presented as mean ± standard deviationStatistical analyses were performed using GraphPad software Differences between groups were analyzed usingStudent' ttest or chisquare test Statistical significancewas set at P ResultsGenome Methylation Profile in GliomaA total of glioma tissues and adjacent normal sampledata publicly available at GEO were analyzed to revealthe global DNA methylation patterns of glioma Firstdensity plots of β values generated from each samplewere used identifying a poor performance of methylatedsignals for raw data Figure 1A Infinium Type I and TypeII probe assays also showed somewhat different distribution of β value ranging from unmethylated sites to fully methylated sites Figure 1B Therefore these datawere regulated by normalization procedures to reduce thepotential impact later Multidimensional scaling MDSplot and dendrogram exhibited a differential clusteringbetween tumor and normal groups which distinguishedglioma from adjacent normal tissues Figure 1C and DFurther heatmap of the top differentially methylatedCpG sites indicated a visible difference in DNA methylation profiles between the tumor and normaltissueand general hypermethylation occurred insamplesglioma Figure 1EDistribution of Genomic Regions forDifferentially Methylated CpG SitesBased on the position relative to gene [1st exon ² untranslated region UTR ² UTRs body transcription start sites bp TSS1500 and TSS200] as well as CpG islands andneighborhood content shores shelves islands and sea the distribution of genomic regions for the differentiallymethylated CpG sites was exhibited More than and methylation differences occurred in bodyIGR and CpGisland sea respectively which was obviously higherthan that in other regions Figure 2A and B From thesubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alFigure Analysis of the IRAK3related gene expression level A Based on the differential genes in the disease data the distribution of upregulated genes anddownregulated genes in the GOrelated pathway was enriched B The expression level of top MAPK signaling pathway“related genesAbbreviation MG malignant gliomaperspective of both genetics and epigenetics gene and CpGcontent regions were combined for more analyses whichshowed that CpG sites in genetic bodyisland sea andIGRisland sea were differentially methylated betweenglioma and adjacent normal samples Figure 2CIRAK3 Was Hypermethylated in GliomaThe heatmap was used to present the top hypermethylationgenes in glioma compared with normal tissues so as to figureout the most characteristic gene with methylation change intumors indicating that the IRAK3 was hypermethylated theCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressmost in glioma Figure 3A Then the methylation differentialdistribution of the IRAK3 was upregulated at each siteFigure 3B Furthermore the β value of CpG sitesIRAK3such as cg cg cg cg cg cg cg andso on on the IRAK3 was significantly higher in the tumorindicating that IRAK3group than in the normal groupwas hypermethylated in the tumor group due to CpGsFigure 3C“I The aforementioned results showed that theIRAK3 CpG sites were highly methylated in glioma tissuesthan in normal tissuestissues and the top signaling pathwayrelated genesenriched in the glioma were listed Figure 4A and B Theresults suggested that the MAPK signaling pathway washighly expressed and activated Figure 4C and D Based onthe differential genes in the disease data the distribution ofupregulated genes and downregulated genes in the GOrelatedpathway was enriched Figure 5A The expression level ofMAPK signaling pathwayrelated genes in glioma is shown inFigure 5B The results of Figures and manifested thatIRAK3related MAPK signaling pathway was highly activatedin glioma However whether any correction existed with thedevelopment of glioma remained to be verifiedMAPK Signaling Pathway Expression Leveland State in GliomaFor the identification of gliomarelated signaling pathwaysthat might be influenced by aberrant DNA methylation thetop IRAK3related signaling pathways in glioma and normalMethylation and Expression Level of theIRAK3 in Glioma Tissues and CellsThe impact of hypermethylation on the expression of IRAK3in glioma tissues and cells was elucidated The IRAK3 wasFigure Methylation level and expression level of the IRAK3 in glioma tissues and cells A The IRAK3 was highly methylated in glioma tissues compared with adjacent tissues BThe IRAK3 was less expressed in glioma tissues than in adjacent tissues C The IRAK3 in the five glioma cells SHG44 U251 HS683 SF539 and GOS3 was hypermethylatedcompared with U251 glioma cells D The IRAK3 was less expressed in the five glioma cells than in normal glioma cells The difference was significant P submit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alFigure Effect on glioma cells after the overexpression and demethylation of the IRAK3 A The expression level of the IRAK3 was significantly higher in the glioma U251 cells of thepcDNA31IRAK3 and 5azadC groups than in the control group B and C The protein expression level of the IRAK3 significantly increased in the pcDNA31IRAK3 and 5azadC groupscompared with the control group D The expression level of the inflammatory factor IL6 significantly decreased in the pcDNA31IRAK3 and 5azadC groups compared with thecontrol group E The activity significantly decreased in the pcDNA31IRAK3 and 5azadC groups compared with the control group F and G The apoptosis rate significantlyincreased in the pcDNA31IRAK3 and 5azadC groups compared with the control group H and I The migration capability was significantly lower in the pcDNA31IRAK3 and 5azadC groups compared with the control group J and K The invasiveness capability was significantly lower in the pcDNA31IRAK3 and 5azadC groups than in the the control group Allthe mentioned differences were significant P The IRAK3 had a suppressive effect on glioma cells in vitroCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepresshighly methylated and less expressed in glioma tissues than inadjacent normal tissues Figure 6A and B Next the IRAK3 infive glioma cell lines SHG44 U251 HS683 SF539 andGOS3 also showed hypermethylation and less expressioncompared with normal gliocytes Figure 6C and D U251cells were selected for subsequent experiments because themethylation level of the IRAK3 in U251 cells was the highestFigure 6COverexpression or Demethylation of theIRAK3 Inhibited the Development of GliomaCellsA systematic test with the overexpression or demethylation ofthe IRAK3 was conducted to understand whether the correctionof abnormal IRAK3 methylation and expression affected thedevelopment of glioma After using pcDNA31IRAK3 tooverexpress or 5azadC to demethylate the IRAK3 the proteinexpression level of the IRAK3 in glioma U251 cells increasedin the tumor group compared with the normal group Figure7A“C IL6 an inflammatory factor secreted by the MAPKsignaling pathway [PMID ] so that its level in theculture medium could be used to represent the status ofMAPK signaling pathway activation decreased in thepcDNA31IRAK3 and 5azadC groups compared with thecontrol groups Figure 7D Surprisingly the viability ofglioma cells significantly decreased while the apoptosis rateincreased Figure 7E“G and the migration and invasivenesscapability decreased in the pcDNA31IRAK3 and 5azadCFigure Effect on glioma cells after treatment with the MAPK signaling pathway inhibitor PD325901 pcDNA31IRAK3 and 5azadC A The expression level of MAPKsignaling pathway“related genes expression level was significantly lower in the PD325901 pcDNA31IRAK3 and 5azadC groups than in the control group B and C Theexpression level of MAPK signaling pathway“related proteins significantly decreased in the PD325901 pcDNA31IRAK3 and 5azadC groups compared with the controlgroup D The level of inflammatory factor IL6 was significantly lower in the PD325901 pcDNA31IRAK3 and 5azadC groups than in the control group E The activitysignificantly decreased in the PD325901 pcDNA31IRAK3 and 5azadC groups compared with the control group F and G The apoptosis rate significantly increased inthe PD325901 pcDNA31IRAK3 and 5azadC groups compared with the control group All the mentioned differences were significant P P submit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et algroups Figure 7H“K which elucidated the associationbetween IRAK3 and the development of glioma The aforementioned results verified that the overexpression or demethylation of the IRAK3 inhibited the development of glioma cellsin vitroMAPK Signaling Pathway SuppressionAlone or Combined with theOverexpression or Demethylation of theIRAK3 Inhibited the Development ofGlioma CellsThe MAPK signaling pathway inhibitors PD325901pcDNA31IRAK3 and 5azadC were used alone or incombination in vitro to confirm the influence of the IRAK3and MAPK signaling pathways on glioma In PD325901pcDNA31IRAK3PD325901 and 5azadCPD325901groups the mRNA and protein expression levels of theMAPK signaling pathwayrelated genes such as MEK1ERK and cFos as well as the level of IL6 decreased inglioma Figure 8A“D The cell viability significantlythe apoptosis rate increased Figure 8E“Gdecreasedand the migration and invasiveness capability significantlydecreased in PD325901 pIRAK3PD325901 and 5azadCPD325901 groups Figure 9A“C The aforementioned results verified that the MAPK signaling pathwaysuppression alone or combined with the overexpression ordemethylation of IRAK3MAPK Signaling Pathway SuppressionAlone or Combined with theOverexpression or Demethylation of theIRAK3 Inhibited Glioma in vivoFinally whether MAPK signaling pathway suppression aloneor combined with the overexpression or demethylation of theFigure Effect on the migration and invasiveness capability of glioma cells after treatment with the MAPK signaling pathway inhibitor PD325901 pcDNA31IRAK3 and5azadC A and B The migration capability was significantly lower in the PD325901 pcDNA31IRAK3 and 5azadC groups than in the control group A and C Theinvasiveness capability was significantly lower in the PD325901 pcDNA31IRAK3 and 5azadC groups than in the control group All the mentioned differences weresignificant P Cancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressFigure Effect on mice after treatment with the MAPK signaling pathway inhibitor PD325901 pcDNA31IRAK3 and 5azadC A The tumor in the transplantationPD325901 p IRAK3 PD325901 and 5azadC PD325901 treatment groups after and days B The tumor volume was smaller in the PD325901pcDNA31IRAK3PD325901 and 5azadCPD325901 groups compared with the control group C The tumor weight followed the same trend as the volume D Theexpression level of the IRAK3 was lower in the transplantation PD325901 pcDNA31IRAK3 PD325901 and 5azadC PD325901 treatment groups compared with thetransplantation control group E and F The protein expression level of IRAK3 was lower in the transplantation PD325901 pcDNA31IRAK3 PD325901 and 5azadC PD325901 treatment groups than in the transplantation control group All the mentioned differences were significant P P IRAK3 had a suppressive effect on glioma in vivo was studiedAfter transplanting glioma U251 cells treated with PD325901pcDNA31IRAK3 PD325901 or 5azadC PD325901the tumorigenesis significantly decreased compared with thatin the control group mice Figure 10A“C Finally mRNAand protein expression levels ofthe MAPK signalingsubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alpathwayrelated genes in xenograft glioma were detectedthey were found to be significantly decreased in thePD325901 pcDNA31IRAK3 PD325901 or 5azadC PD325901 group Figure 10D“FDiscussionAccording to the World Health anization the overallsurvival of patients with cancer has increased significantlyover the years with the improvement in diagnosis and treatment However glioma is still a highly fatal disease1Therefore this study aimed to elucidate the detail of itsprogression mechanisms and search for new therapeutic strategies One recent study reported that the use of MGMTpromoter methylation test in hospitals had a strong influenceon the prognosis of glioma17 suggesting a significant clinicalapplication prospect of DNA epigenetic regulation Hencethe main focus of the present study was on the relationshipbetween DNA methylation and glioma IRAK3IRAK3 belongs to the IL receptorassociated kinaseIRAK family involved in inhibiting Tolllike receptorsignaling by changing the activity of other members ofthe IRAK family to decrease inflammatory response1819Increasing evidence supported thatthe expression ofIRAK3 in tumorassociated macrophages led to compromised immune surveillance of cancer cells when it profitably prevented excessive inflammation contributing tocancer development Therefore the growth of transplantable cancer cells could be inhibited by enhancing hostimmune responses in IRAK3deficient mice20 A smallnumber oftumor cellintrinsicIRAK3 could also support the progression of tumor cellsin colorectal and lung cancers depending on the dysfunctional innate immune system indirectly Interestingly theexpression of IRAKM in colorectal and lung cancers correlated with poor prognosisin patients with thesecancers2122 However little is known about the molecularmechanism of the IRAK3 in glioma This study revealedthe downregulation of IRAK3 glioma tissues and cellsthrough DNA methylation analysis which seemed to becontrary to the previous findings on other cancers Thisstudy investigated whether a special signaling pathwayexisted that connected IRAK3 and glioma progressionshowed thatstudiesAn ongoing study has validated that all members of theIRAK family mediate the activation of MAPK and nuclearfactorκB NFκB signaling pathways23 MeanwhileIRAK3 a general negative regulator was confirmed toinhibit MAPK and NFκB activation2425 Likewisethese results identified that the MAPK signaling pathwaywas highly activated in glioma and its related geneexpression level was downregulated after overexpressionof the IRAK3 In fact upstream genomic events andordifferent extracellular stimuli could activate the MAPKsignaling pathway mediating a wide range of cellularprocesses26 The activation of the MAPK signaling pathway often led to abnormal cell growth and tumorigenesisThe predominant effect relied on the signal intensity andthe context or tissue in which the signal occurred2728Zhang revealed that aberrant DNA methylation inthe promoters of MMPTIMP axis genes upregulated theMAPK signaling pathway promoting the progression oftumor cells Correction of the abnormal DNA epigenotypes attenuated the migration and invasion of tumorcells in vitro and reduced tumorigenicity in vivo29Intriguingly these results were consistent with those ofthe present studyConsidering the reverse expression pattern of IRAK3between glioma and other cancers2122 different epigeneticmodification was regarded as the major reason causing thisdistinction In many cancers the DNA methylation patternsbecame aberrant with tissue specificity serving as diagnosticmarkers and therapeutic targets30“ Hence based on thelower expression and the tumorigenic function of the IRAK3in glioma its epigen
2
" mutations in the exonuclease domain of pole a dna polymerase associated with dna replicationand repair lead to cancers with ultrahigh mutation rates most studies focus on intestinal and uterine cancers withpole mutations these cancers exhibit a significant immune cell infiltrate and favorable prognosis we questionedwhether loss of function of other dna polymerases can cooperate to pole to generate the ultramutatorphenotypemethods we used cases and data from cancer types in the cancer genome atlas to investigate mutationfrequencies of different dna polymerases we tested whether tumor mutation burden patient outcomediseasefree survival and immune cell infiltration measured by estimate can be attributed to mutations in polqand polzrev3lresults thirty six percent of colorectal stomach and endometrial cancers with pole mutations carried additionalmutations in polq eq polzrev3l ez or both dna polymerases ezq the mutation burden in thesetumors was significantly greater compared to poleonly e mutant tumors p in addition eq ez and eqz mutant tumors possessed an increased frequency of mutations in the pole exonuclease domain p colorectal stomach and endometrial eq ez and eqz mutant tumors within tcga demonstrated diseasefree survival even if the pole mutations occurred outside the exonuclease domain p however immunescores in these tumors were related to microsatellite instability msi and not pole mutation status this suggeststhat the host immune response may not be the sole mechanism for prolonged diseasefree survival ofultramutated tumors in this cohort results in this study demonstrate that mutations in polq and rev3l in pole mutant tumors shouldundergo further investigation to determine whether polq and rev3l mutations contribute to the ultramutatorphenotype and favorable outcome of patients with pole mutant tumors correspondence beatriceknudsenpathutahedu1department of biomedical sciences cedarssinai medical center losangeles ca usa2samuel oschin cancer research institute socci cedarssinai medicalcenter los angeles ca usafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0chuang bmc medical genetics page of the analysis of thousands of cancers by the cancergenome atlas tcga consortium and academic institutions revealed a small group of cancers with mutationsin pole and an ultramutator phenotype [ ] multiplestudies have found significantly improved survival in patients with pole mutated endometrial cancers [ “]while the survival benefit was not as profound in polemutated colorectal carcinoma the pole geneencodes the catalytic subunit of dna polymerase epsilon which catalyzes the leading strand synthesis duringdna replication pole possesses highfidelity dnapolymerization proofreading and ²² exonuclease activities which promote accurate dna synthesis firstidentified and reported in “ of colorectal carcinomas[ ] pole mutations were also noted at frequencies of “ amongst uterine corpus endometrialcancers [ ] and in gastric adenocarcinoma mutations can be found across the entire pole genebut those in the pole exonuclease domain are mostprevalent in cancers with ultrahigh mutation rates mutmb these cancers exhibit higher mutationrates than microsatellite instable msi tumors associated with mismatch repair abnormalities in additionpole ultramutant cancers also possess a high frequency of ctoa transversions [ ] a tumor associated inflammatory response similar to thatin msitumors has been reported to occur early on duringdevelopment of pole mutated endometrial and colorectal cancers it is thought to be caused by neoantigens that are generated as a result of the high mutation burden [ ] and render pole mutant cancersresponsive to immunotherapy polymerase theta polq is a lowfidelity dna polymerase lacking a ² to ² exonuclease function theenzyme is involved in the alternative nonhomologousendjoining pathway altnhej which is a backupmechanism of double stranded dna break repair thispathway predominates in cancer cells when other dnarepair pathways are missing or when telomere ends aredeprotected [ ] the loss of polq sensitizes cellsto ionizing radiation and polqdeficient mice exhibit increased dna instability and genomic rearrangementssuggesting a role for polq as a guardian of the genome both overexpression and loss of polq increasemutation frequencies [ ] multiple structuralmotives in polq can interact with dna rad51 andbrca1 in addition polq forms a complex withparp1 in a pathway of synthetic lethality with brca1and is thus considered a therapeutic target [ ]however which domains in polq should be targetedremains to be determined rev3l rev3 like dna directed polymerase zetapolz catalytic subunit is involved in dna synthesisthat reads through damaged dna translesion dnasynthesis tls the high efficiency of polz bypassing a broad spectrum of dna lesions led to its recognition as a master tls polymerase polzrev3l has been linked to carcinogenesis in breastlung gliomas and gastric cancers and modulatescisplatin sensitivity [“]because studies describe overlapping functions andsynergy among the over dna polymerases [ ] we undertook an unbiased approach to identifymutations in polymerases within the tcga tumorcompendium this analysis revealed a greater frequencyof mutations in polq and polzrev3l compared toother polymerases of similar gene length therefore wepropose that polq and polzrev3l may cooperatewith pole in generating ultrahigh mutation ratesmethodsdata acquisitionthe data used in this study are based upon the wholeexome sequence data sets generated by the tcgaresearch network httpcancergenomenihgov thelocations and frequencies of somatic mutations msistatus and clinical stage and follow up information intcga provisional datasets were obtained from cbioportal httpwwwcbioportal [ ] up to supplementary table mutation data were obtainedby first selecting the œquery tab from cbioportal thecancer type specific data were chosen from the œtcgapancancer atlas studies we entered the genes ofinterest pole polq and rev3l to retrieve mutationdata from the genes when the result page wasdisplayed we accessed the information underneath theœmutations tab we downloaded the number of mutations in each sample the annotation of protein aminoacid change and the type of mutation the survivalinformation for each case months after diagnosis wasobtained through the œcomparisonsurvival link functional domains of the proteins were provided by pfamdatabase data visualization for mutations wasperformed with mutationmapper in cbioportalcase selection criteriawe searched all cancer types in tcga for those thatpossess or more cases with pole mutations thisyielded tumor types that we named the pancandata set in this study we then determined the frequencyof mutations within additional polymerase within pancan we selected the three adenocarcinomas uterinecorpus endometrial adenocarcinoma colorectal adenocarcinoma and stomach adenocarcinoma with the highesttriple mutated dnapolymerase status for more detailed analysisfrequency of double or 0chuang bmc medical genetics page of studies performedkaplan meier survival plots were generated using clinicalfollowup data available within the tcga database todetermine the global mutational spectrum we classified types of nucleotide transitions or transversions thefrequency of each mutation type was calculateddigital images of all eq mutant tumors and representative cases of tumors with neither pole nor polqmutations and of msi tumors were assessed by one author jr for the amount of tumor immune infiltratethe combination of tumor infiltrating lymphocytes andthe peritumorallymphoid infiltrate was graded on ascale between and tumor associated lymphocyteswere graded as none minimal rare to per highpowered field hpf to per hpf and perhpf peritumoral lymphocytes were assessed at thedeepest advancing tumor front graded at low powernone minimal mild moderate andmarked these visual semiquantitative scores werecompared with the immune scores evaluated using estimate estimation of stromal and immune cells inmalignant tumor tissues using expression data estimate score were obtained from the website ofestimate at md anderson httpsbioinformaticsmdandersonestimatediseasehtml theplatformtype selected was rnaseqv2 data for colorectalstomach and endometrial cancer types were downloaded next we separated the colorectal stomach andendometrial cancer cases into quartiles defined by thehighest intermediate and lowest of mutational counts or msi status and calculated the immunescores for each groupstatistical data analysisall statistical analyses were conducted in r v313 plotswere generated using ggplot2 package in r datavisualization methods were described previously the horizontal lines in the boxplots represent the 1st2nd and 3rd quartiles and whiskers outside the boxshow the interquartile range the significance of thedifferences of data illustrated in the boxplots wascalculated using the wilcoxon ranksum tests the chisquare test was performed to test the significance ofdifferences in frequencies of all tables the significancein the kaplan“meier survival plot was calculated usingthe log rank test statistical significance was accepted atp resultsof the cancer types in the tcga database weidentified cancer types pancan with or morecases that possessed mutations in the pole proteincoding region fig 1a and supplementary table these cancer types contained cases with polemutations anywhere in the exome of these polemutant tumors carried mutations in one or more of the other dna polymerases we observed dna polymerases polq and polzrev3l to be most frequentlymutated fig 1b these two polymerases were mutatedin of tumors with pole mutations in fact thesetwo polymerases were even more commonly mutatedthan pole in our pancan cohort supplementary figure altogether cases with pole and polq mutations eq cases with pole and polzrev3lmutations ez and cases with pole polq andpolzrev3l eqz mutations were identified in thepancan cohort fig 1c mutations in the exonucleasedomain of pole are responsible for causing the ultramutator phenotype in colorectal and uterine corpuscancers [ ] in order to determine the contribution of polq and rev3l to the ultramutator phenotype we compared the mutation frequencies of tumorswith mutations in only pole to eq ez and eqzmutant tumors mutation frequencies in the cellulargenome increased in the following order no polemutations poleonly mutations anywhere with thepole exome eq ez eqz mutations fig 1dthe median mutation count of eqz tumors was morethan 10fold higher p than that of tumors withonly pole mutations e q and e z mutant tumorsalso displayed significantly higher mutation countscompared to eonly mutant tumors suggesting a contribution of mutationally altered polq or polzrev3l tothe overall cancer mutation rates next we determinedthe number of mutations in the exonuclease and polymerase domains of pole in the cancer types withinour pancan compendium fig 1e the percentage ofpole mutations in the exonuclease domain was greaterin eq ez and eqz mutant tumors compared topoleonly mutant tumors p in contrast thepercentage of mutations in the dna polymerase domainwas similar thus our data confirm the notion thatmutations in the exonuclease domain of pole areresponsible for ultrahigh mutation rates in additionmutations in polq and rev3l may further increasethe mutation burden however why mutations in polqand rev3l preferentially increase tumor mutationfrequencies remains elusive endometrialto further investigate a potential role of these mutantdna polymerases in the ultramutator phenotype wefocused on colorectalucec andstomach stad cancers these cancer types containthe highest numbers of tumors with eq ez and eqz amongst the cancer types included in pancanfig 1a supplementary table among these cancertypes we identified cases with eq cases with ezand cases with eqz mutations fig 2a in thesecancers the mutation burden in pole eq ez and e 0chuang bmc medical genetics page of fig cancer types pancan with poleqz mutations in tcga a number of cases with pole only eq ez and eqz mutations in cancertypes cohort referred to as pancan within tcga the xaxis shows the actual number of cases with pole green eq orange ez pink and eqz blue mutations the yaxis displays the cancer types uterine corpus endometrial carcinoma ucec stomach adenocarcinoma stadcolon and rectum adenocarcinoma skin cutaneous melanoma skcm lymphoid neoplasm diffuse large bcell lymphoma dlbc lungadenocarcinoma luad breast invasive carcinoma brca sarcoma sarc cervical squamous cell carcinoma and endocervical adenocarcinomacesc pancreatic adenocarcinoma paad lung squamous cell carcinoma lusc head and neck squamous cell carcinoma hnsc bladderurothelial carcinoma blca kidney renal clear cell carcinoma kirc and liver hepatocellular carcinoma lihc b case numbers with mutationsin polymerase genes the number of cases in pancan with mutations in the following polymerases is displayed on the yaxis dntt pola1polb pold1 polg polh poli polk poll polm poln polq rev1 rev3l c venn diagram displaying the number of cases in pancan withmutations in or pol genes d mutations per mb yaxis of pancan cases without pole mutations other or with pole eq ez and eqzxaxis mutations number of cases in each group are listed in parenthesis e mutation frequencies in pole exonuclease and polymerase domainsas a percentage of total number of mutations in the pole exome œother refers mutations in the entire exome outside the exonuclease orpolymerase domains the cases are grouped by their polymerase mutation status on the yaxis and the number in parenthesis represents thetotal number of pole mutations within each group 0chuang bmc medical genetics page of fig poleqz mutations in colorectal endometrial ucec and stomach stad cancers a venn diagram displaying the number of caseswith mutations in or pol genes b mutation groups of cases without polymerase mutations other or with mutations in pole only eq ez and eqz the number of cases in each group is listed in parenthesis the pvalue for comparison of pole and eq groups is not significantp c number of cases with mutations in pole exonuclease domain in various mutation groups d percentages the ratio of transitions tiand transversions tv are shown on the yaxis for core stad and ucec the xaxis shows the mutation groupsqz mutant tumors paralleled the mutation burden inthe whole pancan cohort compare fig 2b and fig1c supplementary figure 2ad eqz mutant tumors demonstrated on average an 8fold increase inmutation frequencies compared to tumors with onlypole mutations amongst pole mutant tumors tumors carried mutations outside the pole exonuclease domain while tumors carried mutations withinthe exonuclease domain the frequency of poleexonuclease mutationsin fig 2cprovides a valid explanation forthe difference inmutation rates and potential association of poleexonuclease domain mutations with mutationsinpolq and polzrev3l which are the other twomost frequently mutated dna polymerases casesoverall exonuclease domain mutations were identifiedin cases of pole only mutant tumors eqcases ez cases and of eqz cases fig 2cstratified by cancer types pole exonuclease domainmutations occurred in colorectal endometrial and stomach tumors demonstrating cancertype specific frequencies supplementary figure 3a incontrast to the pole gene that demonstrates mutationalhotspots in the exonuclease domain mutational hotspotsin the polq gene are not associated with a functionalprotein domain supplementary figure 3b c dwhile rev3l does not reveal mutational hotspotsapproximately of mutations lead to truncated protein expression supplementary figure 3e f anothercharacteristic of pole mutant tumors are c to a and g 0chuang bmc medical genetics page of to t transversions we observed the greatest increaseof nucleotide transversions in cancers with eqz mutafig 2d consistent with the loss of poletionsexonuclease activity in these tumorssince mutations in pole confer increased disease freesurvival dfs in patients with uterine cancer even inthose patients with highgrade tumors [ ] we investigated the prognostic role of polq and rev3l mutations in pole mutanttumors kaplanmeier curveswere constructed for colorectal endometrial and stomach cancer cases with followup data fig 3a using thetcga annotations of dfs in individual patients nocancer recurrences were observed in the eq ez andeqz mutant groups pole exonuclease domain mutations were observed in cases in the good survivalgroup and case in the poor survival group consistentwith the expected long dfs periods of patients withpole exonuclease domain positive tumors in additionanalysisin the pancan cohort cases with mutations in pole outside the exonuclease domain were in the good survival group of those had concurrent mutations in polq or rev3l orin both polymerases fig 3b furthermore a kaplanmeierrevealedimproved dfs associated with mutations in polq andrev3l the favorable survival outcome was observed incolorectal endometrial and stomach cancers howeverno favorable outcome was observed in diffuse bcelllymphoma p these data provide preliminaryevidence of cancertype specificfavorable survivaloutcomes in tumors with pole mutations that arelocated outside the pole exonuclease domain if concurrent mutations in polq rev3l or in both polymerasesare presentcompared to microsatellite stable tumorsmssmicrosatellite instability msiin colorectal cancerconfers a better prognosis to determine whetherfig survival and clinical characteristics of patients with polymerase mutations colorectal endometrial ucec and stomach stad cancers akaplanmeier curves of diseasefree survival dfs for groups of patients pole only n median followup months green line eqn median followup months orange line ez n median followup months pink line and eqz n median followup months blue line tumors without mutations in pole polq or rev3l exomes grey line the overall pvalue is p individual pvalues eqz vrs pole “ p eqz vrs none “ p eq or ez or eqz vrs pole “ p b polymerase mutation analysis ofcases in the good survival group in panel a the red bar indicates cases with pole exonuclease mutations c cancer typespecific illustration ofmutation count pole polq and rev3l mutations microsatellite instability msi and tumor stage 0chuang bmc medical genetics page of the favorable outcome of eq ez and eqz mutantcancers can be explained by msi or tmn stage we examined the relationship between msi statustumorstage and polymerase mutations in colorectal endometrial and stomach cancers fig 3c and supplementaryfigure and supplementary table despite improveddfs rates the full range of tumor stages was observedamongst eq ez and eqz tumors p supplementary table 2a comparing the poleonly and eqz mutant cancers did not reveal a significant difference in tumor stage but differed in the frequency ofmsi cases p pole mutant tumors were morefrequent in the msi group than in themss group in addition the frequency ofmsi cases in pole mutant tumors differed between the cancer types p supplementary table 2b c although msi is enriched in samples with highmutation levels supplementary table 2d as expectedp were10fold higher mutation countsobserved in cancers with eqz mutations compared tomsi without eqz mutations supplementary figure these results suggest that mutations in eq ez and eqz confer a better prognosis independent of msi statusand tmn stage in colorectal endometrial and stomachadenocarcinomaswe next examined the amount ofthe cancerassociated immune infiltrate the immune scoreobtained through estimate corresponded tothe categorical score of the immune infiltrate derivedfrom digital he images supplementary fiure therefore we used the estimate immune scoresfor further analysis of colorectal endometrial andstomach cancers as shown in fig 4a a significantdifference was observed in the median immunescores between groups with lowintermediate andhigh mutation burden grouped based on mutationburden and not on e q z mutantseemethods and as expectedthe median immunescores increased with total mutation levels surprisingly the immune scores in eqz mutant tumorsdid not differ significantly from tumors with a lowlevel of mutationsfig 4b as expected msitumors possessed higher immune scores than msstumors p immunescores of msi and eqz mutation tumors weresimilar to those in the msi group and higher thanmss and eqz mutation tumors fig 4d withinthe group of tumors with pole exonuclease domainmutations mss tumors possessed lowerimmunescores than msi tumors but the difference was notsignificanttogether results in this tcga cohort demonstratethat the immune response is driven by msi ratherthan pole exonuclease domain mutationssupplementary fig 4c finallystatusp figurediscussionan analysis of dna polymerases in tumors withmutations in the pole revealed additional mutations inspecific polymerases most commonly in polq andpolzrev3l among the cancer types colorectaluterine and stomach cancer were mostfrequentlyafflicted by these mutations cancers with mutations inpole and polq eq pole and polzrev3l ezand in all polymerases eqz were associated withthe highest mutation burden and an excellent prognosisindependent of msi status and tumor stage mutationsin the exonuclease domain were observed in of eqz mutant tumors but only in of poleonly mutant tumors or in of eq ez tumorshowever despite harboring 10fold more mutationsthan msi tumors and 8fold more mutations than themutation frequencies associated with poleonly mutanttumors eqz mutant tumors did not display significantly more inflammationthe main result from that analysis is that patients withcolorectal stomach and endometrial cancers bearing eq ez and eqz mutations have disease free survival dfs at a median follow up time of months incontrast patients with tumors bearing mutations inpole only most of which outside the pole exonucleasedomain had a dfs of at follow up of monthsthe favorable dfs in eq ez and eqz mutated tumors occurred even in tumors with mutations in polethat are located outside the pole exonuclease domainthe contribution of mutations in pole to tmb cž”asubstitutions and cancer type associations are describedin table of raynor using a larger resourceof cases and should be used to interpret the mutationsin the current study listed in supplementary figure as a whole the current study expands the spectrum ofpole mutant tumors with an excellent prognosis thefavorable prognosis included patients with high tumorstage which echoes prior studies demonstrating a favorable outcome of uterine tumors with pole exonucleasemutations despite adverse standard clinicopathologicindicators including high grade high stage and lymphovascular invasion [ ] while the high mutationfrequencies may cause an early growth advantage as tumors evolve they may succumb to high mutationburden as new mutations can no longer be tolerated andcause tumor cell death [ ] or increased sensitivityto therapeutic agentsthe prevailing hypothesis for the favorable prognosisof cancers displaying the hypermutator phenotype is theincreased attack by the immune system evidence insupport of this theory is the observation that tumorinfiltrating and peritumorallymphocytes are increased and that cytotoxic activities in cd8 and cd4are heightened in polelymphocyte populations 0chuang bmc medical genetics page of fig estimate immune scores by mutation frequency quartiles eqz mutation groups and msi in colorectal endometrial ucec and stomachstad cancers a estimate immune scores in cancers within high intermediate and low overall mutation quartiles b immune scores of sampleswith eq ez and eqz mutations compared to the low mutation quartile from panel a c immune scores in groups of cancers separated by msistatus d immune scores in msi and mss eqz cases compared to all other msi cases for each panel the number of cases within each group isincluded in parentheses on the xaxismutated endometrial cancers [“] similar to hypermutated msi tumors this observation has led tothe hypothesis that immune checkpoint inhibitors maybe efficacious in pole ultramutated tumors ourresults question a direct relationship between mutationburden tumor immune response and pdl1 expressionalso raised in a larger study across cases from cancer types in tcga while we observed a concordance between the computational and histological assessments of the immune infiltrate the immune score intumors with eqz mutations depended on msi statusthis result suggests that the immune infiltrate attributable to mutations in eqz mutant tumors may be lessor that its composition may involve immune cells otherthan lymphocytes lesser cd8 and gammainterferongene expression signatures have also been observed ingastrointestinal tumors with a large single nucleotidevariantsnv burden that was attributed largely topole exonuclease mutations perhaps eqz mutations occur at a later point in tumor evolution whenimmunosuppressive factors already dominate we alsocannot rule out the possibility of increased numbers ofcytotoxic lymphocytes intermixed with eqz mutanttumor cells because computational methods and inspection of he images are not sensitive enough to detectsmall differencesinfiltrating lymphocytestils that may have large antitumoral effectsin tumora significant limitation of the study lies in the relatively small number of tumors this limitation cautionsthe generalization of results and seemingly novel insights 0chuang bmc medical genetics page of into the hypermutator phenotype studies by othergroups attributed hypermutator phenotype to specificmutations primarily within the pole exonucleasedomain given the proofreading function of the exonuclease domain it makes sense that mutations outsidethe domain have a lesser effect on tmb in agreementwith this concept our study reveals that compared totumors harboring only a mutation in pole polesinglemutant tumors of cases pole exonucleasedomain mutations are more common in tumors withboth double ez and eq and triple eqz dnapolymerase mutations of cases and doubleand triple mutant tumors have higher mutation countsthan pole single mutant tumors while it cannot befully excluded that polq and polzrev3l mutationsare bystander events in pole mutanttumors weobserve a higher tmb in cases with mutations in allthree polymerases supplementary figure 2a and bmechanistically polq and polz are thought to function in different repair processes polq in alternativemicrohomologymediated nonhomologous dna repair pathway and polz in translesion dna synthesishow these dna repair processes cooperate with thereplicative dna polymerase pole to prevent genomeinstability remains unknown this will be an importantsubjectthe mechanismfor further understanding ofunderlying the hypermutator phenotypesif validated in additional cohorts our findings may haveimportant clinicalimplications they build upon andexpand the previously well documented good prognosticimpact of pole exonuclease mutationsin uterinecancer that have generated intense interest in part dueto the paradox of a favorable prognosis in tumors withpathologic indicators of poor prognosis while in thisstudy prolonged dfs is observed in colorectal endometrial and stomach cancers with eqz mutations thisis not the case in other noncarcinoma cancer typeswithin tcga thus we find that the positive outcomeprediction is cancer type specific altogether resultsfrom this study provide a rationale for including polqandor polzrev3l mutations in clinical outcomestudies of tumors with pole mutations however future validation is required to confirm the concept that isrevealed in the current studycolorectal core stomach stad and endometrial ucec cancers byspecific polymerase mutated groups in tcga data sets supplementaryfigure locations of mutations in pole polq and rev3l exomes inindividual colorectal core stomach stad and uterine cancers ucecsupplementary figure relationships between mutation spectrumand mutation counts pole polq and rev3l exome mutations msi andtumor stage of individual cases supplementary figure mutationrates per mb yaxis of core stad and ucec cases with msi and eq ez and eqz xaxis mutations supplementary figure relationshipbetween pathology inflammation score and estimate immune scores forcore stad and ucec supplementary figure estimate immunescores in colorectal core endometrial ucec and stomach stadcancers supplementary table number of cases with pole polq zrev3l or multiple exome mutations in the pancan cohortsupplementary table contingency tables showing number of casesof colorectal endometrial and stomach cancers in each categoryabbreviationspole polymerase epsilon polq polymerase theta polzrev3l rev3 likedna directed polymerase zeta polz catalytic subunit tcga the cancergenome atlas msi micro satellite instability mss micro satellite stabilitytmb tumor mutation burden tnm tumor lymph node metastasis estimate estimation of stromal and immune cells in malignant tumor tissuesusing expression dataacknowledgementsnot applicableauthors™ contributionsall authors have read and approved the content of this manuscript fhstudy design and data analysis ht data interpretation jr data analysis andmanuscript writing bsk data interpretation and manuscript writingfundingthe prostate cancer foundation funded salaries of fh and bsk forcomputational analysis steven spielberg team science award andr01ca131255 bsk funded salaries for data analysis and paper writing of fhand bsk we also acknowledge the institutional support of salaries throughthe nih g20 rr030860 to the cedarssinai biobank and translational research core for salaries of bsk and fh the content of this publication doesnot necessarily reflect the views or policies of the department of health andhuman services nor does any mention of trade names commercial products or anizations imply any endorsement by the us governmentavailability of data and materialssequencing data can be obtained from national cancer institue gdc dataportal and are published in raw genomic and clinical data can befound at the nci genomic data commons httpsportalgdccancergovlegacyarchive the mc3 mutation annotation file can be accessed at httpsgdccancergovaboutdatapublicationsmc32017 and the processed datafiles can be viewed at httpsgdccancergovaboutdatapublicationspancanatlasethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors do not declare any competing interestssupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12881020010899additional file supplementary figure number of cases polemutations n and mutations in the exomes of dna polymerasegenes in pancan supplementary figure mutation counts forauthor details1department of biomedical sciences cedarssinai medical center losangeles ca usa 2samuel oschin cancer research institute soccicedarssinai medical center los angeles ca usa 3surgerycedarssinai medical center los angeles ca usa 4pathology andlaboratory medicine cedarssinai medical center los angeles ca usa 5department of pathology university of utah salt lake city ut usa 0chuang bmc medical genetics page of received january accepted july referenceskandoth c schultz n cherniack ad akbani r liu y
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"inhibitors by causing immediate and nearly complete intracellular and extracellular thiamine deprivation [6]. In previous studies we have shown that thiaminase has both in vitro and in vivo cytotoxicity activity further supporting the concept that TDEs could represent new targets for novel therapies [6] [7] [8]. We have also previously reported that rapamycin has antagonistic effect on thiaminase-mediated growth inhibition of leukemia cells [7] a surprising finding since rapamycin generally acts as a sensitizing agent in combination with cytotoxic drugs. We now present metabolic and metabolomic observations regarding the anticancer activities and metabolic effects of thiaminase in leukemia and breast cancer cells. We chose to focus on breast and leukemia models because these were the models in which we observed the most promising activity of thiaminase in xenografts. These studies help define thiaminase metabolic effects that may be responsible for its cytotoxic activity. These studies also further elucidate the role of mTOR as an inhibitor of thiaminase-mediated alterations in cellular metabolism and demonstrate the role of mTOR in regulating expression of enzymes involved in thiamine-dependent metabolism. Methods Ethics statement All animal studies were approved by the University of Kentucky Institutional Animal Care and Use Committee. Cell Lines The human breast cancer cell line MCF-7 and the non-malignant breast cell line MCF-10A were obtained from ATCC; human leukemia cell lines Reh and RS4 were originally obtained from ATCC and generously provided by Dr. Terzah Horton Baylor College of Medicine. Cell line authentication was performed by PCR amplification of nine short tandem repeat (STR) loci (Research Animal Diagnostic Laboratory St. Louis MO) and comparing the profile to the ATCC STR database. The STR profile of MCF-7 and RS4 cell lines were identical to the ATCC profile. The Reh cell line matched all alleles in the ATCC Reh profile plus one extra allele at two loci. Cytotoxicity assays Human leukemia cell lines Reh or RS4 were plated in triplicate in 96-well microtiter plates in RPMI-1640 (with 25 mM HEPES) medium containing 10% fetal bovine serum at final densities of 8—104 cells/well. MCF-7 cells were plated in the same medium at the final density of 1000cells/well. Medium containing native thiaminase at a concentration range of 1—10?6 to 4 units/ml was added to cells and incubated for four days. Following incubation an MTT Cell Proliferation Assay (ATCC) was performed according to the ATCC protocol. The IC50 was calculated from the dose response curve as the concentration of drug producing a 50% decrease in the mean absorbance compared to the untreated wells using Prism GraphPad software. The cytotoxicity experiments were repeated a minimum of three times in triplicate. Analysis of primary human ALL specimens was performed by plating cells at a density of 1—106/ml and treating for 24 hours with the indicated concentration of thiaminase. Viability was evaluated by dead cell exclusion labeling with trypan blue dye as well as flow cytometric analyses using Annexin-V labeling as previously described [9]. Xenograft studies RS4 tumor xenografts were established by subcutaneously inoculating 1—107 cells into the right flank of five-week-old female Crl:NU-Foxn1 nude mice (Charles River Laboratories Wilmington MA). For the establishment of MCF-7 xenograft a 17?-estradiol pellet (0.72 mg 60 days release; Innovative Research of America Sarasota FL) was implanted subcutaneously into the neck to facilitate optimal tumor growth for the estrogen receptor“positive MCF-7 cells. The xenografts were initiated by subcutaneously injecting 5—106 MCF-7 cells into the right flank of five-week-old female Crl:NU-Foxn1 nude mice. When palpable tumors had formed mice were treated with native thiaminase at its MTD (2000 units/kg body weight) twice a week at a site distant from the formed tumor for three weeks. Mice were treated with a single dose of thiaminase enzyme conjugated with 1k linear chain PEG (1K-LCPTE) at its MTD (50 U/kg) at a site distant from the formed tumor. Mice treated with N3-pyridyl thiamine (N3PT) received an intraperitoneal (i.p.) dosage of 80 mg/kg daily for five days. Mice treated with both 1K-LCPTE and N3PT received a single dose of 1K-LCPTE at 50 U/kg first then after 5five days mice received N3PT at 80 mg/kg daily for five days. Tumors were measured twice a week in a blinded manner by measuring perpendicular diameters with a digital caliper and tumor volumes (mm3) were calculated using the following formula: volume ?=? width — width — length — ?/6. The predetermined endpoint was a tumor volume of 1500 mm3. The control mice were injected with MCF-7 or RS4 cells and left untreated and results combined with a previous control group of the same xenograft [7]. Kaplan-Meier survival curves and statistical analysis was performed with GraphPad Prism software. Primary lymphoblastic leukemia xenograft methods A primary lymphoblastic leukemia specimen was transplanted by IV injection into sub-lethally irradiated immune deficient NOD/SCID/IL2Rg mice. When the tumor burden was established in the bone marrow (day 17) animals received treatment with thiaminase 2000 units/kg on days 17 20 and 24 administered by subcutaneous injection (the longer interval between treatments in these mice in comparison to the Crl:NU-Foxn1 nude mice was due to tolerability). The animals were sacrificed on day 33 and marrow cells were isolated and examined by flow cytometry using human-specific antibodies for CD45 and CD19 to determine the level of human leukemia cell engraftment as previously described [9]. Mitochondrial bioenergetics measurements Oxygen consumption was determined using the Seahorse Extracellular Flux (XF-96) analyzer (Seahorse Bioscience Chicopee MA). The XF-96 measures the concentration of oxygen and free protons in the medium above a monolayer of cells in real-time. Thus the rates of oxygen consumption and proton production can be measured across several samples at a time. To allow comparison between experiments data are presented as oxygen consumption rate (OCR) in pMoles/min/104 cells and the extracellular acidification rate (ECAR) in mpH/min/104 cells. RS4 and Reh leukemia cells were seeded at 125000 cells/well into gelatin-coated Seahorse Bioscience XF microplates cultured in the presence or absence of 2 g/L D-glucose and then centrifuged to adhere to the bottom of the wells while for the MCF-7 and MCF-10A about 45000 cells were plated and allowed to adhere overnight. OCR was measured four times and plotted as a function of cells under the basal condition followed by the sequential addition of oligomycin (1 µg/ml) FCCP (1 µM) and rotonone (1 µM). The ATP-linked OCR was calculated as the basal OCR minus the OCR measured after the addition of oligomycin. The OCR maximal capacity was the direct rate measured after the addition of FCCP. The reserve capacity is the FCCP OCR minus the basal OCR. For the ECAR measurements cells were washed following overnight incubation and changed to assay media lacking glucose. Basal ECAR were measured four times and plotted as a function of cells under the basal condition followed by the sequential addition of glucose (25 mM) oligomycin (1 µg/ml) and 2-deoxyglucose (25 mM). The rate of glycolysis was determined by subtracting the basal ECAR from the ECAR after the addition of glucose. Glycolytic reserve was determined by subtracting the ECAR following the addition of oligomycin from the ECAR following the addition of glucose. Differences between treatment groups were calculated using the Kruskal Wallis test. Metabolomic studies RS4 leukemia cells and MCF-7 breast cancer cells were analyzed under six conditions: control for 24 hrs (C-24); incubation in thiaminase for 24 hrs (T-24); control for 48 hours (C-48); thiaminase for 48 hrs (T-48); rapamycin for 48 hrs (R-48); and both rapamycin and thiaminase for 48 hrs (R+T-48). Four independent samples were produced for each time point for each cell line and cell pellets were stored at ?80°C until the separate experiments were all completed. Metabolomic studies were performed at Metabolon Inc. (Durham NC). The non-targeted metabolic profiling platform consisted of three independent instrumental methods: ultrahigh performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS2) optimized for basic species; UHPLC/MS/MS2 optimized for acidic species; and gas chromatography/mass spectrometry (GC/MS). The detailed process of the platform; including sample processing instrument configuration data acquisition as well as metabolite identification and quantitation were published previously [10] [11]. Three hundred and forty two metabolites were identified by automated comparison of the ion features in the experimental samples to a reference library of chemical standard entries that included retention time molecular weight (m/z) preferred adducts in-source fragments and associated MS spectra [12]. Instrument variability was determined by calculating the median relative standard deviation (RSD) for the internal standards that were added to each sample prior to injection into the mass spectrometers. Overall process variability was determined by calculating the median RSD for all endogenous metabolites (i.e. non-instrument standards) present in 100% of a set of technical replicates of pooled samples. Values for instrument and process variability meet Metabolon™s acceptance criteria with instrument variability of 3% and overall process variability of 12%. Following normalization to total protein (Bradford assay) log transformation and imputation with minimum observed values for each compound Welch™s two-sample t-tests were used to identify biochemicals that differed significantly between experimental groups. The entire metabolomic data sets for RS4 and MCF-7 cells with statistical results are included in data tables (Table S1). Immunoblot analysis Cells were treated with thiaminase (0.001 U/ml) and/or rapamycin (0.1 µM) for 96 hours. Cells were lysed with a triple-detergent lysis buffer (50 mM Tris pH8.0 150 mM NaCl 1% NP-40 0.5% DOC 0.1% SDS 0.02% sodium azide 100 µg/ml PMSF protease inhibitors (Roche) and phosphatase inhibitors (Thermo Scientific)). Equal amounts of protein were loaded into each well and separated by SDS-PAGE gel followed by transfer onto nitrocellulose membranes. The membranes were blocked incubated with the indicated primary antibodies at 4°C overnight and the appropriate horseradish peroxidase“conjugated secondary antibody was added for 1 hour at room temperature. Immunoblots were developed by use of SuperSignal West Pico chemiluminescent substrate (Thermo Scientific) according to the manufacturer™s protocol and analyzed by FujiFilm LAS-4000 luminescent image analyzer (Multigauge software). The primary and secondary antibodies used in this study are listed as follows. Anti- PKM1; anti-BCAT2; anti-TPK1 and anti-THTPA antibodies were purchased from Proteintech Group Inc. (Chicago IL). Anti- PKM2 and anti-CPT1A antibodies were from Cell Signaling Technology (Danvers MA). Anti-BCKDE1 and anti-phospho-BCKDE1 antibodies were obtained from Bethyl laboratories (Montgomery TX). Anti-BCAT1 anti-?-actin and all secondary antibodies were obtained from Sigma-Aldrich (St. Louis MO). Results Evidence of antitumor activity of thiaminase in leukemia and breast cancer tumor models is shown in Figure 1. Figure 1A is a Kaplan-Meier plot of MCF-7 subcutaneous xenografts treated with thiaminase showing a prolongation in the time to endpoint (pre-defined tumor volume) (TTE) from 41 days in the mock treated cohort to 59 days in the treated cohort (p?=?0.03). In Figure 1B RS4 subcutaneous xenografts show an increase in median TTE from 16.5 days from the start of treatment to undefined TTE after 60 days (p<0.001). We have also previously shown evidence of thiaminase activity against MDA231 breast cancer [8]. In Figure 1C the activity of thiaminase is shown against primary human leukemia cells. The most sensitive primary leukemia specimens appear to be lymphoblastic specimens with MLL-gene re-arrangements. This was of interest as the RS4 cell line is also an MLL-rearranged cell line. Figure 1D shows flow cytometric analysis of bone marrow of a primary MLL-rearranged leukemia cell xenograft treated with thiaminase demonstrating a decrease in leukemia cell proportion after treatment. These studies along with previous studies [7] [8] provided the rationale for performing further detailed examination of the metabolic effects of thiaminase in the breast cancer cell line MCF-7 and in the RS4 leukemia cell line. In addition for further points of comparison we included selected studies in two additional cell lines Reh leukemia cells another lymphoblastic leukemia cell line and MCF-10A a non-malignant breast cell line. 10.1371/journal.pone.0085702.g001 Figure 1 In vivo evidence of thiaminase anticancer activity. A. A Kaplan-Meyer plot of time to pre-defined tumor volume endpoint for subcutaneous MCF-7 breast cancer xenografts treated with thiaminase 2000 units SC QOD or buffer control. The median time to endpoint was 41 "
1
netosis is a type of regulated cell death dependent on the formation of neutrophil extracellular traps net where netlike structures of decondensed chromatin andproteases are produced by polymorphonuclear pmn granulocytes these structuresimmobilise pathogens and restrict them with antimicrobial molecules thus preventing theirspread whilst nets possess a fundamental antimicrobial function within the innate immunesystem under physiological circumstances increasing evidence also indicates that netosisoccurs in the pathogenic process of other disease type including but not limited to atherosclerosis airway inflammation alzheimer™s and stroke here we reviewed the role ofnetosis in the development of an injury including injury to the brain lung heart kidneymusculoskeletal system gut and reproductive system whilst therapeutic agents in blockinginjuries induced by netosis in its primitive stages were also discussed this review providesnovel insights into the involvement of netosis in different an injuries and whilstpotential therapeutic measures targeting netosis remain a largely unexplored area thesewarrant further investigationkey words netosis neutrophil an injury cell death inflammation cell death is commonly segregated into necrosisand apoptosis apoptosis being programmed cell death anaesthetics pain medicine and intensive care department of surgeryand cancer faculty of medicine imperial college london chelsea andwestminster hospital fulham road london sw10 9nh uk department of anesthesiology shanghai fengxian district central hospital shanghai jiao tong university affiliated sixth people™s hospitalsouth campus fengxian district shanghai china to whom correspondence should be addressed at anaesthetics painmedicine and intensive care department of surgery and cancer faculty of medicine imperial college london chelsea and westminsterhospital fulham road london sw10 9nh uk emaildmaimperialacukfor instance during development and physiological cellular turnover whilst necrosis predominantly takesplace in an unregulated manner netosis like necrosis is a mode of cell death that involves the loss ofmembrane integrity during netosis decondensationof chromatin is thought to be initiated by peptidyl arginine deiminase pad4 its subsequent releasetogether with granule contents is vital in the innateimmune response to infection and inflammation recentstudies suggest that net formation is of central topathogenesis of an injury this review will summarise the current understanding of the molecular mechanisms of netosis and the therapeutic approaches underdevelopment targeting netinduced an injury the authors this is an open access publication 0cmolecular mechanism of netformationalthough netosis is closely associated with netformation not all net formation requires the process ofcell death to take place beforehand according to nomenclature committee on cell death the term ˜netosis™should only be used in the context of cell death and notjust based on the presence of net formation two main pathways of net formation have beendescribed and categorised according to their dependenceon the activity of nicotinamide adenine dinucleotide phosphate nadph oxidase pathway fig nadph oxidasedependent net formationthe nadph oxidasedependent molecular pathwayof net formation begins with activation of neutrophilsurface receptors by stimuli derived from pathogenic ornonpathogenic sources such as cholesterol or urate andends with cellular lysis these stimuli trigger calciumrelease from the endoplasmic reticulum er resulting inthe activation of protein kinase c pkc and the assemblyof the nadph oxidase complex generating reactive oxygen species ros following this neutrophil elastasene a protease stored in the cytoplasmic granules migrates to the nucleus in a myeloperoxidase mpodependent manner and cleaves histones to initiate chromatindecondensation this is promoted by the citrullinationof histone arginine residues by peptidylarginine deiminaseiv pad4 finally mixing of the chromatin andgranule proteins takes place as cellular membranes arebroken down interestingly there have been reports ofmitochondrial dna mtdna instead of nuclear dnabeing the source of the dna fibres in nets with observations of mtdna being released from granulocytes inresponse to disease states such as trauma and systemiclupus erythematosus sle [ ] moreover it seemsthat histone citrullination is not always required for netformation as observed by kenny and colleagues in theirstudy of neutrophils activated by the pkc agonist phorbol12myristate 13acetate pma this highlights the diversity of pathways for net formation following their induction degradation of nets can take place through severalpathways for example by dnases or endocytosed bymacrophages factors that influence net formation include phco2 and hco3ˆ’ levels which modulate neutrophil activation this explains why nets form more readily in thecahilog zhao wu alam eguchi weng and maperiphery of an inflammatory site where the ph is morealkaline this may influence treatment efficacy asnets can seal off the affected area an acidic environmenthas been hypothesised to reduce nadph oxidasedependent net formation by reducing neutrophil glycolysis nadph oxidasedependent net formation also requires neutrophils to be in the cell cycle necessitating theactivation of cyclindependent kinases cdk phosphorylating the retinoblastoma proteinnadph oxidaseindependent net formationthis mechanism of net formation is more relevant inthe context of infection as inducers of netosis here arecalcium ionophore a23187 and the potassium ionophorenigericin which are products of streptomyceschartreusensis and streptomyces hygroscopicus respectively how this pathway leads to net release ispoorly understoodnetosis and inflammationnets under physiological conditions are central topathogen clearance when there is excessive formation orsuboptimal nets are able to initiate further destructivesignalling through interaction with other tissue constituentsand the immune system moreover the antimicrobial histones and peptides within the net structure impose adirect cytotoxic effect on tissues to date there havebeen numerous accounts of netosis being present indiseases of major ans understanding of netosis inpathophysiology may offer unique opportunities for clinical managementnetosis in an injurythere is an expanding body of research describingnetosis in infectious and noninfectious an injurysummarised in fig although it is valid that in thesescenarios nuclear dna released during necrotic cell deathcan contribute to tissue injury and exacerbate the extent ofan damage here we focus on the contribution by aberrant net formation and the implication of understandingits underlying pathogenesis for therapeutic interventions 0crole of neutrophil netosis in an injuryfig type of cell death for neutrophil in an injury during solid an injury neutrophils could be prompted to undergo caspasedependent apoptosiswhich results in controlled dissolution of cell into apoptotic bodies containing cellular debris to prevent immune and inflammatory responses neutrophilextracellular traps nets form via two pathways the first is through lytic netosis a cell death pathway characterised by nuclear delobulation anddisintegration of the nuclear envelope which precedes loss of cellular polarisation chromatin decondensation and plasma membrane rupture the secondmechanism involves the nonlytic form of netosis which is not associated with cellular death but prompts expulsion of nuclear chromatin together withrelease of granule proteins through degranulation these components can assemble in the extracellular space into nets leaving behind enucleated cytoplaststhat continue to ingest microanisms in addition neutrophils could undergo unregulated necrosis that does not involve specific molecular pathwayswith uncontrolled release of cellular debris acting as dangerassociated molecular patterns damps to trigger proinflammatory responsebrainalzheimer™s disease alzheimer™s disease ad is acommon disorder of neurodegeneration characterised bygradual loss of cognitive functions in ad patients neutrophils have been observed to invade the brain parenchyma and release nets causing destruction of neural cellsand the bloodbrain barrier stroke it is well known that the adaptive immunesystem is altered after a stroke predisposing patients toinfections [“] interestingly netosis has also beendescribed as significantly impaired up until on day inthose with an ischaemic stroke though netosis inhaemorrhagic stroke patients has yet to be documented ithas been noted that the generation of ros a keyrequirement for chromatin decondensation is suppressedin these patients for up to days lungcystic fibrosis it has been well established that chronic infections in cystic fibrosis cf patients are due to thehighly viscous mucus production harbouring microbialgrowth although impaired clearance of mucus has beenprincipally named responsible there is increasing evidencethat the high viscosity is also due vast amounts of freedna found in sputum samples which was in concordance with the high concentration of neutrophil and 0ccahilog zhao wu alam eguchi weng and mafig involvement of netosis in an injury accumulating evidences now point to an important role of netosis in infectious and noninfectious solidan injury neutrophil invasion into brain parenchyma and release of neutrophil extracellular traps nets have been established in the pathophysiology ofalzheimer™s disease to cause destruction to neural cells and bloodbrain barrier abnormal netosis activity and reactive oxygen species ros response akey element to netosis initiation were observed in stroke patients the degree of neutrophil infiltration net formationcomponent eg cellfreenucleosomes and netosis have been found to correlate with the severity of a range of lung diseases including cystic fibrosis acute lung injury aliacute respiratory distress syndrome ards and lung infection netosis was also shown to be involved in allergic asthma chronic obstructive pulmonarydisease and pulmonary hypertension wherein degree of net formation correlates with disease severity during liver ischaemiareperfusion tolllikereceptordependent net release has been suggested to mediate liver inflammation and injury conversely deficiency in net release was reported indecompensated cirrhotic liver disease and could explain susceptibility to bacterial peritonitis infection in those patients net formation and netosis havefurther been implicated in atherosclerosis and myocardial infarction wherein net was found in thrombi and infarct lesion and correlate with disease severityin rheumatoid arthritis enhanced net release and netosis are observed in synovial tissue rheumatoid nodules and skin whilst proinflammatory cytokinesand autoantibodies further aggravate neutrophil infiltration and netosis neutrophils could also be potently activated by monosodiumurate msu crystals ingout joints and point to a potential role of netnetosis in gout pathogenesis moreover neutrophil activation and net deposition were also observed incolon mucosa of ulcerative colitis excessive neutrophil activation net formation and netosis could also be responsible different pregnancyrelateddisorders including preeclampsia wherein net deposition and netosis in the intervillous space may damage maternal endothelium and impair foetaloxygen exchangenets found in cf lungs the source was believed to befrom necrotic neutrophils but is now considered to besecondary to netosis additionally net productionwas found to be promoted by bacterial infection in cfairways and was defective in clearance of the bacteriapseudomonas aeruginosa nets are also named as a facilitative factor for biofilm formation there is evidencethat surfactant protein d spd responsible foropsonising pathogens and dying cells for clearance byalveolar macrophages is essential for net clearancethrough binding directly to the chromatin within the netsspd levels are decreased in cf patients and the level ofdecrease is proportional to the degree of inflammationthrough accumulation of nets 0crole of neutrophil netosis in an injurylung infection neutrophils migrated into the affectedsite and initiate the cascade of antimicrobial mechanismsincluding net generation to combat microanismsthis happens more readily in the lungs compared with inother tissues with neutrophils found to exist in higherconcentrations in pulmonary vasculature compared withsystemic blood vessels a prominent pathway leadingto net formation in infection is through the interaction oflipopolysaccharide lps with tolllike receptor tlr4found on neutrophils in patients with communityacquired pneumoniacap increased levels of cellfree nucleosomes in serumsamples used as surrogate markers of netosis werefound this was associated with prolonged hospitalisationand a greater 30day allcause mortality this findingsuggests nets could function as a novel marker of prognosis in capacute lung injury and acute respiratory distresssyndrome the degree of neutrophil influx into the lungsand net release during ali and ards positively correlates with disease progression and severity with neutrophil depletion conferring protection in a transfusionrelated ali animal model netosis seems to be akey component of ventilatorinduced lung injury vili as evidenced by detection of citrullinated histone3suggesting that this was a mode of cell death independentfrom apoptosis and necrosis the authors suggestedthat this may be due to increased levels of il1β andhmgb1 which have been both shown to be able to inducenetosisallergic asthma patients with neutrophilic asthmahave a greater severity of disease and reduced response tocorticosteroid treatment compared with the eosinophilictype the increased expression of neutrophilchemoattractant il8 in airway smooth muscle cells couldbe contributing to disease severity through inducingnetosischronic obstructive pulmonary disease netosis hasbeen documented as an integral part of chronic obstructivepulmonary disease copd pathophysiology unlike asthma where neutrophils are important in certain subtypesnetosis has been directly linked to disease severity incopd [ ] tlr4 expression one of the main potentiators for net formation is increased during copd exacerbations pulmonary hypertension nets are also able to potentiate dysregulated angiogenesis as seen in patients withchronic thromboembolic pulmonary hypertension and idiopathic pulmonary hypertension as plasma levels of dnane and mpo levels are significantly elevated moreovernets also seem to destabilise intercellular junctions andincrease endothelial cell motility through direct contactwith endothelial cells nets were found to induce theactivity of the proinflammatory transcription factor nfκbby approximately 3fold moreover nets increase thesurface expression of von willebrand factor and plateletadhesion thereby producing a prothrombotic state kidneyglomerulonephritides nets have been visualised upon immunostaining renal biopsies from patients with sleand antineutrophil cytoplasmic antibodiesassociated vasculitis aav and may be at least partially responsiblefor activating complement pathways resulting in diseaseexacerbations these autoimmune conditions alsoseem to affect the patient™s ability to degrade nets amplifying their deleterious inflammatory effects increscentic glomerulonephritis neutrophilmediated glomerular damage is worsened by addition of extracellularmpo which could have been released during netosis netosis could also contribute to the loss of immunetolerance through further externalisation of crucialautoantigens during cell death haemolytic uraemic syndrome hus plasma from affected patients exhibited a greater capacity to undergonetosis compared with healthy patients the ensuingdamage has been linked to the proinflammatory cytokinesil6 and il8 released from glomerular epithelial cellsupon stimulation by nets this potentiates microvasculature inflammation and thrombosis precipitating renal failure liverdecompensated cirrhotic liver disease a deficiency innet release has been demonstrated to play a role in theonset of endstage liver disease as neutrophils incirrhotic patients are found to have defective ros production which commonly triggers net release thismay also partially explain why these have a predispositionto recurrent bacterial infections and increased rates ofdecompensatory complications such as spontaneous bacterial peritonitis sbp this is corroborated by defectivenet release from ascitic fluid neutrophils in cirrhoticpatients compared with controls in vitro cirrhoticpatients with sbp were also found to have an increase in 0cpro and antiinflammatory cytokines in peripheral bloodand ascitic fluid ischaemiareperfusion injury ischaemiareperfusioninjury iri is an inherent consequence of liver transplantation hypovolaemia or trauma and results in the release ofdamageassociated molecular patterns damps which inturn cause net formation in a tlrdependent mannerexacerbating inflammation treatment with apeptidylargininedeiminase pad4 inhibitor ordnase has been shown to be significantly hepatoprotectivefollowing liver iri cardiovascular systematherosclerosis nets are a wellknown constituent ofatherosclerotic lesions mpo has been strongly associated with diminishing the cardioprotective effects ofhighdensity lipoprotein cholesterol hdlc through oxidation reactions and an increased enzymatic activity islinked to increased plaque rupture other proteinsfound in nets such as cathelinrelated antimicrobial peptide cramp have also been shown to contribute todisease progression moreover in vitro studies showthat hypercholesterolemia triggers net formation alargescale study in patients with suspected coronary arterydisease revealed that the markers of netosis such asextracellular dna are independently associated with disease severity coronary specimens from patients afteran acute myocardial infarction mi showed the presenceof nets in both fresh and lytic thrombi therefore suggesting netosis happens in the early stages of thrombusevolution furthermore net burden was shown tobe positively correlated with the infarct size in patientsundergoing primary percutaneous coronary interventionspostmi this is supported by increased levels of mpodna and ne in the lesion site therefore nets couldpotentially be considered as a novel biomarker in atherosclerosis diabetes mellitusinduced vasculopathy it has beenshown that neutrophils form peripheral blood of diabetesmellitus dm patients showed increased spontaneousnetosis interestingly metformin reduces the deleterious effects of netosis in a mechanism independentlyfrom glucose control one recent study showed that month treatment with metformin in predm patients reduced levels of components of nets whereas glycaemiccontrol with other medication such as insulin saw nodifference when compared with placebo controls thiscahilog zhao wu alam eguchi weng and mahas been attributed to a direct effect of metformin oninhibiting the activation of nadph oxidase musculoskeletal systemrheumatoid arthritis neutrophils from the peripheralblood and synovial fluid of patients with rheumatoid arthritisra exhibit increased netosis compared with healthy controls and patients with osteoarthritis the externalisation ofcitrullinated proteins during the process of netosis wasfound to initiate and perpetuate the aberrant immune responsein ra moreover the autoantibodies and inflammatory cytokines commonly seen in ra promote netosis resulting in avicious cycle of tissue destruction gout gout is an inflammatory disease that involvesthe deposition of monosodiumurate msu crystals injoints during acute gout there is increased movement ofneutrophils into the synovial fluid msu is a known neutrophil stimulus and it has been shown that acute gout isassociated with an increase in il1β levels a keyplayer in net formationgutulcerative colitis there is prominent neutrophil infiltration in the colon mucosa in ulcerative colitis uc and this correlates with disease severity in uc the inflammatory environment promotes neutrophil activation andil1β expression in contrast nets do not seem toplay a key role in crohn™s disease this may explain whymesalazine a known inhibitor of il1β production and thefirstline treatment for uc flareups is not therapeutic incrohn™s patients per se reproductive systempreeclampsia placentas from women diagnosed withpreeclampsia showed increased neutrophil infiltration andnetosis when compared with nonhypertensive pregnantcontrols [ ] and are probably involved in causingwidespread damage to the maternal endothelium placental and endothelial injury during pregnancy aberrantneutrophil activity during pregnancy is also associated withother severe complications including recurrent foetal loss one recent study indicated neutrophils in pregnant womenseem to have an increased propensity to undergo netosissecondary to an increase in granulocyte colonystimulating 0crole of neutrophil netosis in an injuryfactor during pregnancy progesterone has been shown toattenuate neutrophilmediated ros production whereas 17βestradiol induces intracellular ros generation in a dosedependent manner associated an injury associated with netosis fig examples of recent publications on potential therapeutic compounds targeting netosis are summarised in table netosis as a therapeutic targettargeting critical steps in net formation and degradation is critical for developing treatment strategies for netosistlr inhibitorsdexamethasone dex has been shown in vitro toreduce netosis in neutrophils that are stimulated withstaphylococcus aureus but not in those stimulated withpma the tlrs involved in s aureusinduced net formation seem to be tlr2 and tlr4 as agonists of thesereceptors rescued dex inhibition interestingly althoughfig therapeutic strategies targeting net formation stimulation of neutrophil receptors eg fc γ receptor tolllike receptor by microanisms orsterile signals leads to release of calcium ca2 from the endoplasmic reticulum er cellular ca2 overload results in activation of protein kinase c pkcassembly of the nicotinamide adenine dinucleotide phosphate nadph oxidase complex andor mitochondrial activation thus stimulating reactive oxygenspecies ros production oxidative stress promotes myeloperoxidase mpodependent migration of granular neutrophil elastase ne into the nucleus tocleave histones subsequent activation of peptidylarginine deiminase pad induces histone citrullination to cause chromatin decondensation the last stepinvolves nuclear membrane degradation and extrusion of a mixture of chromatin and granular proteins into extracellular space whereby extracellular dnaseeventually digests and removes neutrophil extracellular traps nets in this regard modulation of critical steps in net formation and degradation shown byblocking arrows might be beneficial for the treatment of inflammatory disorders figure modified and reproduced with permission fcγr fc γ receptortlr tolllike receptor 0cdex reduced net formation it did not significantly affectros production calcineurin inhibitorscalcineurin is a calciumdependent serinethreonineprotein phosphatase that is important for neutrophil activity many stages of netosis induction depend upon calcium mobilisation hence modulators of the calcineurinpathway are potential pharmacological inhibitors of netformation cyclosporine a csa an antagonist of thecalcineurin pathway has been shown to reduce the effectof key physiological activators of neutrophils this effecton netosis may in part explain csa™s efficacy in ra and steroidresistant uc patients pmainducednetosis seems to be calciumindependent as this wasnot inhibited by csa cahilog zhao wu alam eguchi weng and mapad inhibitorsusing a murine model of atherosclerosis knight andcolleagues have shown that pharmacological inhibition ofpad4 using weeks of daily clamidine injections reduced netinduced vascular damage with delayed plaqueprogression in the carotid arteries the same groupalso showed that pad inhibitors reduce disease activity inmurine models of sle by reducing endothelial dysfunction it is worth mentioning that the possibility of padinhibition as a therapeutic avenue to be pursued in netinduced an damage in glomerulonephritis has beenrecently challenged by the work of gordon and colleagueson murine models on sle with pad4 deletion theyshowed that this did not reduce endan damage asmeasured by proteinuria suggesting that mechanismsother than net formation are implicated in this complexautoimmune conditionros scavengersdnase therapythe mitochondria are a powerful source of ros ros scavengers such as nacetyl cysteine nac reducenet formation and severity of sle in patients troloxand tempol are two antioxidants which have also beenshown to prevent netosis of pmastimulated humanneutrophils in a dosedependent manner and have beenrecommended for treatment of autoimmune and inflammatory pathologies dnase therapy has been proposed to improve outcomes in cf patients through reducing mucous viscosityhowever it appears that recombinant dnase does notreduce the load of dnaprotein complexes seen innetosis one solution to this is to combine elastase withdnase in order to degrade histones and provide dnaseaccess to chromatin this combination has been shown toenhance solubilisation of sputum drug classstudymain findingstable potential therapeutic approaches targeting netosistlr inhibitorswan t et al calcineurin inhibitorsgupta ak et al dexamethasone reduced netosis in neutrophils stimulated with s aureusagonists of tlr2 and tlr4 rescued dexamethasone inhibitionros production was unaffected by dexamethasonecyclosporine a reduced the effect of key physiological stimuli that activate neutrophilssuch as il8 and suppressed netosisros scavengerspatel et al vorobjeva nv and pinegin bvnacetyl cysteine reduced net formation and severity of sle in patientsantioxidants trolox and tempol prevent netosis of in stimulated human neutrophils in apad inhibitorsknight js et al weeks of daily clamidine injections reduced netinduced vascular damage and area ofdosedependent mannerlesions in a murine model of atherosclerosispad inhibition dampens disease activity by reducing endothelial dysfunction in a murinemodel of slednase therapypapayannopoulos v staab delastase combined with dnase therapy enhances solubilisation of sputum in cystic fibrosistetrahydroisoquinolines martinez ne et al tetrahydroisoquinolines selectively target net overproduction at micromolarzychlinsky a patientsconcentrations possibly at multiple stages of net formation without compromisingnormal neutrophil function 0crole of neutrophil netosis in an injurytetrahydroisoquinolinesin contrary to the aforementioned mechanisms ofnetosis inhibitors tetrahydroisoquinolines thiqs area new class of net formation inhibitors that do not targetros formation or granular protein activity as functionalneutrophils are paramount to maintaining immune reactivity this difference offers an advantage to selectively targetnet overproduction without impairing normal functionthe exact molecular mechanisms of thiqs are yet to bedetermined however it is known that thiq inhibition ofnetosis take place at micromolar concentrations and possibly at different stages of net formation conclusionwhen regulated as part of normal physiology netsare antimicrobial and fundamental to the innate immunesystem dysregulated net formation contributes to thepathogenesis of a plethora of diseases this review hassummarised the role of netosis in pathologies of multiplebody systems as well as highlighted the stages of netosisthat has so far been investigated as emerging pharmacological targets these putative strategies seem to hold therapeutic potential and warrant further investigationauthors™ contributionsdm designed and reviewed the manuscript zc andhz wrote the first draft of the paper all authors readrevised and approved the final manuscriptcompliance with ethical standardscompeting interests the authors declare that they haveno competing interestsethics approval and consent to participate notapplicableconsent for publication not applicableopen access this is licensed under a creativecommons attribution international license whichpermits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicateif changes were made the images or other third partymaterial in this are included in the 's creativecommons licence unless indicated otherwise in a creditline to the material if material is not included in the's creative commons licence and your intended useis not permitted by statutory regulation or exceeds thepermitted use you will need to obtain permission directlyfrom the copyright holder to view a copy of this licencevisit httpcreativecommonslicensesby40references vandenabeele p l galluzzi t vanden berghe and g kroemer molecular mechanisms of necroptosis an ordered cellularexplosion nature reviews molecular cell biology “ httpsdoi101038nrm2970 lewis hd j liddle je coote sj atkinson md barker bdbax kl bicker rp bingham m campbell yh chen cwchung pd craggs rp davis d eberhard g joberty kelind k locke c maller k martinod c patten o polyakovace rise m rüdiger rj sheppard dj slade p thomas jthorpe g yao g drewes dd wagner pr thompson rkprinjha and dm wilson inhibition of pad4 activity issufficient to disrupt mouse and human net formation naturechemical biology “ httpsdoi101038nchembio1735 galluzzi lorenzo ilio vitale stuart a aaronson john m abramsdieter adam patrizia agostinis emad s alnemri et al molecular mechanisms of cell death recommendations of the nomenclature committee on cell death cell death and differentiation “ httpsdoi101038s414180170012 gupta s and mj kaplan the role of neutrophils andnetosis in autoimmune and renal diseases nature reviews nephrology “ httpsdoi101038nrneph201671 papayannopoulos v neutrophil extracellular traps in immunity and disease nature reviews immunology “httpsdoi101038nri2017105 kobayashi sd and fr deleo role of neutrophils ininnate immunity a systems biologylevel approach wiley interdisciplinary reviews systems biology and medicine “httpsdoi101002wsbm32 metzler kd c goosmann a lubojemska a zychlinsky andv papayannopoulos a myeloperoxidasecontaining complex regulates neutrophil elastase release and actin dynamics duringnetosis cell reports “ httpsdoi101016jcelrep201406044 tessarz p and t kouzarides histone core modificationsregulating nucleosome structure and dynamics nature reviewsmolecular cell biology “ httpsdoi101038nrm3890 yousefi s c mihalache e kozlowski i schmid and husimon viable neutrophils release mitochondrial dna toform neutrophil extracellular traps cell death and differentiation “ httpsdoi101038cdd200996 wang haiting ting li sheng chen gu yueying and shuang ye neutrophil extracellular trap mitochondrial dna and its 0cautoantibody in systemic lupus erythematosus and a proofofconcept trial of metformin arthritis rheumatology “ kenny ef a herzig r kruger a muth s mondal prthompson v brinkmann hv bernuth and a zychlinsky diverse stimuli engage different neutrophil extracellular trappathways elife httpsdoi107554elife24437 hakkim a bg furnrohr k amann b laube ua abed vbrinkmann m herrmann re voll and a zychlinsky impairment of neutrophil extracellular trap degradation is associatedwith lupus nephritis proceedings of the national academy of sciences of the united states of america “ httpsdoi101073pnas0909927107 farrera c and b fadeel macrophage clearance of neutrophil extracellular traps is a silent process journal of immunology “ httpsdoi104049jimmunol1300436 maueroder c a mahajan s paulus s gosswein j hahn dkienhofer mh biermann et al 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"When we analyzed the effects conferred by ibuprofen on the translocation of Bax to mitochondria in cisplatin-treated cells we observed an approximately 1.3-fold increase in the amount of translocated Bax (b). To exclude an effect of ibuprofen unrelated to the inhibition of Hsp70 we performed RNAi for a selective knock-down of Hsp70 and we studied its effects on the activation of Bax. Consistent with the earlier data presented for ibuprofen the depletion of Hsp70 increased the activation of Bax in cisplatin-treated cells although its extent was greater with Hsp70 RNAi than with ibuprofen (c). These observations confirmed that (a) ibuprofen promotes the activation of Bax dependent on cisplatin and its translocation to the mitochondria in A549 cells and (b) its mechanism of action is mediated by the inhibition of Hsp70. Ibuprofen facilitates events occurring upstream and downstream of mitochondrial disruption in cisplatin-mediated apoptosis Previous studies have shown that Hsp70 can inhibit apoptosis by acting downstream of the mitochondria.121314 15 Hsp70 interacts directly with Apaf-1 to prevent the formation of cytochrome c-mediated apoptosome and subsequent activation of caspase-9. To examine whether ibuprofen also influences the downstream mitochondrial events we measured its effects on the cleavage of procaspase-9 in the apoptosis mediated by cisplatin. With an anti-active caspase-9 antibody fully processed caspase-9 was predominantly identified in cisplatin-treated A549 cells (a lane 3) over untreated cells (a lanes 1 and 2). It is noteworthy that treatment with ibuprofen increased >4-fold the amount of active caspase-9 in cells treated with cisplatin compared with cells incubated with cisplatin alone (a lane 4). As as reported earlier the highest increases in the activation of Bax and release of cytochrome c by ibuprofen were <2-fold these observations suggest that ibuprofen also facilitates the post mitochondrial process taking place between the release of cytochrome c and the activation of caspase-9. To verify that this is a specific effect we studied the effect of Hsp70 knock-down on the activation of caspase-9 mediated by cisplatin. The caspase-9 activity in cells depleted of Hsp70 with cisplatin was fourfold greater than in control (scrambled) siRNA-treated cells (b). We obtained similar results when we measured the activity of caspase-9 in cells treated with ibuprofen (c) or siRNA against Hsp70 (d) by a fluorometric assay using a synthetic substrate. Overall these observations confirmed unambiguously that ibuprofen intensified the apoptosis induced by cisplatin by its effects on the events occurring downstream of the mitochondria by inhibiting Hsp70 although whether it stimulated the formation of apoptosome (essential for the recruitment of procaspase-9) remains to be determined. We conclude that ibuprofen promotes the apoptosis induced by cisplatin at multiple stages of the mitochondrial cascade by attenuating the expression of Hsp70 in A549 cells. Discussion We found that compared with non-malignant bronchial epithelial cells human lung cancer cells overexpressed Hsp70. This is an important observation as targeting the expression or function of Hsp70 has been suggested as an effective treatment strategy in several cancers based on the hypothesis that higher levels of Hsp70 protect against cell death and increase the survival rate against modalities used in chemotherapy.11 15 In fact it is well documented that the expression of Hsp70 is significantly increased in cancer tissues and/or serums obtained from patients with non-small cell lung cancer (NSCLC)34353637 38 and its overexpression correlates with poor prognosis in NSCLC.36 Several reports have indicated that functionally related small molecules that inhibit Hsp70 decrease the viability of colo-rectal or pancreatic cancer cells by promoting apoptosis via the downregulation of Hsp70 and may be a promising new class of cancer chemotherapeutics.1921 22 We showed that ibuprofen a relatively non-toxic and widely used NSAID significantly decreased the expression of Hsp70 in lung adenocarcinoma cell lines. We also clearly demonstrated that the inhibitory mechanisms of ibuprofen on Hsp70 are due to a decrease in HSF1 expression. Although the fundamental mechanism behind the reduction in HSF-1 expression is unknown a previous study has indicated that the nuclear factor 1 family member NFIX which codes for site-specific DNA-binding proteins known to have multiple roles in replication signal transduction and transcription exerts a transcriptional repressive effect on the expression of HSF1 in cancer cells.39 Whether NFIX is indeed involved in the inhibition of HSF1 expression evoked by ibuprofen is applicable in further studies. To the best of our knowledge this is the first study of the inhibitory effects of NSAID on the cellular expression of Hsp70. In addition we showed that ibuprofen does not influence the cell viability without additional stimuli unlike its maximal effect on the expression of Hsp70. The lack of inhibitory efficacy of ibuprofen against tumours is consistent with a previous study which showed that low-dose ibuprofen did not induce apoptosis in mouse and human colorectal cancer cell lines.29 Similar observations were made following RNAi of Hsp70 suggesting that the attenuation of Hsp70 per se is insufficient to cause the death of A549 and perhaps other cells. It has been shown that the knockdown of Hsp70 has no effect on the viability of several cancer cell lines although sensitized them to anticancer drugs.40 41 Therefore the therapeutic potential of ibuprofen combined with chemotherapeutic agents needs to be explored. Cisplatin is one of most effective chemotherapeutic drugs against NSCLCs.42 It is noteworthy that damage to DNA caused by cisplatin enables apoptosis involving mitochondrial pathways which is negatively regulated by Hsp70. As ibuprofen prominently suppressed the expression of Hsp70 in A549 and H358 cells we examined the possible synergistic activity of ibuprofen and cisplatin against cancer. As expected ibuprofen potentiated synergistically the anti-proliferative effect of cisplatin in A549 and H358 cells. Despite its potent antitumoural properties the therapeutic use of cisplatin in oncology is seriously limited by dose-dependent adverse effects and frequent development of drug resistance.43 Therefore our findings may make useful contributions toward the development of new and less toxic chemotherapy against NSCLCs. We also examined the molecular mechanisms of these synergistic properties of ibuprofen. Hsp70 protects cells against mitochondria-dependent apoptosis at different levels although the precise mechanism remains hypothetical because of regular contradictory descriptions of Hsp70 function. Earlier reports have shown a protective effect of Hsp70 against cellular apoptosis by inhibition of the apoptosome function a protein complex comprising Apaf-1 and cytochrome c.12 13 However recent reports have questioned this repression of apoptosis downstream of the mitochondrial membrane permeabilization. Several studies have suggested that Hsp70 functions upstream of the mitochondria by preventing the release of cytochrome c instead of inhibiting the apoptosome or other downstream points in the caspase cascade.16 17 Some of this confusion may be due to different experimental systems used to evaluate apoptosis or reflects the variability of apoptotic pathways among different cell lines. In this study we found that the inhibition of Hsp70 by ibuprofen facilitates the activation of Bax induced by cisplatin and its translocation to the mitochondria in A549 cells. This finding is consistent with the previous observation of blockade of Bax activation being one of the upstream sites of action of Hsp70. On the other hand the role played by Hsp70 in the A549 cellular mitochondrial apoptotic pathway is likely to be more complex than described earlier because in cisplatin-treated cells the decreased expression of Hsp70 caused by ibuprofen amplified the activation of caspase-9 significantly compared with that of Bax. Furthermore the similar increase in the activation of cisplatin-dependent Bax and release of cytochrome c by ibuprofen suggests that Hsp70 also inhibits the post mitochondrial steps between the release of cytochrome c and the activation of caspase-9. If Hsp70 were acting downstream of the mitochondria one would predict that it interferes with the activation of caspase-9 in response to cytochrome c either by inhibiting the formation of the apoptosome or by preventing the binding of pro-caspase-9 to this complex. When we studied the effects of Hsp70 on the formation of and recruitment of pro-caspase-9 to the apoptosome the cell lysates were immunoprecipitated although Hsp70 failed to migrate with Apaf-1 cytochrome c or caspase-9 (data not shown). These results may be supported by previous report that no association between Hsp70 and Apaf-1 or apoptosome complex was observed even under in vitro activation of caspase by the addition of cytochrome c and dATP.44 Furthermore we were unable to identify a new target for Hsp70 in the process of caspase-9 activation indicating that its inhibitory activity is attributable to another indirect effect instead of a direct one as previously reported. Altogether the data presented here are the first evidence of cell death inhibition by Hsp70 by its targeting of both upstream and downstream mitochondrial processes while the precise mechanisms by which it interferes with the activation of caspase-9 remains to be clarified. In conclusion ibuprofen potentiated the antitumoural properties of cisplatin in the cells of lung adenocarcinoma via a mechanism of action mediated by the suppression of Hsp70. These findings may promote the development of a new strategy to increase the effectiveness of cisplatin in the treatment of NSCLCs as well as highlight the putative merits of developing anticancer treatments targeting Hsp70. Materials and Methods Materials The mouse monoclonal anti-Hsp70 the rat monoclonal anti-Hsc70 and rabbit polyclonal HSF-1 antibodies purchased from Stressgen – Enzo Life Sciences Inc. Plymouth Meeting PA USA. Anti-Bax rabbit polyclonal (N-20) and anti-VDAC-1 goat polyclonal (N-18) antibodies were purchased from Santa Cruz Biotechnology Inc. Santa Cruz CA USA. Anti-cytochrome c mouse monoclonal antibody (556433) was obtained from BD Pharmingen Inc. San Diego CA USA. Anti-caspase 9 and -ERK antibodies were acquired from Cell Signaling Technology Inc. Danvers MA USA. The mouse monoclonal antibody against actin was obtained from Chemicon International Inc. Temecula CA USA. Anti-Bax 6A7 monoclonal antibody and other reagents were purchased from Sigma-Aldrich St. Louis MO USA. Cell culture and viability assay A549 and H358 lung cancer cell lines were cultured in Dulbecco's modified Eagle's medium containing 10% foetal bovine serum at 37?°C. BEAS-2B cells were grown in bronchial epithelial basal medium. All NSAID and cisplatin were dissolved in dimethyl sulphoxide and added to the medium at indicated concentrations. The activity of mitochondrial dehydrogenase 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) assay was used to measure cell death/survival. The reaction product was measured at A570 and the relative viability of cells treated with reagents versus untreated cells was calculated. TUNEL staining The TUNEL assay was performed using an in situ cell death detection kit (F Hoffmann-La Roche Basel Switzerland) according to the manufacturer's instructions. The ratio of TUNEL-positive cells to the total number of cells was calculated. Immunoprecipitation and cell fractionation A549 cells were lysed in RIPA buffer (50?mM Tris-HCl pH 7.5 150?mM NaCl 1?mM sodium orthovanadate 1?mM EDTA 0.1% NP-40 10?mM NaF) containing the Calbiochem Protease Inhibitor Cocktail Set III (Merck KGaA Darmstadt Germany). The cell lysates and immunoprecipitates were resolved in Laemmli sample buffer. The samples underwent sodium dodecyl sulphate-polyacrylamide gel electrophoresis were transferred to a polyvinylidene difluoride membrane reacted with the respective antibodies and detected with an ECL chemiluminescence detection kit (GE Healthcare Fairfield CT USA). For the immunoprecipitation the cell lysates were incubated with the indicated antibodies for 1?h at 4?°C. Protein G-sepharose beads were added to collect the immunocomplexes for an additional 1?h of incubation. The pellets were washed three times with lysis buffer. The mitochondria and cytosol fractions were prepared as described previously.45 Chromatin immunoprecipitation (ChIP) assay ChIP assays were performed as described previously46 using an EZ ChIP kit (Upstate Biotechnology Inc. Waltham MA USA). Briefly after adding formaldehyde A549 cells were suspended in SDS lysis buffer and the chromatin DNA was disrupted by sonication. For the immunoprecipitation the lysate was incubated with anti-HSF-1 antibody followed by immobilization on salmon sperm DNA/Protein G agarose. The protein/DNA complexes extracted with elution buffer were heated to 65?°C for 6?h to reverse cross-links then digested with proteinase K. DNA fragments were amplified in PCR with the ChIP assay primers containing the heat shock element sites in human Hsp70 promoter. PCR primers for the ChIP assay were as follows: Hsp70 (?103/+7) (F) 5?-TGATTGGTCCAAGGAAGGCT-3? and (R) 5?-AAAAAGGTAGTGGACTGTCGC-3?. Reverse transcriptase-PCR RT-PCR was carried out using a Qiagen (Valencia CA USA) One-Step RT-PCR Kit. We used the following primer pairs to amplify. Hsp70 (F) 5?-ATGAAGCACTGGCCTTTCCA-3? (R) 5?-TTGTTCTGGCTGATGTCCTT-3? Hsc70 (F) 5?-TGGAACTATTGCTGGTCTCAA3? (R) 5?-AGAACCACCAACCAGGACAAT-3? HSF-1 (F) 5?-TTCGACCAGGGCCAGTTT-3? (R) 5?-AGAGCTGGCCACAGCATCA-3? actin (F) 5?-AGAGGCATCCTCACCCTGA-3? (R) 5?-CATCTCTTGCTCGAAGTCCA-3?. The products were examined by agarose gel electrophoresis after 23 cycles. RNA interference The sequences of the sense strands used to generate specific siRNA were obtained as follows: HSF-1 5?-AAGTACTTCAAGCACAACAA-3? 5?-AAGAGTGAAGACATAAAGAT-3? 5?-AAGTCGTCAACAAGCTCATT-3?. The siRNAs were synthesized using the Silencer siRNA construction kit (Ambion; Applied Biosystems Inc. Carlsbad CA USA). Double-stranded Hsp70 and control siRNA duplex were synthesized as followed by Qiagen: Hsp70-specific sequence 5?-CCAUUGAGGAGGUAGAUUAdTdT-3?. A549 cells were transfected with each siRNA (10?nmol/l) using the Lipofectamine 2000 (Invitrogen; Applied Biosystems Inc.) and grown for 72?h to allow an effective decrease in the expression of the respective target molecules. Quantification of apoptosis by flow cytometry A549 cells were washed with Annexin V staining buffer (10?mM HEPES pH 7.4 150?mM NaCl 5?mM KCl 1?mM MgCl2 1.8?mM CaCl2) and incubated with CF488A-Annexin V and propidium iodide (Biotium Inc. Hayward CA USA) in staining buffer for 30?min at 37?°C in the dark. Fluorescence was measured using a FACSCalibur (BD Biosciences San Jose CA USA) and the data were analyzed with CellQuest software (BD Biosciences). JC-1 staining and quantification A549 cells were cultured at 37?°C for 48?h on glass chamber slides and treated with ibuprofen and cisplatin at the specified concentrations for 48?h. Mitochondrial permeability transition was determined by staining the cells with 55?66?-tetrachloro-1133?-tetraethyl- benzimidazolylcarbocyanin iodide (JC-1; Molecular Probes Invitrogen Carlsbad CA USA) in the dark. The cells were subsequently washed with assay buffer according to the manufacturer's protocol and immediately imaged using a fluorescence microscope (Keyence Corporation Osaka Japan) with the red (?excitation: 560±40?nm band pass filter ?detection"
1
"primary bone tumors in early adolescence with unsatisfied prognosisAberrant DNA methylation had been demonstrated to be related to tumorigenesis and progression of multiple cancers andcould serve as the potential biomarkers for the prognosis of human cancers In this study identified downregulated hypomethylation genes and upregulated hypomethylation genes in OS by integrating the analysis theGSE97529 and GSE42572 datasets Bioinformatics analysis revealed that OSspecific methylated genes were involved inregulating multiple biological processesincluding chemical synaptic transmission transcription response to drug andregulating immune response KEGG pathway analysis showed that OSspecific methylated genes were associated with theregulation of Hippo cAMP calcium MAPK and Wnt signaling pathways By analyzing R2 datasets this study showed that thedysregulation of these OSspecific methylated genes was associated with the metastasisfree survival time in patients with OSincluding CBLN4 ANKMY1 BZW1 KRTCAP3 GZMB KRTDAP LY9 PFKFB2 PTPN22 and CLDN7 This study provideda better understanding of the molecular mechanisms underlying the progression and OS and novel biomarkers for the prognosisof OS IntroductionOsteosarcoma OS is one of the most common types of primary bone tumors in early adolescence which was characterized by an aggressive osteolytic or osteoblastic appearancewith a periosteal reaction [] Chemotherapy and surgeryare the most important treatments for patients with OS [] The survival rate of primary OS patients after treatmentsremains at “ [] However the prognosis of patientswith progressive or recurrent OS was less than [] Inthe past decades emerging studies reported that multiple factors are associated with the tumorigenesis and progression ofOS including germline genetic variants [] dysregulation ofoncogenes or tumor suppressors [] and the abnormal epigenetics change [ ] A few proteins had been revealed tobe related to the progression of OS For example GFRA1was reported to promote autophagy and cisplatininducedchemoresistance in OS [] The isoform of TMIGD3 suppressed OS progression though downregulating NFκB []Understanding the mechanisms related to OS developmentcould provide new targets for OSDNA methylation could aï¬ectthe gene expressionthough suppressing transcription [] Aberrant DNA methylation had been demonstrated to be involved in regulatingtumorigenesis and progression of multiple cancers [ ]In OS DNA methylationmediated suppression of miR449c could promote cell cycle though inhibiting cMyc inOS [] Hypomethylation of IRX1 was found to promote 0cComputational and Mathematical Methods in Medicineamocras laivonySamocras laivonySamocras laivonySamocras laivonySamocras laivonySamocras laivonySamocras laivonySamocras laivonySamocras laivonySamocras laivonySamocras laivonySamocras gnwEamocras gnwEamocras gnwEamocras gnwEamocras gnwEamocras gnwEamocras gnwEamocras gnwEamocras gnwEamocras gnwEiiiiiiiiiiamocrasoetsamocrasoetsamocrasoetsamocrasoetsamocrasoetsamocrasoetsamocrasoetsamocrasoetsamocrasoetsamocrasoetsamocrasoetsamocrasoetsamocrasoetsamocrasoetsamocrasoetsOOOOOOOOOOOOOOO“““Figure OSspecific methylated genes were identified by using the public dataset GSE97529 a DNA methylation status of CpGsites in Ewing™s sarcoma synovial sarcoma and OS samples were included in this datasetabOS metastasis by activating CXCL14NFκB signaling []Very interestingly recent studies showed that aberrantDNA methylation was associated with the prognosis of OSFor example the DNA methylation level of WNT6 was negatively correlated to the prognosis of children with osteosarcoma [] The hypermethylation of ESR1 was correlated tothe worse overall survival of OS [] These results suggestedthat the DNA methylation status could be potential diagnostic and therapeutic targets for OSThe present study analyzed the GSE97529 [] dataset toidentify OSspecific methylated genes In silico analyses wereperformed to explore the functions of OSspecific methylatedgenes Next the GSE42572 dataset was used to validate theexpression levels of OSspecific methylated genes [] Ofnote we found that these OSspecific methylated genes werecorrelated to the prognosis of patients with OS By thesemethods it is hopeful that novel aberrant methylation genesand pathways will be screened in the OS and an understanding ofthe underlying molecular mechanisms will beenhanced Materials and Methods Microarray Data The present study is aimed at identifying dysregulated OSspecific methylated genes in OS by analyzing public databases with bioinformatics analysis Thuswe screened the GEO databases The candidate databaseswere selected according to standards the candidatedatabase should contain clinical OS samples the numberof clinical samples should be more than cases and thecandidate database was not noncoding RNA datasets Finallyonly the SE97529 and GSE42572 datasets were selected forfurther analysis We have included this information in Materials and Methods The GSE97529 dataset was used to identify OSspecific methylated genes which was downloadedfrom the NCBI GEO database GSE97529 A total of Ewing™s sarcoma synovial sarcoma and OS sampleswere included in this dataset The GSE42572 dataset was analyzed to identify diï¬erently expressed genes in OS comparedto normal samples which was also downloaded from theNCBI GEO database GSE42572 Diï¬erentially expressedgenes DEGs and diï¬erentially methylated genes DMGswere identified by applying GEO2R P and ˆ£foldchange ˆ£ ‰¥ is set as the cutoï¬ criterion Functional and Pathway Enrichment Analyses TheDAVID system was used to predict the potential biologicalprocesses and KEGG pathways involved in target genes inthis study [] P was set as the cutoï¬ criterion ProteinProtein Interaction PPI Network Analysis Inthe present study PPI networks were used to reveal the interactions among diï¬erentially expressed OSspecific methylated genes using the STRING database stringdb PPI was visualized using Cytoscape [] Survival Analysis Survival analysis was performed usingthe OS microarray dataset mixed osteosarcoma mesenchymalKuijjer127vstilmnhwg6v2 from the R2 GenomicsAnalysis and Visualization Platform httpr2amcnl Themedian expression of targets was selected as the cutoï¬ todivide all OS samples into the high or low group 0cComputational and Mathematical Methods in MedicineTranscription from RNA polymerase II promoterNervous system developmentResponse to drugChemical synaptic transmissionCell migrationPotassium ion transmembrane transportNeuron differentiationMovement of cell or subcellular componentAnteriorposterior pattern specificationSkeletal muscle cell differentiationRegulation of synaptic plasticityLongterm memoryHindbrain developmentGland developmentQ valueCountaSignal transductionGprotein coupled receptor signaling pathwayImmune responseInflammatory responseDetection of chemical stimulusDefense response to bacteriumSodium ion transportPositive regulation of PI3K signalingMonocyte chemotaxisAcutephase responseDefense response to fungusCountbPathways in cancerHTLVI infectionMAPK signaling pathwaycAMP signaling pathwayHippo signaling pathwayRap1 signaling pathwayCalcium signaling pathwayTranscriptional misregulation in cancerWnt signaling pathwayEpsteinˆ’Barr virus infectionMelanogenesisColorectal cancerEndometrial cancerBasal cell carcinomaCocaine addictionType I diabetes mellitusThyroid cancerQ valueQ valueCountcFigure Continued 0cComputational and Mathematical Methods in MedicineOlfactory transductionCytokineˆ’cytokine receptor interactionPhagosomeCell adhesion molecules CAMsStaphylococcus aureus infectionComplement and coagulation cascadesInflammatory bowel disease IBDHematopoietic cell lineageBile secretionArrhythmogenic right ventricular cardiomyopathy ARVCViral myocarditisAutoimmune thyroid diseaseType I diabetes mellitusFat digestion and absorptionAllograft rejectionEther lipid metabolismGraftˆ’versusˆ’host diseaseAsthmaFatty acid biosynthesisQ valueCountdFigure Bioinformatics analysis of hypermethylation genes and hypomethylation genes a GO analysis of OSspecific hypermethylationgenes b GO analysis of OSspecific hypomethylation genes c KEGG pathway analysis of OSspecific hypermethylation genes dKEGG pathway analysis of OSspecific hypomethylation genes The gene ratio was present in the Xaxis Results Identification of OSSpecific Methylated Genes The public dataset GSE97529 was used to identify OSspecific methylated genes DNA methylation status of CpG sitesin Ewing™s sarcoma synovial sarcoma and OS samples were included in this dataset Figure 1a Totally weidentified OSspecific methylated genes including hypermethylation genes and hypomethylation genesin OS samples compared to Ewing™s sarcoma or synovial sarcoma samples Figure 1a GO and KEGG Pathway Enrichment Analyses GO analysis showed that hypermethylation genes were significantlyassociated with biological processes BP of the nervous system development chemical synaptic transmission transcription from RNA polymerase II promoter anteriorposteriorpattern specification regulation of synaptic plasticity neurondiï¬erentiation movement of cell or subcellular componentskeletal muscle cell diï¬erentiation response to drug potassium ion transmembrane transport hindbrain developmentgland development and cell migration Figure 2a Hypomethylation genes were significantly related to immuneresponseinflammatory responseacutephase response sodium ion transport monocyte chemotaxis detection of chemical stimulus defense responseto fungus positive regulation of PI3K pathway cell chemotaxis chemotaxis neutrophil chemotaxis innate immuneresponse ion transmembrane transport and cell adhesionFigure 2btransductionsignalKEGG pathway analysis showed that significant pathways of hypermethylation genes in OS included the Hippopathway cAMP signaling thyroid cancer pathways in cancer calcium signaling endometrial cancer Rap1 signalingpathway transcriptional misregulation in cancer MAPK signaling pathway EpsteinBarr virus infection Wnt signalingpathway cocaine addiction and basal cell carcinomaFigure 2c And hypomethylation genes in OS were associated with Staphylococcus aureus infection olfactory transductioninflammatory bowel disease IBD complementand coagulation cascades allograft rejection fat digestionand absorption graftversushost disease phagosome viralmyocarditis and fatty acid biosynthesis Figure 2d OSSpecific Methylated Genes Were Diï¬erentiallyExpressed in OS Subsequently an independent public dataset GSE42572 was used to identify diï¬erentially expressedgenes in OS As shown in Figure 3a we identified upregulated genes and downregulated genes in OS compared to healthy control samples Figure 3a AmongDEGs a total of downregulated hypomethylation geneswere screened out from overlapping hypermethylationand downregulated genes while upregulated hypomethylation genes were screened out from overlapping hypomethylationgenesFigure 3b The diï¬erentially expressed OSspecificmethylated genes were presented by heat map Figure 3cdownregulatedand Construction of PPI Network to Identify HubDiï¬erentially Expressed OSSpecific Methylated Genes Furthermore we constructed a PPI network to identify a hub differentially expressed OSspecific methylated gene using theSTRING database As presented in Figure a total of nodes and edges were included in this network The hubgenes included NPSR1 PTAFR LPAR5 PTGER3 NPY5RKCNK3 KRTDAP HCN4 KRT38 KCNIP2 KCNJ5 andKRTCAP3 Figure The Survival Time Analysis of Diï¬erentially ExpressedOSSpecific Methylated Genes The above analysis was conducted with the GSE97529 and GSE42572 datasets Unfortunately the clinical information about metastasisfree survivaltime was not included in both databases Thus we analyzedan independent database R2 dataset httpr2amcnl to 0cComputational and Mathematical Methods in MedicineDownregulatedLow methylatedHigh methylatedUpregulated“““aFigure Continuedb 0cComputational and Mathematical Methods in MedicineCAMK2AKIAA1211CLDN7CECR1DCCCALCBLY9KLHL32FCRL3KRT38PIK3AP1CR2IFT122KCNJ5SV2CHPSE2NPY5RPTAFRGAS2IL22PCSK6PPP2R2BAQP9PCDH21GADL1ARHGAP9MPP2LILRA4MDM2KRTDAPEPHA10KCNIP2PTGER3C10orf55NOS1NETO1NPSR1MARCOGZMBLPAR5ARSIKCNK3SAMSN1ACSM1FUT6PTPN22BREMRPL39OR2J2PCDHA12PFKFB2STAB2ZAKHCN4NR2E3IQSEC3CBLN4PCDHGB5RHOBTB1RAB3DBZW1ANKMY1MACF1C19orf12PRXCCDC149PLEC1KRTCAP3MEOX1GJA4RHOBTB11ˆ’ˆ’CSM tneitap amocrasoetsOCSM tneitap amocrasoetsOCSM tneitap amocrasoetsOCSM tneitap amocrasoetsOCSM tneitap amocrasoetsOCSM tneitap amocrasoetsOCSM tneitap amocrasoetsOcCSM ronodyhtlaeHCSM ronodyhtlaeHCSM ronodyhtlaeHCSM ronodyhtlaeHCSM ronodyhtlaeHFigure GSE42572 was analyzed to identify diï¬erently expressed genes in OS compared to normal samples a induced genes and reduced genes in OS compared to healthy control samples b Among DEGs a total of downregulated hypomethylation genes and upregulated hypomethylation genes were screened out c The diï¬erentially expressed OSspecific methylated genes were presented byheat mapfurther evaluate the prognostic value of OSspecific methylated genes The median expression of candidates in all OSsamples was selectedAs the cutoï¬ is used to divide OS samples into the highand low groups it was shown that higher expression ofCBLN4 P was associated with longer metastasisfree survival time in patients with OS as well as ANKMY1P BZW1 P and KRTCAP3 P However higher expression of GZMB P KRTDAPP LY9 P PFKFB2 P PTPN22P and CLDN7 P was associated withshorter metastasisfree survival time in patients with OSFigure DiscussionThe mechanisms underlying OS progression remainedlargely unclear It has been widely accepted that DNA 0cComputational and Mathematical Methods in MedicineIFT122STAB2FCRL3MARCOCALCBGAS2PLECNPSR1PTPN22LY9NPY5RPTGER3LPAR5PTAFRMPP2NOS12AMDM2RHOBTB1ARHGAP9KRTDAPKRT38KCNK3KCNIP2HCN4KRTCAP3KCNJ5GZMBIL22Figure PPI network analysis PPI network was used to identify a hub diï¬erentially expressed OSspecific methylated gene using theSTRING databasemethylation was involved in regulating the tumorigenesisand developmentthough modulating gene expressionDNA methylation has been shown to play an important rolein gene regulation and implicated in various types of cancerEmerging studies revealed that the cancerspecific CpGhypermethylation could turn oï¬ the expression of tumorsuppressors however cancerspecific CpG hypomethylationcould activate the expression of oncogenes [] Identification of aberrantly methylated genes in OS would be helpfulto identify new diagnostic and therapeutic biomarkers forOS The present study identified OSspecific methylatedgenes from Ewing™s sarcoma or synovial sarcoma samplesBioinformatics analysis revealed that OSspecific methylatedgenes were involved in regulating multiple biological processes including chemical synaptic transmission transcription response to drug and regulating immune responseFurther validation indicated that OSspecific methylatedgenes were dysregulated in OS samples and correlated tothe prognosis of patients with OSOS together with Ewing™s Sarcoma EWS and synovialsarcoma SS was the most common pediatric sarcomas[] These types of sarcomas occur in similar anatomicallocations however the treatments for these sarcomas differed depending on the tumor type The accurate diagnosisof OS remained to be a big challenge Emerging studies demonstrated that aberrant DNA methylation was associatedwith the prognosis of human cancers including OS Forexample DNA methylation level of WNT6 and ESR1 wasrelated to the prognosis of OS The present study is aimedat identifying OSspecific methylated genes A total of OSspecific methylated genes were identified including hypermethylation genes and hypomethylation genesin OS samples compared to Ewing™s sarcoma or synovial sarcoma samples Furthermore GO and KEGG pathway analyses were further used to predict the potential roles of OSspecific methylated genes Of note our predictions showedthat these methylated genes were associated with the Hipposignaling and Wnt signaling Hippo pathway aberrationshad been demonstrated in OS by multiple studies andinvolved in regulating primary tumor growth angiogenesisepithelial to mesenchymal transition and metastatic dissemination [] The Hippo signaling played an important rolecontrolling cancer cell proliferation and apoptosis [] Multiple studies indicated YAP was overexpressed in OS samplesand knockdown of YAP significantly inhibits OS cell growthand invasion [] Sox2 as a YAP upstream regulator wasreported to be required for tumor development and cancercell proliferation in OS [] This study provided a potentialmechanism to elucidate how the Hippo signaling activated inOS Many studies support an aberrant activation of thecanonical Wnt signaling pathway in osteosarcoma cells Forexample two recent studies described a high βcatenin levelin osteosarcoma tissues compared to adjacent healthy tissuesassociated with poor prognosisand lung metastatic 0cComputational and Mathematical Methods in Medicine lavivrus eerfsisatsateM lavivrus eerfsisatsateMRaw P Bonf P Time monthsCBLN4 highCBLN4 lowaRaw P Bonf P Time monthsGZMB highGZMB lowd lavivrus eerfsisatsateM lavivrus eerfsisatsateM lavivrus eerfsisatsateM lavivrus eerfsisatsateMRaw P Bonf P Time monthsANKMY1 highANKMY1 lowbRaw P 62e03Bonf P Time monthsKRTCAP3 highKRTCAP3 loweFigure ContinuedRaw P Bonf P Time monthsBZW1 highBZW1 lowcRaw P Bonf P Time monthsKRTDAP highKRTDAP lowf 0cComputational and Mathematical Methods in Medicine lavivrus eerfsisatsateM lavivrus eerfsisatsateM lavivrus eerfsisatsateM lavivrus eerfsisatsateMRaw P 71e03Bonf P Time monthsLY9 highLY9 lowgRaw P Bonf P Time monthsPTPN22 highPTPN22 lowiRaw P Bonf P Time monthsPFKFB2 highPFKFB2 lowhRaw P Bonf P Time monthsCLDN7 highCLDN7 lowjFigure The prognostic values of diï¬erentially expressed OSspecific methylated genes were calculated by using the R2 Genomics Analysisand Visualization Platform a“j Higher expression of CBLN4 a was associated with longer metastasisfree survival time in patients withOS as well as ANKMY1 b BZW1 c and KRTCAP3 e However higher expression of GZMB d KRTDAP f LY9 g PFKFB2h PTPN22 i and CLDN7 j was associated with shorter metastasisfree survival time in patients with OSdissemination Wnt signaling pathway played a crucial rolein tumorigenicity and metastasis via regulation oftheimmune system bone remodeling angiogenesis hypoxiaresponse and EMT []Of note this study showed that OSspecific methylatedgenes were significantly diï¬erentially expressed in OS samples A total of downregulated hypomethylation genesand upregulated hypomethylation genes were identifiedin this study PPI network analysis was constructed to revealthe relation among these genes Totally nodes and edges were included in this network By analyzing R2 datasets we found the dysregulation of these OSspecific methylated genes were associated with the metastasisfree survivaltime in patients with OSincluding CBLN4 ANKMY1BZW1 KRTCAP3 GZMB KRTDAP LY9 PFKFB2PTPN22 and CLDN7 Among these regulators BZW1 is atranscription factor related to the regulation of cell cycleand proliferation [] LY9 was a member of SLAM familyof immunomodulatory receptors [] and interacted withthe adaptor molecule signaling lymphocyte activationmoleculeassociated proteins A previous study showed LY9was related to the cancer progression and correlated to overall survival of the patients with breast cancer PFKFB2 is anenzyme involved in regulating the Warburg eï¬ect alsotermed as glycolysis [] PFKFB2 had been found to havea key role in regulating tumor growth and survival in multiple cancer types including gastric cancer gliomas and osteosarcoma [“]Several limitations were also exited in this study Firstour studies revealed several hub OSspecific methylatedgenes However the roles of these genes remained to beunclear The gain or loss of function assays should be performed to further explore their roles in OS Next the expression levels and methylation levels of hub OSspecificmethylated genes in OS samples should be confirmed usingclinical samples Third the direct interaction among thesehub genes has not been confirmed using experimental assays ConclusionIn this study identified downregulated hypomethylation genes and upregulated hypomethylationgenes in OS and a series biological processes and pathwaysregulated by aberrantly methylated genes PPI network analysis revealed the interactions among these genes Moreoverthe present study showed that the dysregulation of OSspecific methylated genes wascorrelated with themetastasisfree time in patients with OS including CBLN4 0cComputational and Mathematical Methods in MedicineANKMY1 BZW1 KRTCAP3 GZMB KRTDAP LY9PFKFB2 PTPN22 and CLDN7 This study provided a betterunderstanding of the molecular mechanisms underlying theprogression and OS and novel biomarkers for the prognosisof OSData AvailabilityThe data used to support the findings of this study are available from the corresponding author upon requestConflicts of InterestThe authors declare that they have no conflicts of interestAuthors™ ContributionsFei Wang and Guoqing Qin are cofirst authorsAcknowledgmentsThis work was supported by the Special fund for the key laboratory of Science and Technology Department of Jilin Province 20190201282JCReferences[] E Simpson and H L Brown œUnderstanding osteosarcomasJAAPA vol no pp “ [] G Bacci P Picci P Ruggieri œPrimary chemotherapyand delayed surgery neoadjuvant chemotherapy for osteosarcoma of the extremities The Istituto Rizzoli experience in patients treated preoperatively with intravenous methotrexate high versus moderate doses and intraarterial cisplatin Cancer vol no pp “ [] A Luetke P A Meyers I Lewis and H Juergens œOsteosarcoma treatment where do we stand A state of the art reviewCancer Treatment Reviews vol no pp “ [] P Picci œOsteosarcoma osteogenic sarcoma OrphanetJournal of Rare Diseases vol no p [] W Wang H 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œSarcomas of soft tissueand bone Progress in Tumor Research vol pp “[] S Morice G Danieau F Redini B BrounaisLeRoyer andF Verrecchia œHippoYap signaling pathway a promisingin bone paediatric cancers Cancerstherapeutic targetBasel vol no p [] S L Teoh and S Das œThe emerging role of the hippo pathway in lung cancers clinical implications Current Drug Targets vol no pp “ [] H Wang Y C Du X J Zhou H Liu and S C Tang œThedual functions of yap1 to promote and inhibit cell growth inhuman malignancy Cancer Metastasis Reviews vol no pp “ 0cComputational and Mathematical Methods in Medicine[] Y A Chen C Y Lu T Y Cheng S H Pan H F Chen andN S Chang œWw domaincontaining proteins yap and Taz inthe hippo pathway as key regulators in stemness maintenancetissue homeostasis and tumorigenesis Frontiers in Oncologyvol p [] P McQueen S Ghaï¬ar Y Guo E M Rubin X Zi and B HHoang œThe Wnt signaling pathway implications for therapyin osteosarcoma Expert Review of Anticancer Therapyvol no pp “ [] S Li Z Chai Y Li œBzw1 a novel proliferation regulatorthat promotes growth of salivary muocepodermoid carcinoma Cancer Letters vol no pp “ [] A Angulo M Cuenca P MartinezVicente and P EngelœViral Cd229 Ly9 homologs as new manipulators of hostimmunity Journal of Leukocyte Biology vol no pp “ [] S C Ozcan A Sarioglu T H Altunok œPfkfb2 regulatesglycolysis and proliferation in pancreatic cancer cells Molecular and Cellular Biochemistry vol no pp “[] Q Cheng and L Wang œLncRNA XIST serves as a ceRNA toregulate the expression of ASF1a BRWD1M and PFKFB2 inkidney transplant acute kidney injury via sponging hsamiR2123p and hsamiR1225p Cell Cycle vol no pp “ [] H Liu K Chen L Wang œmiR613 inhibits Warburgeï¬ect in gastric cancer by targeting PFKFB2 Biochemicaland Biophysical Research Communications vol no pp “ [] M Camargo BarrosFilho L Barreto Menezes de LimaM Bisarro dos Reis œPfkfb2 promoter hypomethylationas recurrence predictive marker in welldiï¬erentiated thyroidcarcinomas 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as one of the most common gynecological malignant tumors cervical cancer is the fourth leadingcause of cancerrelated death among women worldwide although eï¬orts including periodiccancer screening prompt surgical treatment chemotherapy and radiotherapy have been madeto decrease the mortality of cervical cancer the prognosis of patients is still poor and cervicalcancer remains an important public health issue the pathogenesis of cervical cancer has notbeen clearly illustrated but it is confirmed that the activation of tumorpromoting genes and theinactivation of tumor suppressor genes participate in the progression of cervical cancer toscreen for novel abnormally expressed genes functioning in cervical cancer may provide potentialprognostic markers and therapeutic targets for treatmentedited byihab youniscarnegie mellon university inqatar qatarreviewed byweifeng dingnantong university chinamassimo brogginimario negri pharmacologicalresearch institute irccs italycorrespondencelin xuxulin83njmueducnemei lulem13705179888sinacnbinhui renrobbishren163com these authors have contributedequally to this workspecialty sectionthis was submitted tocancer geneticsa section of the frontiers in oncologyreceived april accepted june published august citationzhu b wu y luo j zhang qhuang j li q xu l lu e and ren b mnx1 promotes malignantprogression of cervical cancer viarepressing the transcription ofp21cip1 front oncol 103389fonc202001307frontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancermnx1 motor neuron and pancreas homeobox also knownas hb9 hlxb9 is a member of homeobox gene family andencodes a nuclear protein the homeobox genes are agroup of genes containing homeobox a base pairs longdna sequence and encode homeodomain proteins that actas transcription factors many homeobox genes have beenproved to be implicated in various human cancers acting asoncogenes or tumor suppressors “ mnx1 was firstly foundto be expressed in lymphoid and pancreatic tissues and definedas a novel human homeobox gene in early studiesshowed that mnx1 was involved invertebrate and pancreaticdevelopment and motor neuronal diï¬erentiation defects in this gene result in currarino syndrome an autosomicdominant congenital malformation in followup studymnx1 was found to be abnormally expressed in several cancertypes including prostate cancer hepatocellular carcinoma andacute myeloid leukemia “ furthermore recent studiesconfirmed that mnx1 played oncogenic roles in colorectalcancer breast cancer and bladder cancer “the aim of this study is to identify the expression andfunction of mnx1 in cervical cancer our results revealedthat mnx1 was significantly upregulated in cervical cancerand correlated with poorer prognosis the knockdown oroverexpressed mnx1 inhibited or promoted aggressiveness ofcervical cancer including proliferation migration and invasioncapacities by enhancing or repressing the transcription of p21cip1thus regulating the g2m cell cycle transition these findingssuggested that mnx1 might be a potential diagnostic marker andtherapeutic target for cervical cancermaterials and methodsbioinformaticsthe tcga dataset termed tcga_cesc_exp_hiseqv22015 was downloaded from the ucsc cancer browser genomecancerucscedu to evaluate the expression ofmnx1 in cervical cancer and adjacent normal tissues gepiagene expression profiling interactive analysis httpgepiacancerpku cnindexhtml was used to analyze the expressionof mnx1 with disease free survival dfs of cervical cancerpatients the cbioportal website httpwww cbioportal was utilized to obtain highly coexpressed genes with mnx1totally genes highly correlated with mnx1 pearson score table s1 were submitted to david bioinformaticsresources httpdavidabccncifcrfgov for geneontology go kyoto encyclopedia of genes and genomeskegg and reactome pathway analysis and we analyzed thebinding site of mnx1 and p21cip promoters through the jaspardatabase httpjaspardevgeneregnet human cervical cancer cell linesthe human normal cervical celllines hacat and cervicalcancer cell lines hela siha caski and c33a were purchasedfrom american type culture collection atcc usa helasiha c33a and hacat cells were incubated in dmem mediumkeygen nanjing china and caski cells were cultured inrpmi1640 keygen nanjing china medium containing fetal bovine serum gibcobrl invitrogen carlsbad causa and cultured at —¦c in a humidified incubator containing co2human cervical cancer tissuesthe pairs of cervical cancer tissues and adjacent tissues wereselected from the affiliated cancer hospital of nanjing medicaluniversity and informed consent was obtained from all subjectsall tumors and paired nontumor tissues were confirmed byexperienced pathologists and no patients received chemotherapyor radiotherapy before surgery the mrna expression ofmnx1 and p21cip1 in cervical cancer tissues was detected byqrtpcr collection of human tissue samples was conductedin accordance with the international ethical guidelines forbiomedical research involving human subjects cioms thisstudy was approved by the ethics committee of the nanjingmedical university affiliated cancer hospitaltissue microarrayspaired cervical cancer tissue microarrays were obtained fromshanghai outdo biotech co ltd cat no odctrputr03 and odctrputr03006 totally pairs of paraffinembedded human cervical cancer sections were analyzed formnx1 expression all tissues were examined by two experiencedpathologists and the tnm stage was confirmed in each patientwith blinded methods the sections were incubated with an antimnx1 primary antibody abcam ab79541 the ihcscores were calculated according to intensity and percentage ofpositive cells the staining intensity was evaluated as the basis offour grades negative staining 1weak staining moderatestaining or strong staining the product percentage ofpositive cells and respective intensity scores was used as the finalstaining scores a minimum value of and a maximum valueof rna preparation reverse transcriptionand qrtpcrtrizol reagent invitrogen carlsbad ca usa was used toextract total rna from tissue samples or cultured cells accordingto the manufacturer™s protocol a reverse transcription kittakara cat rr036a keygen was utilized to generate cdnaqrtpcr was performed with sybr select master mix appliedbiosystemscat keygen nanjing china and primersare shown in table s2western blottinglysis buï¬er ripa keygen containing protease inhibitorspmsf keygen was used to extract protein of cells andtissues and protein concentration was detected with a bcakit keygen protein samples µg were loaded into sodium dodecyl sulfate polyacrylamide electrophoresissdspage gels and transferred onto a pvdf membraneafter electrophoresis the membrane was blocked with nonfatmilk for h and incubated overnight with antibodies againstrespective antibodies mnx1 abcam ab79541 p21cip1cell signaling technology pthr161cdk1 cellsignaling technology cdk1 cell signalingfrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancertechnology p27kip1 cell signaling technology cyclinb1 abcam ab72 cycline1abcam ab3927 cycline1 abcam ab3927 cyclind1santa cruz biotechnology sc246 actinabcam ab15265 sirna and plasmid transfectionthe sirnas targeting mnx1 and p21cip1 were conductedand purchased from ribobio guangzhou china all sirnasequences are shown in table s3 the fulllength cdnaof human mnx1 were pcramplified and cloned intothe expression vector pgpu6gfpneo vigene biosciencesshandong china the sirnas and overexpression plasmidswere transfected into cervical cancer cells according to thelipofectamine reagent invitrogen carlsbad ca usaprotocol nonsense rnai sinc and empty plasmids oencwas used as negative controls²analysiscell proliferation assaythe cell proliferation assays were performed h aftertransfection for real timexcelligencesystemrtca cells100 µl were seeded in eplates and theplates were locked into the rtca dp device in the incubator tocalculate the œcell index value in colony formation assay a totalof cells were placed in afresh 6wellplate and the cells werestained with crystal violet solution after “ days for5ethynyl2deoxyuridine edu assay keyfluor488 clickitedu kit ribobio guangzhou china the transfected cells wereplaced in 96wellplates cellswell overnight in a co2incubator then cells were incubated with µlwell of µmedu for h at —¦c and fixed with µl paraformaldehydecontaining pbs for min at room temperature subsequentlythe cells were cultured for min with µl of mgmlglycine and then washed with µl bsa in pbs afterpermeabilization with triton x100 for min the cellswere cultured with × clickit reaction solution for minat room temperature in dark conditions after that cells wereincubated with µlwell of × hoechst solutionsfor min at room temperature in the dark after washing with µl of pbs the cells were then imaged using fluorescencemicroscopy and proliferation cell ratios were counted fromfive random fields in every well each experiment was repeatedthree times a total of cells in a fresh sixwellplates weremaintained in medium containing fbs the medium wasreplaced every or days after weeks the cells were fixedwith paraformaldehyde and stained with crystal violeteach experiment was repeated three timesmigration and invasion assayfor wound healing assay cells were growing on the 6wellplate then artificial scratch on a confluent monolayer of cellswas created with a µl pipette tip the medium wasreplaced with the serumfree and cells imaged h later fortranswell and matrigel assay totally transfected cells wereadded to the upper chamber of transwell assay inserts µmpet 24well millicell or a matrigel coated membrane bdbiosciences containing µl serumfree dmem media thelower chambers were filled with µl dmem media containing fbs after a 24h migration assay or 48h invasionassay incubation the cells were fixed with polyformaldehydestained with crystal violet and imaged migration and invasionwere assessed by counting cell nuclei from five random fields onevery filter each experiment was repeated three times rtcawas also used to evaluated the ability of migration and invasioncimplates installation and baseline measurement was carriedout according to the instructions add µl of mixed serumfree cell suspension × cells to the upper chamber in cimplates and the plates were locked into the rtca dp device in theincubator to calculate the œcell index valuecell cycle analysiscells were digested with trypsinedta and fixed with ethanol for h at —¦c the ethanolsuspended cells werecentrifuged and stained with pi staining solution for minin the dark at —¦c a facscalibur flow cytometer was usedto detect cell cycle distribution the percentage of the cells ing0“g1 s and g2“m were counted and comparedchromatin immunoprecipitation chipcells were crosslinked in paraformaldehyde and the reactionwas quenched with glycine after two washes with cold pbscells were added with precooling pbs containing cocktailhalttm protease inhibitor cocktail thermo scientific and scraped into a centrifuge tube the cells were centrifugedfor min at g at —¦c then added with µl celllysis buï¬er containing µl cocktail and incubated onice for min cells were then centrifuged for min at × g —¦c and cell precipitates were resuspended in µlnucleus lysis buï¬er containing µl cocktail the cellswere sonicated amplitude on ice for min and solublechromatin was obtained by centrifuging for min at g at—¦c five micrograms of antimnx1 antibody sigmaaldrichsab2101494 coupled to magnetic beads resin m2 sigmashanghai china was used to immunoprecipitate the dnaprotein complex and the igg antibody was used as a negativecontrol the immunoprecipitation products were washed with µl low salt buï¬er high salt buï¬er lici buï¬er and tebuï¬er successively all for min at —¦c the chip elution buï¬ercontaining proteinase k was used for dna purification thebeads were wiped out on a magnetic frame and the dna waseluted with elution buï¬er c from adsorption column chipdna samples were subjected to pcr amplification with primersspecific to p21cip1 promoter region pcr products were then usedfor agarose gel electrophoresis the sequence of primers used areshown in table s4 and gapdh was used as a controlluciferase reporter assaythe p21cip1 cdkn1a promoter region ˆ’ bp wasamplified and cloned into luciferase reporter plasmid pgl3basic the p21cip1 promoter wildtype plasmids or mutanttype plasmids were cotransfected with cmvmnx1 expressionplasmids in hek293t cells and cmvempty vectors were usedas a negative control relative luciferase activity was corrected forfrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerrenilla luciferase activity of pgl3basic and normalized to theactivity of the controlxenograft modelall animal studies were conducted in accordance with nihanimal use guidelines and protocols were approved by nanjingmedical university animal care committee sixteen femalenude mice “ weeks old were purchased from nanjingmedical university school of medicine™s accredited animalfacility the mice were randomly divided into two groupsusing random number generator in each group × exponentially growing cervical cancer cells were injected inaxilla subcutaneously before tumor transplantation cells weretransfected with shrnas or overexpression plasmids thetransfection was performed by transient transfection accordingto the specification of lipofectamine invitrogen carlsbadca usa the shnc and empty vector pcdna31 were usedas controls and totally µg plasmid vectors were transfectedinto cells for each group the sequences of shrnas are shown intable s5 tumors were harvested at weeks after injection theweight of tumor was measured on the scale and tumor volumewas estimated using calipers [length × width2] and tissueswere immunohistochemically stained with mnx1 abcamab79541 ki67 abcam ab79541 and p21cip1abcam ab109520 western blotting was performed aspreviously described no blinding was done in the animal studiesstatistical analysisresults are presented as the mean ± standard deviation sdstatistical analyses were performed using spss statistics version chicago ill and graphpad prism software graphpadsoftware inc la jolla ca usa p was consideredstatistically significantresultsoverexpression of mnx1 correlates withpoorer prognosis and more aggressiveclinical featuresanalysis of tcga dataset revealed that the mrna expressionof mnx1 was remarkably upregulated in cervical cancer tissuescompared with paratumor tissues p figure 1a ingepia gene expression profiling interactive analysis websitepatients with higher expression of mnx1 bore a worse diseasefree survival nhigh nlow p figure 1b theexpression of mnx1 in cervical cancer tissues were significantlyhigher than adjacent tissues in of cervicalcancer patients p figures 1cd ihc assays based ontissue microarrays tmas were performed to detect the proteinexpression of mnx1 in paired human cervical cancer tissuesand paratumor tissues and results showed that staining scoresof mnx1 were higher in cancer tissues p figure 1ecombined with the patients™ clinical information the expressionof mnx1 was higher in patients with more advanced tnm stagestage i“ii vs iii“iv p figure 1f t stage t1 vst2“t3 p figure 1g and n stage n0 vs n1 p figure 1h moreover mnx1 staining scores were linkedto higher pathological grade level ii vs iii p figure 1iand larger tumor maximum diameter d vs ‰¥ cm p figure 1j and ihc images of two patients with diï¬erentclinical stages were presented figure 1kknockdown of mnx1 inhibited progressionof cervical cancer in vitroto evaluate the expression of mnx1 in cell lines qrtpcr andwestern blotting were performed and results showed that mnx1was generally upregulated in cervical cancer cell lines comparedwith normal human cervical cell lines hacat figures 2ab tofurther investigate the biological function of mnx1 in cervicalcancer two specific sirnas targeting mnx1 were transfectedinto hela and siha cells both two sirnas showed favorablesuppression efficiency in hela figures 2cd and siha cellsfigures 2ef the rtca proliferation assay figure 2g eduassay figure 2h and colony formation assay figure 2ishowed that knockdown of mnx1 inhibited the proliferationability of cervical cancer in hela and siha cells moreover rtcamigration assay figure 2j transwell assay and matrigel assayfigure 2k and wound healing assay figure 2l revealed thatsilencing mnx1 inhibited the ability of cervical cancer cells tomigrate and invade these results suggest that mnx1 plays a vitalrole in the malignant phenotype of cervical cancerectopic expression of mnx1 enhancedaggressiveness of cervical cancer in vitroto further verify the biological role of mnx1 in cervical cancer apcdna31 plasmid to overexpress mnx1 was constructed andtransfected into c33a and hela cells the plasmid eï¬ectivelyupregulated the expression of mnx1 confirmed by qrtpcrand western blotting figures 3ab consistently our resultsshowed that ectopic expression of mnx1 promotes proliferationmigration and invasion figures 3c“g of cervical cancer cellssimnx1 induced g2m cell cycle arrestand upregulated the expression of p21cip1two hundred and eight genes highly correlated with mnx1were used for go kegg and reactome pathway analysisresults showed that mnx1 may participate in œtranscriptionand œmetabolism pathway figure 4a cell cycle detectionshowed that knockdown of mnx1 induced g2m cell cyclearrest in hela and siha cells figure 4b furthermore weexamined the eï¬ect of mnx1 on the expression of cell cyclekeyrelated genes including p15ink4b p16ink4a p21cip1 p27kip1cdk1 cdk2 cdk4 cyclinb1 cyclind1 and cycline1 bothin hela and siha cells knockdown of mnx1 upregulated theexpression of p21cip1 which has been confirmed as a tumorsuppressor gene in multiple cancers figure 4c and westernblotting results suggested that knockdown of mnx1 increasedthe expression of p21cip1 while decreased the expression ofphosphorylated cdk1 pthr161cdk1 a downstream eï¬ectorof p21cip1 figure 4d consistently with these results ectopicexpression of mnx1 decreased the expression of p21cip1 whileincreased the expression of pthr161cdk1 in c33a and helacells figures 4ef it suggested that mnx1 might exerted itsbiological function via modulating the expression of p21cip1frontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure mnx1 is upregulated in cc tissues and positively correlates with aggressive clinical characteristics a mnx1 is upregulated in cc tissues compared withadjacent normal tissues in tcga dataset p b patients with high expression of mnx1 have poor disease free survival dfs in cc p cd themrna expression of mnx1 in cervical cancer tissues was significantly higher than that in adjacent normal tissues in patients p e the mnx1staining score was upregulated compared with that in adjacent normal tissues p f the mnx1 staining score was positively correlated with tnm stage p g t stage p h lymph node metastasis p i tumor differentiation p and j local primary tumor diameter p incc patients k representative ihc staining images in tmas were shown error bars represent the mean ± sd values ns no significance represents p frontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure knockdown of mnx1 suppressed the proliferation migration and invasion in cc cells ab mnx1 mrna and protein level are upregulated in cc celllines c“f two specific sirna si1 and si2 of mnx1 were designed and the transfection efficiencies of sirnas in hela and siha cells were analyzed by qrtpcrand western blot g“i the proliferation abilities were evaluated by xcelligence system assay edu incorporation assay and colony formation assay were inhibitedafter knockdown of mnx1 in hela and siha cells j the xcelligence system assay k transwell and matrigel assay and l wound healing assay indicated thatmigration and invasion capacities were suppressed after simnx1 in hela and siha cells error bars represent the mean ± sd values of three independentexperiments p p p ns no significancefrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure ectopic expression of mnx1 enhanced aggressive abilities in c33a and hela cells ab the pcdna31mnx1 was synthesize and the transfectionefficiencies were analyzed by qrtpcr and western blot the proliferation functions were measured by c the xcelligence system assay d colony formationassays and e edu incorporation assays were elevated in oemnx1 c33a and hela cells f the transwell assay and matrigel invasion assay g wound healingassay also showed that oemnx1 strengthened migration and invasion capacities error bars represent the mean ± sd values of three independent experiments p p p ns no significancemnx1 suppressed the expression ofp21cip1 via binding to its promoter regionour previous results showed that knockdown or ectopicexpression of mnx1 altered the expression of p21cip1 to furtherverify the mechanism we analyzed the correlation betweenmnx1 and p21cip1 in cases of cc samples and the resultswere shown that mnx1 and p21cip1 had a negative correlationn p figure 5a as transcription factors usuallybind to sequencespecific dna to regulate transcription weutilized jaspar database to predict the binding site betweenmnx1 and the promoter region upstream bp of codingregion of cdkn1a the gene symbol of p21cip1 it turnedout that mnx1 was predicted to have four binding sites withthe promoter region of cdkn1a of which “ bpfrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure knockdown of mnx1 expression induced g2m phage arrest by regulating the p21cip1 expression a many genes were enriched in regulation oftranscription by go analysis most of the genes were enriched in the metabolic pathways by kegg and reactome pathway analysis b knockdown of mnx1generated g2m stage arrest in hela and siha cells were measured by flow cytometry cf the p21cip1 mrna levels were upregulated or downregulated after si oroemnx1 in cc cell lines de the protein level of p21cip1 was upregulated or downregulated while the expression of pthr161cdk1 was decreased or increased afterknockdown or ectopic mnx1 of cc cells the expression of cdk1 ccne1 ccnd1 and ccnb1 had no obvious changes error bars represent the mean ± sdvalues of three independent experiments p p p ns no significanceaacaataaat and “ bp gcccattaat showedhigher combination scores figure 5b accordingly the wildcdkn1a promoter region and mutant types 226mt and1371mt were generated and cloned into luciferase reportervector pgl3basic figure 5c and in luciferase reporterassay overexpression of mnx1 inhibited the transcriptionalactivity of the wild cdkn1a promoter but not mutant typefigure 5d moreover chip assay also revealed that mnx1bound to the p21cip1 promoter region in hela and sihacells figures 5effrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure mnx1 bounds to the p21cip1 promoter region and suppresses p21cip1 transcription a the expression of mnx1 and p21cip1 is negatively correlated in cervical cancer tissues p b the jarspar database indicates that mnx1 has several binding sites with the promoter region of p21cip1 c schematicdiagram shows that the two sites with the highest score of mnx1 on p21cip1 promoter and the mutant p21cip1 promoter were selected d overexpression of mnx1remarkably decreased wild type but not mutant p21cip1 promoter luciferase activity p21cip1226 p p21cip11371 p e chromatinimmunoprecipitation chip assays using normal igg or antimnx1 demonstrated that mnx1 directly binding to p21cip1 promoter region f the results of chippcrproduct electrophoresis were showed that a clear band was observed in the antimnx1 group while almost no band was detected in the igg control groupp p frontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure downregulation of p21cip1 partially recovered the malignant phenotypes of simnx1 cells a the transfection efficiency of p21cip1 was determined byqrtpcr and si1p21cip1 was chosen to further experiments b“d the proliferative abilities were partially rescued after knockdown p21cip1 in simnx1 hela cellswere measured by the xcelligence system assay colony formation assay and edu incorporation assay ef the invasion and migration capacities have also beensignificantly improved after knockdown p21cip1 in simnx1 cells compared with simnx1 alone cells g the protein level of p21cip1 and pthr161cdk1 were partiallyreversed when knockdown of p21cip1 in simnx1 compared with simnx1 alone error bars represent the mean ± sd values of three independent experiments p p p ns no significancefrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure knockdown or overexpression of mnx1 inhibited or promoted tumor growth in vivo ab the transfection efficiency of shmnx1 was measured byqrtpcr and western blot c a total of eight nude female mice were sacrificed and xenograft tumors were collected after injection with shmnx1 cells weeksde tumor volume and weight were reduced in the shmnx1 group compared with those in the shnc group f the expression of mnx1 and ki67 wasdownregulated and p21cip1 was upregulated in shmnx1 xenograft tumors analyzing by ihc staining g the protein level of mnx1 pthr161cdk1 weredownregulated and p21cip1 was upregulated in shmnx1 mouse xenograft tumors analyzed by western blot h a total of eight nude female mice were sacrificed andxenograft tumors were collected after injection with oemnx1 cells weeks jk tumor volume and weight was increased in the oemnx1 group compared withthose in the oenc group i the expression of mnx1 and ki67 was upregulated and p21cip1 was downregulated in oemnx1 xenograft tumors analyzing by ihcstaining error bars represent the mean ± sd values p p p ns no significancesilencing p21cip1 rescued the function ofsimnx1to determine whether the function of mnx1 was relied onp21cip1 we designed three sirnas table s3 to knockdownthe expression of p21cip1in hela cells the si1p21cip1showed the best transfection efficiency figure 6a and it wasused for the following experiment rtca proliferation assaycolony formation assay edu assay transwell assay matrigelassay and would healing assay revealed that silencing p21cip1partially rescued the decreased proliferation migration andinvasion ability of hela cells caused by knockdown of mnx1figures 6b“f and western blotting showed that the proteinlevel of p21cip1 and pthr161cdk1 were partially reversed bysilencing p21cip1 figure 6gmnx1 promoted tumor growth of cervicalcancer in vivothe xenograft models were used to explore the function ofmnx1 in vivo the shrnamnx1 shrnanc as control wastransfected into hela cells and the knockdown efficiency wasconfirmed by qrtpcr and western blotting figures 7abresults showed that knockdown of mnx1 inhibited tumrowth measured by tumor weight and volume in vivofigures 7c“e ihc staining and western blotting of harvestedtumors revealed that knockdown of mnx1 upregulated theprotein level of p21cip1 and downregulated ki67 and pthr161cdk1 in vivo figures 7fg moreover ectopic expression ofmnx1 promoted tumor growth and altered the expression ofp21cip1 and ki67 in vivo figures 7h“kfrontiers in oncology wwwfrontiersinaugust volume 0czhu discussionin this study we identified mnx1 a transcription factor ofhomeobox family was significantly upregulated and involvedin the progression of cervical cancer the overexpression ofmnx1 correlated with advanced clinical stages and poorerprognosis of cervical cancer patients furthermore mnx1exerted its oncogenic role via modulating the expression ofp21cip1 especially by targeting the promoter region of p21cip1thus to repress its transcriptionin accordance with ourfindings a recent showed that mnx1 had a role in theprogression of cervical cancer partially through upregulating cellcycle regulators ccne1 and ccne2 and mnxas1 theantisense lncrna of mnx1 was also reported to promote theinvasion and metastasis of gastric cancer through repression ofcdkn1a all this results indicated that mnx1 played acritical role in cancer growth and cell cycle progression andmnx1 might serve as a useful diagnostic and treatment targetfor cervical cancermnx1is a member of homeobox gene family which allcontain a homeobox a dna sequence around basepairs long and encode homeodomain protein products astranscription factors this cluster of genes has beenidentified to participate in the regulation of development andmorphogenesis in animals fungi and plants for examplecdx1 which is stably expressed in the human intestine playsan important role in embryonic epicardial development and the protagonist of our study mnx1 participates inmotor neuron development and neurodegenerative processesof zebrafish and moreover controls cell fate choice inthe developing endocrine pancreas in recent years moreand more researches uncovered the role of developmentrelatedhomeobox genes in carcinogenesis and these genes show greatapplication prospect in tumor diagnosis and prevention asthe role of carcinoembryonic antigen cea in gastroenterictumors and alpha fetal protein afp in liver cancer “for instance pdx1 is a key regulator in pancreatic developmentand cell function and meanwhile dynamically regulatespancreatic ductal adenocarcinoma initiation and maintenance hoxc13 a highly conserved transcription factor involvedin morphogenesis of all multicellular anisms is aberrantlyexpressed and associated with cancer progression in esophagealcancer lung adenocarcinoma and liposarcomas likewise mnx1 has been reported to promote sustainedproliferation in bladder cancer by upregulating ccne12 and to act as a novel oncogene in prostate cancer and in ourstudy mnx1 was also confirmed to be upregulated in cervicalcancer and enhance the progression of cervical cancerin terms of mechanism we found that mnx1 promotedtumor growth of cervical cancer via accelerating the progressionof the cell cycle especially by modulating the expression ofp21cip1 cell cycle is a vital process by which a cellleadsto duplication and disorders of the cell cycle regulation maylead to tumor formation the cell cycle progress isdetermined by two types of regulatory factors cyclins and cyclindependent kinases cdks active cyclincdk complexesphosphorylate proteins to elevate the expression levels of cyclinsmnx1 enhances progression of cervical cancerand enzymes required for dna replication converselythe cell cycle progression can be prevented by inhibitors bybinding to and thus inactivating cyclincdk complexes suchas p21cip1 p27kip1 p16ink4a and so on the p21cip1also known as cyclindependent kinase inhibitor cdkn1ahas been identified as a regulator of cell cycle and a tumorsuppressor in multiple kinds of cancers our results provedthat mnx1 repressed the transcription of p21cip1 by directlytargeting its promoter region and furthermore promoted thephosphorylation of downstream cdk1 the mnx1p21cip1pthr161cdk1 axis played crucial roles in the progression ofcervical cancer and meanwhile provided new evidence forthe pathogenesis of cervical cancer moreover the associationbetween cervical cancer and hpv has long been identified as asexually transmitted agent hpv are involved in transformationand maintaining of transformed status many studies havereported that hpv can also alter the expression of p21 “thus we searched the geo dataset to seek for information aboutthe relationship between mnx1 and hpv viral infection weanalyzed the gse dataset and found that there were nosignificant changes in the expression of mnx1 nm_005515 inhacat cells infected with hpv11e6 or hpv18e6 in gse3292gds1667 hpv positive or negative head and neck squamouscell carcinoma hnscc showed no expression diï¬erences ofmnx1 figure s1 this information suggests that mnx1 mightnot be directly involved in hpv carcinogenesis and furtherinvestigations are needed in the future in addition cervicalcancer i
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purpose pancreatic ductal adenocarcinomas exhibit a high degree of desmoplasia due to extensive extracellular matrix deposition encasement of mesenteric vessels by stroma in locally advanced pancreatic cancer lapc prevents surgical resection this study sought to determine if the addition of a monoclonal antibody to connective tissue growth factor pamrevlumab to neoadjuvant chemotherapy would be safe and lead to improved resectability in this surgically adverse patient populationmethods in this phase iii trial patients with lapc were randomised to gemcitabinenab paclitaxel plus arm a n24 or minus arm b n13 pamrevlumab those who completed six cycles of treatment were assessed for surgical eligibility by protocol defined criteria resection rates progression free and overall survival were evaluatedresults eighteen patients in arm a and seven in arm b completed six cycles of therapy with similar toxicity patterns in arms a and b carbohydrate antigen “ response as defined by ‰¥ decline from baseline occurred in and respectively sixteen per cent of patients were radiographically downstaged by national comprehensive cancer network criteria in arm a and in arm b positron emission tomography normalised in vs of patients in arm a vs arm b respectively and correlated with surgical exploration eligibility for surgical exploration was vs p00019 and resection was achieved in vs of patients in arm a vs arm b p01193 respectively postoperative complication rates were not different between armss neoadjuvant chemotherapy with pamrevlumab holds promise for enhancing resection rates in patients with lapc without added toxicity this combination merits evaluation in a larger patient cohortintroductionpancreatic cancer is currently the third leading cause of cancer death in the usa1 and by it will likely become the second leading cause of cancer related death after key questionswhat is already known about this subject –º pamrevlumab is anti ctgf1 inhibitor highly safe when given to patients with pancreatic cancer and potentially adds to the activity of gemcitabine and erlotinib when given in metastatic diseasewhat does this study add –º this study examines a the safety and activity of pamrevlumab when added to gemcitabine and nab paclitaxel in locally advanced pancreatic cancer b the impact of this regimen and surgical resection and postoperative safety c explores the utility of a novel study design for locally advanced pancreatic cancerhow might this impact clinical practice –º this study has the potential to lead to practice changing activity in locally advanced pancreatic cancer via the eventual approval of pamrevlumab for use in this situation the promulgation of a new study design for locally advanced pancreatic cancer and increased potential for surgical resection and thus prolonged os curability in locally advanced pancreatic cancerlung cancer surpassing breast and colon cancer2 surgical resection is generally necessary for treatment with curative intent or to extend life expectancy3 however only of patients have disease amenable to upfront curative resection at the time of diagnosis4 approximately “ of patients are diagnosed with locally advanced disease5 determined surgically unresectable per national comprehensive cancer network nccn guidelines6 patients with locally advanced pancreatic cancer lapc have a prognosis similar to those with metastatic disease with a historical median overall survival os of picozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen access“ months with recent trials demonstrating median os of months7 recent single institution retrospective studies have reported the potential for resection of lapc with neoadjuvant therapy irrespective of imaging findings with promising results8 however these are limited by significant selection bias lack of strict radiographic classification and chemotherapy standardisation current prospective trials have documented resection rates of lapc in the range of to therefore novel approaches are needed to improve patient outcomesthe tumour biology inherent to pancreatic ductal adenocarcinoma pdac significantly contributes to the poor outcomes seen in this disease notably pdac exhibits a high degree of desmoplasia often in association with elevated connective tissue growth factor ctgf expression12 ctgf appears to play a central role in the biology of pancreatic cancer affecting both its cellular biology and its extracellular matrix composition this leads to many simultaneous biological effects that promote pancreatic cancer growth including increased cellular proliferation and differentiation increased cellular adherence and migration antiapoptosis vascular permeability angiogenesis and suppression of tumour immunological responses13 this stroma may also contribute to the radiographic imaging findings of mesenteric vessel involvement or encasement that is used to determine resectability of pancreatic tumours executing a pharmacological intervention on the pancreatic cancer stromal environment is therefore a major goal of the development of novel pancreatic cancer therapeuticspamrevlumab is a human monoclonal antibody that targets ctgf preclinical studies showed that ctgf overexpression is associated with both desmoplasia and gemcitabine resistance in the kpc pancreatic cancer mouse model14 when pamrevlumab was used in combination with gemcitabine sensitivity to gemcitabine was enhanced which correlated with inhibition of xiap an antiapoptotic protein15 when tested in patients with advanced pancreatic cancer stage iv and locally advanced stage iii treated with gemcitabine and erlotinib in a phase iii study n75 pamrevlumab displayed multiple favourable outcomes16we hypothesised that through inhibition of the downstream effects of ctgf overexpression on tissue adhesion and other mechanisms pamrevlumab may influence resectability of pdac tumours with this in mind this novel phase iii randomised multicentre trial was designed to explore the safety and efficacy of pamrevlumab in combination with gemcitabinenab paclitaxel in lapc with special emphasis on surgical eligibility and safetymethodsstudy designthis was a phase iii randomised trial of safety and efficacy in patients with lapc who received gemcitabine and nab paclitaxel with or without pamrevlumab as neoadjuvant therapy the randomisation was preplanned and blinded to the investigator the study was approved by individual institutional review boards at nine us institutions and conducted according to the declaration of helsinki the trial was registered at clinicaltrials gov as nct eligibilitykey protocol eligibility requirements included biopsy proven diagnosis of pdac radiographic staging consistent with locally advanced unresectable disease as defined nccn guidelines v2 clinical stage confirmed by diagnostic laparoscopy radiographically measurable disease per response evaluation criteria in solid tumors recist v11 eastern cooperative oncology group ecog performance status of or adequate haematological renal and hepatic function no prior therapy for pdac and no concomitant cancer diagnosis within the past yearsstudy schemaeligible patients were randomised to arm a or arm b to receive a total of six treatment cycles “ weeks of therapy figure patients in arm a received pamrevlumab mgkg by intravenous infusion on days and of each day cycle with an additional dose given on day in the first cycle patients in both arms a and b received gemcitabine mgm2 by intravenous infusion on days and of each day treatment cycle nab paclitaxel mgm2 by intravenous infusion on days and of each day cycle doses for gemcitabine and nab paclitaxel were modified for haematological and non haematological toxicity as per standard of care soc15 patients remained on therapy for six treatment cycles “ weeks unless they had disease progression an intolerable adverse event ae or toxicity withdrew consent or were withdrawn at the investigator™s discretion all patients were followed for drug toxicity until days after the last drug dose patients undergoing surgery were followed for days following hospital discharge for surgical complications ctgf levels were obtained prior to treatment from all patients plasma samples for pamrevlumab level determination were obtained from all patients receiving this drug after all protocol specified therapy was completed patients were followed for disease progression survival and additional oncological therapy postoperative complications including day readmissions and day mortality were notedresponse assessmentpatients were evaluated for response by the following measures carbohydrate antigen ca “ measured at baseline first day of each cycle and end of treatment eot recist v11 read based on full body ct imaging high resolution dual phase fine cut ct imaging at baseline and every weeks thereafter fluorodeoxyglucose fdg positron emission tomography pet imaging and nccn v2 resectability criteria at baseline and eotpicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen accessfigure patient flow and surgery outcomes in arm a four of the eligible subjects had their surgeries cancelled 1portal vein thrombosis 3medical issues precluding surgery in arm a four eligible subjects underwent surgery but resection was not achieved 3metastatic disease discovered 1extensive sma encasement in arm b one eligible subject underwent surgery but resection was not achieved 1extensive vascular encasement sma superior mesenteric arterysurgical assessmentsubjects who finished six cycles of combination chemotherapy were evaluated for eligibility for surgical exploration per protocol pp defined criteria given that patients included in the trial were determined to be initially unresectable by radiographic imaging and nccn criteria objective criteria were developed to standardise attempts at surgical resectionpatients were deemed eligible for surgery if one or more of the following criteria were met reduction in plasma ca “ level by ‰¥ at eot compared with baseline reduction in fdg pet maximum standardised uptake value suvmax by ‰¥ at eot compared with baseline radiological tumour response per recist of partial response pr or complete response cr at eot or met the definition of resectable or borderline resectable per nccn guidelines subjects were classified as ineligible for surgical exploration if any of the following occurred development of distant metastases or local progression on ct scan tumour anatomy precluding vascular reconstruction unreconstructible local complications preventing surgery eg portal vein pvsplenic vein thrombosis pancreatitis or decline in performance status to a karnofsky score ‰¤ or picozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668absolute contraindication to surgery from comorbidity eg recovery from myocardial infarction or uncontrolled diabetes the final decision regarding whether resection was to be performed was made by the treating surgeonendpointssafety endpoints included serious adverse events sae during neoadjuvant therapy and surgical complications postresection the efficacy endpoints included surgical eligibility r0 resection r0r1 resection median os progression free survival pfs and year survival rate all patients were followed and data analysis was stratified by pp population and intention to treat itt cohortstatistical considerationsthe comparison between selected clinical characteristics toxicity profiles and eligibility for surgical exploration or completed surgical resection was performed using the χ² test exact cis for the point estimates as well as the treatment difference were obtained from the sas proc freq procedure with the exact option the two treatment arms were compared using the cochran mantel haentzel test controlling for baseline factors tnm stage ecog ca “ pet suvmax 0copen accesssuperior mesenteric artery sma involvement coeliac abutment and so on as prespecified in the protocol all cause mortality was used in determining os which was analysed by the kaplan meier method survival status was updated within month before the data cut off date data from patients who were alive at the cut off date were censored for survival analysis all statistical tests were performed at the significance level of α005 using two sided testsresultspatient characteristics and dispositionthirty seven patients were randomised to study treatment to arm a pamrevlumabgemcitabinenab paclitaxel and to arm b gemcitabinenab paclitaxel alone patient characteristics at baseline are summarised in table all patients enrolled were unresectable by nccn criteria patients had tumour arterial involvement sma encasement ° coeliac abutment table patient characteristicsbaseline demographics “ years “ years ‰¥ years median male femaleage group sex bmi kgm2 mean sd median min maxecog grade grade tnm stage t3 n0 m0 t3 n1 m0 t4 n0 m0 t4 n1 m0 t4 nx m0location of the tumour in the pancreas non resectability per nccn criterion head body tail median tumour size mm ° sma encasement any coeliac abutment inferior vena cava invasion or encasement unreconstructible smvportal occlusion aortic invasion and encasementarm agnppn24 arm bgnpn13 totaln37 to to to · ok as isnot mutually exclusivebmi body mass index ecog eastern cooperative oncology group g gemcitabine n number of subjects nccn national comprehensive cancer network np nab paclitaxel p pamrevlumab pv portal vein sma superior mesenteric artery smv superior mesenteric veinpicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen accesswithout gastroduodenal artery involvement or aortic involvement inferior vena cava invasion and unreconstructible pvsuperior mesenteric vein smv occlusion a higher percentage of patients with sma encasement ° were randomised to arm a vs arm b patient disposition is summarised in figure twenty four patients in arm a received gemcitabinenab paclitaxel and pamrevlumab patients completed six treatment cycles six patients discontinued treatment early due to progressive disease three patients aes two patients or physician decision one patient thirteen patients in arm b received gemcitabinenab paclitaxel patients completed six treatment cycles six patients discontinued treatment early due to progressive disease two patients aes two patients or patientphysician decision two patientssafetysaes are summarised in table forty one per cent of patients had a treatment emergent sae arm a arm b no individual toxicity category occurred with frequency except systemic infection patients there was no demonstrable increase in any toxicity with the addition of pamrevlumab to gemcitabinenab paclitaxel chemotherapytable summary of treatment emergent serious adverse eventssystem organ classpreferred term ascites nausea pancreatitis vomiting device occlusion drug withdrawal syndrome feverno of patients with any treatment emergent saeblood and lymphatic disorders haemolytic uremic syndrome lymphadenopathycardiac disorders cardiac failure supraventricular tachycardiagastrointestinal disorders general disorders and administrative site conditions hepatobiliary disorders infections sepsis cellulitis urinary tract infectioninjury poisoning and procedural complications respiratory thoracic and mediastinal disorders skin and subcutaneous disorders cholangitis hyperbilirubinaemia craniocerebral injury pneumonitis pulmonary embolism rasharm an24n arm bn13n overalln37n · ok as ispicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen accessresponse to therapyin arm a had ‰¥ ca “ decline at eot response by recist pr ‰¥ decline in pet suvmax and were radiographically downstaged by nccn criteria during the treatment period the median ca “ decline was patients were non secretors seven out of patients had best objective recist response crpr some patients had ˜exceptional™ responses defined as normalisation or ‰¥ decline of ca “ patients or normalisation pet suvmax in in arm b had ‰¥ ca “ decline at eot response by recist pr ‰¥ decline in pet suvmax and were radiographically downstaged by nccn criteria four out of patients had best objective recist response cr pr in arm b of patients had an œexceptional ca “ response and had an ˜exceptional™ pet response as defined by either ‰¥ normalized ca response normalized suv max andorradiographic downstaging post therapy completion surgical evaluationoverall of the total study patients were eligible for surgical exploration using protocol defined criteria arm a arm b p00019 resection was completed in of the patients arm a arm b p01193 details of the nine resected patients are shown in table in arm a of the patients were eligible for surgical exploration in the itt population and of the patients were eligible in the pp population patients who completed six cycles of treatment in arm a out of eligible patients ultimately underwent surgical exploration for resection five operations were cancelled four due to drug toxicitymedical comorbidity sepsis gallbladder perforation declined performance status and fever and one patient declined eight out of patients in arm a were resected r0 r1 the remaining four patients who were explored were not resected due to progression or unresectable disease intraoperatively in arm b of the patients were eligible for surgical exploration in the itt population and were eligible in the pp population of the two subjects found to be surgically eligible only one was resected as the other patient had local progressionpredictors of resectionhigh ca “ response ‰¥ decline andor normalisation was contributive to surgical eligibility vs p03 normalisation versus non normalisation of pet suvmax was predictive of surgical eligibility vs p0013 and successful resection vs p0002 combining these two criteria was highly predictive for surgical eligibility vs p0003 and completed vs p0002 resection all nine successful resections were identified by one or both of these criteria table summary of resected patientssitesubject idtreatmentarmresponse to treatmentnccnbaselinenccnend of treatmentresection status“““““““““aaaaaaaab unresectablecoeliacunresectablesma smvunresectablecoeliacunresectablecoeliacunresectablesmvunresectablesmaunresectablesma smv coeliacunresectablesmaunresectablecoeliacunresectablecoeliacunresectablesma smvunresectablecoeliacborderline resectableunresectablesmvunresectablesmaunresectablecoeliacunresectablesmaunresectablecoeliacr0r1r0r0r1r1r1r0r0protocol defined criteria ca “ decrease fdg pet suvmax decrease ‰¥ recist v11 response pr or cr nccn resectable or borderline resectable criteriaca carbohydrate antigen cr complete response fdg fluorodeoxyglucose nccn national comprehensive cancer network pet positron emission tomography pr partial response recist response evaluation criteria in solid tumors sma superior mesenteric artery smv superior mesenteric vein suvmax maximum standardised uptake valuepicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0cconversely radiographic features of response did not correlate with operative potential neither recist response nor radiographic downstaging per nccn criteria statistically correlated with completed resectionsurgical complicationspostoperative complications were summarised according to the clavien dindo classification posthoc analysis ischaemic gastritis and ulceration and right lower lung lobe collapse were reported for one patient in arm a grade ii there was one episode of clinically significant pancreatic leak in each arm grade iiia no reoperations and no day or day surgical mortality were noted one patient in arm b had a delayed gastric perforation following a distal pancreatectomy with coeliac axis resection likely due to thermal injury and was treated non operatively grade iiib no wound complications or superficial site infections were noted in either group four out of patients and out of patients in arm a and b respectively were readmitted within days and the difference between treatment arms was not clinically or statistically significantsurvivalas of the data cut off date of evaluable patients were known to be alive with a median length of follow up at approximately months pfs was months ci to and months ci to in arm a and arm b respectively one year survival and median os were and months ci open accessto in arm a and and months ci nr in arm b the median os for all patients who were eligible for surgical exploration arm a arm b vs ineligible arm a arm b was months ci nr vs months ci to p00766 the median os for resected arm a arm b vs non resected patients arm a arm b was not reached ci nr vs months ci to p00141 figure discussionthe treatment of lapc with neoadjuvant therapy remains challenging and there is no established soc several high volume centres have reported their single centre experiences with varying neoadjuvant chemotherapy and chemoradiation strategies8 the combination of more active regimens delivered over an extended period and surgeons™ comfort with resecting and reconstructing major mesenteric vessels has led to an increase in resection rates a meta analysis of studies using folfirinox has demonstrated resection rates ranging from to in lapc17 one of the larger studies including patients with lapc reported a resection rate of that was more dependent on duration of therapy months than chemotherapy regimen folfirinox or gemcitabine based18 recently a single institution and single arm prospective study of neoadjuvant folfirinox and losartan with selective use of radiation in patients with lapc reported an r0 resection rate of figure overall survival resected vs non resected patientspicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen access however the lack of randomisation makes it difficult to determine what aspect of therapy was responsible for this effect and only of resected patients needed any type of vascular reconstruction perhaps suggesting more favourable outcome than usually seen in locally advanced disease these retrospective studies contain significant interpretative challenges including selection bias treatment variables including radiation and the use of different definitions of lapcthe anti ctgf mechanism of action with respect to gemcitabine based therapy a recent large scale prospective trial of patients with lapc treated with induction gemcitabine with or without erlotinib followed by radiation only patients were able to undergo resection10 furthermore the addition of erlotinib to gemcitabine did not improve any downstaging capacity and there was no difference in survival with the addition of radiation more recently the la pact trial examining the role of induction gemcitabine nab paclitaxel found that only out of patients with lapc were able to undergo surgery following neoadjuvant therapy with combination gemcitabine and nab paclitaxel for six cycles by investigator™s choice11 last although folfirinox has been the most studied induction combination chemotherapy regimen in this population recent randomised data from european patients who received neoadjuvant folfirinox versus gemcitabinenab paclitaxel prior to resection20 showed no clear difference with respect to r0r1 to resection rate vs p0135 or os vs months p0268given for pamrevlumab its use in the neoadjuvant setting has the potential to impact tumour regression and modulate the desmoplastic niche and possibly affect tumour margins allowing for improved resection rates previous studies have looked at the ability of gemcitabinenab paclitaxel to reduce cancer associated fibroblasts resulting in a ˜softening™ of tumours by endoscopic ultrasound elastography21 this stromal depletion also translated into a decrease of suv uptake on pet22 in the study reported herein we combined gemcitabine and nab paclitaxel with pamrevlumab to explore its effect in terms of therapeutic response the impact on eligibility for surgical exploration and improved resection rates in locally advanced patientsthe protocol specified therapeutic response criteria ca “ pet suvmax recist and nccn criteria were used as criteria to determine eligibility for surgical exploration in lapc this is a novel approach specific to the protocol and allows participating patients to be explored for resection when otherwise they may not qualify by current treatment standards nccn criteria for example by nccn conversion alone ie converted from unresectable to borderline resectable only of patients in arm a would have been eligible for surgical exploration however by protocol criteria of patients in arm a were eligible for surgical exploration a higher percentage of patients were eligible for surgical exploration by the above criteria in arm a vs arm b vs respectivelyoverall the rate of successful resections in the pamrevlumab treated group was higher than in the control group but this did not reach statistical significance most probably due to small sample size of the nine subjects that were successfully resected in this trial only one was converted by nccn criteria to borderline resectable prior to surgical exploration despite this phenomenon the data support the hypothesis that pamrevlumab a human monoclonal antibody with anti ctgf mechanism of action could alter tumour characteristics allowing resection in otherwise unresectable patients this hypothesis needs to be confirmed and patients should be stratified by coeliac andor sma involvementthe most common predictive factors for eligibility for surgical exploration and resection were ca “ decline and pet suv max response which are indicators of tumour response to treatment the combination of these two factors proved to be a highly sensitive objective readout for prediction of potential surgical success both the ability of ca “ response and the inability of radiographic response recist and nccn criteria of resect ability to predict surgical outcome has been observed by others3 and these observations deserve further examination in subsequent clinical trials in the mpact study both ca “ and pet response correlated to improved survival in metastatic patients treated with gemcitabine and nab paclitaxel23 recent surgical series of patients with borderline resectable and lapc have also corroborated their impact in the localised setting25 correlation of clinical response with plasma levels of endogenous ctgf and pamrevlumab exposure as shown in the prior study by picozzi et al16 may provide added prognostic and predictive insightwith regard to safety no major incremental toxicity in any category was noted with the addition of pamrevlumab to gemcitabinenab paclitaxel in addition a higher number of patients were able to complete six cycles of the three drug combination including pamrevlumab when compared with gemcitabinenab paclitaxel alone pamrevlumab is well tolerated and considered safe compared with the soc drugs for patients with pdac these observations represent a very favourable attribute when considering potential neoadjuvant chemotherapy in a patient population with the frequency of medical problems typically seen in lapc in addition there were no signals of increased surgical morbidity or wound healing problems with ctgf blockade by pamrevlumab in fact there were only two clinically significant pancreatic leaks one in each arm which is comparable to national outcome data from high volume pancreatic surgery centres similarly readmissions following resection were comparable between arms and reflected the complexity of this challenging patient populationfinally while survival data are not yet mature both patients who were eligible for surgery and those that picozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0cwere ultimately resected had longer pfs and os highlighting the importance of surgical resection of the tumour therefore more investigation into newer agents targeting lapc and increased consideration of candidacy for surgery in those patients who do not progress on therapy or suffer toxicity should be of utmost importance to improve outcomes in this diseasein this is the first prospective randomised multicentre trial examining the role of neoadjuvant therapy in lapc with prespecified criteria for surgical exploration the use of pamrevlumab in combination with gemcitabine and nab paclitaxel showed a potential to enhance tumour response and increase resection rates further evaluation of this drug combination in the neoadjuvant treatment setting for lapc is warranted and a larger phase iii trial with resection and survival endpoints is ongoingcontributors fibrogen inc was the study sponsor that designed the study in consultation with the principal investigator vp and surgical co investigator fgr all authors except those of the sponsor contributed patients to the study fibrogen was responsible for data collection and analysis all authors reviewed the manuscript and signed off on its accuracyfunding the study was funded by fibrogen inc san francisco cadisclaimer the corresponding author has the right to grant on behalf of all authors and does grant on behalf of all authors an exclusive licence or non exclusive for government employees on a worldwide basis to the bmj publishing group ltd and its licensees to permit this article if accepted to be published in esmo open editions and any other bmjpgl products to exploit all subsidiary rights as set out in our licencecompeting interests mc mz sp ek and ec are employees of fibrogen and hold stock andor stock options in fibrogenpatient consent for publication not requiredprovenance and peer review not commissioned externally peer revieweddata availability statement data are available on reasonable request all data relevant to the study are included in the article or uploaded as supplementary information data will be available as presented in this manuscriptopen access this is an open access article distributed in accordance with the creative commons attribution non commercial cc by nc license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited any changes made are indicated and the use is non commercial see a0http creativecommons org licenses by nc orcid idewa a0carrier http orcid org references american cancer society cancer facts figures available httpswww cancer org content dam cancer org research cancer facts and statistics annual cancer facts and figures cancer facts and figures pdf rahib l smith bd aizenberg r et a0al projecting cancer incidence and deaths to the unexpected burden of thyroid liver and pancreas cancers in the united states cancer r
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properly citedIntroduction Endogenously produced antiganglioside antibodies could aï¬ect the evolution of cutaneous melanoma Epidemiologicaland experimental evidence suggest œchronic ‚ammation to be one of the hallmarks in skin cancers The aim of the study was tocharacterize the relation between antiganglioside antibodies and ‚ammation in cutaneous melanoma focusing on gangliosidesGM1 GM2 GM3 GD1a GD1b GT1b GQ1b Material and Method We performed an observational study that included subjects subdivided into three groups patients with metastatic melanoma cases patients with primary melanoma casesand healthy subjects subjects The assessment of antiganglioside antibodies IgG and IgM classes against GM1 GM2GM3 GD1a GD1b GT1b GQ1b was performed using immunoblot technique EUROLine kit Results The presence of IgGand IgM antiganglioside antibodies in primary melanoma was as follows antiGM1 and GM2 and GM3 and GD1a and GD1b and GT1b and GQ1b and In metastaticmelanoma the level of antiganglioside antibodies was significantly lower compared with primary melanoma p while inthe control group they were absent Antiganglioside antibodies antiGM1 and GD1a were positively correlated while antiGM3GD1b and GT1b were negatively associated with the ‚ammatory markers interleukin IL8 and C reactive protein CRPConclusions Tumour ganglioside antigens generate an immune response in patients with primary melanomas The host™s ability toelaborate an early antiganglioside response could be considered as a defence mechanism directed toward eliminating a dangersignal from the tumour microenvironment Antiganglioside antibodies associated with ‚ammation markers could be used asdiagnostic monitoring and treatment tools in patients with cutaneous melanoma IntroductionGangliosides are a group of bioactive glycolipids located onthe outer face of cell membranes These glycolipids play amajor role in cell proliferation diï¬erentiation migrationapoptosis signal transduction cell adhesion modulatinggrowth factor or hormone receptor antigen recognition protein trafficking viral transformation and oncogenesis [“]Atypical expression of some ganglioside antigens associatedwith certain tumours neuroblastomas melanomas gliomaslymphomas small cell lung cancer and prostate cancer andfurthermore could play an importantrole in cancer 0cJournal of Immunology Researchimmunotherapy [“] Gangliosides that are released inextracellular spaces could have dual action antitumor andprotumour eï¬ect [“] Data regarding the endogenousimmune response directed toward tumour gangliosides andthe significance of this response are limited A series of studiesperformed in in vivo experimental models and in vitro inmurine and human cancer cells have shown that monoclonalantiganglioside antibodies have antitumor potential Theseantibodies exert numerous antitumor eï¬ects through variousmechanisms An important mechanism is the translocationof gangliosides from the plasmatic membrane into theintracellular spaces so binding of antibodies to the surfaceof the tumor cells and complement activation that leads to celllysis mediated by complementdependent cytotoxicity andantibodymediated cellular cytotoxicity [ ] Antiganglioside antibodies modulate ceramide synthesis [ ]reception and transduction of the cytotoxic signal [] theyare involved in suppression or induction of cell death throughdiï¬erent pathways apoptosis necrosis oncogeneslike structural and functional changes of mitochondria accumulationof reactive oxygen species acetylation of gangliosides accumulation of sphingosine sphingamine ceramides [ ]Proteomic studies showed that antiganglioside antibodiescould induce changes like the disruption of signalling systemsP38MAPK PARP JNK123 METc ERK12 P13KAKTand FAK modulation of the level and function of transcription factors P53 SP1 MYCN and HSF1 regulating the balance between apoptosisinducing and apoptosissuppressingfactors cysteineaspartylproteases Bax Bcl2 [ “]These antibodies stimulate the cytotoxicity of chemotherapeutic drugs and small molecule inhibitors [ ] As a result antiganglioside antibodies could be used as diagnostic monitoringand treatment tools in cancer patients [ ]Ganglioside levels are increased in malignant melanocytes and represent an important topic of research [ ]Several researchers have emphasized the role of glycolipidsas markers of melanoma A study analysing the expressionof gangliosides in melanocyte lines and melanoma cell linesfound out an increased expression of GD3 synthase genesin melanoma cells but not in melanocytes The same resultswere obtained for GM2GD2 synthase [] It seems thatgangliosides induce cell proliferation and invasion throughp130Cas and paxillin in melanoma cells []Inflammatory mechanisms play an important role inmelanoma Multiple studies have shown that plasma levelsof C reactive protein CRP increase during tumor proliferation and several relations have been evaluated CRPsurvivalrelationship CRPresponse therapy CRP‚ammationNowadays CRP is considered a true marker for assessing‚ammation in melanoma as well as a marker for responseto treatment Prospective studies have provided consistentresults in the predictive value of CRP in neoplastic diseaseproving high sensitivity and specificity [] In addition inmelanoma elevated levels of CRP may reflect the amountand activity of circulating pro‚ammatory cytokines eginterleukin IL8 IL8 plays a crucial role in regulating cellfunction for host defence and for developing natural immunity [ ] Moreover IL8 is released by various cell typesincluding polymorphonuclear neutrophils PMNs monoSerum dilution Incubation with fixed on strip antigens degrees min with balanceWashing with universal tampon times min each with balanceIncubation with conjugated enzyme degrees min with balanceWashing with universal tampon times min each with balanceIncubation with fixed on strip antigens degrees min with balanceWashing with universal tampon times min each with balanceEUROLine scan evaluationFigure Antiganglioside detectioncytes T lymphocytes and endothelial cells upon exposureto ‚ammatory stimuli Melanoma cells have been reportedto express IL8 and this ‚uences their oncogenic properties[ ] IL8 follows the evolution of melanoma progression and regression under treatment reflecting the stage ofthe disease [“]Based on these accumulating data we have investigatedantiganglioside antibodies in correlation with other ‚ammatory markers IL8 CRP and the clinical evolution of the melanoma patients Clarifying these relations could significantlyimprove the prediction of clinical outcomes Furthermore itcan lead to the development of appropriate therapeutic strategies in patients with cutaneous melanoma Material and Method Patients We performed an observational prospectivestudy during years in Clinical Hospital for Infectious andTropical Diseases œVictor Babes”Dermatology Department Bucharest The study was approved by the Ethics Committee of the Hospital All participants agreed to be includedin research studies without prejudice of the diagnosis orpersonal image and signed the informed consent accordingto the Declaration of HelsinkiThe study included adult patients with cutaneous melanoma with no other pathologies and no treatment for theprimary disease Exclusion criteria were age under yearspregnancy alcohol use melanoma under treatmentWe performed an observational study that included subjects subdivided in three groups patients with metastaticmelanoma cases patients with primary melanoma cases and healthy subjects with matching sex and age subjects Patients were selected and examined accordingto ESMO Clinical Practice Guidelines for melanoma 0cJournal of Immunology ResearchTable Test strips coated with parallel lines of purified antigensAntigenGM1GM2GM3GD1aGD1bGT1bGanglioside typeSourceMonosialoganglioside GM1Monosialoganglioside GM2Monosialoganglioside GM3 Dog erythrocytesBovine brainBovine brainDisialoganglioside GD1aDisialoganglioside GD1bTrisialoganglioside GT1bBovine brainBovine brainBovine brainGQ1bTetrasialoganglioside GQ1bBovine brainStructureGal3GalNAc4[Neu5Ac3]Gal4GlcCerGalNAc4[Neu5Ac3]Gal4GlcCerNeu5Ac3Gal4GlcCerNeu5Ac3Gal3GalNAc4[Neu5Ac3]Gal4GlcCerGal3GalNAc4[Neu5Ac8Neu5Ac3]Gal4GlcCerNeu5Ac3Gal3GalNAc4[Neu5Ac8Neu5Ac3]Gal4GlcCerNeu5Ac8Neu5Ac3Gal3GalNAc4[Neu5Ac8Neu5Ac3]Gal4GlcCer Glcglucose Gal galactose GalNAc Nacetylgalactosaminediagnosis based on clinical histopathological immunohistochemical and imagistic data All the events related to theprogression of the disease were recorded relapse metastasisneurotoxicity hyper reactivation of the immune system upontreatment The group characteristics were similar for age andsex the primary melanoma group included women and men with a mean age of ± years the metastaticmelanoma group included women and men with a meanage of ± years and the control group included women and men with mean age of ± years Materials and Reagents In this work the assessment ofantiganglioside antibodies was made by the immunoblottechnique using EUROLine kits Figure This methodallows the evaluation of antibodies IgG and IgM classesagainst GM1 GM2 GM3 GD1a GD1b GT1b and GQ1bfrom serumplasma The kit contains strips marked withpurified antigens Table Theevaluation ofantigangliosideantibodies wasperformed using the EUROLine Scan software After readingthe signal intensity on the strips marked with ganglioside antigens the results were evaluated and the results are presented asoptical sensibility The assessment of IL8 was performed bythe ELISA method using Enzo Life Science reagents withTECAN analyser and the results are presented as pgdl CRPwas assessed by immunoturbidimetry using Human reagentsand HumaStar300 analyser the results are presented as mgdl Statistical Analysis All the results were analysed usingIBM SPSS Statistics We evaluated the normality of datadistribution using the KolmogorovSmirnov test The variationbetween groups was determined using the parametric teststtest when two groups were compared or ANOVA test whenmore groups were compared and nonparametric tests likeMannWhitney or Wilcoxon The correlation between groupswas evaluated using linear regression and Pearson coefficientp was considered with statistical significance ResultsAntiGM1 GM2 GM3 GD1a GD1b GT1b andGQ1b autoantibodies determined in primary metastaticmelanoma had diï¬erent serological profiles compared tothe control group The presence of IgG and IgM antiganglioside antibodies in primary melanoma was as followsantiGM1 and antiGM2 and antiGM3 and antiGD1a and antiGD1b and antiGT1b and antiGQ1b and Inmetastatic melanoma IgG and IgM antiganglioside antibodies had the following profile antiGM1 and antiGM2 and antiGM3 and antiGD1a and antiGD1b and antiGT1b and and antiGQ1b and In the control group antiganglioside autoantibodies were absentThe assessment for IgG antiGM1 antiGM2 antiGM3antiGD1a antiGD1b antiGT1a and antiGT1b showedextremely low signal intensity in all groups Figure Whencomparing the mean of signal intensity for IgG no statisticaldiï¬erences were observed between groups We obtained a statistically significant diï¬erence in IgM antiGM1 antiGM2antiGM3 antiGD1a antiGD1b antiGT1a and antiGT1b when comparing primary melanoma respectively metastatic melanoma to the control group and once more whencomparing primary versus metastatic melanoma Table To evaluate if the presence of IgM antibodies was associatedwith melanoma development we determined their relation to‚ammatory factors IL8 and CRP recommended by AJCCfor melanoma staging Table IL8 levels were statistically significantly increased in primary melanoma ± pgmland in metastatic melanoma ± pgml when compared with the control group ± pgml CRP levelswere found in primary ± ngml and metastaticmelanoma ± ngml significantly higher when compared with the control group ± ngml IL8 andCRP had no statistically significant variation when comparedto primary versus metastatic melanoma groups Positive correlations with statistical significance were determined betweenantiGM1 and CRP respectively IL8 between antiGD1aand CRP respectively IL8 Figure Negative significant correlations were observed between antiGM3 antiGT1b andCRP respectively IL8 Figure High levels of CRP and IL were associated with an increase in antiGM1 antiGD1aand a decrease in antiGM3 antiGM2 antibodies of IgM typeTable DiscussionsMelanoma the most aggressive skin tumour is a multifactorial cancer being the result of the interplay between geneticimmunological and environmental factors [“] Gangliosides due to their expression on tumor cells have beeninvolved in tumor biology and immunogenicity and hence 0cJournal of Immunology ResearchAntiGM1 IgM and IgG signalintensity in all groups AntiGM2 IgM and IgG signalintensity in all groups ControlPrimary melanomaMetastatic melanomaControlPrimary melanomaMetastatic melanomaAntiGM1 IgM Ž AntiGM1 IgGaAntiGM3 IgM and IgGintensity levels in all groups AntiGM2 IgM Ž AntiGM2 IgGbAntiGD1a IgM and IgGintensity signal in all groups ControlPrimary melanomaMetastatic melanomaControlPrimary melanomaMetastatic melanomaAntiGM3 IgM Ž AntiGM3 IgGcControlPrimary melanomaMetastatic melanomaAntiGD1b IgM and IgG signalintensity in all groupsControlPrimary melanomaMetastatic melanomaAntiGD1b IgM Ž AntiGD1b IgGAntiGD1a IgM Ž AntiGD1a IgGdAntiGT1b IgM and IgGintensity levels in all groups AntiGT1b IgM Ž AntiGT1b IgGefAntiGQ1b IgM and IgGintensity levels in all groupsControlPrimary melanomaMetastatic melanomaAntiGQ1b IgM Ž AntiGQ1b IgGgFigure Antiganglioside signal intensity in all groups ˆ—p have been considered as targets for cancer immunotherapy[“] The probability thatsome tumourassociatedganglioside determinants induce a human immune responsegenerated much interest in medical research [ “]If endogenously synthesized antiganglioside antibodies reactonly with human cancer cells these antibodies could play animportant role in the host™s protective immunity to thetumor There is little information about quantitative variations of serum antigangliosides their origin and progressionof melanoma Tumour ganglioside antigens generate a significantly increased immune response in patients with primarymelanoma versus metastatic melanoma In our study thehost™s ability to generate an early antiganglioside responseis supported by a significantly increased titter of IgMantibodies in patients with primary versus metastatic melanoma and the control groupfor antiGM1 antiGM2antiGM3 antiGD1a antiGD1b antiGT1b and antiGQ1b Figure The range of antiganglioside antibodiescould serve as an indicator of diï¬erentiation between patientswith primary melanoma and metastatic melanoma Based on 0cJournal of Immunology ResearchTable IgM antiganglioside signal intensity in all groupsAntibodies Classp significancePM vs MM PM vs control MM vs controlIgMAntiGM1IgMAntiGM2IgMAntiGM3AntiGD1aIgMAntiGD1b IgMAntiGT1bIgMAntiGQ1b IgMMM metastatic melanoma PM primary melanoma psignificancestatisticalTable IL8 and CRP in all studied groupsStudy groupPrimarymelanomaMetastaticmelanomaControlgroupCRPmgdl ± ± ± psignificance”IL8pgml ± ± ± psignificance”our findings we estimate that the levels of the antigangliosideantibodies could provide information regarding the clinicalstaging of melanomaIn addition the capacity of patients to develop an antiganglioside response in the early stage of development of melanoma could be understood as a mean of defence of the bodythrough eliminating a danger signal from the tumour microenvironment represented by the stimulation of glycosphingolipid synthesis [ ] Synthesis of antiganglioside antibodiescould confer a survival advantage in patients with primarymelanoma [] In metastatic melanoma patients we observeda reduction of antiganglioside antibody synthesis a result thatcould suggest the immunosuppressive eï¬ect exerted by theoverproduction of gangliosides associated with tumour metastasis andor due to the overall decreased immune responseIt has been shown in a previous study that in patients withuntreated primary melanoma there is a significant statisticalcorrelation between antiGM1 type IgM level and clinicalstage of the disease Breslow index Clark level tumour localization histologic type presenceabsence of ulceration [ ]In our study patients with cutaneous melanoma had detectable levels of antiGM1 in primary stages The presence of apositive significant relationship between IgM antiGM1 leveland IL8 and CRP in our study justified our statement regarding the involvement of these antibodies in tumour proliferation by stimulating ‚ammation [ “] The presentstudy is the first one that evaluated the relation betweenantiganglioside antibodies and IL8 and CRP based on theirrole in melanoma diagnosis progression and outcome [“] in metabolic disorders [ ]Previous studies in patients with prostate cancer [] orsarcoma [] have shown that antiGM1 antibodies had nodiagnostic or prognostic value in these pathologies In patientswith diï¬erentiated thyroid cancer antiGM1 type IgG andIgM were associated with carcinogenesis but the lack of correlation between antibody level and clinical status indicated thatantiGM1 had no diagnostic value in diï¬erentiated thyroidcancer []Another study performed by our group showed thatpatients with primary melanoma with a high level of IgMantiGM3 had a favourable prognosis compared withpatients displaying a low antibody titer [] In a study onpatients with primary untreated melanoma stages I andII lymph nodes clear of metastasis it was shown that theantiGM2 antibody titer for IgMtype was not diï¬erentiatedin correlation to the tumor thickness For antiGM3 it wasobtained a direct relationship between the serum titer andthe thickness of the tumor [] AntiGM2 and antiGM3antibodies have no diagnostic significance in thyroid cancerdue to the low prevalence of these antibodies [] In ourpresent study antiGM3 negative correlations with IL8and CRP are suitable with the hypothesis that patients withprimary melanoma with a high level of IgM antiGM3 havea favourable prognosisGD1a was thought to generate an immune response inpatients with earlystage melanoma [] In patients withT1T2 stage prostate cancer there were identified increasedIgM antiGD1a values compared to the T3T4 stage whichsustains the development of an early endogenous immuneresponse able to eliminate the danger signal from the tumormicroenvironment These data support the role of antiGD1ain the early diagnosis of localized prostate disease [] TheantiGD1a IgMtype titer was defined as a negative predictivefactor of survival in patients with soft tissue sarcoma [] andin patients with primary melanoma [] In patients™ serumdiagnosed with ovarian cancer an increased titer of IgMantiGD1 was found the authors pointing out that theseantibodies could represent immunological markers associated with ovarian cancer progression [] In cutaneousmelanoma IgM antiGD1a could be considered a markerassociated with melanoma progression based on the negativecorrelation with CRP and IL8 as shown in our studyThe antiGD1b immune response in patients with gastricneoplasm can be used as a prognostic marker [] On thecontrary the lack of correlation between the presence ofantiGD1b and the clinical status of patients with thyroidcancer has indicated that antigangliosides do not have diagnostic significance in this neoplasm []The antiGT1b titer can be an overall positive factor associated to global survival in sarcoma [] AntiGD1b GT1band GQ1b antibodies that are negatively correlated with IL8and CRP suggest that they could indirectly suppress tumorgrowth and angiogenesis AntiGD1b GT1b and GQ1bIgM type ‚uence the progression of melanoma [ ]soft tissue sarcomas [ ] Ehrlich subcutaneous solidtumors [ ] Ehrlich carcinomas accompanied by ascites[ ] and gastric cancer []Our study limitations could be considered the semiquantitative assessment method of antiganglioside antibodies as aquantitative determination technique could oï¬er more sensitive data ing the door for further studies One more 0cJournal of Immunology ResearchMGitnAMGitnAaDGitnA CRP CRP CRP MGitnAMGitnAaDGitnAIL8IL8IL8Figure AntiGM1 antiGM3 and antiGD1a in relation to CRP and IL8 in the primary melanoma groupimportant limitation of the study is that we evaluated thecorrelation between antiganglioside antibodies and ‚ammation markers in melanoma IL8 and CRP only after orbetween the surgical treatment of melanoma newer therapiesbeing also in place This could be the first study which otherresearchers and clinicians can use and analyze in order toevaluate the ‚uence of diï¬erent melanoma treatments onantiganglioside antibodies The pathogenic mechanismsinvolved in melanoma are complex [“] therefore theevaluation during the followup period of melanoma patientsat diï¬erent points is needed for a better antigangliosideprofile characterization 0cJournal of Immunology ResearchTable AntiGM1 GM2 GM3 GD1a GD1b GT1b andGQ1b relation with ‚ammatory markers in primary melanomagroupAntiGM1AntiGM2AntiGM3AntiGD1aAntiGD1bAntiGT1bAntiGQ1bCRPr p ‰¤ r ˆ’p r ˆ’p r p ‰¤ r ˆ’p ‰¤ r ˆ’p ‰¤ r ˆ’p IL8r p ‰¤ r ˆ’p r ˆ’p r p ‰¤ r ˆ’p ‰¤ r ˆ’p ‰¤ r ˆ’p ConclusionsAntiganglioside antibodies antiGM1 and GD1a werepositively correlated while antiGM3 GD1b and GT1bwere negatively associated with the ‚ammatory markersIL8 and CRP The host™s ability to elaborate an early antiganglioside response could be considered as a defence mechanism directed toward eliminating a danger signal from thetumour microenvironment Moreover our results suggest thattumour ganglioside antigens generate a significantly increasedimmune response in patients with primary versus metastaticmelanoma Antiganglioside antibodies associated with ‚ammation markers could be used as diagnostic monitoring andtreatment tools in patients with cutaneous melanomaData AvailabilityThe data used to support the findings of this study areincluded within the articleConflicts of InterestThe authors declare no conflicts of interestAuthors™ ContributionsAll authors have equally contributed to the writing and editing of the manuscriptAcknowledgmentsThis research and article processing charges were funded by agrant of the Romanian Ministry of Research and InnovationCCCDIUEFISCDI project number 61PCCDI2018 PNIIIP112PCCDI2017034References[] YH Xu S Barnes Y Sun and G A Grabowski œMultisystem disorders of glycosphingolipid and ganglioside metabolism Journal of Lipid Research vol no pp “ [] I Horwacik and H Rokita œTargeting of tumorassociatedgangliosides with antibodies aï¬ects signaling pathways andleads to cell death including apoptosis Apoptosis vol no pp “ [] J L Daniotti R D Lardone and A A Vilcaes œDysregulatedexpression of glycolipids in Tumor cells from negative modulator of Antitumor immunity to promising targets for developing therapeutic agents Frontiers in Oncology vol p [] I Nicolae A Caragheorgheopol S Schipor œGnagliosides and sex hormones in human melanoma Acta Endocrinologica vol no pp “ [] Y Ohmi M Kambe Y Ohkawa œDiï¬erential roles ofgangliosides in malignant properties of melanomas PLoSONE vol no article e0206881 [] M H Ravindranath S Muthugounder N Presser A D Santin R S Selvan and D L Morton œGanglioside GD1a present in ovarian cancer cells ascites and sera of patients elicitsendogenous IgM response Proceedings of the American Association for Cancer Research vol p [] V B Doronin T A Parkhomenko M Castellazzi et alœComparison of antibodies hydrolyzing myelin basic proteinfrom the cerebrospinal fluid and serum of patients with multiple sclerosis PLoS One vol no article e107807 [] C D Ene and I Nicolae œGangliosides and Antigangliosidesin Malignant Melanoma Melanoma Current Clinical Management and Future Therapeutics M Murph Ed MandiMurph Intech ISBN [] N AlvarezRueda S Leprieur B Clemenceau œBindingactivities and antitumor properties of a new mousehumanchimeric antibody specific for GD2 ganglioside antigen Clinical Cancer Research vol no pp 5613s“5620s [] K Bennaceur I Popa J A Chapman œDiï¬erent mechanisms are involved in apoptosis induced by melanoma ganglicellsosidesGlycobiology vol no pp “ on human monocytederived dendritic[] C D Nicolae and I Nicolae œAntibodies against GM1 gangliosides associated with metastatic melanoma Acta Dermatovenerologica Croatica vol no pp “ [] S GrouxDegroote Y Guerardel and P Delannoy œGangliosides structures biosynthesis analysis and roles in cancerChembiochem vol no pp “ [] C Tringali I Silvestri F Testa œMolecular subtyping ofmetastatic melanoma based on cell ganglioside metabolismprofiles BMC Cancer vol no p [] M Neagu C Constantin C Caruntu C Dumitru M Surceland S Zurac œInflammation a key process in skin tumorigenesis Oncology Letters vol no pp “ [] R Takeuchi M Kambe M Miyata œTNFαsignal andcAMPmediated signals oppositely regulate melanomaassociated ganglioside GD3 synthase gene in human melanocytes Scientific Reports vol no p [] A H Otake R de Freitas Saito A P M Duarte A F Ramosand R Chammas œGD3 gangliosideenriched extracellular vesiclesetstimulate melanocyte migration Biochimica 0cJournal of Immunology ResearchBiophysica Acta Molecular and Cell Biology of Lipidsvol no pp “ [] K Hamamura K Furukawa T Hayashi œGangliosideGD3 promotes cell growth and invasion through p130Casand paxillin in malignant melanoma cells Proceedings of theNational Academy of Sciences of the United States of Americavol no pp “ [] M Neagu C Constantin C Caruntu M Surcel D Boda andS Zurac œCytokine pattern for improving immunoscore inmelanoma patients European Journal ofImmunologyvol pp [] M Neagu C Constantin and C Longo œChemokines in themelanoma metastasis biomarkers portrait Journal of Immunoassay Immunochemistry vol no pp “ [] K A Timani B Gyorï¬y Y Liu K S Mohammad and J J HeœTip110SART3 regulates IL8 expression and predicts theclinical outcomes in melanoma Molecular Cancer vol no p [] S J Wigmore K C H Fearon J P Maingay P B S Lai andJ A Ross œInterleukin8 can mediate acutephase protein production by isolated human hepatocytes American Journal ofPhysiologyEndocrinology and Metabolism vol no pp E720“E726 [] M Bickel œThe role of interleukin8 in ‚ammation andJournal of Periodontologyregulationmechanisms ofvol no pp “ [] M Neagu C Constantin and S Zurac œImmune Parametersin The Prognosis and Therapy Monitoring of Cutaneous Melanoma Patients Experience Role and Limitations BioMedResearch International vol Article ID pages[] S R Georgescu M R Ioghen M I Sarbu œBiologicaltherapy in the treatment of melanoma Journal of Mind andMedical Sciences vol no pp “ [] A V Dumitru M Tampa S R Georgescu œImmunohistochemical mismatch in a case of rhabdomyoblastic metastaticmelanoma Romanian Journal of Morphology and Embryology vol no pp “ [] S N Pavri J Clune S Ariyan and D Narayan œMalignantMelanoma Plastic and Reconstructive Surgery vol no pp 330e“340e [] M Rastrelli S Tropea C R Rossi and M Alaibac œMelanoma epidemiology risk factors pathogenesis Diagnosis andClassification In Vivo vol no pp “ [] L LugovićMihić D Ćesić P Vuković G Novak BilićM Å itum and S Å poljar œMelanoma development currentknowledge on melanoma pathogenesis Acta Dermatovenerologica Croatica vol no pp “ [] M Costache A V Dumitru O M Pătraşcu œA challenging case of ocular melanoma Romanian Journal of Morphology and Embryology vol Suppl pp “ [] R Ancuceanu and M Neagu œImmune based therapy for melanoma Indian Journal of Medical Research vol no pp “ [] C A Perez M H Ravindranath D Soh A Gonzales W Yeand D L Morton œSerum antiganglioside IgM antibodies insoft tissue sarcoma clinical prognostic implications CancerJournal vol no pp “ [] B Mondal and S Sahal œInhibition of subcutaneous growth ofEhrlich ascites carcinoma EAC tumor by postimmunizationwith EACcell gangliosides and its antiidiotype antibody inrelation to tumor angiogenesis apoptosis cell cycle and ltration of CD4 CD8 lymphocytes NK cells suppressorcells and APCcells in tumor Indian Journal of ExperimentalBiology vol no pp “ [] S Sahal and S Mondal œSupression of Ehrlich subcutaneoussolid tumor growth by immunization with ganglioside GT1bof its origin its IgM antibody or antiidiotype antibody Journal of Experimental Clinical Cancer Research vol no p [] M M Konstandoulakis K N Syrigos M LeandrosA Charalabopoulos A Manouras and B C GolematisœAutoantibodies in the serum of patients with gastric cancertheir prognostic importance Hybridoma vol no pp “ [] M H Ravindranath S Muthugounder X Ye and D L Morton œInnate immune response to gangliosides of primary melanoma favors danger hypothesis Proceedings of the AmericanAssociation for Cancer Research vol p [] M H Ravindranath S Muthugounder and N Presser œGanglioside signatures of primary and nodal metastatic melanomacell lines from the same patient Melanoma Research vol no pp “ [] A Lewartowska T Pacuszka G Adler M Panasiewicz andW Wojciechowska œGanglioside reactive antibodies of IgGand IgM class in sera of patients with diï¬erentiated thyroidcancer Immunology Letters vol no pp “ [] I Nicolae C D Nicolae O A Coman M StefanescuL Coman and C Ardeleanu œSerum total gangliosides levelclinical prognostic implication Romanian Journal of Morphology and Embryology vol no pp “ [] C Nicolae and I Nicolae œHeterogeneity of gangliosides inmelanocytic tumors Acta Endocrinologica vol no pp “ [] S GrouxDegroote M RodríguezWalker J H Dewald J LDaniotti and P Delannoy œGangliosides in cancer cell signaling Progress in Molecular Biology and Translational Sciencevol pp “ [] I Nicolae C D E Nicolae and E Ceauşu œInvestigation onantigangliosides antibodies in asymptomatic HIV patientsBMC Infectious Diseases vol no S4 p [] G N Tzanakakis M Neagu A M Tsatsakis and D NikitovicœProteoglycans and immunobiology of cancer therapeuticimplications Frontiers in Immunology vol p [] Q Li M Sun M Yu œGangliosides profiling in serum ofbreast cancer patient GM3 as a potential diagnostic biomarker Glycoconjugate Journal vol no pp “[] C D Ene A E Anghel M Neagu and I Nicolae œ25OHvitamin D and interleukin8 emerging biomarkers in cutaneous melanoma development and progression Mediators ofInflammation vol Article ID pages [] N R Sproston and J J Ashworth œRole of Creactive proteinat sites of ‚ammation and infection Frontiers In Immunology vol p [] A E Anghel C D Ene M Neagu and I Nicolae œThe relationship between interleukin8 and Ki67 in cutaneous malignant melanoma HVM Bioflux vol no pp “[] C D E Nicolae and I Nicolae œInterleukin 8serumconcentration but not lactate dehydrogenase activity positively correlates to CD34 antigen in melanoma tumors Journal of 0cJournal of Immunology ResearchImmunoassay and Immunochemistry vol no pp “ [] A E Anghel C D Ene I Nicolae V A Budu C Constantinand M Neagu œInterleukin major player in cutaneous melanoma metastasic process Romanian Biotechnological Letters vol no pp “ [] M Neagu œMetabolic traits in cutaneous melanoma Frontiers in oncology vol p [] C D Ene A E Anghel M Neagu and I Nicolae œInterleukin and diabetic nephropathy Human and Veterinary Medicine vol no pp “ [] M Neagu C Constantin I D Popescu œInflammationand metabolism in cancer cell “ mitochondria key playerFrontiers in Oncology vol p [] C Nicolae I Nicolae and O Coman œGD1b GT1b
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" exosomes are extracellular vesicles containing a variety of biological molecules including micrornasmirnas we have recently demonstrated that certain mirna species are selectively and highly enriched inpancreatic cancer exosomes with mir1246 being the most abundant exosome mirnas have been shown tomediate intercellular communication in the tumor microenvironment and promote cancer progression thereforeunderstanding how exosomes selectively enrich specific mirnas to initiate exosome mirna signaling in cancercells is critical to advancing cancer exosome biologyresults the aim of this study was to identify rna binding proteins responsible for selective enrichment ofexosome mirnas in cancer cells a biotinlabeled mir1246 probe was used to capture rna binding proteins rbpsfrom panc1 cells among the rbps identified through proteomic analysis srsf1 eif3b and tia1 were highlyassociated with the mir1246 probe rna immunoprecipitation rip and electrophoretic mobility shift assay emsaconfirmed the binding of srsf1 to mir1246 lentivirus shrna knockdown of srsf1 in pancreatic cancer cellsselectively reduced exosome mirna enrichment whereas gfpsrsf1 overexpression enhanced the enrichment asanalyzed by next generation small rna sequencing and qrtpcr mirna sequence motif analysis identified acommon motif shared by of srsf1associated exosome mirnas emsa confirmed that shared motif decoysinhibit the binding of srsf1 to the mir1246 sequences we conclude that srsf1 mediates selective exosome mirna enrichment in pancreatic cancer cells bybinding to a commonly shared mirna sequence motifkeywords srsf1 exosome mirna mir1246 pancreatic cancer exosomes are endosomederived extracellular vesiclesevs that can be transferred from cancer cells tostromal cells in the tumor microenvironment [ ]these membrane vesicles are “ nm in size andcontain proteinsincludinglipids and nucleic acids correspondence weiqundingouhscedu1department of pathology university of oklahoma health sciences centeroklahoma city stanton l young blvd bmsb 401a oklahoma city ok usa6stephenson cancer center university of oklahoma health sciences centeroklahoma city ok usafull list of author information is available at the end of the small rnas such as micrornas mirnas[ ]exosomemediated intercellular communication betweencancer cells endothelial cells [ ] fibroblasts [ ] orimmune cells [ ] can facilitate tumor progressionfurthermore cancer exosomes are released into the circulation and contribute to premetastatic niche formation in distant ans [ ]how cancer exosomes interact with stromal cells topromote tumor progression has been extensively investisignaling eventgated one criticalin the tumormicroenvironmentisthe exosome mirnamediatedintercellular communication [ “] studies haveshown that exosome mirna signaling promotes tumor the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cxu cell communication and signaling page of progression in various model systems [ ] notablyit has been reported that mirnas contained in exosomes are delivered to recipient cells in the tumormicroenvironment or distant ans where they canregulate target gene expression and promote tumorangiogenesis and metastasis [ ]in the context of exosome mirna signaling we andothers have reported that certain mirna species areselectively enriched in cancer exosomes as compared toexosomes derived from normal epithelial cells [ “] results from several studies have also indicated thatselective enrichment of exosome mirnas is relevant totumor progression for example exosome sortingof mir193a was found to promote colon cancer progression likewise mir122 a cancer exosomeenriched mirna [ ] was shown to reprogram glucose metabolism in a premetastatic niche to facilitatemetastasis in a breast cancer model system moreover the exosome enriched mir1246 was reportedto promote tumor invasion in both breast cancer and oral squamous cell carcinoma it seems clearthat selective enrichment of exosome mirnas drivescancer exosome mirna signaling in the tumor microenvironment which in turn reinforces tumor invasiveness and progression however how exosome mirnasare enriched or how exosome mirna signaling is initiated in cancer cells remains largely unknown elucidating the mechanisms ofselective exosome mirnaenrichment in cancer cells may help identify new cancertherapeutic opportunities that are urgently neededrecentreports have indicated that certain rnabinding proteinsrbps are involved in exosomemirna sorting in eukaryotic cells and the type ofrbps involved seems to differ among various modelsystems [ ] suggesting that exosome mirnasorting is a tissue or celltype specific processfurthermore there have been no reports on the identification of rbps that regulate exosome mirna sorting in pancreatic cancer cells we have recentlycharacterized the biogenesis of exosome mir1246 which is the most highly enriched mirna inpancreatic cancer cellderived exosomes the aimof this study was to utilize our established cell modelsystems to identify rbps that are involved in exosomemirna loading in pancreatic cancer cells using alabeled mir1246 probe as œbait we fished out several rbpsincludingserine and arginine rich splicing factor srsf1eukaryotic translation initiation factor subunit beif3b and t cellrestricted intracellular antigen tia1 we found that srsf1 a recently claimedoncoproteininregulating exosome mirna enrichment in pancreaticcancer model systemsfrom pancreatic cancer cellspredominantlyinvolved ismethodscell culturelines panc1the human pancreatic cancer celllinemiapaca2 and bxpc3 and breast cancer cellmdamb231 were obtained from the american typeculture collection atcc manassas va usa cellswere cultured following atcc™s instructions except thatexosomedepleted fetal bovine serum fbs and horseserum wereapplied whenever needed exosomedepleted fbs and horse serum were prepared by pelleting the serum exosomes at —g for h at °ccells were routinely incubated in a humidified environment at °c and co2exosome isolationexosomes were isolated from the culture medium utilizing a combination of centrifugation ultracentrifugationand filtration as we recently described [ ] withminor modifications in brief the culture medium ofpanc1 cells was precleared by g centrifugationfor min at °c and the resulting supernatant was filtered through a μm pvdf centrifuge filter thelarge size evs were trapped in the filter and recovered inpbs the filtered supernatant was then applied to a μm pvdf centrifuge filter the medium size evswere trapped in the second filter and resuspended inpbs the small size evs exosomes in the final supernatant were recovered by ultracentrifugation g min at °c the isolated exosomes were verified bywestern blot detecting positive and negative exosomemarker proteins and nanop analysis nanosightns300 system malvern instruments uk measuringboth sizes and concentrations of the isolated exosomesfig mirna binding protein pulldownpulldown experiment was performed using the pierce„¢magnetic rnaprotein pulldown kit thermo fisherscientific briefly pmol of biotinlabeled mir1246or polya rna oligonucleotides integrated dna technologies were hybridized to μl streptavidin magnetic beads prod1862766 thermo fisher scientificthe mir1246biotinstreptavidin beads were incubatedwith panc1 lysate for min at °c the lysatebeadmixture was washed three times with washing bufferfrom the abovementioned kit to elute bound proteins μl of elution buffer was applied and a magnetic separator was applied to separate the beads from the elutedprotein following the manufacturer™s protocol pierce„¢magnetic rnaprotein pulldown kit thermo fisherscientific proteins were separated by sdspage beforemass spectrometry ms analysis 0cxu cell communication and signaling page of fig verification of the exosomes derived from panc1 cells a representative western blot analysis of cd63 nonreducing condition cd81flotillin and calnexin in the evs isolated from panc1 cells positive exosome markers are only detected in small evs exosomes b representativenanop tracking analysis of exosomes small evs derived from control and srsf1 knockdown panc1 cells three individual experimentswere performed for both a and bliquid chromatography“mass spectrometry lcmsmassspectrometry ms measurementthe experiment was performed by the laboratory formolecular biology and cytometry research core facilityat ouhsc proteins were digested with trypsin according to the fasp protocol briefly the eluate was buffer exchanged in m urea the proteins were reducedwith mm dithiothreitol and then alkylated with mm iodoacetamide the peptides were eluted dried andresuspended liquid chromatography tandem mass spectrometry was performed by coupling a nanaoacquityuplc waters corp manchester uk to a qtofsynapt g2s instrument waters corp manchesteruk each protein digest about ng of peptide wasdelivered to a trap column μm — mm nanoacquity uplc nanoease column μm beh c18 waterscorp manchester uk at a flow rate of μlmin in solvent a mm ammonium formate ph inhplc grade water tandem mass spectra were generated in the trapping region of the ion mobility cell byusing a collisional energy ramp from v low massstartend to v high mass startend the pusherionmobility synchronization for the hdmse method wasperformed using masslynx v41 and driftscope v24lockspray of glufibrinopeptideb mz wasacquired every s and lock mass correction was appliedpost acquisitionprotein identificationraw ms data were processed by plgs proteinlynxglobal server waters corp manchester uk for peptide and protein identification msms spectra weresearched against the uniprot human database containing reviewed sequences with the followingsearch parametersfull tryptic specificity up to twomissed cleavage sites carbamidomethylation of cysteineresidues was set as a fixed modification and nterminalprotein acetylation and methionine oxidation were set asvariable modificationssmall rna library preparation and next generationsequencingtotal rna was extracted from cell and exosome pelletsusing the trizol reagent invitrogenlife technologiescarlsbad california the small rna libraries were constructed and run on the illumina miseq platform as werecently described [ ]rna immunoprecipitation assaypanc1 cells or mdamb231 celllysates were prepared using ip buffer mm trishcl ph mmnacl mm edta mm pmsf and triton x the lysate was sonicated for min on ice andinsoluble material was removed by centrifugation supernatants were collected and protein concentrations weremeasured the supernatant was precleared by proteing dynabeads„¢thermo fisher scientific and thenmixed with antibody srsf1 santa cruz sc33652eif3b santa cruz sc137214 tia1 santa cruz sc gapdh promab and igg santacruz sc2025 in a ratio of at °c overnight withgentle rotation to capture the antibodyproteinrnacomplexes μl of protein g magnetic beads wereadded and the complexes were rotated for h at °cthe sample was separated by magnetic separation trizol reagent invitrogenlife technologies was appliedto isolated rna from the complex the mirna expression was analyzed by qrtpcr 0cxu cell communication and signaling page of coimmunoprecipitation coipcoimmunoprecipitation coip using panc1 cell lysate and antibody of srsf1 santa cruz sc33652eif3b santa cruz sc137214 tia1 santa cruz sc gapdh promab and igg santacruz sc2025 was performed as described previously and the protein complex was detected by westernblotwestern blot analysiswestern blot was performed as we recently described[ ] primary antibodies raised against srsf1 santacruz sc33652 eif3b santa cruz sc137214 tia1santa cruz sc166247 betaactin a5441 and glyceraldehyde 3phosphate dehydrogenase gapdh santacruz sc47724 were used for detection nuclear andcytoplasmic protein extraction was extracted followingrockland nuclear cytoplasmic extract protocol and verified by histoneh3 cst 4499s andgapdh santa cruz sc47724 detection antibodiesused for exosome marker detection include cd63cd81 santa cruz bio technology inc ca usaflotillin1 and calnexin cell signaling technology incma usaquantitative realtime reverse transcription polymerasechain reaction qrtpcrqrtpcr was performed as we described [ ] withspecific primers cel54 ²gcgcgcccgtaatcttcataatcc3² mir1246 ²gcgcgatggatttttggagcag3² mir320c ²gcaaaagcuggguugagagggu3² and mir320d ²gcgaaaagcuggguugagagga3²srsf1 shrna expression plasmid constructiontarget specific oligonucleotides were designed using online tool rnai codex cold spring harbor laboratoryand were synthesized integrated dna technologieswith the addition of overhangs according to the cuttingsite of bamh1 and ecori the shrna expression plasmid was constructed by annealing the oligonucleotidesto psihh1 vector following the user manual of psihh1 shrna system sbi system bioscience the oligonucleotide sequences for shrna of srsf1 eif3b ortia1 are provided in supplemental table 3rd generation packaging plasmidslentivirus transductionlentiviral ps were produced as previously described using the shrna expression plasmid andthepmd2gaddgene plasmid pmdlrregp addgeneplasmid and prsvrev addgene plasmid the packaging plasmids were cotransfectedwith the lentiviral expression vector into t cellsusing the polyethyleneimine polysciences inc to produce replication deficient lentivirus after transfectionthe supernatant was pooled and filtered with a μmmembrane and concentrated by ultracentrifugation toacquire lentivirus infection was performed by usinglentivirus in the presence of μgml polybrene sigmaaldrich approximately h postinfection cells wereselected by treating with μgml puromycin invivogen san diego cagfpsrsf1 expressionthe gfpsrsf1 expression plasmid was a gift from drmassimo caputi dna transfection was performedusing lipofectamine thermo fisher scientific topanc1 cells and the expression of gfpsrsf1 wasverified by western blotgstsrsf1 protein purificationbl21 thermofisher scientific c600003 competentcells transformed with pgex6psrsf1 dna addgeneplasmid were cultured at °c for hand after od600 reached to “ bacteria weretreated with mm isopropyl βd1thiogalactopyranoside for h at °c gsttaggedsrsf1 was purifiedwith glutathione sepharose beads ge health carethe purity of the recombinant proteins was determinedby sds“page with coomassie blue stainingelectrophoretic mobility shift assay emsaird800 labeled mir1246 μm integrated dnatechnologies was mixed with μl of gst slurry stsrsf1 in binding buffer tris ph mm kcl mm mgcl2 mm np40 dtt mm glycerol and incubated at room temperature for minavoiding light 5x loading buffer kcl mm tris ph mm glycerol xylene cyanol bromophenol blue was then added and the complexwas separated on a native gel polyacrylamide m tris ph m glycine m edta apstemed at voltage for min the signal was detected using the licor odyssey imaging systemlicor inc usadesign of decoy motif mimicsthe decoy motif mimics were designed by permutationand combination of the identified motif sequences in thelength of nucleotides the secondary structure of thedesigned sequences was analyzed in rnafold webserveruniversityselfcomplementary were selected decoy mimics ²uuggacuaggacuaggau3² decoy mimics ²aggaaggaaggaagga3²sequences withoutof vienna 0cxu cell communication and signaling page of bioinformatics analysisthe mirna motif analysis was performed using memesuite the protein profile analysis for the result ofmass spectrometry was performed using david bioinformatics abcc at saicfrederick inc the rnabinding protein and mirna sequence binding analysiswas performed using the database of rnabindingspecificities rbpdb srsf1 expression in cancertissues was examined using oncomine thecorrelation of gene expression with cancer patient survival was extracted from the human protein atlasscilifelab sweden statisticsstatistical analyses were performed using graphpadprism software graphpad software inc la jolla causa the heatmap was made in rstudio rstudio incwith the ggplot2 package student™s ttest was applied to determine significant differences among controland experimental groupsresultsidentification of mir1246 associated proteinsbecause rbps are involved in exosome mirna sortingwe first sought to identify proteins that bind to mirnashighly enriched in cancer exosomes mir1246 the mosthighly enriched mirna in pancreatic cancer exosomeswas biotinlabeled and incubated with a cellular lysatefrom panc1 cells the biotinmir1246 probe wascaptured with streptavidincoated magnetic beads biotinlabeled polya mimics were used as control themirnaprotein complexes were eluted and the proteinswere analyzed by liquid chromatographymass spectrometry in triplicate table there were total of proteins specifically pulled down by the mir1246 probeinterestingly about half of the proteins that associatewith mir1246 are vesicleassociated proteins supplement fig 1a based on the intensity of detection rnabinding property and cancer relevance we ranked therbps using œthe database for annotation visualizationand integrated discovery david this resulted in tencandidate rbps that complex with the mir1246 sequenceexosomestable among them srsf1 also called sfrs1 waspredictedsequencethe mir1246and arerelevantto eukaryotictobindtotable over view of the result of mass spectrometryexperiments™ conditionpoly a panc1number of proteins detectedpoly a mdamb231mir1246 panc1mir1246 mdamb231table mir1246 rna binding protein candidates obtainedfrom the mass spectrometric analysisprotein full nameprotein symbolsrsf1serineargininerich splicing factor park7eif3bthoc4acocddx5tia1if5a1eif2aimdh2parkinson disease protein eukaryotic translation initiation factor subunit btho complex subunit alyref export factorcytoplasmic aconitate hydrataseprobable atpdependent rna helicase ddx5tcellrestricted intracellular antigen1eukaryotic translation initiation factor 5a1eukaryotic translation initiation factor 2ainosine5²monophosphate dehydrogenase supplement fig by in silico analysis using the database of rnabinding specificities rbpdb levelsverification of srsf1 binding to mir1246rna immunoprecipitation rip was performed to verifythe association of several identified rbps with mir1246including srsf1 eif3b and tia1 igg and gapdhantibody was used as controls for immunoprecipitationas shown in fig 2a mir1246 expression is more than12fold higher in the srsf1precipitants as compared tothat of igg precipitants indicating a specific associationof srsf1 with mir1246 mir1246 expression wasmoderately increased in the tia1precipitants and nearigg controlin the eif3b precipitants coimmunoprecipitation coip experiments were performed to verify the immunoprecipitation proceduresdata not shown to directly determine the binding ofsrsf1 to the mir1246 sequence glutathionestransferase gst conjugated human srsf1 protein wasexpressed in bl21 competent e coli captured by glutathione sepharose beads and eluted by glutathione thepurity of eluted gstsrsf1 protein was shown by sdspage and coomassie blue staining supplement fig the binding of gstsrsf1 to a fluorescenttaggedmir1246 probe was determined by rna emsa asshown in fig 2b binding of the labeled probe was specific to gstsrsf1 but not gst and increased withgreater protein input the specific binding of gstsrsf1 to the mir1246 probe was evident as the unlabeled mir1246 probe effectively competed with thelabeled mir1246 probe in a concentrationdependentmanner fig 2c the detected bands were semi quantified and the kd was calculated from the detected signalsfig 2d these data confirmed the direct binding ofsrsf1 to the mir1246 sequence 0cxu cell communication and signaling page of fig srsf1 binds to mir1246 a qrtpcr detection of mir1246 in igg gapdh srsf1 eif3b and tia1 immunoprecipitants of panc1 lysaten p student ttest bc emsa detection of the srsf1mir1246 complex hot probe ird800 labeled mir1246 mimics cold probemir1246 mimics n direct binding of gstsrsf1 and mir1246 b and concentrationdependent competition between the cold and hotmir1246 probe for binding to gstsrsf1 c d semiquantification of srsf1 and mir1246 binding in c and calculated dissociationconstant n exosome mirna enrichment by srsf1 in cancer cellsbecause srsf1 is a key splicing factor that is essential toeukaryotic cells a knockout model could not beestablished thereforeto determine whether srsf1mirna binding activity is relevant to exosome mirnaenrichment we established a lentivirus srsf1 shrnaconstruct to knockdown srsf1 expression in panc1cells fig 3a interestingly though srsf1 protein wasdetected both in the nucleus and cytoplasm the knockdown was more pronounced in the cytoplasm fig 3bknockdown of srsf1 did not significantly alter the concentration and size distribution of the exosomes releasedby panc1 cells fig 1b cellular and exosome rnafrom control and srsf1shrna cells were isolated andsmall rna sequencing was performed among the highly enriched panc1 exosome mirnas expressionof mirnas was significantly downregulatedin exosomes derived from srsf1shrna panc1 cellsas compared to exosomes derived from control panc1cells fig 3c strongly indicating the involvement ofsrsf1 in exosome mirna enrichment a heatmapshowing the expression of the top mirnas enrichedin panc1 exosomes demonstrates the dramatic dropin expression levels of mirnasin srsf1shrnapanc1 exosomes compared to panc1 exosomesfig 3d notably mir1246 was the highest enrichedexosome mirna data not shown and its expressionin exosomes wassignificantly reduced by srsf1knockdown fig 3d on the other hand among of the mirnas less enriched in exosomes only were expressed at lower levels in exosomesderived from srsf1 knockdown cells as compared toexosomes derived from wild type panc1 cells fig3c suggesting that srsf1 knockdown mainly affectsexosome enriched mirnasto further confirm the effect of srsf1 knockdown onexosome mirna enrichment the expression levels ofseveral representative mirnas were quantified by qrtpcr srsf1 knockdown in panc1 cells significantlyreduced exosome levels of mir1246 mir320c andmir320d confirming the small rna sequencing resultsfig 3e in contrast knockdown of eif3b or tia1 didnot reduce exosome mir1246 expression suggestingthat these rbps may not promote exosome mirna 0cxu cell communication and signaling page of fig see legend on next page 0cxu cell communication and signaling page of see figure on previous pagefig cellular and exosome mirna profiles after srsf1 knockdown in panc1 cells a detection of srsf1 knockdown by shrnas in panc1 cellsb panc1 srsf1 protein levels in nuclear and cytoplasmic fractions nc normal control c venn diagram of overlap of mirnas detected by nextgeneration small rna sequencing in srsf1 knockdown and control panc1 cells and exosomes d heatmap showing the expression of top exosome mirnas in cells and exosomes after srsf1 knockdown e qrtpcr analysis of mir1246 mir320c and mir320d in exosomes derivedfrom control and srsf1 knockdown panc1 cells p student™s ttest shown are representatives of three independent experiments aeenrichment supplement fig and our observationswere extended to two additional pancreatic cancer celllines miapaca2 and bxpc3 fig 4af in additionexpression of let7c which is less enriched in exosomeswas unchanged in exosomes after srsf1 knockdowndata not shownto verify the involvement of srsf1 in exosomemirna enrichment in cancer cells we also exogenouslyoverexpressed srsf1 in panc1 cells a gfpsrsf1 expression plasmid was introduced into panc1 cells andsrsf1 overexpression was confirmed by western blotfig 5a expression of mir1246 mir320c and mir320d in the exosomes derived from gfpsrsf1 panc1cells was analyzed by qrtpcr fig 5bc as shown infig 5b overexpression of gfpsrsf1 increased exosome expression of mir1246 and rescued mir1246levels in exosomes derived from srsf1shrna cellslevels of mir320c and mir320d were also increased inexosomes derived from the gfpsrsf1 cellsfurthersupporting the involvement of srsf1 in exosomemirna enrichment fig 5cdidentification of rna sequence motifs involved inexosome mirna enrichmentaccording to the rbpdb srsf1 binds specifically to amotif present in the mir1246 sequence supplementfig qrtpcr analysis of mir1246 mir320c mir320d expression in exosomes derived from srsf1 knockdown bxpc3 and miapaca2 cells ac qrtpcr detection of mir1246 mir320c and mir320d in exosomes derived from srsf1 knockdown bxpc3 cells n p student ttest df qrtpcr detection of mir1246 mir320c and mir320d in exosomes derived from srsf1 knockdown miapaca2 cells n p student™s ttest 0cxu cell communication and signaling page of fig qrtpcr analysis of exosome enriched mirnas derived from srsf1 overexpression panc1 cells a confirmation of gfpsrsf1overexpression in panc1 cells b qrtpcr detection of mir1246 in exosomes derived from wild type and srsf1 knockdown panc1 cells withgfpsrsf1 overexpression c qrtpcr detection of mir320c in exosomes derived from gfpsrsf1 overexpression panc1 cells d qrtpcrdetection of mir320d in exosomes derived from gfpsrsf1 overexpression panc1 cells p student™s ttest n for bdfig to understand the contribution of specific rnamotifs involved in exosome mirna enrichment we applied an unbiased approach to identify the rna motifsthat contribute to exosome mirna enrichment for thispurpose we analyzed the rna sequences of the mirnas highly enriched in cancer exosomes and regulatedby srsf1 using the bioinformatics tool meme suite a 6bp length motif was found to be shared in of the exosome enriched mirnasincluding mir fig ac to test whether the binding of srsf1to mir1246 depends on this motif two decoy mimicswere designed according to the shared motif sequencesand their secondary structure determined with thernafold webserver httprnatbiunivieacatcgibinrnawebsuiternafoldcgi the binding of the decoymimics to srsf1 protein was determined by rnaemsa analysis addition of decoy motif did not alterthe binding of srsf1 to the mir1246 probe fig 6dwhereas decoy motif competed with mir1246 binding to srsf1 in a concentrationdependent manner fig6df indicating that srsf1 directly interacts with thissequence motifdiscussionthe role of exosome mirna signaling in promotingcancer progression has been intensely investigated andwell recognized in recent years [ ] the higher enrichment of certain mirnas in cancer exosomes [“] indicates that exosome mirna encapsulation isan active cellular process that initiates exosome mirnasignaling in the tumor microenvironment however thespecificselectivecellular processresponsiblefor 0cxu cell communication and signaling page of fig see legend on next page 0cxu cell communication and signaling page of see figure on previous pagefig srsf1associated exosome mirna sequence motif analysis a the motif commonly shared among srsf1associated exosome mirnas bvenn diagram showing the number of srsf1associated exosome mirnas that share the motif c list of mirnas sharing the common motif demsa analysis demonstrating the inhibition of gstsrsf1 binding to mir1246 by rna decoys d1 decoy ²uuggacuaggacuaggau3² d2decoy ²aggaaggaaggaagga3² e concentrationdependent inhibition of gstsrsf1 binding to mir1246 by d2 f semiquantification ofthe detected bands in fig 5e and the calculated dissociation constantexosome mirna enrichment has not been well established in eukaryotic cells the most significant findingfrom the present study is that we have identified srsf1as a mediator of exosome mirna enrichment in pancreatic cancer cells a specific mirna sequence motif wasalso identified that may be involved in the exosomemirna enrichment process these findings provide newinsight into how mirnas are enriched in cancer cellexosomesexosomemediated mirnasignalinginitiatetowe recently reported that exosome mir1246themost highly enriched mirna in pancreatic cancer cellderived exosomes is derived from rnu2“ a smallnuclear rna important for mrna splicing alongthis line of our research we sought to determine howthis mirna is enriched in cancer exosomes using ourestablished model systems in the present study we haveprovided severallines of evidence demonstrating thatsrsf1 a vital splicing factor and established oncoprotein is significantly involved in exosome mirnaenrichment in pancreatic cancer cells the first line ofevidence indicating srsf1 involvementin exosomemirna enrichment was obtained from the biotinlabeled mir1246 pulldown experimentfollowed byproteomic analysis among the rbps identified severalwere selected based on their detection intensity relevance to extracellular vesicles and reported connectionsto human cancer including srsf1 eif3b andtia1 of note srsf1 was the only rbp among themthat was also predicted by the rbpdb to bind to a motifin the mir1246 sequence furthermore the direct binding of srsf1 to the mir1246 sequence was verified byrip and rna emsa analysis strongly indicating thephysical interaction of srsf1 and not eif3b or tia1with the mir1246 sequence the most convincing evidence demonstrating the involvement of srsf1 in cancer exosome mirna enrichment was the observationthat knockdown of srsf1 significantly reduces exosomemirna enrichmentfor a majority of the selectivelyenriched exosome mirnas without altering the expression levels of less enriched exosome mirnas these results were based on small rna sequencing andconfirmed by rtpcr analysis the observations werealso extended to additional human pancreatic cancer celllines including miapaca2 and bxpc3srsf1 was initially identified as a splicing factor ineukaryotic cells but srsf1 was later revealed toindependent ofshuttle between the nucleus and cytoplasm to regulate rna metabolism mirna procession and othercellular eventsthe mrna splicingprocess importantly srsf1 is overexpressed indifferent cancer types and is considered a potent oncogene [ ] moreover srsf1 over expression in different types of cancer is associated with worse prognosissupplement fig while the full spectrum of srsf1function remains to be determined our results revealthat srsf1 binds to specific mirnas and is significantlyinvolved in exosome mirna enrichment in cancer cellsthis function is likely independent ofthe splicingprocess as the reduced expression of the detected exosome mirnas after srsf1 knockdown is greater thantheir expression change in the cells because exosomemirna signaling contributes to tumor developmentthrough intercellular communication in the tumormicroenvironmentthe involvement ofsrsf1 in exosome mirna signaling initiation likelyrepresents a part of its oncogenic action which maylead to new therapeutic strategies to intervene withexosome mirna signaling in cancer several rbpshave been previously identified as mediators of exosome mirna sorting in various model systemsincluding major vault protein in colon cancer cells hnrnpa2b1 in t cells and ybx1 in hek293tcells the identification of srsf1 involvement inexosome mirna enrichmentin pancreatic cancercells further supports the notion that the cellular exosome mirna sorting process in eukaryotic cells maydiffer among different cell types[ ]we have also identified a mirna motif commonlyshared by the srsf1associated exosome mirnasusing the meme suite program memesuitethis motif was specifically bound by srsf1 as evidenced by our rna emsa analysis a similar motifalbeit slightly shorter was identified in our recentreport that describes exosome mir1246 enrichmentin pancreatic cancer cells our results reinforcethe concept that specific mirn
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" drug resistance leads to tumor relapse and further progression during chemotherapy in lung cancer close homolog of l1 chl1 has been identified as a tumor suppressor in most malignancies however to the best of our knowledge whether chl1 mediates chemoresistance remains unknown the present study observed that chl1 was significantly downregulated in cisplatin ddp‘resistant cells a549ddp and paclitaxel ptx‘resistant cells a549ptx compared with a549 cells when treated with or without ddp and ptx silencing of chl1 in a549 cells promoted the cell survival rate and clone formation and decreased apoptosis whereas overexpression of chl1 in a549ddp and a549ptx cells impeded the cell survival and clone formation and promoted apoptosis additionally chl1 overexpression enhanced the chemosensitivity of a549ddp cells to ddp in vivo notably the chemoresistance induced by chl1 depletion was reversed by the akt inhibitor sc66 in a549 cells the results of the present study demonstrated that chl1 enhanced sensitivity of lung cancer cells by suppressing the akt pathway which suggested that chl1 may be a potential target for overcoming chemoresistance in lung cancerintroductionlung cancer is the most common human malignancy accounting for of all cancer‘associated deaths worldwide during in addition its morbidity and mortality rank the highest among all malignant tumor types worldwide according to the differentiation degree and morphological correspondence to dr rimao huang department of cardiothoracic surgery xiangya changde hospital moon avenue west of langzhou north road changde hunan pr chinae‘mail xyhuangrm163comkey words lung cancer close homolog of cisplatin paclitaxel chemosensitivitycharacteristics of cancer cells lung cancer can be roughly clas‘sified into non‘small‘cell lung cancer nsclc and small‘cell lung cancer among patients with lung cancer nearly are diagnosed as nsclc which manifests with earlier diffu‘sion and metastasis currently resection chemotherapy radiotherapy and targeted therapy are the primary treatments for lung cancer for patients with advanced nsclc or those who are clinically incapacitated for surgery chemo‘therapy is a remarkably important treatment cisplatin ddp is widely applied in the treatment of several malignan‘cies and it exhibits a broad spectrum of antitumor effects by inducing dna damage and hindering dna damage repair paclitaxel ptx another commonly used chemotherapeutic agent in the clinic targets the microtubule cytoskeleton and impedes cell division the majority of patients have a good initial response to chemotherapy agents however subse‘quent relapse is common and largely due to the emergence of drug resistance thus chemoresistance is considered one of the main factors of poor prognosis in patients with advanced nsclc therefore there is an urgent need to investigate the target and mechanism of chemoresistance in nsclcclose homolog of l1 chl1 is a member of the l1 family of nerve cell adhesion molecules and is located on the 3q26 locus as a nerve cell adhesion molecule chl1 serves an important role in the development regeneration and plasticity of the nervous system the absence or mutation of chl1 can trigger 3p syndrome and schizophrenia the abnormal expression of chl1 may lead to reduced working memory and social behavior mental damage and abnormal behavior chl1 has been reported to be involved in carcinogenesis and progression in a variety of human cancers in esophageal squamous cell carcinoma escc chl1 downregulation is associated with invasion lymph node metastasis and poor overall survival functional studies revealed that chl1 has anti‘proliferation and anti‘metastasis abilities the expression of chl1 is downregulated by hypermethylation in human breast cancer and its negative expression contributes to breast tumorigenesis and progression in thyroid cancer and colonic adenocarcinoma chl1 impedes cell proliferation and invasion and acts as a tumor suppressor in lung cancer hÓ§tzel evaluated chl1 expression 0ccai chl1 enhances the chemosensitivity of lung cancer cellsin nsclc cases based on a tissue microarray and it was reported that chl1 expression is associated with t stage in adenocarcinomas as well as with metastatic lymph node status and improved survival additionally by analyzing the gene expression omnibus dataset gse21656 submitted by sun microarray results demonstrated that chl1 expression in ddp‘resistant h460 cells is significantly lower compared with that in parental cells suggesting that chl1 may be involved in nsclc chemoresistance however to the best of our knowledge the underlying mechanism remains unknownin the present study the expression of chl1 in ddp‘ and ptx‘resistant a549 cells and the parental cells was assessed functional studies of chl1 were performed to investigate its potential role in chemoresistancematerials and methodsdata processing the human gse21656 microarray dataset was downloaded from the ncbi gene expression omnibus geo database wwwncbinlmnihgovgeo the available dataset gse21656 was based on the gpl6244 platform affymetrix human gene st array affymetrix thermo fisher scientific inc this data includes h460 cells and ddp‘resistant h460 cells sample and each cell has three repeats samples the online tool geo2r httpwwwncbinlmnihgovgeogeo2r was used to determine the differen‘tially expressed genes in h460 and ddp‘resistant h460 cells p005 and log2fold‘change‰¥ were set as cut‘off standardscell culture the human nsclc cell line a549 the ptx‘resistant cell line a549ptx and the ddp‘resistant cells a549ddp were purchased from procell life science technology co ltd the cells were cultured in ham's f‘12k medium supplemented with fetal bovine serum both purchased from thermo fisher scientific inc uml penicillin and uml strep‘tomycin cat no thermo fisher scientific inc in a ˚c humidified incubator with co2cell transfection the resistant cells a549ptx and a549ddp cells were transfected with µg chl1 recombinant expres‘sion plasmid cat no hg10143‘ny sino biological inc empty vector pcmv3‘sp‘n‘ha was used as the control a549 cells were transfected with pmol small interfering sirnas the sirna sequence for chl1 guangzhou ribobio co ltd were sirna‘ '‘gga gcu aau uug acc aua utt‘' sirna‘ '‘cag caa uau uag cga gua utt‘' and scrambled control '‘uuc ucc gaa cgu guc acg utt‘' plasmids and sirnas were transfected into cells using lipofectamine® thermo fisher scientific inc following the manufacturer's instructions the time interval between transfection and subsequent experimentation was h for the rescue experiments the chl1 silenced a549 cells were treated with the akt inhibitor sc66 cat no s5313 selleck chemicals along with ddp µgml or ptx ngml both purchased from selleck chemicals for h at ˚crna extraction and reverse transcription‘quantitative pcr rt‘qpcr assay total rnas were isolated using trizol reagent thermo fisher scientific inc according to the manufacturer's instructions and the mixed dnas were elim‘inated by dnase i new england biolabs inc first‘strand cdna synthesis was conducted using the goscripttm kit promega corporation according to the manufacturer's instructions the reaction conditions for reverse transcription were ˚c for min ˚c for min and ˚c for min the sybr green real‘time pcr master mix thermo fisher scientific inc was used to perform rt‘qpcr using a lightcycler480 system roche diagnostics gmbh the chl1 primer sequences were as follows forward '‘ggc ttg gtc tct tgc ttt cc‘' and reverse '‘atc ttc cct ccc ttt gca cg‘' and ‘actin forward '‘ttc ctt cct ggg cat gga gtc ‘' and reverse '‘tct tca ttg tgc tgg gtg cc‘' the following thermocycling conditions were used for qpcr min at ˚c followed by cycles at ˚c for sec sec at ˚c and a final extension at ˚c for sec each reaction was conducted in triplicate relative expression levels were calculated using the ‘δδcq method cell viability cell viability was detected by mtt assay a cell suspension µl was seeded into ‘well plates at a density of 1x104 cellswell and incubated overnight at ˚c the concentrations of ddp used to treat a549 cells were and µgml while the concentrations of ptx used to treat a549 cells were and ngml the concentrations of ddp used to treat a549ddp cells were and µgml while the concentrations of ptx used to treat a549ptx cells were and ngml after treating with different concentrations of ddp or ptx for h at ˚c µl mtt mgml solution was added to each well and incubated for h at ˚c subsequently µl dmso was added to each well to dissolve the blue formazan crystals and the absorbance was measured using a microplate reader biotek instruments inc at nmclone formation assay a total of 1x103 cells were seeded into a ‘mm dish in triplicate and maintained in f‘12k medium with or without ddp or ptx at ˚c for h a total of weeks later cells were fixed in paraformalde‘hyde for min at room temperature and stained with crystal violet dye at room temperature for min the rate of colony formation was calculated using the following equation number of coloniesnumber of seeded cells x100flow cytometry apoptosis was detected using a fitc annexin v apoptosis kit bd pharmingen bd biosciences according to the manufacturer's protocol cells 1x105 were collected and washed twice with pbs prior to being suspended in µl binding buffer subsequently cells were incubated with µl annexin v‘fitc and µl propidium iodide in the dark for min at room temperature and apoptosis was analyzed using a cytoflex flow cytometer beckman coulter inc data were analyzed using cytexpert software beckman coulter inc the ratio between early and late apoptosis was calculatedwestern blotting cells were collected washed twice with pbs and lysed with ripa lysis buffer thermo fisher scientific inc proteins were isolated from the cell lysis buffer and 0concology letters quantified using the piercetm„¢ bca protein assay kit cat no thermo fisher scientific inc with bovine serum album as a standard equal amount of protein µg proteins were separated by sds‘page gel next the proteins were transferred onto a polyvinylidene membrane thermo fisher scientific inc blocked with bsa thermo fisher scientific inc for h at ˚c and incubated overnight at ˚c with primary antibodies against chl1 cat no ‘‘ap proteintech inc multi‘drug resistance gene mdr1 cat no ‘‘ap proteintech inc multidrug resistance‘associated protein mrp cat no ‘‘ig proteintech inc low‘density lipoprotein receptor‘related protein lrp cat no ‘‘ap proteintech inc phosphorylated p‘akt cat no ab38449 abcam and akt cat no ab227385 abcam after washing three times with pbs the membrane was incubated with horseradish peroxide‘conjugated goat anti‘rabbit cat no ab6271 abcam_or rabbit anti‘mouse cat no ab6728 abcam secondary antibodies for h at room temperature and the blots were detected with enhanced chemiluminescence reagent thermo fisher scientific inc protein expression was quantified using image‘pro plus software media cybernetics incanimal experiments the animal experiments were approved by the medical ethics committee of xiangya changde hospital approval no and were performed in compliance with all regulatory institutional guidelines for animal welfare the national institutes of health publications no ‘ a total of male balbc‘nu mice ‘week‘old ± g hunan sja laboratory animals center of the chinese academy of sciences were used in this study all animals were kept at the spf level laboratory at ‘Ëšc a relative humidity of ‘ a h lightdark cycle and timesh of fresh air exchange all mice were given free access to food and water the bedding materials drinking water feeding cages and other items in contact with the animals were all autoclaved prior to use a549ddp cells 1x107 transfected with empty vector and chl1 overexpression vector using lipofectamine® reagent thermo fisher scientific inc were subcu‘taneously injected into the nude mice to establish xenograft models following anaesthesia with chloral hydrate mgkg xenografts were allowed to grow to mm3 over weeks and the mice were randomly divided into four groups n3group as follows i vector group a549ddp cells transfected with empty vector and treated with µl saline solution ii vector‘ddp group a549ddp cells trans‘fected with empty vector and treated with mgkg ddp iii chl1 group a549ddp cells transfected with chl1 overexpression vector and treated with µl saline solu‘tion and iv chl1‘ddp group a549ddp cells transfected with chl1 overexpression vector and treated with mgkg ddp ddp was administered by intraperitoneal injection every days for weeks the mice were observed daily and the tumors were measured by a vernier caliper every days the tumor volumes were calculated as length x width22 a total of weeks post‘injection mice were euthanized with co2 at volume displacement rate vdr per min using a programmable logic controller barry‘wehmiller design group inc mice were monitored continuously and once the mice were immobile except for breathing for min the vdr was provided at for min the animals remained in the euthanasia chamber for min and were then observed for an additional min breathing and heart rate were monitored to determine deathstatistical analysis all experiments were performed in tripli‘cate and data are presented as the mean ± standard deviation all experiments were performed at least three times paired student's t‘test was performed for comparisons between two groups and one‘way analysis of variance followed by tukey's multiple comparison post‘hoc analysis was performed for comparisons between multiple groups spss ibm corp was used to perform the analysis p005 was considered to indicate a statistically significant differenceresultschl1 is downregulated in a549ddp and a549ptx‘resistant cells in order to investigate the mechanism of chemore‘sistance in lung cancer the lung adenocarcinoma cell line a549 the ddp‘resistant cells a549ddp and ptx‘resistant cells a549ptx were used in the present study cells were exposed to different concentrations of ddp ‘ µgml and ptx ‘ ngml and mtt assay was used to detect the cell survival rate a549ddp and a549ptx cells demonstrated higher resistance to ddp and ptx compared with a549 cells fig 1a the half maximal inhibitory concentration ic50 of ddp was significantly higher in a549ddp cells ± µgml compared with a549 cells ± µgml and the ic50 of ptx was significantly higher in a549ptx cells ± ngml compared with a549 cells ± ngml fig 1b in addition the expression levels of the drug‘resistant markers mdr1 mrp and lrp were significantly higher in a549ddp and a549ptx cells compared with a549 cells fig 1c additionally the mrna and protein expression levels of chl1 were significantly lower in a549ddp and a549ptx cells compared with those in a549 cells fig 1d and e and this was also observed in h460 ddp‘resistant cells obtained from the geo dataset gse21656 fig 1f these results suggested that chl1 may be involved in regulating ddp and ptx resistance in nsclcknockdown of chl1 enhances resistance to ddp and ptx in a549 cells as chl1 was upregulated in a549 cells chl1 was silenced in a549 cells using sirnas chl1 expres‘sion was significantly reduced in the chl1 sirna groups compared with that of the scrambled control group fig 2a as sirna‘ demonstrated the greatest interference efficiency it was selected for use in the following experiments notably chl1‘knockdown enhanced the resistance to ddp and ptx in a549 cells fig 2b and c colony formation assay revealed that compared with the control group chl1‘knockdown significantly increased the rate of colony formation in the absence of chemotherapeutics and enhanced the resistance to ddp and ptx fig 2d flow cytometry results demon‘strated significantly reduced apoptosis in chl1‘knockdown cells after ddp and ptx treatment compared with that of the control group fig 2e 0ccai chl1 enhances the chemosensitivity of lung cancer cellsfigure chl1 is downregulated in ddp and ptx‘resistant a549 cells a cell survival of a549 and a549‘resistant cells a549ddp and a549ptx treated with increasing concentrations of ddp and ptx as assessed by mtt assay b the ic50 values of ddp in a549ddp and a549 cells and the ic50 values of ptx in a549ptx and a549 cells p005 vs a549 cells c western blotting demonstrated the expression of drug resistance‘related proteins mdr1 mrp and lrp in a549 cells and a549‘resistant cells a549ddp and a549ptx p005 vs a549 cells the protein and mrna expression levels of chl1 in a549 cells and a549‘resistant cells a549ddp and a549ptx were analysed by d western blotting and e reverse transcription‘quantitative pcr respectively p005 vs a549 cells f the mrna expression of chl1 in h460 and h460ddp cells in the gse21656 dataset p005 vs h460 cells chl1 close homolog of l1 ddp cisplatin ptx paclitaxel mdr1 multi‘drug resistance gene mrp multidrug resistance‘associated protein lrp low‘density lipoprotein receptor‘related protein ic50 half maximal inhibitory concentration chl1 overexpression enhances the sensitivity of a549 resistant cells to ddp and ptx as chl1 is downregu‘lated in a549ddp and a549ptx cells the present study successfully overexpressed chl1 in these cells using chl1 recombinant expression plasmids fig 3a the results demonstrated that chl1 overexpression alleviated the resis‘tance to ddp and ptx compared with that of the control group fig 3b and c in addition chl1 overexpression inhibited colony formation in the absence or presence of ddp and ptx fig 3d additionally flow cytometry results demonstrated that restoration of chl1 expression promoted apoptosis in resistant cells following ddp and ptx treat‘ment fig 3eto further validate the effects of chl1 overexpression on ddp or ptx sensitivity xenograft mice model experi‘ments were performed the results demonstrated that chl1 overexpression or ddp treatment significantly impeded the tumor growth fig 3f and decreased the tumor weight fig 3g in addition chl1 overexpression further aggra‘vated ddp‘mediated repression on tumor growth fig 3f and g these data suggested that chl1 overexpression suppressed tumor growth and enhanced the chemosensitivity in nsclcchl1 mediates chemosensitivity by inhibiting akt activity recently studies have confirmed that chl1 inhibits akt activity in escc and neuroblastoma cell lines thus the present study investigated whether chl1 mediates chemoresistance via the akt pathway in nsclc in a549 cells compared with the scrambled group chl1‘knockdown elevated the expression of p‘aktser473 fig 4a by contrast restoring chl1 expression in a549ddp and a549ptx cells inhibited the akt phosphorylation compared with the control group fig 4a suggesting chl1 mediates chemosensitivity via the akt pathway subsequently chl1‘silenced a549 cells were treated with the akt inhibitor sc66 and it was demon‘strated that inhibiting akt activity significantly reduced the promotive effects on cell survival fig 4b and clone forma‘tion fig 4c and the inhibitory effects on apoptosis fig 4d induced by chl1‘depletion these results confirmed that chl1 mediates chemosensitivity in nsclc by inhibiting the akt pathway 0concology letters figure chl1‘knockdown increases a549 cell resistance to ddp and ptx a western blotting was performed to validate the efficiency of transfection with chl1 sirnas p005 vs scramble mtt assays were performed to determine the survival rate of chl1‘knockdown a549 cells treated with b ‘ µgml ddp or c ‘ ngml ddp d colony formation assay of a549 cells transfected with chl1 sirna in the presence or absence of µgml ddp or ngml ptx e flow cytometry analysis was used to detect apoptosis in a549 cells transfected with chl1 sirna in the presence or absence of µgml ddp or ngml ptx p005 p0001 chl1 close homolog of l1 ddp cisplatin ptx paclitaxel si small interfering discussionthe results of the present study demonstrated that chl1 was significantly downregulated in a549ddp and a549ptx cells compared with a549 cells the knockdown of chl1 in a549 cells facilitated the cell survival and clone formation and decreased apoptosis when treated with or without ddp and ptx whereas chl1 overexpression in a549ddp and a549ptx cells inhibited cell survival and clone formation and increased apoptosis the results of the present study also demonstrated that chl1 enhances nsclc chemosensitivity through inhibition of the akt pathway these data suggested that chl1 may be a promising target to improve the efficacy of chemosensitivity in nsclcchl1 belongs to the l1 family of nerve cell adhesion molecules it was initially cloned in mice and its expression in mouse development was analyzed by senchenko through cell‘cell interactions and mediating cell‘cell and cell‘matrix interactions chl1 has an important effect on the development regeneration and plasticity of the nervous system previous reports have demonstrated that chl1 also participates in carcinogenesis ‘ chl1 was observed to be significantly downregulated in up to types of tumor tissues compared with their adjacent normal tissues in most tumors chl1 is a potential tumor suppressor gene whose silencing is associated with tumor growth invasion and metastasis ‘ for example knockdown of chl1 expression results in enhanced cervical cancer cell invasion and migration a low expres‘sion of chl1 in patients with neuroblastoma predicts a poor prognosis and enhancing chl1 expression suppresses tumor progression in contrast chl1 has been reported to promote cell proliferation metastasis and migration in human gliomas however to the best of our knowledge research on chl1 and tumor chemoresistance has rarely been reported 0ccai chl1 enhances the chemosensitivity of lung cancer cellsfigure overexpression of chl1 increases the sensitivity of resistant a549 cells to ddp and ptx a western blotting was performed to detect chl1 expression in a549ddp and a549ptx cells transfected with chl1 expression plasmids p005 vs vector effect of chl1 overexpression on resistant a549 cell survival rate when treated with b ‘ µgml ddp or c ‘ ngml ptx as determined by mtt assay d colony formation assays demon‘strated the number of colonies of resistant a549 cells transfected with chl1 expression plasmids in the presence or absence of µgml ddp or ngml ptx e flow cytometry analysis was performed to assess apoptosis in resistant a549 cells transfected with chl1 expression plasmids in the presence or absence of µgml ddp or ngml ptx chl1 overexpression enhanced chemosensitivity of a549ddp cells to ddp in vivo which was demonstrated by the effect of ddp treatment or chl1 overexpression on the f growth and g weight of xenografts derived from a549ddp cells p005 p001 chl1 close homolog of l1 ddp cisplatin ptx paclitaxel the present study examined the differentially expressed genes in nsclc ddp‘resistant cells in a geo dataset chl1 was demonstrated to be upregulated in ddp‘resistant cells compared with parental cells suggesting that chl1 may be involved in nsclc chemotherapy resistance similarly a study that compared and analyzed the differentially expressed genes in chemosensitive tumors and chemoresistant ovarian adenocarcinomas tissues reported that the expression of chl1 in chemotherapy‘sensitive tumor tissues is higher compared with that in drug‘resistant tissues suggesting that chl1 may help to predict the efficacy of chemotherapy for ovarian cancer in addition aberrant methylation of chl1 may be associated with the recurrence of colorectal cancer crc following chemotherapy ‘azadc treatment restores ‘fluro‘uracil sensitivity in vitro which also suggests that chl1 may be involved in crc chemotherapy resistance the results of the present study demonstrated that chl1 was down‘regulated in a549ddp cells additionally as multiple drug resistance is a common characteristic another type of resistant cells a549tax cells were also used in the current study the results also demonstrated that chl1 was downregulated in a549ptx cells compared with control cells overexpression of chl1 significantly increased the sensitivity of cells resistant to ddp and ptx whereas knockdown of chl1 expression in 0concology letters figure chl1 mediates ddp and ptx sensitivity by inhibiting akt activity a western blotting was performed to detect the expression of p‘akt and total akt in chl‘silenced and ‘restored cell models p005 vs scramble or vector b mtt assays were performed to detect cell survival rates of a549 cells treated with chl1 sirna and akt inhibitor sc66 p005 c colony formation assays were performed in a549 cells treated with chl1 sirna and the akt inhibitor sc66 in the presence of ddp µgml or ptx ngml p005 vs si‘chl1 d apoptosis were measured in a549 cells treated with chl1 sirna and akt inhibitor sc66 in the presence of ddp µgml and ptx ngml p005 vs si‘chl1 chl1 close homolog of l1 ddp cisplatin ptx paclitaxel si small interfering p‘ phosphorylated parent a549 cells displayed the opposite results to the best of our knowledge this study is the first study to suggest that chl1 may be involved in chemosensitivity in lung cancer the concentration of ddp used in vivo is mgkg however this may not be in line with the concentrations that would be used in a clinical setting in a clinical trial the human initial dose was calculated from the no observed adverse effect levels noaels verified in animal experiments noael is the maximum dose level without significant adverse reactions the noael verified in animal experiments can be converted to a human equivalent dose according to the body surface area conversion which is based on the area standardization mgm2 proportional among different species in the present study the concentration of ddp used in vivo was not the noael thus it was not consistent with the concentrations used in clinical settingsakt is a serinethreonine protein kinase that is activated by phosphorylation as a key molecule of the pi3kakt signaling pathway p‘akt regulates cell survival cell growth cell motility and angiogenesis and prevents apoptosis additionally akt activation is associated with tumor chemore‘sistance the results of the present study demonstrated that compared with the control groups the expression of p‘akt was increased in chl1‘knockdown a549 cells and its expres‘sion was reduced in chl1 overexpressed a549ddp and a549ptx cells when akt activity was inhibited by the akt inhibitor the sensitivity to ddp and ptx in chl1‘knockdown a549 cells was restored this finding suggested that chl1 enhanced the chemosensitivity of nsclc by inhibiting the akt pathway considering numerous studies have confirmed that the akt pathway mediates chemoresistance via regulation of atp binding cassette abc members ‘ the present study didn't further investigate the specific abc members and mechanisms which was a of the limitation to the present study thus this research should be further investigated in vivoin summary the present study demonstrated that chl1 was downregulated in resistant cells a549ddp and a549ptx and upregulation of chl1 enhanced the chemosensitivity of nsclc via inhibiting the akt pathway to the best of our knowledge this was the first study to confirm the function and 0ccai chl1 enhances the chemosensitivity of lung cancer cellsmechanism of chl1 in mediating chemosensitivity in cancer thus the development of chl1‘based therapeutic strategies may improve the efficacy of chemosensitivity in nsclcacknowledgementsthe authors of the present study would like to thank mr dingliang li xiangya hospital changsha china for his guidance and assistance in flow cytometric analysisfundingno funding was receivedavailability of data and materialsthe datasets used andor analyzed during the present study are available from the corresponding author upon reasonable requestauthors' contributionsrh conceived and designed the present study xc bh yh and pl performed experiments and collected the data sl zz and zh analyzed and interpreted the data ml and lz analyzed the data and prepared the figure xc ml and lz drafted the initial manuscript and revised it for intellectual content all authors read and approved the final manuscriptethics approval and consent to participatethe animal experiments were approved by the medical ethics committee of xiangya changde hospital changde china approval no patient consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreferences parascandola m and xiao l tobacco and the lung cancer epidemic in china transl lung cancer res suppl s21‘s30 chen w cancer statistics updated cancer burden in china chin j cancer res oser mg niederst mj sequist lv and engelman ja transformation from non‘small‘cell lung cancer to small‘cell lung cancer molecular drivers and cells of origin lancet oncol e165‘e172 thatcher n faivre‘finn c blackhall f anderson h and lorigan p sequential platinum‘based chemotherapy‘thoracic radiotherapy in early stage non‘small cell lung cancer clin cancer res suppl s5051‘s5056 yano t okamoto t fukuyama s and maehara y therapeutic strategy for postoperative recurrence in patients with non‘small cell lung cancer world j clin oncol ‘ fang z chen w yuan z liu x and jiang h lncrna‘malat1 contributes to the cisplatin‘resistance of lung cancer by upregulating mrp1 and mdr1 via stat3 activation biomed pharmacother ‘ cai y dong zy and wang jy lncrna nnt‘as1 is a major mediator of cisplatin chemoresistance in non‘small cell lung cancer through mapkslug pathway eur rev med pharmacol sci ‘ han ml zhao yf tan ch xiong yj wang wj wu f fei y wang l and liang zq cathepsin l upregulation‘induced emt phenotype is associated with the acquisition of cisplatin or paclitaxel resistance in a549 cells acta pharmacol sin ‘ liu j meisner d kwong e wu xy and johnston mr translymphatic chemotherapy by intrapleural placement of gelatin sponge containing biodegradable paclitaxel colloids controls lymphatic metastasis in lung cancer cancer res ‘ hassan wa yoshida r kudoh s kameyama h hasegawa k niimori‘kita k and ito t notch1 controls cell chemoresistance in small cell lung carcinoma cells thorac cancer ‘ tang h jiang l zhu c liu r wu y yan q liu m jia y chen j qin y loss of cell adhesion molecule l1 like promotes tumor growth and metastasis in esophageal squamous cell carcinoma oncogene ‘ liu h focia pj and he x homophilic adhesion mechanism of neurofascin a member of the l1 family of neural cell adhesion molecules j biol chem ‘ tassano e biancheri r denegri l porta s novara f zuffardi o gimelli g and cuoco c heterozygous deletion of chl1 gene detailed array‘cgh and clinical characterization of a new case and review of the literature eur j med genet ‘ morellini f lepsveridze e kahler b dityatev a and schachner m reduced reactivity to novelty impaired social behavior and enhanced basal synaptic excitatory activity in perforant path projections to the dentate gyrus in young adult mice deficient in the neural cell adhesion molecule chl1 mol cell neurosci ‘ he lh ma q shi yh ge j zhao hm li sf and tong zs chl1 is involved in human breast tumorigenesis and progres‘sion biochem biophys res commun ‘ martín‘sánchez e mendaza s ulazia‘garmendia a monreal‘santesteban i blanco‘luquin i córdoba a vicente‘garcía f pérez‘janices n escors d megías d chl1 hypermethylation as a potential biomarker of poor prog‘nosis in breast cancer oncotarget ‘ zhu h fang j zhang j zhao z liu l wang j xi q and gu m mir‘ targets chl1 and controls tumor growth and invasion in papillary thyroid carcinoma biochem biophys res commun ‘ yu w zhu k wang y yu h and guo j overexpression of mir‘‘5p promotes proliferation and invasion of colon adeno‘carcinoma cells through targeting chl1 mol med hötzel j melling n müller j polonski a wolters‘eisfeld g izbicki jr karstens kf and tachezy m protein expression of close homologue of l1 chl1 is a marker for overall survival in non‘small cell lung cancer nsclc j cancer res clin oncol ‘ sun y zheng s torossian a speirs ck sc
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the incidence and death rate of nonsmall cell lung cancer nsclc in china ranks the first among the malignant tumors circular rna circrna was reported to be involved in the progression of nsclc our study aimed to investigate the underlying mechanism of circ_0020123 in nsclc progressionmethods quantitative realtime polymerase chain reaction qrtpcr was used to detect the expression of circ_0020123 mir5905p and thrombospondin thbs2 in nsclc tissues and cells cell proliferation and migration were examined by cell counting kit8 cck8 assay and transwell assay respectively flow cytometry assay was used to detect the apoptosis of nsclc cells the protein levels of ki67 matrix metalloprotein9 mmp9 cleavedcaspase9 cleavedcasp9 and thbs2 were detected by western blot the targets of circ_0020123 and mir5905p were predicted by starbase and targetscan and then confirmed by dualluciferase reporter assay and rna immunoprecipitation rip assay the animal experiment showed the effect of circ_0020123 on tumor growth in vivoresults the expression of circ_0020123 was upregulated in nsclc tissues and cells functionally circ_0020123 downregulation inhibited the proliferation and migration and promoted the apoptosis of nsclc cells interestingly circ_0020123 directly targeted mir5905p and inhibition of mir5905p reversed the knockdown effects of circ_0020123 on nsclc cells more importantly thbs2 was a target of mir5905p and thbs2 overexpression reversed the effects of circ_0020123 knockdown on cell proliferation migration and apoptosis in nsclc cells finally suppression of circ_0020123 inhibited tumor growth in vivo through mir5905pthbs2 axis circular rna circ_0020123 regulated thbs2 by sponging mir5905p to promote cell proliferation and migration and inhibit cell apoptosis in nsclc cellskeywords nsclc circ_0020123 mir5905p thbs2highlights circ_0020123 was upregulated and downregulation of circ_0020123 inhibited cell proliferation migration and promoted cell apoptosis in nsclc cellscorrespondence bskrju163comdepartment of thoracic surgery lianyungang second people™s hospital no hailian east road haizhou district lianyungang jiangsu china circ_0020123 directly targeted mir5905p and mir5905p downregulation reversed the knockdown effects of circ_0020123 on nsclc progression thbs2 acted as a target of mir5905p and overthe effects of expression of thbs2 reversed circ_0020123 knockdown on nsclc progression downregulation of circ_0020123 suppressed tumor growth in vivo through mir5905pthbs2 axis the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the ™s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the ™s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cwang a0et a0al cancer cell int page of lung cancer has the highest incidence of total cases and is the most common cause of cancer death of total cancer deaths in worldwide lung cancer can be divided into several histological subtypes according to the location and the tendency of metastasis small cell lung cancer sclc accounts for about of all lung cancer cases however nonsmall cell lung cancer nsclc accounts for of lung cancer and the a0years overall survival rate os is only about therefore it is important to find the effective treatment and potential molecular targets of nsclc progressioncircular rna circrna is a single stranded rna molecule with a closed circular structure recently amounts of circular dna have been discovered and most of which were thought to be the byproducts of typical splicing [ ] previous reports indicated that the expression of circrna was tissuespecific and the change of its expression intensity was associated with some diseases [“] furthermore circrna was involved in the occurrence and development of the disease and might be used as a potential biomarker in clinical diagnosis prognosis and treatment of diseases [ ] for example circ_0039569 facilitated cell proliferation and migration of renal cell carcinoma by sponging mir34a5p to regulate cc chemokine ligand ccl22 meanwhile hsa_circ_0043256 participated in the progression of nsclc cells by mediating the cinnamaldehyde treatment a previous report suggested that circ_0020123 acted as an oncogene in nsclc and circ_0020123 regulated zincfingerenhancer binding protein zeb1 and enhancer of zeste homolog ezh2 by competitively binding with mir144 to induce cell progression and migration these reports suggested that circ_0020123 was a vital factor in the pathogenesis of nsclc and its function and molecular mechanism need to be further studiedas a small endogenous rna microrna mirna is essential in regulating gene expression and plays a potential role in the exploitation of biomarkers recently some aggregated mirnas have been found in prostate cancer such as mir221222 mir143145 mir23b27b241 and mir1133a which were downregulated and had tumor inhibiting functions a previous study found that circulating mir5905p could be used as routine diagnostic tools for lung cancer and as a potential prognostic marker for liquid biopsy besides overexpression of mir5905p reduced the development of nsclc cells and regulated the expression of epithelialmesenchymal transformation emtrelated proteins by targeting the signal transducers and activators of transcription stat3 however the precise mechanism by which mir5905p affects nsclc needs further investigationthrombospondin thbs2 as a secreted protein was confirmed to be highly expressed in different cancers including cervical cancer colorectal cancer and nsclc a previous report suggested that thbs2 was involved in the proliferation apoptosis and antiautophagy regulation of cervical cancer cells by mir20a tian et a0al found the expression and clinicopathological features of thbs2 in colorectal cancer were significantly correlated with the prognosis of cancer and might be used as a biomarker of prognosis however the molecular function of thbs2 in nsclc remains poorly definedin this study the targeting relationship between circ_0020123 and mir5905p was firstly detected the effects of circ_0020123 on cell proliferation migration apoptosis and tumor growth were performed by gain and lossoffunction experiments and molecular biology techniquesmaterials and a0methodspatients and a0specimensnsclc tissues and the adjacent healthy lung tissues were taken from nsclc patients in the lianyungang second people™s hospital all volunteers signed written informed consents nsclc tissues and the adjacent tissues were immediately frozen in liquid nitrogen and kept at ˆ’ a0 °c for further experiments this research was approved by the ethics committee of lianyungang second people™s hospitalcell culture and a0cell transfectiontwo nsclc cell lines a549 and h1299 and one normal lung cell line imr90 were obtained from the beijing concorde cell library beijing china a549 h1299 and imr90 cells were cultivated in dulbecco™s modified eagle medium dmem hyclone logan ut usa supplementing with fetal bovine serum fbs hyclone and cultured in an incubator at a0„ƒ with co2small interfering rna sirna targeting circ_00201231 sicirc_00201231 and sicirc_00201232 short hairpin rna shrna targeting circ_0020123 shcirc_0020123 mir5905pinhibitors sirna negative control sinc shnc and ncinhibitors were all obtained from biomics biotech jiangsu china full length of thbs2 cdna sangon biotech shanghai china was subcloned into pcdna31 plasmid ekbioscience shanghai china then cell transfection was performed by lipofectamine thermo fisher scientific waltham ma usa 0cwang a0et a0al cancer cell int page of rna isolation and a0quantitative real‘time polymerase chain reaction qrt‘pcrthe trizol reagent invitrogen carlsbad ca usa was used for extracting the total rnas next the reversed transcription was carried out by rtpcr kit invitrogen the qrtpcr was performed using the abi sybr green master mix invitrogen the primers in our study were as follows f5²ttc gga cga ccg tca aac at3² and r5²agg atc cct gca cca caa tg3² for circ_0020123 f5²tga aag acg tga tgg cac ac3² and r5²ctt cca ttt tgg for mir5905p f5²aga agg ggt ttt tgg3 ² ctg ggg ctc att tg3² r5²agg ggc cat cca cag tct tc3² for glyceraldehyde3phosphate dehydrogenase gapdh f5²gcg gct ggg tct att tgt c3² and r5²gca gga ggt gaa gaa cca tc3² for thbs2 f5²att gga acg ata cag aga agatt3² and r5²gga acg ctt cac gaa ttt g3² for u6 gapdh and u6 were the internal parameterscell counting kit‘ cck‘ assaythe proliferation viability of a549 and h1299 cells were detected by the cellcounting kit8 msk wuhan china a549 and h1299 cells were cultivated into a 96well plate with a density of × a0cellswell and incubated in a0°c for or a0h then a0μl fresh medium and cck8 solution was added after incubation at a0°c for a0h the od values were detected by the multiskan ascent microplate reader abcam cambridge ma usatranswell assaytranswell chamber corning life sciences corning ny usa was used to detect cell migration firstly the serumfree dmem thermo fisher scientific was fixed with cell suspension cells and seeded into the upper chamber and the dmem containing serum was put into the lower chamber after incubation for a0h the cells in lower surface of the upper chamber were treated with formaldehyde solution for a0 h and then thoroughly washed finally the migrated cells were stained with crystal violet corning life sciences and observed by using a microscopeflow cytometryfirstly a549 and h1299 cells were cultured and pbs was used for washing cells then the binding buffer was used to resuspend cells and the annexin vfluorescein isothiocyanate vfitcpropidium iodide pi apoptosis detection kit thermo fisher scientific was used to stain cells finally cell apoptosis was detected by flow cytometry thermo fisher scientificwestern blot analysisthe total proteins of nsclc tumors or cells were collected by ripa lysis buffer sangon biotech then the proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis sdspage and transferred to polyvinylidene fluoride pvdf membranes thermo fisher scientific the skimmed milk was added and incubated with primary antigapdh antibody invitrogen carlsbad ca usa antiβactin antibody invitrogen antiki67 antibody invitrogen antimatrix metalloprotein9 mmp9 antibody invitrogen anticleavedcaspase9 cleavedcasp9 antibody invitrogen or antithbs2 antibody invitrogen at a0°c overnight finally the membranes were incubated with the secondary antibody for a0 h at room temperature the results were viewed using kodak film developer fujifilm japandual‘luciferase reporter assaysthe wild type circ_0020123 sequences circ_0020123wt mutant circ_0020123 sequences circ_0020123mut wild type thbs2 ²utr sequences thbs2wt mutant thbs2 ²utr sequences thbs2mut were cloned into pgl3 luciferase reporter plasmid promega madison wi usa then the plasmid and mir5905p or mirnc were cotransfected into a549 and h1299 cells by lipofectamine thermo fisher scientific after transfection for a0h the dualluciferase reporter assay system promega was performed to detect the luciferase activityrna immunoprecipitation ripfirstly the magna rip rnabinding protein immunoprecipitation kit gzscbio guangzhou china was performed to verify the relationship between circ_0020123 and mir5905p in brief the magnetic beads and antiago2 antibody abcam were added into cells and incubated for a0h then the proteinase k and the phenol“chloroformisoamyl alcohol reagent were added for purifying rnas finally qrtpcr was used to measure circ_0020123 enrichmentanimal experimentsthe 4weekold balbc male nude mice vitalriver beijing china were raised in a sterile environment for 0cwang a0et a0al cancer cell int page of experiments then pbs was used to suspend a549 cells × transfected with shcirc_0020123 or shnc next the nude mice were divided into two groups n a549 cells transfected with shcirc_0020123 or shnc were shcirc_0020123 or shnc inoculated into the nude mice the tumor volume was detected every a0 days after a0days the nude mice were euthanatized and the tumor weight was detected besides the tumor tissues from each group were collected to detect the expression of circ_0020123 mir5905p and thbs2 the animal experiment was approved by the animal experimentation ethics committee of lianyungang second people™s hospitalstatistical analysisthe software graphpad prism was performed for statistical analysis the data was displayed as mean ± standard deviation sd the significant difference was calculated by student™s t test and oneway analysis of variance anova p was considered as statistically significantresultscirc_0020123 was a0upregulated in a0nsclc tissues and a0cellsto begin with qrtpcr was used to detect the expression of circ_0020123 the result showed that circ_0020123 was significantly upregulated in nsclc tissues compared with the adjacent healthy tissues fig a0 1a similarly the expression of circ_0020123 in nsclc cells a549 and h1299 was markedly higher than that in normal cells imr90 fig a01b from these data it is speculated that circ_0020123 might be acted as an oncogene in nsclcfig circ_0020123 was upregulated in nsclc tissues and cells a qrtpcr was used to detect the expression of circ_0020123 in adjacent healthy tissues n and tumor tissues n b the expression of circ_0020123 in normal cell line imr90 and nsclc cell lines a549 and h1299 was detected by qrtpcr p downregulation of a0circ_0020123 inhibited the a0proliferation migration and a0induced apoptosis of a0nsclc cellsto investigate the functional effects of circ_0020123 on nsclc cells sicirc_00201231 and sicirc_00201232 were obtained and transfected into a549 and h1299 cells firstly the transfection efficiency was detected by qrtpcr fig a02a next cck8 was used to detect the proliferation and the results showed that the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was reduced fig a0 2b moreover the migration of a549 and h1299 cells was significantly downregulated by circ_0020123 knockdown fig a02c in addition the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was obviously higher than transfected with sinc fig a02d finally the protein levels of cell proliferationrelated protein ki67 and cell migrationrelated protein mmp9 were inhibited while cell apoptosisrelated protein cleavedcasp9 was upregulated in nsclc cells transfected with sicirc_00201231 or sicirc_00201232 fig a02e these data suggested that inhibition of circ_0020123 suppressed cell proliferation migration and promoted apoptosis in nsclc cellscirc_0020123 directly targeted mir‘‘5pby searching in the online software starbase the potential binding sites between circ_0020123 and mir5905p were detected fig a0 3a to confirm that the dualluciferase reporter assay was performed the results showed that the luciferase activity of circ_0020123wt reporter plasmid was reduced by mir5905p mimic while the circ_0020123mut reporter plasmid activity was not changed in a549 and h1299 cells fig a03b furthermore the expression of mir5905p was lower in a549 and h1299 cells compared with that in imr90 cells fig a0 3c in contrast mir5905p expression was elevated in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 fig a0 3d finally the rip assay was also used to confirm the targeting relationship between circ_0020123 and mir5905p and the results showed that circ_0020123 and mir5905p were enriched in antiago2 group fig a03emir‘‘5p downregulation reversed the a0inhibition effects of a0circ_0020123 on a0nsclc cellsto further explore the functional effects between circ_0020123 and mir5905p mir5905pinhibitor was established qrtpcr was used to detect the transfection efficiency fig a0 4a interestingly mir5905p was upregulated in a549 and h1299 cells transfected with sicirc_00201231 while the expression of mir5905p was recovered in cells transfected with 0cwang a0et a0al cancer cell int page of fig downregulation of circ_0020123 inhibited the proliferation and migration and induced the apoptosis of nsclc cells a the transfection efficiency of sicirc_00201231 and sicirc_00201232 in a549 and h1299 cells was detected by qrtpcr b cck8 assay was used to detect the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 c the migration of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was measured by transwell assay d flow cytolysis assay was used to detect the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 e the protein levels of cell proliferation related protein ki67 cell migration related protein mmp9 and cell apoptosis related protein cleavedcasp9 were detected by western blot p fig a0sicirc_00201231 mir5905pinhibitors 4b moreover circ_00201231 knockdown inhibited cell proliferation and migration while the mir5905p inhibitor reversed these effects fig a0 4c d in addition the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 was increased which was abolished by mir5905pinhibitor fig a0 4e similarly mir5905p inhibitors reversed the effects on the protein levels of ki67 mmp9 and cleavedcasp9 in a549 and h1299 cells transfected with sicirc_00201231 fig a0 4f these results that mir5905p downregulation reversed the effects of circ_0020123 downregulation on the proliferation migration and apoptosis of nsclc cellsindicated mir‘‘5p targeted thbs2 in a0nsclc cellsthe thbs2 ²utr was predicted to contain the binding sites of mir5905p through the online software targetscan fig a05a then the dualluciferase reporter assay was used to confirm the targeting relationship the results showed that cotransfection of mir5905p and thbs2wt significantly limited the luciferase activity in both a549 and h1299 cells the luciferase activity was not altered in cells cotransfected with mir5905p and thbs2mut fig a05b importantly the mrna and protein level of thbs2 was enahnced in nsclc cells fig a05c d we further explored whether circ_0020123 affected the functions of thbs2 in nsclc cells the mrna and protein expression of thbs2 were repressed in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 fig a05e f 0cwang a0et a0al cancer cell int page of fig circ_0020123 directly targeted mir5905p a the binding site between circ_0020123 and mir5905p was detected by the online software starbase b the luciferase activity of circ_0020123wt or circ_0020123mut reporter plasmid in a549 and h1299 cells transfected with mirnc or mir5905p was detected by dualluciferase reporter assay c qrtpcr was used to detect the expression of mir5905p in a549 and h1299 cells d the expression of mir5905p in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was detected by qrtpcr e rip assay was used to confirm the relationship between circ_0020123 and mir5905p p thbs2 overexpression reversed the a0effects of a0circ_0020123 knockdown on a0the a0proliferation migration and a0apoptosis of a0nsclc cellsbased on the work ahead of us the pcdna31thbs2 was constructed then the qrtpcr and western blot were used to detect the transfection efficiency and the thbs2 expression was increased in a549 and h1299 cells transfected with pcdna31thbs2 fig a0 6a b in addition the proliferation and migration rates of a549 and h1299 cells transfected with sicirc_00201231 pcdna31thbs2 were higher than that transfected with sicirc_00201231 fig a0 6c d meanwhile a similarly phenomenon was also observed in cell apoptosis the pcdna31thbs2 significantly reversed the promotion effect of circ_0020123 deletion on cell apoptosis fig a0 6e furthermore the effects of circ_0020123 deletion on ki67 mmp9 and cleavedcasp9 protein levels were also reversed by thbs2 overexpression fig a0 6f these data suggested that overexpression of thbs2 could reverse the effects of circ_0020123 downregulation on cell proliferation migration and apoptosisreduction of a0circ_0020123 suppressed tumor growth in a0vivo through a0circ_0020123mir‘‘5pthbs2 axisto further explore the function of circ_0020123 in nsclc cells the shcirc_0020123 was constructed and the xenograft tumor was established then a549 cells transfected with shcirc_0020123 or shnc were injected into the nude mice the xenograft tumor volume was measured every a0 days after injection and the results showed that tumor volume was smaller shcirc_0020123 group than that in shnc group fig a07a moreover tumor weight was inhibited by circ_0020123 knockdown fig a0 7b furthermore the expression circ_0020123 and thbs2 was decreased while the mir5905p was increased in xenograft tumor transfected with shcirc_0020123 fig a0 7c western blot assay also revealed that the protein level of thbs2 was repressed by circ_0020123 knockdown fig a07d finally the digital tomosynthesis dts was used to detect the number of lung metastatic nodules in xenograft tumor and it was reduced in shcirc_0020123 group fig a07e the results suggested that downregulation of circ_0020123 inhibited tumor growth in a0vivodiscussionclinically only a few nsclc patients were diagnosed at an early stage and treated by surgical resection more than of nsclc patients were diagnosed with the advanced stage or metastatic tumors thus finding novel biomarkers and therapeutic targets were necessary for the effective diagnosis and treatment of nsclcrecently circrna was no longer considered as a random product in the rna shearing process and its biological significance and function in malignant tumors 0cwang a0et a0al cancer cell int page of fig mir5905p downregulation reversed circ_0020123 knockdown effects in nsclc cells a qrtpcr was used to detect the expression of mir5905p in a549 and h1299 cells transfected with mir5905pinhibitors b the expression of mir5905p in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors was detected by qrtpcr c the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors was tested by cck8 assay d transwell assay was used to measure the migration of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors e flow cytolysis assay was used to detect the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors f the protein levels of ki67 mmp9 cleavedcasp9 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors were detected by western blot p had received more and more attention previous reports revealed that circ_0020123 was involved in the development of nsclc moreover the level of circ_0020123 was elevated in nsclc cells consistently we found that the expression of circ_0020123 was markedly higher in nsclc tissues and cells moreover this research indicated that inhibition of circ_0020123 suppressed the proliferation migration and induced apoptosis of nsclc cells in a0 vitro besides circ_0020123 promoted tumor growth in a0vivoendogenous circrnas could act as microrna sponges to inhibit their function and some studies linked mirna sponges to human diseases including cancer a previous study indicated that circrna ctransferrin receptor ctfrc regulated tfrc by sponging mir107 to facilitate bladder carcinoma development mir5905p was studied in different cells such as airway smooth muscle cells colon epithelial cells and nsclc cells however the potential relationship between mir5905p and circrna has not been researched in this study circ_0020123 directly targeted mir5905p and mir5905p inhibition reversed the effects of circ_0020123 knockdown on nsclc progression these data provided a clue to the therapeutic strategy for nsclc 0cwang a0et a0al cancer cell int page of fig mir5905p targeted thbs2 in nsclc cells a the potential binding site between thbs2 ²utr and mir5905p was predicted by the online software targetscan b dualluciferase reporter assay was used to measure the luciferase activity of thbs2wt or thbs2mut reporter plasmid in a549 and h1299 cells transfected with mirnc or mir5905p c qrtpcr was used to detect the mrna expression of thbs2 in nsclc cells d the protein level of thbs2 in nsclc cells was tested by western blot e the mrna expression of thbs2 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was detected by qrtpcr f western blot was used to measure the protein level of thbs2 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 p our study also confirmed that mir5905p could target thbs2 directly in nsclc cells thbs2 is a calciumbinding protein that binds to and inactivates matrix metalloproteinase mmp genes involved in tissue formation and repair [ ] a previous document suggested that thbs2 acted as a target of mir2213p and participated in lymph node metastasis in cervical cancer the data in this research showed that the expression of thbs2 in nsclc cells was markedly higher than normal healthy cells furthermore overexpression of thbs2 reversed the effects of circ_0020123 knockdown on proliferation migration and apoptosis of nsclc cells suggesting that circ_0020123 promoted the progression of nsclc cells through mir5905pthbs2 axisin our research showed that the expression of circ_0020123 was higher in nsclc tissues and cells than control and downregulation of circ_0020123 inhibited the proliferation migration and promoted apoptosis of nsclc cells and also suppressed tumor growth in a0 vivo moreover circ_0020123 directly targeted mir5905p while mir5905p inhibition reversed the effects of circ_0020123 knockdown on nsclc cells more importantly circ_0020123 regulated the expression of thbs2 by sponging mir5905p and upregulation of thbs2 reversed the effects of circ_0020123 knockdown on nsclc cells therefore our research demonstrated that circ_0020123 enhanced proliferation migration and inhibited 0cwang a0et a0al cancer cell int page of fig overexpression of thbs2 reversed the effects of circ_0020123 knockdown on proliferation migration and apoptosis of nsclc cells a b the mrna and protein expression of thbs2 in a549 and h1299 cells transfected with pcdna31thbs2 was detected by qrtpcr and western blot c cck8 assay indicated the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 d the migration of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 was measured by transwell assay e the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 was detected by flow cytolysis assay f the protein levels of ki67 mmp9 cleavedcasp9 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 were detected by western blot p apoptosis of nsclc cells by sponging mir5905p to regulate thbs2results and develop the manuscript all authors read and approved the final manuscriptabbreviationsnsclc nonsmall cell lung cancer circrna circular rna qrtpcr quantitative realtime polymerase chain reaction cck8 cell counting kit8 mmp9 matrix metalloprotein9 cleavedcasp9 cleavedcaspase9 cleavedcasp9 cleavedcaspase9 rip rna immunoprecipitation zeb1 zincfingerenhancer binding protein ezh2 zeste homolog stat3 signal transducers and activators of transcription thbs2 thrombospondin acknowledgementsnot applicableauthors™ contributionslw collaborated to design the study lz were responsible for experiments analyzed the data yw wrote the paper all authors collaborated to interpret fundingnoneavailability of data and materialsplease contact corresponding author for data requestsethics approval and consent to participatethis research was approved by the ethics committee of lianyungang second people™s hospital the animal experiment was approved by the animal experimentation ethics committee of lianyungang second people™s hospitalconsent for publicationall listed authors have actively participated in the study and have read and approved the submitted manuscript 0cwang a0et a0al cancer cell int page of fig reduction of circ_0020123 suppressed the tumor growth in vivo through circ_0020123mir5905pthbs2 axis a a total of × a549 cells transfected with shcirc_0020123 or shnc were injected into nude mice to establish the xenograft tumor tumor volume was measured every d after injection b tumor weight was measured on d c the expression of circ_0020123 mir5905p and thbs2 in xenograft tumor was measured by qrtpcr d the protein level of thbs2 in xenograft tumor was evaluated by western blot e the number of lung metastatic nodules in xenograft tumor was detected by digital tomosynthesis dts p competing intereststhe authors declare that they have no competing interestsreceived april accepted july references bray f ferlay j soerjomataram i siegel rl torre la jemal a global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries ca cancer j clin “ abe h takase y sadashima e fukumitsu c murata k ito t kawahara a naito y akiba j insulinomaassociated protein is a novel diagnostic marker of small cell lung cancer in bronchial brushing and cell block cytology from pleural effusions validity and reliability with cutoff value cancer cytopathol “li c zhang l meng g wang q lv x zhang j li j circular rnas pivotal molecular regulators and novel diagnostic and prognostic biomarkers in nonsmall cell lung cancer j cancer res clin oncol “ belousova ea filipenko ml kushlinskii ne circular rna new regulatory molecules bull exp biol med “ zhang z xie q he d ling y li y li j zhang h circular rna new star new hope in cancer bmc cancer li l chen y nie l ding x zhang x zhao w xu x kyei b dai d zhan s guo j zhong t wang l zhang h myodinduced circular rna cdr1as promotes myogenic differentiation of skeletal muscle satellite cells biochim biophys acta gene regul mech “ greco s cardinali b falcone g martelli f circular rnas in muscle function and disease int j mol sci weng xd yan t liu cl circular rna_larp4 inhibits cell migration and invasion of prostate cancer by targeting foxo3a eur rev med pharmacol sci “ deng n lei d huang j yang z fan c wang s circular rna expression profiling identifies hsa_circ_0011460 as a novel molecule in severe preeclampsi
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recently the current pandemic of coronavirus disease covid characterized by a pulmonary infection in humans is caused by a novel virus strain from family coronaviridae known as severe acute respiratory syndrome coronavirus sarscov2 the previous outbreak of severe acute respiratory syndrome sars in “ and middle east respiratory syndrome mers in has demonstrated the lethality of coronaviruses when they cross the species barrier and infect humans so far six coronaviruses infecting humans have been identified and the novel coronavirus is the seventh one described to date as being responsible for a respiratory infection sarscov and merscov and the new sarscov2 belong to the betacoronavirus family [“] the coronaviruses have the largest genome around k among the rna viruses sarscov2 was closely related from “ identity to two batderived severe acute respiratory syndrome sarslike coronaviruses batslcovzc45 and batslcovzxc21 but it was more distant from sarscov from “ and merscov about furthermore the performed bioinformatic analysis showed that the nucleotide sequence of sarscov2 is similar to those of other betacoronaviruses with nucleotide identities of ‰¥ there are currently no effective licensed therapies for human coronaviruses hcov infections and existing treatment strategies are generally limited to symptomatic treatment and supportive care email addresses kuzunovahqtchaikapharmacom k uzunova efilipovahqtchaikapharmacom e filipova vpavlovahqtchaikapharmacom corresponding author v pavlova tvekovmuplevenabvbg t vekov 101016jbiopha2020110668 received may received in revised form august accepted august biomedicinepharmacotherapy1312020110668availableonline24august2020075333222020theauthorspublishedbyelseviermassonsasthisisanopenaccessundertheccbyncndlicensehttpcreativecommonslicensesbyncnd40 0csuch as solidarity who recovery k uzunova in the absence of a specific treatment for this novel virus the effort of researchers is focused on understanding and controlling the disease and on preventing and controlling the replication and spread of the virus to devise therapeutic strategies to counteract the sarscov2 infection numerous potential treatment options are being evaluated in ongoing clinical trials many antiviral and immunological treatments being investigated against coronaviruses are summarized by who in landscape analysis of therapeutics as of march the realtime dashboard of completed ongoing and planned clinical trials for covid includes drugs and promising therapies such as remdesevir lopinavirritonavir hydroxychloroquine il6 inhibitors tocilizumab and sarilumab convalescent plasma therapy stemcell transfusion vaccine candidates traditional chinese medicines which are of top interventions of the presented network among them remdesivir an analogue of adenosine seems to have a more promising future due to proven in vitro and in vivo antiviral efficacy till the beginning of june promising therapies involving lopinavirritonavir and chloroquine or hydroxychloroquine were part of treatment guidelines in many countries but currently they are excluded from covid19 treatment protocols because of uncertainty regarding their risks and benefits and it is recommended that they should be used only in the context of clinical trials [“] in spite of its known in vitroin vivo efficacy and safety profiles some trials evaluating these drugs for covid19 infection treatment uk ntc04381936 and discovery inserm ntc04315948 discontinued hydroxychloroquine and lopinavirritonavir arms the interim trial results showed that hydroxychloroquine and lopinavirritonavir produced little or no reduction in the mortality of hospitalized covid19 patients when compared to standard of care nevertheless some countries worldwide continue to recommend chloroquinehydroxychloroquine as a treatment option [“] the existing drugs that target viral proteins associated with enzymatic activities or blocking viral replication machinery or host proteins involved in viral life cycle regulating the function of the immune system or other cellular processes in host cells have great potential and are available on the market our review aims to highlight the potential molecular mechanisms of the therapeutic options available for the cure of other health conditions and their repurposing for the treatment of this novel coronavirus sars cov2 selected treatments of sarscov2 remdesivir gs5734 “ polymerase inhibitor deltacoronavirus genus pdcov which have the most divergent rdrp of known cov as compared to sars and merscov an in silico test of the covid19 rdrp built model suggested the effectiveness of remdesivir as a potent drug sarscov and sarscov2 both belong to the betacoronaviruses of the b lineage and the rdrp amino acid sequences of the two viruses are identical whereas merscov belongs to the betacoronaviruses of the c lineage and is only identical with sarscov2 another in vitro and in vivo proof came from sheahan who examined if gs5734 could inhibit replication of sarscov and mers cov in primary human airway epithelial hae cell cultures they found out a dosedependent reduction in replication with average ic50 values of μm sarscov and μm merscov moreover the compound inhibits a broad range of diverse cov including circulating human zoonotic bat cov and prepandemic zoonotic cov with both prophylactic and therapeutic 1dpi dosing of gs5734 a reduction in replication below a diseasecausing threshold in mouse model of sars cov pathogenesis was demonstrated therapeutic gs5734 substantially reduced the sarscov induced weight loss in infected animals and significantly suppressed virus lung titers p thus demonstrating that therapeutic administration of gs5734 can reduce disease and suppress replication during an ongoing infection furthermore remdesivir has the potential to block sarscov2 infection in vitro at lowmicromolar concentration and in treatment of merscov and sarscov infections in vivo it demonstrated a significant improvement of pulmonary pathology in mice the rnadependent rna polymerase rdrpmediated mechanism of cov inhibition by gs5734 is proven even in the setting of intact exoribonuclease exonmediated proofreading using the model coronavirus murine hepatitis virus mhv it was demonstrated that gs5734 dramatically inhibited viral replication and viral rna synthesis in wildtype wt virus while an nsp14 exon mutant lacking proofreading demonstrated increased susceptibility to gs5734 45fold more active this suggests that gs5734 is recognized at least partially by a functional exon but that the exon activity is not sufficient to prevent potent inhibition of cov replication the results provide strong evidence that rdrp is the target for remdesivir and support the hypothesis that gs5734 directly inhibits viral rna synthesis the mechanism of inhibition of rdrp of merscov by remdesivir was studied by gordon et al they coexpressed the merscov nonstructural proteins nsp5 nsp7 nsp8 and nsp12 rdrp in insect cells as a part of a polyprotein coexpression of the mers nsp5 protease with nsp7 nsp8 and nsp12 in insect cells yielded a stable complex composed of nsp8 and nsp12 the triphosphate form of the inhibitor rdvtp is utilized as a substrate and competes with its natural counterpart atp and they observed that incorporation of the nucleotide analogue was significantly more efficient once added into the growing rna chain the inhibitor does not cause immediate chain termination the presence of the ²hydroxyl group allows the addition of three more nucleotides until rna synthesis is arrested at position i3 therefore the main possible mechanism of action is delayed rna chain termination recently the same authors obtained almost identical results with sarscov merscov and sarscov2 rdrps they provided evidence that all three coronavirus rdrp complexes terminated rna synthesis at position i3 almost all viruses encode polymerases in the central steps of replication and transcription therefore polymerases are becoming the most attractive and suitable targets for antiviral development there are two major types of polymerase inhibitors i nucleoside and nucleotide substrate analogs and ii allosteric inhibitors nucleoside analogs are first triphosphated by the host cell to produce the active inhibitor and then act as an inhibitor by competing with the natural nucleoside triphosphates and terminating the growing viral nucleic acids to date most of the approved antiviral drugs for antihiv therapy utilize this mechanism remdesivir is a nucleotide analogue with a proved mechanism of action as an inhibitor of rnadependent rna remdesivir rdv is an investigational compound with a broad spectrum of antiviral activities against rna viruses including sarscov and merscov gs5734 was originally developed for the treatment of the ebola virus disease gs5734 the single sp isomer of the 2ethylbutyl lalaninate phosphoramidate prodrug effectively bypasses the rate limiting first phosphorylation step of the nuc nucleoside ribose analogue the mechanism of action of nuc requires intracellular anabolism to the active triphosphate metabolite ntp which is expected to interfere with the activity of viral rnadependent rna polymerases rdrp gs5734 selectively inhibits ebola virus replication by targeting its rdrp and inhibiting viral rna synthesis following efficient intracellular conversion to ntp in nonhuman primates this compound shows a broad spectrum of antiviral activities against several rna viruses including respiratory syncytial virus rsv junín virus lassa fever virus and middle east respiratory syndrome virus but was inactive against alphaviruses or retroviruses furthermore remdesivir dosedependently inhibits endemic human cov229e and covoc43 replications which typically cause upper respiratory infection in children but can cause more severe lower respiratory infection in adults with underlying respiratory conditions ie asthma copd and the elderly as well as a member of the biomedicinepharmacotherapy13120201106682 0c lopinavirritonavir “ protease inhibitor the proteases encoded by most viruses play a crucial role in the viral life cycle the protease inhibitors pis bind competitively to the substrate site of the viral protease this enzyme is responsible for the post translational proteolysis of a polyprotein precursor and the release of functional viral proteins allowing them to function correctly and individually in replicationtranscription and maturation inhibition results in the production of immature virus ps coronavirus proteases of which there are two in sarscov a papainlike cysteine proteinase plpro nsp3 and a 3clike proteinase 3clpro or mpro nsp5 and three in several other coronaviruses cleave the orf1 polypeptide as it is translated enabling the formation of the viral replication complex the substratebinding pockets are highly conserved among cov 3clpro suggesting the possibility for a widespectrum inhibitor design targeting this region in the 3clpro of all covs it is postulated that the 3clproinhibiting activity of lopinavirritonavir contributes at least partially to its anticov effects in silico binding studies of the drugs using the identified crystal structure of mpro and employing the hex program to conduct the docking of the ligands to the sarscov main proteinase revealed that lopinavir and ritonavir could basically bind to the active site of sars main proteinase but the efficacy of lopinavirritonavir was predicted to be poor according to the latest report of the structure of 3clpro from sarscov2 pdb code 6lu7 and the available structure of 3clpro from sarscov pdb code 1uk4 the two main proteases differ by only amino acids comparing ligand binding free energies for the main proteases has confirmed that good binders for sarscov are in general and sarscov k uzunova polymerases this mechanism is probably involved in an antiviral activity against sarscov2 biochemical data provided by gordon suggested a unifying mechanism of inhibition of sarscov merscov and sarscov2 fig and future emerging covs may be similarly susceptible to the inhibition by remdesivir comparable replication with also good binders for sarscov2 3clpro protease inhibitors a class of drugs best known for success against hiv block the final step of virion assembly in the treatment of human immunodeficiency virus infection with proven efficacy the combination of lopinavir with ritonavir is widely used as a boosted protease inhibitor in the treatment of hiv infection because of low oral bioavailability of lopinavir and its extensive metabolism by the cyp3a4 isoenzyme lopinavir needs to be coadministered with ritonavir to achieve drug concentrations high enough to inhibit viral replication [ “] so far the reported results from studies in different cell lines animal models and patients for lopinavirritonavir are not so convincing in their inhibition action in human coronaviruses screening the library of fdaapproved drugs for antimerscov activity in cell culture has identified four compounds chloroquine chlorpromazine loperamide and lopinavir which inhibit merscov replication effective concentration ec50 3cid0 μmoll in vitro lopinavir inhibited mers cov efficacy ec50 μm and a maximal protective effect were observed at a dose of μm it was previously shown that lopinavir but not ritonavir inhibit sarscov chymotrypsinlike 3cl protease at the concentration of μm moreover it was suggested that lopinavir blocks a postentry step in the merscov replicative cycle in vitro the detectable antiviral activities of ribavirin rimantadine lopinavir and baicalin were shown by using the frhk4 cell line and in vero e6 cells infected with sarscov2 lopinavir inhibit replication with ec50 at μm during the sars outbreak treatment with lopinavir in combination with ritonavir was explored with some success in nonrandomized clinical trials patients with sarscov treated with lopinavirritonavir showed a progressive decrease of viral load and reduction of the composite adverse outcome at day recently the antiviral activity of remdesivir and ifn was found to be superior to that of lopinavirritonavirifn against merscov in vitro and in vivo the efficacy of lopinavirritonavir with or without ribavirin is evaluated in sarscov2 patients under randomized control trials currently it was demonstrated that this combination has no benefits in adult patients with severe covid19 although protease inhibitors are a common class of medication used in the treatment of hiv1 infection their efficacy in human coronavirus infections is not convincing moreover several antihiv pis are also known to influence other intracellular pathways it was demonstrated that hiv protease inhibitors indinavir saquinavir and lopinavir independently from any viral infection can hinder lymphocyte apoptosis by influencing mitochondrial homeostasis in view of the weak antiviral activity of protease inhibitors further studies should be done to ascertain whether the clinical benefit could be attributed to their antiapoptotic rather than their antiviral activity hence even if the molecular target of lopinavirritonavir is the main protease 3clpro in sarscov2 infected cells fig there are no biochemical and molecular studies confirming the interaction and associating this with clinical efficacy of the protease inhibitor chloroquinehydroxychloroquine chloroquine chq was introduced into clinical practice in as a prophylactic treatment for malaria hydroxychloroquine hcq differs from chloroquine by the presence of a hydroxyl group at the end of the side chain the nethyl substituent is hydroxylated currently chq and its hydroxyl form hcq are used as antiinflammatory agents for the treatment of rheumatoid arthritis lupus erythematosus and amoebic hepatitis in addition chq has been studied as a potent antiviral agent against hiv1aids [“] hcov229e sarscov [ ] influenza a h5n1virus influenza a and b and many other rna and dna viruses many recent reports and published studies suggested that chq and hcq were associated with reduced fig inhibition of viral infection by lopinavirritonavir and remdesivir biomedicinepharmacotherapy13120201106683 0ck uzunova progression of the covid19 and decreased duration of the symptoms [“] there are in fact overall more than trials currently underway around the world on its impact either as a prophylactic or treatment for covid19 it is noteworthy that the usefulness of hydroxychloroquine and chloroquine is intensively investigated chloroquine was found to exert an antiviral effect during pre and postinfection conditions suggesting to have both prophylactic and therapeutic advantages timeofaddition assay demonstrated that chq functioned at both entry and postentry stages of the sarscov2 infection in vero e6 cells however it did not reduce viral replication in sarscov infected mice hydroxychloroquine is significantly more potent than chq in vitro ec50 values and μm respectively and has a lower potential for drugdrug interactions than chloroquine pharmacokinetic models demonstrate that hydroxychloroquine sulfate is significantly superior days in advance to chloroquine phosphate in inhibiting sarscov2 in vitro and was demonstrated to be much less toxic than chq in animals on the other hand data presented by liu demonstrated that the antiviral effect of hcq against sarscov2 infection was comparable with chq in vitro cc50 μm and μm for chq and hcq respectively moreover they suggested that both chq and hcq blocked the transport of sarscov2 from early endosomes ees to endolysosomes els and caused noticeable sizemorphological changes in ees and els they surmised that endosome maturation might be blocked at intermediate stages of endocytosis resulting in failure of further transport of virions to the ultimate releasing site hydroxychloroquine shares the same mechanism of action as chloroquine apart from the probable role of chq and hcq as antiviral agents their mechanisms of action are not fully understood and it was demonstrated that they have multiple effects on mammalian cells ace2 is known to be a cell receptor for sarscov the high similarities of the amino acid sequences and predicted protein structures of the receptorbinding domain of sarscov2 and sarscov suggest that sarscov2 may efficiently use human ace2 as a receptor for cellular entry and employ the cellular serine protease tmprss2 for s protein priming zhou confirmed that sarscov2 used the ace2 receptor to enter cells and did not use other coronavirus receptors such as aminopeptidase n apn and dipeptidyl peptidase dpp4 so the primary mechanism by which cell infection is prevented by these drugs may be at the stage of binding with the surface receptor and endosomemediated viral entry two independent in vitro studies confirmed that chq inhibits the replication of the sarscov chloroquine inhibits the early stage of sarscov replication in vero e6 cells with a effective concentration of ± μml the antiviral activity of chq was indicative at the time point at virus attachment or penetration vincent established that the drug might interfere with terminal glycosylation of the cellular receptor ace2 when chq was added prior to infection the impairment of terminal glycosylation of ace2 may result in reduced binding affinities between ace2 and sarscov spike protein and negatively influence the initiation of sarscov infection when chq or nh4cl were added after infection these agents could rapidly raise the ph and interrupt ongoing fusion events between the virus and endosomes thus inhibiting the infection on the basis of in vitro experiments they suggested that the primary mechanism by which infection was prevented was the poor affinity of sarscov spike protein to underglycosylated ace2 in vitro studies with hiv infected cells also identified that inhibition of glycosylation might be a possible mechanism of action of chq chq inhibits hiv replication at a postintegration stage resulting in the production of immature virions it was demonstrated that the sole mechanism explaining the antihiv activity of chq was a decrease in the infectivity of the newly produced virus associated with defective production of the heavily glycosylated 2g12 epitope of gp120 according to in vitro results the antiretroviral effects of chq are attributable to the inhibition of viral p glycosylation these effects appeared to be specific since the chq concentrations effective in vitro neither affected any other step in hiv1 replication nor were cytotoxic thus there is direct evidence that chq is an inhibitor of glycosylation of gp120 and these alterations may be responsible for the decreased infectivity of hiv grown in the presence of chloroquine when added after the initiation of infection these drugs might affect the endosomemediated fusion and subsequent virus replication sarscov pseudoviruses may enter cells via receptordependent clathrin and caveolaeindependent phsensitive endocytosis likely through a process involving lipid rafts a later study however suggests that the entry of coronaviruses into the host cells occurs through clathrinmediated endocytosis murine hepatitis virus mhv a prototypic member of the cov family requires trafficking to lysosomes for proteolytic cleavage at the fp proximal position of its spike s protein membrane fusion to occur many authors indicated that s protein cleavage is an important step for fusion activity and subsequent internalization of the sarscov virus genome into cells [“] adding chq prior to infection results to inhibition of endosome maturation and strongly decreased mhv infection and fusion which was not observed when the drug was added at hpi indicating that the compound mainly affects mhv entry chloroquine is a weak base that is known to increase the ph of lysosomal and transgolgi network tgn vesicles leading to the dysfunction of enzymes necessary for proteolytic processing and posttranslational modifications of newly synthesized viral proteins chloroquine is able to prevent the processing of prm protein to m protein in flavivirusinfected mammalian and mosquito cells by raising the ph of the postgolgi vesicles in which this cleavage occurs as a result virions from infected cells which had been treated with acidotropic amines late in the virus replication cycle contained prm protein rather than m protein and this reduced the infectivity of the virus the chloroquinemediated rise in endosomal ph modulates iron metabolism in a variety of cell types decreasing in intracellular concentration of iron affects the function of several cellular enzymes involved in pathways leading to the replication of cellular dna and to the expression of different genes [“] autophagy is a lysosomedependent degradative pathway chq and its analogue hcq are known clinically relevant autophagy inhibitors chq is a weak base that inhibits lysosomal acidification which prevents the fusion of autophagosomes with lysosomes and subsequent autophagic degradation inhibition of autophagy with chq stimulates superoxide generation ubiquitinconjugated protein accumulation and apoptosis in a colon cancer xenograft model chq treatment clearly inhibited autophagy in mouse lung and efficiently ameliorated acute lung injury and dramatically improved the survival rate in mice infected with live avian influenza a h5n1 virus h5n1 virusinduced autophagic cell death in alveolar epithelial cells through a pathway involving the kinase akt the tumor suppressor protein tsc2 and the mammalian target of rapamycin and autophagyblocking agents might be useful as prophylactics and therapeutics against infection of humans by the h5n1 virus furthermore prentice suggested that authophagy was induced by the coronavirus mouse hepatitis virus mhv and was required for formation of double membranebound mhv replication complexes which significantly enhanced the efficiency of replication replication of the virus was impaired in atg5 knockout embryonic stem cells the same authors also examined the sarscovs and found out similar colocalization of the key viral replication proteins with endogenous lc3 a protein marker for autophagosome it could be assumed that autophagy inhibitors like chq could inhibit virus replication at present the exact role of autophagy in cov infection remains debatable and there is much evidence suggesting that the endocytic pathway plays a key role in mediating viral entry for many covs including sarscovs merscovs and possibly sarscov2 the antiinflammatory properties of chqhcq should also be considered several studies have suggested that multiple an failure biomedicinepharmacotherapy13120201106684 0chas not yet been identified in sarscov2 infected patients and probably multiple pathways could be involved fig conclusion the sarscov2 is the cause of the coronavirus disease covid19 that has been declared a global pandemic by the world health anization who in despite some clinical characteristics that differentiate covid19 from sarscov merscov and seasonal influenza the pathogen sarscov2 has the same phylogenetic similarity to sarscov and mers cov most of the encoded proteins exhibited high sequence identity between sarscov2 and the related batderived coronaviruses batslcovzc45 and batslcovzxc21 a notable difference was a longer spike protein encoded by sarscov2 compared with the bat sarslike coronaviruses sarscov and merscov in addition sarscov2 was distinct from sarscov in a phylogeny of the complete rnadependent rna polymerase rdrp gene moreover the receptorbinding domain of sarscov2 which directly engages the ace2 receptor for cell entry was more closely related to those of sarscovs “ amino acid identity since the outbreak researchers have released many agents that could have potential efficacy against covid19 there is currently no clinically proven specific antiviral agent available for sarscov2 infection like sarscov and merscov certain agents like chloroquine hydroxychloroquine lopinavirritonavir and remdesivir are being used in ongoing clinical trials all over the world with hopes to further delineate their role in the treatment and prophylaxis of covid19 furthermore due to their availability and using for decades and proven safety records it is reasonable to suggest that they may be appropriate for treatment of covid19 remdesivir an adenosine analogue with wellstudied mechanism of action in cov infections can target the rnadependent rna polymerase and block viral rna synthesis and has been a promising antiviral drug antiviral studies in cell culture and animal models the available human safety data as well as the clear mechanism of action characterize rdv as a directacting antiviral since some authors found that lopinavir“ritonavir treatment did not significantly accelerate clinical improvement hence antiviral effects as an inhibitor of the sarscov main 3cl protease should be further investigated although chq and hcq are wellknown drugs for the treatment of k uzunova observed in fatal cases are most likely associated with not only the direct viral infection and destruction of susceptible cells eg endothelial cells but also the effects of proinflammatory cytokines chemokines and other mediators released from infected and activated cells such as monocytes and macrophages the clinical worsening of individuals with sars in week is apparently unrelated to uncontrolled sars coronavirus replication but may be related to immunopathological damage another study reveals that the presence of viral elements within endothelial cells and the accumulation of inflammatory cells led to endotheliitis in several ans as a direct consequence of viral involvement and to host inflammatory response moreover chq has immunomodulatory effects suppressing the productionrelease of tumour necrosis factorα and interleukin6 which mediate the inflammatory complications of several viral diseases chloroquinehcq was reported to inhibit the production of soluble mature tnf in macrophage cell line inhibit tnfα receptor in human histocytic u937 cells inhibit tnfα ifnγ and il6 in peripheral blood mononuclear cells pbmc reduce tnfα production and lipopolysaccharide lpsinduced il1 release in human monocytic cells it is suggested that chq exerts antiinflammatory and immunomodulatory effects predominantly by pretranslational and nonlysosomotropic mechanisms chloroquineinduced inhibition of tnf and il6 production is not mediated through a lysosomotropic mechanism and chloroquine probably acts on tnf secretion by disrupting iron homeostasis besides its antiviral activity and due to its suppressive effects on the productionrelease of tnfα and interleukin chqhcq may be effectively used in the treatment of viral infections characterized by symptoms associated with inflammatory processes andor immunehyperactivation antiinflammatory effects of chq remain poorly understood regulation of proinflammatory cytokines chq can also act on the immune system through cell signaling chq inhibits the activation of p38 mapk in hcov229einfected cells and evokes the activation of erk independently of infection these results suggested that chq may inhibit cov replication by suppressing the p38 activation additionally chq strongly inhibited phosphorylation of mitogenactivated protein kinase mapk p38 and to a lesser extent cjun nterminal kinase and extracellular signalregulated kinase ½ chloroquine could also inhibit innate immune responses trough downregulation of tlr9 signaling pathways requiring endocytosis and acidification of endosomes within plasmacytoid dendritic cells pdcs and act as novel antagonists to chemokine receptor cxcr4 that suppress pancreatic cancer cell proliferation on the other hand another hypothesized mechanism of chq is via the inhibition of antigen degradation and improving the crosspresentation efficiency of dcs in vitro in vivo evidence suggested that a short course of treatment with chq followed by a booster dose of a soluble antigen immunization can efficiently enhance human cd8 t cell responses and single vaccination with inactivated influenza virus combined with chloroquine treatment elicits a higher t cell immunity in mice regulation of nlrp3 inflammasome activation may offer a promising therapeutic approach by inhibiting or slowing down the process of acute respiratory distress syndrome hcq is a known nlrp3 inhibitor and its potential clinical effectiveness is certainly based on the downregulation of il1 expression the major proinflammatory cytokine interleukin1beta il1 is elevated in plasma from hospitalized covid19 patients and its associated signaling pathway seems to drive sarscov2 pathogenicity il1 secretion is primarily initiated by inflamm
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Pathway‘specific model estimation for improved pathway annotation by network crosstalkMiguel Castresana‘Aguirre Erik L L SonnhammerPathway enrichment analysis is the most common approach for understanding which biological processes are affected by altered gene activities under specific conditions However it has been challenging to find a method that efficiently avoids false positives while keeping a high sensitivity We here present a new network‘based method ANUBIX based on sampling random gene sets against intact pathway Benchmarking shows that ANUBIX is considerably more accurate than previous network crosstalk based methods which have the drawback of modelling pathways as random gene sets We demonstrate that ANUBIX does not have a bias for finding certain pathways which previous methods do and show that ANUBIX finds biologically relevant pathways that are missed by other methodsImprovements in molecular biology have led to an increase in highthroughput data which typically produces lists of differentially expressed genes or proteins These lists are useful for identifying genes with important roles in certain conditions However more insight about the biological mechanisms is often needed eg which functional gene sets are related to genes in the result list The study of the relation between a query gene set differentially expressed gene list and functional gene sets pathways is called pathway enrichment analysisImprovements in molecular biology have led to an increase in highthroughput data which typically produces lists of differentially expressed genes or proteins These lists are useful for identifying genes with important roles in certain conditions However more insight about the biological mechanisms is often needed eg which functional gene sets are related to genes in the result list The study of the relation between a query gene set differentially expressed gene list and functional gene sets pathways is called pathway enrichment analysisThere are four generations of pathway enrichment analysis approaches Overrepresentation analysis ORA calculates how many genes from a list of genes extracted based on a threshold or criteria eg differentially expressed genes are in a certain pathway1 Statistical significance is assessed repeating this process with a background list of genes eg all the genes in the microarray This is known as Gene Enrichment Analysis GEA and famous tools like DAVID2 use it Similar but taking into account all the genes in the experiment and the gene expression values is the Functional Class Scoring algorithms FCS3 for which known algorithms include Gene Set AnalysisGSA4 and Gene Set Enrichment Analysis GSEA5 However both FCS and ORA have limitations They both consider genes as independent which is often not true only taking into account their overlap and not their associations or interactions6 Another issue with overlapbased methods is their low coverage since they are heavily dependent on pathway knowledge which is still incomplete leading to a high rate of false negatives7 Pathway topologybased methods use the same steps as FCS with additional pathway topology information However the reliance on gene overlap leads to similar limitations as ORA and FCSWe could consider the network crosstalk enrichment tools as the fourth generation They rely on a network such as a functional association network like Funcoup8 or STRING9 These networks integrate different experiments from different data types into a single network providing information about gene to gene functional associations which is translated into links in the network With this limitations such as gene independency and low coverage of overlapbased methods are overcome Association between two sets is measured in terms of links between them in the network known as crosstalk In the past few years different ways to assess enrichment between two gene sets have been published like NEA10 EnrichNet11 CrosstalkZ12 NEAT13 NEArender14 BinoX7 and GeneSetDPGeneSetMC15 EnrichNet defines a network enrichment score based on network distances between two gene sets using random walks with restart but is not able to calculate statistical significance Department of Biochemistry and Biophysics Science for Life Laboratory Stockholm University Box Solna Sweden email eriksonnhammerdbbsuseScientific RepoRtS 101038s4159802070239zVol0123456789wwwnaturecomscientificreports 0cof the enrichment The tools NEA and CrosstalkZ assess significance using statistical tests assuming that crosstalk between nonenriched gene sets is normally distributed but this is often not the case Moreover they rely on network randomizations to obtain null model parameters which makes them computationally very slow Computational time is reduced in BinoX which also applies network randomization but uses the binomial distribution to calculate statistical significanceThe methods NEAT NEArender and GeneSetDPGeneSetMC do not use network randomization NEAT calculates the expected number of links between two gene sets based on their degrees and then uses the hypergeometric distribution to assess statistical significance NEArender computes the expected number of links in the same way as NEAT but uses a chisquare test to assess statistical significance GeneSetDP uses dynamic programming to calculate an exact distribution of the expected number of links to a pathway for a certain gene set size GeneSetMC does this approximately using MonteCarlo sampling which is faster These two algorithms are however not implemented to allow large scale pathway enrichment analysisThe null model assumption of NEAT NEArender and BinoX is that compared gene sets are expected to behave like random gene sets For real pathways that are very nonrandom eg highly intraconnected this can lead to underestimating the expected level of crosstalk and produce a high false positive rate FPR To avoid this it is important that the method can cope with the nonrandomness of pathways To this end we have developed a novel networkbased pathway enrichment analysis algorithm called ANUBIX Adaptive NUll distriButIon of Xtalk which is based on scoring random gene sets against real pathways to build its null model We show that ANUBIX clearly outperforms recent network crosstalk methods like BinoX NEArender and NEAT in terms of avoiding False Positives FP showing that it can model expected network crosstalk to pathways more preciselyMaterial and methodsOur networkbased pathway enrichment analysis tool ANUBIX depends on a global functional association network We used the network Funcoup version with a link confidence cutoff of containing genes and links With those genes cid31g1 g2 gnˆ’ gncid30 ˆˆ S and all the pairwise links between them form a symmetric matrix A with dimensions SxS such thataij 1if gi is connected to gj and i � j otherwise aij A gene set Q and a pathway P are a subset of the total number of genes for a certain proteome such that Q P Š† S Notice that S Š† Q we can have some genes from the proteome that are not in the network The crosstalk between Q and P is measured with the degree k cid31iˆˆQcid31jˆˆPaijThe null model is built based on the expected crosstalk between a random gene set of the same size as the original gene set Q and pathway P Since the network connections are binary each link is considered as a Bernoulli trial Y ˆ¼ Bcid31pcid30 where p is the probability of observing a link We also calculate n QP ˆ’ Q ˆ P all the possible links between Q and P We count the links each gene from Q has to the pathway P meaning that if two linked genes are in Q and also in the P we count that link twice boosting the cases where we find overlap Each of these Bernoulli trials are assumed to be independent and the sum of them follows a binomial distributionIn the binomial distribution the mean and variance are defined as µ np and Var npcid311 ˆ’ pcid30 respectively This means that µ ‰¥ Var which may not be true when the random variable is overdispersed leading to an underestimation of its variance16The betabinomial distribution has been extensively used as an alternative to handle overdispersed binomiallike random variables1718 Here the probability of success p is not fixed as it is in the binomial distribution but follows a beta distribution Betaα β with parameters α and β The marginal distribution of the betabinomial is described in Eq a0fcid31kn α βcid30 cid29 nk cid28 Bk α n ˆ’ k βBα βTo estimate the optimal parameters of the betabinomial we use maximum likelihood estimation MLE19 where the loglikelihood is Eq a0lcid31kn α βcid30 logLcid31kn α βcid30 logcid29 n logcid29 nk cid28 logBα k β n ˆ’ k ˆ’ logBα βk cid28 logŴα k logŴβ n ˆ’ kˆ’logŴα β n ˆ’ logŴα ˆ’ logŴβ logŴα βThe negative loglikelihood is optimized with the Nelder and Mead method20 The factorial term in the loglikelihood is removed since it does not depend on the parameters to be optimized Once we have the betabinomial parameters α β of our null distribution we calculate if the crosstalk between Q and P is enriched The null and alternative hypotheses areH0 No more links between Q and P than expected by chanceH1 More links between Q and P than expected by chanceBecause of the discrete nature of the null distributions ordinary pvalues are conservative and therefore mid pvalues were used2122 Mid pvalue is defined as half the probability of the observed statistic plus the probability of observing more extreme values22 The workflow of the ANUBIX algorithm is depicted in Fig a0Scientific RepoRtS 101038s4159802070239zVol1234567890wwwnaturecomscientificreports 0cFigure a0 Workflow of ANUBIX The algorithm assesses the significance of the network crosstalk between a query gene set and a pathway A null distribution is generated for each pathway to model the expected crosstalk of random gene sets of the same size as the original gene set This distribution is then fit to a betabinomial distribution to calculate the probability of reaching at least the number of observed links or more between the query gene set and the pathway Software Inkscape version inksc apeIt is important to point out that the networkbased approaches ANUBIX NEAT NEArender and BinoX test three different types of null hypothesis ANUBIX which takes only enrichment into account computes a one tailed test NEAT computes two onetailed tests for enrichment and depletion and takes the minimum pvalue of them multiplied by to emulate a twotailed test BinoX and NEArender compute both enrichment and depletion but only perform one onetailed test since the hypothesis test changes depending on whether the observed number of links is above or below the expected crosstalkPathways To generate the false positive and true positive benchmarks we used KEGG v70123 pathways and REACTOME v6224 pathways for Homo sapiens REACTOME pathways have a deep hierarchical structure including many small pathways on the lower levels that are very specific To reduce Reactome™s specificity we resolved its hierarchy by collapsing lower level pathways below a certain pathway size to their parents until obtaining an average pathway size similar to KEGG pathways genes per pathwayPerformance measures In the FP benchmark we generated random gene sets and tested them against KEGG and REACTOME pathways To make these gene sets representative of real experiments we took the average size of MSigDB25 gene sets which is genesIn the True Positive TP benchmark we bisected the KEGG pathways and REACTOME pathways into two parts Each part gets a similar number of genes and links7 To be able to benchmark GEA we emulated some overlap between the two bisected parts This overlap corresponded to the average overlap between the MSigDB gene sets and the pathway measured individually for each of the pathways in KEGG and REACTOMECorrection for multiple hypothesis testing was done using the Benjamini“Hochberg procedure26Stability Our null distributions are based on random sampling We take random samples of genes from the genome This stochastic procedure makes the null distributions different every time they are generated Since the pvalues are computed from the null distribution their values may change To analyze stability we generated the null distribution times for the crosstalk between the same gene set to the same pathways for increasing numbers of random samples For each sample size we computed the coefficient of variation CV which is the ratio between the standard deviation SD and the mean We required a CV lower than to limit the dispersion of the mean of the null distribution and this was reached at random samples Once the number of random samples were chosen we measured how much the pvalues were varying in each run For that we ran a randomly selected MSigDB gene set times To compute the confidence interval of the pvalues we used the central limit theorem and applied normal distribution statistics to compute themUsed programs ANUBIX bitbu cketsonnh ammer group anubi xBinoX bitbu cketsonnh ammer group binox NEAT cranrproje ctwebpacka gesneatneatpdfNEArender cranrproje ctwebpacka gesNEAre nderNEAre nderpdfGeneSetDP githu bcomstati stica lbiot echno logygenes etdpScientific RepoRtS 101038s4159802070239zVol0123456789wwwnaturecomscientificreports 0cFigure a0 Overdispersion of KEGG and REACTOME pathways null distributions when sampling random gene sets of size from the proteome The dispersion for each pathway is calculated as the ratio between the variance and the mean of the crosstalk null distribution For each pathway database we illustrate the dispersion values through a boxplot and also by showing the dispersion distribution Software R version wwwrproje ctFigure a0 Observed crosstalk distribution fit with binomial and betabinomial distributions random gene sets of size were used to generate a null distribution of crosstalk to the A œBetaalanine metabolism B œProstate cancer and C œAlzheimer™s disease pathways Betabinomial shows a much better fit to the observed link distribution than the binomial Software R version wwwrproje ctResultsTo correctly assess the statistical significance of an observed network crosstalk between two gene sets eg one experimental gene set and one known pathway it is paramount that the null distribution appropriately models the crosstalk of random query gene sets Note that it is not necessarily appropriate to assume that the pathway gene set behaves like a random gene set ie the null distributions need to model crosstalk between random query gene sets versus real pathway gene sets It is also paramount to model the expected crosstalk distribution with an appropriate distribution Previous methods such as BinoX or NEAT use binomial and hypergeometric distributions respectively which are not appropriate for overdispersed distributions since they do not allow the variance of the distribution to be greater than the mean To showcase this we generated null distributions for KEGG and REACTOME pathways by sampling gene sets of size from the proteome In Fig a0 we show the dispersion for each pathway as the ratio between the variance and the mean of the crosstalk null distribution We observe that almost all of these distributions suffer from overdispersion meaning that the variance of the distribution is greater than the mean Therefore statistical models that cannot cope with overdispersion are not appropriate to model the null distribution of most pathwaysTo visualize the overdispersion in detail we chose pathways that are in different quartiles of the dispersion distribution We show their null distributions in Fig a0 Figure a03A shows the œBetaalanine metabolism Scientific RepoRtS 101038s4159802070239zVol1234567890wwwnaturecomscientificreports 0cFigure a0 Pvalue uniformity test of ANUBIX Binox GEA GeneSetDP NEArender and NEAT random gene sets of genes were tested for crosstalk enrichment against the KEGG pathway œProstate cancer A Reported pvalues are plotted against theoretical quantile rank A perfect method should adhere to the diagonal B Distributions of the FPR for all KEGG and REACTOME pathways tested with ANUBIX BinoX NEArender NEAT and GEA Green distribution for enriched tests and red distribution for depleted The dashed line at FPR denotes the expected FPR level The black triangle and circle represent the mean FPR for enrichment or depletion respectively Software R version wwwrproje ctpathway whose dispersion value is in the first quartile Figure a03B shows the œProstate cancer pathway with a dispersion in the second quartile and Fig a03C shows the œAlzheimer™s disease pathway with a dispersion in the fourth quartile The high variance relative to the mean gives a very poor fit with the binomial distribution yet the betabinomial distribution gives a very good fit This underestimation of variance by the binomial distribution would lead to many false positives With a few pathways there is no overdispersion in the data but these can fit a betabinomial equally well as a binomialBenchmark for false positives Since the null model in ANUBIX is based on random gene sets we expect the pvalue distributions when tested with random query gene sets to behave uniformly for any pathway For almost all pathways we observed a virtually perfectly uniform distribution when plotting ANUBIX pvalues of random gene sets against each KEGG pathway full results at Supplementary Fig a0 A few pathways deviated somewhat from uniform which is the result of the betabinomial fit not being able to model the null distribution with enough precision A second type of deviation from perfect uniform distribution is caused by staggering of observed pvalues This is relatively frequent and arises because the support of the test statistics is limited to a few values and therefore unavoidable We also generated the pvalue distributions for gene sets of size and size against each KEGG pathway Supplementary Fig a0 and respectively which gave similar results However some pathways seem to be affected by the size of the gene set ANUBIX was compared to the top networkbased methods BinoX NEAT and NEArender and a recently published method GeneSetDP For comparison we also tested a popular overlapbased pathway enrichment method GEA Because GeneSetDP and GenesetMC are too computationally heavy for large scale analysis we first tested all the gene sets against one individual pathway We only used GeneSetDP because GeneSetMC produces similar pvalues Pvalues were plotted versus quantiles of a uniform distribution For an unbiased method the pvalues would lie on the diagonal y x Figure a04A shows that for the œProstate cancer pathway Pvalues of ANUBIX adhere to the diagonal much better than for BinoX NEAT NEArender and GEA while performing equally well as GenesetDPFor crosstalk to random gene sets we expect of the pvalues to be lower than However for the œProstate cancer pathway BinoX had of its pvalues lower than NEAT and NEArender GEA whose coverage is small7 had of its pvalues below and highly discrete taking on only four possible values for œProstate cancer due to few overlapping genes ANUBIX and GeneSetDP find a correct fraction of the pvalues with and GeneSetDP under respectivelyWe also ran ANUBIX BinoX NEAT NEArender and GEA for the random gene sets against all pathways in the KEGG database and REACTOME database Full results in Supplementary Data and Data respectively GeneSetDP was not included as it is not implemented to run at a large scale NEAT NEArender and BinoX can also give statistical significance when gene sets have fewer links to a pathway than expected by chance known as depletion To make a more consistent benchmark where all methods can be compared equally we only considered enrichment and depleted pathways were treated as nonsignificant The average FPR for all KEGG pathways was with ANUBIX with BinoX with NEAT with NEArender and with GEA For REACTOME almost the same FPR values were obtained ANUBIX BinoX NEAT NEArender and GEA Roughly the same FPR levels came from significant depletions for BinoX NEAT and NEArender However the averaging of the FPR levels for all pathways does not show the real problem of these methods Some pathways could give very nonconservative pvalues while other pathways could give very conservative pvalues To show how each method performs for each of the pathways we plot the distribution of the FPR fraction of pvalues below for each pathway as violin plots in Fig a04B Since GEA Scientific RepoRtS 101038s4159802070239zVol0123456789wwwnaturecomscientificreports 0cand ANUBIX cannot test for depletion they only have the enriched case A perfect method would have all points close to the dashed line at FPR ANUBIX produces FPR values close to this line meaning that the model is robust GEA greatly underestimates FDR and produces almost no false positives but this leads to very poor sensitivity as shown below NEArender NEAT and BinoX produce similar FPR distributions that are very spread out ie the FPR tends to be very different for different pathways For the tests performed per pathway some pathways reach an FPR of for enriched cases and similar for depleted Summing these two can lead to a total FPR above if we take both enriched and depleted cases into account which is very nonconservative The plot also shows that for some pathways these methods are overly conservative giving considerably lower FPR than they should In other words methods like BinoX NEAT and NEArender have a huge variation in the quality of their pvalues depending on the pathway under studyBinoX is implemented in a web server called PathwAX27 where users can submit a query gene set to test for network crosstalk enrichment By analogy we studied false positive rates assuming independence between gene sets where each user submits a single gene set ie multiple testing correction is only performed for number of pathways each query is compared to random gene sets were used against the KEGG database A FDR threshold of was used and enrichment and depletion were grouped separately as shown in Fig a05A The top pathways with highest FPR for BinoX were plotted full results in Supplementary Data all having a highly nonconservative behaviour for BinoX NEAT and NEArender Every time a user submits a random gene set the chance of getting one of these pathways is very high on average if we take both enriched and depleted cases into account In contrast ANUBIX and GEA have less than FPR We observed a very high correlation between perpathway FPR values for BinoX NEAT and NEArender above for each pairwise comparison This indicates that the pathway enrichment analysis results obtained with these methods are highly similar They all had low Pearson correlation to ANUBIX with for BinoX for NEAT and for NEArender The corresponding Spearman correlations were and As for the pathways we noticed that there is a high overlap between some of them For instance the œAlzheimer™s disease and œParkinson™s disease pathways share of their genes The œAlzheimer™s disease the œParkinson™s disease and the œHuntington™s disease pathways have of the genes in common from the union between them Further the œOxidative phosphorylation the œNonalcoholic fatty liver disease the œAlzheimer™s disease the œParkinson™s disease and the œHuntington™s disease have of the genes in common from the union between them Therefore if there is significant crosstalk to one of them crosstalk to the other pathways is very likely The high dependency between some pathways points to opportunities for further improvement of pathway definitions Further exploration was performed in these pathways™ topologies to understand their tendency to generate many FPsWe computed the fraction of intralinks for each pathway as the ratio between the number of internal links and the total number of links We plotted this ratio against the FPR Fig a05B A higher fraction of intralinks means that more links are within the pathway than to the outside suggesting a more isolated pathway The Spearman correlation coefficient between the fraction of intralinks and FPR for BinoX was indicating that the fraction of internal links plays a major role in causing false positives This dependence is also observed with NEAT with a correlation of and with NEArender at However ANUBIX had a correlation of only and GEA This indicates that methods like NEAT NEArender and BinoX cannot deal properly with pathways that are clearly not random and behave more as isolated communitiesAdditionally we calculated the number of maximal cliques each of the KEGG pathways has and we observed a correlation with the FPR for BinoX with a spearman correlation of These maximal cliques were computed using the igraph package in R We considered cliques as all complete subgraphs and a clique is considered maximal if we cannot add more nodes to it This indicates that the higher the number of maximal cliques in a pathway meaning a less random pathway in terms of topology the higher the FPR isBenchmark of true positives Besides a correct FPR it is also important to verify that the power of the method is sufficient for a high true positive rate TPR To this end we devised a benchmark by splitting each KEGG and REACTOME pathway into two parts and then measured each method™s ability to reconnect these parts The splitting into parts included giving an amount of gene overlap between the two parts emulated based on the average overlap between MSigDB gene sets and KEGG and REACTOME pathways We compared the methods by their Receiver Operating Characteristic ROC curves Figure a06A shows only the tests that are statistically significant FDR and only considering enrichment ANUBIX has a TPR of of the enrichment tests as significant without having any FP BinoX has a TPR of with FPR NEArender a TPR of with FPR and NEAT a TPR of with FPR GEA whose coverage is low gives only TPR and no FPs Figure a06B shows the ROC curve for all the enriched tests performed also including insignificant results This shows the coverage of each method ANUBIX recovers of the TP tests without suffering any FPs BinoX NEArender and NEAT have similar curves recovering and of the enriched TP tests respectively GEA can here maximally find of the TP tests since only those tests have some gene overlap This benchmark shows that GEA has very low coverage of what it can potentially find We note that the maximal TPR obtained by GEA corresponds to the amount of significantly enriched crosstalks obtained when running all of MSigDB against KEGG pathways see Pathway annotation of MSigDB gene setsStability and robustness Considering that the null distributions are based on random sampling we studied the number of iterations required to reach a coefficient of variation CV of Figure a07A shows how many pathways pass that threshold depending on different amounts of random samples of the pathways had a CV lower than when using random samples to model the null distribution To verify that this number of random samples is sufficient for every pathway we computed the enrichment of one randomly selected MSigDB Scientific RepoRtS 101038s4159802070239zVol1234567890wwwnaturecomscientificreports 0cFigure a0 Analysis of why certain pathways are very prone to produce false positives random gene sets of genes were tested independently for crosstalk enrichment against the KEGG pathways A The top ten pathways that produce the highest false positive rate FPR with BinoX and the FPR obtained with other methods B Fraction of intralinks for each of the KEGG pathways against FPR The size of the point reflects the total number of links in each pathway Software R version wwwrproje ctgene set to all KEGG pathways times The null distributions are thus generated times for each pathway and we would expect some changes in the pvalues between runs Figure a07B shows the standard deviation of the pvalues We observe that the pvalues almost did not vary showing that random samples are enough Moreover because of sampling the pvalue is not an exact pvalue but a point estimate of it we also provide with the confidence interval of each of the pvalues Supplementary Data Compute time Our method relies on random sampling to model the null distribution which makes ANUBIX computationally intensive To benchmark its speed we did runs each time with a randomly chosen biological gene set extracted from MSigDB against KEGG REACTOME and KEGG plus REACTOME We measured the compute time for each of the networkbased methods see Fig a0 With this benchmark we can show that ANUBIX is fast when running single gene sets One should take into account that ANUBIX and BinoX need a precomputation step before running the actual analysis However the ANUBIX precomputation step takes around a0s whereas in BinoX it takes around a0min To compute the randomized network for BinoX Scientific RepoRtS 101038s4159802070239zVol0123456789wwwnaturecomscientificreports 0cFigure a0 Receiver Operating Characteristic ROC curve For the TP tests each KEGG and REACTOME pathway is divided into two and a TP is interpreted as the crosstalk between two parts from the same pathway For the FP tests random gene sets of size are tested for enrichment against KEGG and REACTOME pathways A ROC curve for only the significantly enriched tests FDR B ROC curve for all enriched tests Software R version wwwrproje ctFigure a0 Stability analysis of ANUBIX A Fraction of KEGG pathways with Coefficient of variation CV below for different number of iterations B ANUBIX pvalues are stable”their variance is low and proportional to the magnitude of the pvalue A randomly chosen MSigDB gene set DAIRKEE_CANCER_PRONE_RESPONSE_BPA was run times against KEGG pathways Standard deviation of the logpvalue is plotted against the meanlogpvalues for each pathway Software R version wwwrproje ctwe used iterations A drawback for ANUBIX compared to methods like BinoX or NEAT is that the computation time for large scale analyses take more time For instance the time required to compute the large scale pathway annotation study for the MSigDB gene sets against KEGG pathways took a0min for ANUBIX using cores a0min for NEArender a0min for BinoX and a0min for NEAT Compute times were measured on an i77700 CPU a0GHz with a0Gb RAMPathway annotation of MSigDB gene sets We carried out a largescale pathway analysis study by running MSigDB gene sets against KEGG pathways using ANUBIX BinoX NEAT NEArender and GEA Full results are in Supplementary Data In total crosstalk tests were done per method and to get a more fair comparison between different methods we only considered enriched crosstalk considering that ANUBIX and GEA can only test for enrichmentNEArender BinoX and NEAT found the highest number of significantly FDR enriched crosstalks with and of all pairs respectively followed by ANUBIX with and GEA with Many MSigDB gene sets thus appear to have a high occurrence of pathway enrichments Even if we do not know whether those enrichments are TPs or FPs we show above Figs a0 and 5A that BinoX NEArender and NEAT are prone to produce FPsThe Venn diagram in Fig a0 shows that the overlap between BinoX NEAT and NEArender is very high having of their significant pathway annotations in common This was expected since all these methods consider pathways as random The overlap is even higher between NEAT and NEArender because they compute Scientific RepoRtS 101038s4159802070239zVol1234567890wwwnaturecomscientificreports 0cFigure a0 Compute time when running a random experimental gene set from MSigDB different gene sets were tested against KEGG REACTOME and KEGG plus REACTOME pathways for each of the networkbased methods Since ANUBIX allows parallelization we also added another run with cores The error bars show the variability in compute time for each of the methods in each of the databases The BinoX precomputation step is not included since it takes a0min Software R version wwwrproje ctFigure a0 KEGG pathway annotation for MSigDB gene sets with five methods The Venn diagram shows the number of shared pathway annotations at FDR Note that ANU
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"Later in the text we introduce the global improver which can often overcome both problems.Our ˜global improver™ is based on the procedure in (17). Let M = {M1 ¦ Mn} be a collection of disjoint node sets (e.g. a set can be a single gene or not linked to any other set). Given sets (UV) U V M and x U the significance of the linkage of x with V is calculated using Wilcoxon rank-sum test by comparing the edge weights WG between x and V to the edge weights between x and all nodes not in V. Such P-values are calculated for all nodes in U and V and they are combined using Stoufer™s method (28). If the final P-value p(UV) is at most ? then U and V are connected by a ˜link™ in the map. Let L = {(U1V1) ¦ (UpVp)} be the resulting set of links.The ˜global score™ of the solution is the sum WH of edge weights within each Mi plus the sum of WG edge weights between the linked node sets: The improvement stage merges a pair of node sets (two modules or a module and a single gene) if the global score increases and the new link passes the significance test. Considering a merge that creates a new module Y requires recalculating p(YZ) for all other modules Z in M in order to calculate the global score. This process is done greedily: iteratively the merge that yields the best improvement is performed until no possible merge can improve the global score.We modified the aforementioned method to allow for fast analysis of large graphs as follows. First when calculating p(UV) we consider the links in G™ (the unweighted version of G). We use a hypergeometric test to evaluate if a node has significant number of edges in G™ to the opposite set (e.g. from a node v V to the set U) and then all node P-values are merged using Fisher™s method (29). The sets U and V are linked if the resulting value ??. This test is much faster and provides maps of equal quality to using the Wilcoxon test on G (see Supplementary Text). Weighted tests such as the Wilcoxon test are not always appropriate for detecting linkage among gene modules. For example in the DC graphs a strong link must contain many positive edges whereas the Wilcoxon test only looks at the ranks of the edge scores.Second we set another parameter ?>>? and if at some point the P-value for the possible link between two sets is at least ? we say that the sets are ˜anti-linked™. In the original algorithm when considering merging two sets U and V into W possible links between W and every other set Y must be calculated. However if U and Y are anti-linked or V and Y are anti-linked then we mark W and Y as anti-linked avoiding the need to consider the possible link (WY). In practice we used ? = 0.005 for the yeast data as suggested in (17) and tested several options in the gene expression data (see Supplementary Text). In all cases we used ? = 0.2. Finally we perform multiple merge steps simultaneously in a single iteration in a way that guarantees that the global score improves (see Supplementary Text). This provides a speed up of two-fold or more in practice without loss of solution quality.SimulationsWe constructed initially empty 500-node graphs H and G and then added edges creating a perfect module map in which modules are cliques in H and links are bicliques in G. The module map topology (ML) was a random tree with |M| = 6. We then added two H-cliques and two G-bicliques to the graphs to represent additional ˜decoy™ structures that are not part of the map. Clique biclique and module sizes were randomly selected in the range 10“20 with uniform distribution and disjoint node sets. Call the resulting edge sets EH* and EG*. Finally we modified these graphs by introducing random noise: each edge in G and H was deleted with probability P and each non-edge was replaced by an edge with probability P. All reversal steps were done independently. For creating weighted graphs the same procedure was used but all possible edges are present in the final H and G: w(uv) is sampled from N(1?) if (uv) is in EH* or EG* and from N(?1?) otherwise. We also generated in this manner 1000 node graphs with 10 or 20 modules and five decoys (cliques and bicliques).Analysis of negative genetic interactions and protein-protein interactions in yeastThe PPIs and the negative GIs were downloaded from BIOGRID (30). These networks were used to find epistatic relations among protein complexes. The PPI network was used as H and the GI network was used as G (see Supplementary Table S1).Analysis of DNA damage-specific genetic interactions dataWe used the data of (21) in which all pairwise GIs among 418 genes were tested and of (31) which tested GIs between 55 query genes and 2022 genes. A ˜DNA damage-specific positive GI™ was defined as one that had S < 0 in the untreated cells S > 0.5 in the treated cells and the P-value for differential GI was <0.01. This analysis yielded 840 interactions from (21) and 1677 interactions from (31). We additionally defined a positive GI as ˜stable™ if it had S > 1.5 both in the untreated cells and in the DNA damage cells. "
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" preproof 0c highlights f0b7 propolis made by bees from bioactive plant resins has antiviral activity f0b7 propolis potentially can interfere with host cell invasion by sarscov2 f0b7 propolis blocks proinflammatory pak1 a kinase highly expressed in covid19 patients propolis a resinous material produced by honey bees from plant exudates has long been used in traditional herbal medicine and is widely consumed as a health aid and immune system booster the covid19 pandemic has renewed interest in propolis products worldwide fortunately various aspects of the sarscov2 infection mechanism are potential targets for propolis compounds sarscov2 entry into host cells is characterized by viral spike protein f0b7 propolis is a safe widely consumed functional food with medicinal properties f0b7 standardized propolis has consistent properties for lab and clinical research preprooffactor in advanced covid19 disease propolis has also shown promise as an aid in the treatment of various of the comorbidities that are particularly dangerous in covid19 patients including induced lung inflammation fibrosis and immune system suppression propolis components have inhibitory effects on the ace2 tmprss2 and pak1 signaling pathways in addition antiviral activity has been proven in vitro and in vivo in preclinical studies propolis promoted α this immunoregulation involves monocytes and macrophages as well as jak2stat3 nfkb and inflammasome pathways reducing the risk of cytokine storm syndrome a major mortality interaction with cellular angiotensinconverting enzyme ace2 and serine protease tmprss2 this mechanism involves pak1 overexpression which is a kinase that mediates coronavirusimmunoregulation of proinflammatory cytokines including reduction in il6 il1 beta and tnfrespiratory diseases hypertension diabetes and cancer standardized propolis products with consistent bioactive properties are now available given the current emergency caused by the covid19 pandemic and limited therapeutic options propolis is presented as a promising and 0crelevant therapeutic option that is safe easy to administrate orally and is readily available as a natural supplement and functional food keywords propolis sarscov2 covid19 antiviral antiinflammatory pak1 blocker introduction the covid19 pandemic is of grave concern due its impact on human health and on the deadly disease most require more robust data in clinical trials before they can be widely and safely used isolation and stayathome measures do not effectively protect essential workers economy it is much more deadly than influenza and other types of diseases that recently have had worldwide impact forcing countries to take unusual measures such as limiting travel closing schools businesses and other locations where many people can come into contact with each other various public healthcare strategies have been adopted in an attempt to reduce the impact of the disease but with limited effectiveness as the virus continues to spread often through asymptomatic patients unfortunately tests to determine if people are infectious or were previously infected are not widely available often are costly and frequently do not provide timely and accurate results various therapeutic alternatives have been proposed and tested however preproofœnonessential professions return to the workplace they become more at risk for infection in this scenario any options that could help ameliorate disease progression and its consequences even marginally would be useful the world needs safe alternatives to help reduce the impact of this especially health care personnel who have become infected and are dying at alarming rates economic and other necessities limit how well and how long these isolation measures can be maintained especially in poor countries and in poor communities such as slums and favelas [ ] as populations gradually try to get back to normalcy reducing social distancing and people in natural products which have historically been widely used to help avoid and alleviate diseases are among the options being considered as an adjuvant treatment for sarscov2 infection because they are generally inexpensive widely available and rarely have undesirable side effects some have proven antiviral activity an important advantage of using natural remedies is that people who have other health problems or who have mild flurelated 0c infection infection by sarscov2 the virus that causes covid19 is characterized by binding symptoms but do not have the means or courage to visit an already overcrowded medical facility could take simple and inexpensive measures to help reduce the impact of infection with sarscov2 considering the large number of deaths and other types of damage that the covid19 pandemic is causing there is an urgent need to find treatments that have been approved as safe and potentially able to inhibit the new coronavirus reduce its infectivity andor alleviate the symptoms of infection [ ] along this line propolis and its components emerge as potential candidate materials that could help to reduce the pathophysiological consequences of sarscovfactors increased pak1 levels also suppress the adaptive immune response facilitating viral replication [ ] sarscov2 infection is associated with increased levels of chemokines and activated proinflammatory cytokines that lead to the development of atypical pneumonia with rapid respiratory impairment and pulmonary failure immunologicalinflammatory between viral spike proteins and angiotensinconverting enzyme ace2 activation of the spike protein is mediated through proteases such as tmprss2 which play important roles in the viral infection after entry followed by endocytosis coronavirus infection causes pak1 upregulation a kinase that mediates lung inflammation lung fibrosis and other critical mortality preproofphenomena such as cytokine release syndrome have been shown to be important in the spectrum of sarscov2 infection these mechanisms are associated with an dysfunction more than the viral load per se along this line a retrospective observational study found higher serum levels of proinflammatory cytokines such as il6 il1 and tnfα in patients with severe inflammatory diseases by affecting various metabolic cycles recently several studies have shown that propolis extract and some of its components act against several important targets in the pathophysiological context of the disease caused by sarscov2 such as reducing tmprss2 expression and reducing ace2 anchorage which would otherwise facilitate entry of covid19 compared to individuals with mild disease there is considerable evidence that propolis can reduce and alleviate the symptoms of the virus into the cell this is in addition to immunomodulation of monocytes macrophages reducing production of and eliminating il1 beta and il6 reduction of the transcription factors 0cviral replication and antiinflammatory action [ ] propolis and its properties are particularly relevant to sarscov2 infection such as immune system fortification reduced nfkb and jak2 stat3 and blocking pak1 which determine inflammatory activities and fibrosis caused by covid19 various comorbidities have been associated with severe covid19 symptoms and a greater chance of patients requiring intensive care these include hypertension and diabetes also mortality rates of covid19 patients are much higher in those with cardiovascular disease chronic respiratory disease and diabetes [ ] there is considerable evidence that these conditions could be alleviated by treatment with propolis this includes research in animal models of diabetes [ ] hypertension [ ] and cardiovascular disease [ ] propolis has properties that preproofthese plant products to make propolis which they use to protect the colony the production and use of propolis by honey bees evolved to the point that these social bees have considerably fewer immune genes than solitary insect species bees in colonies that produce more propolis are healthier and live longer and propolis consumption by the bees augments their immune [ ] as this variability can affect their medicinal properties standardized propolis products have been developed to help meet the need for a product that does not vary in the main bioactive components and is safe with minimal interaction with pharmaceutical drugs and proven efficacy in clinical trials in recent decades it has been shown to have antimicrobial including the composition of propolis varies according to the plant species available in each region propolis is a product derived from resins and plant exudates plants defend themselves from pathogens mainly by producing phytochemicals many of which have been extracted and used in medicine plant defense substances collected by bees include phenols and terpenoids phytochemical compounds that show promise for the inhibition of coronavirus in humans include quercetin myricetin and caffeic acid all components of propolis honey bees and many other species of social bees recognize these antimicrobial properties and selectively collect and process response to bacterial challenge antiviral antiinflammatory immunomodulatory antioxidant and anticancer properties propolis has historically been widely used to alleviate various diseases [ ] it also has been considered among other natural alternatives as an adjuvant treatment for sarscov2 infection 0ccompared to the propolis that the bees make from these plant materials because it is generally inexpensive widely available and rarely causes undesirable side effects some types of propolis that are highly valued for their medicinal properties such as brazilian green propolis are mainly produced by bees from materials they collect from specific plants in this case baccharis dracunculifolia after the botanical origin of the propolis has been identified extracts of the plant can be made to develop useful products such as a medicinal mouthwash however the medicinal properties of these plant extracts are often inferior can be safely consumed these propolis products and the raw material for their manufacture are extensively exported by brazilian companies especially to asian countries including japan south among natural medicine alternatives propolis has been widely studied and is already extensively consumed in many countries [ ] for example propolis products such as throat sprays and extracts are produced by hundreds of companies in brazil and are sold as a health aid in nearly every pharmacy throughout the country demonstrating on a practical basis that they preproofin fact propolis has a wide spectrum of pharmacological properties and is a dietary supplement that is commonly consumed by both healthy and sick people as a preventative precaution and for treatment it is also used in veterinary medicine due its antibacterial antifungal antiviral korea and china [ ] the importance of propolis in chinese japanese russian and korean medicine is reflected in the number of patents for propolis containing products registered by including about by china and “ each for japan russia and korea since about new propolisrelated patents were applied for in the us patent office it is a key ingredient in traditional chinese medicine japanese scientists have isolated and patented various brazilian propolis components for cancer treatment demonstrating their usefulness why propolis may be a good fit for dealing with covid19 antiparasitic hepatoprotective and immunomodulatory activities in the wake of the coronavirus outbreak south korea has seen a boon in the use of functional foods according to their ministry of food and drug safety œhealth functional foods are nutrients that have been proven to be beneficial to health in march of this year in response to the coronavirus pandemic the ministry eased regulations for propolis which is considered a 0ccould help fight against the covid19 pandemic active site of this enzyme is a relevant target for drug discovery functional food and allowed new oral formulations however despite considerable evidence that propolis can reduce and alleviate disease symptoms its acceptance as a healthpromoting supplement in human medicine has been limited in many countries such as the usa because of a relevant criticism that propolis products are not standardized and vary in their components and biological activity in part this is because propolis varies with the species of plants available in each region from which the bees collect resins to produce it [ ] however standardized propolis products have recently become available to help fill the need for a product that does not vary in the main bioactive components and effectiveness [ ] one such option a standardized brazilian propolis extract blend has been tested for safety and effectiveness in clinical trials for treating kidney disease and diabetes denture stomatitis and burn patients therefore propolis as a nutraceutical or functional food should be considered as a resource that preproofcaffeic acid phenethyl ester cape galangin chrysin and caffeic acid substances found in several different types of propolis around the world appeared as potential drugs against this viral target table specifically cape was predicted to interact with sarscov2 mpro in a potential targets in order to inactive the virus and reduce the damage that it causes the main protease of coronavirus sarscov2 mpro 3chymotrypsinlike cysteine enzyme is essential similar study therefore although it will be necessary to run in vitro assays to evaluate the potential antisarscov2 effects of propolis andor its constituents these in silico results are along this line hashem evaluated various natural compounds with an in silico approach molecular docking to try to find useful options for treating sarscov2 infection curiously for coronavirus processing of polyproteins and for its life cycle and therefore inhibition of the some propolis compounds can potentially interact with sarscov2 mpro the research community has examined the genetic code of coronavirus and the mechanisms underlying the damages caused by sarscov2 to help search for drugs andor well boding propolis can interact with ace and tmprss2 potentially blocking or reducing sarscov2 invasion of the host cell 0c sarscov2 strongly binds to angiotensinconverting enzyme ace2 using this enzyme as a receptor for invasion and replication in the host cell [ ] causing damage and increasing interpersonal transmission [ ] consequently ace inhibitors have been considered as useful drug alternatives however potential deleterious effects on users of angiotensinconverting enzyme ace inhibitors and angiotensin receptor blockers arbs have tool against potential cardiovascular events inhibition of ace2 enzyme is an important target for treatment against sarscov2 myricetin caffeic acid phenethyl ester hesperetin and pinocembrin rutin interacts with zinc fingers of the active sites of ace2 a metalloprotease that presents the same zinc finger in ace1 emerged as a concern for treatment of covid19 patients an observational study involving patients did not confirm this suspicion and therefore these classes of drugs remain an important infection [ ] güler et al prepared an alcoholic extract of propolis and identified some hydroxycinnamic acids caffeic acid pcoumaric acid tcinnamic acid and cape the flavanons rutin and myricetin and the flavones hesperidin chrysin and pinocembrin using molecular docking evaluations they found that rutin had the highest binding energy to ace2 followed by preproofvarious characteristics including inhibition of ace they found strong inhibition for most of the propolis types they studied with higher than ace inhibition the best results were found in addition to the in silico evidence osés et al evaluated several types of propolis for with the propolis components catechin and pcoumaric acid ace2 and tmprss2 transmembrane serine protease on the surface of host cells are used by sarscov2 via interaction with spike glycoproteins in order to proceed with invasion and replication vardhan sahoo studied several molecules commonly found in medicinal herbs using molecular docking procedures with relevant targets such as rnadependent rna polymerase rdrp ace2 and spike glycoproteins and compared the resulting scores with those of hydroxychloroquine limonin was the most active compound however quercetin and kaempferol also propolis compounds gave high docking scores kaempferol was studied in prostate cancer models and the expression of tmprss2 was reduced showing a potential mechanism of action for an antitumoral effect kaempferol could be an important propolis component for use against covid19 since it is involved in the inhibition of tmprss2 0c potentially interacting with ace2 rdrp and spike glycoprotein sgp besides its antiviral activity table propolis blocks pak1 potentially avoiding lung fibrosis and restoring a normal immune response among the possible targets for controlling covid19 damage the major œpathogenic contributes to their suppression pak1 inhibitors can both help combat the virus and restore a normal immune response kinase pak1 is key it is an essential component in malaria and viral infections but it is also involved in a wide variety of other diseases and disease conditions including cancer inflammation and immunosuppression when abnormally activated consequences of pak1 activation include lung fibrosis which is an aggravating factor in covid19 pak1 is activated by rac xu et al demonstrated that caffeic acid and its ester cape components of propolis can inactivate rac consequently inhibiting pak1 the inactivation of pak1 directly or upstream can potentially attenuate coronavirus pathogenesis bcells and tcells are lymphocytes that produce specific antibodies against viruses and other intruders and pak1 preproofimprove the immune response its components increase neutralizing antibody titers activate phagocytosis and increase ifnγ levels and the number of lymphocytes an increase in ifnγ levels was also detected by shimizu et al who evaluated the mechanisms involved in the cape caffeic acid phenethyl ester is a potent inhibitor of activation of nfkb in myelomonocytic cells anse et al demonstrated that propolis cape quercetin hesperidin and some other propolis flavonoids can inhibit the cytokine production of th1 and th2 type t cells while increasing tgfbeta an important antiinflammatory cytokine moreover cape can attenuate oxidative stress and inflammation through downregulation of jak2stat3 signaling propolis from europe and temperate asia usually made by bees from resins collected from poplar trees has predominantly flavonoid compounds while green propolis from baccharis dracunculifolia a propolis exclusively found in brazil has various kinds of flavonoids and prenylated phenylpropanoids such as artepellin c baccharin and drupanin these and all other types of propolis can inactivate pak1 artepillin c selectively inhibits pak1 table some studies have shown that propolis can act as an immunostimulant with the ability to effects of some types of propolis in a herpes simplex animal model 0c as well as having an immunomodulatory effect in which cape inhibits il6 phosphorylation and stat3 which are important for proinflammatory th17 development besides the antiinflammatory effect of cape and kaempferol paulino et al evaluated the antiinflammatory effect of artepellin c in rat paw edema and in cell cultures demonstrating that the activity was at least in part mediated by prostaglandin e2 and no inhibition through nfkb modulation artepillin c is an important biomarker of brazilian green propolis botanical source baccharis dracunculifolia immune modulation is desirable since coronavirus infection dysregulates the immune response in the initial phases of infection which facilitates viral replication however in later stages of covid19 the body develops an exaggerated inflammatory response which can greatly damage the lungs and other ans propolis different from typical immunosuppressants can help avoid immunosuppression during the initial phases of disease and in later stages reduce an exaggerated host inflammatory response inhibiting excess il6 il2 and jak signaling cape a propolis component is also known as an immunemodulating agent and should be considered as an alternative to help reduce an exaggerated inflammatory response in a mouse model propolis had immunomodulatory action in vivo on tolllike receptor expression and on preproofreplication and infectivity potentially decreasing lung inflammation due to antiinflammatory properties while promoting immune system fortification these are useful properties that could there is ample evidence for interference of propolis andor its components with viral propolis has been tested against various viral disease anisms initial successes have prompted research to determine the most useful components which may be modified to produce more active and specific pharmaceuticals viruses that were controlled by propolis in animal models with suggestion for control in humans include influenza [ ] herpes simplex virus type and hiv [ ] shimizu et al evaluated three different types of propolis in ethanol extracts using a murine model of herpes simplex virus type despite the chemical differences proinflammatory cytokine production help minimize the symptoms and deleterious effects of covid19 figure figure propolis as an antiviral substance 0cdue to the different plant origins of the resins the bees used to produce the propolis baccharis dracunculifolia baccharis eriodata and myrceugenia euosma all three propolis extracts not only had direct antihsv1 effects but also stimulated immunological activity against intradermal hsv1 infection in mice antiviral activity of propolis has been reported for dna and rna viruses poliovirus herpes simplex virus and adenovirus in an in vitro model cultured cells the best results were obtained against poliovirus and herpes virus with inhibition of the latter at a propolis concentration of ugml the propolis components chrysine and kaempferol caused a concentrationdependent reduction of intracellular replication of herpesvirus strains when host cell monolayers were infected and subsequently cultured in a drugcontaining medium quercetin another propolis component had the same effect but only at the highest concentrations tested ugml against various human herpes simplex virus strains with a intracellular replication reduction of approximately while it reduced the infectivity of bovine herpes virus human adenovirus human coronavirus and bovine coronavirus about the reduction was in the preproofimpairment and pulmonary failure inflammatory response to infection sarscov2 infection is associated with increased levels of chemokines and activated proinflammatory cytokines that lead to the development of atypical pneumonia with rapid respiratory immunologicalinflammatory phenomena such as cytokine release syndrome have been shown to be important mortality factors in sarscov2 infection higher serum levels of proinflammatory cytokines such as il6 il1 and tnfα are found in patients with severe covid compared to those of individuals with mild disease molecular mechanisms involved in this immune process are the targets of various synthetic medicines being tested in patients including ciclesonide hydroxy chloroquine ivermectin and ketorolac which are pak1 blockers pak1 raccdc42activated kinase is overexpressed in the lung in response to sarscov2 infection and is a critical mediator of the cytokine storm that frequently results in mortality the most critical cases of covid19 which require ventilatorassisted intensive care and often result in prolonged ventilator dependency and death are a result of an exaggerated case of rotavirus antiinflammatory and immunomodulatory properties of propolis 0cin hospitalized patients fortuitously propolis components are effective pak1 blockers table there is considerable evidence that propolis can reduce and alleviate the symptoms of inflammatory diseases and has immunomodulatory properties [ ] however these properties can vary according to the plant origin of the propolis as well as the extraction processsolvent used and the inflammatory protocol cell culture animal models induction by lipopolysaccharides when the propolis extracts are tested tests with animal models have shown that propolis can reduce the levels of il6 and tnfalpha which are key proinflammatory mediators and increase the levels of the regulatory cytokine il10 kaempferol a propolis component reduces il6 tnfalpha and vegf vascular endothelial growth factor via the preproofifnγ in serum fernandes et al found that propolis exerted a positive adjuvant effect on vaccines that were developed against canine coronavirus they assayed ifnγ which is an effective way to measure the cellular response induced by a vaccine in a mouse model propolis added as an adjuvant to inactivated swine herpesvirus type vaccine stimulated increased cellular propolis is considered a safe immunostimulant and a potent vaccine adjuvant propolis has been widely tested as a vaccine adjuvant because it induces an earlier immune response and provides a longer protection period it is also included as an adjuvant ingredient in traditional chinese medicine propolis flavonoids have potential as adjuvants enhancing igg il4 and propolis has potential as a vaccine adjuvant erknfkbcmycp21 pathway table tests on macrophage cell cultures also demonstrated that propolis inhibits the production of il1 beta an important component of the inflammasome inflammatory pathway in diseases such as rheumatoid arthritis lupus and other autoimmune diseases although the mechanisms of action are not well elucidated these propolis components have potential as complementary supplements in the preventive treatment of chronic inflammatory diseases and humoral responses increasing ifnγ [ ] propolis enhanced the immune response to inactivated porcine parvovirus vaccine in guinea pigs when added to a trichomonas vaginalis protein vaccine propolis increased the igg antibody response times in mice 0ccancer is considered a relevant comorbidity factor for covid19 cancer patients have a patients cancer patients compared to the protein alone propolis was also effective as an adjuvant in the immunization of cattle with bovine herpesvirus it improved the humoral and cellular responses in mice inoculated with inactivated virus vaccines propolis as an adjuvant gave a similar immune response increasing ifnγ levels to alum and freund™s adjuvant in mice vaccinated with an hiv1 polytope vaccine candidate with less risk of undesirable side effects comorbidities and evidence of how propolis can help reduce their impact in covid19 to risk a visit to a clinic or hospital to determine if they have cancer alternative therapies could help retard cancer or reduce the impact of cancer and cancer treatment in covid19 times higher risk of progressing to severe covid19 disease than patients without comorbidities also the hospital environment during the coronavirus pandemic can interfere with or delay the treatment that cancer patients should receive patients with symptoms may choose not preproofacid cape quercetin and chrysin propolis and its components normally have little impact on normal cells displaying differential cytotoxicity in liver cancer melanoma and breast cell carcinoma cell lines [ ] propolis enhances the activity of tumor various types including bladder blood brain breast colon head and neck kidney liver pancreas prostate and skin cancers propolis could help prevent cancer progression in various parts of the world it is considered an alternative therapy for cancer treatment propolis extracts have been found to inhibit tumor cell growth both in vitro and in vivo including inhibition of angiogenesis demonstrating potential for the development of new anticancer drugs various components of propolis have been shown to inhibit cancer cell growth including cinnamic propolis has potential as a complementary therapy for cancer it has shown efficacy against necrosis factor related apoptosis inducing ligand trail in cancer cells hypertension and cardiovascular disease 0c hypertension and cardiovascular disease are considered relevant comorbidities for covid19 propolis has demonstrated antihypertensive effects in rat models [ ] in cameroon it is used in popular medicine to treat various ailments including high blood pressure propolis has been widely used as a dietary supplement for its health benefits including cardiovascular protective effects [ ] in a human trial consumption of propolis improved critical blood parameters including hdl gsh and tbars levels demonstrating that obesity a natural antiobesity agent it could contribute to a reduced risk for cardiovascular disease obesity is a major comorbidity and predictor of increased mortality in covd19 patients obesity and sarscov2 both induce an inflammatory process exacerbating sarscov2 infection in the obese propolis reduced inflammation and prevented hyperlipidemia and metabolic syndromes in highly caloric diet induced obesity in mice body weight gain visceral adipose tissue liver and serum triglycerides cholesterol and nonesterified fatty acids were all reduced in the propolis fed mice [ ] caffeic acid phenethyl ester a propolis component is preproofplatelet aggregation was reduced by propolis in tests on human blood in vitro and in other in vitro tests caffeic acid phenethyl ester cape a wellstudied bioactive propolis component inhibits collagen induced platelet activation thromboembolism thrombosis and microthrombosis microthrombosis disseminated intravascular coagulation and consequent multian failure are common in severely affected covid19 patients with associated high mortality rates anticoagulants are sometimes prescribed to such patients because they can reduce mortality tang et al an elevated level of plasminogen activator inhibitor1 pai1 is a biomarker and risk factor for thrombosis and atherosclerosis [ ] various types of evidence demonstrate that propolis can reduce platelet aggregation and other thrombosisrelated parameters propolis decreased thrombotic tendencies in mice by suppressing lipopolysaccharideinduced increases in pai1 levels [ ] propolis downregulated plateletderived growth factor and platelet endothelial cell adhesion molecules in lowdensity lipoprotein knockout mice 0c old age the elderly are more often affected by chronic inflammation characterized by systemically increased levels of proinflammatory cytokines which can contribute to development of a cytokine storm a major cause of covid19 mortality propolis has antioxidant properties which could help retard o
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"associated with lung cancer with squamous differentiation. J Clin Oncol. 2013;31(10):e161“e16423358982 5. PaoWGirardN New driver mutations in non-small-cell lung cancer. Lancet Oncol. 2011;12(2):175“18021277552 6. ShigematsuHTakahashiTNomuraM Somatic mutations of the HER2 kinase domain in lung adenocarcinomas. Cancer Res. 2005;65(5):1642“164615753357 7. StephensPHunterCBignellG Lung cancer: intragenic ERBB2 kinase mutations in tumours. Nature. 2004;431(7008):525“52615457249 8. BargmannCIHungMCWeinbergRA Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain of p185. Cell. 1986;45(5):649“6572871941 9. BocharovEVMineevKSVolynskyPE Spatial structure of the dimeric transmembrane domain of the growth factor receptor ErbB2 presumably corresponding to the receptor active state. J Biol Chem. 2008;283(11):6950“695618178548 10. FleishmanSJSchlessingerJBen-TalN A putative molecular-activation switch in the transmembrane domain of erbB2. Proc Natl Acad Sci U S A. 2002;99(25):15937“1594012461170 11. MineevKSBocharovEVPustovalovaYEBocharovaOVChupinVVArsenievAS Spatial structure of the transmembrane domain heterodimer of ErbB1 and ErbB2 receptor tyrosine kinases. J Mol Biol. 2010;400(2):231“24320471394 12. BaselgaJSwainSM Novel anticancer targets: revisiting ERBB2 and discovering ERBB3. Nat Rev Cancer. 2009;9(7):463“47519536107 13. EngelmanJA Targeting PI3K signalling in cancer: opportunities challenges and limitations. Nat Rev Cancer. 2009;9(8):550“56219629070 14. MountziosGPlanchardDBesseB Mitogen-activated protein kinase activation in lung adenocarcinoma: a comparative study between ever smokers and never smokers. Clin Cancer Res. 2008;14(13):4096“410218593986 15. PlanchardDCamara-ClayetteVDorvaultNSoriaJCFouretP p38 Mitogen-activated protein kinase signaling ERCC1 expression and viability of lung cancer cells from never or light smoker patients. Cancer. 2012;118(20):5015“502522415779 Oncotarget Oncotarget ImpactJ Oncotarget 1949-2553 Impact Journals LLC 24519909 3996653 Research Paper Sp1-mediated microRNA-182 expression regulates lung cancer progression Yang Wen-Bin 1 Chen Ping-Hsin 2 Hsu Tsung-I 3 Fu Tzu-Fun 4 Su Wu-Chou 5 Liaw Hungjiun 6 Chang Wen-Chang 7 Hung Jan-Jong 1 2 3 7 1 Institute of Bioinformatics and Biosignal Transduction College of Bioscience in Biotechnology National Cheng Kung University Tainan 701 Taiwan 2 Department of Pharmacology College of Medicine National Cheng Kung University Tainan 701 Taiwan 3 Center for Infectious Disease and Signal Transduction Research National Cheng Kung University Tainan 701 Taiwan 4 Department of Medical Laboratory Science and Biotechnology College of Medicine National Cheng Kung University Tainan 701 Taiwan 5 Department of Internal Medicine College of Medicine and Hospital National Cheng Kung University Tainan 701 Taiwan 6 Department of Life Sciences College of Bioscience in Biotechnology National Cheng Kung University Tainan 701 Taiwan 7 Graduate Institute of Medical Sciences College of Medicine and Center for Neurotrauma and Neuroregeneration Taipei Medical University Taipei 110 Taiwan Correspondence to:petehung petehungmail.ncku.edu.tw 2 2014 25 1 2014 5 3 740 753 18 11 2013 24 11 2014 Copyright: 2014 Yang et al. 2014 This is an open-access distributed under the terms of the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original author and source are credited. Our recent study indicated that overexpression of Sp1 enhances the proliferation of lung cancer cells while represses metastasis. In this study we found that the transcriptional activity of FOXO3 was increased but its protein levels decreased following Sp1 expression. Sp1 increased expression of miR-182 which was then recruited to the 3'-untranslated region of FOXO3 mRNA to silence its translational activity. Knockdown of miR-182 inhibited lung cancer cells growth but enhanced the invasive and migratory abilities of these cells through increased N-cadherin expression. Repression of FOXO3 expression in the miR-182 knockdown cells partially reversed this effect suggesting that miR-182 promotes cancer cell growth and inhibits cancer metastatic activity by regulating the expression of FOXO3. The expression of several cancer metastasis-related genes such as ADAM9 CDH9 and CD44 was increased following miR-182 knockdown. In in the early stages of lung cancer progression Sp1 stimulates miR-182 expression which in turn decreases FOXO3 expression. This stimulates proliferation and tumor growth. In the late stages Sp1 and miR-182 decline thus increasing FOXO3 expression which leads to lung metastasis. Sp1 miR-182 FOXO3 Lung cancer INTRODUCTION Post-transcriptional regulation plays an important role in diverse cellular processes such as development neurogenesis and cancer progression [1-3]. MicroRNAs (miRNAs) have emerged as important post-transcriptional regulators that inhibit mRNA translation or induce mRNA cleavage by base pairing with a seed region in the 3'-untranslated region (3'-UTR) of target genes [4 5]. Recent studies have shown that dysregulation of miRNAs contributes to the initiation progression metastasis and drug resistance of cancer [6 7]. For example miR-200c targets Kras to regulate Kras expression during tumorigenesis [8]. Furthermore several upregulated and downregulated miRNAs have been identified in lung cancer the most frequently diagnosed cancer and the most common cause of cancer-related death worldwide [9-11]. Identification of early-detection biomarkers and precise diagnosis are necessary if lung cancer patients are to receive efficacious therapeutic treatment quickly. Several factors such as USP17 have been identified as potential biomarkers for lung cancer [12 13]. Circulating miRNAs could also serve as useful clinical biomarkers for the screening of high-risk populations and the detection solid tumors in the early stages of cancer progression [14 15]. miRNAs offer new targets for cancer therapy [16 17]. Therefore a detailed understanding of the mechanisms underlying miRNA production and function is important. Identification of miRNA target genes and the use of gene set enrichment analysis have clarified the function role of miRNAs. However the molecular mechanisms that regulate of miRNA biogenesis are still largely unknown. Recent studies have shown that transcription factors (TFs) regulate not only the expression of protein-encoding genes but also miRNA biogenesis through RNA polymerase II-dependent transcription [18]. Several TFs including p53 c-myc and HIF1? that directly recognize miRNA promoters and regulate miRNA transcription have been reported [19-21]. Specificity protein 1 (Sp1) which belongs to the specificity protein/ Kr¼ppel-like family was the first TF identified in mammalian cells. Sp1 contains three Cys2His2-type zinc finger DNA binding motifs that recognize GC-rich promoter sequences [22]. Sp1 regulates thousands of coding genes such as those encoding cyclin A2 p21cip1/waf1 E-cadherin and Sp1 itself. These genes are involved in a variety of physiological processes including cell cycle progression and cell migration [23-26]. Sp1 also regulates the expression of noncoding genes. Sp1 forms a complex with NF-?B to downregulate miR-29b expression through the recruitment of histone deacetylase (HDAC) 1 and HDAC3 in leukemia and thereby contributes to the growth of leukemia cells [27]. Sp1 also forms a complex with HDAC4 to downregulate miR-200a expression in hepatocellular carcinoma and contributes to cell proliferation and migration [28]. In addition Sp1 is an activator of miR-34c miR-132 and miR-365 expression [29-31]. However no studies have assessed whether Sp1 regulates the expression of miRNAs involved in lung tumorigenesis. Because the accumulation of Sp1 is required for lung tumor growth further investigation of Sp1-mediated miRNA regulation is needed. In this study we showed that Sp1 suppressed FOXO3 expression via post-transcriptional regulation. To elucidate whether miRNAs were involved in this process we used a systematic screening approach to identify Sp1-regulated miRNAs. We identified a novel Sp1-regulated miRNA miR-182 in lung cancer cells and demonstrated that Sp1 downregulated FOXO3 expression by upregulating miR-182 expression. Our results show that miR-182 functions as an oncomiR to enhance cancer cell proliferation and acts as a tumor suppressor to inhibit cancer metastasis. RESULTS Sp1 regulates miR-182 expression Our previous studies demonstrated that Sp1 is involved in KrasG12D-induced lung tumorigenesis [23 32]. Using cDNA microarray analysis we found that Sp1 increased oncogene expression and decreased tumor suppressor gene expression. In the present study we initially used software to analyze the promoters of all identified miRNAs. According to the miRBase database the human genome contains 1600 miRNA genes. We investigated whether Sp1 participates in the regulation of intergenic miRNAs. First we screened the upstream (-1 kb) flanking sequences of intergenic miRNAs. Using the TFSEARCH program we identified 205 intergenic miRNAs that contained potential binding sites for Sp1. Because Sp1 is upregulated in lung cancer and the expression of its target genes is altered we next examined the expression of these miRNAs in lung cancer. According to previous studies the expression patterns of 22 miRNAs differed significantly in lung cancer tissue and normal lung tissue (Supplementary Table S1). "
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99mtc‘labeled nanocolloid drugs development methodsVladimir Sadkin1 Viktor Sкuridin1 Evgeny Nesterov1 Elena Stasyuk1 Alexander Rogov1 Natalya Varlamova1 Roman Zelchan2The work considers the problem of obtaining nanocolloid radiopharmaceuticals RPs and studying their functional suitability for diagnosing sentinel lymph nodes SLN in cancer patients Two principal approaches to the formation of technetium99mlabeled ps based on inanic and anic matrices were considered when carrying out research to develop methods for the production of nanocolloid RPs The composition of the reagents and the conditions for obtaining nanocolloid radiopharmaceuticals were determined The functional suitability of new RPs for scintigraphic diagnostics of sentinel lymph nodes has been studiedThe identification of sentinel lymph nodes”the first nodes that stand in the way of metastasizing of malignant neoplasms attracts increasing interest in modern oncological practice1“ It is believed that if the SLN is not affected by the metastatic process then all other regional lymph nodes are intact so the results of biopsy of these nodes are an objective diagnostic criterion for the spread of malignant process Fig a0 The optimal method of detecting SLN is the use of colloid nanomaterials labeled with technetium99m for scintigraphic or radiometric determination of node localization6“ Not so much the chemical nature of such ps but their size is the determining factor in the choice of the indicator in this case Thus according to Schauer14 a colloid with a p size of less than a0nm can accumulate not only in the SLN but also at nodes of and orders of magnitude Ps with the sizes of more than a0nm slowly migrate from the injection site The colloid with the p size from to a0nm was recognized as the optimal one for detecting SLNThe simplest method of obtaining colloids with given sizes and properties is immobilization of 99mTc on the surface of nanosized materialsTechnetium99m is by far the most popular radionuclide for conducting diagnostic studies practically in all fields of medicine15“ This is primarily due to its nuclearphysical characteristics a relatively short halflife a0h and Îradiation energy of a0meV providing a low exposure dose and at the same time sufficient penetrating power for radiometric measurementsToday the Tc99m Tilmanocept radiopharmaceutical is widely used which has proven itself well and gives good results But its production is quite timeconsuming and requires expensive components We offer a less laborious method from the simple components1920Materials and methodsMaterials All the reagents were purchased from SigmaAldrich ACS grade and used without further purification Technetium99m was obtained from chromatographic 99Mo99mTc generator œ99mTcGTTOM produced by Tomsk Polytechnic University TPU”Tomsk RussiaThree types of nanops were selected to obtain nanocolloids labeled with 99mTc The first type of colloids was created on the basis of metal chelates with chemically modified complexons of diethylenetriaminepentaacetic acid DTPA It should be noted that the DTPA molecule itself like its complexes with metals is hydrophilic and does not tend to form colloidal ps The introduction of hydrophobic fragments into its structure allowed the preparation of waterinsoluble modified DTPA complexes21 The original substance of the modified DTPA DTPAmod was synthesized in Tomsk Polytechnic University Preparation of colloid solution DTPAmod was produced using the following method A sample of modified DTPA with the mass of a0mg was quantitatively transferred to a volumetric flask of a0ml and dissolved in a0ml of NaHCO3 solution by heating to a0°C After that the volume was adjusted with the same solvent up to the mark In order to reduce the p size the container with suspension was heated in water to a0°C and treated with ultrasound for a0min 1Tomsk Polytechnic University Lenina Avenue Tomsk Russia 2Tomsk National Research Medical Center Russian Academy of Sciences Kooperativny street Tomsk Russia email sadkintpuruScientific RepoRtS 101038s41598020709912Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Scheme for determining the sentinel lymph node using nanocolloid radiopharmaceuticals radiopharmaceutical sentinel lymph node detectorFigure a0 The general scheme for the synthesis of 99mTcDTPAmodwhich reduced the average p radius up to a0nm The general scheme for the synthesis of 99mTcDTPAmod is shown in Fig a0The second type of colloids is iron nanops coated with a carbon shell of FeC Fig a03a These ps were obtained from the Institute of Metal Physics UrB RAS Ekaterinburg Russia In order to impart lipophilic properties to ironcarbon ps and to increase their stability in solution in the form of a colloid a technique for preliminary deposition of anic radicals aryldiazonium tosylates ADT onto the surface of these ps has been developed An effective method for the synthesis of ADT followed by their application onto the carbon surface of ps was developed at the Tomsk Polytechnic University22 The general scheme for the synthesis of FeC ps and their subsequent interaction with 99mTc is shown in Fig a03bIn the third type of colloids technetium99m was adsorbed on aluminum oxide powder A powder of lowtemperature cubic modification of gammaoxide Al2O3 prepared from aluminum hydroxide powder AlOH3 by its calcination in a muffle furnace was used The substance was synthesized in Tomsk Polytechnic UniversityA reducing agent”tin II chloride dihydrate was used in order to obtain complexes of 99mTc with colloidsGelatin powdered Ph Eur USPNF pure pharma grade CAS Number was purchased from AppliChem GmbH Darmstadt GermanyMethods Method for preparation of 99mTc labeled nanocells The introduction of the radioactive label 99mTc into a colloidal substance was carried out by mixing of the selected substance with the reducing agent SnCl2ˆ™2H2O “ a0mgml in different ratios and then adding a a0ml of eluate of 99mTc “ a0MBqml to the mixtures The mixtures were incubated for a0min at a temperature of “ a0°C The preparation is ready for use after cooling at room temperature The reducing agent SnCl2ˆ™2H2O was used as a hydrochloric acid solution The amount of a0g of tin chloride II is added to the vial and a0ml of a0M hydrochloric acid HCl Scientific RepoRtS 101038s41598020709912Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A Carbon encapsulated iron nanop B the general scheme for the synthesis of FeC psis then added for its preparation After dissolution the volume is adjusted with distilled water to a0ml Dissolution was carried out in an inert gas argon mediumDetermination of the size of 99mTc labeled colloidal ps The determination of the size of the labeled nanocolloids was carried out by spectroscopy on a Nanophox p size analyzer œSympatec GmbH Germany and also by a technique based on measuring the activity of the suspension before and after filtering it successively through filters with predetermined pore sizes and a0nm Three samples were taken with a volume of a0μl from each initial solution and filtrates for the subsequent measurement of their activity Calculations of the yield of products with different p sizes were determined according to the formulas given belowC220 Avc ˆ’ A1Avc C100 A1 ˆ’ A2A1 C50 A2 ˆ’ A3A2where Avc is the activity of the initial suspension prior to filtration A1 is the activity measured after filtration through a a0nm filter A2 is the activity after filtration through a0nm filter A3 is the activity measured after filtration through a0nm filterIn parallel determination of the radiochemical purity RCP of preparations by thin layer chromatography method was carried outThin‘layer chromatography TLC procedure To determine radiochemical purity of 99mTc“nanocolloid a0 µl of prepared sample was spotted on silicagel impregnated strip Sorbfil Russia — a0 cm To determine Scientific RepoRtS 101038s41598020709912Vol0123456789wwwnaturecomscientificreports 0cAmount of SnCl2ˆ™2H2O mgA[Sn99mTc]A ATcVIIA Table Change in relative activities of the complex [Sn99mTc] and 99mTc VIIpertechnetate content of the radiopharmaceutical sample first strip was developed using acetone as the mobile phase time of chromatography a0min In this system pertechnetate migrated with the front of the mobile phase Rf To determine the colloid content of the preparations the second strip was developed using ethanolwaterammonium hydroxide as the mobile phase time of chromatography a0min In this system the 99mTc“nanocolloid migrated with the front of the mobile phase Rf Stability The stability of 99mTc“nanocolloid was studied in a0vitro by mixing of a0ml of normal serum and a0ml of 99mTc“nanocolloid following by incubation at a0°C for a0h At different time points a0h a0h and a0h a0ml aliquots of complex were removed and evaluated for radiochemical purity using TLC24Determination of the functional suitability of preparations for scintigraphic detection of SLN A study to assess the functional suitability of new nanocolloid RPs was performed in series of experiments involving white Wistar male rats weighing “ a0g Injection of RP in a dose of “ a0MBq was performed between the first and second fingers of the rat™s hind paw The animals were anesthetized with ether before the subcutaneous injection and during the scintigraphic study Since the introduction the kinetics of radiopharmaceutical distribution throughout ans and tissues was recorded by a framebyframe recording for a0min one frame” a0s in a — pixel matrix Static scintigraphy was performed after and a0h in the anterior and posterior projections in a matrix of — with a set of pulses scintigraphy of animals was performed on an ECAM Signature gamma camera Siemens Germany The results of scintigraphic studies determined the percentage of accumulation of RP in the lymph node and the injection site The maintenance and participation of the animals in the experiment was carried out in accordance with the rules adopted by the œEuropean Convention for the Protection of Vertebrates Used for Experiments or Other Scientific Purposes Strasbourg The experimental protocols were approved by Cancer Research Institute Biomedical Ethics Committee Protocol number All invasive manipulations with animals were performed using inhalation or drug anesthesiaStatistical analysis All mean values are expressed as IDg ± SD Data were analyzed statistically using methods of general statistics with a commercially available software package œStatistics for Windows StatSoft Inc Version Results and discussionTo carry out the labeling of colloids 99mTc extracted from a standard generator in the form of pertechnetate ions contained in a NaCl solution was used It has a higher degree of oxidation VII in this chemical form and is not prone to complex formation A reducing agent”tin II chloride dihydrate widely used for the preparation of various compounds labeled with 99mTc to was used to reduce its valence state in order to obtain complexes with nanoscale ps25 As a result of the reaction of these components the appearance of an untargeted colloid is also possible due to the hydrolysis of excess SnCl2·2H2O or the additional formation of a complex of reduced 99mTc with tin26 All this required preliminary experimental studies to establish the necessary and sufficient amount of SnCl2·2H2O in the reaction mixtureDuring the experiments it was found that the optimal concentration of Sn II in the composition of the reaction mixture when it interacts with 99mTc should be in the range of “ a0mgml Table a0It was found that when the eluate with the preliminarily reduced 99mTc VII was added to the nanops the Sn II concentration introduced in the RP was CSn a0mgml almost the entire amount of 99mTc has time to enter the composition of the largesize complex with tin even before its mixing with nanops This means that the sequence of the introduction and mixing of the reagents has a great influence on the labeling process especially it concerns the introduction of the Sn II solution into the reaction mixture In this connection the reduction of 99mTc with tin II was carried out in the presence of the selected substance In this case we can observe a competitive redistribution of the radionuclide between the substance and the tin complex The technique of applying of the 99mTc label to the surface of nanosized ps is given in the previous sectionAs a result of the studies reagent compositions and conditions for obtaining three nanocolloid RPs were determined Table a0 shows their components as well as the radiochemical purity and yield of the target colloid with p sizes of “ a0nmProceeding from the chromatograms it follows that the content of radiochemical impurity of unreduced 99mTc VII in the obtained preparations is “ Preliminary tests of these preparations on experimental animals showed that accumulation in lymph nodes is practically not observed although colloids have p sizes in the required range from to a0nm Scintigrams of rats obtained after subcutaneous administration of a technetium99m labeled nanocolloid based on aluminum oxide are shown in Fig a0Scientific RepoRtS 101038s41598020709912Vol1234567890wwwnaturecomscientificreports 0cComposition of the preparation per a0mlDTPAmod a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg n FeC a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg n Al2O3 a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg n Colloid yield “ a0nm RCP ± ± ± Table Composition of reagents for production of technocium99 a0m nanocolloidsFigure a0 Distribution of the preparation in the rat when the preparation is administered [Al2O3 99mTc Sn II] A immediately after the administration of the drug B a0min after the administration C a0min after the administrationComposition of preparations per a0mlAl2O3 a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg G a0mg n DTPAmod a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg G a0mg n FeC a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg G a0mg n Yield of colloid “ a0nm RCP ± ± ± Table Indicators of RCP and the yield of a colloid with a fraction of “ a0nm after the introduction of gelatin in the reagentsScintigrams showed that the drug remains at the injection point for a0h without significant accumulation of 99mTc in the blood of animals which indicates a strong fixation of the radionuclide on the surface of the nanocolloid Along with this positive point it should be noted that accumulation of the preparation in the lymph nodes is not observed Gelatin G was introduced into the reaction mixture in this connection to increase the œmobility of the labeled ps and increase the speed of their movement through the lymph system Matrix systems based on gelatin provide a fairly uniform distribution of the immobilized substance and in this case prevents the formation of a large tin colloid with 99mTc The results of the experiments showed that the addition of gelatin “ a0mgml to the reagent additionally provides an increase in the yield of the target colloid with p sizes of “ a0nm Table a0In addition the size of these ps was determined on a Nanophox p analyzer The obtained dependence of the change in the density of the distribution of the number of ps on their size constructed from the results of a threedimensional measurement of the preparations is shown in Fig a0 A B C The average p size diameter is and a0nm respectivelyStability test showed that complex 99mTc“nanocolloid was stable in normal serum at least for a0h Radiochemical purity of the tracer at the end of the experiment was ± A study of the functional suitability of the obtained radioactive colloids for the scintigraphic imaging of the sentinel lymph nodes showed that these preparations provide a good level of accumulation in the sentinel lymph nodes Fig a0 Table a0 displays the Al2O3 99mTc DTPAmod 99mTc FeC 99mTc biodistribution data at different time points postinjectionThe level of accumulation of preparations in the lymph nodes is “ of the total injected activityconclusionAs a result of the studies the composition of the reagents and the conditions for the synthesis of three nanocolloid RPs were determined An experimental dependence of the change in the content of 99mTc VII impurities on the concentration of tin II was established and its minimum amount a0mgml was determined to reach a RHP greater than In this case the yield of the target colloid with p sizes of ± a0nm is “ Preliminary tests of the developed preparations on experimental animals showed that accumulation of RP in lymph nodes is practically not observed although the sizes of colloidal ps are in the required range Increase in the speed of transportation of colloids through the lymphatic system was achieved by the introduction of gelatin in the composition “ a0mgml In addition there was an increase in the yield of the colloid Scientific RepoRtS 101038s41598020709912Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Change in the density of the distribution of the number of ps from their size in radiopharmaceuticals A œ99mTcAl2O3 B œ99mTcFeC C œ99mTcDTPAmodwith p sizes of “ a0nm to “ with radiochemical purity of the preparations of “ Repeated studies in experimental animals have shown that all synthesized nanocolloid preparations provide a good level of Scientific RepoRtS 101038s41598020709912Vol1234567890wwwnaturecomscientificreports 0cStomachTime h99mTcAl2O399mTcDTPAmod99mTcFeC ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Figure a0 Distribution of the preparation in the rat with injection of suspension [Al2O3 99mTc Sn II Gelatin] A immediately after the administration of the preparation B a0min after the administration C a0min after the administration D a0min after the administration The numbers indicate ”lymph node ”site of preparation administrationg ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Liver ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Spleen ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± BloodmlHeart ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Lungs ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Table Biodistribution data up to a0h after injection of “ a0MBq of 99mTc in healthy male rats Data represent the average value n accumulation in the SLN Thus the level of accumulation of RP œ99mTcDTPAmod and RP œ99mTcFeCADT in the SLN is and respectively At the same time the accumulation level of the preparation based on aluminum oxide is of the total input activityReceived March Accepted July References Jakobsen J K Sentinel node biopsy in urooncology A history of the development of a promising concept Urol Oncol “ Weixler B et al Sentinel lymph node mapping with isosulfan blue or indocyanine green in colon cancer shows comparable results and identifies patients with decreased survival A prospective singlecenter trial World J Surg 101007s0026 Beasley G M et al Sentinel Lymph node biopsy for recurrent elanoma A multicenter study Ann Surg Oncol Moser J et al Sentinel node biopsy in melanoma A singlecentre experience with consecutive patients Br J Dermatol 101245s1043 “ Buda A et al Optimizing strategies for sentinel lymph node mapping in earlystage cervical and endometrial cancer Comparison of realtime fluorescence with indocyanine green and methylene blue Int J Gynecol Cancer “ Scientific RepoRtS 101038s41598020709912Vol0123456789wwwnaturecomscientificreports 0c“ Sahbai S et al Pericervical injection of 99mTcnanocolloid is superior to peritumoral injection for sentinel lymph node detection of endometrial cancer in SPECTCT Clin Nucl Med “ Hoogendam J P et al 99mTcnanocolloid SPECTMRI fusion for the selective assessment of nonenlarged sentinel lymph nodes in patients with earlystage cervical cancer J Nucl Med “ Stoffels I Leyh J P¶ppel T Schadendorf D Klode J Evaluation of a radioactive and fluorescent hybrid tracer for sentinel lymph node biopsy in head and neck malignancies Prospective randomized clinical trial to compare ICG99mTcnanocolloid hybrid tracer versus 99mTcnanocolloid Eur J Nucl Med Mol Imaging “ Beisani M et al Initial experience in sentinel lymph node detection in pancreatic cancer Rev Esp Med Nucl Imagen Mol Schubert T Uphoff J Henke R P Wawroschek F Winter A Reliability of radioisotopeguided sentinel lymph node biopsy in penile cancer Verification in consideration of the European guidelines BMC Urol “ Jaukovic L et al Lymphoscintigraphy and sentinel lymph node biopsy in cutaneous melanoma staging and treatment decisions Hell J Nucl Med “ Subramanian S Pandey U Shah S Rangarajan V Samuel G An indigenous singlevial kit formulation of human serum albumin nanocolloid for use in sentinel lymph node detection Nucl Med Commun “ RuizDom­nguez J M IbarzServio L Garc­ade Manuel G Calaf Peris© O Intraoperative injection of 99mTcnanocolloid for localization of nonpalpable intratesticular tumours in ansparing surgery Actas Urol “ Schauer A J Specific developments in sentinel node labling using 99mTccolloids In The Sentinel Lymph Node Concept Springer Berlin Wang Y et al Gasphase chemistry of technetium carbonyl complexes Chem Phys “ O™Connor M K et al Comparison of Tc99m maraciclatide and Tc99m sestamibi molecular breast imaging in patients with Wang J Zhang R Evaluation of 99mTcMIBI in thyroid gland imaging for the diagnosis of amiodaroneinduced thyrotoxicosis suspected breast cancer EJNMMI Res Br J Radiol Costa P et al Scintigraphic imaging with technetium99Mlabelled ceftizoxime is a reliable technique for the diagnosis of deep sternal wound infection in rats Acta Cir Bras “ Vera D R Wallace A M Hoh C K Mattrey R F A synthetic macromolecule for sentinel node detection 99mTcDTPAmannosyldextran J Nucl Med “ Hoh C K Wallace A M Vera D R Preclinical studies of [99mTc]DTPAmannosyldextran Nucl Med Biol “ Skuridin V et al Modified DTPA moleculebased nanocolloid radiopharmaceuticals J Radioanal Nucl Chem “ Filimonov V D et al Unusually stable versatile and pure arenediazonium tosylates Their preparation structures and synthetic applicability Lett “ Lukasz K Thin Layer Chromatography in Drug Analysis “ CRC Press London Skuridin V et al Radiopharmaceutical drug based on aluminum oxide Indian J Sci Technol 1017485 ijst2015v8i36 Sazonova S I et al Synthesis and experimental study of norfloxacin labeled with technecium99m as a potential agent for infection imaging Iran J Nucl Med “ Skuridin V S et al Synthesis and biological characterization of 99mTclabeled ciprofloxacin Pharm Chem J “ AcknowledgementsThis work was financially supported by Ministry of Education and Science of the Russian Federation RFMEFI57514X0034Author contributionsVS Conducting experimental research analysis and interpretation of the data Final approval for manuscript publication VS development of the concept and direction of research analysis and interpretation of the data Validation of critical intellectual content final approval for manuscript publication EN development of the concept and direction of research analysis and interpretation of the data Validation of critical intellectual content final approval for manuscript publication ES development of the concept and direction of research experimental research development of analytical control methods for the developed kits and radiopharmaceuticals analysis and interpretation of the data Validation of critical intellectual content final approval for manuscript publication AR conducting experimental research analysis and interpretation of the data Final approval for manuscript publication NV conducting experimental research analysis and interpretation of the data Final approval for manuscript publication RZ conducting tests of the functional suitability of drugs Preparation of the section Evaluation of the functional suitability of the preparation by determining its pharmacokinetic characteristics and Figures „– Final approval of the manuscript for publication of the manuscriptCompeting interests The authors declare no competing interestsAdditional informationCorrespondence and requests for materials should be addressed to VSReprints and permissions information is available at wwwnaturecomreprintsPublisher™s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsScientific RepoRtS 101038s41598020709912Vol1234567890wwwnaturecomscientificreports 0c Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons license unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this license visit httpcreat iveco mmons licen sesby40 The Authors Scientific RepoRtS 101038s41598020709912Vol0123456789wwwnaturecomscientificreports 0c'
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"‚ammatory bowel disease ibd is a long life disease with remission and relapse periods ibd arises as a result of inappropriateimmune response to intestinal commensal anisms in individualswith genetic predisposition and consequently causes ‚ammation andintestinal ulcers in addition ibd has a complex pathogenesis andmany factors such as dysbiosis oxidative stress and epigenetics thatmay also be involved in disease pathogenesis [“] ulcerative colitisuc and crohn's diseases cd are known as two main forms of ibdaccordingly these diseases cause intestinal ulcers and some annoyingsymptoms such as diarrhea abdominal pain and rectal bleeding occasionally the severity of these symptoms is very high which can leadpatients to be hospitalized in this regard therapeutic approaches totreat these diseases mainly focus on prolonging remission and are almost similar however diï¬erential diagnosis can also help to treat thedisease in a more eï¬ective way for example 5asa which is acommon drug in the treatment of ibd is less eï¬ective on maintainingremission in cd patients on the other hand antibiotic therapy is notrecommended for the treatment uc but it can be eï¬ective on cd patients diï¬erential diagnosis is a serious challenge because cdand uc have significant similarities in terms of their clinical endoscopic and histological features however there are some diï¬erencesbetween uc and cd which are summarized in table1 in addition tointestinal complications uc and cd also have significant extraintestinal manifestations for example it was shown that uc is significantly associated with primary sclerosing cholangitis and cd is alsoassociated with cholelithiasis especially in cases that the ileum is involved furthermore cd can cause fistulas to the urinary systemwhich leads intestinal bacteria to enter the urethra and recurrent urinary tract infections both cd and uc can cause several disorderssuch as arthritis erythema nodosum pyoderma gangrenosum andanemia which are known as the most important extraintestinal manifestations of ibd the latest statistics showed that the globalŽ corresponding author at department of clinical biochemistry and laboratory medicine faculty of medicine tabriz university of medical sciences daneshgahstreet po box tabriz iranemail address vagharimtbzmedacir m vagharitabari101016jcca202008025received july received in revised form august accepted august available online august elsevier bv all rights reserved 0cf khakikhatibi table1clinical endoscopic and histological features of cd and ucclinical featuresfeaturesrectal bleedingabdominal painfevermucus defectionintestinal obstructionperineal diseasepostoperative recurrenceasca positiveanca positiveendoscopic featurescdoccasionallyfrequentlyfrequentlyoccasionallyyesyesyesfrequentlynot commonucfrequentlyoccasionallynot commonfrequentlynonononot commonfrequentlyfeaturescduclocationmucosal involvementdepth of ulcerationfistulacobblestone appearanceaphthous ulcerationmucosal friabilityhistological featuresfeaturesgranulomascrypt abscessespatchinessany part of gi tractdiscontinuousdeepyesyesfrequentlynot commoncdfrequentlynot commonfrequentlycolon and rectumcontinuoussuperficialnonooccasionallyfrequentlyucrarefrequentlynot commonprevalence of ibd currently is on the rise and it is not an exaggerationif we consider it as a global serious health problem according to areport published in ibd has the highest prevalence rate ineurope and its prevalence in the newly industrialized countries of asiaafrica and south america also appears to be increased over the pastthree decades unfortunately the peak of the disease is at the young age of“ years old therefore in addition to the suï¬ering from ‚icts on the patients it also has many negative eï¬ects on societymoreover many financial burdens are annually imposed on countriesfor controlling and treating this chronic disease the invasive diagnosticand therapeutic measures are currently undertaken to diagnose andmanage ibd which are unpleasant for patients as well as having thehigh associated costs now the gold standard method for diagnosingibd and monitoring patient status is performing colonoscopy examination and histopathologic evaluation which are invasive measures therefore in recent years many studies have been conducted tofind a suitable laboratory marker with sufficient sensitivity and specificity for the purpose of diagnosing and noninvasive management ofibd a high proportion of these studies have investigated the efficacy offecal calprotectin in diagnosing and monitoring patients althoughsome of these studies reported auspicious results there are still somedoubts on the eï¬ectiveness of fecal calprotectin on diagnosing andmonitoring ibd patients so in this review we addressed the advantages and limitations of fecal calprotectin for the diagnosis andmanagement of ibd the role of fecal calprotectin in diagnosis and management ofibdthe efficacy of fecal calprotectin as an laboratory marker in various areas of ibd diagnosis and management have been studied including ibd diï¬erentiation from irritable bowel syndrome ibs evaluation of endoscopic activity of the disease evaluation of histologicalactivity of the disease and prediction of disease recurrence andclinica chimica acta “response to treatment in following after a brief introduction andmentioning the important points regarding laboratory measurement offecal calprotectin we reviewed the most interesting findings in all ofthe abovementioned areas calprotectin a clinically valuable proteincalprotectin is an antimicrobial protein mainly secreted by neutrophils this protein competes with bacteria over zinc thus kills thebacteria however this is not the only contribution that it has to antimicrobial activity moreover this protein has many potential clinicalapplications such as the elevated serum levels that have been observedunder various immunological and immunopathological conditionsserum calprotectin levels rapidly increase in response to bacterial infections in the kidney and heart or during transplant rejection at theearly stages of ‚ammation of the lung serum calprotectin can also beconsidered as a reliable marker besides plasma levels of calprotectinappear to be useful in reflecting disease activity in ‚ammation of thejoints in addition it has been demonstrated that serum calprotectin levels are increased in patients with bacterial sepsis so it can beconsidered as a reliable biomarker in neonatal sepsis the serumlevel of calprotectin increases as well as a sensitivity of and aspecificity of that have been reported for serum calprotectin indiagnosis of neonatal sepsis it has been recently shown thatserum calprotectin levels increase in patients with aneurysmal subarachnoid hemorrhage and higher levels in the first of onset areassociated with a poor prognosis at the first three months serumcalprotectin levels also increase in patients with rheumatoid arthritisand even in patients with a moderate to high disease activity who havenormal or low crp levels so they appear to be more efficient at reflecting disease activity some studies have also investigated the efficacy of serum calprotectin in the diagnosis of cancers correspondingly in one of thesestudies it was shown that serum calprotectin levels significantly increased in patients with laryngeal carcinoma compared with healthyindividuals and those with benign laryngeal pathologies moreover inthis study a direct relationship was also observed between serum levelsof calprotectin and stage of cancer another study showed that theserum level of calprotectin increased in patients with papillary thyroidcarcinoma but it significantly decreased after operation alsoregarding the efficacy of serum and saliva calprotectin for the diagnosisof ibd impressive results have been reported a study onpatients with ibd both uc and cd have shown that serum calprotectinlevels were directly correlated with fecal calprotectin levels and weremore potent in ibd diagnosis compared to crp and albumin this studyalso indicated that the combination of serum calprotectin with crp oralbumin can be helpfulin the prediction of treatment escalationespecially in patients with cd however no significant correlationwas observed between serum calprotectin and fecal calprotectin levelsin patients with cd and uc as well as a slight correlation betweenserum calprotectin level and crp that was observed only in patientswith uc another study showed that the serum level of calprotectin was significantly higher in patients with cd compared to healthyindividuals in addition although a significant correlation was observedwith the clinical activity of the disease no significant correlation wasfound between the level serum calprotectin and endoscopic activity ofthe disease the efficacy of salivary calprotectin in the diagnosisof ibd has also been studied which showed that salivary calprotectinsignificantly increased in patients with ibd compared to healthy individuals in this study auc values for unstimulated saliva and stimulated saliva to distinguish ibd patients from healthy individualswere reported to be and respectively however thepopularity of calprotectin is mainly due to the use of fecal calprotectinin the diagnosis and management of ibd that is discussed in the following 0cf khakikhatibi clinica chimica acta “ laboratory measurement and reference intervalfecal calprotectin is a stable protein that remains stable for “ daysat room temperature this property is an excellent advantage for alaboratory marker also it seems that keeping the specimen at refrigerated temperature °c can increase the stability of fecal calprotectin however evidence has been obtained regarding thatthe stability of this protein decreases after staying for three days atroom temperature on the other hand it is not also recommended tokeep samples in the refrigerator for more than days it seemsthat fecal calprotectin remains stable up to one year at ˆ’ °c measurement of fecal calprotectin can be done both qualitatively andquantitatively accordingly in the qualitative measurement monoclonal antibodies are used to detect fecal calprotectin and the positiveresults are characterized by the appearance of colored lines on the testcassette however in the qualitative one only positive or negative results are reported and despite of sensitivity test specificity in theevaluation of disease activity was reported to be only it seems thatthe main application of this test is to diï¬erentiate healthy individualsfrom ibd patients rapidly however some studies have shown that it isnot accurate enough in this case as well nevertheless asignificant concordance has been reported between home test resultsibdoc and fecal calprotectin laboratory measurement results whenquantum blue calprotectin elisa kit was used notably the agreements between results were and depending on the selectedcutoï¬s several commercial kits are also available for fecal calprotectin qualitative test known as rapid calprotectin these tests reportpositive results ranged from to µgg there are also severalcommercial kits that can be used for the quantitative measurement offecal calprotectin these kits are usually designed in terms of the elisamethod and some have a measurement range between and µgg moreover the chemiluminescence immunoassays cliamethod can also detect values between and µgg fluoro enzyme immunoassays feia and particle enhanced turbidimetric immunoassays petia can also be used for the measurement of fecalcalprotectin in this regard one of the most serious challenges to thelaboratory evaluation of fecal calprotectin is the determination of theupper limit in healthy individuals among healthy adults there is asignificant agreement on µgg as an upper limit one study suggested values up to µgg in people over years old and up to µgg in children aged between and years old as referenceranges of fecal calprotectin in healthy individuals fecal calprotectin levels appear to be higher in healthy infants andchildren under four years old than in adults and further studies areneeded in this regard to determine the acceptable upper limit for diagnosis of pediatric ibd table lists the median levels of fecalcalprotectin in healthy individuals with diï¬erent ages reported in somestudies according to these reports age can aï¬ect fecal calprotectinlevels fecal calprotectin and ibd diagnosisonly a small percentage of patients complaining of abdominal painand diarrhea have ibd in many cases ibs as a functional gastrointestinal disorder is known as the cause of such clinical symptomspatients with ibs have normal colonoscopy results while ibd patientsindicate abnormal colonoscopy results and have intestinal ulcersunfortunately the significant prevalence of ibs and the overlap between clinical symptoms and ibd can increase the colonoscopy ratetherefore a noninvasive diagnostic marker can be very helpful in thisregard notably the first evidence of the efficacy of fecal calprotectin inthe diagnosis of ibd was obtained in the 1990s røseth in proposed a method for measuring calprotectin in stool specimens one of the first and most interesting studies regarding fecal calprotectinutility in ibd diagnosis was the study by røseth published in in this study patients with ulcerative colitis were studied and according to their results fecal calprotectin levels are higher in patientswith ulcerative colitis compared to healthy controls this study havealso shown that even patients with low disease activities had higherlevels of fecal calprotectin compared to healthy individuals subsequent studies somehow confirmed and complemented the findings of this study in another study published in auc values of ci “ were reported for fecal calprotectin in thediagnosis of colorectal ‚ammation moreover in a study onchildren with ibd it was shown that the level of fecal calprotectin washigher in these patients compared to healthy children so it can beconcluded that it is also directly correlated with esr levels in astudy published in kolho reported auc values of ci “ for fecal calprotectin in the diagnosis of pediatric ibd in a study on patients with crohn disease a sensitivity of and a specificity of at cutoï¬ of μgg have been reportedfor fecal calprotectin in diagnosis of the disease the results of ourrecent study along with other studies showed that fecal calprotectin ispreferred over traditional ‚ammatory biomarkers such as crp andesr in the diagnosis of ibd diamanti reported a sensitivity of and a specificity of for fecal calprotectin at a cutoï¬ of μgg in ibd diagnosis in our recent study a sensitivityof and a specificity of at a cutoï¬ of μgg were observed for fecal calprotectin in the diagnosis of ibd however oursample size was and the majority of patients were in the active phaseof the disease in another study conducted on patients with ulcerative colitis asensitivity of and a specificity of at cutoï¬ of μgg havebeen reported in this regard in one study it was shown that fecalcalprotectin in cutoï¬ of μgg is able to distinguish patients withibd from patients without ibd patients with diseases other than ibdpatients with ibs and healthy persons with sensitivity and specificity caviglia in their study reported a sensitivity of and a specificity of at a cutoï¬ of μgg for fecalcalprotectin in diï¬erentiating between ibs and ibd howeversome studies have reported significantly lower values accordingly in astudy on patients with ulcerative colitis kalantari reported asensitivity of and a specificity of at a cutoï¬ of μgg besides there is a considerable agreement between fecal calprotectinand capsule endoscopy findings in patients with crohn's disease asensitivity of and a specificity of have also been reported at acutoï¬ of mgkg for fecal calprotectin in predicting ce findings anddiagnosis of crohn's disease in another study lower sensitivityand specificity rates sensitivity specificity were reportedfor fecal calprotectin in this regard furthermore in one studythat examined the efficacy of fecal calprotectin in predicting wirelesscapsule endoscopy findings a sensitivity of and a specificity oftable reported median levels of fecal calprotectin in healthy individuals of diï¬erent agesagesmedian levels of fecal calprotectin range µggnumber of subjectsused kitup to monthchildren “ yearschildren “ yearsadultsover years “ “ “ “ “bühlmann elisabühlmann elisacalpro® calprotectin elisa test alpphicalphicalreference 0cf khakikhatibi were reported for this biomarker at μgg in the diagnosis ofsmall bowel ‚ammation in crohn's disease given these findings it seems that fecal calprotectin has no ideal sensitivity and specificity for the diagnosis of ibd where the small intestine is involvedbesides there are some preanalytical limitations which are explainedin the next sections therefore optimistically speaking fecal calprotectin measurement can eliminate the need for colonoscopy howeverin a metaanalysis performed to evaluate the efficacy of fecal calprotectin and some other ‚ammatory markers to diï¬erentiate betweenibd and ibs the probability of ibd was less than at fecal calprotectin values lower than µgg or crp values lower than mgdl therefore it seems that fecal calprotectin can be helpful at leastin ruling out the possibility of ibd in patients with ibslike symptoms aswell as reducing the rate of colonoscopy moreover it should be notedthat although a systematic review has reported pooled sensitivity andspecificity above for fecal calprotectin to diï¬erentiate between ibdand ibs it emphasized more on the possibility of falsepositive resultsin low cutoï¬ points hence performing extensive studies indiï¬erent countries on the healthy population and the ibd patient is beneeded to determine a suitable cutoï¬ with maximum sensitivity andspecificity and minimum falsepositive resultstable summarizes the results of various clinical investigationsregarding fecal calprotectin utility in the diï¬erential diagnosis of ibdfrom ibs and table4 summarizes some metaanalysis results in thisregard as shown in table the most important limitation of the majority of clinical studies conducted to date is the small sample size alarge global study may be helpful in providing a more precise evaluation of fecal calprotectin clinical value in discrimination between ibdand nonibd diseases fecal calprotectin and endoscopic and histologic activity evaluationundoubtedly one of the most serious challenges in the managementof ibd is evaluating the endoscopic and histologic activities of thedisease nowadays colonoscopy and histopathologic examinations arethe routine tools for the assessment of mucosal healing in patients withibd as noted earlier several scoring systems have been devised toscore disease activity based on the findings of colonoscopy and histopathologic examinations in recent years many promising results havebeen reported regarding the correlation between these scores and fecalcalprotectin levels in addition many studies have been performed inthe last decade all of which cannot be reviewed in this article the firstevidence of a link between fecal calprotectin and disease endoscopicactivity was obtained in the late 1990s in one of the first studiesroseth found a significant correlation between fecal calprotectinlevels and endoscopic and histologic activities in patients with ulcerative colitis furthermore in another study they observed that ibdpatients who were in remission clinically and had normal fecal calprotectin levels less than mgl had normal colonoscopy results these interesting findings indicate that fecal calprotectin can beconsidered as a biomarker in the evaluation of endoscopic activity andclinica chimica acta “table4summarized results of some metaanalysis regarding the utility of fecal calprotectin in discrimination between patients with ibd and without ibdsample sizepooled sensitivitypooled specificityreferences mucosal healing in ibd patients also these studies were the startingpoint of extensive studies that have been conducted up to now in astudy conducted on patients with crohn's disease sipponen investigated the sensitivity and specificity of fecal calprotectin in predicting endoscopic activity of crohn's disease correspondinglythe researchers used the crohn's disease endoscopic index of severitycdeis scoring system in their study to evaluate the endoscopic activity of crohn's disease as a result they found that there was a significant correlation between the endoscopic activity of the disease andthe level of fecal calprotectin besides the findings of this study demonstrated that fecal calprotectin at µgg cutoï¬ can predict theendoscopic activity of crohn's disease with sensitivity and specificity in another study cdeis and mayo disease activity indexmdai were used to evaluate the endoscopic activity of crohn's diseaseand ulcerative colitis respectively according to the results of thatstudy on ibd patients there was a significant correlation between fecalcalprotectin levels and disease endoscopic activity another studyshowed that fecal calprotectin is more strongly correlated with theendoscopic activity of the disease in ulcerative colitis compared to therachmilewitz clinical activity index in addition in this study theoverall accuracy of fecal calprotectin for endoscopically active diseaseidentification was obtained as some studies have also shown the superiority of fecal calprotectinover traditional ‚ammatory markers like crp besides one studyfound that fecal calprotectin was more strongly correlated with thesimple endoscopic score for crohn's disease sescd compared to thecrp and even crohn's disease activity index cdai the modifiedbaron index was also used in another study to evaluate the endoscopicactivity of ulcerative colitis as a result it was shown that calprotectinis more strongly correlated with the endoscopic activity of ulcerativecolitis compared to crp and clinical activity of the disease in thisregard similar results were also observed in our recent study in whichthe ulcerative colitis endoscopic index of severity uceis and sescdwere used therefore fecal calprotectin appears to be superior totraditional ‚ammatory markers in the prediction of ibd endoscopicactivity the high values of sensitivity and specificity that were mentioned earlier have raised the hope that using fecal calprotectin canreduce colonoscopy rate for patients™ monitoring however severalrecent studies have reported some significantly lower values accordingly in a recent study in which mayo endoscopic score [mes] wasused to evaluate the endoscopic activity of ulcerative colitis atable summary of the results of some studies regarding the utility of fecal calprotectin in discrimination between patients with ibd and without ibdnumber of ibd patientsage grouplocationcut oï¬sensitivityspecificity cd and uc cd and uc cd and uc and unclassified68cd and uc cd and uc and unclassified cd and uc cd and uc uc cd ucadultsadultsadultsboth adult and pediatricpediatricadultspediatricadultsadultsboth adult and pediatrictaiwanchinaitalyspainfinlandiranitalyirandenmarkindia48µgg µgg150µgg150µgg595µgg784µgg160µgg164µgg150µgg188µggaucreferences spsrefidbib60 0cf khakikhatibi clinica chimica acta “table summary of the results of some studies regarding the correlation of fecal calprotectin with endoscopic activity in ibd patientsage groupstudylocationusedendoscopicactivity indexcorrelationcoefficientrreferencenumberof ibdpatients cd uc uc cd ucadultsadultsadultsadultsadultsfinlandiranswitzerlandswitzerlandswitzerland modifiedcdeisuceisrachmilewitzsescd uc cdadultsadults uc cd uc cd cd uc ucadultsadultsadultsadultsadultsadultsadultsbaron scorerachmilewitzsescdgermanyusa andcanadajapanitalyitalybrazilfrancefrancesouth korea uceismattssescdmayo scoresescdcdeismayo score sensitivity of and a specificity of were reported for fecalcalprotectin at µgg to diï¬erentiate active endoscopic from inactivemes or from mes or in another study the sensitivityand specificity of fecal calprotectin at a cutoï¬ of µgg for diï¬erentiating mes ‰¤ in patients with ulcerative colitis were and respectively overall as presented in table several studiesperformed in diï¬erent countries reported the correlation between fecalcalprotectin and ibd endoscopic activity although some of these studies reported a strong correlation some others reported a relativelyweak correlation as noted earlier there are significant diï¬erencesbetween the reports on the sensitivity and specificity of fecal calprotectin to predict the endoscopic activity of ibd undoubtedly a widerange of factors from sample size and the inclusionexclusion criteriato preanalysis variables and indexes used to evaluate the endoscopicactivity may also contribute to these diï¬erences however fecal calprotectin does not appear to be a very reliable marker for the predictionof ibd endoscopic activity so currently it seems a bit optimistic toconsider fecal calprotectin as a reliable alternative for colonoscopy inthis regard further studies are still needed however under some certain circumstances such as pregnancy or pandemics the use of fecalcalprotectin to evaluate ibd endoscopic activity can be helpfulpregnant patients with ibd have serious limitations for colonoscopyexamination and it has been recommended that colonoscopy should beonly performed in the second trimester of pregnancy and where there isa strong indication therefore noninvasive markers such as fecalcalprotectin can be helpful during pregnancy in one study physicianglobal assessment [pga] which is a clinical symptombased criterionwas used to evaluate ibd activity and subsequently the associationbetween fecal calprotectin and this criterion was investigated in pregnant women with ibd the results of this study showed a significantcorrelation between fecal calprotectin and pga levels at prepregnancyduring pregnancy and postpartum stages in another study asignificant association was reported between fecal calprotectin levelsand clinical activity of ibd in pregnant women moreover it was shownthat stool calprotectin at a cutoï¬ of mgkg had a sensitivity between and as well as a specificity between and in the assessment of ibd clinical activity at diï¬erent stages ofpregnancy a recently published systematic review has also confirmed the conclusions obtained from these studies according tothese results it seems that fecal calprotectin is not aï¬ected by physiological changes during pregnancy however it is significantly correlatedwith ibd clinical activity during pregnancy therefore from the viewpoint of relatively acceptable sensitivity and specificity in predictingthe endoscopic activity of ibd fecal calprotectin may be considered as anoninvasive biomarker for the evaluation of ibd endoscopic activity inpregnant women in addition under pandemic conditions fecal calprotectin can be very helpful following the covid19 pandemicwhich began in late and is still ongoing healthcare systems indiï¬erent countries were forced to impose significant limitations oncolonoscopy therefore noninvasive ibd management and fecal calprotectin as a noninvasive laboratory marker have become moreimportant than before the combination between disease clinical activity and fecal calprotectin has been recommended as a noninvasiveapproach that can help in making decisions on treatment duringcovid19 pandemic therefore it seems that fecal calprotectincan be considered as an alternative for colonoscopy used for ibd endoscopic activity evaluation during pandemic fecal calprotectin appears to be associated with ibd histologic activity as well given thedifficulty in the evaluation of the histologic activity of crohn's disease some studies have been focused on the ulcerative colitis andmany scoring systems have been devised so far correspondingly thesesystems score the disease's histologic activity based on histologic observationstherefore for this purpose a biopsy of the intestinal tissue is required which can be prepared by colonoscopy and then sent to thelaboratory in this regard one of these histologic scoring systems isrobert™s score that was used in one of our recent studies where weobserved a significant correlation between the level of fecal calprotectinand the histologic activity of ulcerative colitis which was calculatedbased on the robert™s scoring system theede also used themodified harpaz index and performed some interesting studies in thisregard in one of their studies fecal calprotectin was found to be significantly associated with the histologic activity of the ulcerative colitisand it was shown that it could predict histological mucosal healingauc ci95 “ sensitivity specificity andcutoï¬ mgkg in another study on patients with endoscopically inactive ulcerative colitis mayo endoscopic score the researchers showed that patients with ulcerative colitis who were inendoscopic remission but had histologically active disease had higherlevels of fecal calprotectin compared to patients with no histologicallyactive disease versus mgkg p also despite thehigh specificity the sensitivity of fecal calprotectin in theprediction of score of histological activity was achieved as at mgkg in a recent study the geboes index has been used toevaluate histologic activity in patients with clinically quiescent ulcerative colitis as a result this study reported relatively low sensitivityand specificity for fecal calprotectin in the prediction of geboesscore sensitivity specificity and cut oï¬ µgg in another recent study the nancy index has been used toevaluate the histologic activity of ulcerative colitis and a high sensitivity and a low specificity were finally reported for fecalcalprotectin at a cutoï¬ of µgg in the prediction of histologic activity however some studies have reported both high sensitivityand specificity for fecal calprotectin in the prediction of histologicalremission for example one study reported a sensitivity of aswell as a specificity of for fecal calprotectin at a cutoï¬ of µggin the prediction of gs it seems that the cause of theseconflicts should be explored in the endoscopic and clinical activity ofthe disease the inclusion and exclusion criteria of these studies andpossibly in the diï¬erent indexes used for the evaluation of the histologicactivity of the disease another notable issue is that all of these studieshave been conducted on a relatively low number of patients with ulcerative colitis so the need for a study with a large sample size is stillstrongly felt moreover a large global study may be helpful in this regard prediction of relapse and response to treatmentas mentioned previously ibd has recurrence and relief periods sopredicting response to treatment and relapse is one of the significant 0cf khakikhatibi clinica chimica acta “challenges in ibd management the first evidence of the efficacy offecal calprotectin to predict recurrence dates back to the early 2000saccordingly a study published in by tibble is one of the firststudies in this regard in this study ibd patients in clinical remissionwere followed up for one year for the assessment of recurrence afterpreparing a stool sample to measure calprotectin this study has alsoshown that fecal calprotectin levels were higher in ibd patients withrecurrent disease and it was found that fecal calprotectin had a sensitivity of and a specificity of at a cutoï¬ of mgl to predictibd recurrence in a study published in costa showedthat the sensitivity and specificity of fecal calprotectin to predict ulcerative colitis recurrence are more than that of crohn's disease asensitivity of and a specificity of versus a sensitivity of and a specificity at a cutoï¬ of μgg another studyconducted on patients with ulcerative colitis has also reported appropriate sensitivity and specificity rates for fecal calprotectin in the prediction of relapse a sensitivity of and a specificity at a cutoï¬of μgg however another study was conducted on patientswith ulcerative colitis who have been followedup for year and finally a lower sensitivity was reported this study have shown that fecalcalprotectin at cutoï¬ of μgg could predict diseas
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" mirnas regulate a multitude of cellular processes and their aberrant regulation is linked to humancancer however the role of mir4255p in lung cancer lca is still largely unclear here we explored the role ofmir4255p during lca tumorigenesismethods cell proliferation was evaluated by cell counting kit8 and colony formation assay western blot and realtime pcr were accordingly used to detect the relevant proteins mirna and gene expression luciferase reporterassays were used to illustrate the interaction between mir4255p and ptenresults we demonstrate that mir4255p is overexpressed in lca tissue and enhances the proliferative and colonyformation capacity of the lca cell lines a549 and ncih1299 through predictive binding assays pten wasidentified as a direct gene target and its exogenous expression inhibited the procancer effects of mir4255pthrough its ability to downregulate pten mir4255p activated the pi3kakt axis we conclude that mir4255p promotes lca tumorigenesis through ptenpi3kakt signalingkeywords mir4255p lung cancer pten pi3kakt signaling pathways lung cancer lca is a leading cause of cancer relatedmortality across the globe lca is prevalent in males and asymptomatic during early disease stages as manyas in every cases are at an advanced stage iii or ivwhen diagnosed and the 5year survival rates remainlow particularly for those with metastatic lca improved lca therapeutics is thus urgently requiredmicrornas mirnas regulate many cell processesincluding differentiation metabolism and tumorigenesis[“] emerging evidence suggests that mirnas are keyplayers during lca tumorigenesis [“] the aberrantexpression of mir4255p is linked to hepatocellular carcinoma hcc gastric cancer gca and colorectal correspondence kjhhev163comcardiothoracic surgery the fourth affiliated hospital zhejiang universityschool of medicine shangchen road no1 of yiwu zhejiang chinacancer crc [“] here we report the upregulationof mir4255p in lca and highlight its contribution tolca development we further identify pten as a novelmir4255p target that is inhibited in lca to promoteptenpi3kakt signalingmethodspatient specimenslca samples n and adjacent healthy tissue at least cm from the resection margin were collected from thefourth affiliated hospital zhejiang university school ofmedicine the study was fully supported by the institutional review board of the fourth affiliated hospitalzhejiang university school of medicine no2015009all participants provided consent for sample analysisand anything about their identities will not be includedin the data the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhou bmc pulmonary medicine page of lca cell lines cell culture and cell transfectionhuman lung cancer cell lines a549 ncih1299 ncih460 hcc827 and normal human lung epithelial cellline beas2b were obtained from the cell bank of thechinese academy of sciences shanghai china a549 hcc827 cells were cultured in dmem plus fbsand penstrep ncih1299 ncih460 cells weregrown in rpmi1640 plus fbs at °c in co2beas2bs were cultured in clonetics„¢ mediathe mir4255p mimics and negative control mirnanc were chemically synthesized by shanghai genepharma co ltd songjiang shanghai china lipofectamine invitrogen eugene or usa was usedfor transfection according to the manufacturer™s protocol pi3k activity was assayed as previously described pi3k inhibitor ly294002 was obtained from abcamto analyze the effects of mir4255p on pi3kakt indicated lca cells were cultured in the presence or absence ofthe workingconcentrations of ly294002 were μm for experiments with ly294002 treatmentsindicated lca cellswere pretreated with ly294002 for h prior to exposure to proteasome inhibitorsthe drugs for h at °ccell growth assayslca viability was assessed using cell counting kit8from shanghai haling biotechnology co ltd shanghai china as per the manufacturer™s protocols brieflyafter incubating the transfected cells for one full daythey were collected after trypsinization and seeded cellswell into 96well plates ten microliters ofcck8 solution were added per well and kept for h at °c the absorbance of the mixture was estimated in amicroplate readerinchercules usa at nmfrom biorad laboratoriescolony formation assaythe colony formation assays were performed as previous each group of treated cells — per well wasseeded into cm culture dish and cultured for weeksfinally colonies were stained using crystal violet andthe number of cell colonies was countedqrtpcr analysistotal rna was isolated by trizol® reagent from invitrogen thermo fisher scientific inc and a nanodropnanodrop technologies thermo fisher scientific incwas used to estimate its quality and concentration theexpression of mir4255p was done by reverse transcription using the mirx„¢ mirna firststrand synthesis kitfrom takara biotechnology co ltd dalian chinaand quantitative evaluation of the synthesized cdnawas done by quantitative pcr rtqpcr using themirx„¢ mirna qrtpcr tb green® kit from takaraforwardbiotechnology as an endogenous control the small nuclear rna u6 normalized the expression of mir4255pthe ˆ’δδcq system was used to evaluate all genes expressions and the primer sequences were shown as fol² ggggagttaglows mir4255pgattaggtc3² reverse ² tgcgtgtcgtggagtc3² u6 forward ²ctcgct tcggcagcaca² reverse ²aac gct tca cga att tgc gt3²pten forward ² tggattcgacttagacttgaccˆ’ ² reverse ² aggatattgtgcaactctgcaa² gapdh forward ²catcaccatcttccaggagcg3² reverse ™tgaccttgcccacagcctt3²to getdual luciferase assaysthe design and synthesis of pten fragments containingbinding sites for wt wildtype and mut mutant onmir4255p was done by shanghai genepharma thesewere cloned into the target expression vector pmirglo dualluciferase from promega corporation wiusathe reporter plasmids wtpten andmutpten one night prior to transfection seeding ofcells “ confluence was done in plates with wells transfection of these cells was done with reporterplasmids harboring wt or mut in the presence ofmirnc or mir4255p mimic post h of transfectionluciferase activity of the cells was estimated as per instructions using the dualluciferase reporter assay system from promega corporation promega fitchburgwi usa the data normalization was done by the activity of renilla luciferasewbcell lysates ripa lysates were resolved on sds pageand transferred to pvdf membranes membraneswere blocked for h in milk plus tbstincubated with primary antibodies against pten dilution cat no ab170941 abcam pi3k dilution cat no ab32089 abcam pakt dilution cat no ab81283 abcam akt dilution cat no ab32505 abcam pakt dilution cat no ab81283 abcam actin dilution cat no ab8226 abcam at °c overnights andlabeled with hrpconjugated secondary™s for h atroom temperature bands werevisualized usingchemiluminescent hrp substrate and analyzed usingimage lab tm softwarestatistical analysesdata analysis was performed using spss treatmentgroups were compared using a oneway anova pvalues were taken as significant experiments wereperformed on at least three occasions data representthe mean ± sd 0czhou bmc pulmonary medicine page of resultsmir4255p is upregulated in lcawe compared mir4255p levels in paired lcaand normal lung tissue samples by qrtpcr analysismir4255p was upregulatedspecimensfig 1a and expressed to high levels in a549 ncih1299 ncih460 and hcc827 cells compared tonormal human lung epithelial cellline beas2bfig 1b consistent with previous findings in othercancer types previous results indicated that mir4255p is upregulated in lcain lcamir4255p enhances proliferation and inhibits apoptosisin lca cellsto dissect the role of mir4255p in lca its expressionwas manipulated using mir4255p mimics figure 2acshows that mir4255p upregulation enhanced cell survival meanwhile enhanced cell colony formation abilityfig 2d and e taken together above results indicatedmir4255p is thus an oncogene in lca cellsmir4255p targets ptenfrom targetscan pten was identified as a predictedmir4255p target fig 3a in pten ²utr reporterassays mir4255p suppressed wt pten expressionfig 3bc but had little effect on mutated pten ²utr fragments fig 3bc the levels of pten werelower in cells transfected with mir4255p mimics fig3de mir4255p also negatively related pten mrnalevels in lca tissue p r2 fig 3f thesedata implicate pten as a cellular target of mir4255pmir4255p promotes lca via ptento define whether mir4255p regulate pten in lcawe firstly overexpression pten in lca fig 4a wbanalysis demonstrated that mir4255p reduces ptenlevels which could be recovered by exogenous expression fig 4b cell viability assays showed that mir4255p enhances lca proliferation which could be reversedby pten transfection fig 4cd suggesting mir4255pmediates its activities via pten this indicates that mir4255p targets pten to mediate its protumor effectsmir4255p activates ptenpi3kakt signalingit has been reported ptenpi3kakt signaling wasclosely related to cell proliferation and apoptosis [“] next we explored the effect of mirna4255p onptenpi3kakt signaling as shown in fig 5aincomparison to nc groups pten was down regulated inresponse to mir4255p mimics whilst pi3k and paktlevels increased in addition nsclc cells were treatedwith the pi3kakt inhibitor ly294002 or ly294002 mir4255p mimics mimic fig 5b kinase activity assays further showed that pi3k activity in nsclc transfected with ly294002 was significantly lower than thattransfected with mimic control p and pi3k activity in nsclc cells transfected with both ly294002and mir4255p mimic was significantly higher than thattransfected with ly294002 p take together thisimplicates ptenpi3kakt signaling in the protumorigenic effects of mir4255pdiscussionthe poor prognosis of lca highlights the need for urgent therapeutic strategies mirnas are novel targets forfig mir4255p in highly expressed in lca a qrtpcr of mir4255p b qrtpcr of a549 ncih1299 ncih460 hcc827 and beas2b cellsdata mean ± sd p p p 0czhou bmc pulmonary medicine page of fig mir4255p promotes lca a mir4255p expression in a549 and ncih1299 cells b c cck8 assays in mir4255p mimic transfectedcells d e colonyforming assay in mir4255p mimic transfected cells the raw data of all colony formation experiments was listed insupplemental table data mean ± sd p p p fig mir4255p targets pten in lca a predicted binding of mir4255p and pten b c relative luciferase activity of ptenwt ptenmut inlca cells expressing mir4255p mimic d pten mrna levels are significantly lower after transfection with mir4255p mimic e pten expressionassessed by wb fulllength blotsgels are presented in supplementary figure f mir4255p and pten negatively correlate in lca tissue datamean ± sd p p p 0czhou bmc pulmonary medicine page of fig mir4255p promotes lca growth by targeting pten a qrtpcr of pten in indicated lca cell lines b wb analysis of pten expression fulllength blotsgels are presented in supplementary figure c d cck8 analysis of cell viability in the indicated cell lines with empty vector mir4255p mimic andor pten in a549ncih1299 cells data mean ± sd p p p fig effects of mir4255p on ptenpi3kakt a wb analysis of pten pi3k pakt and akt in lca cells fulllength blotsgels are presented insupplementary figure b the pi3k kinase activity was determined in nsclc cells transfected with ly294002 was significantly lower than thattransfected with mimic control and the pi3k kinase activity in nsclc transfected with both ly294002 and mir4255p mimic was significantlyhigher than that transfected with ly294002 data mean ± sd p p p 0czhou bmc pulmonary medicine page of cancer therapies and their dysregulation occurs in lcatissue [“] duan showed that mir203 bindsto zeb2 to suppress emt yuan showed thatmir30a inhibits eya2 migration and invasion andli showed that mir1304 inhibits lca cell divisionthrough heme oxygenase1 the cellular roles ofmir4255p in lca are poorly understoodin thepresent work we further explore the underlying mechanisms of mir4255pinduced lca cell progressionin the present study we confirmed that mir4255p isoverexpressed in lca cell lines and tissues implicating arole in lca tumorigenesis upregulating mir4255plevels enhanced the cell survival and colony formationability of lca cells in vitro implicating it as a novel lcaoncogene in the mechanism using the algorithms targetscan website tools we identified pten as the potential target of mir4255p furthermore we performedluciferase reporter assays and the results showed thatmir4255p may directly target pten3™utr the resultof qrtpcr and western blot also confirmed that overexpression of mir4255p could suppress the expressionlevel of pten all the above suggested that pten was apotentialtarget of mir4255p moreovermir4255p also negatively related pten mrna levelsin lca tissue rescue experiment indicated that exogenous pten expression inhibited the procancer effects ofmir4255p pten was downregulated in lca tissuepten is a tumor suppressor with wellcharacterizedphosphatase activity pi3kakt promotes cell cycleprogression inhibits apoptosis and is known to be overactive in a multitude of human cancers [ ] ptencan suppress pi3kakt signaling and thus displays anticancer effects upon assessment of the molecularmechanisms of mir4255p in lca cells its procancereffects were found to be mediated through manipulationof the ptenpi3kakt signaling axisfunctionalin we highlight mir4255p as an oncogenein lca that promotes an oncogene phenotype by inhibiting pten these findings enhance our knowledge of therole of mir4255p and reveal new therapeutic strategiesfor the diagnosis and treatment of lca angiogenesisand metastasis biological experiments will clarify thefunctions and roles of mir4255p in lcasupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12890020012610additional file supplement figure s1 uncropped images of blotsand gels related to fig supplement figure s2 uncropped imagesof blots and gels related to fig supplement figure s3 uncroppedimages of blots and gels related to fig table s1 the raw data of allcolony formation experimentsabbreviationslca lung cancer pten gene of phosphate and tension homology deletedon chromsome ten pi3k phosphatidylinositol 3kinase nsclc nonsmallcell lung cancer gca gastric cancer crc colorectal cancerhcc hepatocellular carcinoma emt epithelialmesenchymal transitionacknowledgementsnot applicableauthors™ contributionsjsz zsy syc and qf did patient recruitment and assessment andperformed the experiments jhy and cjh were responsible for statisticalanalysis jsz zsy and syc interpreted the data and wrote the manuscriptall authors contributed to the subsequent drafts and approved the finalversionfundingthis research was funded by zhejiang provincial natural science foundationof chinaexploration program lq19h020010 core talent program ofdepartment of health of zhejiang province 2013rca018 zhejiangprovincial natural science foundation of chinageneral programly15ho2004 the funders had no role in the design of the study andcollection analysis and interpretation of data and in writing the manuscriptavailability of data and materialsthe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participateethics approval for this study was obtained from the fourth affiliatedhospital zhejiang university school of medicine no2015009 all patientsgave written informed consent for participation in the studyconsent for publicationnot applicablecompeting interestsall authors declare no conflicts of interestreceived march accepted august referenceschen w zheng r zuo t zeng h zhang s he j national cancer incidenceand mortality in china chin j cancer res “aberle dr berg cd black wc the national lung screening trialoverview and study design radiology “ni js zheng h huang zp microrna1973p acts as a prognosticmarker and inhibits cell invasion in hepatocellular carcinoma oncol lett“zhang tj wang yx yang dq downregulation of mir186 correlateswith poor survival in de novo acute myeloid leukemia clin lab ““ wang b teng y liu q microrna153 regulates nrf2 expression and isassociated with breast carcinogenesis clin lab ““lin h huang zp liu j mir4943p promotes pi3kakt pathwayhyperactivation and human hepatocellular carcinoma progression bytargeting pten sci rep yang j li j le y zhou c zhang s gong z pfklmir128 axis regulatesglycolysis by inhibiting akt phosphorylation and predicts poor survival inlung cancer am j cancer res “ wang r chen x xu t mir326 regulates cell proliferation andmigration in lung cancer by targeting phox2a and is regulated by hotairam j cancer res “qin q wei f zhang j wang x li b mir134 inhibits nonsmall cell lungcancer growth by targeting the epidermal growth factor receptor j cellmol med “ hu h xu z li c mir145 and mir203 represses tgfbetainducedepithelialmesenchymal transition and invasion by inhibiting smad3 in nonsmall cell lung cancer cells lung cancer “ 0czhou bmc pulmonary medicine page of fang f song t zhang t cui y zhang g xiong q mir4255p promotesinvasion and metastasis of hepatocellular carcinoma cells through scaimediated dysregulation of multiple signaling pathways oncotarget “ cristobal i madozgurpide j rojo f garciafoncillas j potential therapeuticvalue of mir4255p in metastatic colorectal cancer j cell mol med “ zhang z wen m guo j clinical value of mir4255p detection and itsassociation with cell proliferation and apoptosis of gastric cancer pathol respract “ huang z xing s liu m deng w wang y huang z huang y huang x wuc guo x pan x jiang j feng f li t mir26a5p enhances cellsproliferation invasion and apoptosis resistance of fibroblastlikesynoviocytes in rheumatoid arthritis by regulating ptenpi3kakt pathwaybiosci rep meng j liu gj song jy chen l wang ah gao xx wang zj preliminaryresults indicate resveratrol affects proliferation and apoptosis of leukemiacells by regulating ptenpi3kakt pathway eur rev med pharmacol sci“liu hy zhang yy zhu bl feng fz yan h zhang hy zhou b mir21regulates the proliferation and apoptosis of ovarian cancer cells throughptenpi3kakt eur rev med pharmacol sci “ yu g wang c song x liu s zhang y fan l yang y huang y song jformaldehyde induces the apoptosis of bmcs of balbc mice via the ptenpi3kakt signal transduction pathway mol med rep “sun xh wang x zhang y hui j exosomes of bonemarrow stromal cellsinhibit cardiomyocyte apoptosis under ischemic and hypoxic conditions viamir4865p targeting the ptenpi3kakt signaling pathway thromb res“ mou x liu s mir485 inhibits metastasis and emt of lung adenocarcinomaby targeting flot2 biochem biophys res commun “su tj ku wh chen hy oncogenic mir137 contributes to cisplatinresistance via repressing casp3 in lung adenocarcinoma am j cancer res“liu y wang f xu p mir590 accelerates lung adenocarcinoma migrationand invasion through directly suppressing functional target olfm4 biomedpharmacother “ yang l luo p song q fei x dnmt1mir200agolm1 signaling pathwayregulates lung adenocarcinoma cells proliferation biomed pharmacother“ duan x fu z gao l direct interaction between mir203 and zeb2suppresses epithelialmesenchymal transition signaling and reduces lungadenocarcinoma chemoresistance acta biochim biophys sin shanghai“ yuan y zheng s li q overexpression of mir30a in lungadenocarcinoma a549 cell line inhibits migration and invasion via targetingeya2 acta biochim biophys sin shanghai “li cg pu mf li cz microrna1304 suppresses human nonsmall celllung cancer cell growth in vitro by targeting heme oxygenase1 actapharmacol sin “li dm sun h ptenmmac1tep1 suppresses the tumorigenicity andinduces g1 cell cycle arrest in human glioblastoma cells proc natl acad sciu s a “ bellacosa a chan to ahmed nn akt activation by growth factors is amultiplestep process the role of the ph domain oncogene “li jw wang xy zhang x gao l wang lf yin xh epicatechin protectsagainst myocardial ischemiainduced cardiac injury via activation of theptenpi3kakt pathway mol med rep “ qin y huo z song x chen x tian x wang x mir106a regulates cellproliferation and apoptosis of colon cancer cells through targeting theptenpi3kakt signaling pathway oncol lett “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"
0
] we have filtered only research s published in english language and selected the following keywords air pollution and covid19 or sarscov2 particulate matter or pm and covid19 or sarscov2 nitrogen dioxide or no2 and covid19 or sarscov2 we choose as inclusion criteria all the available epidemiological studies aimed to identify any temporal and spatial association between reported covid19 cases andor deaths and air pollution data related to pm25 pm10 and no2 thus excluding any letter opinion commentary review or nonrelevant s we obtained a total of eligible published research s in their final version and paper in its preprint version for some of them we chose to include only principal findings that clearly fit the aim this review particulate matter and covid19 atmospheric particulate matter pm is originated by a wide range of anthropogenic and natural sources kim it consists of a heterogeneous mixture of solid and liquid ps suspended in air that varies continuously in size and chemical composition including nitrates sulphates elemental and anic carbon anic compounds biological compounds and metals who it has been associated with increased respiratory morbidity and mortality liu especially in susceptible people due to cardiorespiratory events including asthma chronic obstructive pulmonary disease and thrombosis li rhee in vitro and in vivo studies highlighted its role in the exacerbation of respiratory viral infections becker and soukup recently the research group of setti gave first preliminary evidence that sarscov2 rna can be present on outdoor particulate matter thus suggesting that in conditions of atmospheric stability and high concentrations of pm it could represent a potential early indicator of covid19 although it does not give information regarding covid19 progression or severity several observations report a significant association between ambient concentrations of pm25 adhikari and yin bashir fattorini and regoli frontera jiang li vasquezapestegui wu yao zhu zoran 2020a and pm10 bashir coccia 2020b fattorini and regoli jiang li yao zhu zoran 2020a with covid19 pandemic across the most affected countries china italy and usa see table first evidences on the temporal association between air pollution and covid19 were reported in china where the outbreak was first identified zhu explored the relationship between particulate matter and the viral infection caused by the novel coronavirus in cities in china the authors included over of dailyconfirmed new cases in the whole of china between january 23rd and february 29th they applied a generalized additive model gam to examine the effects of meteorological factors and air pollution on covid19 incidence applying a movingaverage approach to capture the cumulative lag effect of ambient air pollution and considering population size and density as potential confounders they observed that the effect of pm25 on daily confirmed cases was greater than pm10 in particular they found that a 10μgm3 increase lag0“ in pm25 and pm10 was associated with a ci to and ci to increase in the daily counts of covid19 confirmed cases respectively jiang focused their attention on three most affected cities of china wuhan xiaogan and huanggang collecting data of daily cases and ambient air pollutant from jan 25th to feb 29th the authors by applying a multivariate poisson regression revealed a significant temporal association between pm25 increased and covid19 incidence in all the considered cities especially in huanggang wuhan rr ci “ xiaogan rr ci “ huanggang rr ci “ conversely an increase in pm10 concentrations was associated with a decrease of covid19 incidence these results were partially confirmed by findings of li who conducted a simple linear regression to compare covid19 incidence with pm concentrations in wuhan and xiaogan from jan 26th to feb 29th in they found that an increase in pm25 was correlated with an increase of covid19 incidence in both cities wuhan r2 p xiaogan r2 p while for pm10 only in xiaogan r2 p the spatial distribution of particulate matter and case fatality rate cfr of covid19 was studied by yao in cities of china including wuhan collecting data up to march 22nd first they found a significantly positive global spatial autocorrelation of covid19 cfr global moran™s index i p highlighting a high cfr clustering located in hubei province with a multiple linear regression they adjusted their results for several effect modifiers and confounder factors such temperature relative humidity gross domestic product gdp per capita hospital beds per capita local indicators of spatial association lisa map values city size and population or proportion of people older than years it was found that for every μgm3 increase in pm25 and pm10 the cfr increased by “ and “ respectively and the risk estimates increased to “ and “ with every μgm3 increase in average concentrations of pm25 and pm10 in “ respectively some studies describe the association between air pollution and covid19 across italy the second country of the world where the infection spread significantly at the beginning of the pandemic and suddenly has reached many other european countries the 28th of july italy recorded more than total confirmed cases and deaths who most of which were distributed in the regions of northern italy especially the lombardy it is recognized as one the most air polluted areas of europe eea where the frequent pm10 annual exceedances of the who threshold of μgm3 are responsible for attributable deaths per year corresponding to attributable community rates of deaths per inhabitants per year baccini bontempi 2020bfocused the attention on two of the most affected regions of northern italy lombardy and piedmont the authors based on pm10 daily exceedances and covid19 confirmed cases on march 12th thus before the italian sanitary crisis observed that pm10 concentration was exceeded only few times among the lombard cities that at the beginning of the epidemic were most affected on the contrary among some piedmont cities suffering of severe pm10 pollution events covid19 incidence was lower based on their results the authors concluded that covid19 diffusion by airborne pm10 is hard to demonstrate nevertheless several research revealed how pm in particular pm25 could had a role in accelerate and vast diffusion of covid19 in northern italy for example coccia 2020b by analyzed data on italian province capitals and data of infected individuals up to april 7th revealed a relationship between air pollution of cities measured with days exceeding the limits set for pm10 in previous years and covid19 diffusion in particular cities with more than days of pm10 exceedances showed a very high average number of infected individual about infected individuals on 7th april whereas cities having less than days of pm10 exceedances showed a lower average number of infected about infected individuals frontera gave also evidences on the role of pm25 as a contributing factor of covid19 outbreak in northern italy where environmentalresearch19120201101293 0cc copat table summary table reporting reviewed results on the association between covid19 casesdeaths and air pollution pm25 pm10 and no2 references zhu data analysis generalized additive model gam aim temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 spatial association between fatality rate and air pollution pm25 and pm10 spatial association between deaths counts and air pollution no2 temporal association between total cases daily confirmed cases and total deaths and air pollution pm25 and pm10 temporal association between total cases daily confirmed cases and total deaths and air pollution no2 spatial description of pm10 exceedances versus covid19 cases multivariate poisson regression simple linear regression multiple linear regression descriptive analysis percentage of deaths in three no2 μmol m2concentration range “ “ “ pearson coefficient correlation pearson coefficient correlation descriptive analysis number of days of pm10 exceeding μgm3 and covid19 incidence area of study cities of china period from jan 23rd to feb 29th jiang li yao ogen zoran 2020a zoran 2020b bontempi 2020b from jan 25th to feb 29th from jan 26th to feb 29th in data up to march 22nd data up to the end of feb from jan 1st to apr 30th from jan 1st to apr 30th from feb 10th to march 12th wuhan xiaogan and huanggang china wuhan and xiaogan cities of china administrative regions in italy spain france and germany milan italy milan italy provinces of lombardy italy provinces of piedmont italy coccia 2020b data up to april 7th italian provinces fattorini and regoli data up to april 27th italian provinces pm25 a 10μgm3 pm25 increase lag0“ was associated with a increase of daily confirmed new cases pm10 a 10μgm3 pm10 increase lag0“ was associated with a increase of daily confirmed new cases wuhan rr ci1032“ xiaogan rr ci “ huanggang rr ci “ wuhan r2 p xiaogan r2 p wuhan rr ci “ xiaogan rr ci “ huanggang rr ci “ wuhan r2 p xiaogan r2 p χ2 p a μgm3 increase in pm25 was associated with a “ increase in fatality rate χ2 p a μgm3 increase in pm10 was associated with a “ increase in fatality rate no2 a 10μgm3 no2 increase lag0“ was associated with a increase in daily confirmed new cases wuhan rr ci “ xiaogan rr ci “ huanggang no association found wuhan r2 p xiaogan r2 p of fatality cases are associated with no2 μmolm2 r cid0 r r cid0 r cid0 r r cid0 r cid0 r cid0 r cid0 lombardy pm10 exceeding between and covid19 incidence between and piedmont pm10 exceeding between and covid19 incidence between and covid19 in north italy has a high association with air pollution of cities measured with days exceeding the limits set for pm10 r2 p r2 p continued on next page hierarchical multiple regression model pearson regression coefficient analysis r2 p spatial association between confirmed cases and air pollution pm10 spatial association between total confirmed cases and air pollution pm25 pm10 and no2 environmentalresearch19120201101294 0cc copat table continued references frontera frontera wu adhikari and yin bashir bashir vasquezapestegui vasquezapestegui vasquezapestegui period data up to 31st march data up to 31st march data up to april 04th from march 1st to apr 20th from march 4th to april 24th from march 4th to april 24th data up to june 12th data up to june 12th data up to june 12th area of study italian regions italian regions counties in the usa queens county new york usa california california districts of lima perù districts of lima perù districts of lima perù aim spatial association between total confirmed cases and air pollution pm25 spatial association between deaths and air pollution pm25 prediction of risk of covid19 deaths in the long term average exposure to fine particulate matter pm25 temporal association between daily confirmed cases and total deaths and air pollution pm25 association between confirmed cases and air pollution pm25 pm10 and no2 association between deaths and air pollution pm25 pm10 and no2 spatial association between total confirmed cases and air pollution pm25 spatial association between deaths and air pollution pm25 spatial association between case fatality rate and air pollution pm25 data analysis pearson regression coefficient analysis pm25 r2 p pm10 pearson regression coefficient analysis r2 p longterm exposure increase of μgm3 in pm25 is associated with a increase in the covid19 death rate estimate on cases values cid0 ci “ estimate on deaths value cid0 ci “ kendall r cid0 spearman r cid0 zeroinflated negative binomia models negative binomial regression model spearman and kendall correlation tests spearman and kendall correlation tests no2 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 multivariate regression model crude coefficient p multivariate regression model crude coefficient p multivariate regression model crude coefficient cid0 p mortality was found significantly higher than less polluted italian regions by collecting data up to march 31st for all italian regions and performing a pearson correlation analysis they found a strong positive association both with the total number of confirmed cases r and deaths r other than with hospitalized cases r the italian situation was further highlighted by the study of fattorini and regoli in italian provinces they explored the spatial association between air pollution and covid19 cases with data up to april 27th by applying the pearson regression coefficient analysis they revealed a positive association both with pm25 and pm10 r2 p and r2 p respectively a focus on the most affected city of italy milan was conducted by zoran 2020a this city is located in the po valley basin known hotspot for atmospheric pollution at the continental scale eea the authors performed a temporal association between covid19 total cases daily new positive cases and total deaths and particulate matter from jan 1st and apr 30th by applying a person correlation in accordance with other studied they found a positive association between daily confirmed cases and pm25 r and pm10 r although they did not consider any delay time from infection to covid19 onset nevertheless they found a negative association between total cases and total deaths and particulate matter but the assumption of a temporal linear correlation may be inaccurate because the above mentioned variables could have more complex nonlinear relationships to date the usa have more than million confirmed cases and thousand deaths who here ambient concentrations of pm and o3 were found responsible to cause between and premature deaths fann the association between air pollutants and covid19 cases and deaths was studied by bashir in the state of california from march 4th to april 24th corresponding to the beginning of the covid19 outbreak in usa based on their significant correlation found the authors state that a limited human exposure to these pollutants will contribute to defeating covid19 this conclusion seems unclear because they found a negative correlation with pm25 and pm10 environmentalresearch19120201101295 0cc copat by applying both the kendall rank correlation and spearman™s one and it is not clear if they normalized covid19 cases by population size and if they performed a day by day association or a spatial association across the country a focus on the queen county new york usa was provided by adhikari and yin they retrieved data of pm daily concentrations from two ground monitoring stations and collected data of confirmed covid19 cases and numbers of related deaths from usafacts in the period from march to april the authors elaborated their data with a negative binomial regression model and considered the cumulative lag effect of pm25 on disease outcomes over the past days they found a significant negative association among pm25 and new daily confirmed covid19 cases cid0 ci “ and deaths cid0 ci “ low pm concentrations in this area of study mean μgm3 are likely to have played a less central role in the spread of infection than in other areas such as italy where pm25 monthly concentrations reached values higher than μgm3 fattorini and regoli frontera or in china where pm25 monthly concentrations reached values higher than μgm3 zhu jiang as said by the authors other gaseous pollutants such as no2 and so2 could have influenced transmission and pathogenesis of covid19 in the united states wu investigated whether longterm average exposure to fine particulate matter pm25 increases the risk of covid19 deaths by considering approximately counties in the united states of the population with an exposure prediction model the authors calculated the county level longterm exposure to pm25 averaged for to and collected covid19 deaths counts up to april 04th they conducted a strong and robust statistical analysis with zeroinflated negative binomial mixed models adjusting their results by several potential confounders such as sociodemographic socioeconomic behavioural and meteorological factors they found that a small longterm exposure increase of only μgm3 in pm25 is associated with a increase in the covid19 death rate confidence interval ci vasquezapestegui recently reported first evidences on the spatial relationship between particulate matter and covid19 outbreak from latin america the authors described the situation occurred in districts of lima located in the second most affected country of latin america peru in particular by applying a multivariate regression model they evaluated the association between the population exposure to pm25 concentrations in the previous years “ and cases deaths and casefatality rates of covid19 with data up to june 12th a significant association has been found both with cases and deaths crude coefficient with p and with p respectively but not with case fatality rate all these studies highlight the role of pm in triggers of the covid19 disease and how government measures targeting to sustainable growth such as the reduction of urban and industrial emissions could have a positive impact on the prevention of health outcomes reducing mortality rate as well the burden on health care systems nitrogen dioxide no2 and covid19 induced lung damage hence viral infection becomes more common after exposure to no2 zhu furthermore no2 is associated with other several health effects such as elevated risks for asthma allergic rhinitis and eczema in children to increase of outpatient visits and hospitalizations due to bronchitis and asthma exacerbation bahrami asl kowalska increase of chronic obstructive pulmonary disease copd ghanbari ghozikali pfeffer and increase of pulmonary heart disease related mortality chen a recent study explored the possible role of no2 in interference in angiotensin converting enzyme ace2 the expression of ace2 is high on lung alveolar epithelial cells and it is the human cell receptor of virus agent of covid19 alifano first observations report an association between ambient concentrations of no2 and covid19 pandemic across europe china and usa bashir fattorini and regoli jiang li et al ogen zhu et al zoran et al 2020b conversely to the other papers findings of zoran 2020b and bashir provides different findings reporting no association or a negative one between no2 and daily deaths counts in china zhu by applying the same method explained for pm observed that a 10μgm3 increase lag0“ in no2 is associated with a ci “ increase in the daily counts of covid19 confirmed cases in cities of china these findings are confirmed by jiang and li et a who applied the same method described for pm jiang revealed a significant positive association between no2 and covid19 both in wuhan and xiaogan wuhan rr ci1053“ xiaogan rr ci “ but did not found any significant association in huanggang li found a significant linear correlation both in wuhan r2 p and xiaogan r2 p ogen presented evidences on the relationship between exposure to no2 including the months of january and february shortly before the covid19 spread in europe and novel coronavirus fatality in the most affected european countries concluding that longterm exposure to no2 may be a potential contributor to mortality caused by sarscov2 he collected data concerning the number of fatality cases from administrative regions in italy spain france and germany and correlated mortality with tropospheric no2 concentrations measured by the sentinel5 precursor spaceborne satellite the major tropospheric no2 hotspot identified was located in the northern italy in all european regions considered gas concentrations ranged between and μmolm2 with airflows directed downwards results showed that out of the fatality cases by march were in five regions located in north italy and central spain furthermore by analysing mortality trends it was revealed that the highest percentage of deaths occurred in regions where the maximum no2 concentration was above μmolm2 with a significant decrease where the maximum concentration was between and μmolm2 and below μmolm2 the methodology used by ogen cannot support a longterm exposure investigation surely a validation of the satellite measure with those of the ground ™ones the adjustment of the results according to the different population size of each country could have made their results more robust nevertheless the study provide new insights for future investigation the italian situation was further studied by fattorini and regoli who collected data of covid19 incidence up to april 27th from italian provinces they revealed a strong spatial correlation with no2 mean levels concentrations “ pearson coefficient r2 p confirming the northern italy being a hotspot of no2 in addition to urbanized cities of central and southern italy such as rome and naples a focus on the temporal association between ground levels of no2 and covd19 cases total cases daily new positive cases and total deaths was performed by zoran 2020b for the city of milan italy in the period pre and postlockdown measures the authors nitrogen dioxide is a nastysmelling gas formed by reaction in the atmosphere of nitrogen oxides nox with other chemicals nox is naturally produced in atmosphere by lightning kang et al volcanoes oceans and biological decay thurston the major outdoor anthropogenic sources of nox are primarily emissions from transportation and fuel combustion in particular in urban areas they comes from vehicle exhaust gases and domestic heating grange maawa the nitrogen dioxide has mainly effect on the respiratory system because an increase of the outdoor concentration of no2 may significantly increase the risk of respiratory tract infection this phenomenon is particularly evident in children as they are more susceptible to no2 environmentalresearch19120201101296 0cacknowledgments c copat found no2 negative correlated with all the considered epidemiological data but the methodology used has some limitations as the delay time from infection to the covid19 onset or covid19 death was not considered as well the significant reduction of air pollution due to lockdown measures since midmarch in usa the association was also studied by bashir for the state of california as discussed above for pm the authors found a negative correlation also between no2 levels and covid19 cases and mortality nevertheless they stated that this pollutant contributes to the spread of the disease based on these scientific evidences in addition to confirming that exposure to no2 is harmful to human health and increases the risk of incurring respiratory diseases it can be stated that exposure to no2 may be one of the most important trigger for the spread and fatality caused by the covid19 disease declare references adhikari a yin j shortterm effects of ambient ozone pm25 and the authors declare no conflict of interest we have no funding to bontempi e 2020b first data analysis about possible covid19 virus airborne alifano m alifano p fez p iannelli a reninangiotensin system at the meteorological factors on covid19 confirmed cases and deaths in queens new york int j environ res publ health httpsdoi103390 ijerph17114047 heart of covid19 pandemic biochimie httpsdoi101016j biochi202004008 baccini m biggeri a grillo p consonni d bertazzi pa health impact assessment of fine p pollution at the regional level am j epidemiol “ httpsdoi101093ajekwr256 bahrami asl f leili m vaziri y salahshour arian s cristaldi a oliveri conti g ferrante m health impacts quantification of ambient air pollutants using airq model approach in hamadan iran environ res “ httpsdoi 101016jenvres201710050 bashir mf ma bj bilal komal b bashir ma farooq th iqbal n bashir m correlation between environmental pollution indicators and covid19 pandemic a brief study in californian context environ res https doi101016jenvres2020109652 becker s soukup jm exposure to urban air particulates alters the macrophage mediated inflammatory response to respiratory viral infection j toxicol environ health “ httpsdoi101080009841099157539 bontempi e 2020a commercial exchanges instead of air pollution as possible origin of covid19 initial diffusion phase in italy more efforts are necessary to address interdisciplinary research environ res httpsdoi101016j envres2020109775 diffusion due to air particulate matter pm the case of lombardy italy environ res httpsdoi101016jenvres2020109639 bontempi e vergalli s squazzoni f understanding covid19 diffusion requires an interdisciplinary multidimensional approach environ res httpsdoi101016jenvres2020109814 bremner sa anderson hr atkinson rw mcmichael aj strachan dp bland j m bower js shortterm associations between outdoor air pollution and mortality in london occup environ med “ httpsdoi 101136oem564237 cai qc lu j xu qf guo q xu dz sun qw yang h zhao gm jiang qw influence of meteorological factors and air pollution on the outbreak of severe acute respiratory syndrome publ health “ https doi101016jpuhe200609023 carugno m dentali f mathieu g fontanella a mariani j bordini l milani g p consonni d bonzini m bollati v pesatori ac pm10 exposure is associated with increased hospitalizations for respiratory syncytial virus bronchiolitis among infants in lombardy italy environ res “ https doi101016jenvres201806016 chen h chen y lian z wen l sun b wang p li x liu q yu x lu y qi y zhao s zhang l yi x liu f pan g 2020a correlation between the migration scale index and the number of new confirmed coronavirus disease cases in china epidemiol infect e99 httpsdoi101017 s0950268820001119 chen j zeng j shi c liu r lu r mao s zhang l associations between shortterm exposure to gaseous pollutants and pulmonary heart diseaserelated mortality among elderly people in chengdu china environ health httpsdoi 101186s1294001905008 chen s prettner k kuhn m geldsetzer p wang c b¨arnighausen t bloom de 2020b covid19 and climate global evidence from countries medrxiv prepr serv health sci httpsdoi1011012020060420121863 coccia m 2020a how high wind speed can reduce negative effects of confirmed cases and total deaths of covid19 infection in society ssrn scholarly paper no id social science research network rochester ny httpsdoi 102139ssrn3603380 coccia m 2020b factors determining the diffusion of covid19 and suggested strategy to prevent future accelerated viral infectivity similar to covid sci total environ httpsdoi101016jscitotenv2020138474 balakrishnan k brunekreef b dandona l dandona r feigin v freedman g hubbell b jobling a kan h knibbs l liu y martin r morawska l pope ca shin h straif k shaddick g thomas m van dingenen r van donkelaar a vos t murray cjl forouzanfar mh estimates and year trends of the global burden of disease attributable to ambient air pollution an analysis of data from the global burden of diseases study lancet lond engl httpsdoi101016s0140673617305056 “ conticini e frediani b caro d can atmospheric pollution be considered a co factor in extremely high level of sarscov2 lethality in northern italy environ pollut barking essex httpsdoi101016jenvpol2020114465 croft dp zhang w lin s thurston sw hopke pk van wijngaarden e squizzato s masiol m utell mj rich dq associations between source cohen aj brauer m burnett r anderson hr frostad j estep k conclusion the scientific evidences collected in the literature highlight the important contribution of chronic exposure to air pollution on the covid19 spread and lethality although the potential effect of airborne virus exposure it has not been still demonstrated in particular it seems that pm25 and no2 are more closely correlated to covid19 than pm10 the lower correlation of pm10 with covid19 incidence and mortality can be due to the impossibility of particulate matter greater than μm to reach type ii alveolar cells where is located the cell entry receptor ace2 for sarscov2 nevertheless differences between countries such as the implementation of different lockdown restrictions stage of infection topographic sociodemographic and socioeconomic characteristics level of air pollution and meteorological factors may have contributed to obtain some contrasting finding although most of the revised studies support the relationship between air pollution and covid19 the manifold limitations of this review are the small number of papers collected and the great diversity of methodologies used sometimes lacking in some parts which makes the results difficult to compare the authors who first investigated this association although with great effort and rapidity of analysis dictated by a global emergency sometimes do not include all confounding factors whenever possible such as control policy urbanization rate availability of medical resources population size weather lifestyles sociodemographic and socioeconomic variables in addition to date incidence data are underestimated in all countries and to a lesser extent mortality data for this reason the cases included in the considered studies cannot be considered conclusive more studies are needed to better clarify the role of air pollution during the covid19 pandemic particularly studies that consider the multiplepollutants to strengthen scientific evidences and support firm conclusions useful to implement pandemic application plans to adequately prevent new health emergencies for a long time we have known that reducing outdoor and indoor air pollution in cities or countries can have a significant effect on health almost immediately and the benefits can far outweigh the costs surely the health emergency that the world is experiencing right now highlights how environmental research is a fundamental reference point to improve the knowledge concerning diseases of infectious origin and how all the intellectual and economic resources are to be spent to accelerate actions aimed to implement environmental policies act to reduce air pollution and develop new urban planning interventions influences or multidisciplinary studies declaration of competing interest the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper environmentalresearch19120201101297 0cc cop
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"The 24-week metrics (albeit with higher c-index point estimate) were not meaningfully better than the 12-week metrics. None of the metrics did particularly well for breast cancer. Conclusion Alternative cut points to RECIST standards provided no meaningful improvement in OS prediction. Metrics assessed at 12 weeks have good predictive performance. J Thorac Oncol J Thorac Oncol JTO Journal of Thoracic Oncology 1556-0864 1556-1380 Lippincott Williams & Wilkins 24787965 4132045 00005 10.1097/JTO.0000000000000157 Original s Translational Oncology A Comparison of Immunohistochemical Assays and FISH in Detecting the ALK Translocation in Diagnostic Histological and Cytological Lung Tumor Material Le Quesne John MA (Cantab) PhD MBBS FRCPath * Maurya Manisha PhD   Yancheva Slaveya G. FRCPath * O™Brien Mary MD ¡ Popat Sanjay FRCP PhD ¡ Wotherspoon Andrew C. MBBCh FRCPath § de Castro David Gonzalez PhD FRCPath   Nicholson Andrew G. MBBS DM FRCPath * *Department of Histopathology Royal Brompton and Harefield NHS Foundation Trust London;  Centre for Molecular Pathology The Royal Marsden Hospital Sutton Surrey; ¡Department of Oncology The Royal Marsden Hospital; and §Department of Histopathology Royal Marsden Hospital Chelsea London United Kingdom. Address for correspondence address: Andrew G. Nicholson MBBS DM FRCPath Department of Histopathology Royal Brompton Hospital Sydney St London SW3 6NP United Kingdom. E-mail: a.nicholsonrbht.nhs.uk. 6 2014 30 5 2014 9 6 769 774 Copyright 2014 by the International Association for the Study of Lung Cancer 2014 This is an open-access distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivitives 3.0 License where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially. Introduction: Detection of the ALK rearrangement in a solid tumor gives these patients the option of crizotinib as an oral form of anticancer treatment. The current test of choice is fluorescence in situ hybridization (FISH) but various cheaper and more convenient immunohistochemical (IHC) assays have been proposed as alternatives. Methods: Fifteen FISH-positive cases from patients seven with data on crizotinib therapy and clinical response were evaluated for the presence of ALK protein using three different commercially available antibodies: D5F3 using the proprietary automated system (Ventana) ALK1 (Dako) and 5A4 (Abcam). A further 14 FISH-negative and three uncertain (<15% rearrangement detected) cases were also retrieved. Of the total 32 specimens 17 were excisions and 15 were computed tomography-guided biopsies or cytological specimens. All three antibodies were applied to all cases. Antibodies were semiquantitatively scored on intensity and the proportion of malignant cells stained was documented. Cutoffs were set by receiver operating curve analysis for positivity to optimize correct classification. Results: All three IHC assays were 100% specific but sensitivity did vary: D5F3 86% ALK 79% 5A4 71%. Intensity was the most discriminating measure overall with a combination of proportion and intensity not improving the test. No FISH-negative IHC-positive cases were seen. Two FISH-positive cases were negative with all three IHC assays. One of these had been treated with crizotinib and had failed to show clinical response. The other harbored a second driving mutation in the EGFR gene. Conclusions: IHC with all three antibodies is especially highly specific (100%) although variably sensitive (71%-86%) specifically in cases with scanty material. D5F3 assay was most sensitive in these latter cases. Occasional cases are IHC-positive but FISH-negative suggesting either inaccuracy of one assay or occasional tumors with ALK rearrangement that do not express high levels of ALK protein. Pulmonary adenocarcinoma ALK Immunohistochemistry Fluorescence in situ hybridization Crizotinib OPEN-ACCESS TRUE Rearrangements of the anaplastic lymphoma kinase (ALK) gene drive the malignant phenotype in 3% to 7% of primary lung adenocarcinomas.1“5 The resulting fusion protein most often a fusion with echinoderm microtubule-associated protein-like 4 (ELM4) has a constitutively active tyrosine kinase domain. The small molecule drug crizotinib is a specific inhibitor of this kinase6 and cases with the rearrangement respond to crizotinib treatment.7 Therefore accurate rapid and inexpensive identification of tumors growing under the influence of translocated ALK is needed. Currently the only test approved by the FDA is fluorescence in situ hybridization (FISH) using œbreak-apart probes (Vysis Abbott Molecular Abbott Park IL). This test is regarded as the œgold standard for detection of re-arrangements and is recommended by CAP/International Association for the Study of Lung Cancer/AMP.8 However FISH is technically demanding expensive and many diagnostic laboratories lack either the expertise or the facilities to perform the test. Even in ideal circumstances the results are often difficult to interpret requiring the scrutiny of large numbers of individual cells by a highly experienced diagnostician. Furthermore there are rare circumstances (such as small intrachromosomal inversion) in which the FISH test is negative but the tumor nevertheless expresses EML4-ALK fusion protein.59“11 A cheaper and potentially more widely applicable method is immunohistochemistry (IHC); indeed overexpression of ALK protein has been used in the diagnosis of anaplastic large-cell lymphoma for many years. Although early studies in lung cancer lacked sensitivity45 more recent studies have shown greater specificity and sensitivity8“11 and recent international guidelines (CAP/International Association for the Study of Lung Cancer/AMP) have recommended that if clinically validated IHC may be used as a screening test for FISH testing.8 However there have been few comparative studies on the most appropriate antibody to use. The aim of this study was therefore to compare three different immunohistochemical assays two being routine methods using antibodies widely used in the diagnosis of lymphoma with the third being a proprietary system including signal amplification that is currently being promoted as an alternative to FISH (Ventana). We also evaluated the relationships between ALK rearrangement as detected by FISH IHC and patient response to therapy. MATERIALS AND METHODS Clinical Samples The diagnostic archives from the Royal Brompton and Harefield NHS Foundation Trust and Royal Marsden hospitals from 2007 onwards were reviewed to identify cases with a diagnosis of lung adenocarcinoma that tested positive for an ALK rearrangement (>15% positive cells) and a randomly selected complementary group of cases with a normal ALK locus for comparison. We had been testing all primary lung tumors regardless of stage as part of a feasibility study which led to a large number of early stage cases being included. More recently our current policy is only to test advanced cases of non-squamous non“small-cell carcinoma using IHC screening with confirmatory FISH as per recently published guidelines.8 The cases under study are summarized in . TABLE 1. Summary Data of All Cases Included in the Study Paraffin blocks from a total of 32 diagnostic cases were retrieved; 15 of these had tested positive for the ALK rearrangement by FISH three were uncertain (with <15% of cells showing rearrangement) and the remaining 14 cases were negative. All but two blocks dated from 2011 or later. Seventeen cases were blocks from tumor excisions (six of these were FISH positive) and the remainder were cytological or core biopsy/endobronchial ultrasound samples. Data on treatment with crizotinib and response were retrieved from patient records. Cases with at least partial response to treatment defined according to the Response Evaluation Criteria in Solid Tumors criteria12 (i.e. at least 30% decrease in the sum of the longest diameters of target lesions) were designated as œresponsive. The study was evaluated and classified as a service evaluation by the Imperial College Heads of Consortia and as such was exempt from Research Ethics Committee review. Fluorescent In Situ Hybridization Unstained 2 ?m FFPE sections were put through deparaffinization and protease pretreatment steps before being denatured and hybridized overnight with the commercially available Vysis ALK dual color break apart probe (Abbott Molecular). Tissue sections then underwent SSC washes and were mounted in 4'6-diamidino-2-phenylindole for nuclei counterstaining. Results were analyzed and interpreted in accordance with probe manufacturer™s instructions. Non-rearranged ALK showed as fused (yellow) signals. Rearranged ALK appeared as split 3? (red) and 5? (green) signals or an isolated 3? (red) signal. The recommended cutoff of 15% was used to interpret samples as positive or negative for ALK rearrangements in 200 nuclei. Immunohistochemistry An additional five sections were cut per case. Three were used for the immunohistochemical assays and the remaining two for negative controls. Immunohistochemical assays were optimized using the monoclonal antibodies D5F3 (Ventana) ALK1 (Dako) and 5A4 (Abcam). The D5F3 assay was performed using the Ventana autostainer and a tyramide amplification step as specified in the manufacturer™s protocol. The other assays were performed using a Dako autostainer with conventional polymer-based diaminobenzidine staining (no tyramide amplification). Details of the antibodies and conditions employed are given in Table 2. TABLE 2. Immunohistochemical Assay Conditions Used Scoring Immunohistochemically stained sections were examined without knowledge of FISH status by two pathologists independently. Scores for proportion and intensity of immunohistochemical staining were assigned by consensus. The predominant intensity of staining was recorded on a scale of 0“3 (0 = negative 1 = weak 2 = moderate 3 = strong). As the Ventana stain was more intense due at least partly to the signal amplification step the visual cutoffs for intensity scoring with this antibody were different (e.g. a œmoderate degree of intensity seen with the Ventana stain would usually be interpreted as œstrong on a section stained with 5A4). The proportion of malignant cells staining positive was recorded as per œAllred estrogen receptor scoring in breast cancer on a scale of 0“5 (0 = 0% 1 ? 1% 2 = 1“10% 3 = 11“33% 4 = 34“66% and 5 ? 66%). A composite score (intensity + proportion) was also derived. Statistical Analysis Statistical analyses were performed using the STATA/IC package. RESULTS Fluorescence In Situ Hybridization Slides were scored according to the manufacturer™s recommendations. Representative FISH images are shown in Figure 1A. The 15 positive cases all showed greater than 15% cells with rearranged ALK genes. Three cases were classified as œindeterminate; these were all scanty biopsy or cytological samples with 10% to 15% of positively rearranged FISH signals. Seventeen further cases were FISH negative. FIGURE 1. (A) Representative fluorescence in situ hybridization images showing normal fused signals (neg) and nuclei with multiple separated red signals (pos). (B) Three representative excision specimens of adenocarcinoma. Case 1 is negative with all three immunohistochemical assays; the D5F3 assay shows relatively high background presumably because of the tyramide signal amplification (TSA) step. Cases 2 and 3 are positive with all three immunohistochemical assays with clear cytoplasmic staining. The markedly reduced signal seen with the 5A4 and ALK1 assays in case 2 was typical and again probably related to the absence of tyramide amplification. Case 3 demonstrates that occasional cases show strong staining using the non-TSA assays. Immunohistochemistry No signal was observed in negative controls. The intensity of staining between the three antibodies varied (Fig. 1B). IHC was impossible to assess in three cases with very scanty material (two FISH negative and one FISH positive). "
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" microwave ablation mwa is widely used to treat unresectable primary and secondary malignanciesof the liver and a limited number of studies indicate that ablation can cause not only necrosis at the in situ site butalso an immunoreaction of the whole body this study aimed to investigate the effects of mwa on cytokines inpatients who underwent mwa for a hepatic malignancymethods patients admitted to the oncology department in the first affiliated hospital of soochow universitybetween june and february were selected peripheral blood was collected from patients with a hepaticmalignancy treated with mwa the levels of cytokines il2 ifnÎ tnfα il12 p40 il12 p70 il4 il6 il8 il10and vascular endothelial growth factor vegf were detected with a milliplex® map kit the comparison times wereas follows before ablation h after ablation days after ablation and days after ablation data were analyzedusing a paired sample ttests and spearman™s correlation analysisresults a total of patients with hepatic malignancies were assessed there were significant differences in il2il12 p40 il12 p70 il1 il8 and tnfα at h after mwa significant increases 2fold vs before ablation wereobserved in il2 il1 il6 il8 il10 and tnfα after mwa elevated il2 and il6 levels after ablation werepositively correlated with energy output during the mwa procedures wa treatment for hepatic malignancies can alter the serum levels of several cytokines such as il2 and il6keywords microwave ablation hepatic malignancy cytokines il2 il6 immunoregulation primary and secondary malignancies of the liver have asubstantial impact on morbidity and mortality worldwidein china hepatocellular carcinoma hcc has the secondhighest mortality rate of malignancies the treatmentof primary and secondary hepatic malignancies via correspondence lengbengsudaeducn jing zhao qiang li and merlin muktiali contributed equally to this work2department of oncology the first affiliated hospital of soochow universitysuzhou china5division of neurosurgery city of hope beckman research institute duartecalifornia usafull list of author information is available at the end of the interventional imaging therapy is undertaken by investigators in the field of interventional radiology and possibly bya smaller group of practitioners known as interventionaloncologists whose major focus is cancer care via minimally invasive approaches [ ] recently percutaneous ablation therapy has been widely accepted as a radicaltreatment method for hcc and its fiveyear survival rateis similar to that of resection microwave ablationmwa is widely used to treat unresectable hcc and recurrent hcc and has the advantages of minimal invasiona good curative effect and no side effects due to radiationor chemotherapy immune checkpoint inhibitors icis the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhao bmc cancer page of such as pd1pdl1 and ctla4 antibodies have beenwidely applied in several cancers and studies have indicated that ici treatment could enhance the effect of ablation evidence hasindicated that hyperthermicdestruction causes the release of a large population of heterogeneous tumor antigens and inflammatory cytokinesmay play crucial roles in this process cytokines aremediators that regulate a broad range of processes involved in the pathogenesis of cancer several cytokineswhich can arise from either tumor cells or immunocytes such as tumor necrosis factor tnfα interleukinil1 il6 il8 il10 and vascular endothelial growthfactor vegf have been linked with cancers and can either promote or inhibit tumor development the serumlevels of cytokines differ during cancer development although cytokines have been found to be altered after anticancer treatment such as chemotherapy and radiotherapy[ ] few investigations have focused on cytokines beforeand after mwa it is still unknown whether the above cytokines changed before andor after mwa in patientswith hepatic malignancies in this study we investigatedthe effects of mwa on the serum levels of cytokines inpatients with hepatic malignanciesmethodspatients and samplesthe patient population examined in this study was derivedfrom the first affiliated hospital of soochow universitypatients were admitted to the oncology department between june and february the total number ofpatients was with liver metastases and primaryliver cancers the inclusion criterion was a tumor locatedat a hepatic site either primary or metastases all patients with metastatic hepatic malignances should be givensystematic treatments chemotherapy or target therapyand get at least stable disease sd or partial responsepr for more than days informed consent for blooddraw and the relevant therapy was obtained from all patients the protocol was approved by the human ethicscommittee of the first affiliated hospital of soochowuniversity and was conducted in accordance with thedeclaration of helsinki all written informed consent wasobtained from all participants and clearly stated wholeblood ml was drawn into edta anticoagulant tubeson days ˆ’ to before and h days and days afterablation mostly on the last day of the course for cytometry and cytokine analysesablation procedurethe ablation procedure used in this research was mwathe puncture site and pathway were determined underthe guidance of a computed tomography ct scanlocal infiltration anesthesia was achieved by using lidocaine the placement of microwave ablation probeswas guided by a ct scan or ultrasonic device and allprobes were placed at the maximum diameter layerdouble probes were employed when the maximumdiameter of the tumor was up to cm the power andtime of ablation were designed for each patient in therange of w and min respectively basedon the size number and position of the tumor theboundaries of ablation zones were designed as extended cm upon the tumor sitecytokine detectiona milliplex map kit with human cytokinechemokinepanels that measured ifnÎ il2 il6 il8 il10 il12p40 il12 p70 il1 tnfα and vegf was utilized according to the manufacturer™s instructions briefly chemically dyed antibodybound beads were mixed withstandard or sample incubated overnight at °c washedand then incubated with a biotinylated detection antibodyafter the beads were washed they were incubated with astreptavidin phycoerythrin complex and the mean fluorescent intensities were quantified on a luminex analyzer luminex corporation all samples were measured in duplicate standard curves of known concentrations of recombinant human cytokineschemokines wereused to convert fluorescence units to cytokine concentration units pgml the minimum detectable concentrations were as follows ifnÎ pgml il2 pgmlil12 p40 pgml il12 p70 pgml il1 pgml il6 pgml il8 pgml il10 pgml tnfα pgml and vegf pgml all resultsbelow the minimum concentrations were processed as theminimum concentrationsstatistical analysisibm spss statistics software was used for the statistical analysis along with graphpad prism for figurecreations normally distributed numerical data areexpressed as the mean ± standard deviation and nonnormally distributed numerical data are expressed as themedian and confidence interval ci cytokinesat different times were compared using a onetailedpaired ttest spearman™s correlation analysis was executed to determine the correlation between clinical indexes and cytokine levels p indicates a significantdifferenceresultsclinical characteristics of the enrolled patientsas shown in table a total of patients with tumorslocated on the liver liver metastases primary livercancers were analyzed the patients™ cytokine levelswere compared according to time before treatment h after treatment days after treatment and daysafter treatment 0czhao bmc cancer page of table clinical characteristics of the patients enrolled n characteristicsexmalefemaleagepathogenesisprimarysecondaryprimary site for metastatic hepatic malignancescolon rectalpancreasstomachebreastothersmaximum tumor length mmablation probe usedablation time minaverage power per probe w ± ± ± ± average energy time × power time × power–¼–¼ time and power indicate the time and power respectively ofdifferent probes used during the operation ± ifnÎ il12 p40 and il12 p70 were slightly increasedafter mwa treatmentas shown in table and fig the median level ofifnÎ before the mwa treatment was pgml ci “ pgml at days and days after themwa treatment there was a slight increase comparedto that premwa with median levels of pgml ci “ pgml and pgml ci“ pgml respectively the median level of il p40 before the mwa treatment was pgml ci “ pgml there was a slight increase to pgml ci “ pgml days postmwathe median il12 p70 level before the mwa treatmentwas pgml ci “ pgml and increasedto pgml ci “ pgml days afterthe mwa treatment and to pgml ci “ pgml days postmwa no significant alteration in the vegf median level was detected after themwa treatmentil2 il1 il6 il8 and il10 were elevated over 2foldafter the mwa treatmentas shown in table fig and fig the median levelof il2 before the mwa treatment was pgml ci “ pgml there was a significant increase at h postmwa with a median level of pgml ci “ pgml the median level ofil1 before the mwa treatment was pgml ci “ pgml and a significantincrease wasnoted days after the mwa treatment pgml ci “ pgml the median level of il6before the mwa treatment was pgml ci“ pgml and significantly increased daysafter the mwa treatment pgml ci “ pgml the median level ofil8 before themwa treatment was pgml ci “ pgml and increased significantly to pgml ci“ pgml days after the mwa treatmentthe median level of il10 before the mwa treatmentwas pgml ci “ pgml and increasedsignificantly days after the mwa treatment pgml ci “ pgml the median level oftnfα before the mwa treatment was pgml ci “ pgml and increased significantlyto pgml ci “ pgml days afterthe mwa treatmentlevelselevated il2 and il6 levels after ablation were positivelycorrelated with energy output during mwato further evaluate the relationship between the increased cytokineand mwa treatment weemployed the concept of œenergy time × power time × power time and power indicated thetime and power of different probes used in the operation to reflect total hyperthermic damage to hepatictissues during the mwa procedure as shown in table and fig the il2 levels at h postmwa and the il levels at days postmwa illustrated significant correlations with energy the relative indexes were and respectivelydiscussionas technology continues to develop other types of localtherapy such as radiotherapy chemical ablation andhyperthermal ablation for primary and metastatic livercancer are increasingly being used mwa for liver malignances is reserved for patients who cannot undergosurgical removal or for whom other treatments havefailed a consensus guideline was recently developed to address indications for mwa in these patientsthermal ablation is a process that heats the target tissueto a temperature that causes immediate coagulative necrosis usually over °c terminal treatment requiresthat a necrotic area surrounds the target site with anadditional “10mm margins however in the liverhigh tissue perfusion and large blood vessels can cause aœheat sink effect around the ablation zone making itdifficult to achieve terminal ablation the heat sink 0czhao bmc cancer page of table median levels of cytokines before and after mwacytokineifnÎil2premwa pgml ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ h postmwa pgml ci “ ci “ –¼ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “il12 p40il12 p70il1il6il8il10tnfαvegf p vs premwa –¼ 2fold vs premwa days postmwa pgml ci “ ci “ ci “ ci “ ci “ –¼ ci “ –¼ ci “ –¼ ci “ –¼ ci “ –¼ ci “ days postmwa pgml ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “effect can lead to sublethal temperatures and the retention of malignant cells thereby increasing the likelihoodof local tumor progression ltp however an incompletely ablated zone containing immune cells andcancer cells as well as functional vessels could establisha serious inflammatory site that may provide tumorspecific antigens cytokines and activated immune cellsin our study significant increases in the secretion ofchemokines il8 proinflammatory cytokines il1il12 ifnÎ and tnfα and antiinflammatory cytokines il10 were observed after mwa il8 is mainlyproduced by macrophages the classical biological activity of il8 is to attract and activate neutrophils whichcan lead to a local inflammatory response however recent studies have indicated that il8 both macrophageand cancer cellderived can recruit myeloidderivedsuppressor cells mdscs into the tumor microenvironment eventually inhibiting antitumor immunity andpromoting cancer progression [ ] il1 is mainlyproduced by macrophages b cells and nk cells couldproduce il1 under certain circumstances generallycells can only synthesize and secrete il1 after beingstimulated by foreign antigens or mitogens il1 couldpromote the th1 response promoting the activation ofdendritic cells dcs and cytotoxic t lymphocytesctls il12 is mainly produced by b cells and macrophages human il12 is a heterodimer with two subunits p40 kd and p35 kd which areinactivated in isolated form in general il12 functionsas a combination of two subunits il12 p70 while p40alone possesses partial functions of il12 p70 it™s mentionable that il12 p40 and p35 are not expressed inequal proportions so the amounts of il12 p40 and il p70 are different in one cell il12 can stimulate theproliferation of activated t cells and promote the differentiation of th0 cells into th1 cells moreover il12could induce the cytotoxic activity of ctls and nk cellsand promote the secretion of several cytokines such asifnÎ and tnfα previous research indicatedthat tnfα may play a crucial role in mwa in combination with immunotherapy notably our data illustrated that the il12 results were consistent with thoseof ifnÎ after the ablation operation but not with thoseof tnfα this result indicated that upregulation ofifnÎ may be a major effect of the il12 increase aftermwa on the other handan antiinflammatory and immunosuppressive cytokine wasevaluated after mwa il10 is a multicellularderivedmultifunctional cytokine that regulates cell growth anddifferentiation and could participate in inflammatoryand immune responses il10 was reported to increaseafter thermal ablation in the literature [ ] strategiesto inhibit il10induced immunosuppression after thermal ablation treatment would be of interestil10asablation therapy can mediate antitumor immunity astumor tissue necrosis caused by ablation may release various antigens that eventually form a kind of œin situ vaccination moreover ablative therapy can not onlydirectly kill cancer cells in situ but also regulate immunecells and promote the immune function of patients withliver cancer [ ] many immunoregulatory cytokineswere released or expressed after thermal ablation notablythe cytokines released after thermal ablation can regulatethe positive and negative aspects of the cancer immunecycle previously researchers demonstrated that proinflammatory cytokines such as il1 il6 il8 il18 andtnfα were increased several hours or days after thermalablation [ ] to our knowledge terminal tumorthermal ablation may not only cause local heat injury intissues surrounding the tumor site but also induce a systemic reaction this systemic reaction would becaused by different mechanisms first interventional operation may result in trauma to the liver although this procedure is very minimally invasive the healing process maycause alteration of some cytokines second heat injurycould cause acute thermal necrosis in liver and tumor 0czhao bmc cancer page of fig levels of cytokines before and after mwa treatment slightly increased ifnÎ il12 p40 and il12 p70 levels after mwa treatment over fold enhancement of il2 h postmwa and of il1 il6 il8 il10 and tnfα d postmwa p 0czhao bmc cancer page of fig trends in cytokines significantly altered after mwa treatment the levels of il2 at h postmwa il1 at d postmwa il6 at dpostmwa il8 at d postmwa and il10 at d postmwa were elevated over 2fold compared to the levels premwatable correlation between the ablation energy and significantly elevated cytokinesenergyvsil2 h postmwaenergyvsil1 d postmwaˆ’energyvsil6 d postmwaenergyvsil8 d postmwaenergyvsil10 d postmwaenergyvstnfα d postmwaspearman™s rp value onetailed p 0czhao bmc cancer page of fig correlation between the ablation energy and the serum levels of il2 and il6 the serum levels of il2 at h postmwa and il6 at dpostmwa were positively correlated with energy output during the mwa procedureand nonspecifictissues and release of necrotic tissue fragments into bloodcould cause immunological reactions including nonspecific and specific reactions generally cytokines affectedby wound healingimmunologicalreactions do not last longer than those affected by specificimmunologicalreactions ablation treatmentinducedspecific immunological reactions are more complicatedand could affect more immunocytes [ ] which wouldmake this process last longer than other reactions theseexplanations may be the reason why the cytokine changeslasted different durations moreover cytokines affected bythe second manner would be positively correlated withthe ablation scale which is why we employed the œenergyindex in our ablation operation design to receive a terminal ablation larger tumor would cost higher energy including higher power and longer duration time terminaltumorthermal ablation would release tumorrelatedneoantigen to blood circulation eventually induce a systemic reaction this reaction is dependent on the scale ofthermal injury and the local immunological microenvironment of the tumor our findings indicated that il2 andil6 were significantly altered after the ablation procedureand positively correlated with mwa energy il2 is commonly derived from activated t cells primarily th1 cellsil2 can stimulate t cells to proliferate and differentiateactivate natural killer nk cells and macrophages and enhance the functions of cytotoxic t lymphocytes ctls our data illustrated that il2 is significantly increased at h after mwa indicating that il2 may induce a nonspecific immune response after mwa but il decreased after h postmwa in our study suggesting that the il2induced immune response may not belong lasting mentionable many cytokines detected il8il1 il12 were mainly derived from macrophagewhich was a widely distributed antigen presenting cellthis result support the theory that mwa could releasefragment of cancer cells into blood as neoantigen macrophages could response to this proceed and cause a systemic immunoreaction additional cytokines alterationsuch as il6 after ablation may be no anspecific inliver evidences indicate that increase of il6 was not onlyoccurred in liver ablation researches focus on lung cancerincluding primary lung cancer and pulmonary metastasesdemonstrated that serum il8 il1 il6 il10 il12and tnfα were significantly raised after radiofrequencythermal ablation moreover joseph found that imageguided thermal ablation of tumors located in lung liver orsoft tissues increases plasma levels of il6 and il10 another question remain unveiled was if our result wasœcancerspecific we checked literature about cytokinemodulation after thermal ablation in benign diseases andonly got limit evidences based on benign thyroid nodules and adenomyosis according to these literatureil6 levels did not show any significant difference aftertreatment compared with pretreatment values indicatingthat elevation of il6 may be caused by tumour antigenreleased by ablation treatment however the ablationenergy used in thyroid nodules was much lower thanliver and lung which would lead to a false negativein cytokine detection to the research about adenomyosis on the other hand experiment design was determined to followup the il6 at months afterhifu ablation as our data demonstrated mostly cytokines were return to premwa level after monthdetection after months may miss the modulation ofil6 overall few evidences support that some of thecytokines were altered in a œcancerspecific mannerwhile no solid results could confirm that further animal experiments were required to make a clarifieddata and answer this question 0czhao bmc cancer page of thetumorassociated immunein recent years ablationinduced systemic effects suchasresponse haveattracted increased attention de baere t first reported two cases of spontaneous regression of multiplepulmonary metastases occurring after radiofrequencyablation of a single lung metastasis although growing evidence suggests that thermal ablation can inducespontaneous regression of the socalled œabscopal effecton distant tumors the incidence rate of such an effect israre probably due to uncontested immunological activation caused by one ablation treatment and the lack ofimmuneamplification management in it was described that in situ tumor destruction can provide a useful antigen source forthe induction of antitumorimmunity however clinical studies could not sufficiently utilize such an effect until the development ofimmune checkpoint inhibitors icis [ ] icis suchas pd1pdl1 and ctla4 antibodies are widely applied in several cancers and studies have indicated thatici treatment could enhance the effect of ablation evidence indicates that hyperthermic destruction causesthe release of a large population of heterogeneous tumorantigens and inflammatory cytokines may play crucialroles in this process however opposite evidence indicated that incomplete radiofrequency ablation couldinduce inflammation which may accelerates tumor progression and hinders pd1 immunotherapy suggesting that ablation treatment may promote tumorprogression our data demonstrated that il6 was significantly increased after mwa treatment il6 is derived from monocytes macrophages dcs th2 cells andsometimes cancer cells and it plays a key role in t cellproliferation and survival the role of il6 appearsto be rather complex korn classified il6 as œdifferentiation factor which could involve in differentiation ofth17 cells however il6 does not direct the commitment to the th1 or th2 cell lineage but controls theproliferation and survival of immunocytes cooperatingwith other cytokines such as tgf tnf or il21 for instance il6 activated stat3 pathway in naivecd4 t cells in the presence of the morphogen tgfbpromotes the population expansion of th17 cells recent evidence indicates that il6 plays an indispensable role in t cellinfiltration to the tumor sitewhich could benefit immunomodulatory therapy incontrast il6 can increase mdscs inhibit the development and maturation of dendritic cells dcs and inhibit the polarization of th1 cells eventuallyresulting in negative immunomodulatory effects according to muneeb ahmed™s work the adjuvant uses ofa nanop smallinterfering rna sirna can besuccessfully used to target the il6mediated locoregional and systemic effects of thermal ablation il6 knockout via a nanop antiil6 sirna in mice coulddecrease the local vegf level at the ablation site therefore how to utilize the positive effect of il6 whileavoiding the negative effect after mwa needs further investigation preclinical research indicated that il6 andpdl1 blockade combination therapy reduced tumorprogression in animal models [ ] thus an antiil strategy after ablation should be considered whencombined with ici therapy previous studies and ourshave demonstrated that most cytokine levels returned topretreatment levels days after ablation this resultsuggests that h to days after ablation may be optimal timing for additional immunomodulatory therapysour results reported here support the evidence for terminal tumor thermal ablation could cause heat injury totissues surrounding the tumor site and release neoantigento blood circulation eventually induce a systemic reactionthis reaction could lead to a detectable alteration of cytokine levels further investigation is required to revealwhether the cytokines altered by mwa treatment couldaffect cancer progression whether positive or negativeabbreviationsmwa microwave ablation hcc hepatocellular carcinoma icis immunecheckpoint inhibitors tnf tumor necrosis factor il interleukinvegf vascular endothelial growth factor sd stable disease pr partialresponse ct computed tomography ci confidence interval ltp likelihoodof local tumor progression mdscs myeloidderived suppressor cellsctls cytotoxic t lymphocytes nk natural killer sirna small interfering rnaacknowledgementsnot applicableauthors™ contributionsjz conceptualization data curation writing“original draft and writing“review and editing ql conceptualization and writing“review and editingmm conceptualization and writing“review and editing brconceptualization and writing“review and editing and collect samples yhexecute milliplex assay and collect data dpl patient enrollment executemwa ablation and collect samples zl execute mwa ablation and collectsamples dml patient enrollment execute mwa ablation and collectsamples yx execute milliplex assay and collect data mt conceptualizationand writing“review and editing rl conceptualization data curation formalanalysis visualization writing“original draft and writing“review and editingall authors have read and approved the manuscriptfundingthis work was supported by the national natural science foundation ofchina the natural science foundation ofjiangsu province of china bk20140295 the jiangsu governmentscholarship for oversea studies js2018179 and the œsix one projects forhighlevel health personnel in jiangsu province lgy2018077availability of data and materialsthe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethe protocol was approved by the human ethics committee of the firstaffiliated hospital of soochow university and was conducted in accordancewith the declaration of helsinki patients were informed that the bloodsamples were stored by the hospital and potentially used for scientific 0czhao bmc cancer page of research and that their privacy would be maintained all written informedconsent was obtained from all participants and clearly statedconsent for publicationnot applicablecompeting intereststhere is no financial or personal relationship with other people oranizations that could inappropriately influence bias this workauthor details1department of radiation oncology the first affiliated hospital of soochowuniversity suzhou china 2department of oncology the first affiliatedhospital of soochow university suzhou china 3department of lymphatichematologic oncology jiangxi cancer hospital nanchang china4department of interventional radiology the first affiliated hospital ofsoochow university suzhou china 5division of neurosurgery city of hopebeckman research institute duarte california usareceived january accepted august referencesfu j wang h precision diagnosis and treatment of liver cancer in chinacancer lett “bruix j han kh gores g llovet jm mazzaferro v liver cancer approachinga personalized care j hepatol suppls144“rognoni c ciani o sommariva s bargellini i bhoori s cioni r facciorussoa golfieri r gramenzi a mazzaferro v transarterial radioembolizationfor intermediateadvanced hepatocellular carcinoma a budget impactanalysis bmc cancer nault jc sutter o nahon p gannecarrie n seror o percutaneoustreatment of hepatocellular carcinoma state of the art and innovations jhepatol “yin j dong j gao w wang y a case report of remarkable response toassociation of radiofrequency ablation with subsequent atezolizumab instage iv nonsmall cell lung cancer medicine baltimore 20189744e13112shi l chen l wu c zhu y xu b zheng x sun m wen w dai x yang m pd1 blockade boosts radiofrequency ablationelicited adaptiveimmune responses against tumor clin cancer res “lippitz be cytokine patterns in patients with cancer a systematic reviewlancet oncol 2013146e218“jin yb zhang gy lin kr chen xp cui jh wang yj luo w changes ofplasma cytokines and chemokines expression level in nasopharyngealcarcinoma patients after treatment with definitive intensitymodulatedradiotherapy imrt plos one 2017122e0172264kim mj jang jw oh bs kwon jh chung kw jung hs jekarl dw lee schange in inflammatory cytokine profiles after transarterial chemotherapy inpatients with hepatocellular carcinoma cytokine “ gillams a goldberg n ahmed m bale r breen d callstrom m chen mhchoi bi de baere t dupuy d thermal ablation of colorectal livermetastases a position paper by an international panel of ablation expertsthe interventional oncology sans frontieres meeting eur radiol “ ahmed m solbiati l brace cl breen dj callstrom mr charboneau jwchen mh choi bi de baere t dodd gd 3rd imageguided tumorablation standardization of terminology and reporting criteriaa 10yearupdate radiology “ chiang j hynes k brace cl flowdependent vascular heat transfer duringmicrowave thermal ablation conf proc ieee eng med biol soc “ huang hw influence of blood vessel on the thermal lesion formationduring radiofrequency ablation for liver tumors med phys najjar yg rayman p jia x pavicic pg jr rini bi tannenbaum c ko jhaywood s cohen p hamilton t myeloidderived suppressor cellsubset accumulation in renal cell carcinoma parenchyma is associated withintratumoral expression of il1beta il8 cxcl5 and mip1alpha clin cancerres “ alfaro c teijeira a onate c perez g sanmamed mf andueza mp alignanid labiano s azpilikueta a rodriguezpaulete a tumorproducedinterleukin8 attracts human myeloidderived suppressor cells and elicitsextrusion of neutrophil extracellular traps nets clin cancer res “kundu m roy a pahan k selective neutralization of il12 p40 monomerinduces death in prostate cancer cells via il12ifngamma proc natl acadsci u s a “ onishi h kuroki h matsumoto k baba e sasaki n kuga h tanaka mkatano m morisaki t monocytederived dendritic cells that capture deadtumor cells secrete il12 and tnfalpha through il12tnfalphanfkappabautocrine loop cancer immunol immunother “ yu z geng j zhang m zhou y fan q chen j treatment of osteosarcomawith microwave thermal ablation to induce immunogenic cell deathoncotarget “ yang w wang w liu b zhu b li j xu d ni y bai l liu gimmunomodulation characteristics by thermal ablation therapy in cancerpatients asia pac j clin oncol 2018145e490“erinjeri jp thomas ct samoilia a fleisher m gonen m sofocleous ctthornton rh siegelbaum rh covey am brody la imageguidedthermal ablation of tumors increases the plasma level of interleukin6 andinterleukin10 j vasc interv radiol “ den brok mh sutmuller rp van der voort r bennink ej figdor cg ruerstj adema gj in situ tumor ablation creates an antigen source for thegeneration of antitumor immunity cancer res “ zerbini a pilli m laccabue d pelosi g molinari a negri e cerioni sfagnoni f soliani p ferrari c radiofrequency thermal ablation forhepatocellular carcinoma stimulates autologous nkcell responsegastroenterology “ zhang h hou x cai h zhuang x effects of microwave ablation on tcellsubsets and cytokines of patients with hepatocellular carcinoma minim
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" levels of physical activity change throughout the year however little is known to what extentactivity levels can vary based on accelerometer determined sedentary and physicallyactive time the aim of thislongitudinal study was to examine older adults™ activity changes from a nonsnowfall season to a subsequentsnowfall season with consideration of the codependence of domains of time usemethods participants were older japanese adults women aged “ years living in a rural area ofheavy snowfall who had valid accelerometer active style pro hja750c data during nonsnowfall and snowfallseasons activity was classified as sedentary behavior sb lightintensity pa lpa and moderatetovigorous pamvpa compositional changes from the nonsnowfall to the snowfall season were analyzed using aitchison™sperturbation method the ratios of each component in the composition such as [sbsnowsbnonsnow lpasnowlpanonsnow mvpasnowmvpanonsnow] for seasonal changes were calculated and were then divided by thesum of these ratiosresults in men the percentages of time spent in each activity during the nonsnowfallsnowfall seasons were for sb for lpa and for mvpa these corresponded to mean seasonal compositionalchanges δsb δlpa δmvpa of and respectively in women the percentages of time spent ineach activity during the nonsnowfallsnowfall seasons were for sb for lpa and formvpa these corresponded to mean seasonal compositional changes δsb δlpa δmvpa of and respectively the degree of seasonal change was greatest in mens in older adults activity behaviors were changed unfavorably during snowfall season particularly so formen the degree of seasonal change was greatest for sb development of strategies to keep rural older adultsactive during the snowfall season may be needed for maintaining a consistentlyactive lifestyle for their healthkeywords accelerometry aging environment exercise sedentary lifestyle correspondence inouetokyomedacjp1department of preventive medicine and public health tokyo medicaluniversity shinjuku shinjukuku tokyo japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0camagasa bmc public health page of global physical activity guidelines state that older adultsleast min per week of moderateshould do atintensity physical activity atleast min vigorousintensity physical activity or an equivalent combinationof both in bouts lasting min or more physical inactivity increases the risk of many adverse health outcomes including mortality cardiovascular disease type diabetes several types of cancer and cognitive decline[“] in recent years there has been significant growthin research investigating the detrimental health effects ofsedentary behavior sb put simply too much sitting reducing sb and increasing physical activity are keyingredients of initiatives to reduce the global burden ofnoncommunicable diseases [ ]there is a growing body of evidence identifying the importance of natural environments the attributes of whichcan vary greatly by season as well as built environments asinfluences on physical activity [ ] one systematic review of studies from eight different countries showed thatlevels of physical activity tended to be lowest during winter to date however most of these studies have relied onselfreport physical activity assessments and have not focusedon older adults objective assessment via accelerometers can now overcome problems of recall and reportingbias and allow more accurate and finergrained assessmentsof activity behavior patterns sb lightintensity physical activityactivitymvpa there is limited evidence on how season affects devicebased activity behaviors [“] compositionaldata analysis coda allows consideration ofthe codependence of time spent in all behaviors arising within aday or other fixed period [ ] to date no previous studyhas investigated seasonal changes of activity behaviors takingtime spent in each activity behavior into accountlpa and moderatetovigorous physicalwe compared times spent in accelerometermeasuredactivity behaviors during a nonsnowfall and a snowfallseason in a sample of communitydwelling older japanese adults using the coda approach we also exploredwhich activity behaviors were most affected by seasonmethodsstudy sample and data collectionwe used longitudinal data from the neuron to environmental impact across generations study neige study themethods of this study are described in previous study participants were older adults without longterm care livingin tokamachi city japan tokamachi is a rural city officiallyregistered as a heavy snowfall region during winter locatedin the southernmost region of niigata prefecture participants were from two areas matsunoyama mountain areaor tokamachi area downtown the mountain area mmaximum snow depth had more snow than the downtownarea maximum snow depth during winter theaverage temperature at tokamachi city in february was ˆ’ °c lowest ˆ’ °c highest °c brieflyin a total of residents were recruited from a resident registry using stratified randomsampling in the nonsnowfall season autumn of we conducted a questionnaire survey and health examination of older adults who agreed to enrollment inthe neige study at the same time they were asked towear an accelerometer of these participants agreedto also wear an accelerometer during the snowfall seasonwinter of accelerometermeasured activity behaviorshabitual time spent in activity behaviors were evaluatedby active style pro hja750c omron healthcarekyoto japan active style pro is a validated accelerometer [“] and comparable to the devices most commonly used in studies conducted in western countries[ ] its measurement algorithm has been explainedin detail elsewhere [ ] participants were instructedto wear an accelerometer over the waist on an elasticated belt for seven consecutive days except duringsleep and waterbased activities during snowfall andnonsnowfall season respectively in the survey duringsnowfall season participants were mailed an accelerometer no acceleration signal detected for longer than consecutive minutes was defined as œnonwear participants with a wear time corresponding to at least hduring waking time per day collected over four ormore valid days were included in the analysis thedata were collected in 60s epochs activity behaviorswere classified into three intensity categories based onmetabolic equivalents mets sb ‰ mets lpa“ mets and mvpa ‰¥ mets [ ]sociodemographic biological and psychological factorsparticipants reported their age gender living arrangement with others alone and selfrated health verygood good fair poor in autumn we classified participants between the ages of and years as youngoldand those between ages and years as oldold bodymass index bmi was calculated from height and weightkgm2 measured by body composition analyzer mc780a tanita corporation tokyo japanstatistical analysesall analyses were performed using r version r foundation for statistical computing vienna austria we usedr package œcompositions for coda approach for all analyses pvalues were considered statistically significantanalyses were applied stratified by gender since activity behavior patterns were significantly different between men andwomen 0camagasa bmc public health page of the chisquare test or t test was performed to compare participant characteristics mcnemar™s test wasused to compare nonsnowfall and snowfall season™sproportions of those adhering to physical activity guidelines ie ‰¥ minweek of mvpa in bouts of at least min a minbout mvpa was defined as ormore consecutive minutes above the moderate intensitythreshold with the allowance of “ min interruptionintervals we described activity behaviors during the nonsnowfalland snowfall season using coda approach as all participantsspent some time in every behavior there was no need for amethod to deal with zeros compositional changes [δsbδlpa δmvpa] were then determined by aitchison™s perturbation method [“] the ratios of each component inthe composition such as [sbsnowsbnonsnow lpasnowlpanonsnow mvpasnowmvpanonsnow] for seasonalchanges were calculated and were then divided by the sumof these ratios an equal composition of these three activitiesin the nonsnowfall and snowfall seasons would result in acompositional change of[ ] compositionalchanges were plotted as ternary diagrams with some significant guide values illustratedresultsparticipant enrollment and characteristicsof the older adults who agreed to wear an accelerometer during both the nonsnowfall and snowfallseasons response rate were excluded for notmeeting wearing time criteria n bone fracturen unreturned accelerometer n and malfunction n the analytic sample was who had validaccelerometer datawhen compared to participant characteristics betweenthose who had valid accelerometer data in snowfall season and those who did not a significant difference wasfound in age groups youngold oldold chisquare test participants who participated in thesurvey in the snowfall season were significantly morephysically active approx more minutes of mvpaper day than those who did not ttesttable presents the characteristics of the participantsthe mean sd age was years womenand most of the participants were living with others kgm2 bmi and good perceived health comparedto women men were more likely to be from the mountain area be living with others and to have attained‰¥ minweek of mvpa there were no significant seasonal differences in proportion of those adhering to global physical activity guidelines nonsnowfall seasonoverall men women snowfall season overall men women activity behaviors in snowfall and nonsnowfall seasonmean accelerometer wear time was minday innonsnowfallseason and minday in snowfalltable participant™s characteristics at baseline nonsnowfall seasonoverall n mean ± sd n ± men n mean ± sd n ± women n mean ± sd n ± ± ± pvalueage yrsage categoriesyoungold “ yrsoldold ‰¥ yrsarea of residencemountain areadowntownliving arrangementliving with othersliving alonebmi kgm2bmi categoriesnormal kgm2obese ‰¥ kgm2perceived healthgood very good good ± poor fair poorpvalue was calculated using t test or chisquare test as appropriate 0camagasa bmc public health page of season older adults spent most of their time on sb andlpa table presents the descriptive statistics of timespent in each activity behavior in men time spent ineach activity sb lpa and mvpa was and respectively during the nonsnowfall season and and respectively during the snowfall season corresponding to mean seasonal change of forsb for lpa and for mvpa fig sincelarger distance from indicates greater change in timespent in activity the largest seasonal change was observed in sb compared to lpa and mvpa in womentime spent in each activity was and respectively during nonsnowfall season while that was and respectively during snowfall seasoncorresponding to mean seasonal change of for sb for lpa and for mvpa significant unfavorable seasonal changes in activity behaviors were observed in both men and women but the degree ofchange was larger in men in every behavior if we contrast the changes the ratios between men and womensimilar findings were observed after stratification byresidential area but the degree of change in activity behaviors was larger in mountain area than in downtownregardless of genderdiscussionwe identified seasonal differences of accelerometer determined activity behaviors in communitydwelling olderadults in a rural snowfall area using the coda approacholder adults had more unfavorable activity behavior patterns during the snowfall season compared to nonsnowfall season with the magnitude of these differencesgreater in men no significant seasonal differences werefound in the proportions adhering to global physical activity guidelinesour findings are consistent with previous studies usingselfreport [ ] and pedometer accelerometer [“] data where levels of adults™ physical activity duringwinter was less than the other seasons a study iniceland found that both older men and women weremore active during summer than winter with less timespent in accelerometer determined sb in men in women a study in the uk found that onlytime spent in lpa was significantly higher during springthan that during winter among older adults winter ± hday spring ± hday summer ± hday and autumn ± hday we found thatthe magnitude of decline during winter seems to belarge compared to these icelandic and uk studies thismay result from winter conditions including amount ofsnowfall furthermore our study participants were relatively more active and less sedentary during autumn thenonsnowfall season more highly active participantsmay experience activity decline to a greater degree compared to low activity participants in this sample therewere no significant seasonal changes of adherence toglobal physical activity guidelines this may be due tovery little time spent in mvpa in bouts of more than mingender differences of activity patterns were in linewith our previous findings [ ] that japanese olderwomen were more physically active than older men provided that all activity behaviors were assessed in thissample men experienced greater seasonal activity declines than did women in japan women are more responsible for household chores and thus engage in acertain amount of activities throughout the year theremay be needed to develop a strategy to combat this seasonal decrease in physical activity for menin the current study activity behavior patterns wereunfavorably changed with approximately increasein sb and decrease in mvpa the degree of the changein activity behaviors may be significant for health a previous study found decline of physical activity during winter unfavorably affected the physical performance levelafter one year in communitydwelling older women particularly its effect on maximum walking speed given that the rate of muscle mass decline is higher inolder adults compared to middleaged adults maintaining physical activity may be required to keep physicaltable compositional geometric means of time spent in activity behaviors during the nonsnowfall and snowfall seasonnonsnowfall season minday wear timewear timesb lpa snowfall season minday wear timewear timesb lpa mvpa mvpa overallmenmountain areadowntownwomenmountain areadowntownabbreviation sb sedentary behavior lpa lightintensity physical activity mvpa moderatetovigorous physical activity 0camagasa bmc public health page of fig changes in sedentary behavior sb light intensity physical activity lpa and in moderatetovigorous physical activity mvpa from thenonsnowfall to snowfall season in ruraldwelling older japanese adults a stratified by gender blue men red women b stratified by genderand residential area blue mountain area green downtown abbreviation sb sedentary behavior lpa lightintensity physical activity mvpamoderatetovigorous physical activityability and prevent a negative cycle of frailty another study showed shortterm decreased physical activity with increased sb caused impairment of metabolism which increases risk of cardiovascular disease therefore promoting physical activity during wintermay be one way to tackle agerelated diseases and lossof physical functioningstrengths and limitationsstrengths of this study included objective assessment ofactivity behaviors and a novel statistical approach eventhough there has been increasing research using objective methods [“ ] no study has treated with consideration to the codependence of time spent in activitybehaviors moreover we provided resultsfrom aseldomstudied elderly population in a rural snowfallarea however several limitations should be consideredto interpret the findings we may underoverestimate ofsb and lpa since active style pro cannot provide posturalinformation also some activities eg snow removal and skiing may be not captured accuratelysecond we may underestimate the decline of activitylevel since most of the days that participants wore an accelerometer were relatively good weather in spite ofheavy snowfall area it has previously been observed thatlonger day length is associated with increased devicebased physical activity in the older population [ ]third as tokamachi city is a rural area where heavysnowfall is common during winter it is not necessarilyrepresentative ofjapanese rural areas with different 0camagasa bmc public health page of climates people in areas with less snowfall may experience a smaller decline of physical activity during snowfall season more research is needed in the differentclimate zones from different geographic areas finallyselection bias may have occurred in general accelerometry respondents have been more active and healthierthan nonparticipating older adults in this studythose had valid accelerometer data in snowfall seasonwere more physically active than those who did notimplications for future research policy and practiceour findings suggest several implications in terms of bothdevelopment of interventions to protect against seasonalphysical activity decline and physical activity surveillancemonitoring further research regarding how to stay activeduring winter may be required for health promotion particularly in regions that experience long winters and withsevere weather eg heavy snow given that approximately of the land in japan has snow and a cold winter and that a quarter of the population lives in thoseareas leading an active lifestyle during winter potentially has a significant public health impactsince sb is the most affected by season it may be better tofocus on developing strategies to reduce time spent in sittingolder adults might be particularly influenced by seasonaloutside conditions due to reduced physical functioning andmobility and spend much of their time indoors it thus maybe effective to provide supplementary resources for indooractivities eg gymnastic exercise programs sharing ofhousehold chores and making educational instrumentalsupports for safe snow removal further approach includes replacing mentallypassive with mentallyactive sbmay be effective for health particularly for preventing cognitive decline [ ] and depression [ ]as for physical activity surveillance monitoring investigators should be aware of the potential for under oroverestimation of levels of activity especially when theinterest is in its betweenindividual variation includingcommunitylevel and countrylevel comparisons seasonality also should be considered when interventionstudies are performedsaccelerometer determined activity behaviors were greatlyaffected by season in communitydwelling older adults ina rural snowfall area resulting in unfavorable changesparticularly in sb time during snowfall season development of strategies to keep rural older adults active duringthe snowfall season may be needed for maintaining aconsistentlyactive lifestyle for their healthabbreviationssb sedentary behavior lpa lightintensity physical activitymvpa moderatetovigorous physical activity coda compositional dataanalysis mets metabolic equivalentsacknowledgementswe thank the city workers and the participants for their time and effort inthe neige studyauthors™ contributionsys hm si and tf developed the neige study ys hm si tf hk nf mmand sa collected the data sa performed the analysis and prepared themanuscript sc and no gave critical feedback and revised the manuscript allauthors advised on the data analysis and interpretation and reviewed themanuscriptfundingthis study was funded by grant from ˜the policy research institute ministryof agriculture forestry and fisheries in japan™ and ˜the pfizer healthresearch foundation™ and by jsps kakenhi grant number 16h03249 k19794 k10829 and 19h03910 the funding bodies were not involved inany portion of the study design data collection and analysis interpretationof the results and preparation of the manuscriptshiho amagasa is supported by jsps research fellowships for youngscientists neville owen is supported by a national health and medicalresearch council of australia nhmrc centre of research excellence grant nhmrc senior principal research fellowship and thevictorian government™s operational infrastructure support programavailability of data and materialsthe datasets used and analyzed during the current study are available fromthe corresponding author on reasonable requestethics approval and consent to participatethe university ethics committees niigata university and tokyo medicaluniversity granted ethical approval written informed consent was obtainedfrom all participantsconsent for publicationnacompeting intereststhe authors declare no conflict of interestauthor details1department of preventive medicine and public health tokyo medicaluniversity shinjuku shinjukuku tokyo japan 2institute ofgerontology the university of tokyo hongo bunkyoku tokyo “ japan 3department of global health promotion tokyo medical anddental university yushima bunkyoku tokyo japan 4school ofhealth and life science institute of applied health research glasgowcaledonian university cowcaddens road glasgow uk 5department ofsport and movement science ghent university ghent belgium6behavioral epidemiology laboratory baker heart diabetes institute level commercial road melbourne vic australia 7centre for urbantransitions swinburne university of technology po box hawthornmelbourne australia 8division of international health niigata universitygraduate school of medical and dental sciences asahimachidoriniigata city japan 9department of active ageing niigatauniversity graduate school of medical and dental sciences asahimachidori niigata city japanreceived february accepted august references world health anization global recommendations on physical activity forhealth httpappswhointirisbitstream106654439919789241599979_engpdf accessed jan lee im shiroma ej lobelo f puska p blair sn katzmarzyk pt effect ofphysical inactivity on major noncommunicable diseases worldwide ananalysis of burden of disease and life expectancy lancet “kyu hh bachman vf alexander lt mumford je afshin a estep k veermanjl delwiche k iannarone ml moyer ml physical activity and risk ofbreast cancer colon cancer diabetes ischemic heart disease and ischemic 0camagasa bmc public health page of stroke events systematic review and doseresponse metaanalysis for theglobal burden of disease study bmj 2016354i3857aune d norat t leitzmann m tonstad s vatten lj physical activity andthe risk of type diabetes a systematic review and doseresponse metaanalysis eur j epidemiol “arem h moore sc patel a hartge p berrington de gonzalez avisvanathan k campbell pt freedman m weiderpass e adami ho leisure time physical activity and mortality a detailed pooled analysis of thedoseresponse relationship jama intern med “sofi f valecchi d bacci d abbate r gensini gf casini a macchi c physicalactivity and risk of cognitive decline a metaanalysis of prospective studiesj intern med “young dr hivert mf alhassan s camhi sm ferguson jf katzmarzyk ptlewis ce owen n perry ck siddique j yong cm sedentary behavior andcardiovascular morbidity and mortality a science advisory from theamerican heart association circulation biswas a oh pi faulkner ge bajaj rr silver ma mitchell ms alter dasedentary time and its association with risk for disease incidence mortalityand hospitalization in adults a systematic review and metaanalysis annintern med “ding d lawson kd kolbealexander tl finkelstein ea katzmarzyk pt vanmechelen w pratt m the economic burden of physical inactivity a globalanalysis of major noncommunicable diseases lancet “tucker p gilliland j the effect of season and weather on physical activity asystematic review public health “ barnett dw barnett a nathan a van cauwenberg j cerin e builtenvironmental correlates of older adults' total physical activity and walking asystematic review and metaanalysis int j behav nutr phys act shephard rj tudorlocke c the objective monitoring of physical activitycontributions of accelerometry to epidemiology exercise science andrehabilitation new york springer international publishing davis mg fox kr hillsdon m sharp dj coulson jc thompson jlobjectively measured physical activity in a diverse sample of older urbanuk adults med sci sports exerc “ arnardottir ny oskarsdottir nd brychta rj koster a van domelen drcaserotti p eiriksdottir g sverrisdottir je johannsson e launer lj comparison of summer and winter objectively measured physical activityand sedentary behavior in older adults age geneenvironmentsusceptibility reykjavik study int j environ res public health yasunaga a togo f watanabe e park h park s shephard rj aoyagi y sexage season and habitual physical activity of older japanese the nakanojostudy j aging phys act “ chastin sf palareaalbaladejo j dontje ml skelton da combined effectsof time spent in physical activity sedentary behaviors and sleep on obesityand cardiometabolic health markers a novel compositional data analysisapproach plos one 201510e0139984 dumuid d stanford te martinfernandez ja pedisic z maher ca lewis lkhron k katzmarzyk pt chaput jp fogelholm m compositional dataanalysis for physical activity sedentary time and sleep research statmethods med res “shobugawa y murayama h fujiwara t inoue s cohort profile of the neigestudy in tokamachi city japan j epidemiol “japan meteorological agency historical weather data search httpswwwjmagojpjmaindexehtml accessed jul ohkawara k oshima y hikihara y ishikawatakata k tabata i tanaka s realtime estimation of daily physical activity intensity by a triaxial accelerometerand a gravityremoval classification algorithm br j nutr “ oshima y kawaguchi k tanaka s ohkawara k hikihara y ishikawatakata ktabata i classifying household and locomotive activities using a triaxialaccelerometer gait posture “ park j ishikawatakata k tanaka s bessyo k tanaka s kimura t accuracyof estimating step counts and intensity using accelerometers in olderpeople with or without assistive devices j aging phys act “ nagayoshi s oshima y ando t aoyama t nakae s usui c kumagai stanaka s validity of estimating physical activity intensity using a triaxialaccelerometer in healthy adults and older adults bmj open sport exercmed 20195e000592kurita s yano s ishii k shibata a sasai h nakata y fukushima n inoue stanaka s sugiyama t comparability of activity monitors used in asianand westerncountry studies for assessing freeliving sedentary behaviourplos one 201712e0186523 murakami h kawakami r nakae s nakata y ishikawatakata k tanaka smiyachi m accuracy of wearable devices for estimating total energyexpenditure comparison with metabolic chamber and doubly labeledwater method jama intern med “tudorlocke c camhi sm troiano rp a catalog of rules variables anddefinitions applied to accelerometer data in the national health andnutrition examination survey prev chronic dis 20129e113troiano rp berrigan d dodd kw masse lc tilert t mcdowell m physicalactivity in the united states measured by accelerometer med sci sportsexerc “ haskell wl lee im pate rr powell ke blair sn franklin ba macera ca heathgw thompson pd bauman a physical activity and public health updatedrecommendation for adults from the american college of sports medicineand the american heart association circulation “ pate rr o'neill jr lobelo f the evolving definition of œsedentary exercsport sci rev “ aitchison j ng kw the role of perturbation in compositional data analysisstat model “ boogaart kg tolosanadelgado r analyzing compositional data with rberlin springerverlag berlin heidelberg winkler eah chastin s eakin eg owen n lamontagne ad moodie m dempsey pckingwell ba dunstan dw healy gn cardiometabolic impact of changing sittingstanding and stepping in the workplace med sci sports exerc “ uitenbroek dg seasonal variation in leisure time physical activity med scisports exerc “ matthews ce freedson ps hebert jr stanek ej 3rd merriam pa rosal mcebbeling cb ockene is seasonal variation in household occupational andleisure time physical activity longitudinal analyses from the seasonalvariation of blood cholesterol study am j epidemiol “ amagasa s fukushima n kikuchi h takamiya t oka k inoue s light andsporadic physical activity overlooked by current guidelines makes olderwomen more active than older men int j behav nutr phys act amagasa s inoue s ukawa s sasaki s nakamura k yoshimura a tanaka akimura t nakagawa t imae a ding d kikuchi h tamakoshi a are japanesewomen less physically active than men findings from the dosanco healthstudy j epidemiol httpsdoi102188jeaje20200185 mizumoto a ihira h makino k saitoh s ohnishi h furuna t physical activityduring winter in oldold women associated with physical performance afterone year a prospective study curr gerontol geriatr res volpi e nazemi r fujita s muscle tissue changes with aging curr opin clinnutr metab care “fried lp tangen cm walston j newman ab hirsch c gottdiener jseeman t tracy r kop wj burke g mcburnie ma frailty in older adultsevidence for a phenotype j gerontol series a 200156m146“ bowden davies ka sprung vs norman ja thompson a mitchell klhalford jcg harrold ja wilding jph kemp gj cuthbertson dj shorttermdecreased physical activity with increased sedentary behaviour causesmetabolic derangements and altered body composition effects inindividuals with and without a firstdegree relative with type diabetesdiabetologia “ ormazabal v nair s elfeky o aguayo c salomon c zuñiga fa associationbetween insulin resistance and the development of cardiovascular diseasecardiovasc diabetol wu yt luben r wareham n griffin s jones ap weather day length andphysical activity in older adults crosssectional results from the europeanprospective investigation into cancer and nutrition epic norfolk cohortplos one 201712e0177767schepps ma shiroma ej kamada m harris tb lee im day length isassociated with physical activity and sedentary behavior among olderwomen sci rep inoue s ohya y odagiri y takamiya t kamada m okada s tudorlocke cshimomitsu t characteristics of accelerometry respondents to a mailbasedsurveillance study j epidemiol “ ministry of land infrastructure transport and tourism snow disaster preventionhttpwwwmlitgojproadbosaifuyumichiprojecthtml accessed jan chastin sf de craemer m lien n bernaards c buck c oppert jm nazareja lakerveld j o'donoghue g holdsworth m the sosframeworksystems of sedentary behaviours an international transdisciplinaryconsensus framework for the study of determinants research priorities andpolicy on sedentary behaviour across the life course a dedipacstudy int jbehav nutr phys act 0camagasa bmc public health page of kesseguyot e charreire h andreeva va touvier m hercberg s galan poppert jm crosssectional and longitudinal associations of differentsedentary behaviors with cognitive performance in older adults plos one20127e47831 bakrania k edwardson cl khunti k bandelow s davies mj yates tassociations between sedentary behaviors and cognitive function crosssectional and prospective findings from the uk biobank am j epidemiol“ hallgren m owen n stubbs b zeebari z vancampfort d schuch fbellocco r dunstan d trolle lagerros y passive and mentallyactivesedentary behaviors and incident major depressive disorder a 13yearcohort study j affect disord “ hallgren m nguyen tt owen n stubbs b vancampfort d lundin adunstan d bellocco r lagerros yt crosssectional and
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"Nonsmall cell lung cancer is the most common cause of cancer death worldwide highlighting the need fornovel therapeutic concepts In particular there is still a lack of treatment strategies for the group of elderly and frail patientswho are frequently not capable of receiving standard therapy regimens Despite comprising the majority of lung cancerpatients this group is underrepresented in clinical trials This applies also to elderly and frail patients suffering fromunresectable stage III NSCLC who are unfit for chemotherapy and therefore cannot receive the standard therapycomprising of radiochemotherapy and the recently approved subsequent durvalumab consolidation therapy These patientsoften receive radiotherapy only which raises the concern of undertreatment The TRADEhypo trial aims at optimizingtreatment of this patient group by combining radiotherapy with concomitant durvalumab administration therebyemploying the immunepromoting effects of radiotherapy and determining safety feasibility and efficacy of this treatmentMethods design In this prospective phase II clinical trial durvalumab therapy will be combined with either conventionallyfractionated CONgroup or hypofractionated HYPOgroup thoracic radiotherapy A safety stopandgo leadin phase willassess safety of hypofractionated radiotherapy with respect to severe pneumonitis in small patient cohorts before ing fullenrollment Tumor tissue blood and stool samples will be collected before and during the study period to investigate theimmunological mechanisms responsible for checkpoint inhibitor efficacy and immunepromoting effects of radiotherapyContinued on next page Correspondence FarastukBozmehrmeduniheidelbergde1Department of Thoracic Oncology Thoraxklinik at University Hospital ofHeidelberg R¶ntgenstraŸe Heidelberg Germany2Translational Lung Research Center Heidelberg TLRCH Member of theGerman Center for Lung Research DZL Im Neuenheimer Feld Heidelberg GermanyFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cBozmehr BMC Cancer Page of Continued from previous pageDiscussion Preclinical data suggests that irradiationinduced immunogenicity can be further increased if applied in ahypofractionated setting potentially boosting the expected synergistic effect with immune checkpoint inhibition in restoringthe immune antitumor response If proven safe and efficient a hypofractionated radiation schedule can provide a considerablymore practicable option for the patient Taking into consideration the intend to develop a combination treatment strategy thatcan be made available to patients soon after proving to be efficient and the potentially elevated toxicity of a hypofractionatedradiotherapy approach this trial was designed as a twotrialsinone design An accompanying translational research program isplanned striving to gain insights into the tumorhost biology and to identify suitable biomarkers to predict therapy responseTrial registration Clinicaltrialsgov NCT04351256 Registered April EudraCT “ Registered October Keywords Nonsmall cell lung cancer NSCLC Radioimmunotherapy Immune checkpoint inhibition AntiPDL1 monoclonalantibody Hypofractionated radiation Geriatric risk profileBackgroundLung cancer is the most common cause of cancer deathworldwide with nonsmall celllung cancer NSCLCrepresenting “ of cases [ ] Improving therapeutic strategies is thus of imminent importance especially considering elderly and frail patients With a medianage of about years at diagnosis lung cancer clearly is adisease of the elderly yet this group is underrepresentedin clinical trials and these patients are frequently not capable of receiving standard treatment protocols due to ageand tobaccoassociated comorbidities [“]attracting both immunocompetentIn recent years the advent of immunotherapy has pavedthe way for novel therapeutic concepts including the combination of radiotherapy with immune checkpoint inhibitioneg Programmed cell death ligand PD1 PDL1 Thisapproach is of particular interest as it utilizes synergistic effects While immune checkpoint inhibitors can restore thepatients™ antitumor immunity through T cell activationradiotherapy may further boostimmunemediated anticancer mechanisms by exposing tumorassociated antigensand byantigenpresenting cells and tumoricidal effector cells [ ] Indeedfor patients with unresectable stage III NSCLC the PACIFICtrial has revealed a profound clinical benefit treatment withthe antiPDL1 monoclonal antibody durvalumab after chemoradiotherapy with remarkably low toxicities [ ] As aresult sequential treatment with durvalumab after chemoradiotherapy has become the new standard treatment for locally advanced unresectable NSCLC However about ofpatients do not receive chemotherapeutic agents presumablydue to significantly higher rates of age and comorbidityrelated adverse events AE under chemoradiotherapy [] Thus elderly and frail patients often receive radiotherapyalone raising the serious concern of undertreatment and theneed for new therapeutic concepts [ ]Considering the immunepromoting effects of radiotherapy a combination with durvalumab therapy mayimprove response rates in these potentially undertreatedpatients Moreoverif applied early concomitant localdatathatsuggestradiotherapy with systemic immunotherapy may particularly increase control of distant micrometastases Preclinicalirradiationinducedimmunogenicity can even be further increased if appliedin a hypofractionated setting with single doses ‰¥ GrayGy in line with a radiation dosedependent abscopal[“] While a hypofractionated radiationeffectschedule is also considerably shorter and more convenient for the patient safety of concurrent immunoradiotherapy is a concern as both therapy modalities maycause severe pneumonitisIn this prospective phase II clinical trial we thereforeaim to determine feasibility and treatment efficacy ofdurvalumab treatment combined with thoracic radiotherapy TRT in previously untreated NSCLC stage IIIpatients unable to receive radiochemotherapy Strivingto develop a combination treatment strategy thatifproven safe and efficient can be quickly made availableto patients a twotrialsinone design was chosen thatcombines durvalumab with either conventionally fractionated CONgroup or hypofractionated thoracicradiotherapy HYPOgroup This study not only aims toincrease the efficacy of radiotherapy by utilizing theimmunesensitizing effects elicited by PDL1 inhibitionbut will also provide biomaterials that will be analyzedwith respect to immunological mechanisms responsiblefor checkpoint inhibitor efficacy and immunepromotingeffects of radiotherapy as well as potential biomarkersMethodsdesignStudy designThe TRADEhypo trialis a prospective randomized label multicenter phase II trial with a safety stopandgo leadin phase Fig During the leadin phasepatients in the HYPOgroup who will receive durvalumab in combination with hypofractionated thoracicradiotherapy will be closely evaluated with regard totoxicity defined as pneumonitis ‰¥ grade within 0cBozmehr BMC Cancer Page of Fig Study design of the TRADEhypo trial Patients will be enrolled according to eligibility criteria and treated with either a hypofractionated TRTregimen HYPOgroup or conventionally fractionated TRT CONgroup in combination with durvalumab For the HYPOgroup a safety stopandgophase with a design precedes full enrollment Whenever this arm is for recruitment patients will be allocated to this arm until the cohort isclosed whenever HYPOarm is closed for Stop Go decision evaluation based on the toxicity assessment of this regimen weeks after the end of TRTpatients are allocated to the CONarm When the study proceeds to expansion phase patients will be allocated to treatment arms by randomizationusing œbiased coin algorithm An efficacy interim analysis will be performed after patients have been enrolled in each armweeks after radiotherapy in small cohorts n beforeproceeding with full enrollment into this arm Fig respectrelated biomarkersto treatmentinduced changes and immuneStudy settingThe TRADEhypo trial will recruit patients from participating centers across Germany over a period of months Start of recruitment was planned for April but was delayed to May due to the Covid19pandemic A full list of sites can be obtained at clinicaltrialsgov NCT04351256Study objectivesThe primary objective of this study is to evaluate safetyand tolerability of conventionally fractionated CONgroup and hypofractionated HYPOgroup TRT incombination with durvalumab in patients with unresectable stage III NSCLC unfit for chemotherapy Moreoverefficacies of the two modes of radiotherapy will be evaluated with respect to response rates Further parameterswill be determined in order to assess efficacy safety andquality of life QoL in both treatment arms by recordingincidence and severity of adverse events AEs as well asspecific laboratory abnormalitiesExploratory endpoints include assessment of vulnerability and analyses of tumor tissue blood and stoolsamples that are collected during the clinical trial withCharacteristics of participantsA total of patients will be included into this studyPatients potentially eligible for trialinclusion will beapproached and asked to participate as they present inthe clinic Before a patient™s participation in the clinicalstudy the investigator must obtain written informedconsentEach participant must be eligible regarding all inclusion and exclusion criteria set for this trial Table Key inclusion criteria include diagnosis of unresectablestage III NSCLC and nonfeasibility of sequential chemoradiotherapy due to increased oxygenindependentvulnerability as reflected by fulfilling at least one of thefollowing criteria i Performance status Eastern Cooperative Oncology Group [ECOG] scale ii ECOG and Charlson comorbidity index CCI ‰¥ or iii age ‰¥ Moreover participants must have at least one measurable site of disease as defined by RECIST as wellas adequate bone marrow hepatic and renal functionPatients having received prior immunotherapy other investigational agents or thoracic radiotherapy within thepast years will be excluded from the study Additionally participants must not have an active or recent history of a known or suspected autoimmune disease or 0cBozmehr BMC Cancer Page of Fig Cohort design of the safety stopandgo leadin phase HYPOgroup The safety leadin phase follows a design in order to carefullyevaluate the toxicity of the treatment in the HYPOgroup with respect of the occurrence of a grade pneumonitis œevent within weeksafter the end of TRT Two events in the first six patients two events in the first or two events in the first patients will result in terminationof the HYPOgroup œStop If no event is observed within the first two safety cohorts ie the first patients the HYPOarm will be ed forfull enrollment with close toxicity assessment with respect to pneumonitis grade and terminated as soon as two events are reported withinthe subsequent six patients œGo Full enrollment in the HYPOarm will only take place if the criteria for the nontoxicity scenario are met ie ‰¤ event in n patients œGoany other medical condition conflicting with the studyinterventions and not have used immunosuppressivemedication For a full list of the inclusion and exclusioncriteria see Table TreatmentDurvalumabPatients will be enrolled based on the inˆ’ exclusion criteria Two treatment groups will be evaluated Onegroup will receive durvalumab immunotherapy combined with conventionally fractionated TRT CONgroup and the other one with hypofractionated TRTHYPOgroup In both groups durvalumab will be administered intravenously at a fixed dose of mg onday and every weeks thereafter for a maximum of months maximum doses last dose at week untilconfirmed disease progressioninacceptable toxicitywithdrawal of consent or end of the study Fig andTable RadiotherapyAll patients are subjected to preparation of individualpositioning devices and CTbased planning Motionmanagement may comprise either 4DCT or midbreathing CT image acquisition Further imaging modalitiessuch as FDGPETCT may be used when deemed necessary Gross tumor volumes GTV will be contouredand expanded by adequate clinical CTV and planningPTV safety margins in order to account for subclinicaldisease and positioning errors No elective nodal irradiation will be performed As for ans at risk bothlungs spinal cord heart and esophagus must be contoured In the HYPOarm no more than of œbothlungs minus GTV should receive Gy in the CONarm no more than of œboth lungs minus GTVshould receive GyIn the HYPOarm fractions of Gy will be administered total dose Gy corresponding to GyBED αβ In the CONarm fractions of Gywill be administered total dose Gy corresponding to GyBED αβ Dose deviations of ± are acceptable when clinically indicated Radiotherapy isscheduled to start within h after the first administration of durvalumab Dose prescription will follow international reports ICRU and Both 3Dconformal and modulated photon radiation techniquessuch as IMRT and VMATRapdArc are acceptable Allparticipating institution are encouraged to perform regular if no daily positioning control using either portalimaging or onboardCT devicesStudy proceduresIn order to minimize the risk exposure of patients whensubjected to the hypofractionated radiation regimen a 0cBozmehr BMC Cancer Page of Table Complete list of inclusion and exclusion criteriaInclusion criteriacid129 Fullyinformed written consent and locally required authorization obtained from the patient legal representative prior to performing any protocolrelated procedures including screening evaluationscid129 Age ‰¥ yearscid129 Histologically documented diagnosis of unresectable stage III NSCLCcid129 Nonfeasibility of sequential chemoˆ’radiotherapy as determined by the site™s multidisciplinary tumor board if there is no tumor board then thisdecision will be made by the investigator in consultation with a radiation oncologist if the investigator is not a radiation oncologist or by the investigator in consultation with an oncologist if the investigator is not an oncologistcid129 Fulfills at least one of the following criteria—‹ ECOG —‹ ECOG and CCI ‰¥ —‹ Age ‰¥ yearscid129 Must have a life expectancy of at least weekscid129 FEV1 ‰¥ of predictedcid129 DLCO ‰¥ of predictedcid129 FVC or VC ‰¥ of predictedcid129 At least one measurable site of disease as defined by RECIST criteriacid129 Adequate bone marrow renal and hepatic functioncid129 Female patients with reproductive potential must have a negative urine or serum pregnancy test within days prior to start of trialcid129 Evidence of postmenopausal status or negative urinary or serum pregnancy test for female premenopausal patientscid129 The patient is willing and able to comply with the protocol for the duration of the study including hospital visits for treatment and scheduledfollowup visits and examinationsExclusion criteriacid129 Concurrent enrollment in another clinical study or enrollment within days prior to first dose of treatment unless it is an observational noninterventional clinical study or during the followup period of an interventional studycid129 Prior immunotherapy or use of other investigational agentscid129 History or current radiology suggestive of interstitial lung diseasecid129 Oxygendependent medical conditioncid129 Any concurrent chemotherapy investigational product IP biologic or hormonal therapy for cancer treatmentcid129 Prior thoracic radiotherapy within the past years before the first dose of study drugcid129 Major surgery within weeks prior to enrollment into the study patients must have recovered from effects of any major surgery Local nonmajorsurgery for palliative intent is acceptablecid129 Active or prior documented autoimmune or inflammatory disorders with the following exceptions Patients with vitiligo or alopecia patients withhypothyroidism stable on hormone replacement or any chronic skin condition that does not require systemic therapy Patients without activedisease in the last years may be included but only after consultation with the study physiciancid129 Active uncontrolled inflammatory bowel disease Patients in stable remission for more than year may be includedcid129 Uncontrolled intercurrent illness including but not limited to ongoing or active infection symptomatic congestive heart failure uncontrolledhypertension unstable angina pectoris uncontrolled cardiac arrhythmia interstitial lung disease gastrointestinal conditions associated with diarrheaor psychiatric illnesssocial situations that would limit compliance with study requirement substantially increase risk of incurring AEs or compromisethe ability of the patient to give written informed consentcid129 History of another primary malignancy except for a malignancy that has been treated with curative intent and was not active for ‰¥ years beforethe first dose of IP and of low potential risk for recurrence or adequately treated nonmelanoma skin cancer or lentigo maligna without evidence ofdisease or adequately treated carcinoma in situ without evidence of diseasecid129 History of leptomeningeal carcinomatosiscid129 History of active primary immunodeficiencycid129 History of allogenic an or tissue transplantationcid129 Clinical diagnosis of active tuberculosiscid129 Positive testing for hepatitis B virus surface antigen or hepatitis C virus RNA indicating acute or chronic infection or for human immunodeficiencyviruscid129 Current or prior use of immunosuppressive medication within days before the first dose of durvalumab The following are exceptions to thiscriterion Intranasal inhaled topical steroids or local steroid injections systemic corticosteroids at physiologic doses not to exceed mgday ofprednisone or its equivalent steroids as premedication for hypersensitivity reactionscid129 Receipt of live attenuated vaccine within days prior to the first dose of IPcid129 Female patients who are pregnant or breastfeeding or male or female patients of reproductive potential who are not willing to employ effectivebirth controlcid129 Known allergy or hypersensitivity to any of the IPs or any of the constituents of the productcid129 Any coexisting medical condition that in the investigator™s judgement will substantially increase the risk associated with the patient™s participationin the studycid129 Patient who has been incarcerated or involuntarily institutionalized by court order or by the authorities § Abs S Nr AMGcid129 Patients who are unable to consent because they do not understand the nature significance and implications of the clinical trial and thereforecannot form a rational intention in the light of the facts [§ Abs S Nr 3a AMG]safety leadin phase with stopandgo design will precedefull enrollment into the HYPOgroup Fig Toxicitywill be evaluated with a design that is based on thestatistical assumption that ‰¤ event in n patientsconforms to a nontoxicity scenario with œevent beingdefined as the occurrence of pneumonitis grade ‰¥ within weeks after end of TRT Consequently twoevents in the first six patients or two events in the first 0cBozmehr BMC Cancer Page of Table Schedule of assessmentsProcedure Point in timeInformed Consent eligibility criteria demographicsmedical and historyAllocation RandomizationPrior and Concomitant Medication ReviewDurvalumab administrationRadiotherapy CONa or HYPObAEsFull Physical ExaminationDirected Physical ExaminationVital Signs O2 Saturation and WeightPulmonary function tests12lead ECGECOG Performance StatusPregnancy Test CBC with Differential Serum ChemistryPanel Thyroid function testHBV HCVUrinanalysisTumor ImagingFACTL and G8 screening questionnaireTissueScreening Treatment Cycles Q4WScreening C1D1 C1D4 C2D1 C3D1 C4D1XC5D1C13D1EOT PostTreatmentEOT Safety FU FU Q12WXXXXXXXXXXXXXXXXXXXXXXXXXXXXXcXX cXXXXXXXXXXXXXtogether with staging XWhenever clinically indicatedXXXXXXXXXXXXXXXXWhenever clinically indicatedXdXdXeQ8WeXfXXXtogether with staging XXXgXhXXiOptional ReBiopsy at time of progressionXiXXBlood and stoolaCONgroup radiation scheme Patients receive conventional fractions of — Gy Gy within weeks of TRT to be started within h after start ofdurvalumab treatmentbHYPOgroup radiation scheme Patients receive hypofractionated thoracic radiotherapy consisting of — Gy Gy within weeks of TRT to be startedwithin h after start of durvalumab treatmentcTo be performed on C2D1 and C3D1 if in accordance with local standarddChest Xray to be performed on cycles and if in accordance with local standardeFirst onstudy CT imaging to be performed weeks after first durvalumab administration Further onstudy imaging to be performed Q8W days ± daysfOnly applicable if EOT not according to already detected disease progressiongIn patients with EOT not due to disease progression tumor imaging will be performed until the start of a new anticancer treatment disease progression deathwithdrawal of consent or the end of the studyhQuestionnaires will be collected until disease progression only and may be collected by telephone callsiBiomarker sample to be taken prior to first study drug medication either during screening or C1D1 visitX or two events in the first patients will result in termination of the HYPOgroup Fig During this safety stopandgo phase patients will beallocated to treatment arms asfollows While theHYPOarm is recruiting patients will exclusively enterthis treatment group During safety evaluation of the sixpatients of a HYPOcohort Stop Go decision patientswill be allocated to the CONgroup only If safety criteria in the HYPOcohort are met the HYPOarm willbe re ed to continue toxicity assessment and patients will solely be allocated to this arm Fig Inorder to avoid any bias patients will be allocated centrally by the study management during this phase If thenontoxicity criteria in the safety cohorts are met thestudy will proceed to theandremaining patients will be randomized into the twotreatment arms using an interactive web responseexpansion phasesystem integrated in the electronic case report formeCRF A possibly uneven distribution of patients between the treatment groups at this stage will be compensated by a randomization strategy based on the œbiasedcoin method [ ] In the randomization phase patients will be stratified according to tumor stage stageIIIA vs stage IIIBIIICIn total patients will be enrolled per group Aftern patients have been enrolled to the HYPO orCONtreatment arm respectively an interim efficacy analysis for the respective arm will be conducted based on theobjective response rate ORR at weeks after first durvalumab administration In case of insufficient efficacy ofone of the arms ie the number of patients who haveachieved a response is eight out of or lower this treatment arm will be terminated Recruitment will be halteduntil results of the interim efficacy analysis are available 0cBozmehr BMC Cancer Page of Tumor response will be assessed according to RECIST by CT and or MRI scans at baseline weeks afterdurvalumab initiation and then every weeks Safetymeasures willinclude physical examinations performance status ECOG clinical laboratory profiles and continuous assessments of AEs An overview of all studyprocedures is presented in Table An Independent Safety Monitoring Board ISMB willensure the continued safety of participants throughoutthe trial Data management and data quality assurancewill be conducted following the Standard OperationalProcedures of the Institute of Clinical Cancer ResearchIKF at Northwest Hospital GmbH Frankfurt GermanyAn eCRF will be carefully maintained for each participant for data collection also reporting serious and nonserious AEs following the common criteria for adverseevents CTCAE version throughout the entire trialAfter the end of the study participants will be proactively followed up regarding treatmentrelated AEsuntil resolved returned to baseline or considered irreversible until lost to followup or withdrawal of studyconsent All subjects will be followed for survival Patients who decline to return to the site for evaluationswill be offered a followup FU by phone every months as an alternative At the end of the study treatment the investigators are responsible for the furthertreatment of the patient and must ensure that he or shereceives appropriate standard of care or other appropriate therapiesSampling for translational researchIf patients participate in the translational research program blood samples will be collected prior to cycles and and at the time of disease progression or end oftreatment EOT along with stool samples Table Samples of untreated tumor lesions serving as baselinespecimens will be collected as paraffinembedded tissueIf rebiopsies are taken at the time of progression samples should also be also submitted for translationalresearchStudy endpointsThe primary endpoint of the study will be toxicity defined by the occurrence of treatmentrelated pneumonitis grade ‰¥ The ORR evaluated weeks after firstdurvalumab administration according to RECIST isset as the primary efficacy endpoint Secondary endpoints ofthe occurrence oftreatmentrelated AEs and serious AEs SAEsfrequency of prespecified abnormal laboratory parametersprogressionfree survival PFS and duration of clinicalbenefit metastasisfree survival overall survival OSand QoLcomprisethestudyPatient vulnerability and its association with survivaland outcome will be assessed as an exploratory endpoint To this end the G8screening questionnaire asimple and fast screening tool for identifying geriatricrisk profiles with a strong prognostic value for functionaldecline and OS in older populations with cancer will beused [] Furthermore biomaterials will be collectedduring the trial for correlation analyses on selected molecular parameters and clinical data in order to identifymolecular biomarkers predictive for response to therapyThisto obtainhypothesisgenerating data for future research due to theexplorative character of this trialapproach is deemed appropriateStatistical analysisSample size justificationSafety runin phase HYPOgroup With regard to thepneumonitis grade ‰¥ rate this phase is designed to distinguish between a toxicity scenario pTox and anontoxicity scenario pTox A sample size ofn will yield a probability of to correctly detectthe toxicity scenario while the nontoxicity scenario willcorrectly be detected with a probability of Theseprobabilities are based on the decision rule that if thenumber of patients with pneumonitis grade ‰¥ in thiscohort is ‰¥ recruitment to the HYPOgroup will beterminatedanalysisInterim efficacyregarding ORR andexpansion phase An interim efficacy analysis based onthe ORR will be conducted after n patients in eacharm have completed radiotherapy and the 18th patienthas undergone first radiographic assessment at weeksafter first durvalumab dosePreviously an ORR of after radiotherapy alonehas been reported in a Japanese population of elderly patients with unresectable stage III NSCLC [] Based onthis and the observation that Asian ethnicity is associated with a favorable prognosis we assume for theTRADEhypo trial that an ORR higher than in bothtreatment arms can be demonstrated ie the null hypotheses for arm HYPO and CON are defined as H0HYPO Ï HYPO ‰¤ and H0CON Ï CON ‰¤ where ÏHYPO and Ï CON denote the actual ORR in arm HYPOand CON respectively [ ] Under the alternativehypothesis it is assumed that both Ï HYPO and Ï CONamount to Using an optimal Simon™s twostage design with a onesided significance level of α and apower of 1β for each hypothesis test n patients per arm are required while the interim analysis isconducted after n patients per arm have been recruited to the trial After successfully passing the safetyanalysis in the HYPOgroup if among patients in the 0cBozmehr BMC Cancer Page of HYPO or CONarm the number of patients who haveachieved a response is eight or lower the respective armwill be closed Otherwise an additional number of patients will be enrolled into the respective arm Thenull hypothesis of the respective arm can be rejected ifat least of all patients per arm achieve a responseSample size calculation was done using the R packageœOneArmPhaseTwoStudy []To account for an estimated dropout rate of fourpatients will additionally be recruited to each treatmentarm Deviations from planned sample sizes will be handled as described by Englert Kieser allowing strictcontrol of the aspired type I error rate in each arm []Methods of statistical analysis The primary populationfor evaluating all efficacy endpoints and subject characteristics is defined as all patients enrolled according toinitial allocationrandomization intentiontotreat population IIT Secondary efficacy analyses will be carriedout on the perprotocol PP population The safetypopulation comprising all patients who received at leastone dose of the study medication will be used for allsafety endpoints and will be analyzed according to theactual treatment receivedResponse rates with confidence intervals CI and pvalues in both study arms will be estimated taking intoaccount the twostage nature of the design [ ] Secondary endpoints will be evaluated descriptively All toxicities will be summarized by relative and absolutefrequency and severity grade based on CTCAE V50 AEand SAE summary tables will provide the number andpercentage of patients with AEs and the ClopperPearson type CIs All analyses will be done using SASversion SAS Institute Cary NC or higherTrial statusAs of July 15th eight study sites are initiated Firstinitiation coincided with the beginning of the Covid19pandemic in Germany Therefore recruitment of patients was withheld On May 8th recruitment wasresumed after consultation with the ISMB The first patient was enrolled on July 13th renal carcinoma and NSCLCDiscussionIn recent years the concept of restoring the patients™ antitumor immunity by immune checkpoint inhibition hasrevolutionized cancer therapy especially in advancedmelanomaImmunecheckpoint molecules efficiently regulate T cell activation and thus enable tumor cells to evade the immunesystem for example by hijacking the PD1 PDL1 interaction to downregulate effector T cells [ ] To dateseveral human monoclonal antibodies pharmacologicallyblocking these interactions have been implemented incancer therapy such as the antiPD1 antibody pembrolizumab that has been approved in combination withchemotherapy for nonsquamous NSCLC irrespective ofPDL1 expression [ ]Several studies have shown that immune checkpointinhibition and radiotherapy in combination can act synergistically to further boost antineoplastic effects [“] Although in a large phase III trial no benefit ofblockade of cytotoxic T lymphocyteassociated antigen CTLA4 after radiotherapy was observed in metastaticprostate cancer [] clinical trials such as NICOLASNCT02434081 and DETERRED NCT02525757investigating concurrent PDL1directed immunotherapyand chemoradiot
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neprilysin nep is a neutral endopeptidase it is also known by different functional names such as common acute lymphoblastic leukemia antigen calla the cluster of differentiation cd10 endoprotease endopeptidase and membrane metalloen dopeptidase nep is a member of m13 family of zinc peptidase in the body nep cleaves many peptides such as atrial natri uretic peptides btype natriuretic peptides angiotensins i ii ii en ix bradykinin substance p endothelin i ii amyloid dorphin neurotensin vasopressin etc [“] the progression of various pathological conditions such as kidney and heart disease obesity diabetes [ ] few malignancies such as colon can a ˆ—corresponding author email address anoopkishoremanipaledu a kishore 101016jmolstruc2020129073 elsevier bv all rights reserved cer lung cancer and melanomas [“] etc is associated with the peptidase activity of nep in the us food and drug ad ministration fda approved sacubitrilvalsartan the combination of a neprilysin inhibitor and an angiotensin receptor blocker arb respectively commonly known as angiotensin receptor neprilysin inhibitor arni for heart failure with reduced ejection fraction further in clinical trials involving sacubitrilvalsartan treatment groups performed well in the renal failure population as compared to treatment with an arb valsartan alone there fore nep has gained considerable attention in the last decade for its peptide degrading property and its inhibition has therapeutic potential in multiple diseases but the known and available nep inhibitors are limited hence drug repurposing using different in silico tools can aid in speeding up the process of drug discovery for the development of new nep inhibitors the role of nep has been extensively studied in various dis eases the study report of the paradigm trial highlighted the role 0c r sankhe e rathi and s manandhar of molecular structure of nep inhibitors in the population of heart failure with reduced ejection fraction in an invivo study of subtotal nephrec tomy the renoprotective effect of sacubitrilvalsartan was found to be stronger as compared to valsartan alone according to the result of the uk harpiii trial the combination of sacubi trilvalsartan is effective and is welltolerated in the chronic kidney disease population similarly various studies are focussed on the importance of nep on chronic kidney and cardiovascular dis eases nep inhibition in streptozotocininduced diabetic mice im proved outcomes of cardiac function for heart failure with reduced ejection fraction in diabetic nephropathy the combination of the nep inhibitor thiorphan with an angiotensin receptor blocker and an angiotensinconverting enzyme ii activator showed significant improvement in the condition by modulating components of the reninangiotensin system and natriuretic peptide system the activation of the leptinaldosteroneneprilysin axis contributes to the pathogenesis of cardiac complications in obese patients in obesity and type diabetes nep inhibition showed improve ment in insulin sensitivity and glycaemic control the inhibition re sults in modulation of several peptides with glucoregulatory prop erties such as bradykinin cholecystokinin glycogen like peptide glucosedependent insulinotropic peptide secretin and vasoactive intestinal polypeptide leading to improved glucose homeostasis and weight loss a study conducted to evaluate the effect of nep on nociception concluded that nep inhibition can be a good strategy for pain management in cancers such as colon cancer [ ] lung cancer [ ] and melanomas the increased levels of nep is correlated with neoplastic progression the peptidase ac tivity of nep and its interaction with akt focal adhesion kinase is assumed to contribute to the pathogenesis of colon cancer in aggressive melanomas cd10 nep is the biomarker for detec tion a recent report has highlighted the role of arni in enhanc ing anti‚ammatory and natriuretic peptide systems in covid patients [ ] additionally the use of arni is also recom mended for patients suffering from covid19 all these find ings highlighted the need for designing novel nep inhibitors but de novo drug development is resource intensive and time consum ing hence drug discovery by repurposing the existing drugs can be an attractive strategy with the benefit of reduced developmen tal risk especially in the case of nep inhibitors the computation repurposing is known as ˜ insilico drug re purposing™ in in the us approximately of drugs ap proved was through the drug repurposing approach the con cept of drug repurposing has been already practiced in cardio vascular disorders cancer obesity erectile dysfunction smoking cessation stress psychosis etc drug repurposing using al ready approved drugs reduces the time and money on preliminary screening toxicity studies clinical trials bulk manufacturing and formulation development on the other hand the establishment of new drug candidates requires lots of time and resources a good example is the case of allopurinol which was originally approved for cancer and is now available for the treatment of gout in this context we decided to identify a series of inhibitors for nep using insilico drug repurposing the protein structure of the extracellular domain of nep with sacubitralat the active metabo lite of sacubitril was used in the current study the inhibitor bind ing pocket in the protein structure of the extracellular domain of human nep pdb id 5jmy has already been revealed by schier ing nikolaus the inhibitor binding pocket contains the catalytically essential triad of his583 his587 and glu646 for our drug repurposing study the structures of fda approved drugs were downloaded from the zinc database based on the binding pocket of the nep inhibitor the high throughput virtual screening of existing fda approved drugs was done to find out a new se ries of nep inhibitors to the best of our knowledge this is the first study based on drug repurposing approach that is being re ported and employed for the development of nep inhibitors using receptorinhibitor complex materials and methods in the current study the maestro molecular platform version by schrodinger llc was used to perform molecular dock ing and simulation studies on an hp desktop system with linux ubuntu lts platform intel haswell graphics card 8gb ram and intel core i34160 processor protein preparation and grid generation xray crystallographic structure of the extracellular domain of human nep pdb id 5jmy was downloaded from the rcsb pro tein data bank the pdb id 5jmy has a resolution of ˚a prior to docking and simulation studies the biological unit of protein was prepared using ˜protein preparation wizard™ in schrodinger suite during the process of protein preparation the protein was subjected to import and refine review and modify and minimize processes in protein preparation wizard missing side chains and residues were filled using the prime tool the active site and cat alytically important residues were retained in the protein structure the water molecules beyond ˚a were deleted and stages were generated for hetero atoms to generate low energy state protein energy minimization was done using opls3e optimized potential for liquid stimulation force field and the prepared protein was used for molecular modelling to generate a grid around the lig and the receptor grid generation workflow was used by keeping all functional residues in the grid ligand preparation the structures of fda approved drugs from zinc database were downloaded for ligand preparation the lig prep tool was employed the lowest energy 3d structures with cor ± under the opls3e related chiralities were generated at ph force field in this process all the ligands were preprocessed which includes generation of tautomers ionization state at ph ± using epik addition of hydrogen bond charged group neu tralization and ligand geometry were optimized ligand docking all the molecular docking studies were carried out using the ligand docking tool glide gridbased ligand docking with ener getics module the glide module was used for predicting ligand protein binding modes and ranking different scoring functions are involved in glide such as highthroughput virtual screening htvs standard precision sp and extra precision xp first all the drugs were docked with htvs mode but computationally htvs docking does not use descriptor and explicit water technol ogy as used in the xp mode hence to avoid falsepositive results few drugs were reanalyzed using sp and xp modes [ ] free ligand binding energy calculation the prime module was used to determine absolute ligand binding affinities to nep using mmgbsa molecular mechanics energies generalized born and surface area continuum solvation method the mmgbsa assay of top eight xp docked drugs was performed using pose viewer file of glide xp mode the prime mmgbsa method is dependent on the vsgb solvation model that uses a variabledielectric generalized born model and water as a solvent under the opls3e force field to calculate binding energy 0cr sankhe e rathi and s manandhar of molecular structure adme analysis for the assessment of the adme profile the qikprop tool from the maestro modeling platform was used the qikprop tool helps in the prediction of the druggable property of best four hits based on adme analysis during this process various descriptors such as molecular weight cardiotoxicity qplogherg predicted octanolwater partition coefficient qplogpow permeability qp pcaco polar surface area psa human oral absorption oral absorption and lipinski rule of five were calculated induced fit docking ifdsp table docking score and prime mmgbsa score of top eight drugs sr no drug dock score xp kcalmol mmgbsa 01g bind sacubitrilat zinc000001533877 zinc000000001427 zinc000001851195 zinc000000402909 zinc000000601283 zinc000000000797 zinc000003831594 zinc000028973441 ifdsp was carried out using the inducedfit docking module from maestro molecular modelling platform based on the xp glide docking score binding energy crucial residues involved and adme analysis four zinc0 zinc0 zinc0 and zinc0 drugs were selected for ifd“sp docking in ifd based on the bfactor side chains were trimmed with receptor and van der waals scaling of and respectively and a maximum of poses were set for each ligand further prime sidechain prediction and minimization were performed in which refinement of all residues within ˚a of the ligands™ pose and side chains were performed this pro cess allows the ligand structure and conformation to accommodate nearby reorienting side chains the ligands and residues were min imized in inducedfit protein structure all the ligands were rigor ously docked and ifd score for each was calculated using the for mula ˆ— prime_energy ˆ— glide score ˆ—ifd score glide_ecoul molecular dynamics md simulation the flexibility of the receptor is restricted in gridbased dock ing systems like xp and ifd these do not mimic the actual bio logical systems where the protein and drug are solvated in wa ter hence to tackle this problem md simulation was performed based on the glide docking score free binding energy and ifd score four drugs were selected for md simulation for 20ns for md simulation three steps were performed viz system builder mini mization and md simulation the docked complex of protein and ligand were selected and the system model was made by prede fined spc solvent under orthorhombic boundary conditions next the system model was subjected to energy minimization until a gradient threshold reached kcalmol ˚a balanced at k tem perature and bar pressure via npt ensemble in the final step minimized ligandprotein complex were subjected to md simula tion bioisostere replacement for optimization of adme and biological properties of top two selected compounds zinc0 and zinc0 the bioisostere replacement of functional group was performed the bioisosteric replacement tool from maestro molecular modelling platform was employed to create bioisosteric structures of better potency and adme profile further the results of the generated bioisosteres were analysed through interaction of ligands with crucial amino acid residues xp glide docking score free binding energy and adme analysis results nep was prepared at a neutral ph of αhelical α subdomains were present in the extracellular domain both helical subdomains of nep are connected with the linker region ± two and essential catalytic triad are present in the central cavity of both subdomains in the central cavity the catalytically impor tant zinc atom is coordinated with the side chains of amino acid residues his583 his587 and glu646 [ ] in the protein the cocrystallized ligand sacubitrilat is bound to the active site of nep and showed crucial interactions with his583 his587 and glu646 residues a fourth interaction was provided by the car boxylate oxygen adjacent to the p1 methyl group of sacubitri lat to generate a receptor grid receptor grid generation workflow was used and the cubic box of specific dimensions was generated around sacubitrilat to perform molecular docking studies ligand docking around ligands from zinc database were screened with htvs docking mode of glide panel htvs docking mode utilizes a small period to a large set of drugs by reducing the final torsional refinement and comprehensive sampling but during htvs dock ing mode the number of intermediate conformational sampling is limited hence a total of drugs with dock scores less than kcalmole were filtered and reanalyzed in sp docking mode after performing sp docking around drugs were subjected to an extensive xp docking mode of glide panel xp docking mode is more accurate avoids the possibility of falsepositive results and gives an appropriate correlation between a good pose of drugs and a good dock score finally based on xp dock score and pivotal interactions eight active drugs zinc0 zinc0 zinc0 zinc0 zinc0 zinc0 were identified for further screening the docking score of cocrystalized ligand sacubitralat was found to be all the eight selected drugs showed docking scores between to given in table zinc0 zinc0 all the eight drugs showed similar interaction as sacubitri lat schiering nikolaus et al had reported that the hydropho bic interaction of sacubitrilat with phe544 was towards the shal low s1 pocket of nep protein the charge positive interac tion with arg717 and polar interaction with asn542 were found to be common in sacubitrilat and selected eight drugs even in this study all the eight drugs showed hydrophobic interactions with phe544 sacubitrilat also showed interactions with asn542 arg717 arg110 and arg102 our eight selected drugs showed in teractions with atleast two of the aforementioned residues insilico docking studies also showed that all the eight drugs showed in teraction with his711 which then formed a hydrogen bond with zinc causing the stabilization of zinc transition state this in teraction with zinc and its stabilization might result in decreased catalytic activity of nep as it is a zinc dependent endopeptidase nep degrades various peptide substrates at the amino sides of hydrophobic amino acids according to the reports the pro tein structure of nep consists of a large hydrophobic pocket con taining the side chains ala543 ile558 phe563 met579 val580 0c r sankhe e rathi and s manandhar of molecular structure his583 val692 and trp693 the cocrystalized ligand sacu bitrilat showed hydrophobic interaction with ala543 ile558 phe563 met579 val580 val692 and trp693 the eight se lected drugs also showed hydrophobic interaction with ala543 ile558 phe563 met579 val580 val692 and trp693 but the hydrophobic interaction with ile558 met579 and trp693 were missing in interactions of zinc0 zinc0 and zinc0 respectively sacubitrilat and the selected eight drugs showed polar pipi stacking and cation interaction with his583 the interactions with side chains of ala543 ile558 phe563 met579 val580 his583 val692 and trp693 may con tribute to inhibition of the peptidase activity of nep according to previous reports amino acid residue glu584 is important for peptidase activity and residues such as ala543 and asn542 are important for nep inhibition in the current study all eight selected drugs possess interaction with glu584 asn542 and ala543 the 2d interaction diagrams with a summary of all non bonding interactions are given in table free ligand binding energy calculation the primemmgbsa was employed to calculate the binding en ergy of the top eight drugs with selected docked poses all the 01g bind eight drugs showed stability in the docked pose with 01g bind ing energy kcalmol described in table the ing energy of cocrystallized drug sacubitrilat was found to be 9651kcalmol the cocrystalized ligand and the eight drugs were found to be stable with docked pose this finding indicates that the selected drugs may act as nep inhibitors induced fit docking ifdsp after the virtual docking studies based on the ligand interac tion and binding energy of the eight drugs four ligands showing good values were taken forward for induced fit docking ifd in virtual docking protocol the interactions occur between the bind ing site of the rigid protein and the flexible ligand but this is not the case with the actual ligandprotein interactions in the body where the target protein undergoes backbone or sidechain move ments after binding with ligands this induces alteration in binding sites of the protein also in the body the ligand binding site on the proteins conforms to the ligand shape and binding mode ifd was conducted to resolve the shortcomings of rigid docking pro tocols ifd has two main applications first it generates the most accurate active complex structure of ligand which is not possi ble in virtual molecular docking with rigid protein structure sec ond ifd avoids falsenegative results of virtual docking in virtual docking screening of the ligands was done with the single confor mation of ligands however in ifd confirmers were generated for each ligand hence ifdsp was carried for zinc0 zinc0 zinc0 and zinc0 and a maximum of conformers were generated for each ligand based on molecular docking and binding energy further the ifd score and ligand interaction were analyzed for selected drugs the ifd score and 3d ligand interactions are given in fig zinc0 showed similar nonbonding interactions as predicted in xp docking the zinc0 exhibits a new hbond interaction with his711 with similar nonbonding interactions as observed in xp docking in ligand interactions of zinc0 the new hbond interaction was observed with his711 and lost with glu584 the hydrophobic interaction with ala543 val580 met579 phe689 val692 trp693 phe563 and phe106 was also lost similarly new hydrophobic interaction was observed with ile718 and lost with ile558 and phe544 the new pipi stacking interactions were observed with trp693 and phe106 and missing with amino acid residue his583 the pipi cation interaction with arg717 was retained and lost with arg110 as predicted in xp docking zinc0 retained hbond interaction with his711 and glu584 showed new hbond inter action with trp693 and lost hbond interaction with arg717 the new pipi stacking interaction was observed with phe106 zinc0 also showed new hydrophobic interaction with phe689 and met579 and hydrophobic interaction missing with tyr545 it also showed similar hydrophobic interaction patterns with other amino acid residues as predicted in xp docking adme analysis adme properties of the four drugs were analyzed using the quikprop module the adme profile was assessed using vari ous descriptor calculations such as molecular weight qplogherg qplogpow qppcaco human oral absorption psa and lipinski rule of five given in table all the selected drugs obey the lip inski rule of five molecular dynamics md simulation molecular dynamics is used to simulate ligandprotein com plexes in presence of systems with biological relevance it includes the explicit solvent representation with the entire protein the main advantage of md stimulation is that it represents the actual conditions of the biological system it provides a highly dynamic protein structure and the ligandprotein complex is solvated with water as happens in the biological system ifd however pro vides limited flexibility which is insufficient to mimic the actual conditions of a biological system hence md simulation studies were carried out to get insights into the top four drugs in terms of binding stability and nonbonding interactions with crucial amino acid within the drugbinding pocket of nep protein in a dynamic state in md simulation the frame was captured for 20ps which results in the generation of frames for 20ns stimulation time and saved in a trajectory further rmsd root mean square devi ation for nep protein and ˜lig fit prot™ for the ligands were com puted to estimate the stability of ligandprotein complex based on molecular docking score binding energy and ifd score the md simulation was carried out for four ligand protein complexes viz zinc0 01427nep docked complex complex zinc0 01533877nep docked complex complex zinc0 0601283nep docked complex complex and zinc0 03831594nep docked complex complex for com plex rmsd values for protein and ligand were found to be ˚a and ˚a respectively the rmsd values were found to be in the acceptable range ˚a but the drift in the ligandprotein complex was observed for a period of 05ns20ns in case of complex the ligandprotein stabilization was observed from 022ns and 59ns respectively and drift was observed for 720ns in complex the rmsd values are ˚a and ˚a for protein and ligand respectively for complex the rmsd values were found to be ˚a for both the complex was initially stable but there was drift for 313ns and eventually stabilization was observed for 1320ns according to the results obtained from md simulation complex is possibly more stable than complex and similarly complex showed rmsd value of ˚a for both the protein and the ligand the com plex showed initial drift from to 13ns but eventually stabi lized from 1320ns overall better stability in protein and ligand was observed in complex and compared to complexes and the rmsd plot of selected ligandprotein complexes are given in fig further the binding pattern and nonbonding interactions were analyzed for all four complexes the binding pattern was found to be different for all four complexes in complex the signifi 0cr sankhe e rathi and s manandhar of molecular structure table 2d interaction diagrams of top eight drugs with a summary of all nonbonding interactions sr no drug 2d ligand interaction diagrams nonbonding interaction sacubitrilat zinc000001533877 zinc000000001427 zinc000001851195 hbond glu584 his711 arg717 arg102 asn542 hydrophobic met579 val580 ile558 phe689 val692 trp693 phe563 phe106 ile718 ala543 phe544 polar his583 his587 asn542 salt bridge zn806 arg102 pipi stacking trp693 his583 charged positive arg102 his711 arg717 arg110 charged negative asp650 glu646 glu584 hbond arg717 glu584 ala543 asn542 hydrophobic ile718 phe689 val692 trp693 ala543 phe544 met579 val580 phe106 ile558 phe563 polar thr721 his587 his583 asn542 salt bridge zn806 his711 arg110 pipi cation his583 charged positive his711 arg717 arg110 charged negative glu646 asp650 glu584 hbond ala543 his711 glu584 hydrophobic ile558 phe544 ala543 val580 met579 ile718 phe689 val680 trp693 phe563 phe106 polar asn542 his583 his587 salt bridge zn806 pipi stacking his583 trp693 charged positive arg717 his711 charged negative asp650 glu646 hbond glu584 his711 ala543 trp693 hydrophobic ile718 ile558 ala543 phe544 phe689 ala690 val692 trp693 met579 val580 phe563 phe106 polar thr721 his587 his583 asn542 salt bridge zn806 pipi stacking trp693 charged positive arg717 his711 arg110 charged negative asp650 glu646 glu584 zinc000000402909 hbond his711 glu584 hydrophobic ile718 ala543 phe544 phe689 val692 trp693 met579 val580 phe106 phe563 polar his587 his583 asn542 pipi stacking phe106 his583 salt bridge zn806 charged positive arg717 his711 charged negative asp650 glu646 glu584 continued on next page 0c r sankhe e rathi and s manandhar of molecular structure table continued sr no drug 2d ligand interaction diagrams nonbonding interaction zinc000000601283 zinc000000000797 hbond his711 glu584 hydrophobic phe544 ala543 trp693 val692 phe689 val580 met579 phe106 ile558 phe563 polar his587 his583 asn542 salt bridge zn806 pipi stacking his583 pipi cation arg717 arg110 charged positive arg102 arg110 his711 arg717 charged negative asp709 glu646 glu584 asp650 hbond asn542 hydrophobic ile718 val580 met579 phe689 val692 trp693 ile558 ala543 phe544 phe563 phe106 polar his587 his583 asn542 salt bridge zn806 pipi stacking his711 phe544 his583 pipi cation his711 charged positive arg717 his711 charged negative glu646 glu584 asp650 zinc000003831594 hbond glu584 his711 arg717 hydrophobic val580 ala543 phe544 tyr545 phe106 phe563 ile558 trp693 val692 polar his587 his583 asn542 salt bridge zn806 charged positive arg717 his711 arg110 arg102 charged negative glu646 glu584 zinc000028973441 hbond glu584 his711 hydrophobic met579 val580 phe544 ala543 phe106 trp693 val692 phe563 ile558 polar his587 his583 asn542 salt bridge zn806 pipi stacking phe106 pipi cation arg110 his711 charged positive arg717 his711 arg110 arg102 charged negative glu646 glu584 asp650 0cr sankhe e rathi and s manandhar of molecular structure fig 3d ifd ligand interactions and scores of the top four selected drugs ligand interaction of a zinc0 b zinc0 czinc0 0601283d zinc0 with different amino acid residues of nep fig rmsd plot of ligandprotein complexes rmsd plot of a zinc0 b zinc0 c zinc0 d zinc0 with the active site of nep 0c r sankhe e rathi and s manandhar of molecular structure table adme analysis of top four selected drugs using qikprop compound id molecular weight qplogp ow qplogherg qplogs qppcaco oral absorption psa rule of five sacubitrilat zinc000001533877 zinc000000001427 zinc000000601283 zinc000003831594 fig ligandprotein interaction diagram obtained after md stimulation ligand interaction of a zinc0 b zinc0 c zinc0 d zinc0 with different amino acid residues of nep cant hbond interactions were observed with amino acid residues glu584 ala543 and his711 and pipi interaction with his583 and trp693 as predicted in xp docking the hydrophobic interac tions with ala543 trp693 met579 and phe689 were retained in md simulation on the other hand hydrophobic interactions with ile558 phe544 and phe563 were missing in md simulation the hydrophobic interaction with ala543 val580 ile718 val692 and phe106 was weaker affecting the stability of the ligand protein complex similarly the water bridgetype interaction with glu584 was observed in complex strong hbond interaction was shown by asn542 arg717 glu584 and ala543 additional hbond interactions were also observed with his711 and glu646 the hydrophobic interaction with ala543 ile718 phe689 trp693 met579 val580 ile558 phe106and phe563 were weakly con tributing to the stability of ligandprotein complex and the inter action was lost with the amino acid residue phe544 additional water bridge type of interaction was shown by asn542 glu646 and ala543 the pipi cation interactions were retained with his583 as predicted in xp docking in complex hbond interac tion was retained with glu584 and his711 and new hbond inter action was observed with asp709 and arg110 in md simulation complex showed weak hydrophobic interaction with ala543 phe544 val580 trp693 phe563 ile558 and phe106the hy drophobic interaction was lost with amino acid residues met579 phe689 and val692 the new pipi stacking interaction was ob served with his711 however pipi stacking interaction was missing with his583 the new pipi cation interaction was observed with arg717 and pipi cation interaction was missing with arg110 as compared to xp docking the additional water bridge type of inter action was shown by asp709 and glu584 in complex hbond interaction was retained with his711 and arg717 new hbond in teractions were found with trp693 and ala543 whereas hbond interaction was lost with glu584 complex showed strong hy drophobic interaction with trp693 and ala543 whereas weak hy drophobic interaction with val680 phe106 phe563 ile558 and val692 in contrast to xp docking similarly hydrophobic interac tion was missing with amino acid residues phe544 and tyr545 the additional water bridge type of interaction was observed with ala543 among all four complexes complexes and were found to more stable the additional hbond interactions in complexes and may contribute to the stability of the ligandprotein com plexes the ligandprotein md interaction diagrams and histograms of selected complexes are given figs and bioisostere replacement the zinc0 indomethacin a nonsteroidal anti ‚ammatory drug and zinc0 tyropanoic acid a ra diocontrast agent were found to be more stable in md simulation for 20ns the zinc0 is anti‚ammatory antipyretic 0cr sankhe e rathi and s manandhar of molecular structure fig histogram of ligandprotein complexes histogram of a zinc0 b zinc0 c zinc0 d zinc0 with different amino acid residues of nep and analgesic in nature it is commonly used in rheuma toid arthritis acute shoulder pains osteoarthritis spondylitis and acute gouty arthritis zinc0 is known as sodium tyropanoate which is employed in xray diagnosis and imaging of gallstones though they exhibit good binding affinity for nep one of the major disadvantages of zinc0 is its rapid elimination from the body [ ] therefore bioisostere re placement of zinc0 and zinc0 was per formed to enhance biological activity and surpass rapid excretion bioisosteres are the molecules which are generated by replace ment an atom or a group of atoms from the parent drug with other functional groups two main advantages associated with bioisostere replacement are first it will result in generation of new bioisostere molecules with similar biological characteristics of the parent drug second bioisosteres can overcome various prob lems associated with the parent drug™s activity pharmacokinetics and toxicity during the bioisosteric replacement and bioisosteric structures of zinc0 and zinc0 respec tively were generated out of these the top two bioisosteres were identified based on the ligand interactions with the crucial amino acid residues of nep docking score the binding energy calculated employing mmgbsa and adme parameters the top two selected bioisosteres of zinc0 and zinc0 are il lustrated by fig the docking scores of the bioisosteres of zinc0 structure structure are and with binding en ergies and kcalmol respectively similarly the dock ing scores of structure and of zinc0 were found to be and with binding energies and Ï Ïkcalmol respectively table further assessment was done based on the ligand interactions with crucial amino acid residues of the protein compared to the parent drugs table structure of zinc0
0
ulcerative colitis is a type of ‚ammatory bowel disease thatcan potentially lead to cancer e age of onset of colitis istypically “ years old and it can seriously threaten thequality of life of patients e immunopathogenesis andimmunosuppressive treatment of colitis are currently theresearch topics of significant interest e research goals areto diagnose and treat colitis in order to prevent exacerbationof the disease e drugs used to treat colitis in the clinicoften have adverse eï¬ects after their longterm applicationanother crucial area of colitis research is focused on thediscovery of functional foods that can prevent colitis withoutside eï¬ects natural plants including aegle marmeloslinn also have the intervention eï¬ect on colitis a recentstudy has shown that the intestinal flora is closely related tocolitis and that the intestinal flora participates in the mucosalimmune response bacteria are an important promoter of‚ammatory bowel disease e symptoms of colitis can bealleviated by regulating the intestinal flora preventing floralimbalance and increasing the number of probiotics yak yoghurt is a natural fermented food that is rich innutrients and is common in the minority areas of theqinghai tibet plateau previous research has suggested thatyak yoghurt exerts various physiological activities such asantioxidationimmunitycholesterolreductionand 0cevidencebased complementary and alternative medicineenhancement e qinghai tibet plateau has a specificclimate and unique environment for the fermentation of yakyoghurt additionally the availability of yak milk and specialtibetan fermentation utensils eg certain fermentationmicroanisms can make the flavor and quality of yakyoghurt diï¬er greatly from ordinary fermented milk aprior study on the intestinal physiological activity of lacticacid bacterial species in yak yoghurt showed that the lacticacidproducing bacteria isolated from yak yoghurt hadantioxidant and constipation preventing eï¬ects in this studythe potential eï¬ects of lactobacillusplantarum ys4 lpys4 on oxazolidoneinduced colitiswere investigated for the first time e findings provide apossible foundation for further development of lpys4especially its application in functional food or medicineratio � solution massnormal group were daub treated with ml of ethanolwith those in the remaining four groups being daub treated with ml of oxazolidonesolvent � after treatment for days the mice wereanesthetized en the blunt head of a silicone tube wasinserted into the intestinal tract from the anus of the mouse at adepth of cm e mice in normal group were administeredwith ml of ethanol solution while those in theremaining four groups were administered with ml of oxazolidone solution mass ratio � solvent � ethanoltwenty seconds later the catheters were removed and the micewere lifted up by their tails for half a minute on the last dayof treatment day all the mice were sacrificed by decapitation and their plasma samples and colon tissues were collected e length and weight of the colon were documentedexperiment animal materials materials and methodsand reagentsexperimental strain the strain was isolated from yak yoghurt in the yushu area of qinghai province china by ourteam it was named lpys4 and stored in china center fortype culture collection cctcc wuhan china nom2016750 e negative control strain lb was purchasedfrom the cctcc no ab fifty male balbc mice weeks old were purchasedfrom the experimental animal center of chongqingmedical university certificate no syxk yu “oxazolone was purchased from sigmaaldrich co llcusa il2 il10 et1 sp ss and vip serum cytokine kitswere purchased from biolegend inc usa gsh sodmpo and mda kits were purchased from nanjing jiancheng bioengineering institute nanjing china trizol reagent oligodt18 rnase dntp mlv primer bca proteinquantitative kit aps temed sdspage pvdf membrane first antibody and second antibody were purchasedfrom ermo fisher scientific inc usa instruments and equipment imark microplate readerwas purchased from biorad usa steponeplus pcrinstrument was purchased from ermo fisher scientificinc usa tanon chemiluminescence imager waspurchased from tanon science and technology co ltdchina sas v91 statistical software package was purchasedfrom sas institute inc usalb animal grouping and intervention a total of balbcmale mice were assigned to five groups model groupnormal group lactobacillus bulgaricustreatmentgroup and highdose lpys4h and lowdoses lpys4l treatment groups and there were mice in each groupe mice in lb lpys4h and lpys4l groups were fedwith — — and — cfukg ml livingbacteria physiological saline of each corresponding straindaily by oral gavage for consecutive days and normal andmodel groups were fed with ml physiological salineafter days of treatment the abdomens of all mice wereshaved with an area of cm — cm e mouse abdomens in detection of endothelin1 et1 substance p spsomatostatin ss and vasoactive intestinal peptide vipconcentrations in serum samples e whole blood samplesof mice were allowed to clot at room temperature for h andthen centrifuged at rpmmin for min after collecting the serum samples the concentrations of et1 sssp and vip were detected using commercial kits determination of interleukin2 il2 and interleukin10il10 levels in serum samples e mouse serum sampleswere prepared according to section en the serumlevels of il2 and il10 cytokines were assessed usingcommercial kits determination of glutathione gsh malondialdehydemda myeloperoxidase mpo and superoxide dismutasesod activities in colon tissues a mixture of g colontissue and ml normal saline was prepared at weightratio after homogenizing the mixture the activities of gshmda mpo and sod in colon tissues were evaluated usingcommercial kits pathological observation of he staining e lesionsite — — cm of colon was cut with a scalpel etrimmed tissue and corresponding label were placed in neutral formalin solution for h e colon tissue wasdehydrated embedded sliced dewaxed stained and thendehydrated transparent and sealed finally the pathologicalstate of colon tissue was observed under a microscopebx43 olympus tokyo japan qpcr assay total rna was isolated using rnazol andthen diluted to the final concentration of μgμl for cnasynthesis μl of the diluted rna extract was taken andcdna was prepared using a reverse transcriptase kit enthe cdna template μl was added into μl of sybrgreen pcr master mix and μl of forward primer andreverse primer each table qpcr amplification wascarried out for cycles under the reaction conditions of95oc s °c s °c s and °c s followed by 0cevidencebased complementary and alternative medicineckitinosenosnnossequencegene nametable sequences of primers used in this studyforward ²agagagatcgggttcaca3²reverse ²cacagaactgagggtaca3²forward ²tcgtccaacttctgggctctt3²reverse ²ccttctcttcctcccctctcttc3²forward ²tcagccatcacagtgttccc3²reverse ²atagcccgcatagcgtatcag3²forward ²catagcccaggtaaagcacaat3²reverse ²gaacactccagaatcgtcaactcforward ²tcagggactacgctgcgaaag3²reverse ²aagagctggcagaccgactca3²forward ²tgcaccaccaactgcttag3²reverse ²gatgcagggatgatgttc3²Î´ctdetection gene δctgapdh measured according to the following equation ˆ’δct �cycle under the reaction conditions of °c s and°c s gapdh was used as internal control for thisdetermination and the relative mrna expression was²gapdhscf western blotting mg of tissue samples was mixedwith μl of pmsf and ml of ripa and then homogenized at rmin 4oc for min protein quantificationwas conducted using the bca protein quantitative kit andthe protein samples were diluted to μgml en thediluted protein and sample buï¬er were mixed at heatedat 100oc for min and icebathed for min subsequentlyacrylamide starting buï¬er resolving buï¬er temed aps and diï¬erentiated water were mixed in specific proportions in order to prepare sdspage separation glue andconcentration glue e prestained samples and proteinladder were placed into the sample hole of the rubber sheetrespectively and then the proteincontaining sdspageglue was subjected to vertical gel electrophoresis for minafter activation with methanol for min the pvdf wereblocked with skimmed milk in — tbst solution for hen the blocked pvdf membranes were rinsed with — tbst followed by incubation with the primary antibodyat °c for h after washing with — tbst for times thesecondary antibody was incubated at °c for h lastly theprotein bands were visualized by supersignal west picoplus chemiluminescent substrate and the images werecaptured using a chemiluminescence imager multiple comparisons were conducted using onewayanova followed by tukey™s test statistical analysis e average value of three experimental results was determined and the statistical softwaresas was used to analyze whether there was a significantdiï¬erence between each group at the level of p longest in normal group ± cm while being the results eï¬ect of lpys4 on colon parameters e experimentalresults demonstrated that the length of mouse colon was thepared to those in the remaining four groups in contrast thethe ratio of colon weightlength was the highest in normalpgml and the lowest concentrations were found for sslpys4h group and these eï¬ects were better than those of eï¬ect of lpys4 on the serum contents of et1 sp ss andvip in mice it can be seen from figure that the serumml was decreased in normal mice compared to that of theremaining four groups e colitis model mice exhibited theopposite results in which the highest concentrations wereserum concentrations of ss and vip in colitis mice andmarkedly decrease those of et1 and sp more importantlythe eï¬ects were better after treatment with lpys4hshortest in the colitis model group ± cm similarlygroup ± while being the lowest in the colitis modelgroup ± figure it was found that lpys4 couldsignificantly p attenuate the decline in colon lengthand weightlength ratio induced by colitis ± cm and± for lpys4l group ± cm and ± forlb ± cm and ± concentrations of ss ± pgml and vip± pgml in normal mice were increased comlevel of et1 ± pgml and sp ± pgobserved for et1 ± pgml and sp ± ± pgml and vip ± pgml interestingly lpys4 could significantly p improve the± ± ± and ± pglb ± ± ± and ±± pgml in colitis model mice was signifigroups p following the treatment with lpys4 anhigher in lpys4h ± pgml and lpys4ltreated mice ± pgml than in lbtreated mice± pgml and the serum levels of il10 werelower in lpys4h ± pgml and lpys4ltreated mice ± pgml than in lbtreated mice± pgml p gsh ± μmolmg and sod ± μmolhighest while those of mpo ± mumg and mda± nmolmg were the lowest among the five± μmolmg and sod ± μmolgprotwere significantly reduced p while those of mpo± mumg and mda ± nmolmg wereremarkably increased p in the model group mice eï¬ect of lpys4 on the serum concentrations of il2 andil10 in mice it can be seen from figure that the serumcontentand il10± pgml eï¬ect of lpys4 on the activities of mpo sod gsh andmda in mouse colon as shown in figure the activities ofincrease in il2 and a decrease in il10 cytokine level wereobserved notably the serum levels of il2 were markedlyml and the eï¬ects of lpys4 were better than those ofgroups after induced by oxazolidone the levels of gshcantly reduced and raised compared to the remaining fourgprot in the colon tissue of the normal group were the pgmlofil2 0cevidencebased complementary and alternative medicinebacmice treated with a low concentration of lactobacillus plantarum ys4 — cfukg lpys4h mice treated with a high concentrationof lactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillus bulgaricus — cfukgfigure ac colon length and colon weightcolon length of each group of mice data are presented as the mean± standard deviationa“d diï¬erent letters mean values are significantly diï¬erent p according to tukey™s honestly significantly diï¬erent test lpys4land mda levels p such eï¬ect was stronger comparedto lbtreated mice ± μmolmg ± μmolgprot ± mumg and ± nmolmg more± μmolgprot in colon tissue and markedly decreased those of mpo ± mumg and mda± nmolmg when compared to colitis model groupand the goblet cells were increased compared with the modelgroup among them lpys4h had the most obvious eï¬ecton improving colon tissue which indicated that lpys4 couldreduce the colon injury caused by dss and the high efficiencyeï¬ect was also enhanced with the increase of concentrationlpys4 could markedly attenuate the decline in gsh andsod levels and prevented colitisinduced increase in mpo± μmolmgspecifically treatment with lpys4h significantly increasedthesodlevelsandof pathological observation as shown in figure in thenormal group the epithelial cells of colon mucosa were intactthe ‚ammatory cells were normal without ltration andthe goblet cells were arranged orderly without congestionand edema in the model group the epithelial cells of colontissue were obviously damagedthe intestinal wall wasthickened and edema ‚ammatory cell ltration andgoblet cells were reduced after treatment with lb and lpys4 congestion edema and cell ltration were alleviated eï¬ect of lpys4 on mrna and protein expression inmouse colon as shown in figures and colitis inductioncould lead to the upregulated mrna and protein expressionof inos in mouse colon but downregulated the relativeexpression of ckit enos nnos and scf treatment withlpys4h could increase the relative expression of nnosenos ckit and scf and decrease that of inos in the colontissues of colitis mice such eï¬ects were stronger than thoseof lpys4l or lb treatment group discussione ratio of colon weightlength is employed as a vitalstandard for assessing colitis in vivo e colon length ofnormalmodellblpys4llpys4hadccb0020406080100normalmodellblpys4llpys4hcolon length cmcolon lengthadccb00100200300400500normalmodellblpys4llpys4hcolon weightcolon lengthcolon weightcolon length 0cevidencebased complementary and alternative medicineabfigure a et1 b ss c sp and d vip serum levels of each group of mice data are presented as the mean± standard deviationa“d diï¬erent letters mean values are significantly diï¬erent p according to tukey™s honestly significantly diï¬erent test lpys4lmice treated with a low concentration of lactobacillus plantarum ys4 — cfukg lpys4h mice treated with a high concentrationof lactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillus bulgaricus — cfukgdcfigure a il2 and b il10 serum levels of each group of mice data are presented as the mean± standard deviation a“e diï¬erentletters mean values are significantly diï¬erent p according to tukey™s honestly significantly diï¬erent test lpys4l mice treatedwith a low concentration of lactobacillus plantarum ys4 — cfukg lpys4h mice treated with a high concentration oflactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillus bulgaricus — cfukgbadabbc0050100150200250normalmodellblpys4llpys4het1 level pgmlet1aedcb00100200300400500600700normalmodellblpys4llpys4hss level pgmlssvipdabbc00100200300400500600700normalmodellblpys4llpys4hsp level pgmlspaedcb00100200300400500600700normalmodellblpys4llpys4hvip level pgmlvipadccb050100150200250300normalmodellblpys4llpys4hil2 level pgmlil2eabcd02004006008001000normalmodellblpys4llpys4hil10 level pgmlil10 0cevidencebased complementary and alternative medicineabfigure a mpo b no c gsh and d mda colon tissue levels of each group of mice data are presented as the mean± standarddeviation a“e diï¬erent letters mean values are significantly diï¬erent p according to tukey™s honestly significantly diï¬erent testlpys4l mice treated with a low concentration of lactobacillus plantarum ys4 — cfukg lpys4h mice treated with a highconcentration of lactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillus bulgaricus — cfukgcdcolitis mice was shorter on average than that of control miceand the ratio of colon weightlength was lower in colitis micethan in control mice howeverit appeared thattreatment with lpys4 could attenuate the decline in colonlength and weightlength ratio induced by colitisa prior study has shown that vasoconstriction of theendothelin can lead to colonic mucosa erosion and ulceration which in turn can exacerbate the progression of colitis ss however can reduce gastrointestinal ‚ammationby suppressing the production of gastric acid and othergastrointestinal fluids us a decrease in ss level can inducethe secretion of gastrointestinal fluids thus aggravatingcolitis excessive accumulation of sp can induce colitisbut after antagonizing it has been shown to relieve colitis invivo vip inhibits no production by regulating thetranscriptional activity of inos in the colon tissue thusprotecting the intestinal mucosa besides vip can also ‚uence certain immune aspects of colitis in this studylpys4 inhibited colitis by downregulating the levels of etand sp and upregulating those of ss and vipil2 is an eï¬ector cytokine produced by cells whichhas been closely associated with colitis cells regulate the‚ammatory response that causes colitis and il2 is involved in the suppression of ‚ammatory process andseverity reduction of colitis by ‚uencing cells il is another cytokine produced by treg cells with immunoinhibitory eï¬ects which plays a significant role in thedevelopment of colitis ‚ammatory bowel diseaseibd is a chronic refractory intestinal ‚ammatory diseasemainly including ulcerative colitis uc and crohn™s diseasecd although the etiology of ibd is still unclear theimbalance between and has been recognized as themain cause of mucosal damage in ibd under the action ofdiï¬erentiation factor il10 regulatory t cells derived fromintestinal mucosa associated lymphoid tissue can correct deviation by secreting high level of il10 andmedium level of tgfβ so as to achieve the purpose oftreating ‚ammatory bowel disease to a certain extent it was observed that lpys4 could increase the level of il2thus regulating immunity and alleviating colitis and lpeabcd00100200300400normalmodellblpys4llpys4hmpo level mumgmpoadccb00100200300400normalmodellblpys4llpys4hsod level μmolgprotsodaedcb0020406080100120normalmodellblpys4llpys4hsgh level μmolmggshdabbc000510152025normalmodellblpys4llpys4hmda level nmolmgmda 0cevidencebased complementary and alternative medicinefigure observation of colon pathology in mice by he stainingys4 could inhibit the colitis and reduce the secretion of ile aggregation of neutrophils began to decline afterintestinal ‚ammation and a large amount of them enteredinto the circulation and migrated to tissues at the sametime free radicals such as reactive oxygen species and reactive nitrogen species gathered in large quantities which inturn led to damage and toxicity of colon tissue and furtheraggravated colitis after colitis the levels of sod andgsh were reduced in colon tissue while those of mda andmpo were elevated our findings also indicated that colitiscould lead to decrease in gsh and sod levels as well asincrease in mda and mpo levels [ ] in addition lpys4 attenuated colitis by inhibiting the transcriptional responses to oxidative stressnos can be divided into nnos enos and inos it hasbeen reported that no produced by enos plays a key rolein response to colonic tissue damage and excessive nogenerated by inos promotes colitis damage enoscontrols the production of no to keep the colonic tissue ina normal state which plays an important role in reducingcolitisinduced colonic injury e presence of excessiveno aggravates colon damage nnos can also controlthe level of no in tissue and protect the tissue from beingdamaged by excessive no in this study lpys4upregulated the expression of enos and nnos in the colonand downregulated that ofthereby attenuatingcolitisinosulcerative colitis not only shows hematochezia anddiarrhea but also presents colonic motility disorders it hasbeen proved that interstitial cells of cajal icc are related tocolonic motility dysfunction and directly participate in theprogression of colitis as a specific marker of gastrointestinalicc ckit is a transmembrane glycoprotein specificallyexpressed on icc cell membrane ckit gene located onchromosome 4q1213 belongs to protooncogene and itsproduct is tyrosine kinase type iii as a receptor of scfckit can regulate the proliferation and diï¬erentiation ofhematopoietic stem cells through a series of signalingpathways [ ] scf exerts a direct eï¬ect on ‚ammatorybowel disease by regulating the function and number oficc scf can interact with its ligand ckit and the dysregulation of scfkit signaling pathway may decrease theproliferation and diï¬erentiation of icc thus exacerbatingcolitis [ ] e abnormal expression of scfckitsignaling pathway can also change the physiologicalfunction of icc weaken gastrointestinal motility andaggravate intestinal dysfunction in the present studylpys4 could inhibit colitis by regulating the expressionlevels of scf and ckiticc autophagy regulation has become a new target forthe treatment of intestinal motility disorder in ulcerativecolitis because the drug treatment of colitis often hasside eï¬ects and once stopped it is easy to relapse ereforethe use of natural harmless substances through the regulation of icc prevention and treatment of colitis canmaintain longterm health a study has shown thataurantii fructusimmaturus and atractylodis macrocephalae rhizoma can inhibit the autophagy of cajal stromalcells induced by glutamate which may play an inhibitoryrole in colitis meanwhile there is also a study showingthat lactic acid bacteria can regulate icc thus regulatingnormallblpys4llpys4hmodel 0cevidencebased complementary and alternative medicineacbdthe mean± standard deviation a“e diï¬erent letters mean values are significantly diï¬erent p according to tukey™s honestlyfigure eï¬ect of lpys4 on a nnos b enos c inos d ckit and e scf mrna expression in mouse colon data are presented asesignificantly diï¬erent test lpys4l mice treated with a low concentration of lactobacillus plantarum ys4 — cfukg lpys4hmice treated with a high concentration of lactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillusbulgaricus — cfukgintestinal function and protecting the intestine isstudy also confirmed that the lpys4 can regulate the scfckitsignaling pathway and the scfckit signalingpathway is an important icc regulatory pathway erefore the lpys4 may also inhibit the colon by regulating iccaedcb001020304050normalmodellblpys4llpys4hrelative to multiple of model groupnnosadccb001020304050normalmodellblpys4llpys4hrelative to multiple of model groupenoseabcd00020406081012normalmodellblpys4llpys4hrelative to multiple of model groupinosaedcb0010203040normalmodellblpys4llpys4hrelative to multiple of model groupscfaedcb0010203040normalmodellblpys4llpys4hrelative to multiple of model groupckit 0cevidencebased complementary and alternative medicineacbdmean± standard deviation a“e diï¬erent letters mean values are significantly diï¬erent p according to tukey™s honestly sigfigure af eï¬ect of lpys4 on nnos enos inos ckit and scf protein expression in mouse colon data are presented as thenificantly diï¬erent test lpys4l mice treated with a low concentration of lactobacillus plantarum ys4 — cfukg lpys4hmice treated with a high concentration of lactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillusbulgaricus — cfukgef conclusionin this study oxazolone was used to induce colitis in balbcmice and the inhibitory eï¬ects of lpys4 on colitis weredetected rough the observation of colon tissues and serum samples of mice it was found that lpys4 treatmentcould alleviate colitis by restoring the levels of ‚ammatoryindicators closer to those measured in healthy control miceis work suggests that lpys4 is superiorquality lactic acidbacteria with a potential role in colitis treatment and provides a foundation for further research and developmentabbreviationslpys4 lactobacillus plantarum ys4lbqpcr quantitative polymerase chain reactionhelactobacillus bulgaricushematoxylineosinnormalmodellblpys4llpys4hckitenosinosnnosscfβactinadcbcb0010203040normalmodellblpys4llpys4hrelative to multiple of model groupnnosaedcb00102030405060normalmodellblpys4llpys4hrelative to multiple of model groupenoseabcd00020406081012normalmodellblpys4llpys4hrelative to multiple of model groupinosadccb0010203040506070normalmodellblpys4llpys4hrelative to multiple of model groupscfadccb00102030405060normalmodellblpys4llpys4hrelative to multiple of model groupckit 0cevidencebased complementary and alternative medicineendothelin1substance psomatostatinvasoactive intestinal peptideinterleukin2interleukin10et1spssvipil2il10mpo myeloperoxidasesodgshmda malondialdehydeenosnnos neuronal nitric oxide synthaseinducible nitric oxide synthaseinosscfstem cell factorsuperoxide dismutaseglutathioneendothelial nitric oxide synthasedata availabilityno data were used to support this studyconflicts of intereste authors of this manuscript state that they do not haveconflicts of interest to declareauthors™ contributionsruokun yi and fang tan contributed equally to this workruokun yi and fang tan performed the majority of theexperiments and wrote the manuscript huayi suo wenfengli xianrong zhou and jianfei mu contributed to the dataanalysis xin zhao and peng xie designed and supervised thestudy and read the final manuscriptacknowledgmentsis research was funded by national key rd program ofchina 2018yfd0502300 children™s research institute ofnational center for schooling development programmeand chongqing university of education csdp19fs01103theand technology research program ofchongqing municipal education commission kjzdk201901601 and research project of chongqing universityof education ky2015tbzc chinasciencereferences r k yi f tan w liao et al œisolation and identification oflactobacillus plantarum hfy05 from natural fermented yakyogurt and its eï¬ect on alcoholic liver injury in mice microanisms vol no p j liu f tan x h liu et al 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a
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Circulating tumor cells CTCs derived from primary tumors andor metastatic tumors aremarkers for tumor prognosis and can also be used to monitor therapeutic efficacy andtumor recurrence Circulating tumor cells enrichment and screening can be automatedbut the final counting of CTCs currently requires manualintervention This not onlyrequires the participation of experienced pathologists but also easily causes artificialmisjudgment Medicalimage recognition based on machine learning can effectivelyreduce the workload and improve the level of automation So we use machinelearning to identify CTCs First we collected the CTC test results of patientsAfter immunofluorescence staining each picture presented a positive CTC cell nucleusand several negative controls The images of CTCs were then segmented by imagedenoising image filtering edge detection image expansion and contraction techniquesusing python™s CV scheme Subsequently traditional image recognition methodsand machine learning were used to identify CTCs Machine learning algorithms areimplemented using convolutional neural network deep learning networks for trainingWe took cells from patients for training and testing About cells wereused for training and the others were used for testing The sensitivity and specificity ofrecognition reached and respectively We will further revise our modelshoping to achieve a higher sensitivity and specificityKeywords circulating tumor cells CTCs imFISH machine learning image segmentation CNN networkINTRODUCTIONThe metastasis of cancers is a complex and multistage process The circulating tumor cells CTCsare the œseeds shed from the primary tumor andor metastatic lesions and rooted in a new œsoiltransferred by the circulatory system Paget Circulating tumor cell is an intermediate stageof cancer metastasis correlated with cancer aggressiveness and the likelihood of metastasis andtherefore can be used to predict disease progression and survival on a realtime basis by liquidbiopsy Lindsay Praharaj Anand and Roszik Baek Maly Marcuello Pan Riebensahm The molecular subtypesof CTCs not only the CTCs count are interrelated with the prognosis BanysPaluchowski Cristofanilli Dong Stefanovic What™s more the PDL1Edited byCheng GuoColumbia University United StatesReviewed byJuanying XieShaanxi Normal University ChinaKhanh N Q LeTaipei Medical University TaiwanCorrespondenceBinsheng Hehbscsmu163comQiliang Zhou13974942986163comYuebin LiangliangybgeneiscnGeng Tiantianggeneiscn These authors have contributedequally to this workSpecialty sectionThis was submitted toComputational Genomicsa section of the journalFrontiers in Bioengineering andBiotechnologyReceived March Accepted July Published August CitationHe B Lu Q Lang J Yu HPeng C Bing P Li S Zhou Q Liang Yand Tian G A New Methodfor CTC Images Recognition Basedon Machine LearningFront Bioeng Biotechnol 103389fbioe202000897Frontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine Learningexpression in CTCsis correlated with the response toimmunity inhibitors Kloten PDL1EMTCTCs were associated with significantly poorer survival aftercurative surgery showing that PDL1 expression and EpithelialMesenchymal Transition EMT of CTCs are negative survivalpredictors for Nonsmall celllung cancer NSCLC patientsJanning Manjunath Pretreatment PDL1 CTCs are usually associated with a bad prognosis in patientstreated with PD1 inhibitors in NSCLC such as nivolumabGuibert The liquid biopsies worked as an ongoing monitoring systemto assess tumor heterogeneity and make it possible to detect asingle CTC or clusters of cells Wan Merker Praharaj Asante The breakthroughfor CTCdetection is the application of immunomagnetic CTCenrichment combined with flow cytometry which is still theœgold standard of CTCdetection Racila Howeverthis method that lack of the cancer specific markers still remainslots of limitation Grover Ferreira Gabriel Keller Thusthe multimarker immunofluorescence staining is required for recognizeCTCs Antibodies against chromosome centromere duplicationCEP8chromosome centromere duplication CEP17 areused to mark the rapidly dividing tumor cells antibodies againstCD45 as typical leukocytes filaments as well as 4cid486diamidino2phenylindole DAPI for labeling nuclears Koudelakova Lu Liu Lee Although there are great advantages in enrichment technologythe automatic recognition of CTCs still remains problemsManual identification is very timeconsuming and unreliableWith the continuous deepening of the application of CTCsrecognition in various cancer diseases the demand for rapidand automatic identification and counting methods of CTCs isincreasing Several studies have reported the automated screeningprocess Nagrath Yang Kraeft performed a fluorescencebased automated microscope systemREIS for cell detection This scanning can quantify the numberof cells reliably and reproducibly and categorize positive cellsbased on the marker expression profile Ligthart redefined the CTCs by computer algorithms after the manualcounting The stricter definition with the standard deviationof the signal in the CKPE channel the peak signal value inboth the DNADAPI and CD45APC channels and the size ofthe objects used as classifier was well validated CTC by clinicaloutcome using a perfectly reproducing automated algorithmMingxing reported an automated CTC enumerationZhou Allimages with diï¬erent colors weretransferred to a grayscale image and the grayscale images wereused to identify the position and outline of cells Howeverdespite the widely accepted these classification methods stillremain subjective as the rules are set artificially The fixedconditions may not identify the morphologically heterogeneousCTCs integrally What™s more diï¬erent technologies usually usediï¬erent antibodies making comparison and standardizationacross diï¬erent platforms challenging Marcuello With the maturity of artificial intelligence AI recent yearsmachine learning become an exciting field for research TheUS Food and Drug Administration FDA has approved severalcommercial products using machinelearning algorithms in themedical diagnosis and research The cardiovascular MRI analysissoftware of Arterys was the world™s first internet platform formedical imaging AI powered and FDA cleared This software isable to analyze multiple multiperiod MR images to determineblood flow in heart and main vessels The cloud platformwill enable software to collect and analyze the vast amount ofcardiovascular data from MR scanners in real time which willspeed up doctors™ diagnosis This artificial machine is consistentand tireless and is able to identify characters beyond humanperception which provided a substantial interest in the fieldof medical research specifically medical images Dominguez Erickson Lundervold and Lundervold Maier Many algorithms are developed forselecting the best weights for features involving neural networksHornik decision trees Quinlan supportvector machines Cristianini and ShaweTaylor the na¯veBayes Lowd and Domingos knearest neighbors Zhouand Chen and deep learning McBee Wainberg Zou Deep learning as wellas deep neural network learning refers to the use of neuralnetworks with more than layers able to integrate vast datasetslearn arbitrarily complex relationships and incorporate existingknowledge Convolutional neural networks CNNs is a powerfulalgorithm for advancing biomedical image analysis as it assumesthat the input layer has a geometric relationship such as the rowsand columns of images Anthimopoulos Poplin It has been successfully applied in the cancer diagnosisand nuclei or tissue identification Le Le Xing present a novel method for automatednucleus segmentation powered by CNNs The features involvedin the images are considered as a part of the search processand there is no need to limit the features compared to thetraditional machine learning methods which will eliminate thebias created subjective Here we apply deep learning to therecognition of CTCs in order to reduce the artificial errors andimprove accuracyMATERIALS AND METHODSPatients and Samples PreparationA cohort of patients with cancers were enrolled inthis study during “ which was approved by theethics committee of Chifeng Municipal Hospital The clinicalpathological characteristics of patients including age genderCTC number and cancer type are summarized in Table Fourmilliliter of peripheral venous blood was routinely collectedfor every patient The first ml blood samples obtainedafter puncture was discarded in order to avoid the skinepithelial cells contamination Then the blood was placed inanticoagulation tubes and store at room temperature The testwas completed within hAll the patients were divided into two parts according tothe collecting date The earlier patients we collected wereused as the training data the others were used as the independentFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningTABLE Clinical pathological characteristicsClinicopathologic variableAgeGenderSamples typeCTC numberCancer typeCategoryMeanMaleFemaleUnknownPeripheral bloodMeanLung cancerLiver cancerGastrointestinal cancerBreast cancerCarcinoma of thyroidNPCOtherClinical level““testing data Thousand three hundred cells images in the earlierreceived patients were selected to build the CTC recognitionmodel which will be further tested by the cells images of thetest dataset There was no cross part between the two datasets inorder to avoiding the overfittingEnrichment and imFISH Identification ofCTCsThe Cyttel method was used to isolate and enumerate CTCsThe peripheral blood was first centrifuged at g for minto get the precipitation and then washed by CS1 buï¬er CyttelBiosciences Co Ltd Beijing China Then the red blood cellswere lysed by CS2 buï¬er Cyttel After centrifuged at gfor min the precipitate was washed by CS1 buï¬er Thenthe cells were incubated completely with antiCD45 monoclonalantibodyconjugated beads Cyttel for min Three milliliterseparation medium was used to separate the beads and the CTCsby gradient centrifugation at g for min Then the upper rarecell layer was centrifuged at g for min and resuspendedby CS1 The tube was put on a magnetic stand for min Aftersmeared fixed and dried cells were used to perform the imFISHThe slides were fixed dehydrated and then dried at roomtemperature µl CEP8CEP17 antibody was added to the cellsand the slides were placed in a hybridization and denatured for h at —¦C The probe was eluted and the slides were washedtwice in — SSC Then the CD45 fluorescent antibody was addedto the sample area and the slides were put in a wet box andincubate for h at —¦C After incubation CD45 fluorescentantibody was aspirated and µl mounting media containingDAPI was added to the sample area After mounted the cells canbe observed and counted under a fluorescence microscopeThe Manual Interpretation Standard ofCTCs CountingAfter imFISHlots ofimages were acquired with diï¬erentfluorescent colors Usually manual counting is the œgoldstandard but it™s a time consuming and exhausted processionThe Manual interpretation standard of CTCs counting is Eliminates the aggregation superposition and interference ofnucleus or impurity DAPI positive CD45 negative and Three or more than three CEP8CEP17 signal points Itwill be regarded as one signal point if the distance between twosignal points is smaller than the diameter of one pointThe Image Segmentation Method WasUsed to Segment Single Nucleus andGive Labels of Cells Instead of ManualSince the obtained microscopic image is very huge the algorithmwill be limited by the memory and cannot be executed normallyon a conventional computer We first selected part of the imagecontaining one CTC cell and several nonCTC cells around toperform the following test The chosen resolution is — The CV package of python was used to process theCTCs images including conversion of color and morphologicaltransformations The RGB image was converted to the gray image The derivatives were calculated using the CVfunction Sobel from an image Morphological transformations operations based on theimage shapeThe Morphological package of python was used to segmentthe images of CTCs by image denoising image filtering edgedetection image expansion and contractionNuclei were segmented in the blue channel DAPI and theproportion of red in the red channel was detected based onthe position of the nucleus The nucleus with proportion of redhigher than was defined as having a common leukocyteantigen The orange channel was used to detect the number ofCEP8 chromosomes and the green channel was used to detectthe number of centromere probes extracted by CEP17 Diï¬erentcell types were distinguished by diï¬erent colors Figure The CNN Deep Learning Method WasUsed for CTCs IdentificationWith the development of AI machine learning has been wildlyused in the procession of medical images Deep learning is a bigimprovement on artificial neural networks allowing higherlevelfeature extraction and better data prediction with more layersAfter segmentation CNN network were used to identify CTCcells in single nucleus Finally it enters the output layer andoutput the result ie CTCs or nonCTCsOur CNN model was built based on AlexNet which wasfirst introduced in Krizhevsky The networkconsists of eight weighted layers Figure the first five layersare convolution layers and the remaining three layers are fullconnection layers The output of the last full connection layeris the input of the dimensional softmax values which willgenerate the distribution network of two types of labelsThe fivefold cross validation was used to prevent overfittingand select hyperparameters of the model The best crossvalidation score was obtained by searching the hyperparameterspace round and round The final hyperparameters involved inFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningFIGURE The imFISH result and the segmentation of chromosome and nuclear A“C The imFISH result of CEP8 CD45 and DAPI D The merge of panelsA“C E The CTCs were identified by CV segmentation method and marked in red box a“c The CEP8 signal points were identified by CVsegmentation method and marked in red boxour model are activation function kernel regularizer type andregularization factor The workflow is shown belowSp The grid was defined on 3dimensions with eachofthese maps for hyperparameter sets eg hyperparameters activation function kernel regularizer typeregularization factor activation function œsoftmaxœReLU œtanh kernel regularizer type œl1 œl2regularization factor œ œ The range of possible values were defined of eachdimension Allestablishing the best onethe possibleconfigurations weresearched forEvaluation Criteria for ClassificationModelsAfter segmentation some performance evaluation criteria Xie were involved in to evaluate the performance of theclassification model such as sensitivity Se or recall specificitySp precision F1 score and area under the receiver operatingcharacteristic curve AUCSerecall TPTP FNTNTN FPTPprecision TP FPF1 — precision — recallprecision recallIn the equations TP stands for the number of positive CTCcells which are correctly recognized as positive CTC cells FPstands for the number of negative CTC cells that are incorrectlyrecognized as positive CTC cells FN stands for the numberof positive CTC cells incorrectly recognized as negative CTCcells TN stands for the number of negative CTC cells correctlyrecognized as negative CTC cells Table RESULTSPatient CharacteristicsA total of patients were enrolled in this study from January to June The average age is years old Patients withlung cancer count of all patients and the next is breastcancer and gastrointestinal cancer Table Frontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningFIGURE The layers of the CNN model The first five layers are convolution layers and the remaining layers are full connection layersThree SubImages Were Required forManual CountingWe performed imFISH for all the patients and required images of CTCs cells Every image was divided into or channels with diï¬erent color The orange channel representedthe chromosome with CEP8 Figure 1A the green channelthecentromereofthechromosomerepresentedCEP17 Supplementary Figure S1represented the whitethe blueFigure 1C The mergence wasWe then manually labeled all withred channelcell with CD45 Figure 1Brepresented the nuclei with DAPIshown in Figure 1Dthese subimages accordingchannelFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningTABLE Confusion matrix definitionsTABLE The confusion matrix of the models for test datasetConfusion MatrixPredictionMethodConfusion MatrixPredictionPositiveNegativePositiveNegativeTruePositiveNegativeTrue positive TPFalse positive FPFalse Negative FNTrue Negative TN CVALexNetTrueTruePositiveNegativePositiveNegativethetoare CTCs positivestandard AmongourresultspatientsTABLE Tuning of the hyperparameters of AlexNetActivation functionKernel regularizer typeRegularization factorThe Segmentation of Nuclear andIdentifying CTCs by CVSegmentation MethodIn order to avoid the artificial error and save costs we performedthe traditional image identification method for CTCs countingFigure The nucleus was separated in the blue channelDAPI Figure 1E and the red proportion of the red channelwas detected according to the location of the cell nucleus Theproportion higher than was defined as the number of theCEP8 chromosome detected by the common antigen orangechannel of white blood cells Figures 1A“C the number ofcentromeric probes detected by the green channel such as CEP17Supplementary Figure S1After segmentation of nuclear we used CV segmentationmethod to identify CTC cells from single nucleus regions in testing dataset by the manual interpretation standard ofCTCs counting After identification and judgment cells of negative nuclei were recognized as CTC negative About cells of positive nuclei were recognized as CTCnegative The sensitivity and specificity were and while the precision and F1 score reached and respectively Table We also applied the regionbased image segmentationalgorithm such as watershed algorithm in the segmentationprocess The watershed algorithm was implemented the bywatershed function in CV python and CV In this method optimal threshold value was used respectivelyin binaryzation process by setting THRESH_OTSU mode Thetraditional watershed algorithm was sensitive to noise and theaccuracy was lower than our segmentation method on CTCnegative data set in size of Supplementary Table S3The HyperParameters Selected forEvaluating the CNN MethodWe used GridSearchCV class in scikitlearn by providinga dictionary of hyperparameters to determine the hyperparameters of the model After the crossvalidation processactivation function was set to ReLU kernel regularizer type wasset to l2 and regularization factor was set to as shown inTable with the best performance Further the hyperparameterswe selected were used to construct the model on the wholetraining datasetsoftmaxReLUtanhl1l2l1l2l1l2The underline value shows the best result of AUC value in the tuning process of thehyperparameters of AlexNetThe Identification of CTCs by CNNMethodWe got nuclei of patients by segmentation processFigure showed the whole flowchart of the experiment About nuclei were used for training the left were usedfor testing We use the same images for testing cells of negative nuclei were recognized as CTC negative and cells of were recognized as CTC positive The sensitivityand specificity were and while the precisionand F1 score reached and respectively Table and Figure Before that we also compared the performance of AlexNetmodel with others such as ResNet and Xception All ofthem have close AUC values Figure butthe AlexNetwas less timeconsuming in the training and test processSupplementary Table S1DISCUSSIONThis study showed a method for CTC counting powered bymachine learning The use of machine learning for imageinterpretation can capture important image features reduceerrors caused by manually setting interpretation standardsand save time and labor costs Although this method showsa higher sensitivity and specificity in CTC countingisslightly worse than the first method for the data used inthis study Actually we have analyzed that the main reasonis that there are fewer positive samples for training and thealgorithm cannot extract features of more positive samplesin the group were excludedIn additiondue to quality problems Unfortunatelythe CTC imagesincluded in the group doesn™t cover the whole film but asome picturesitFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningFIGURE The flowchart of the whole experimentFIGURE The ROC curve of AlexNet ResNet and Xception modelpicture just focused on a certain CTCpositive cell under themicroscope which results in that the machine learning methodhas no advantage in recognition speed compared with thetraditional image recognition method Enlarging the scope ofimages and collected more samples is also that need to beimproved in the futureDeep learning has already been shown to be suitablefor detection of CTCs because of the high sensitivity andspecificity in CTC counting We had changed the filter sizeand number in all convolution layers in order to find thebest CNN parameters We found diï¬erent filter size andnumber will influence the results largely We changed filternumber from range to in our training process Wefound that the training result was not convergence when thenumber was less than It showed that the range of thefeature number of the image is about “ We tried toincrease the filter size from to but the result was notchanged a lot and the convergence speed even became slowerwhen the filter size higher than From this process wesummarized that the feature size in CTCs could not be greaterthan pixels Furthermore there are many appropriately AImodels such as VGG InceptionV14 We will apply themon the CTCs dataset to establish a more suitable model inthe later testingtumorsCirculating tumor cellis an important marker for earlyscreening and prognosis ofIn addition CTCsoriginating from the primary tumor may be more eï¬ectivefor tumor tissue tracing and molecular classification Imagerecognition can only obtain the characteristics ofthe cellsurface If strict tissue tracing is required other molecularbiological experimental data such as the isolation of CTCcells and single cell sequencing may be required Besidesin this study we also evaluated the performance of AlexNetmodel in variant types of cancers Supplementary Table S2and Figure S2 showed that our model presents a betterperformance in Lung cancer than Gastrointestinal cancer andBreast cancer One of the reasons may be that the trainingdata size of Lung cancer is much larger than those ofGastrointestinal cancer and Breast cancer Furtherpostoperative recurrence may occur in approximately of patients even after complete resection of NSCLC Yano These proteins especially epithelial proteinssuch as EpCAM PIK3CA AKT2 TWIST and ALDH1may have more activitiesHanssen whichwilllead more influence in the morphology of cells andaï¬ecting the recognition performance thereby Therefore themultiimage omicsincluding CT images HE staining andimmunohistochemical images as well as the sequencing datamay be urgently needed at this stageFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCONCLUSIONIn orderIn the present study we established a CTC cell recognitionsoftware based on deep learningto make itmore practical we collected samples from the real worldinstead of using the public databases We performed theCTC enrichment and imFISH experiments and screened thefluorescence images according to the figure™s quality In order toimprove the efficiency we used the machine instead of ngmanual screening First the python™s package was used to mage segmentation The obtained recognition sensitivity andspecificity are and respectively In addition therecognition sensitivity and specificity can also reach to and respectively using CNN instead of manual interventionIn the future studies we willfocus on the improvementof the accuracy and sensitivity with a more suitable deeplearning model promoting this technology to the clinic assoon as possibleDATA AVAILABILITY STATEMENTThe datasets generated for this study are available on request tothe corresponding authorCTCs Recognition by Machine Learninglegal guardiannextstudy was provided by the participants™of kin Written informed consent was obtained from theindividuals and minors™ legal guardiannext of kin for thepublication of any potentially identifiable images or data includedin this AUTHOR CONTRIBUTIONSGT YL BH and QZ conceived the concept of the work BH QLJL PB HY and SL performed the experiments QL and BH wrotethe manuscript CP and HY reviewed the manuscript All authorsapproved the final version of this manuscriptFUNDINGThis research was funded by Hunan Provincial Innovation2018RS3105Platform and Talents Program NotheNaturalNo Science Foundation of Chinathe Natural Science Foundation of Hunan Province No2018JJ3570 and the Project of Scientific Research Fundof Hunan Provincial Education Department Nos 19A060and 19C0185ETHICS STATEMENTSUPPLEMENTARY MATERIALThe studies involving human participants were reviewed andapproved by The Ethics Committee of Chifeng MunicipalHospital Written informed consentto participate in thisThe Supplementary Materialonline202000897fullsupplementarymaterialfor this can be foundhttpswwwfrontiersins103389fbioeatREFERENCESAnand K and Roszik J Pilot study of circulating tumor cells in earlystage and metastatic uveal melanoma Cancers 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increasing relevancy of geospatial technologies such as geographic information system GIS inthe public health domain particularly for the infectious disease surveillance and modelling strategies Traditionally thedisease mapping tasks have faced many challenges” authors rarely documented the evidence that were used to createmap before evolution of GIS many errors aroused in mapping tasks which were expanded extremely at global scalesand there were no fidelity assessment of maps which resulted in inaccurate precision This study on infectious diseasesgeosurveillance is divided into four broad sections with emphasis on handling geographical and temporal issues to help inpublic health decisionmaking and planning policies geospatial mapping of diseases using its spatial and temporalinformation to understand their behaviour across geography the citizen™s involvement as volunteers in giving healthand disease data to assess the critical situation for disease™s spread and prevention in neighbourhood effect scientificanalysis of healthrelated behaviour using mathematical epidemiological and geostatistical approaches with capacitybuilding program To illustrate each theme recent case studies are cited and case studies are performed on COVID19 todemonstrate selected modelsKeywords Geospatial technology 01 Citizen Science 01 Public health 01 COVID19 01 Mathematical epidemiologyIntroductionThe public health sector™s increasing demand for mappinganalytics and visualization had started a date back in thelast years which has resulted in a growing informationage technology for communicable disease surveillance andepidemiology Baker Bos and Blobel Friede Friede Khan Reeder Yu and Edberg This continuous publichealth burden with advances in information technology Sameer SaransameeriirsgovinPriyanka SinghPriyankaiirsgovinVishal KumarVishalkumariirsgovinPrakash ChauhanprakashiirsgovinIndian Institute of Remote Sensing Indian Space Researchanisation Kalidas Road Dehradun Indiacombined with spatial data led to the development ofvarious tools and systems that provides visualization ofdisease data in space and time Dredger Kothari Robertson and Nelson Schriml et alThe first integral definition of public health was given byWinslow as ˜˜science and art of preventing diseaseprolonging life and promoting health through the anized efforts and informed choices of society anizations public and private communities and individuals™™The American Public Health Association APHA mentioned public health as a practice of preventing the spreadof disease and an aim of promoting good health from smallcommunities to across the world Turnock Advances in information technology and spatial features resultedin geospatialtechnology which is acute for mappingsurveillance predicting outbreaks detecting clustering andanalysing spread patterns of infectious diseases with epidemic or pandemic potential in communities and acrossterritories AvRuskin Carpenter Castronovo Dominkovics Gao 0c Heymann and Brilliant Hills Klompas Reis Geospatial technology has provided visualization and analytical tools topublic health professionals and decision makers to executediseases control programs in affected andor suspectedregions and make analysis and predictions possible thatwas once technologically out of reachGeospatial technology includes geographical information systems GIS global positioning systems GPS andsatellitebased technologies such as remote sensing RSGIS is known for geographic data capture input updatemanipulation transformation analysis query modellingand visualization of all forms of geographically referencedinformation through the set of computer programs BonhamCarter GPS provides positioning navigationand timing PNT services by capturing data from satellitesand providing it to users Eldredge and RS isan earth observation instrumentthat delivers regionalinformation on climatic factors and landscape featuresTherefore GPS and RS provide regional and spatialinformation while GIS provides geospatial data integrationas well as accurate geospatial analysis in realtime mannerZhen Geospatial Technology and InfectiousDisease SurveillanceInfectious diseases mostly adapts antimicrobial andmobility features later formed in a shape of pandemic andor epidemic Chen Cheng Lee andNishiura which forced public health authorities tounderstand not only the diseases virulence but also itsdemographic and environmentalfactors that helps inmaking spread patterns though space and time domainCroner For example the global spread of highlypathogenic avian ‚uenza HPAI H5N1 in “ withno effective vaccines led to concern among public healthdecision makers in spite of many international programsRappole and Huba´lek The reason behind theirconcern was they were lacking of disease surveillance toolin its initial stage which caused inaccessibility to populations atrisk and faced difficulties in implementingimmunization strategies at a global scale Kitler Stoto However the impact of environmentaland demographic factors also plays a major role as this caninform about the interaction between hosts and pathogensand patterns of spread in space and timeThe GIS provides dynamic maps to understand geographical distribution of diseases for analysis on frequencyof cases disease mapping spatial cluster of diseases disease association with environmentalfactors networkanalysis etc With such a visualization and analyticalJournal of the Indian Society of Remote Sensingforservice frameworkcapabilities GIS technology is holding a widespreadgrowth in public health Ahmad 2011a b Booman HanafiBojd Kolivras Martin Nykiforuk and Flaman Abdul Rasam Zhang Zhen Theseamless integration of GIS with realtime infectious diseaserelated diverse datasets through webbased mappingto the development of geospatial dashboardleadsgeospatialinfectious diseasesurveillance Dent Gao Yun Theinfectious diseaserelated data mightinclude diseasesurveillance data activeconfirmed cases and health system data hospital visits emergency services availabilitynursedoctor availabilityICUbed availability Many source geospatialstandards of GeospatialConsortium OGC are used as a Web Map ServiceWMS Web Coverage Service WCS Web ProcessingService WPS Web Feature Service WFS etc Bulatovic´ Gao to visualize accesspublish and manipulate geospatial resources Also manyother popular industrial geospatial standards are developedby ESRI Google Yahoo and MapInfo Granell to fetch locationbased data and provide infectiousdisease surveillance dashboard to monitor and control thegeographically spread of disease Zhang TheGeocoded Really Simple Syndication GeoRSS taggedXML files from GeoRSS services can also be used toprovide geocoded infectious disease news from socialmedia platform Tolentino KassHout andAlhinnawi 2013a b Kodong Historical ContextThe mapping of infectious diseases using geospatial andinformation technology to benefit public health is not a newway of tracking the diseases Ahmad Cui Hirsch Hornsby Matthew May Mujica Nicholson and Mather Noble Perl and Moalem Williams The historical disease mapping has faced manychallenges” authors rarely documented the evidencethat were used to create map after mapping had beenimplemented before the beginning of geographical information systems many errors arouse which were expandedextremely at global scales and there were no fidelityassessment of maps which resulted in inaccurate precisionBut nowadays wide range of geospatial applications areavailable in public health community with a possibilities ofvisualization analysis detection of clusters formed andcalculate diseaserelated metrics such as incidence andprevalence rate Beck Clarke Hay Jacquez Kleinschmidt Lawson and 0cJournal of the Indian Society of Remote SensingLeimich Moore and Carpenter Robinson Wilkinson The earliest mapping for visualisation ofthe linkbetween disease and place was done in on plagueepidemic in Italy Dent During cholera outbreak in the study of physician John Snow had made a novelcontribution in history of public health and epidemiologyby using cartography applications and geographic visualization in fighting cholera After years the maps wereidentified as a communication tool in understanding andtracking of infectious diseases such as the ‚uenzapandemic yellow fever and cholera Since then revolutionof webbased tools started in applied health geographyBoulos The trend of infectious disease mappingcould be seen from review of the Health GIS literature which demonstrated that research papers out of were focused on infectious disease mapping Lyseen Covid19The ongoing pandemic outbreak targeting humans™ respiratory system was recently discovered in December by the name of Coronavirus Disease Covid19 WorldHealth anization from a cluster of patients with acuterespiratory distress syndrome in Wuhan Hubei ProvinceChina Huang Lu 2020a b and spreadglobally by March This pathogenic disease is structurally related to the Coronavirus CoV which belongs tofamily Coronaviridae and the order Nidovirales Thisfamily is classified into four genera”AlphacoronavirusBetacoronavirus Gammacoronavirus and Deltacoronavirus on the basis of their phylogenetic and genomicanalysis The species of Alphacoronavirus and Betacoronavirus infect mammals causes respiratory illness inhumans and gastroenteritis in animals while species ofGammacoronaviruses and Deltacoronaviruses infect birdsbut some of them can also infect mammals Woo et alfrom Betacoronavirus The two virusgenus”Severe Acute Respiratory Syndrome SARSCoVor Middle East Respiratory Syndrome MERSCoV”hadearlier demonstrated that coronaviruses can cause significant public threat Ge The COVID19 is categorizedby World Healthanization WHO on the basis of genomic sequencinganalysis ofrespiratory tract samples which isobtained from total of nine patients Huang Lu 2020a b COVID19 has started behaving like theonceinacentury pandemic by affecting healthy adults aswell as elderly people with some health issues and byinfecting others at an exponential rate of increase thanSARS or MERSinto BetacoronavirusspecieslowerGeospatial TechnologyDuring occurrence of diseases geospatial technologies andservices could help in representing the spatiotemporalinformation and in analysing the dynamic spread of diseases As mentioned by Boulos geospatial technologies and services which performs in real time mannerare tremendously relevantto create a ˜˜spatial healthinformation infrastructure™™ In this section a review onmany geospatial technologies with enabled IT services iscarried out to understand and analyse the spread and outbreak of disease with a case study on COVID19 pandemicCitizen Scienceissues and concernsThe expansion of Citizen Science from biodiversity andecological domain Haklay MillerRushing to public health community across spatial extentsmade an urgent need to study its different forms Crowl The indepth report of EU describes taxonomyof Citizen Science in three levels European Commission described in Roy Wiggins andCrowston and Haklay Roy categorized Citizen Science by participant™s number and oftheir spread ˜˜local™™ and ˜˜mass™™ and ˜˜thoroughness™™time and resource investment or King described ˜˜for the people with the people or by the people™™ about Citizen Science activities Wiggins andCrownston classified Citizen Science projects inconservation managing natural resources action addressing localinvestigation answering scientific questions and education providingknowledge to citizens Haklay classified CitizenScience into four levels based on participant™s engagement” level is crowdsourcing in which citizens withless or no knowledge on activity perform as sensors tocomplete computing tasks level is distributed intelligence where citizens are being trained with skills forinterpretation of collected data level is participatoryscience in which citizens decide about research questionsand types of data to be collected and level is extremewhere citizens are fully involved in defining researchstrategies data collection data interpretation and performing scientific analysis Apparentlythe concept ofCitizen Science is rare in public health domain but some ofits contribution seen in some studies which not only helpsin predicting disease risks but also in combating theinfectious diseases CurtisRobles Palmer Smolinski Wilson Another approach similar to Citizen Science is ˜popularepidemiology™ in which experts and laypersons jointly 0ccollect environmental data responsible for particular healthconsequences Brown or ˜street science™ as a process in which general public communities actively engagedin defining problems framing of research questions anddecisionmaking activities about research design CorburnCrowdsourceVGI Mobile AppsDespite technological and computational developments inGeoWeb many web technologies such as jQuery andAJAX mapping APIs like Google and GPS devicesresulted in a new revolution of neogeography Turner where mapping is done by crowd and can bereached by anyone from general public members groupSuch revolution brought a trend of Volunteered GeographicInformation VGI which is first coined and explained byGoodchild 2007a According to Goodchild 2007b VGIhighlighted the human capabilities in collecting geospatialinformation by using five senses and then integrating withexternal sensors of mobile devices like GPS accelerometer camera digital compass and microphone gives valuable datasets which can neither be retrieved from satelliteimagery nor collected with any GPS receivers Anothersuccessful term in geospatial mapping using mobile technology is crowdsourcing Heipke HudsonSmith which was coined by Howe thatinvolves the collection of geospatial information or mapping of any particular activity by an undefined crowd ornetwork of people Both terms VGI and crowdsourcingslightly differ but they are usually recognized as a synonyms or even as a combined term ˜˜crowdsourcing geographic information™™ Sui Over the lastdecade VGIoriented source mobileareEpiCollect Aanensen for ecology and epidemiology NoiseTube Maisonneuve httpnoisetubenet and Noise Battle GarciaMartı´ for noise monitoring Skywatch Windoo httpwindoochfor weather monitoring Mappiness httpwwwmappinessukfor behavioural analysis MacKerron andMourato appsThe source mechanism for data collection usingAndroid devices can be performed by Data KitODK suite107 https datakit which is composed of ODK Collect and ODK Aggregate ODK Collecthttps datakitusecollect provides a customizable framework for geospatial data collection and ODKAggregate is a web application that runs on ApacheTomcat server httptomcatapache to store collecteddata through a synchronization with a databaseforexample PostgreSQL Brunette As suchsuite™s performance can be seen in various activities likeagricultural monitoring Krosing and Roybal Journal of the Indian Society of Remote Sensingmonitoring of deforestation and school attendance documentation of war crimes and health programs Anokwa Digital Contact TracingNowadays COVID19 has become the greatest threat forpublic health in last years and due to such pandemicvarious levels of lockdown are issued across the world tobreak its chain of infection transmission However this isthe first approach to invade the contagion but once itwould be lifted this pandemic would start in a new wayand might reach its highest peak by infecting more andmore population Ferguson Thereforetocombat with such a global pandemic threat anotherapproach is discovered by a group of researchers known asdigital contact tracingSmartphonebased contact tracing is known as a digitalcontact tracing which presents a sustainable solution tolimit the transmission of infectious disease by tracing theirpotential transmission routes in a population howeversuch an app presents significant concerns regarding privacy The digital contact tracing works on the principle of˜crowdsource data™ by measuring the proximity to aninfectious person In previous diseases risk surveillancethe contact tracing apps were used to pool location timestamped data to determine the exposures to risk of infectionsSacks Such data are highly personal and leadmany privacy concerns Smith but they werenot always accurate to infer the exposure risks due to noisydata Farrahi Therefore various smartphoneapps are developed in COVID19 pandemic in which someapps use location for proximity and some of them are notusing location services of mobile device subject to theprivacypreserving natureCOVID19 Contact TracingIn order to illuminate the epidemiology of COVID19 andto characterize its severity Lipsitch there is anurgent need of digital platform that captures realtimeaccurate information on COVID19 patients diseasesdiagnosis treatment and clinical reports and whom theyget interacted at which place to detect clusters and generatealerts Such information may help in understanding riskfactors of infection and in predicting the next generation ofinfectious persons FitzGerald Addressing thisunprecedented challenge many mobile apps have beendeveloped and are being used at large scale and some ofthem are as follows 0cJournal of the Indian Society of Remote Sensing¢ COVID Symptom Study COVID Symptom Tracker”This mobile app is developed in collaboration of ZoeGlobal Ltd a digital health care company and a groupof academic scientists from Massachusetts GeneralHospital and King™s College London which waslaunched in UK on March and becameavailable after days in USA This app enquires aboutage location and other diseases risks and also a selfreporting function is enabled which is associated withCOVID19 infection and exposure Drew This app retrieve updates on healthcare worker™sexperiences who are on COVID19 duty their stressand anxiety and use of personal protective equipmentPPE kits are being surveyed through this app toobserve intensity of health care workers Drew et alappimplemented¢ Aarogya Setu”This mobile app is launched on April by Government of India to aware general publicon COVID19 symptoms government advisory measures online consultation facility and dynamics ofdisease Thiscrowdsourcingapproach by which general public members enter theirdetails for selfassessment and this assessment is thenused to trace the infectious contacts or agents as adigital contact tracing concept This app uses locationservices to geolocate the users and Bluetooth tomaintain the log of contacts when one userdevicecomes in contact with another userdevice and as suchdigital contact tracing activity helps in identifying thecluster of diseases and communities which are at risk ofinfection The Aarogya Setu app was downloadedby million users within days of its launchUpadhyay and by using app™s crowdsourcedata the Indian government detected approx positive casesinformed probable users ofbeing at risk and identified potential clusters TheTimes of IndiaNumerous digital contact tracing apps are in use indifferent parts of world”TrackCOVID Yasaka TraceTogether Bay WeTrace De Carli and Google and Apple™s recently announcedjoint initiative Li and Guo COVID19 Data Visualization and ExploratoryData AnalysisWith early experiences of epidemics such as “SARSCoV Boulos and the “ MERSCoVGikonyo and other seasonal flu™s online realtime or nearrealtime mapping of diseases™ occurrencesusing geospatial technologies and web applications havealways been used as a pivotal webbased tools in trackinghealth threats and combating infectious diseases Thissection described a range of mapping dashboards based ongeospatialtechnologies for tracking and unfolding thecoronavirus disease around the world Some of the globaland national geospatial initiatives with an aim to supplyinformation faster than diseases are as summarized inTable Infectious Diseases ModellingThe intention of infectious diseases surveillance is to detectepidemics in their early stages so that the countermeasurescould be taken for preventing its wide spread Suchsurveillance tasks require many epidemiological and statistical methods with geospatial features in investigatingepidemics preferably from localized areas The reason forpreferring the local areas for investigation is because epidemics generally emerged in small areas and then spreadwidely if they are not controlled However some methodsrequire rigorous conventions in their underlying modelsand are too problematical to be applied on small areasThereforefordetecting diseases prevalence with case studies on smalldatasets which would be more useful for public healthactivitiessection discussessimple methodsthisClusteringClustering deals with the study of spatialtemporal patternsof the spread of communicable diseases and identificationof other diseaserelated aspects allied with heterogeneousgeographical distribution which might be helpful in elucidating the diseases™ spread mechanism Such study andanalysis on spacetime patterns is a kind of diseasesurveillance which involves detecting the outbreak clustersof active cases monitoring of localisation and isolation ofinfectious agents and relative risks assessment of affectedsites at early stage Clements Cromley Kulldorff This study on geographical clustering ofinfectious diseases with temporal features helps in makingstrategies that dynamically update on emergence source ofdisease outbreak to help epidemiologists and decisionmakers for identification of spread and risk zones Thusclustering helps to enable timely prevention and containment measures and timely resource allocation to mitigatethe diffusion of diseasesBased on spacetime surveillance of diseases spacetime scan statistic Kulldorff is one of the clusterdetection tools which is widely used in geographicalsurveillance of diseases during epidemic andor pandemicThe spacetime scan statistic comes with two versions”prospective and retrospective Desjardins 0cTable Summary table for geospatial dashboards for COVID19Project nameDatasetsScopePurposeJournal of the Indian Society of Remote SensingWHO Coronavirus Disease COVID19WHO™s official dataDashboard Dong httpscovid19whointGlobal Visualization of official daily counts ofconfirmed cases and deaths related toCOVID19 with time stamps using EsriArcGIS Online serviceExploratory data analysis using 3D graphto perform countrywise analysis usingpopulation confirmed cases cumulativecases deaths and cumulative deathsProvides daily aggregate case and deathcount in CSVJohns Hopkins University COVID19Aggregated data from WHO EuropeanGlobal Dashboard for visualizing realtimeDashboard Dong httpscoronavirusjhuedumaphtmlCentre for Disease Prevention andControl ECDC WorldoMeters BNONews US CDC 1Point3Arces COVIDTracking Project and DXYmapping of COVID19 with graphs onconfirmed and daily casesCritical trend analysis on new cases perday mortality and fatality analysis inpopulation timeline of outbreak etcISRO™s BHUVAN COVID19 GeospatialSolution httpsbhuvanapp3nrscgovincoronacorona_dashboarddashboarddashboardphptypecitizenCOVID19 data Source MoHFWIndiaTime series visualization of activerecovered and deceased cases fromMarch to till dateGraphical analysis on spread trend ofCOVID19 daywise and statewiseCOVID19INDIA httpswwwCM Health M handles Press Trust ofIndiaVisualization of cumulative and dailycovid19indiaIndia state press bulletins PBI and ANIreportsnumbers of confirmed active recovereddeceased and tested statewise casesthrough maps and graphsProvides daily COVID19 cases in stateand district and cases reassigned to statesthrough APIMapmyIndia COVID19 httpsmapsCOVID19 data Source MoHFWIndiaProvides API on corona dashboards tomapmyindiacomcoronadistricts_containment_zonecontainment_zone_gradientHunger Relief Centres Source MyGovHunger Night Shelter Source MyGovNDMAvisualize cases at district and state levelhotspots treatment centres testing labsquarantine centres containment zoneslockdown issues hunger relief hunterand night sheltersMonthly climate explorer for COVID19httpscdsclimatecopernicuseuappsc3sappc3smonthlyclimatecovid19explorerMonthly COVID19 cases Source JHUGlobal Visualization of COVID19 fatalities withCSSEAtmospheric composition”PM10 and NO2Source CAMS EAC4Meteorological data”humidity hPaand surface air temperature on hourly andmonthly average rate Source ERA5reanalysisclimatic and atmospheric variations onmonthly basisExploratory analysis on correlation ofpollutants and specific humidity withCOVID19 deathsExperimental COVID19 and GlobalCOVID19 cases Source JHU CSSEGlobal Visualize earthquake as a cause of increaseSeismic Risk Map httpsmaps quakemapcovid1920200520v3grm234900Global earthquake risk map Source GEMGlobal Earthquake Modelin COVID19 cases due to people™sdisplacement from damaged buildingsOwusu and difference between both is thatprospective neglects historical clusters which may havepreviously occurred before the most current time period ofanalysis with no health threat Kulldorff Thereforethe prospective version of spacetime scan statistic iscommonly used to detect statistically significant active orevolving clusters of diseases for the present time periodand when more data become available the tool can be rerun to detect new evolving clusters with update on relativerisks for each affected sites Previously the prospectivespacetime scan statistic was used in thyroid cancerKulldorff shigellosis Jones measlesYin syndromic surveillance Yih and many other diseases However cluster analysis of 0cJournal of the Indian Society of Remote Sensingdiseases can be performed through several packages andlibraries in R Go´mezRubio and Pythonsoftware Yeng The contribution of cluster detections and analysis inCOVID19 pandemic is becoming useful nowadays as itdetects active and emerging clusters of COVID19 andnotify epidemiologist decision makers and public healthcare officials which can help in eradicating infections fromaffected sites and improving interventions quarantine andisolation measures The significant applications of clustering with respect to infectious diseases modelling aredemonstrated across the world Zarikas forexample India Bhosale and Shinde USA Desjardins Hohl Brazil Martines Italy Cereda China Ji Liu 2020a b Qiu Zhang Singapore Bhosale and Shinde Pung SouthKorea Shim French Alps Danis Germany Pfefferle Sergipe Andrade etcOutlier AnalysisThe outlier is defined by Hawkins as ˜˜an observationwhich deviates so much from the other observations as toarouse suspicions thatit was generated by a differentmechanism™™ In other words when data generation processstarts behaving abnormal and reflects the abnormalities orerrors in data such abnormalities are known as outliersBansal However the outliers generally holdadvantageous information about the systems unusual characteristics and entities which impact the data generationprocess Some of the useful applications of outliers in diseases are Cleynen Dai and Bikdash Krishnan Lo Prensner Washington Wu and Krishnan Clusteringalgorithms are optimized to find clusters rather than outliersand the accuracy of outlier detection depends on how goodthe clustering algorithm captures the structure of clustersMaximum Entropy Modelling Maxent ApproachIn context of disease systems disease transmission risksdepend on distribution of pathogens species in space andtime in some complex environmental conditions Townsend and as such treatments are focused mainly on spatialdimensions therefore diseases transmission risks are purelyhandled through geographical phenomena Such geographical link with diseases leads to the challenge of spatialmapping of disease transmission which overcame throughthe branches of biodiversity science”ecology and biogeography Such approach of ecological and biogeographical modelling can be seen from various studies on diseasetransmission risks mapping for example Arboleda Deka and Morshed Ferreira Holt Mweya Nakazawa Reeves Samy Qian Zhao Zhu Following recent studies on geographical mapping ofpathogens causing disease transmission machine learningbased maximum entropy method Maxent Elith Phillips is applied on spatial records ofCOVID19 with a set of bioclimatic environmentalvariables from WorldClim Poggio Ramı´rezVillegas and Bueno Cabrera to analyse theirfavourable environmental conditions as shown in Fig and Table required in maintaining its population TheMaxent principle is to estimate the target probability distribution by applying the maximum entropy to distributionwhich is most spread or closest This study is carried out inR software Ihaka and Gentleman and a geographical dataset consists of latitude and longitude of thoseregions which were affected till March Figure depicts the habitat suitability map of virus withprobability range in colour scale to visualize the highsuitability light and dark green colour medium suitabilityyellow and dark brownlow suitability light browncolour and unsuitable grey colour Table lists thefavourable bioclimatic variables and their contribution inpercent in maintaining the suitability of virusSusceptibleInfectiousRecovered SIR ModelEpidemiology deals with the study of pattern and occurrence of diseases in space and time associated with otherfactors such as environment demography and the translation of epidemiology into mathematical equations todescribe the spread of infectious diseases is known asmathematical epidemiology Allen Rayner andBender The mathematical epidemiology model isimplemented to understand the transmission dynamics ofcommunicable diseases by categorizing population intosusceptible infectious and recovered compartments Thefirst basic model known as SusceptibleInfectiousRecovered SIR model was proposed by Kermack andMcKendrick to describe the transmission of epidemic diseases from individual to individual The SIRmodel is a set of nonlinear ordinary differential equationswhich is mathematically defined as follows¼ l N þ SðÞ 00 bSI¼ bSI 00 cI 00 lI¼ cI 00 lRdSdtdIdtdRdtð1Þð2Þð3Þ 0cJournal of the Indian Society of Remote SensingFig Predicted suitability of Betacoronavirus using data till March Table Responsible bioclimatic variables in suitability modellingHereS is the class of susceptible individuals who are not yetcontracted to diseaseI is the class of infectious people who are now infectedwith disease and become infectious to infect others¢ R is class of recovered individuals who have recoverednow and are removed from class S¢ N is a total population size N S I R and t istime in days or weeks¢ b is the contact rate of infected person with suspected¢¢¢Bioclimatic variablesPercent contributionMean temperature of coldest quarterPrecipitation of wettest monthMean diurnal rangeIsothermalityAnnual mean temperatureMax temperature of warmest monthPrecipitation of coldest quarterPrecipitation of wettest quarterAnnual precipitationPrecipitation of driest quarterMean temperature of driest quarterMean temperature of wettest quarterPrecipitation seasonalityTemperature seasonalityPrecipitation of warmest quarterMean temperature of warmest quarterTemperature annual rangePrecipitation of driest monthMin temperature of coldest monthperson per dayc is the infectious period and average infectious periodis 1c¢ l is the per capita death rate which is adjusted by birthrate lNThere are many other compartment models derived fromthe basic epidemic model SIR with more compartmentsand transitions” SusceptibleExposedInfectiousRecovered SEIR Li and Muldowney SusceptibleInfectiousExposedRecoveredDeadSEIRDPiccolomiini and Zama SusceptibleInfectiousExposedRecoveredSusceptible SEIRS Liu and Zhang SusceptibleInfectiousQuarantineRecoveredSIQR Erdem
2
incidence and death rate of nonsmall cell lung cancer NSCLC in China ranks the first among the malignant tumors Circular RNA circRNA was reported to be involved in the progression of NSCLC Our study aimed to investigate the underlying mechanism of circ_0020123 in NSCLC progressionMethods Quantitative realtime polymerase chain reaction qRTPCR was used to detect the expression of circ_0020123 miR5905p and Thrombospondin THBS2 in NSCLC tissues and cells Cell proliferation and migration were examined by Cell Counting Kit8 CCK8 assay and Transwell assay respectively Flow cytometry assay was used to detect the apoptosis of NSCLC cells The protein levels of Ki67 matrix metalloprotein9 MMP9 Cleavedcaspase9 Cleavedcasp9 and THBS2 were detected by Western blot The targets of circ_0020123 and miR5905p were predicted by starBase and TargetScan and then confirmed by dualluciferase reporter assay and RNA immunoprecipitation RIP assay The animal experiment showed the effect of circ_0020123 on tumor growth in vivoResults The expression of circ_0020123 was upregulated in NSCLC tissues and cells Functionally circ_0020123 downregulation inhibited the proliferation and migration and promoted the apoptosis of NSCLC cells Interestingly circ_0020123 directly targeted miR5905p and inhibition of miR5905p reversed the knockdown effects of circ_0020123 on NSCLC cells More importantly THBS2 was a target of miR5905p and THBS2 overexpression reversed the effects of circ_0020123 knockdown on cell proliferation migration and apoptosis in NSCLC cells Finally suppression of circ_0020123 inhibited tumor growth in vivo through miR5905pTHBS2 axisConclusion Circular RNA circ_0020123 regulated THBS2 by sponging miR5905p to promote cell proliferation and migration and inhibit cell apoptosis in NSCLC cellsKeywords NSCLC Circ_0020123 miR5905p THBS2Highlights Circ_0020123 was upregulated and downregulation of circ_0020123 inhibited cell proliferation migration and promoted cell apoptosis in NSCLC cellsCorrespondence bskrju163comDepartment of Thoracic Surgery Lianyungang Second People™s Hospital No Hailian East Road Haizhou District Lianyungang Jiangsu China Circ_0020123 directly targeted miR5905p and miR5905p downregulation reversed the knockdown effects of circ_0020123 on NSCLC progression THBS2 acted as a target of miR5905p and overthe effects of expression of THBS2 reversed circ_0020123 knockdown on NSCLC progression Downregulation of circ_0020123 suppressed tumor growth in vivo through miR5905pTHBS2 axis The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40 The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cWang a0et a0al Cancer Cell Int Page of BackgroundLung cancer has the highest incidence of total cases and is the most common cause of cancer death of total cancer deaths in worldwide [] Lung cancer can be divided into several histological subtypes according to the location and the tendency of metastasis Small cell lung cancer SCLC accounts for about of all lung cancer cases [] However nonsmall cell lung cancer NSCLC accounts for of lung cancer and the a0years overall survival rate OS is only about [] Therefore it is important to find the effective treatment and potential molecular targets of NSCLC progressionCircular RNA circRNA is a single stranded RNA molecule with a closed circular structure Recently amounts of circular DNA have been discovered and most of which were thought to be the byproducts of typical splicing [ ] Previous reports indicated that the expression of circRNA was tissuespecific and the change of its expression intensity was associated with some diseases [“] Furthermore circRNA was involved in the occurrence and development of the disease and might be used as a potential biomarker in clinical diagnosis prognosis and treatment of diseases [ ] For example circ_0039569 facilitated cell proliferation and migration of renal cell carcinoma by sponging miR34a5p to regulate CC Chemokine ligand CCL22 [] Meanwhile hsa_circ_0043256 participated in the progression of NSCLC cells by mediating the cinnamaldehyde treatment [] A previous report suggested that circ_0020123 acted as an oncogene in NSCLC and circ_0020123 regulated zincfingerenhancer binding protein ZEB1 and enhancer of zeste homolog EZH2 by competitively binding with miR144 to induce cell progression and migration [] These reports suggested that circ_0020123 was a vital factor in the pathogenesis of NSCLC and its function and molecular mechanism need to be further studiedAs a small endogenous RNA microRNA miRNA is essential in regulating gene expression and plays a potential role in the exploitation of biomarkers [] Recently some aggregated miRNAs have been found in prostate cancer such as miR221222 miR143145 miR23b27b241 and miR1133a which were downregulated and had tumor inhibiting functions [] A previous study found that circulating miR5905p could be used as routine diagnostic tools for lung cancer and as a potential prognostic marker for liquid biopsy Besides overexpression of miR5905p reduced the development of NSCLC cells and regulated the expression of epithelialmesenchymal transformation EMTrelated proteins by targeting the signal transducers and activators of transcription STAT3 [] However the precise mechanism by which miR5905p affects NSCLC needs further investigationThrombospondin THBS2 as a secreted protein was confirmed to be highly expressed in different cancers including cervical cancer [] colorectal cancer [] and NSCLC [] A previous report suggested that THBS2 was involved in the proliferation apoptosis and antiautophagy regulation of cervical cancer cells by miR20a [] Tian et a0al found the expression and clinicopathological features of THBS2 in colorectal cancer were significantly correlated with the prognosis of cancer and might be used as a biomarker of prognosis [] However the molecular function of THBS2 in NSCLC remains poorly definedIn this study the targeting relationship between circ_0020123 and miR5905p was firstly detected The effects of circ_0020123 on cell proliferation migration apoptosis and tumor growth were performed by gain and lossoffunction experiments and molecular biology techniquesMaterials and a0methodsPatients and a0specimensNSCLC tissues and the adjacent healthy lung tissues were taken from NSCLC patients in the Lianyungang Second People™s Hospital All volunteers signed written informed consents NSCLC tissues and the adjacent tissues were immediately frozen in liquid nitrogen and kept at ˆ’ a0 °C for further experiments This research was approved by the Ethics Committee of Lianyungang Second People™s HospitalCell culture and a0cell transfectionTwo NSCLC cell lines A549 and H1299 and one normal lung cell line IMR90 were obtained from the Beijing Concorde Cell Library Beijing China A549 H1299 and IMR90 cells were cultivated in Dulbecco™s modified eagle medium DMEM HyClone Logan UT USA supplementing with fetal bovine serum FBS HyClone and cultured in an incubator at a0„ƒ with CO2Small interfering RNA siRNA targeting circ_00201231 sicirc_00201231 and sicirc_00201232 short hairpin RNA shRNA targeting circ_0020123 shcirc_0020123 miR5905pinhibitors siRNA negative control siNC shNC and NCinhibitors were all obtained from Biomics Biotech Jiangsu China Full length of THBS2 cDNA Sangon Biotech Shanghai China was subcloned into pcDNA31 plasmid EKBioscience Shanghai China Then cell transfection was performed by Lipofectamine Thermo Fisher Scientific Waltham MA USA 0cWang a0et a0al Cancer Cell Int Page of RNA isolation and a0quantitative real‘time polymerase chain reaction qRT‘PCRThe TRIzol reagent Invitrogen Carlsbad CA USA was used for extracting the total RNAs Next the reversed transcription was carried out by RTPCR kit Invitrogen The qRTPCR was performed using the ABI SYBR Green Master Mix Invitrogen The primers in our study were as follows F5²TTC GGA CGA CCG TCA AAC AT3² and R5²AGG ATC CCT GCA CCA CAA TG3² for circ_0020123 F5²TGA AAG ACG TGA TGG CAC AC3² and R5²CTT CCA TTT TGG for miR5905p F5²AGA AGG GGT TTT TGG3 ² CTG GGG CTC ATT TG3² R5²AGG GGC CAT CCA CAG TCT TC3² for glyceraldehyde3phosphate dehydrogenase GAPDH [] F5²GCG GCT GGG TCT ATT TGT C3² and R5²GCA GGA GGT GAA GAA CCA TC3² for THBS2 [] F5²ATT GGA ACG ATA CAG AGA AGATT3² and R5²GGA ACG CTT CAC GAA TTT G3² for U6 [] GAPDH and U6 were the internal parametersCell Counting Kit‘ CCK‘ assayThe proliferation viability of A549 and H1299 cells were detected by the CellCounting Kit8 MSK Wuhan China A549 and H1299 cells were cultivated into a 96well plate with a density of × a0cellswell and incubated in a0°C for or a0h Then a0μL fresh medium and CCK8 solution was added After incubation at a0°C for a0h the OD values were detected by the Multiskan Ascent microplate reader Abcam Cambridge MA USATranswell assayTranswell chamber Corning Life Sciences Corning NY USA was used to detect cell migration Firstly the serumfree DMEM Thermo Fisher Scientific was fixed with cell suspension cells and seeded into the upper chamber and the DMEM containing serum was put into the lower chamber After incubation for a0h the cells in lower surface of the upper chamber were treated with formaldehyde solution for a0 h and then thoroughly washed Finally the migrated cells were stained with crystal violet Corning Life Sciences and observed by using a microscopeFlow cytometryFirstly A549 and H1299 cells were cultured and PBS was used for washing cells Then the binding buffer was used to resuspend cells and the Annexin Vfluorescein isothiocyanate VFITCpropidium iodide PI Apoptosis Detection Kit Thermo Fisher Scientific was used to stain cells Finally cell apoptosis was detected by flow cytometry Thermo Fisher ScientificWestern blot analysisThe total proteins of NSCLC tumors or cells were collected by RIPA lysis buffer Sangon Biotech Then the proteins were separated by Sodium dodecyl sulphate polyacrylamide gel electrophoresis SDSPAGE and transferred to polyvinylidene fluoride PVDF membranes Thermo Fisher Scientific The skimmed milk was added and incubated with primary antiGAPDH antibody Invitrogen Carlsbad CA USA antiβactin antibody Invitrogen antiKi67 antibody Invitrogen antimatrix metalloprotein9 MMP9 antibody Invitrogen antiCleavedcaspase9 Cleavedcasp9 antibody Invitrogen or antiTHBS2 antibody Invitrogen at a0°C overnight Finally the membranes were incubated with the secondary antibody for a0 h at room temperature The results were viewed using Kodak film developer Fujifilm JapanDual‘luciferase reporter assaysThe wild type circ_0020123 sequences circ_0020123WT mutant circ_0020123 sequences circ_0020123MUT wild type THBS2 ²UTR sequences THBS2WT mutant THBS2 ²UTR sequences THBS2MUT were cloned into pGL3 luciferase reporter plasmid Promega Madison WI USA Then the plasmid and miR5905p or miRNC were cotransfected into A549 and H1299 cells by Lipofectamine Thermo Fisher Scientific After transfection for a0h the DualLuciferase Reporter Assay System Promega was performed to detect the luciferase activityRNA immunoprecipitation RIPFirstly the Magna RIP RNAbinding Protein Immunoprecipitation Kit gzscbio Guangzhou China was performed to verify the relationship between circ_0020123 and miR5905p In brief the magnetic beads and antiAgo2 antibody Abcam were added into cells and incubated for a0h Then the proteinase K and the phenol“chloroformisoamyl alcohol reagent were added for purifying RNAs Finally qRTPCR was used to measure circ_0020123 enrichmentAnimal experimentsThe 4weekold BALBc male nude mice Vitalriver Beijing China were raised in a sterile environment for 0cWang a0et a0al Cancer Cell Int Page of experiments Then PBS was used to suspend A549 cells × transfected with shcirc_0020123 or shNC Next the nude mice were divided into two groups n A549 cells transfected with shcirc_0020123 or shNC were shcirc_0020123 or shNC inoculated into the nude mice The tumor volume was detected every a0 days After a0days the nude mice were euthanatized and the tumor weight was detected Besides the tumor tissues from each group were collected to detect the expression of circ_0020123 miR5905p and THBS2 The animal experiment was approved by the Animal Experimentation Ethics Committee of Lianyungang Second People™s HospitalStatistical analysisThe software GraphPad Prism was performed for statistical analysis The data was displayed as mean ± standard deviation SD The significant difference was calculated by Student™s t test and oneway analysis of variance ANOVA P was considered as statistically significantResultsCirc_0020123 was a0upregulated in a0NSCLC tissues and a0cellsTo begin with qRTPCR was used to detect the expression of circ_0020123 the result showed that circ_0020123 was significantly upregulated in NSCLC tissues compared with the adjacent healthy tissues Fig a0 1a Similarly the expression of circ_0020123 in NSCLC cells A549 and H1299 was markedly higher than that in normal cells IMR90 Fig a01b From these data it is speculated that circ_0020123 might be acted as an oncogene in NSCLCFig Circ_0020123 was upregulated in NSCLC tissues and cells a QRTPCR was used to detect the expression of circ_0020123 in adjacent healthy tissues n and tumor tissues n b The expression of circ_0020123 in normal cell line IMR90 and NSCLC cell lines A549 and H1299 was detected by qRTPCR P Downregulation of a0circ_0020123 inhibited the a0proliferation migration and a0induced apoptosis of a0NSCLC cellsTo investigate the functional effects of circ_0020123 on NSCLC cells sicirc_00201231 and sicirc_00201232 were obtained and transfected into A549 and H1299 cells Firstly the transfection efficiency was detected by qRTPCR Fig a02a Next CCK8 was used to detect the proliferation and the results showed that the proliferation of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 was reduced Fig a0 2b Moreover the migration of A549 and H1299 cells was significantly downregulated by circ_0020123 knockdown Fig a02c In addition the apoptosis of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 was obviously higher than transfected with siNC Fig a02d Finally the protein levels of cell proliferationrelated protein Ki67 and cell migrationrelated protein MMP9 were inhibited while cell apoptosisrelated protein Cleavedcasp9 was upregulated in NSCLC cells transfected with sicirc_00201231 or sicirc_00201232 Fig a02e These data suggested that inhibition of circ_0020123 suppressed cell proliferation migration and promoted apoptosis in NSCLC cellsCirc_0020123 directly targeted miR‘‘5pBy searching in the online software starBase the potential binding sites between circ_0020123 and miR5905p were detected Fig a0 3a To confirm that the dualluciferase reporter assay was performed the results showed that the luciferase activity of circ_0020123WT reporter plasmid was reduced by miR5905p mimic while the circ_0020123MUT reporter plasmid activity was not changed in A549 and H1299 cells Fig a03b Furthermore the expression of miR5905p was lower in A549 and H1299 cells compared with that in IMR90 cells Fig a0 3c In contrast miR5905p expression was elevated in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 Fig a0 3d Finally the RIP assay was also used to confirm the targeting relationship between circ_0020123 and miR5905p and the results showed that circ_0020123 and miR5905p were enriched in antiAgo2 group Fig a03eMiR‘‘5p downregulation reversed the a0inhibition effects of a0circ_0020123 on a0NSCLC cellsTo further explore the functional effects between circ_0020123 and miR5905p miR5905pinhibitor was established QRTPCR was used to detect the transfection efficiency Fig a0 4a Interestingly miR5905p was upregulated in A549 and H1299 cells transfected with sicirc_00201231 while the expression of miR5905p was recovered in cells transfected with 0cWang a0et a0al Cancer Cell Int Page of Fig Downregulation of circ_0020123 inhibited the proliferation and migration and induced the apoptosis of NSCLC cells a The transfection efficiency of sicirc_00201231 and sicirc_00201232 in A549 and H1299 cells was detected by qRTPCR b CCK8 assay was used to detect the proliferation of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 c The migration of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 was measured by Transwell assay d Flow cytolysis assay was used to detect the apoptosis of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 e The protein levels of cell proliferation related protein Ki67 cell migration related protein MMP9 and cell apoptosis related protein Cleavedcasp9 were detected by Western blot P Fig a0sicirc_00201231 miR5905pinhibitors 4b Moreover circ_00201231 knockdown inhibited cell proliferation and migration while the miR5905p inhibitor reversed these effects Fig a0 4c d In addition the apoptosis of A549 and H1299 cells transfected with sicirc_00201231 was increased which was abolished by miR5905pinhibitor Fig a0 4e Similarly miR5905p inhibitors reversed the effects on the protein levels of Ki67 MMP9 and Cleavedcasp9 in A549 and H1299 cells transfected with sicirc_00201231 Fig a0 4f These results that miR5905p downregulation reversed the effects of circ_0020123 downregulation on the proliferation migration and apoptosis of NSCLC cellsindicated MiR‘‘5p targeted THBS2 in a0NSCLC cellsThe THBS2 ²UTR was predicted to contain the binding sites of miR5905p through the online software TargetScan Fig a05a Then the dualluciferase reporter assay was used to confirm the targeting relationship The results showed that cotransfection of miR5905p and THBS2WT significantly limited the luciferase activity in both A549 and H1299 cells the luciferase activity was not altered in cells cotransfected with miR5905p and THBS2MUT Fig a05b Importantly the mRNA and protein level of THBS2 was enahnced in NSCLC cells Fig a05c d We further explored whether circ_0020123 affected the functions of THBS2 in NSCLC cells The mRNA and protein expression of THBS2 were repressed in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 Fig a05e f 0cWang a0et a0al Cancer Cell Int Page of Fig Circ_0020123 directly targeted miR5905p a The binding site between circ_0020123 and miR5905p was detected by the online software starBase b The luciferase activity of circ_0020123WT or circ_0020123MUT reporter plasmid in A549 and H1299 cells transfected with miRNC or miR5905p was detected by dualluciferase reporter assay c QRTPCR was used to detect the expression of miR5905p in A549 and H1299 cells d The expression of miR5905p in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 was detected by qRTPCR e RIP assay was used to confirm the relationship between circ_0020123 and miR5905p P THBS2 overexpression reversed the a0effects of a0circ_0020123 knockdown on a0the a0proliferation migration and a0apoptosis of a0NSCLC cellsBased on the work ahead of us the pcDNA31THBS2 was constructed Then the qRTPCR and Western blot were used to detect the transfection efficiency and the THBS2 expression was increased in A549 and H1299 cells transfected with pcDNA31THBS2 Fig a0 6a b In addition the proliferation and migration rates of A549 and H1299 cells transfected with sicirc_00201231 pcDNA31THBS2 were higher than that transfected with sicirc_00201231 Fig a0 6c d Meanwhile a similarly phenomenon was also observed in cell apoptosis the pcDNA31THBS2 significantly reversed the promotion effect of circ_0020123 deletion on cell apoptosis Fig a0 6e Furthermore the effects of circ_0020123 deletion on Ki67 MMP9 and Cleavedcasp9 protein levels were also reversed by THBS2 overexpression Fig a0 6f These data suggested that overexpression of THBS2 could reverse the effects of circ_0020123 downregulation on cell proliferation migration and apoptosisReduction of a0circ_0020123 suppressed tumor growth in a0vivo through a0circ_0020123miR‘‘5pTHBS2 axisTo further explore the function of circ_0020123 in NSCLC cells the shcirc_0020123 was constructed and the xenograft tumor was established Then A549 cells transfected with shcirc_0020123 or shNC were injected into the nude mice The xenograft tumor volume was measured every a0 days after injection and the results showed that tumor volume was smaller shcirc_0020123 group than that in shNC group Fig a07a Moreover tumor weight was inhibited by circ_0020123 knockdown Fig a0 7b Furthermore the expression circ_0020123 and THBS2 was decreased while the miR5905p was increased in xenograft tumor transfected with shcirc_0020123 Fig a0 7c Western blot assay also revealed that the protein level of THBS2 was repressed by circ_0020123 knockdown Fig a07d Finally the digital tomosynthesis DTS was used to detect the number of lung metastatic nodules in xenograft tumor and it was reduced in shcirc_0020123 group Fig a07e The results suggested that downregulation of circ_0020123 inhibited tumor growth in a0vivoDiscussionClinically only a few NSCLC patients were diagnosed at an early stage and treated by surgical resection More than of NSCLC patients were diagnosed with the advanced stage or metastatic tumors [] Thus finding novel biomarkers and therapeutic targets were necessary for the effective diagnosis and treatment of NSCLCRecently circRNA was no longer considered as a random product in the RNA shearing process and its biological significance and function in malignant tumors 0cWang a0et a0al Cancer Cell Int Page of Fig MiR5905p downregulation reversed circ_0020123 knockdown effects in NSCLC cells a QRTPCR was used to detect the expression of miR5905p in A549 and H1299 cells transfected with miR5905pinhibitors b The expression of miR5905p in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 miR5905pinhibitors was detected by qRTPCR c The proliferation of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 miR5905pinhibitors was tested by CCK8 assay d Transwell assay was used to measure the migration of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 miR5905pinhibitors e Flow cytolysis assay was used to detect the apoptosis of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 miR5905pinhibitors f The protein levels of Ki67 MMP9 Cleavedcasp9 in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 miR5905pinhibitors were detected by Western blot P had received more and more attention Previous reports revealed that circ_0020123 was involved in the development of NSCLC [] Moreover the level of circ_0020123 was elevated in NSCLC cells [] Consistently we found that the expression of circ_0020123 was markedly higher in NSCLC tissues and cells Moreover this research indicated that inhibition of circ_0020123 suppressed the proliferation migration and induced apoptosis of NSCLC cells in a0 vitro Besides circ_0020123 promoted tumor growth in a0vivoEndogenous circRNAs could act as microRNA sponges to inhibit their function and some studies linked miRNA sponges to human diseases including cancer [] A previous study indicated that circRNA ctransferrin receptor cTFRC regulated TFRC by sponging miR107 to facilitate bladder carcinoma development [] MiR5905p was studied in different cells such as airway smooth muscle cells [] colon epithelial cells [] and NSCLC cells [] However the potential relationship between miR5905p and circRNA has not been researched In this study circ_0020123 directly targeted miR5905p and miR5905p inhibition reversed the effects of circ_0020123 knockdown on NSCLC progression These data provided a clue to the therapeutic strategy for NSCLC 0cWang a0et a0al Cancer Cell Int Page of Fig MiR5905p targeted THBS2 in NSCLC cells a The potential binding site between THBS2 ²UTR and miR5905p was predicted by the online software TargetScan b Dualluciferase reporter assay was used to measure the luciferase activity of THBS2WT or THBS2MUT reporter plasmid in A549 and H1299 cells transfected with miRNC or miR5905p c QRTPCR was used to detect the mRNA expression of THBS2 in NSCLC cells d The protein level of THBS2 in NSCLC cells was tested by Western blot e The mRNA expression of THBS2 in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 was detected by qRTPCR f Western blot was used to measure the protein level of THBS2 in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 P Our study also confirmed that miR5905p could target THBS2 directly in NSCLC cells THBS2 is a calciumbinding protein that binds to and inactivates matrix metalloproteinase MMP genes involved in tissue formation and repair [ ] A previous document suggested that THBS2 acted as a target of miR2213p and participated in lymph node metastasis in cervical cancer [] The data in this research showed that the expression of THBS2 in NSCLC cells was markedly higher than normal healthy cells Furthermore overexpression of THBS2 reversed the effects of circ_0020123 knockdown on proliferation migration and apoptosis of NSCLC cells suggesting that circ_0020123 promoted the progression of NSCLC cells through miR5905pTHBS2 axisConclusionIn conclusion our research showed that the expression of circ_0020123 was higher in NSCLC tissues and cells than control and downregulation of circ_0020123 inhibited the proliferation migration and promoted apoptosis of NSCLC cells and also suppressed tumor growth in a0 vivo Moreover circ_0020123 directly targeted miR5905p while miR5905p inhibition reversed the effects of circ_0020123 knockdown on NSCLC cells More importantly circ_0020123 regulated the expression of THBS2 by sponging miR5905p and upregulation of THBS2 reversed the effects of circ_0020123 knockdown on NSCLC cells Therefore our research demonstrated that circ_0020123 enhanced proliferation migration and inhibited 0cWang a0et a0al Cancer Cell Int Page of Fig Overexpression of THBS2 reversed the effects of circ_0020123 knockdown on proliferation migration and apoptosis of NSCLC cells a b The mRNA and protein expression of THBS2 in A549 and H1299 cells transfected with pcDNA31THBS2 was detected by qRTPCR and Western blot c CCK8 assay indicated the proliferation of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcDNA31THBS2 d The migration of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcDNA31THBS2 was measured by Transwell assay e The apoptosis of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcDNA31THBS2 was detected by Flow cytolysis assay f The protein levels of Ki67 MMP9 Cleavedcasp9 in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcDNA31THBS2 were detected by Western blot P apoptosis of NSCLC cells by sponging miR5905p to regulate THBS2results and develop the manuscript All authors read and approved the final manuscriptAbbreviationsNSCLC Nonsmall cell lung cancer circRNA Circular RNA qRTPCR Quantitative realtime polymerase chain reaction CCK8 Cell Counting Kit8 MMP9 Matrix metalloprotein9 Cleavedcasp9 Cleavedcaspase9 Cleavedcasp9 Cleavedcaspase9 RIP RNA immunoprecipitation ZEB1 Zincfingerenhancer binding protein EZH2 Zeste homolog STAT3 Signal transducers and activators of transcription THBS2 Thrombospondin AcknowledgementsNot applicableAuthors™ contributionsLW collaborated to design the study LZ were responsible for experiments analyzed the data YW wrote the paper All authors collaborated to interpret FundingNoneAvailability of data and materialsPlease contact corresponding author for data requestsEthics approval and consent to participateThis research was approved by the Ethics Committee of Lianyungang Second People™s Hospital The animal experiment was approved by the Animal Experimentation Ethics Committee of Lianyungang Second People™s HospitalConsent for publicationAll listed authors have actively participated in the study and have read and approved the submitted manuscript 0cWang a0et a0al Cancer Cell Int Page of Fig Reduction of circ_0020123 suppressed the tumor growth in vivo through circ_0020123miR5905pTHBS2 axis a A total of × A549 cells transfected with shcirc_0020123 or shNC were injected into nude mice to establish the xenograft tumor Tumor volume was measured every d after injection b Tumor weight was measured on d c The expression of circ_0020123 miR5905p and THBS2 in xenograft tumor was measured by qRTPCR d The protein level of THBS2 in xenograft tumor was evaluated by Western blot e The number of lung metastatic nodules in xenograft tumor was detected by digital tomosynthesis DTS P Competing interestsThe authors declare that they have no competing interestsReceived April Accepted July References Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin “ Abe H Takase Y Sadashima E Fukumitsu C Murata K Ito T Kawahara A Naito Y Akiba J Insulinomaassociated protein is a novel diagnostic marker of small cell lung cancer in bronchial brushing and cell block cytology from pleural effusions validity and reliability with cutoff value Cancer Cytopathol “Li C Zhang L Meng G Wang Q Lv X Zhang J Li J Circular RNAs pivotal molecular regulators and novel diagnostic and prognostic biomarkers in nonsmall cell lung cancer J Cancer Res Clin Oncol “ Belousova EA Filipenko ML Kushlinskii NE Circular RNA new regulatory molecules Bull Exp Biol Med “ Zhang Z Xie Q He D Ling Y Li Y Li J Zhang H Circular RNA new star new hope in cancer BMC Cancer Li L Chen Y Nie L Ding X Zhang X Zhao W Xu X Kyei B Dai D Zhan S Guo J Zhong T Wang L Zhang H MyoDinduced circular RNA CDR1as promotes myogenic differentiation of skeletal muscle satellite cells Biochim Biophys Acta Gene Regul Mech “ Greco S Cardinali B Falcone G Martelli F Circular RNAs in muscle function and disease Int J Mol Sc
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" trophoblast cell surface antigen trop2 is overexpressed in many squamous cell carcinomas andpromotes tumor development and invasion the association between trop2 expression and occurrence anddevelopment of oral squamous cell carcinoma oscc remains to be understoodmethods we investigated the role of trop2 in oscc patients using a combination of biophysical approaches atotal of oscc patient specimens with varying degrees of differentiation were subjected to hematoxylin andeosin staining immunohistochemistry kaplanmeier survival curve analysis and atomic force microscopy to analyzetrop2 expression morphology and mechanical properties of oscc tissuesresults trop2 was overexpressed in of poorly differentiated oscc samples high levels of trop2 wereassociated with survival rate lower than and patient age odds ratio [or] p confidence interval [ci “] tumor size or p ci [“] and tnm stageor p ci [“] average surface roughness of low medium and highly differentiatedoscc tissues were ± ± and ± nm respectively the pearson coefficient revealed anegative association between tumor stiffness and trop2 expression r ˆ’ p overexpression of trop2 negatively associated with patient survival degree of tumor differentiationand tissue mechanics taken together our findings demonstrated that trop2 may be an indicator of osccdifferentiation leading to the altered mechanical properties of oscc tissueskeywords oral squamous cell carcinoma trop2 tissue stiffness differentiation survival oral squamous cell carcinoma oscc is a commonsubtype of head and neck and other malignant tumors[ ] the past few decades have shown increased incidence of oscc that is expected to rise further in the future thereforeimperative to determineisit correspondence zhangkllzu163com baoping zhang shuting gao and ruiping li contributed equally to thiswork1department hospital of stomatology lanzhou university donggang westroad lanzhou gansu chinafull list of author information is available at the end of the biological factors associated with the early diagnosis andtreatment of oscchuman trophoblast cell surface antigen trop2 alsocalled tumorassociated calcium signaltransduction2tacstd2 is a surface glycoprotein encoded by tacstd that has extracellular domains a single transmembrane domain and a short tail [ ] trop2 is overexpressed in many human cancers including ovarian [ ]gastric [ ] colorectal pancreatic and laryngealcancers inhibiting trop2 expression has shownpromise in clinical applications [ ] trop2 regulates the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhang bmc cancer page of tumorigenic properties including cancer cell adhesion invasion and migration tang have recentlyshown that trop2 impacts growth and metastasis byactivating pi3kakt signaling this phenomenon hasalso been observed in gallbladder cancer amongtheinvolved intumorigenesis the role of catenin has been studiedextensively [ “] this has shed light on the biological functions of trop2 and its use as a prognostic biomarker for osccvarious biochemical mechanismsatomic force microscopy afm is a powerful toolthat generates surface topographical images with magnifications that range between macro and nanoscalesafm has been used to determine the mechanical properties of tumor tissues in a variety of cancers such asthose of the breast liver and lung parameters for tissue stress such as mechanical phenotypeindex correlate with cancer development and invasion advancements in technology used for determiningbiophysical properties have facilitated the nanolevelanalysis of tumor tissuesthis study aims at investigating the correlation between trop2 expression and clinicopathological characteristics of oscc we have demonstrated the tissuemorphology and mechanics of oscc samples duringtumor development using afm we believe our findingswill help develop trop2 in accurately diagnosing osccin tumors with different grades of differentiationmethodstissue preparationthe protocols in this study were approved by the researchethics committee of lanzhou university tumor sampleswere collected from patients after obtaining written informed consent a total of patients with oral squamous cell carcinoma oscc were registered atthesecond hospital of lanzhou university between january and march among these samples sampleseach showed high moderate and low levels of differentiation the experimental group comprised males and females aged “ years average years all patientswere diagnosed with oscc based on surgery andfig paraffin pathological sections of tissues a d g — 4fold b e h — 10fold c f i — 40fold 0czhang bmc cancer page of fig immunohistochemical staining was performed to detect the expression of trop2 at different stages of osccpathology patients did not undergo radiotherapy chemotherapy or immunotherapy before surgery pathologicalanalysis after tumor biopsy was performed by two experienced pathologists after which the diagnosis of other diseases including inflammation at other sites and secondarytumors were excluded cancer and cervical lymph nodetissues were collected after maxillofacial surgery all specimens were sampled from typical areas of the lesion andfixed with neutralbuffered formalin followed by conventional paraffin embedding among them and fig average optical density of trop2 poorly differentiated squamous cell carcinoma showed high expressionp 0czhang bmc cancer page of table correlation between trop2 expression and clinicopathological characteristicscharactersntrop2 expressionlow or nototalgendermalefemaleage‰¥ localizationmucosatonguedifferentiationwellmoderatepoortumor sizet1 ‰ cm cmt2 ‰ cmt34cmt4blymph node metastasesn0nxdistant metastasesm0m1tnm stagei iiiii ivperineural infiltrationnoyesvascular invasionnoyespearson x2p value highpatients exhibited no and cervical lymph node metastasesrespectively clinical tnm staging was performed according to the 7th edition tnm staging classification standardjointly developed by the international union for cancercontrol and american joint committee on cancer and world health anization guidelines hematoxylin and eosin he stainingoscc tissues were fixed overnight using neutralformalin solarbio beijing china paraffin embeddedsliced into 4μm thick sections dewaxed using xyleneand rehydrated using different concentrations of ethanol the sections were stained with hematoxylin for min and hydrochloric acidethanol and eosin for min followed by gradient dehydration transparentizationresin sealing solarbiobeijing china sections were visualized and imagedusing the olympus bx53 at magnifications of — and sealing and neutral 0czhang bmc cancer page of immunohistochemistryhe sections were subjected to the sp method to detecttrop2 expression the sections were incubated overnight with the primary antibody against trop2 abcam usa at °c followed by incubation withbiotinlabeled goat antirabbitigg abcamusa at °c for h the sections were then developed using dab beijing zhongshan golden bridgebiotechnology china dehydrated transparentizationand film and neutral resin sealed the prepared sections were visualized using microscopy olympusbx53 japanafmfixed tissues were placed in petri dishes containingphosphatebuffered saline all analyses for mechanical properties were performed using the biologicalatomic force microscope bioafm nanowizard iiibruker usa silicon afm probes from the pointprobe®constant of nmcoating nanoworld usa werecontrreflexused the spring constant ofthe probe was calibrated using builtin thermal vibration before measuringandthickness of μm afm was performed using theseries with a khzforcetheresonancefrequencyofcontact model and a scan rate of hzs in airforcedistance curves are generated when the probecontacts the tissue following whichthe structuremorphology and mechanical properties of samplesare measured at μms six random sites wereselected for each sample and each site was measured times we used the modified hertz contact modelto analyze forcedistance curves and calculateyoung™s modulus and roughness of oscc tissueswith varying differentiationstatistical analysisstatistical analyses were performed using spss statistical product and service solutions ibm forcespectrum data were expressed as mean ± standard errorand statistical comparisons were performed using oneway analysis of variance followed by the tukeykramerhsd test for pairwise comparisons pearson chisquaretest was used to analyze clinical features and trop2 expression based on the calculated odds ratios ors and confidence intervalci survival was evaluatedusing kaplanmeier curves and the difference was analyzed using the logrank test p was consideredstatistically significantfig trop2 total survival curve using kaplanmeier survival curves low blue line high green line 0czhang bmc cancer page of resultstissue morphology and trop2 expression across theclinical stages of oscctumor cells from poorly differentiated oscc samples exhibited characteristic atypia poor differentiation and irregular morphology fig howeverthe number volume atypia nuclear pyknosis andmitotic structures decreased in tumor cellsfromhighly differentiated oscc as compared to those inpoorly differentiated cells trop2 primarily localizedin the cytoplasm of tumor cells but not in adjacentnormal epithelial cells we observed that low differentiation and high malignancy of oscc was associated with higher trop2 expression fig theaverage optical density of trop2 among the lowmedium and highly differentiated oscc tissues were ± ± and ± respectively fig table trop2 expression risk factors with clinicopathological featurescharacteristicsntrop2 expressionlow or nototalgendermalefemaleage‰¥ localizationmucosatonguedifferentiationwellmoderatepoortumor sizet1 ‰ cm cmt2 ‰ cmt34cmt4blymph node metastasesn0nxdistant metastasesm0m1tnm stagei iiiii ivperineural infiltrationnoyesvscular invasionnoyesnote a well vs moderate b moderate vs poor c well vs poorp valueor cihigh 005a 0001b 0001c 0czhang bmc cancer page of association between trop2 expression and clinicalcharacteristics of osccwe analyzed the clinicopathological characteristics of patients with oscc with varying degree of differentiationdifferential expression of trop2 was associated with patient age tumor differentiation tumor size tnm stagepercutaneous nerveinvasiontable p patients with poorly differentiated tumors were more likely than patients with well and moderate differentiated tumors to have high trop2 expressionp however there was no association between theexpression of trop2 and patient gender tumor locationlymph node metastasis or distant metastases p and vascularfiltrationtrop2 expression and patient survivalusing kaplanmeier survival curves we observed that anincrease in trop2 expression negatively correlated withthe overall survival of patients fig and lowno oftrop2 expression group™s 3years survival rate was a for high expression group and 5years ratewere and respectively trop2 expression wasassociated with patient age p or ci[“] tumor differentiation well vs moderatep or ci [“] moderate vspoor p or ci [“]well vs poor p or ci [“] tumor size p or ci[“] tnm stage p or ci[“] vascular invasion p or ci [“] and peripheral nerve invasionp or table high trop2 expressionwas detected in older patients with low degree of differentiation larger tumor volume higher tnm staging andvascular and peripheral nerve invasion thereby resultingin lower overall survival thus trop2 may be a prognostic indicator for survival in oscc patientsfig surface morphology of oscc tissue sections via afm detection irregular morphology appeared in the low differentiation 0czhang bmc cancer page of surface morphology and roughness of oscc tissuesthe surface morphologies of oscc tissues with varying degrees of differentiation were analyzed directtopographical imaging using bioafm figure showsthe representative image from each tissue acquiredduring the cantileverbased afm nano indentationtest the tissue interface varied with tumor differentiationindicating that highly differentiated oscc tissues had a regular and flat morphology oscc tissueswith low differentiation exhibited an overall irregularmorphology with distinct modulation and loose tissueanization figure summarizes the roughness ofoscc tissues with varying differentiation the average surface roughness of low medium and highly differentiated oscc tissues were ± ± and ± nm respectively roughness ofthe tissue surface was enhanced with increasing differentiation of oscc tissuesyoung™s modulus of oscc tissueswe used bioafm to determine young™s modulusbased on the mechanical properties of oscc tissues with varying degrees of differentiation we randomly selected six contact points from each slice andeach contact point was measured times forcedistance curves were generated for each slice and thejpk data processing software version was usedto calculate young™s modulus figure shows theaverage variation in stiffness within individual tissuesin the range of “ kpa in the low differentiationsamples we observed low stiffness as compared tothat in high or medium differentiation samples p fig surface roughness results are express as mean ± sem nm 0czhang bmc cancer page of fig afm test average tissue stiffness young™s modulus e was thus confirmed to be a parameter of cell hardness for various cells and tissuepa p thus tissue differentiation was positively associated with its stiffness fig association between mechanical properties and trop2expression in osccthe pearson coefficient showed a negative associationbetween the stiffness of oscc tissues and trop2 expression fig r ˆ’ p thus we detectedan increase in stiffness with varying differentiation in thetumor samplesdiscussiontrop2 belongs to the family of genes involved in calcium signaling associated with tumorigenesis and foundin human trophoblast and chorionic cell lines studieshave shown that overexpression of trop2 is associatedwith tumorigenesis and malignancy [“]in thisstudy trop2 expression was observed to be a highlysensitive and specific marker of tongue squamous cellcarcinoma and tissue stiffness the relative thickness ofsamples helped accurately diagnose and determine thestaging of tongue squamous cell carcinomaimmunohistochemical analysis revealed that the expression of trop2 in poorly differentiated oscc tissueswas significantly higher than that in welldifferentiatedoscc tissues additionally trop2 upregulation wascorrelated with tumors of advanced tnm iii iv staging and poor differentiation than that in tumors withlow tnm i ii staging thus the abnormal expressionof trop2 may be associated with the occurrence anddevelopment of tongue malignancies furthermore hightrop2 expression predicted low survival as comparedto that in the tumors with low trop2 expression previous research has also demonstrated the correlation between shorter patient lifespan and high levels of trop2as compared to that in patients with laryngeal squamouscell carcinoma and low levels of trop2 trop2possesses sites for tyrosineserine phosphorylation thatregulate signal transduction or its expression and activity thereby rendering cancer cells resistant to apoptosis upregulated trop2 correlates with the poor prognosis of thyroid papillary carcinoma colon cancer liver cancer and other malignanciesthere have been an increasing number of studies on thebiological role of trop2 at the molecular level trop2induces the downregulationloss of pten thereby stimulating pi3kakt signaling and tumor development pten is a wellknown tumor suppressor that is a phosphatase and affects the pi3kpkbakt signaling axisduring the dephosphorylation of pip2 and pip3 pi3k signaling is important in regulating tumor cell proliferation migration and invasion [ ] thus pten is anegative regulator of cancer [ ] li have shownthat trop2 activates epithelialmesenchymal transitionvia pi3kakt signaling thereby promoting proliferationmigration and metastasis in gallbladder cancer similarly trop2 expression stimulates the proliferation migration and invasion of osteosarcoma cells hou demonstrated that trop2 regulates jak2stat3 signaling in glioblastoma cells 0czhang bmc cancer page of fig correlation analysis between changes in mechanical stiffness of oscc tissues and trop2 expression note changes have statisticalsignificance p and show a certain negative correlation r ˆ’ functional differentiation oftissues influences themicromorphology and mechanical stiffness of oscccells we detected low surface roughness on oscc tissues with loose structure reduced hardness and enhanced cell adhesion migration and invasion poorlydifferentiated oscc tissues are œsofter than highly differentiated oscc tissues pi3k is an important celladhesion molecule trop2 triggers the synthesis of proteins with homologous domains such as pleckstrinrac tiam and vav tiam and vav activate rac thatleads to reanization of the actin cytoskeleton cellrecognition and adhesion the underlying mechanisms involved in the alterationof micromechanical properties of oscc samples and occurrence development metastasis and invasion ofoscc tumors remain to be elucidated he staining isthe gold standard for tumor diagnosis with the development of biomechanics in the past two decades the mechanical properties of tissues need to be investigated based on biomedical and physical parametersin this study we have assayed the changes in mechanicalproperties at the micronanometer level using afm anddetermined the association between the tnm grademetastasis and stiffness of tumor samplesin we have demonstrated the association between differential expression of trop2 and patient agetumor differentiation tumor size tnm stage percutaneousnerve filtration and vascular invasion moreover high levelsof trop2 correlated with poor overall survival in patientshighly differentiated cancer tissues exhibited increasedsurface roughness and stiffness lastly high trop2 expression resulted in reduced tumor stiffness however thisstudy had some limitations first the cohort used in thisstudy was relatively small second we did not employ molecular methods of analysis such as western blotting orenzymelinked immunosorbent assay thus using a largerpatient cohort and multiple techniques in molecular andcell biology will help validate our findings and developtrop2 as a specific and efficient prognostic biomarker forosccthese findings could promote new methods for the earlyoscc diagnosis depend on the stage of cancer and developing screening methods with high sensitivity andspecificity more detailed studies are needed to determine the feasibility and therapeutic benefit of testing tissue stiffness in human diseaseabbreviationsoscc oral squamous cell carcinoma trop2 trophoblast cell surfaceantigen afm atomic force microscopyacknowledgementswe thank the individual who participated in this studyauthors™ contributionsbz sg and rpl are responsible for conception and design data wascollected by ytl rc jyc and ymg data was analyzed by ew and yh klzrevised the all authors have read and approved the manuscriptfundingthis work was supported by the fundamental research funds for thecentral universities no lzujbky2020cd03 baoping zhang doctoralmaster 0czhang bmc cancer page of students of the second hospital of lanzhou university sdkygg17 lan yangand key laboratory of mechanics on disaster and environment in westernchina the ministry of education of china no “ kailiang zhangavailability of data and materialsthe datasets used and analyzed during the current study are available fromthe corresponding author on reasonable requestethics approval and consent to participatewritten informed consent was obtained from each participant before samplecollection the study was approved by the committee for ethical affairs ofschool of stomatology lanzhou universityconsent for publicationnot applicablecompeting intereststhe authors have no conflicts of interestauthor details1department hospital of stomatology lanzhou university donggang westroad lanzhou gansu china 2institute of biomechanics andmedical engineering lanzhou university lanzhou chinareceived april accepted august referencesiyer s thankappan k balasubramanian d early detection of oral cancerscurrent status and future prospects curr opin otolaryngol head neck surg“caldeira pc soto aml de aguiar mcf martins cc tumor depth of invasionand prognosis of earlystage oral squamous cell carcinoma a metaanalysisoral dis online ahead of printkim y kim jh increasing incidence and improving survival of oral tonguesquamous cell carcinoma sci rep mcdougall ar tolcos m hooper sb cole tj wallace mj wallace trop2from development to disease dev dyn “guan gf zhang dj wen lj yu dj zhao y zhu l prognostic value oftrop2 in human nasopharyngeal carcinoma int j clin exp pathol “stewart d cristea m antibodydrug conjugates for ovarian cancer currentclinical development curr opin obstet gynecol “liu j yang d yin z gao m tong h su y a novel human monoclonaltrop2igg antibody inhibits ovarian cancer growth in vitro and in vivobiochem biophys res commun “zhao w jia l kuai x tang q huang x yang t the role andmolecular mechanism of trop2 induced epithelialmesenchymal transitionthrough mediated betacatenin in gastric cancer cancer med “zhao w jia l zhang m huang x qian p tang q the killing effect ofnovel bispecific trop2pdl1 cart cell targeted gastric cancer am jcancer res “jordheim lp chettab k crosperrial e matera el dumontet c unexpectedgrowthpromoting effect of oxaliplatin in excision repair crosscomplementation group transfected human colon cancer cellspharmacology ““ nishimura t mitsunaga m sawada r saruta m kobayashi h matsumoto n photoimmunotherapy targeting biliarypancreatic cancer withhumanized antitrop2 antibody cancer med “ wang xd wang q chen xl huang jf yin y da p trop2 inhibitionsuppresses the proliferation and invasion of laryngeal carcinoma cells viathe extracellular signalregulated kinasemitogenactivated protein kinasepathway mol med rep “ wanger tm dewitt s collins a maitland nj poghosyan z knauper vdifferential regulation of trop2 release by pkc isoforms through vesiclesand adam17 cell signal “tang g tang q jia l chen y lin l kuai x trop2 increasesgrowth and metastasis of human oral squamous cell carcinomathrough activation of the pi3kakt signaling pathway int j mol med“trerotola m li j alberti s languino lr trop2 inhibits prostate cancer celladhesion to fibronectin through the 1 integrinrack1 axis j cell physiol“li t su y yu x mouniir dsa masau jf wei x trop2 guaranteescardioprotective effects of cortical bonederived stem cells on myocardialischemiareperfusion injury cell transplant “stoyanova t goldstein as cai h drake jm huang j witte on regulatedproteolysis of trop2 drives epithelial hyperplasia and stem cell selfrenewalvia betacatenin signaling genes dev “sun x xing g zhang c lu k wang y he x knockdown of trop2 inhibitsproliferation and migration and induces apoptosis of endometrial cancercells via aktcatenin pathway cell biochem funct lee h jang y seo j nam jm char k nanopfunctionalized polymerplatform for controlling metastatic cancer cell adhesion shape and motilityacs nano “kruse sa smith ja lawrence aj dresner ma manduca a greenleaf jf tissue characterization using magnetic resonance elastographypreliminary results phys med biol “kaneko ts pejcic mr tehranzadeh j keyak jh relationships betweenmaterial properties and ct scan data of cortical bone with and withoutmetastatic lesions med eng phys “ goetz jg minguet s navarrolerida i lazcano jj samaniego r calvo e biomechanical remodeling of the microenvironment by stromalcaveolin1 favors tumor invasion and metastasis cell “edge sb compton cc compton the american joint committee on cancerthe 7th edition of the ajcc cancer staging manual and the future of tnmann surg oncol “ barnes l eveson jw reichart p sidransky d pathology genetics headand neck tumours lyon barness p “ zhang b li l li z liu y zhang h wang j carbon ionirradiated hepatomacells exhibit coupling interplay between apoptotic signaling andmorphological and mechanical remodeling sci rep yan jf huang gy a doublehertz model for adhesive contact betweencylinders under inclined forces philos trans a math phys eng sci kowalsky ca faber ms nath a dann he kelly vw liu l rapid fineconformational epitope mapping using comprehensive mutagenesis anddeep sequencing j biol chem “ zeng p chen mb zhou ln tang m liu cy lu ph impact of trop2expression on prognosis in solid tumors a systematic review and metaanalysis sci rep calvo a xiao n kang j best cj leiva i emmertbuck mr alterationsin gene expression profiles during prostate cancer progression functionalcorrelations to tumorigenicity and downregulation of selenoproteinp inmouse and human tumors cancer res “ju x jiao x ertel a casimiro mc di sante g deng s vsrc oncogeneinduces trop2 proteolytic activation via cyclin d1 cancer res “ cubas r li m chen c yao q trop2 a possible therapeutic target for latestage epithelial carcinomas biochim biophys acta “ zargari n mokhtari m evaluation of diagnostic utility ofimmunohistochemistry markers of trop2 and hbme1 in the diagnosis ofthyroid carcinoma eur thyroid j “ zhao p zhang z tnfα promotes colon cancer cell migration and invasionby upregulating trop2 oncol lett “sin stk li y liu m yuan yf ma s guan xy downregulation of trop2predicts poor prognosis of hepatocellular carcinoma patients hepatolcommun “ zhang y zhang r luo g ai k long noncoding rna snhg1 promotes cellproliferation through pi3kakt signaling pathway in pancreatic ductaladenocarcinoma j cancer “sai j owens p novitskiy sv hawkins oe vilgelm ae yang j pi3kinhibition reduces mammary tumor growth and facilitates antitumor immunityand antipd1 responses clin cancer res “ chen x pang b liang y xu sc xin t fan ht overexpression of zhang xr wang sy sun w wei c isoliquiritigenin inhibits proliferation andepcam and trop2 in pituitary adenomas int j clin exp pathol “metastasis of mkn28 gastric cancer cells by suppressing the pi3kaktmtor signaling pathway mol med rep “ 0czhang bmc cancer page of wise hm hermida ma leslie nr prostate cancer pi3k pten and prognosisclin sci lond “ yuan b zou m zhao y zhang k sun y peng x upregulation of mir130b3p activates the ptenpi3kaktnfκb pathway to defense againstmycoplasma gallisepticum hs strain infection of chicken int j mol sci pii e2172li jw wang xy zhang x gao l wang lf yin xh epicatechin protectsagainst myocardial ischemiainduced cardiac injury via activation of theptenpi3kakt pathway mol med rep “li x teng s zhang y zhang w zhang x xu k trop2 promotesproliferation migration and metastasis of gallbladder cancer cells byregulating pi3kakt pathway and inducing emt oncotarget “ gu qz nijiati a gao x tao kl li cd fan xp trop2 promotes cellproliferation and migration in osteosarcoma through pi3kakt signalingmol med rep “ hou j lv a deng q zhang g hu x cui h trop2 promotes theproliferation and metastasis of glioblastoma cells by activating the jak2stat3 signaling pathway oncol rep “ rivard n phosphatidylinositol 3kinase a key regulator in adherens junctionformation and function front biosci landmark ed “ pankova d jiang y chatzifrangkeskou m vendrell i buzzelli j ryan a rassf1a controls tissue stiffness and cancer stemlike cells in lungadenocarcinoma embo j 20193813e100532 wullkopf l west av leijnse n cox tr madsen cd oddershede lb cancer cells' ability to mechanically adjust to extracellular matrix stiffnesscorrelates with their invasive potential mol biol cell “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"
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"cellular recognition of microbial dna is an evolutionarily conserved mechanism by which the innate immunesystem detects pathogens cyclic gmpamp synthase cgas and its downstream effector stimulator of interferongenes sting are involved in mediating fundamental innate antimicrobial immunity by promoting the release oftype i interferons ifns and other inflammatory cytokines accumulating evidence suggests that the activation ofthe cgassting axis is critical for antitumor immunity the downstream cytokines regulated by cgasstingespecially type i ifns serve as bridges connecting innate immunity with adaptive immunity accordingly a growingnumber of studies have focused on the synthesis and screening of sting pathway agonists however chronicsting activation may lead to a protumor phenotype in certain malignancies hence the cgassting signalingpathway must be orchestrated properly when sting agonists are used alone or in combination in this review wediscuss the dichotomous roles of the cgassting pathway in tumor development and the latest advances in theuse of sting agonistskeywords cgassting innate immunity type i interferon sting agonists antitumor response cancerdevelopmentintroductionthe discovery of phagocytosis in advanced the understanding of innate immunity the first line of host defenses protection againston patternrecognition receptors prrs which recognize microbialproducts coordinate antimicrobial defenses and activateinfection dependsagainstinfection byvarious pathogens correspondence zqliucsueducn juyan zheng and junluan mo contributed equally to this work1department of clinical pharmacology hunan key laboratory ofpharmacogenetics and national clinical research center for geriatricdisorders xiangya hospital central south university changsha people™s republic of china2institute of clinical pharmacology engineering research center for appliedtechnology of pharmacogenomics of ministry of education central southuniversity changsha people™s republic of chinafull list of author information is available at the end of the adaptive immunity abnormal rna or dna rnadna hybridization and cyclic dinucleotides derived frommicrobes are usually considered pathogenassociated molecular patterns pamps [ ] cells associated with innate immunity recognize different microbial pampsthrough specific prrs thereby playing key roles in hostresistance to microbial infection the pathways governing rna recognition such as retinoid acid induciblegene i rigilike receptors have been reviewed elsewhere and will not be covered herein in the case of dnarecognition one of the best known prrs is tolllike receptor tlr9 which senses extracellular cpg hypomethylated dna that has entered the cytosol through thephagosomelysosome system in addition the aim2like receptor aim2 inflammasome can be triggered afterthe entry of doublestranded dna dsdna into the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czheng molecular cancer page of cytosolic compartment which induces the proteolyticmaturation of proinflammatory cytokines such as il1and il18 and the activation of gasdermin d leading topyroptosis [“] nevertheless the most notable prr iscgas a direct cytosolic dsdna sensor which was identified by dr chen™s group in once cgas bindsto dsdna the cgassting pathway is activated to further induce the expression of type i ifns and other inflammatory cytokinesthus triggering innate immuneresponses mounting evidence suggests that cgassting signaling not only plays pivotal roles in the hostdefense against microbialinfection but also modulatestumorigenesis hence in this review we summarize themechanism of cgassting activation and elaboratefindings regarding its dual effects on tumor developmentcurrent advances in the use of sting agonists as a novelstrategy for antitumor therapy are also reviewedinsights into the cgassting signal transductioncascadecgas is an innate immune sensor that identifies variouscytosolic dsdnaincluding dna with viral bacterialmitochondrial micronuclei and retroelement originswhich can be mainly divided into pathogenderiveddna and selfdna table in the cytoplasm cgas isactivated by interacting with dsdna in a sequence[“]independent butstructural and biochemical analyses have revealed thatthe cterminal lobe of cgas contains a conserved zinclengthdependent mannerionbinding module that mediates dna binding andcgas dimerization [ ] dna ligands promotecgas activation primarily by inducing conformationalchanges around the catalytic site and in the dnabinding structures of cgasthe gscontaining loopundergoes conformational change to maintain stabilitywhich is a major mechanism of cgas activation bydna in addition to the primary dnabinding sitementioned above the secondary site located beside theprimary site is a helix formed between strands 78and several surfaceexposed loops the proximity ofthe two dnabinding sites in cgas leads to a cgasdna complex assembly in which two cgas moleculesembrace two molecules of dsdna [ ] the cgasdimers are anized in œheadtohead alignment nextto the dna and thus form stable œladderlike networks between one long curved dsdna helix or two independent dsdna strands [ ] in this way eachindividual cgasdsdna complex can be cooperativelystabilized and can lead to stronger enzymatic activitywhich may provide a possible explanation for longerdsdna as more likely to activate cgas in additionlong dna is more efficient than short dna in drivingthe liquidliquid phase separation of cgas and the formation ofcriticallydependent on the concentration of cgas and dna inthe cytoplasm cgas and dsdna are spatially concentratedcgasdimerization and activation [“] once cgas andcgas liquidlike dropletsin liquiddropletsistofacilitatetable classification of the cytosolic dsdna that activates the cgassting signaling axisclassificationselfdnasource of dsdnamicronucleipossible mechanismsrupture of the micronuclei membrane leads to exposureof chromatin dna that is recognized by cgas whichactivates the cgassting pathwayreferences mitochondrionnuclear rnapathogenderived dnadna virushsv1 hsv2 kshv adenovirus vacciniavirus cytomegalovirus papillomavirusmurine gammaherpesvirus retrovirushiv siv murine leukemia virusrna viruswest nile virus dengue virus vsvsarscov2bacterialisteria monocytogenes mycobacteriumtuberculosis listeria shigella francisellachlamydia and neisseriamitochondrial stress induces mtdna leakage into thecytosol thus activating the sting pathway and inducingproduction of cytokinesfacilitated by endogenous retroelements nuclear rnacan be reversely transcribed into dna that activatescgassting signaling dna viruses invade host cells and release pathogenderiveddna to induce sting activation[“]dna intermediates generated from reverse transcription maybe recognized by cgas to stimulate downstream stingsignaling infection with rna viruses might cause cellular damage andcell death which results in the release of cellular dna andfurther activation of the cgassting axis sarscov2 bindingto ace2 can lead to excessive angiotensin ii signaling thatactivates the sting pathway in mice[“]bacteria produce cdns such as cyclic digmp and cyclicdiamp which can directly bind to and activate sting[ “]hsv1 herpes simplex virus hsv2 herpes simplex virus kshv kaposi sarcoma“associated herpesvirus hiv human immunodeficiency virus siv simianimmunodeficiency virus vsv vesicular stomatitis virus cdns cyclic dinucleotides and sarscov2 severe acute respiratory syndrome coronavirus 0czheng molecular cancer page of dsdna interacts structural switches rearrange the catalytic pocket to enable cgas to catalyze the synthesis of²²cyclic gmpamp ²²cgamp with atp andgtp as substrates the first step in this process is theformation of a linear dinucleotide ²pppg ²²pawith atp serving as the donor and ²oh on gtp serving as the acceptor then the intermediate product flipsover in the catalytic pocket placing gtp at the donorposition and amp at the acceptor position to form asecond ²² phosphodiester bond [ ] notablyalthough dsrna or singlestrand dna ssdna is ableto bind to cgas neither can rearrange the catalyticpocket which may explain the exclusive activation ofcgas by dsdna ultimately cgamp acts as a secondmessenger to bind to and activate sting a small endoplasmic reticulum erlocated protein kd withfour putative transmembrane domains [ ] normally in a resting state sting is retained in the er byinteracting with the ca2 sensor stromalinteractionmolecule stim1 the cytosolic ligandbindingdomain lbd of sting exists as the most functionalunit capable of integrating with ²² cgamp or cdnscyclic dinucleotides such as cdiamp cdigmp or ²²cgamp from bacteria upon interaction the obviousclosure of the ligand binding pocket in the lbd is observed which is related to the activation of sting next sting transforms into a tetramer through a highorder oligomerization reaction and is translocated fromthe er to the perinuclear area facilitated by cytoplasmiccoat protein complex ii copii and adpribosylationfactor arf gtpases [ ] in the golgi sting ispalmitoylated atcys88 andcys91 a posttranslational modification necessary forsting activation modified sting recruits thekinase tankbinding kinase tbk1 in turn the cterminal domains of sting are phosphorylated bytbk1 and then phosphorylated sting recruits interferon regulatory factor irf3 which is also phosphorylated by tbk1 and dimerizes ultimately dimerizedirf3 enters the nucleus and exerts its function in thetranscription of type i ifns and interferonstimulatedgenes isgs in parallel sting can also bind toand stimulate iκb kinase ikk to mediate the production of nuclear factorκb nfκbdriven inflammatorygenes upon signal transduction termination sting istransferred to endolysosomes for degradation considering that cgamp can be transferred through gapjunctions or delivered in viralexosome packages cgassting signaling may be activated in the cytoplasmwithout dsdna [ ] moreover newly produced typei ifns activate heterodimer interferon receptors ifnar1 and ifnar2 through paracrine signaling and thusinduce the transcription of isgs [ ] in summaryonce virusderived dna and selfdna are located intwo cysteine residuesthe cytoplasm they can be sensed by cgas and a cgasdsdna complex is formed to catalyze the synthesis of ²²cgamp with atp and gtp then ²²cgamp and bacteriaderived cdns induce sting activation and mediate the release of downstream type iifns tnfα and il6 which are prerequisites for antimicrobial defense and antitumor effects the wholeprocess shows that the dsdnacgassting axis canlead to the activation of both innate and adaptive immunity fig the antitumor functions of the cgasstingsignaling pathwayrecent evidence has revealed the close association of thecgassting pathway with cancer development thissignaling pathway is generally regarded as a potent regulator of cancer immunity a stingmediated immunesupportive microenvironment can hamper malignancyoccurrence stressbytumor cell cytosolic dsdna induces sting activationunder normal circumstances dna is strictly unaffiliatedwith the cytoplasm in eukaryotic cells to avoid autoimmunity however dna leaks aberrantly in tumorcells [ ] cancer cells share common features including genome instability tumor suppressor gene mutation or deletion oxidativeand vigorousmetabolism under these intense states nuclear andmitochondrial dna is fragile and easily damaged whichleads to eventual dna leakage in the forms of micronuclei chromatin fragments andor free telomeric dna[ ] chromosomal instability cin is the primary source of cytoplasmic dna in malignant cells andis generally associated with tumor progression distantmetastasis and therapeutic tolerance excessive proliferation of cancer cells results in unstable genomes usuallychromosomal missegregation during mitosis due to defects in segregation lagging chromosomes generate micronuclei in a cellcycledependent manner the vulnerable membraneof micronuclei easily exposes the inner dna to the cytoplasm and activates the cgassting signaling axis exogenous stimuli such as chemotherapy and irradiation can also cause dna damage in addition to leakednuclear dna oxidative stressinduced mitochondrialdna leakage is another crucial initiator of sting pathway activation several anticancer treatments that precisely attack mitochondrial membranes result in effluxand cell death therefore the permeabilization of mitochondria membranes provides a reasonable explanationfor mitochondrial dna escape [ ] other sourcessuch as apoptotic cellderived dna exosomal dnaexodna and transposable elements have also beencharacterized 0czheng molecular cancer page of fig the cgassting dna sensing signaling pathway various dna derived from virus dying tumor cells or nucleus and mitochondria binds toand activates the cytosolic dna sensor cgas cgas catalyzes the synthesis of ²²cgamp in the presence of atp and gtp then ²²cgamp bindsto the er adaptor sting which also can be activated by cdns derived from bacteria upon activation sting translocates from er to golgicompartments where it activates tbk1 and ikk which phosphorylate irf3 and iκbα respectively then irf3 and iκbα dimerize and enter nucleusinitiating the transcription of type i ifn tnf and il6 the primary roles of these cytokines are reflected in host defense inflammation andantitumor immunitydemonstrated to evoke cgas“sting activation intumor cells [ ]type i ifns mediators of sting and adaptive antitumoreffectscgassting signaling exerts antitumor functions incancer cells both in an autonomous and nonautonomousmanner on the one hand dna damage can provokeacute sting signal transduction and induce cellularsenescence an irreversible cell cycle arrest state whichthwarts the aberrant proliferation of tumor cells throughacquisition ofsecretoryphenotype sasp which is associated with the releaseof abundantinflammatory mediators proteases andgrowth factors [ ] in contrast to undergoingsenescence tumor cells also directly propel apoptosisprocesses by upregulating proapoptosis protein bcl2associated x bax and downregulating the bcl2 apoptosis on the other hand stingsenescenceassociatedtheregulatoractivation in tumor cells not only facilitates the transcription of downstream type i ifns to induce dendriticcell maturation but also recruits supportive immunecells for direct nonspontaneous tumor elimination sting activation in nonmalignant cells causes tumorsuppressive effects as well sting signaling protectsagainst colitisassociated carcinomas cacs induced byazoxymethane aom and dextran sulfate sodiumdss which induce dna damage in intestinal epithelialcells and further trigger sting activation downstreamcytokines of sting signaling such as il1 and il18prevent neoplastic transformation by facilitating woundrepair more importantly sting signaling can also provoke cytotoxic t cell responses to control tumorigenesis necrotic cancer cells are commonly engulfed byantigenpresenting cells especially the basic leucine zippertranscription factor atflike batf3drivenlineage of dendritic cells dcs batf3 dcs take intumorassociated antigens and migrate towardsthe 0czheng molecular cancer page of tumordraining lymph node via the lymphatic systemwhere they crossprime tumorspecific cd8 t cellsthen cd8 t cells undergo activation and clonal expansion in the lymph nodes and are trafficked throughblood vessels to kill tumor cells in turn damaged cancercells release more antigens that are further captured bydcs the whole process forms a positive feedback loopcalled the cancerimmunity cycle tumor eradication can be achieved by multiple processes in thecancerimmunity cycle including tumor antigen captureand presentation and t cell priming and activation withtumor antigenspecific t cell priming and activationrelying on dcs and type i ifn release the involvement of type i ifns in innate immune sensing and adaptive immunity provides a reasonable hypothesis forexploring candidate prr pathways as potential immunomodulators mice lacking tlr9 myeloid differentiationprimary response gene myd88 cytosolic rna sensor mavs or the purinergic receptor p2x7r maintainintact antitumor immunity responses whereas mice deficient in sting or irf3 present with impaired cd8 tcell priming and activation [ ] in fact dying tumorcells can release multiple damageassociated molecularpatterns damps to trigger innate immune responsesin dcs among these released stimuli tumor cellderiveddna is a pivotal inducer in general the phagocytosis ofapoptotic cells causesimmune silence because ofdnasebased degradation nevertheless tumor cellreleased dna can be preserved in the dc endolysosomal compartment through an unknown mechanism cgas recognizes dna invading the cytoplasm andinduces the activation of sting cascades excretion oftype i ifns and expression of isgs additionally undersome physiological conditions such as hypoxia andacidic environments nuclear or mitochondrial dnamight be packaged in exosomes exosomal dnaexodna animates sting signaling once it is absorbedby tumorinfiltrating dcs finallytumor cellderived cgamp can also be transferred to host dcs bythe folate transporter slc19a1 and then directly bindsto sting activating it in dcs a recent study moredirectly demonstrated that cellautonomous sting promoted the maintenance of stem celllike cd8 t cellsand augmented antitumor t cell responses and mechanistically cgasstingmediated type i interferon signaling reinforced the stem cell“like cd8 t celldifferentiation program mainly by restraining akt activity immune cellderived type i ifns have crucial functions in antitumor immunity control on the one handtype i ifns boost cross presentation by various mechanisms first they stimulate the maturation of dcs secondthey slow the endosomelysosome acidificationprocess to prevent engulfed tumor antigen clearance andelevate the expression of mhc i molecules on the cellsurface [ ] finally they accelerate dc migrationtowardslymph nodes where they can crossprimetumorspecific cd8 t cells on the other handtype i ifns drive the expression of multiple chemokinessuch as cxcl9 and cxcl10 both of which are necessary for cytotoxic t lymphocyte ctl transfer and infiltration similarly type i ifns restrain the defaultimmune suppressive action of regulatory t treg cellsby downregulating phosphodiesterase pde4 and upregulating cyclic amp camp consequently typei ifns serve as bridges linking the cgassting pathway with cd8 t cellmediated antitumor immunitythe antitumor mechanisms of the cgassting signaling axis are illustrated in fig indeed previous studies revealed that sting activation can stimulate antitumor immune responses inleukemia melanoma glioma and hepatocellular carcinoma [“] additionally sting expression is downregulated in a wide variety of tumor tissues and celllines according to a pancancer analysis with a smallproportion of tumors approximately bearing silent sting expression lower sting expressionwas found in hepatic carcinoma and gastric cancer compared with its level in corresponding normal tissues andthis lower expression level was correlated with highertumor stage and poorer prognosis [ ] consistentlycompared with that in the mcfg10a mammary epithelial cell line lower sting expression was detected inmalignant breast cancer cellincluding mcf7hbl100 and t47d cells as well as human melanomacell lines and colorectal adenocarcinoma lines [ ] collectivelythat cgassting signaling might act as a tumor suppressor in certain types of cancersthese findings suggestlinessting pathway agonists as cancer therapeuticsthe immunostimulatory potential of the cgasstingpathway makes it an attractive pharmacological targetsince its activation in the tumor microenvironmenttme can induce efficient crosspriming oftumorspecific antigens and facilitate the infiltration of effectort cells recent drug research has focused on the development of sting agonists because of their potential inanticancer therapy [ ] to date various kinds ofsting agonists have been discovered and they aremainly divided into the following categories cyclic dinucleotides and their derivates dmxaa and its analogsand small molecular agonists in addition some conventional antitumor therapeutics can also indirectly activatesting such as chemotherapy radiotherapy rt andtargeted therapy in addition sting agonists areable to enhance the efficacy of other anticancer therapeutic agents when used in combination sting 0czheng molecular cancer page of fig the antitumor immunity effect of the cgassting pathway dna damage leads to the formation of dsdna in tumor cells upon itsstimulation sting signaling is activated and promotes the release of type i ifn which is crucial for dc maturation sting signaling activation indcs is the core step of the whole cancerimmunity cycle which can be initiated through engulfment of dyingdamaged tumor cells exosometransfer and cgamp gap junctions then dcs migrate towards the tumordraining lymph node and crossprime tumor specific cd8 t cells withthe help of type i ifns finally t cells undergo clonal expansion and traffic through the blood vessel to conduct tumor killingagonists and their synergistic use with other remedies isfurther explored in detail belowcyclic dinucleotides cdnscdns constitute a main type of sting agonist whichmainly originate from bacteria the known naturalcdns consist of exogenous cyclic digmp cdigmpcdiamp ²²cgamp and endogenous ²²cgampamong these cdigmp cdiamp and ²²cgampare synthesized by bacteria and identified as secondarymessengers that mediate sting signal transduction inprokaryotic cells while ²²cgamp functions as theinitiator of sting in mammalian cells the antitumor potential of these natural dinucleotides was firstproven by the finding that cdigmp could inhibit theproliferation of human colon cancer cells in vitro andbasal cell proliferation of human cecal adenocarcinomah508 cells was inhibited with μm cdigmp intraperitoneal ip injection of highdose cdigmpdirectly activated caspase3 and triggered t1 tumoripcell apoptosis in vitro nmol of cdigmp reduced thegrowth of t1 tumor cells in vitro by and nmreduced it by while lowdose cdigmp nmol accelerated the adaptive t cell response by converting a subgroup of myeloidderived suppressor cellsmdscs into immune stimulatory cells producing il12injection of ²²cgamp consistentlymgkg expedited dramatic leukemic elimination in eltcl1 transgenic mice bearing chronic lymphocyticleukemia cll and promoted tumor shrinkage of multiple myeloma in vivo from the perspective of endogenous cdns ²²cgamp mgkg was alsoshown to restrain tumorigenesis and improve the survival rate of mice bearing ct26 colon adenocarcinomain a dosagedependent manner relying on dc activationand t cell crosspriming more recently ohkurit further demonstrated that intratumoral it injection of ²²cgamp μg25 μldose on and days after the injection of tumor cells significantly mitigated tumor growth and prolonged the survival of breast 0czheng molecular cancer page of cancer t1luc squamous cell carcinoma mscc1colon cancer ct26 and melanoma b16f10 mousemodels notably the it injection of ²²cgampinhibited not only tumor growth but also lung metastases in mice bearing b16f10 cellderived tumors suggesting that cgampinduced cd8 tcell priming can drivesystemic antitumor immunity to control local and distant tumor growth termedvaccinestingvaxconsidering the superior properties of sting signaling in activating adaptive immunityit is rational toutilize sting agonists such as cdns as cancer vaccineadjuvants to increase tumor immunogenicity fu investigated the in vivo therapeutic efficacy of acancercomprisinggranulocytemacrophage colonystimulating factor gmcsf and bacteriaderived or synthetic cdns theyobserved that after it injection of stingvax with μg of cdns per vaccine dose the volume of b16melanoma tumors was dramatically reduced in a dosedependent manner compared to mice receiving gmcsf cancer vaccine alone stingvaxtreated mice hadmore infiltrating cd8 ifnγ t cells in the tumormicroenvironment the in vivo antitumor effect of stingvax was also verified in models of colon carcinomact26 pancreatic carcinoma panc02 and upper aerodigestive squamous cell carcinoma sccfvii although natural cdns are able to produce robust antitumor immunity their chemical features might hindertheir future application in the clinical setting first native cdns are easily degraded by enzymes inside the cellor in the bloodstream second their negatively chargedproperty hydrophilicity and phosphate moieties severelyimpede cdns from penetrating cell membranes to activate cytosolic sting leading to low bioavailability andpoor retention of the cdns in specific cells and tissuesthird unintentional toxicities and narrow therapeuticwindows are also unavoidable thus new strategies toimprove therapeutic efficacy and reduce adverse effectsare urgently needed including drug delivery carrier engineering original structural modification and nonnucleotide agonist screening regarding agonistdelivery smith reported that biopolymer implantscodelivering cdigmp μg and chimeric antigen receptor t cart cells resulted in significant tumor regression in mice bearing pancreatic tumors moreoveriv administration of cdigmpysk05lip equivalent to μg of cdigmp aysk05liposome delivery system encapsulating cdigmp led to a tremendous decrease in metastatic lesionsin a b16f10 mouse melanoma model with nearly ofthe injected mice showing resistance against tumor relapsethe adaptive immune responsememory was successfully induced chen alsofound thatliposomalindicating thatinjection ofintravenousintravenousivnanopdelivered cgamp cgampnp could activate the sting axis more effectively than solublecgamp and converted the immunosuppressive tme toa tumoricidal state in a transplanted b16f10 cell melanoma model and in a genetically engineered triplenegative breast cancer model moreover a recentstudy creatively suggested that modified bacteria mightbe exploited as a selective carrier of sting agonistsintroduction of a dinucleotide cyclasecoding gene intothe escherichia coli nissle strain was an attempt at realizing this effect however advancements to the systemare needed tobysnakeapartdigestionresistancecompoundatoms the modifiedfrom improving delivery methods cdnswith superior properties are currently being synthesized and tested for instance to prevent enzymatichydrolysis of cgamp the nonbridging oxygen atomsin cgamp phosphodiester linkages were replaced by²²sulfurcgsasmp showed resistance against degradation byenpp1 a major ²²cgamp hydrolasetherebyleading to a longer halflife and sustained high affinity for human sting hsting syntheticdithio mixedlinkage cdns with both rp rp r rand rp sp r s dithio diastereomers possessed notonlyvenomphosphodiesterase but also enhanced affinityforsting a novel superior modified product ml rrs2 cda also termed adus100 had the potencyto activate all hsting variants and mouse stingmsting adus100 had higher efficiency in activating sting signaling than endogenous or exogenous cdns mainly because of its enhanced stabilityand lipophilicity its powerful tumor elimination effect was extensively demonstrated in multiple murinemodelsincluding b16 melanoma t1 breast cancer and ct26 colon cancer with all treated animalsshowing significant and durable tumorregressionafter itinjection of adus100 three mg doseswhen tumor volumes reached mm3 theremarkableforhsting laid the foundation for its clinical use related clinicaltrials of adus100 are outlined intable in addition to adus100 some other novelsting agonists have been well designed iacs8779and iacs8803 are two highly potent ²²thiophosphate cdn analogs that induced striking systemicantitumorin a b16 melanoma murineinjection μg on and daysmodel after itposttumor implantation compared with adus100or cgamp the characteristics and preclinicalapplications of all these mentioned cnds are summarized in table because of the structural modification and optimization of delivery strategiestheapplication range and efficacy of cdns have beenand high affinityresponsescurativeeffect 0czheng molecular cancer page of table characteristics and preclinical applications of different sting agonistsclassificationcharacteristicsapplicationmodelsnatural cdnagonistscdigmppoor membrane permeabilitysuitable for various codeliverytechnologiescolon cancer h508cells t1 metastaticbreast cancertreatmentinformation μm nmol ip nmol ip nmol ip²²cgamp²²cgamphigher binding affinity formsting than for hstinghigher affinity for hsting thanits lineage isomers binds tovarious sting nucleotidepolymorphisms observed inhumans easily degraded byphosphodiesteraseimpermeable to the cellmembranechronic lymphocyticleukemia mgkg ipmultiple myeloma mgkg ipct26 colonadenocarcinoma mgkgbreast cancer t1lucsquamous cellcarcinomasmscc1 μg25 μldose it μg25 μldose itcolon cancer ct26 μg25 μldose itmelanoma b16f10 μg25 μldose ittherapeutic effects references[ ] [ ]inhibitsproliferation tumorregression tumorregressionaccelerates tcellresponseleukemiaeliminationsuppressesgrowthrestrainstumorigenesisimproves survivalratedelays tumrowthdelays tumrowthdelays tumrowthdelays tumrowthstingvaxsyntheticcdnagonistspotent in vivo antitumor efficacyin multiple therapeutic modelsof established cancercgampnpsbiopolymer scaffolds cdigmp and car t cellscdigmpysk05lip²²cgsasmpadus100iacs8779iacs8803noncdnagonistsfaaliposomal nanops npsdeliver cgamp intracellularlymore effectively than realizedwith soluble cgamperadicates tumors moreeffectively than systemicdeliveryysk05 is a lipid that can efficientlydeliver cdigmp to the cytosolpossesses high fusogenic activitywhich enhances endosomalescapemore resistant to degradation byenpp1 tenfold more potent atinducing ifn secretion potentialuse as a cancer vaccine adjuvantimproves stability and lipophilicityhigher affinity for hsting thannatural cdn agonists capable toactivate all hsting variants andmstingstimulates a superior systemicantitumor response thanadus100 and cgampcauses hemorrhagic necrosisfailed in a phase i clinical trialdue to species specificity μg cdns itreduces tumorvolume b16 melanomacolon carcinomact26pancreaticcarcinoma panc02b16f10 melanomaivtnbccreates atumoricidal state pancreatic cancer μg cdigmptumor regression b16f10 mousemelanoma μg cdigmp ivdecreasesmetastasisthp1 monocytesb16 melanomathree mg doses it t1 breast cancerthree mg doses itmc26 colon cancerthree mg doses itdurable tumorregressiondurable tumorregressiondurable tumorregression b16 melanoma μg on day and posttumor implantationantitumorresponse murine colontumorsextensive tumorrejection[ ]dmxaafirst discovered as a vascularrat mammary mgkg iphigh anticancer[ 0czheng molecular cancer page of table characteristics and preclinical applications of different sting agonists continuedclassificationcharacteristicsapplicationmodelstreatmentinformationinduces proinflammatory cytokinesin a stingdependent mannerhuman fibroblastsantiviral activity selectively induces stingdependentsynthesis and secretion of bioactiveifns no evidence of binding directlyto stingactivates sting in œopenconformation submicromolarlevels induce sting activationand ifn productionhuman fibroblastsantiviral activity colon tumors mgkg iv of a treatedgroup remainedtumor free faa flavone acetic acid dmxaa 56dimethylxanthenone4acetic acid cma 10carboxymethyl9acrid
0
High glucose HG induced podocytes injury plays an important role in diabetes nephropathy DN development Long noncoding RNA cancer susceptibility candidate CASC2 was found to be decreased in serum of DN patients We aimed to explore the function and possible mechanism of CASC2 in HG induced podocytes injuryMethods Under normal glucose NG HG and mannitol stimulated podocyte conditions the levels of CASC2 microRNA95p miR95p and peroxisome proliferatoractivated receptor gamma PPARÎ were examined by quantitative realtime polymerase chain reaction qRTPCR Podocyte injury was evaluated by measuring cell viability and apoptosis of CIHP1 cells were checked by cell counting kit8 CCK8 assay and flow cytometry respectively Western blot was used to detect all protein levels Dualluciferase reporter RNA immunoprecipitation RIP and RNA pulldown assays were performed to confirm the relationship between CASC2 and miR95pResults HG stimulation inhibited the expression levels of CASC2 and PPARÎ but promoted the expression of miR95p HG could restrain cell viability autophagy and facilitate apoptosis in CIHP1 cells while CASC2 overexpression could reverse HGinduced podocytes injury Furthermore CASC2 could be used as a ceRNA to adsorb miR95p and miR95p mimic overturned the effects of CASC2 on cell viability autophagy and apoptosis in HGstimulated podocytes Additionally PPARÎ was a target gene of miR95p and CASC2 could weaken the HGinduced podocytes injury by upregulating PPARÎConclusion CASC2 increased cell viability autophagy and inhibited cell apoptosis by regulating miR95pPPARÎ axis thus reducing the HGinduced podocytes injuryKeywords High glucose Podocyte CASC2 miR95p PPARÎCorrespondence gltxs009163com Department of Nephrology Tai™an Campus of the 960th Hospital of the Chinese People™s Liberation Army No217 Huanshan Road Taishan District Tai™an Shandong ChinaFull list of author information is available at the end of the BackgroundDiabetes is a common endocrine disease among which the prevalence of diabetes nephropathy DN is “ [] It is estimated that the number of DN patients is expected to increase to million by [] DN is characterized by the presence of albuminuria and a decreased glomerular filtration rate [] Podocyte cells podocytes are epithelial cells in the The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cLi a0et a0al Diabetol Metab Syndr Page of visceral layer of renal follicles which play a key role in the pathogenesis of DN and are an important component of glomerular filtration barrier [ ] Several studies have revealed the correlation between podocytes injury death and apoptosis and albuminuria [] and reducing podocyte injury can improve DN [] However the mechanism for alleviating podocytes injury remains unclearLong noncoding RNAs lncRNAs are nonproteincoding RNA molecules longer than nucleotides which are widely regarded as the important regulators in cellular function and disease processes [] Increased evidences suggested that lncRNA could modulate DN progression For instance lncRNAs GM5524 and GM15645 could regulate the HGstimulated podocyte autophagy in DN [] LncRNA PVT1 knockdown repressed podocytes injury and apoptosis via increasing FOXA1 [] However there are still many lncRNAs in DN function and molecular mechanisms have not been studiedLncRNA cancer susceptibility candidate CASC2 located on chromosome 10q26 plays a regulatory role as an anticancer factor in various cancers such as hepatocellular carcinoma [] and pancreatic carcinoma [] Recently Wang et a0al revealed that CASC2 was specifically reduced in serum and renal tissues of type diabetes patients with chronic renal failure and followup identified that the serum of patients with low CASC2 expression had higher incidence of chronic renal failure [] MicroRNA95p miR95p is both a tumor depressor and a tumor promoter [ ] A report demonstrated that miR95p was related to complications of nephropathy in Type and Type diabetes patients [] The mechanism by which lncRNA can serve as the competing endogenous RNA ceRNA for miRNA to modulate the abundance of mRNA has been widely reported [] Peroxisome proliferatoractivated receptor gamma PPARÎ is implicated in several metabolic syndromes including DN Downregulated PPARÎ could activate catenin signaling to destroy podocyte architectural integrity and increase cell apoptosis in DN [] Furthermore lncRNA TUG1 could relieve extracellular matrix accumulation by sponging miR377 and regulating PPARÎ in DN [] Based on the above findings we speculated whether CASC2 can modulate PPARÎ expression by serving as a ceRNA of miR95p in DNIn this work we aimed to explore the effects of CASC2 on cell viability apoptosis and autophagy in high glucose HG induced podocytes and probe the relationship among CASC2 miR95p and PPARÎ providing a new perspective on the molecular mechanism of podocytes injury in DNMaterials and a0methodsCell culture and a0high glucose inductionHuman podocytes CIHP1 Ximbio London USA were cultured in a Dulbecco™s modified Eagle™s medium DMEM Invitrogen Carlsbad CA USA containing fetal bovine serum FBS Gibco Carlsbad CA USA at a temperature of a0°C with CO2 When cells density reached about CIHP1 cells were exposed to normal glucose NG a0mM high glucose HG a0mM or mannitol a0mM and the exposure time was determined by individual experiments requiredCell transfectionCASC2 overexpressed plasmid CASC2 and its control Vector small interfering RNAs against CASC2 and PPARÎ siCASC2 siPPARÎ and matched siNC were provided by GenePharma Shanghai China miR95p mimic miR95p inhibitor antimiR95p and their corresponding references miRNC antiNC were synthesized by Beyotime Beijing China Transfection of podocytes was performed by using Lipofectamine InvitrogenQuantitative real‘time polymerase chain reaction qRT‘PCRThe RNA in CIHP1 cells was extracted by TRIzol Invitrogen and the complementary DNA cDNA was synthesized via reverse transcription using HiScript Q RT Super Mix Vazyme Piscataway NJ USA The reverse transcription was performed at a0°C for a0min and at a0°C for a0s qRTPCR analysis was conducted on RealTime PCR System Applied Biosystems Foster City CA USA using the SYBR premix Ex TaqIIkit TaKaRa Wuhan China Glyceraldehyde3phosphate dehydrogenase GAPDH and U6 were used as endogenous controls for CASC2PPARÎ and miR95p respectively The primers used in this paper were synthesized by GenePharma and the sequences were used as below CASC2 forward ²GCA CAT TGG ACG GTG TTT CC3² reverse R ²CCC AGT CCT TCA CAG GTC AC3² miR95p F ²GTG CAG GGT CCG AGGT3² R ²GCG CTC TTT GGT TAT CTA GC3² PPARÎ F ²AGA GCC TTC CAA CTC CCT CA3² R ²AAC AGC TTC TCC TTC TCG GC3² U6 F ²TTG GTG CTC GCT TCG GCA ² R ²GTG CAG GGT CCG AGGT3² GAPDH F ²GGA GTC CAC TGG TGT CTT CA3² R ²GGG AAC TGA GCA ATT GGT GG3²F Cell viability and a0apoptosis detectionCIHP1 cells were tiled into the 96well plates and exposed to different treatments HG NG HG Vector HG CASC2 and so on At given points in time a0h a0h and a0h a0µL cell counting kit8 CCK8 0cLi a0et a0al Diabetol Metab Syndr Page of Beyotime was added to the cells and cultured for another a0h at a0°C Finally the absorbance at a0nm was measured by BiotekEpoch2 Beijing ChinaThe apoptosis of podocytes CIHP1 was estimated at a0h after exposure to different treatments by using an Annexin V fluorescein isothiocyanate FITC and propidium iodide PI apoptosis detection kit Keygen Beijing China Briefly podocytes were collected and were then suspended in a0µL FITC and a0µL PI in the absence of light for a0 min The apoptosis of CIHP1 cells was checked by a flow cytometer BD Biosciences Franklin Lake NJ USAWestern blot assayTotal protein from CIHP1 cells was extracted by RIPA Beyotime and denatured at a0°C for a0min before separation and then transferred to polyvinylidene difluoride PVDF Beyotime membranes Membranes were sealed with milk for a0 h before incubation with primary antibodies against Bcell lymphoma2 BCL2 Abcam Cambridge MA USA Cleavedcaspase3 Abcam Light chain 3II LC3II Abcam LC3I Abcam Beclin Beyotime PPARÎ Abcam or GAPDH Beyotime overnight at a0 °C HRPconjugated secondary antibody Abcam was employed to incubate the membranes for another a0h And the proteins were visualized by using BeyoECL Moon BeyotimeDual‘luciferase reporter assayCASC2 wild type CASC2wt with miR95p binding sites and its mutant type CASC2mut without binding sites were cotransfected into CIHP1 cells with miR95p or miRNC respectively Transfection was continued for a0h and luciferase activity was evaluated through a Dualluciferase reporter kit Promega Madison WI USA In the same manner PPARÎ ²untranslated region ²UTRwt with miR95p binding sites and PPARÎ ²UTRmut were cotransfected into cells with miR95p or miRNC respectively and the luciferase activity was detectedRNA immunoprecipitation RIP assay and a0RNA pull‘down assayRIP detection was conducted using a Magna RIP RNABinding Protein Immunoprecipitation Kit Millipore Billerica MA USA CIHP1 cells were treated with miR95p or miRNC a0h later cells were lysed in RIP Lysis Buffer containing protease inhibitors Then Argonaute2 Ago2 or ImmunoglobulinG IgG antibody Abcam were added to the cell lysates overnight at a0°C and the immunoprecipitated RNAs were obtained CASC2 and miR95p levels were estimated using qRTPCR analysisCIHP1 cells were transfected with Biotin labeled BiomiR95p and BiomiRNC respectively At a0 h postobtained by using a Pierce„¢ Magnetic RNA PullDown transfection cells were collected and the bound RNA was Kit Thermo Fisher Scientific Waltham MA USA according to the instructions Finally CASC2 enrichment was assessed by qRTPCRStatistical analysisData were acquired from at least three independent repetitions and displayed as mean ± standard deviation SD Difference analysis was conducted by Student™s ttest with two groups and oneway analysis of variance ANOVA with multiple groups using GraphPad Prism The P value less than was regarded as statistically distinctResultsCASC2 alleviated the a0HG‘induced podocytes injuryFirstly we examined the expression of CASC2 in human podocytes treated with NG HG or mannitol by qRTPCR The results showed that HG significantly decreased CASC2 expression in CIHP1 cells compared with NG and mannitol treatment Fig a0 1a In addition a timedependent reduction in CASC2 expression was displayed in HGtreated CIHP1 cells and a0h Fig a01b In view of the expression of CASC2 was substantially reduced at a0h of HG stimulation we then overexpressed CASC2 in HGstimulated CIHP1 cells for a0 h and overexpression efficiency was identified by qRTPCR As shown in Fig a01c CASC2 expression was obviously promoted in HGstimulated CIHP1 cells after transfection of CASC2 for a0 h CCK8 and flow cytometry results indicated that overexpression of CASC2 induced cell viability Fig a01d and retarded apoptosis Fig a01e in HGtreated CIHP1 cells To confirm the results of apoptosis we detected the expression of apoptosis marker proteins BCL2 and Cleavedcaspase3 Western blot assay demonstrated that upregulation of CASC2 enhanced BCL2 expression and silenced Cleavedcaspase3 expression Fig a0 1f which was in agreement with the results of Annexin VFITCPI Furthermore HG could reduce the ratio of LC3IILC3I and Beclin expression in CIHP1 cells and CASC2 overexpression reversed the effects of HG on the expression of autophagy related proteins Fig a01g The above findings indicated that CASC2 could alleviate the HGinduced podocytes injury by affecting cell viability apoptosis and autophagyCASC2 directly interacted with a0miR‘‘5pLncRNA generally functions as a sponge for miRNA in human diseases [] We speculated whether CASC2 could also act as miRNA sponge to regulate 0cLi a0et a0al Diabetol Metab Syndr Page of Fig CASC2 alleviated the HGinduced podocytes injury a The expression of CASC2in CIHP1 cells treated with normal glucose NG high glucose HG or mannitol was detected by qRTPCR b After CIHP1 cells were treated with HG mM for h h and h respectively CASC2 expression was measured by qRTPCR c CIHP1 cells were divided into four groups which were control NG mM HG mM HG vector and HG CASC2 CASC2 expression was detected by qRTPCR d Cell viability was assessed by CCK8 assay e Cell apoptosis was examined by flow cytometry f g Western blot assay was used to determine the expression levels of apoptosisrelated proteins BCL2 and Cleavedcaspase3 and autophagy related proteins LC3II LC3I and Beclin P 0cLi a0et a0al Diabetol Metab Syndr Page of HGinduced podocytes injury As shown in Fig a0 2a we found that miR95p was upregulated in HGtreated CIHP1 cells compared to cells treated with NG or mannitol and miR95p expression was drastically augmented in HGtreated CIHP1 cells in a timedependent manner Fig a0 2b Interestingly there were complementary sites between miR95p and CASC2 by bioinformatics website starBase v20 Fig a0 2c Dualluciferase reporter assay showed that the luciferase activity of CASC2wt was obviously decreased in CIHP1 cells transfected with miR95p than that cells transfected with miRNC whereas it was no significant difference in luciferase activity of CASC2mut Fig a02d RIP assay indicated that the enrichments of CASC2 and miR95p were higher in CIHP1 cells incubated with Ago2 Fig a02e RNA pulldown assay further revealed that the enrichment of CASC2 in BiomiR95p group was aggrandized relative to that BioNC group Fig a02f These results strongly supported that CASC2 could specifically bind to miR95p Meanwhile qRTPCR data showed that CASC2 knockdown in CIHP1 cells elevated miR95p expression and CASC2 overexpression degraded miR95p expression Fig a02g h These results suggested that CASC2 could act as a ceRNA to negatively regulated miR95p expression in podocytesCASC2 regulated the a0HG‘induced podocytes injury via a0targeting miR‘‘5pAs presented in Fig a0 3a miR95p mimic miR95p could reverse the inhibitory effect of CASC2 overexpression on miR95p expression in HGinduced CIHP1 cells As expected the impact of CASC2 on promoting cell activity Fig a03b and inhibiting cell apoptosis Fig a03c in HGstimulated CIHP1 cells was offset by miR95p Simultaneously the inhibition of CASC2 on the protein expression of Cleavedcaspase3 and the promotion of CASC2 on LC3IILC3I ratio as well as the levels of BCL2 and Beclin could be weakened by transfection of miR95p in HGinduced CIHP1 cells Fig a03d e The obtained data proved that CASC2 attenuated the HGinduced podocytes injury by downregulating miR95pFig CASC2 directly interacted with miR95p a The expression of miR95pin CIHP1 cells treated with normal glucose NG high glucose HG or mannitol was measured by qRTPCR b After CIHP1 cells were treated with HG mM for h h and h respectively miR95p expression was examined by qRTPCR c StarBase v20 was used to predict the target miRNAs of CASC2 d“f Dual luciferase reporter RIP and RNA pulldown assays were utilized to assess the combination of CASC2 and miR95p g CASC2 expression in CIHP1 cells transfected with siNC or siCASC2 was determined by qRTPCR h The expression of miR95pin CIHP1 cells transfected with siNC siCASC2 Vector or CASC2 was measured using qRTPCR analysis P 0cLi a0et a0al Diabetol Metab Syndr Page of Fig CASC2 regulated the HGinduced podocytes injury via targeting miR95p The HGtreated CIHP1 cells were divided into four groups Vector CASC2 CASC2 miRNC and HG miR95p a The expression of miR95p was examined by qRTPCR b c Cell viability and apoptosis were evaluated by CCK8 assay and flow cytometry respectively d e The expression levels of apoptosisrelated proteins BCL2 and Cleavedcaspase3 and autophagy related proteins LC3II LC3I and Beclin were detected by western blot assay P CASC2 acted as a0a a0ceRNA by a0sponging miR‘‘5p to a0facilitate PPARÎ expressionAs appeared in Fig a04a“d HG inhibited the mRNA and protein levels of PPARÎ in CIHP1 cells compared to NG and mannitol stimulation At a0 h after the induction of HG the mRNA and protein levels of PPARÎ were dwindled in CIHP1 cells The effect of HG treatment on PPARÎ expression was the opposite of that of miR95p thus we speculated whether there was a connection between miR95p and PPARÎ As presented in Fig a04e there were binding sites for miR95p in the ²UTR of PPARÎ Dualluciferase reporter assay showed that miR95p markedly decreased the luciferase activity of PPARÎ ²UTRwt in CIHP1 cells than that PPARÎ ²UTRmut Fig a0 4f suggesting PPARÎ was the target mRNA of miR95p Then we examined the effect of miR95p on PPARÎ expression the interference efficiency of antimiR95p on miR95p expression was first examined by qRTPCR Fig a0 4g Western blot data showed that the overexpressed miR95p could restrain the protein expression of PPARÎ while the decreased miR95p could raise PPARÎ protein expression Fig a04h Additionally we found that CASC2 depletion reduced the protein expression of PPARÎ and cotransfection of antimiR95p could reverse this effect Fig a04i The above findings revealed that CASC2 positively regulated PPARÎ expression by acting as a ceRNA for miR95p in podocytesCASC2 alleviated the a0HG‘induced podocytes injury by a0increasing PPARÎConsidering CASC2 could act as a sponge of miR95p to regulate the expression of PPARÎ we further investigated whether PPARÎ was involved in regulation of HGinduced podocytes injury mediated by CASC2 Western blot results indicated that cotransfection of siPPARÎ neutralized the promoting effect of CASC2 on PPARÎ protein expression Fig a0 5a The data of CCK8 and Annexin VFITCPI assays indicated that the effects of CASC2 on cell viability Fig a0 5b and apoptosis Fig a0 5c could be abolished by silencing 0cLi a0et a0al Diabetol Metab Syndr Page of Fig CASC2 acted as a ceRNA by sponging miR95p to facilitate PPARÎ expression a The mRNA expression of PPARÎ in NG HG or mannitoltreated CIHP1 cells was analyzed by qRTPCR b qRTPCR assay was used to measure the mRNA expression of PPARÎ in CIHP1 cells treated by HG mM at different times c The protein expression of PPARÎ in NG HG or mannitoltreated CIHP1 cells was analyzed by western blot assay d Western blot assay was used to measure the protein expression of PPARÎ in CIHP1 cells treated by HG mM at different times e StarBase v20 predicted that there were binding sites between miR95p and PPARÎ f Dual luciferase reporter assay was conducted to detect the interaction between miR95p and PPARÎ in CIHP1 cells g The expression of miR95p in CIHP1 cells transfected with antiNC or antimiR95p was measured by qRTPCR h The protein expression of PPARÎ in CIHP1 cells transfected with miRNC miR95p antiNC or antimiR95p was assessed using western blot assay i PPARÎ protein expression in CIHP1 cells transfected with siNC siCASC2 siCASC2 antiNC or siCASC2 antimiR95p was estimated by western blot P PPARÎ in HGinduced CIHP1 cells Similarly the effects of CASC2 on levels of Cleavedcaspase3 BCL Beclin and LC3IILC3I ratio were rescued by siPPARÎ implying PPARÎ knockdown could increase Cleavedcaspase3 protein and decrease the expression levels of BCL2 and Beclin as well as the ratio of LC3IILC3I Fig a05d e To sum up CASC2 alleviated the HGinduced podocytes injury by upregulating PPARÎOverall it could be concluded that HG inhibited cell viability autophagy but promoted cell apoptosis by downregulating CASC2 and PPARÎ expression as well as upregulating miR95p in CIHP1 cells Fig a0DiscussionPodocytes are terminally differentiated visceral epithelial cells which are important components of the glomerular filtration barrier Podocyte viability and apoptosis as well as autophagy can affect glomerular function [] A large number of studies have shown that high glucose induction can cause podocytes injury [“]Several lncRNAs such as lncRNA MALAT1 [] and lncRNA PRINS [] have been found to be involved in the development of DN they regulated mRNA expression at the posttranscriptional level In this study we found that CASC2 expression was prominently downregulated in high glucosestimulated podocytes in a 0cLi a0et a0al Diabetol Metab Syndr Page of Fig CASC2 alleviated the HGinduced podocytes injury by increasing PPARÎ The HGtreated CIHP1 cells were transfected with Vector CASC2 CASC2 siNC and siPPARÎ respectively a PPARÎ protein expression was examined by western blot b c Cell viability and apoptosis were determined by CCK8 assay and Flow cytometry respectively d e The expression levels of BCL2 Cleavedcaspase3 LC3II LC3I and Beclin were checked by western blot assay P timedependent manner and dose“response manner Autophagy is a doubleedged sword and its excessive activation or repression can cause podocytes injury [] Autophagy activity is impaired in DN patients so promoting autophagy to some extent can reduce podocytes injury [] and Beclin LC3I and LC3II have been shown to be autophagy specific proteins [] In accordance with previous data high glucose could inhibit cell viability and autophagy and promote cell apoptosis while overexpression of CASC2 could attenuate the effect of high glucose on podocytes injury suggesting the protective effect of CASC2 on podocytes injury Similarly Yang et a0al observed that CASC2 was enormously decreased in DN patients while there was no significant difference in CASC2 expression in DN patients as compared to those with DM without complication DN [] Besides Wang et a0 al reported that the development of type diabetes might have no significant effects on CASC2 expression in renal tissue whereas CASC2 expression in renal tissues was found to be evidently lower in patients with type diabetes complicated with chronic renal failure [] These data suggested that 0cLi a0et a0al Diabetol Metab Syndr Page of Fig Schema presented the mechanism that HG repressed cell viability autophagy and promoted cell apoptosis by regulating the CASC2miR95pPPARÎ axis in CIHP1 cellsCASC2 inhibition was very likely to be involved in the pathogenesis of DN Moreover lncRNA often functions as ceRNA and we speculated that CASC2 might also be involved in the regulation of podocytes injury by sponging miRNACompared with nondiabetic subjects the level of miR95p was higher in serum of patients with gestational diabetes mellitus [] and serum miR9 might be an underlying marker for poor prognosis of DN [] In our data the abundance of miR95p was increased in high glucoseinduced podocytes and miR95p was validated to be the target miRNA for CASC2 and CASC2 could inversely modulate miR95p expression in podocytes Recovery experiments showed that CASC2 mitigated podocytes injury by decreasing miR95p via acting as a miR95p sponge similar to the work of Zhang et a0al who indicated that lncRNA SOX2OT could reduce the high glucosestimulated podocytes damage by autophagy induction through binding to miR9 [] Therefore it is reasonable to infer that CASC2 regulated podocyte activity apoptosis and autophagy through sponging miR95pPPARÎ agonists have been widely reported to improve glycemic status in diabetes patients [] and PPARÎ has favorable renal protective effects [] As expected high glucose treatment obviously retarded PPARÎ expression Importantly miR95p directly targeted PPARÎ ²UTR and negatively modulated its expression In addition CASC2 could regulate PPARÎ expression by sponging miR95p based on these results we hypothesized whether CASC2 implicated in podocytes injury by regulating PPARÎ The results showed that PPARÎ knockdown neutralized the effect of CASC2 on podocytes injury Besides PPARÎ has been shown to restore podocyte integrity to improve proteinuria []ConclusionIn summary we believed that CASC2 mainly upregulated the expression of PPARÎ by acting as the ceRNA of miR95p thus alleviating HGinduced podocytes injury through increasing cell viability autophagy and reducing cell apoptosis This study provided a new molecular regulatory mechanism for podocytes injury induced by HG in DNAbbreviationsHG High glucose DN Diabetes nephropathy CASC2 Cancer susceptibility candidate NG Normal glucose RIP RNA immunoprecipitation PPARÎ Peroxisome proliferatoractivated receptor gammaAcknowledgementsThe authors sincerely appreciate all members participated in this study Authors™ contributionsFL designed the experiments performed the experiments and analyzed and collected the data wrote the manuscript BD analyzed interpreted the data performed the experiments and wrote the manuscript XN performed the experiments and analyzed the data All authors read and approved the final manuscript FundingThis work was supported by Key Research Development Program of Shandong Province China [Grant No2019GSF108172] Natural Science Foundation of Shandong Province China [Grant No2016ZRA08005] and Science and Technology Development Plans of TCM of Shandong Province China [Grant No ] Availability of data and materialsThe datasets used andor analyzed during the current study are available from the corresponding author on reasonable request 0cLi a0et a0al Diabetol Metab Syndr Page of Ethics approval and consent to participateNot applicablePatient consent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestAuthor details Department of Nephrology Heze Mudan People™s Hospital Heze Shandong China Department of Nephrology Liaocheng People™s Hospital Liaocheng Shandong China Department of Nephrology Tai™an Campus of the 960th Hospital of the Chinese People™s Liberation Army No217 Huanshan Road Taishan District Tai™an Shandong China Received February Accepted July References Dronavalli S Duka I Bakris GL The pathogenesis of diabetic nephropathy Nat Clin Pract Endocrinol Metab “ Ogurtsova K da Rocha Fernandes JD Huang Y Linnenkamp U Guariguata L Cho NH et al IDF Diabetes Atlas Global estimates for the prevalence of diabetes for and Diabetes Res Clin Pract “ Afkarian M Zelnick LR Hall YN Heagerty PJ Tuttle K Weiss NS et al Clinical manifestations of kidney disease among US adults with diabetes “ JAMA “ Ziyadeh FN Wolf G Pathogenesis of the podocytopathy and proteinuria in diabetic glomerulopathy Curr Diabetes Rev “ Berthier CC Zhang H Schin M Henger A Nelson RG Yee B et al 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glucoseinduced mitochondrial fission through the phosphorylation of Drp1 in podocytes J Cell Physiol https doi101002jcp29646 Jiang L Cui H Ding J Smad3 signalling affects high glucoseinduced podocyte injury via regulation of the cytoskeletal protein transgelin Nephrology https doi101111nep13701 Hu M Wang R Li X Fan M Lin J Zhen J et al LncRNA MALAT1 is dysregulated in diabetic nephropathy and involved in high glucoseinduced podocyte injury via its interplay with betacatenin J Cell Mol Med “ Jiao H Xie D Qiao Y LncRNA PRINS is involved in the development of nephropathy in patients with diabetes via interaction with Smad7 Exp Ther Med “ Kim H Dusabimana T Kim SR Je J Jeong K Kang MC et al Supplementation of abelmoschus manihot ameliorates diabetic nephropathy and hepatic steatosis by activating autophagy in mice Nutrients Wu F Li S Zhang N Huang W Li X Wang M et al Hispidulin alleviates highglucoseinduced podocyte injury by regulating protective autophagy Biomed Pharmacother “ Sc
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EFSA for a scientific opinion on the risks for animal and humanhealth related to the presence of glycoalkaloids GAs in feed and food This risk assessment coversedible parts of potato plants and other food plants containing GAstomato andaubergine In humans acute toxic effects of potato GAs asolanine and achaconine includegastrointestinal symptoms such as nausea vomiting and diarrhoea For these effects the CONTAMPanel identified a lowestobservedadverseeffect level of mg total potato GAskg body weight bwper day as a reference point for the risk characterisation following acute exposure In humans noevidence of health problems associated with repeated or longterm intake of GAs via potatoes hasbeen identified No reference point for chronic exposure could be identified from the experimentalanimal studies Occurrence data were available only for asolanine and achaconine mostly forpotatoes The acute dietary exposure to potato GAs was estimated using a probabilistic approach andapplying processing factors for food Due to the limited data available a margin of exposure MOEapproach was applied The MOEs for the younger age groups indicate a health concern for the foodconsumption surveys with the highest mean exposure as well as for the P95 exposure in all surveysFor adult age groups the MOEs indicate a health concern only for the food consumption surveys withthe highest P95 exposures For tomato and aubergine GAs the risk to human health could not becharacterised due to the lack of occurrence data and the limited toxicity data For horses farm andcompanion animals no risk characterisation for potato GAs could be performed due to insufficient dataon occurrence in feed and on potential adverse effects of GAs in these species European Food Safety Authority EFSA Journal published by John Wiley and Sons Ltd on behalfof European Food Safety AuthorityKeywords glycoalkaloids GAs solanine chaconine potato margin of exposure MOE food feedRequestor European CommissionQuestion number EFSAQ201600811Correspondence contamefsaeuropaeu Leon Brimer was a member of the Working Group on Glycoalkaloids in food and feed until August wwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and foodPanel members Margherita Bignami Laurent Bodin James Kevin Chipman Jes 13us del Mazo BettinaGraslKraupp Christer Hogstrand Laurentius Ron Hoogenboom JeanCharles Leblanc Carlo StefanoNebbia Elsa Nielsen Evangelia Ntzani Annette Petersen Salomon Sand Dieter Schrenk TanjaSchwerdtle Christiane Vleminckx and Heather WallaceAcknowledgements The Panel wishes to thank the following for the support provided to thisscientific output Kelly Niermans The Panel wishes to acknowledge all European competentinstitutions Member State bodies and other anisations that provided consumption and occurrencedata for this scientific outputSuggested citation EFSA CONTAM Panel EFSA Panel on Contaminants in the Food Chain Schrenk DBignami M Bodin L Chipman JK del Mazo J Hogstrand C Hoogenboom LR Leblanc JC Nebbia CSNielsen E Ntzani E Petersen A Sand S Schwerdtle T Vleminckx C Wallace H Brimer L Cottrill BDusemund B Mulder P Vollmer G Binaglia M Ramos Bordajandi L Riolo F Rold 13anTorres R and GraslKraupp B Scientific Opinion “ Risk assessment of glycoalkaloids in feed and food in particular inpotatoes and potatoderived products EFSA Journal pp 102903jefsa20206222ISSN European Food Safety Authority EFSA Journal published by John Wiley and Sons Ltd on behalfof European Food Safety AuthorityThis is an access under the terms of the Creative Commons AttributionNoDerivs Licensewhich permits use and distribution in any medium provided the original work is properly cited and nomodifications or adaptations are madeReproduction of the images listed below is prohibited and permission must be sought directly from thecopyright holderFigure Elsevier Figure Springer Figure American Chemical Society SpringerThe EFSA Journal is a publication of the European FoodSafety Authority an agency of the European UnionwwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and foodSummaryThe European Commission asked EFSA for a scientific opinion on the risks for animal and humanhealth related to the presence of glycoalkaloids GAs in feed and food in particular in potatoes andpotatoderived products This risk assessment covers edible parts of potato plants and other foodplants containing GAs in particular tomato and aubergine Nonedible parts of GA containing plantshave not been considered with the exception of potato sprouts The Panel developed the draftscientific opinion which underwent a public consultation from February to April Thecomments received and how they were taken into account when finalising the scientific opinion werepublished in an EFSA Technical Report EFSA GAs are present in many plants of the family of Solanaceae and contribute to plant resistanceagainst pests and pathogens GAs are composed of a steroidal aglycone and an oligosaccharide sidechain In commercial potato cultivars S tuberosum the main GAs are achaconine and asolanineconsisting of the aglycone solanidine and chacotriose and solatriose as oligosaccharide side chainsrespectively The aubergine fruit S melongena contains primarily the GAs asolamargine and asolasonine composed of the aglycone solasodine and chacotriose and solatriose respectively Inlycopersicum atomatine and adehydrotomatine are the major GAs withtomato fruitlycotetraose coupled to the aglycones tomatidine and tomatidenol respectivelySHuman risk assessmentIn experimental animals the potato GAs asolanine and achaconine show a relatively low oralbioavailability with differences between species Hamsters exhibit higher absorption and slowerexcretion rates for both substances when compared to rats Due to the limited information themetabolic profiles of potato GAs in experimental animals could not be characterisedIn humans asolanine and achaconine are systemically absorbed following ingestion For bothsubstances relatively long serum halflives were reported suggesting a possible accumulation The bloodclearance of the respective aglycone solanidine appears to be slow Accordingly levels of solanidine wereregularly detected in the blood of human volunteers in several studies suggesting hydrolysis of GAs Nofurther information is available on metabolism and excretion of potato GAs in humansThere are no toxicokinetic data on tomato and aubergine GAs and their aglycones in experimentalanimals and humansIn acute oral toxicity studies no adverse effects of asolanine were observed at doses of mgkgbody weight bw per day in rats and mgkg bw per day in mice Reliable data on other potatoGAs or tomato and aubergine GAs and their aglycones are missingIn repeated oral dose studies on potato GAs rodents showed nonspecific effects such as reducedbody weight and relative liver weight with indication of similar potencies of asolanine and achaconine Hamsters exhibited these symptoms after a 5day treatment with mg of asolanine ora chaconinekg bw per day while mice showed these effects after one week of daily treatments with mg of asolanine or mg of achaconinekg bw Solanidine however increased the absoluteand relative liver weight at mgkg bw per day in mice suggesting a different effect of theaglycone compared to the GAsThe tomato GA atomatine and its aglycone tomatidine exerted no effects in rats when applied at mgkg bw per day for a period of day At higher doses atomatine reduced the cholesterol uptakeand increased fecal sterol and coprostanol excretion in hamsters and rats In mice a to 2weektreatment with the aubergine GA asolasonine increased the body weight gain at mgkg bw perday while its aglycone solasodine decreased body weight gain and caused gastric gland degenerationand liver toxicity at mgkg bw per dayDevelopmental studies have been performed mainly in hamsters treated with potato GAs and theiraglycones for only one day or for a short very restricted time period during gestation Outcomes weremainly analysed in late gestational embryos and comprised effects in the central nervous systempredominantly exencephaly encephalocele and anophthalmia These malformations occurred at dosesof mgkg bw per day and above for GAs and of mgkg bw per day and above for theaglycones No noobservedadverseeffectlevelLOAEL could be identified from these studies Reduced postnatal survival of pups due to insufficientmilk production was reported when pregnant Holtzman rats had been exposed to mg of asolaninekg bw per day Studies on the male fertility in dogs have been performed only with theaubergine aglycone solasodine Decreased epididymal weight and cauda epididymal epithelial heightand also an epididymal lumen depleted of sperm occurred in dogs after mgkg bw per day givenlowestobservedadverseeffectNOAEL orlevelwwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and foodfor month Similar effects were observed in Rhesus monkeys exposed to mgkg bw per day for monthsFrom the limited number of studies available there was no evidence for genotoxicity of the potatoGAs asolanine and achaconine and the aglycone solanidine as well as for the aubergine GA asolamargine However there is not sufficient information to conclude on the genotoxic potential ofthese GAsNo longterm chronic toxicitycarcinogencity study for potato tomato or aubergine GAs or for therespective aglycones could be identifiedIn humans acute toxic effects following ingestion of potato GAs include gastrointestinal symptomsof varying severity such as vomiting diarrhoea and abdominal pain which may occur from a totalpotato GAs potato TGA intake of mgkg bw or more Further symptoms including drowsinessapathy confusion weakness vision disturbances rapid and weak pulse and low blood pressure maybe the consequence of dehydration following vomiting and diarrhoeaIn severe cases paralysis respiratory insufficiency cardiac failure coma and death have beenreported Doses in the range of “ mg potato TGAskg bw are considered to be potentially lethal forhumans Results from limited volunteer studies suggest possible differences in the human populationwith respect to the individual susceptibility towards adverse effects associated with the intake ofpotato GAsRegarding the mode of action adverse effects of GAs may be due to their ability to complex withmembrane 3bhydroxy sterols thereby causing disruption and loss of integrity of cell membranesAfter oral exposure these effects may affect the mucosa of the gastrointestinal tract and cause thesymptoms observed in intoxicated humans such as nausea vomiting and diarrhoeaGAs inhibit acetylcholinesterase AChE and serum butyrylcholinesterase BuChE by a reversiblecompetitive mode of action The relative potency of inhibition of asolanine and achaconine appearsto be similar The aglycones exert weak or no inhibitory effects The excess of acetylcholine at theneuronal and neuromuscular junctions upon inhibition of the enzymes might also contribute to thesymptoms described for intoxications with GAsAt high doses atomatine may form a nonabsorbable complex with cholesterol and other sterols inthe enteral lumen which may impair the absorption of cholesterol As a consequence blood cholesterollevels were lowered in rodentsThe CONTAM Panel considered that the use of rodent data on acute toxicity was not appropriate toestablish a reference point for acute exposure to potato GAs in humans The CONTAM Panel selectedthe LOAEL of mg potato TGAkg bw per day as the reference point for acute risk characterisationbased on human data from case reports outbreaks and studies in volunteers The available data onacute toxicity were considered insufficient to establish a healthbased guidance value Instead thePanel used the margin of exposure MOE approach to assess a possible health concern from acuteexposure to potato TGAs via foodAssuming the main symptoms to be mainly due to localirritation of the gastrointestinal mucosarather than inhibition of AChE activity the Panel considered that the possible interindividual variabilityin toxicodynamics is more relevant than the interindividual variability in toxicokinetics Accordingly anMOE higher than indicates that there is no health concern This MOE of takes into account theextrapolation from a LOAEL to a NOAEL a factor of and the interindividual variability intoxicodynamics a factor of The experimental data available for repeated dose toxicity are not sufficient to identify a referencepoint for chronic exposure to potato GAs In humans no evidence of health problems associated withrepeated or longterm intake of GAs via potatoes has been identifiedRegarding GAs or aglycones occurring in edible parts of food plants other than S tuberosum nosuitable study for determining a reference point for tomato or aubergine GAs or aglycones wasidentifiedOccurrence data were only available for asolanine and achaconine and mostly for ˜Maincroppotatoes™ and ˜New potatoes™ Few data were available for processed food No data on the occurrenceof tomato and aubergine GAs and their aglycones were submitted to EFSASince the occurrence data on potato GAs did not cover all the food categories containing potatoesin the Consumption Database it was decided that the best approach for the exposure assessmentwould be to use the occurrence data in the raw primary commodities RPC maincrop potatoes andnew potatoes and the RPC Consumption Database The Panel decided to combine the occurrence of˜New potatoes™ with that of ˜Maincrop potatoes™ and the mean upper bound UB occurrence sum ofwwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and foodasolanine and achaconine for these two groups was mgkg and the P95 occurrence was mgkg The minimum and maximum reported concentrations were and mgkg respectivelyThe acute dietary exposure to potato TGAs was estimated using a probabilistic approach includingonly days in which there was consumption of maincrop potatoes As no occurrence data wereavailable for GAs in tomato and aubergine these foods were not included in the exposure assessmentProcessing of potatoes has been reported to reduce the content of GAs in the final processedproduct In general and according to the literature the peeling of potatoes reduced the GA contentby “ boiling in water and blanching of peeled potatoes by “ and frying in oil of peeledpotatoes by “ Microwave and oven baking of unpeeled potatoes may cause a reduction in theGA content by “ and by “ respectively No information has been found about thechemical nature of the GA degradation products For the exposure assessment processing factors forthe major food processing steps comprising peeling and heat processing boiling frying bakingwere applied to the occurrence data as follows processing factors between and wereattributed to the peeling of potatoes between and for frying and deep frying and between and for all other cooking methodsInformation about the peeling of potatoes was not available in the consumption database but itwas assumed that of the potatoes are consumed as peeled Where information of the cookingmethod was not available a cooking method was randomly attributed to the eating event based onthe relative frequency of cooking methods reportedThe mean UB exposure to potato TGAs across surveys ranged from lgkg bw per day inadults to lgkg bw per day in toddlers The 95th percentile exposure ranged from lgkgbw per day in adults to lgkg bw per day in toddlers up to lgkg bw per day in theupper limit of the confidence intervalComparing the LOAEL for potato TGAs of mgkg bw per day with the acute exposure estimatesthe MOEs for the younger age groups indicate a health concern for the food consumption surveys withthe highest mean exposure as well as for the P95 exposure in all surveys For adult age groups theMOEs indicate a health concern only for the food consumption surveys with the highest P95exposuresThe CONTAM Panel calculated the mean percentage of days with potato consumption acrosssurveys per age group on which the potato TGA intake may be below the MOE of The highestnumber of survey days with intake of potatoes below the MOE of was estimated for toddlers followed by children For the other age groups the estimated TGA intake was below the MOEof in up to “ of the survey daysFor tomato and aubergine GAs the risk to human health could not be characterised due to the lackof occurrence data in food and the limited information on the adverse effects in experimental animalsand humansThe CONTAM Panel considered that the impact of the uncertainties on the risk assessment of acuteexposure to potato GAs in food is moderate and that overall the identified uncertainties may eithercause an over or underestimation of the riskFarm animals horses and companion animals risk assessmentInformation on the toxicokinetics of GAs was limited to ruminants for which the data suggest anextensive conversion of asolanine and achaconine to aglycones in rumen and a low potential ofsolanidine to transfer into cows™ milkNo data on the potential adverse effects of potato GAs in horses companion animals cats anddogs or fur animals were identified Due to an insufficient database on the adverse effects of GAs inruminants pigs poultry rabbits and fish an acute reference dose could not be derivedPotatoes are not grown specifically as feed for livestock but when supply exceeds marketrequirements for human consumption whole raw potatoes may be used as feed for ruminants andpigs Some byproducts of potato processing and starch extraction are used as feeds for farmedlivestock principally nonruminants and for companion animalsData on potato GAs in feed were insufficient to perform an exposure assessmentThus no risk characterisation could be performed due to insufficient occurrence data of GAs forfeed and the lack of or limited data on the adverse effects of GAs in farm animals horses orcompanion animalswwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and foodRecommendationsThe following needs have been identified to improve the risk assessment for humans and reducethe uncertaintiescid129Research on the occurrence of GAs and their aglycones and other potentially toxicologicallyrelevant secondary plant metabolites in the potato cultivars available on the market and onnew potato cultivars resulting from breeding experimentscid129 Occurrence data on GAs and their aglycones in potato processed products including foods forinfantscid129 Occurrence data on GAs and their aglycones in tomato and aubergine and products thereofcid129 Data on the toxicokinetics of potato tomato and aubergine GAs and aglycones in experimentalanimals and humanscid129 Data on repeated dose toxicity including reproductive and developmental toxicity of potatotomato and aubergine GAs and aglycones in experimental animalsStudies in humans linking dietary exposure biomarkers of exposure and adverse effectscid129The following needs have been identified to improve the risk assessment for farm animals horsesand companion animals and reduce the uncertaintiescid129 Occurrence data on potato GAs and their aglycones in feedcid129Studies on the kinetics and the potential adverse effects from feed material containing GAs ofpotato GAs in farm animals horses and companion animalswwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and foodTable of contentsAbstractSummaryIntroduction Background and Terms of Reference as provided by the requestor Interpretation of the Terms of Reference Supporting information for the assessment Chemistry Analytical methods Sources Potatoes Tomatoes Aubergine Previous risk assessments Legislation and other standards Data and methodologies Methodology for data collection selection of evidence and study appraisal Food and feed occurrence data submitted to EFSA Data collection and validation Data analysis Food and feed consumption data Food consumption data Feed consumption data Food classification Methodology for Exposure assessment Methodology for Risk characterisation Assessment Hazard identification and characterisation Toxicokinetics Experimental animals aSolanine aChaconine Humans Mixtures of asolanine and achaconine Solanidine Biomarkers of exposure Farm animals horses and companion animals Summary on toxicokinetics Toxicity in experimental animals Acute toxicity studies GAs from edible parts of S tuberosum GAs from edible parts of food plants other than S tuberosum Summary on acute toxicity studies Repeated dose toxicity studies GAs and aglycones from edible parts of S tuberosum GAs and aglycones from edible parts of food plants other than S tuberosum Developmental and reproductive toxicity studies Developmental effects Reproductive effects Immunotoxicity studies Studies on cardiovascular effects Neurotoxicity studies Genotoxicity GAs from edible parts of S tuberosum GAs from edible parts of food plants other than S tuberosum Carcinogenicity studies Studies on metabolic effects GAs from edible parts of S tuberosum GAs from edible parts of food plants other than S tuberosum Observations in humans wwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and food GAs from S tuberosum Reports on intoxications Studies in human volunteers Epidemiological studies Summary GAs from food plants other than S tuberosum Case Reports Adverse effects in farm animals horses and companion animals Ruminants Pigs Poultry Rabbits Fish Horses Companion animals cats and dogs Fur animals Reports on intoxications Mode of action Membrane effects with implications for the gastrointestinal tract Inhibition of cholinesterases ChEs Comparative determination of inhibition of ChEs in vitro Determination of inhibitory constants Ki for GAs on inhibition of ChEs in vitro Inhibition of ChEs in vivo Developmental and reproductive effects of GAs and their aglycones Inhibition of cholinesterases and effects in the immune system Interference with metabolism Considerations of critical effects and doseresponse analysis for the human risk assessment GAs from edible parts of S tuberosum Considerations of critical effects and doseresponse analysis Derivation of a healthbased guidance value HBGV or margin of exposure MOE approach GAs from edible parts of food plants other than S tuberosum Considerations of critical effects and doseresponse analysis Consideration of critical effects and doseresponse analysis for the farm animal horses andcompanion animals risk assessment Occurrence data Occurrence data submitted to EFSA Previously reported occurrence data in the literature Literature on occurrence data on food Occurrence data on GAs in potatoes Occurrence data on GAs in tomatoes Occurrence data on GAs in aubergines Occurrence data on GAs in other food products Literature occurrence data in feed Influence of storage and processing on the content of GAs GAs from S tuberosum Storage of potatoes Processing of potatoes for food consumption Processing of potatoes for feed GAs from food plants other than S tuberosum Summary on the influence of storage and processing on the levels of GAs Exposure assessment Current acute dietary exposure assessment for humans Previously reported dietary exposure assessments Current dietary exposure assessment for farm animals horses and companion animals Risk characterisation Human health risk characterisation GA from edible parts of S tuberosum GAs from edible parts of food plants other than S tuberosum Farm animals horses and companion animal risk characterisation Uncertainty analysis Assessment objectives Exposure scenarioexposure model wwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and foodHazard identification and characterisation Summary of uncertainties Conclusions Hazard identification and characterisation Toxicokinetics Toxicity in experimental animals Observations in humans Adverse effects in farm animals horses and companion animals Mode of action Margin of exposure MOE approach Occurrence and exposure Food Feed Risk characterisation Human health risk characterisation Farm animals horses and companion animal health risk characterisation Recommendations Documentation provided to EFSA References Abbreviations Appendix A “ Major glycoalkaloids and their aglycones present in Solanum species Appendix B “ Identification and selection of evidence relevant for the risk assessment of glycoalkaloids infeed and food Appendix C “ Details of the study design of the toxicokinetic studies Appendix D “ Comparison of developmental toxicity of single dose studies Appendix E “ Inhibition of cholinesterases by GAs Appendix F “ Rapid Alert System for Food and Feed RASFF reports on the presence of Solanum nigrum infood products Appendix G “ Studies on the toxicity of Glycoalkaloids not considered in the risk assessment Appendix H “ Additional scenario for the human risk characterisation Annex A “ Occurrence data in food and feed submitted to EFSA and dietary exposure assessment forhumans wwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and foodIntroductionBackground and Terms of Reference as provided by the requestorBackgroundMany plants in the family Solanaceae contain glycoalkaloids and they are considered to be naturaltoxins The plant glycoalkaloids are toxic steroidal glycosides and the commonest types found in foodplants are asolanine and achaconine Their natural function is probably to serve as stress metabolitesor phytoalexins for the protection of the plant when attacked by insects fungi etcAmongst the most widely cultivated food crops aubergines tomatoes and potatoes are in theSolanaceae family but the levels of glycoalkaloids in tomatoes and aubergines are generally quite lowThe glycoalkaloids of most relevance to food safety are those occurring in the potato Thepredominant toxic steroidal glycosides in potato are asolanine and achaconine They occur in potatotubers peel sprouts berries leaves and blossoms and their concentration in tubers depends on anumber offactors Concentrations ofglycoalkaloids are “ times greater in the peel than in the flesh There is considerable variation inglycoalkaloid content among potato cultivars Storage conditions especially light and temperature aremainly responsible for increases in solanine Although the glycoalkaloid content can increase in thedark the rate of formation is only about the rate of formation in light Increases of solanine inthe potato peel are closely associated with greening synthesis of chlorophyll of the peel Thesebiochemical processes are independent of each other but are both activated by lightsuch as cultivar maturity and environmentalfactorsBitter or burning sensation in the mouth are sensory impressions which may accompanyglycoalkaloid poisoning symptoms from potatoes that include flulike symptoms such as nauseavomiting stomach and abdominal cramps and diarrhoea More severe cases of glycoalkaloid poisoningmay be accompanied by a variety of neurological effects ie drowsiness apathy restlessnessshaking confusion weakness and disturbed vision There are a few reports of deaths beingattributed to glycoalkaloid exposure from the consumption of potatoes potato leaves and potatoberriesPotatoes and potatoderived products are listed in the Catalogue of feed materials1Terms of ReferenceIn accordance with Art of Regulation EC No the European Commission asks theEuropean Food Safety Authority for a scientific opinion on the risks for animal and human healthrelated to the presence of glycoalkaloids in feed and food in particular in potatoes and potatoderivedproductsInterpretation of the Terms of ReferenceThe CONTAM Panel considered that the opinion should cover edible parts of potato plants and alsoof other food plants containing glycoalkaloids GAs eg tomato and aubergine Nonedible parts ofGA containing plants have not been considered with the exception of potato sprouts In particular theCONTAM Panel concluded this Opinion should comprise thea evaluation of the toxicity of GAs in feed and food in particular in potatoes and potatoderivedproducts for farm and companion animals and humans considering all relevant toxicologicalend pointsb evaluation of the alkaloid profile ie composition of the alkaloids and their concentration ofthe food and feed samples submitted to EFSAc estimation of the dietary exposure of the European population to GAs in food in particular inpotatoes and potatoderived products including the consumption patterns of specific groupsof the population if appropriated estimation of the dietary exposure offarm and companion animals to GAs in feedinparticular in potatoes and potatoderived productse assessment of the human health risks for the European population including specific groupsof the population if appropriate as the consequence of the estimated dietary exposure Commission Regulation EU No of January on the Catalogue of feed materials OJL p wwwefsaeuropaeuefsajournalEFSA Journal 0cGlycoalkaloids in feed and foodf assessment of the farm and companion animal health risks in Europe as the consequence ofthe estimated dietary exposure Exposure to GAs from weeds containing GA is only addressedin this Opinion in the context of accidental intake by farm animalsWhen referring to GAs in potatoes the term total GAs TGA refers to a material comprising asolanineand achaconine as major fraction with no specification on the occurrence of minor GAs as well as band cforms of solanine and chaconine Similarly when referring to tomato and aubergine the termTGA refers to the GAs from the corresponding species and forms thereofSupporting information for the assessment ChemistrySolanine is one of the first alkaloids that has been isolated from nature by Desfosses in Friedman et al In Zwenger and Kind reported that solanine contains a glycoside sidechain Zwenger and Kind Only in it was shown that solanine extracted from potato is infact a mixture of two glycoalkaloids GAs asolanine and achaconine that share the same solanidineaglycone Kuhn and L‚¬ow Since then at least different GAs have been isolated and fullystructurally elucidated from over species of the Solanaceae family S 13anchezMata et al AlSinani and Eltayeb The chemical structures and some physical properties of the most importantones are listed in Appendix AGAs are composed of a steroidal aglycone and an oligosaccharide sidechain attached to the 3bhydroxy group of the aglycone see Figure Friedman et al Friedman Milner et al The GAs of relevance can be divided into the i solanidane group with solanidine as thesteroid backbone and the ii spirosolane group with either the solasodine or the tomatidenoltomatidine backbone GAs often contain a double bond between C5 and C6 but the corresponding 5a6hydrogenated forms are also common and in some species eg tomato they constitute the majorcomponents The stereochemistry at carbons C22 and C25 is well definedtheconfiguration is 22R 25Stheitconfiguration is 22S 25S Friedman et al in solanidineis 22R 25R and in tomatidenoltomatidinein solasodineFurther diversification is generated by the composition of the glycoside sidechain Most GAscontain either a trisaccharide chacotriose or solatriose or a tetrasaccharide lycotetraose ascarbohydrate In commercial potato cultivars Solanum tuberosum mostly achaconine and asolaninecomposed ofthe solanidine aglycone and chacotriose and solatriose respectively are presentFigure Wild S tuberosum varieties may contain a much wider range of GAs Friedman et al Distl and Wink The aubergine fruit derived from S melongena contains primarily asolamargine and asolasonine composed of the solasodine aglycone and chacotriose and solatrioselycopersicum varieties atomatine and arespectivelydehydrotomatine are the major compounds composed of the aglycones tomatidine and tomatidenolrespectively coupled to lycotetraose Friedman derived from SIn tomato fruitThe prefix alpha a refers to the intact glycoside while the prefixe
2
neutrophils account for “ of circulating leukocytes and are the first immune cells recruitedto an ‚ammatory site they play an important role in the innate immune response topathogens as patients with neutropenia are highly susceptible to bacterial and fungal infections neutrophils perform numerous functions that target microbes including phagocytosis the releaseof antimicrobial peptidesproteases and netosis interestingly neutrophils have garneredconsiderable interest for their emerging and prominent roles in modulating cancer growth andmetastatic progression the roles played by neutrophils in the cancer setting are diverse andcomplex leading to the concept of neutrophil heterogeneityplasticity and the notion that distinctneutrophil subsets might existgranulopoiesisdiï¬erentiationand mobilization of maturefrom the bone marrow intocirculation this process begins with the commitment of granulocytemonocyte myeloidsegmented neutrophilsregulatedprocessthatinvolvestheisatightlyedited bybrahm segaluniversity at buffalo united statesreviewed byye liuniversity of texas md andersoncancer center united statesconnie jackamancurtin university australiacorrespondencepeter m siegelpetersiegelmcgillcaspecialty sectionthis was submitted tocancer immunity and immunotherapya section of the frontiers in immunologyreceived may accepted july published august citationhsu be shen y and siegel pm neutrophils orchestrators of themalignant phenotypefront immunol 103389fimmu202001778frontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypegmps which progressthrough a series ofprogenitorsneutrophil progenitors myeloblast promyelocyte myelocytemetamyelocyte band cell untilthey become a matureneutrophil in cancer dysregulated granulopoiesis hasled to theidentification of diï¬erent neutrophil subsets that play a role intumor progression preneus comprise a neutrophil precursorpopulation that retain their proliferative capacity and expandin the bone marrow and spleen of tumor bearing mice preneus diï¬erentiate into immature and mature neutrophilswith the former found to accumulate in growing tumors anearly stage committed unipotent neutrophil precursor nep hasalso been identified and their adoptive transfer into humanizedmice promoted solid tumor growth by inhibiting t cellactivation two neutrophil subsets highdensity neutrophilshdns and lowdensity neutrophils ldns were identified invarious tumor models by diï¬erential density centrifugation hdns represent mature segmented neutrophils whereas ldnscomprise a heterogeneous mixture of mature and immatureneutrophils increasing mobilization of ldns into theperipheral blood was associated with enhanced tumor growthand metastasis “functionsand antitumorigenicin addition to the identification of distinct neutrophil subsetsneutrophils exhibit plasticity in response to tumorderivedfactors in a manner similar to macrophages neutrophils havebeen classified into two categories n1 and n2 to describetheir prorespectively in vivo evidence has shown that tumorassociatedneutrophils tans can change their function from a protumor phenotype n2 to an antitumor n1 phenotypewith the addition of a tgf inhibitor arguing that tgfis an important factor driving the n2 phenotype incontrast signals associated with an antitumor n1 phenotypeinclude type iinterferons and those propagated by themet receptor however this categorization is likelyto represent an oversimplification of neutrophil diversityneutrophil polarization similar to macrophages could alsorepresent a continuum of diï¬erent neutrophil phenotypespresent in the tumor microenvironment these advancesregarding the degree of neutrophil heterogeneityplasticityobserved in the cancer setting have sparked an intense andrenewed interestin this cell population while there areongoing discussions in the field regarding the relationshipsubsets we directbetween pmnmdscs and neutrophilto excellentthe readerthatfully discussthese we will briefly discuss antitumorrelationshipsneutrophilreview will primarilyroles of neutrophils and neutrophildiscussassociated functionsgrowth andmetastatic progressionfunctions howeverin promotingthe recentreviewstumorthisantitumor neutrophil functionscan participateantitumorneutrophilsmechanisms thattumor growth or eliminate cancercells a wellstudied neutrophilassociated function isin a variety oflimittheir ability to generate reactive oxygen species ros tolimit tumor progression upon tumor cell contact mousederived neutrophils can release hydrogen peroxide to eliminatemetastatic cancer cells in vitro subsequentlyit wasdemonstrated that expression of trpm2 transient receptorpotential cation channel subfamily m2 on tumor cells increasedtheirsensitivity to neutrophilmediated h2o2dependentcytotoxicity this occurred through a mechanism that involveda transient increase in ca2 mobilization within cancer cells trpm2 upregulation in tumor cells occurred followingan epithelialtomesenchymaltransition emt and cancercells that have undergone an emt were more susceptible toneutrophilmediated killing more recently an interactionbetween the receptor for advanced glycation end productsrage which is expressed on tumor cells and cathepsin gpresent on murine neutrophils was shown to mediate in vitrotumor cell cytotoxicity in a h2o2dependent manner the release of neutrophil ros is also dependent on the tumormicroenvironment in hypoxic tumor microenvironments theability of murine neutrophils to kill tumor cells in vivo throughthe release of ros is greatly diminished thus neutrophilshave the capacity to mediate rosdependent direct tumorcell killingcausingthe interplay of neutrophils with otherimmune celltypes can also indirectly limittumor progression tumorassociated neutrophils suppress the protumorigenic role ofil17 secreting Îδ t cells by inhibiting their proliferationlow glutathione levels in Îδ17 t cells rendered them sensitiveto neutrophilderived rosenhanced oxidativestress and reduced proliferation in earlystage humanlung cancer a subset ofimmature neutrophils have beenidentified as having antigenpresenting functions and act topromote antitumor immunity by stimulating the secretionofinaddition to neutrophilt cellinteractions communicationbetween neutrophils and monocytes can also elicit antitumoreï¬ects nonmetastaticifnÎproducing monocytes to the lungs ifnÎ release activatestmem173sting within neutrophils whichstimulatesneutrophilmediated killing of disseminated cancer cells in thelungs ‚ammatory cytokinesfrom t lymphocytescan mobilizecancercellsneutrophils have been shown to ltrate deposits of prostatecancer cells within bone metastases importantly neutrophilsimpaired bone metastasis progression by inhibiting stat5signal transducer and activator of transcription functionwithin prostate cancer cells resulting in their apoptotic cell death recently neutrophils have been reported to be involvedin antibodymediated trogocytosis a process that mechanicallydisrupts the plasma membrane of antibodyopsinized cancercells leading to a lyticnecrotictype cell death iga antibodiesagainst receptors expressed by cancer cells her2 egfr couldenhance neutrophilmediated trogocytosis of cancer cells if thecd47sirpα innate immune cell checkpoint was simultaneouslyblocked taken together these results demonstrate thatneutrophils can impair tumor growth and metastasis using acombination of direct and indirect cancer cell killing mechanismssupplementary table frontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypefigure neutrophil functions that promote tumor growth and metastasis to support primary tumor growth neutrophils can mediate t cell suppression and altermacrophage differentiation neutrophil release of timp1 enhances tumor cell invasion by inducing epithelialtomesenchymal transition once in circulation circulatingtumor cells interact with neutrophils which enables tumor cell proliferation secretion of various pro‚ammatory markers such as il8 il1 or mmps can mediateincreased tumor cell extravasation in addition neutrophils can inhibit intraluminal nkmediated killing of circulating cancer cells leading to increased extravasation atthe metastatic site various systemic and microenvironmental factors can promote neutrophil ltration neutrophils can awaken dormant cancer cells by promotingecm remodeling and angiogenesis lastly continued growth of the metastatic lesion is facilitated by key neutrophildependent mechanisms which includeangiogenesis proliferation immune suppression and immune exclusion csf1 colony stimulating factor timp1 tissue inhibitor of matrix metalloprotease pdl1programmed death ligand tgf transforming growth factor ros reactive oxygen species mmp matrix metalloproteinases gmcsf granulocyte macrophagecolony stimulating factor angptl2 angiopoetin like2 fgf2 fibroblast growth factor ltb4 leukotriene b4 inos inducible nitric oxide synthase net neutrophilextracellular trap caf cancerassociated fibroblastneutrophil functions thatpromote primary tumor growthneutrophils promote primary tumor growth by variousmechanisms figure netosis is a process that involvesthe extrusion of neutrophilderived chromatin structures thatare decorated with neutrophil granule constituents whichform extracellular structures called neutrophil extracellulartraps nets normally netosis and net productionhave been described in the context of a neutrophil™s ability tocapture and kill bacteria extracellularly however netshave been shown to play an important role in the growth of aprimary tumor tumor microenvironmental changes includingtumorassociated coagulation and enhanced thrombosishave been linked to enhanced tumor growth several recentstudies suggest that netosis may play an important role inthese processes lps stimulation was shown to increase c3arexpression within neutrophils enhance netosis and increasecoagulation these events were correlated with n2 neutrophilpolarization and increased tumor growth interestingly ithas recently been shown that immature neutrophils preferentiallyrespond to cancer cell derived c3a to promote their migration subsequently it was shown that breast cancer cells thatexpressed high levels of gcsf and il1 exhibited highneutrophil counts and tumorassociated thrombosis which wasdependent on net formation pharmacological blockadefrontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypeof il1 receptor signaling reduced net formation attenuatedtumorassociated thrombosis and impaired tumor growth nets can also directly ‚uence cancer cell proliferationneutrophil elastase ne present within nets activates tumorcells to increase mitochondria biogenesis and atp productionthereby further enhancing the growth of cancer cells in addition to the impact of nets neutrophils canalso interact with other immune cells through additionalmechanisms to promote tumor growth neutrophilderived roscan inhibit t cell proliferation creating an immunosuppressiveenvironment that is supportive of tumor growth phenotypiccharacterization and singlecell rna sequencing identified aneutrophil subset that is cd84hi which exhibited potent t cellsuppressive activity and increased ros production in amodel of gastric cancer neutrophils were activated by tumorderived gmcsf that resulted in elevated programmed deathligand pdl1 expression these pdl1 neutrophils wereable to suppress t cell function and promote tumor growth secretion of mmp9 matrix metalloproteinase fromltrating neutrophils activates latent tgf and induces tcell suppression and tumor growth in a colorectal cancer model siglecfhigh neutrophils in lung adenocarcinoma createdan immunosuppressive environment by promoting macrophagediï¬erentiation causing the release of high levels of rosand enabling tumor progression together these findingsindicate that neutrophils that ltrate diverse primary tumorscan modify the local environment in diï¬erent ways to favortumor growthneutrophil functions thatpromote metastasisthe ability of cancer cells to leave the primary tumor anddisseminate to distant ans represents the deadliest aspectof cancer progression indeed the emergence of metastaticcancer accounts for ˆ¼ of cancer related deaths themetastatic cascade represents a series of barriers to cancercells and neutrophils have been found to assist cancer cells insuccessfully navigating several of these distinct steps figure supplementary table local invasionintravasationltrating neutrophils within primary tumors are associatedwith an increase in emt enhanced metastasis and pooroutcomes mechanisticallyof matrixmetalloprotease timp1 secreted by neutrophils induced anemt and consequently increased the migration and invasion oftumor cells cancer cells that had undergone an emt expressedcd90 which enhanced timp1 secretion by neutrophils in acontactdependent manner inhibitortissuesurvival in circulationextravasationthe ability of circulating tumor cells ctcsto surviveis criticalfor metastasis formation the formation ofheterotypic cancer cell”neutrophil clusters was found to greatlyincrease metastatic fitness using a 4t1 breast cancer modelit was demonstrated that ctcneutrophil interactions reliedon vcam1 dependent adhesion which enhanced cancercell proliferation and increased metastasis indirectlyneutrophils can also inhibit nk cellmediated tumor clearancein circulation thereby increasing the intraluminal survival ofdisseminated tumor cells in this study 4t1 breast cancer cellswere injected subcutaneously to mobilize murine neutrophilsly6gfollowing which d2a1 breast cancer cells wereinjected intravenously mice bearing 4t1 cells exhibited reducedclearance of d2a1 cells from the lungs when compared to micethat were not injected with 4t1 cells depletion of nk cellsresulted in enhanced d2a1 cancer cell accumulation in the lungswhile neutrophil depletion had the opposite eï¬ect cancer cells that have survived in circulation must exitthe bloodstream and extravasate into tissue parenchyma neutrophils have been shown to regulate the extravasationprocessthrough several mechanisms neutrophilderivedfactors can diminish the integrity of the endothelial barrierpermitting cancer cellsil8il1 and matrix metalloproteasesmmp8 and mmp9released from neutrophils activated endothelial cells reducedendothelialtransendothelialmigration and accelerated the rate of cancer cell extravasation to extravasate more easilyincreasedfunctionbarriersites“netosis and net constituents can support cancer cellextravasation through enhanced trapping of ctcs withinmetastaticimportantly blocking netosisdecreases cancer cell adhesion and inhibits metastatic spread tothe lung and liver furthermore changes within specificmetastatic microenvironments such as exposure to ozoneor redox imbalance triggered netosis and led to increasedentrapment of cancer cells in the lung and enhanced metastasis collectively these studies show that neutrophils play animportant role in enhancing tumor cell survival and increasedextravasation which promote cancer metastasisrecruitmentearly seedingsurvivalsystemic and tumorderived factors have been implicatedin neutrophilin the premetastatic nichetumorderived il1 induces Îδ t cells to produce il17aand granulocytecolony stimulating factor gcsf whichresults in the recruitment of immunosuppressive neutrophilsto the lung gmcsf and il5 have been shown topromote the expansion and recruitment of prometastaticneutrophils in the lungs of obese mice which promotes lungmetastasis angiopoetinlike2 angptl2 secreted byosteosarcoma cells implanted in the tibia stimulates lungepithelial cells which led to the accumulation of neutrophilsin the lung and enhanced lung metastatic burden in the lung neutrophils secrete ltb4increases theinitiating cellsproliferation of ltb4rpositive metastasis activation of notch1 in colorectalcellsdrives tgf2dependent recruitment of immunosuppressiveneutrophils within the liver which enabled the formation of livermetastases cancerthatnets also support early cancer cell seeding and colonizationof metastases induction of nets by ovarian tumorderivedfactors has been shown to be important in promoting metastasisfrontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypeto the omentum in the liver nets have also beenshown to promote metastasis by activating cancerassociatedfibroblasts growth in the metastatic siteneutrophils have been shown to promote the growth ofmetastases after seeding minor subclones of breast cancer cellsthat secrete il11 and figf cfosinduced growth factor cansupport the formation of polyclonal metastases composed ofdriver and passenger subpopulations these il11 producingsubclones activated il11 responsive mesenchymal stromal cellswhich induced chemokine secretion and subsequent recruitmentof prometastatic neutrophils tumor cellderived gmcsfwas shown to stimulate neutrophils to synthesize and secretetransferrin an iron transport protein which has mitogenicactivity that promotes lung metastatic growth when taken up bycancer cells a recurring function of prometastatic neutrophils is theirability to create an immunosuppressive microenvironmentthat support metastasis within lung metastasesinduciblenitric oxide synthase inos producing neutrophils havebeen shown to limit cd8 t cell dependent antitumorresponses by promoting immune suppression recently p53deficient cancer cells were found to increase the expressionof wnt ligands which in turn upregulated il1 productionfrom tumorassociated macrophages high il1 levelsengaged Îδ17 t cells which subsequently enhanced neutrophilrecruitment that promoted the formation of lung metastases furthermore loss of elf5 e74like transcription factorexpression in triplenegative breast cancer led to increased ifnÎ signaling resulting in the expansion of immunosuppressiveneutrophils in addition to tumorderived factors a lackof systemic testosterone levels can lead to an impairmentof antitumor neutrophil functions a shift toward immatureneutrophils was observed in castrated male mice leading toincreased neutrophilderived ros and suppression of nk cellactivation that promoted increased lung metastatic burden intwo melanoma models recently a role for net formationhas been described for the continued growth of establishedmetastases nets released during cancer progressionwas shown to limitthe ability of nk and cytotoxic tcells to eliminate cancer cells specifically net formationimpaired direct contact between the cancer cells and cytotoxicimmune cells nk and t cells inhibition of netosis with aprotein arginine deiminase pad4 inhibitor synergized withimmune checkpoint inhibitors to control tumor growth andmetastasis proangiogenic functions have long been ascribed forneutrophils which revealed that neutrophilderived proteasessuch as mmp9 could release stored angiogenic factors vegffgfs that were stored in the local environment to enable bloodvessel formation recently a diï¬erent mechanism bywhich neutrophils enhance angiogenesis has been describedthe synthesis and secretion of fibroblast growth factor fgf2 by neutrophils in the liver microenvironment drivesangiogenesis and growth of nascent colorectal cancerderivedhepatic metastases dormantresidual disease andtherapy resistanceneutrophils have also been implicated in awakening dormantcancer cells lpsinduced tissue ‚ammation led to metastaticoutgrowth of dormant tumor cells in a neutrophildependentmanner mmp9 produced by neutrophils can trigger thegrowth of dormant cancer cells by remodeling extracellularmatrix and releasing potent angiogenic factors ne andmmp9 which are enzymes associated with nets can cleavethe extracellular matrix ecm leading to integrinmediatedsignaling which awakens dormant cancer cells and promotescancer cell growth severalstudies have shown that neutrophils promoteresistance to therapy doxorubicin and paclitaxel resistant breastcancer cells express more il17 and cxcr2 ligands whichincreases neutrophil recruitment a neutrophilenrichedsubtype characterized in triple negative breast cancer tnbcdetermined that neutrophils were largely immunosuppressiverendering these tumors resistant to immune checkpoint blockadetherapy in a genetically engineered mouse model ofsarcoma neutrophils promote resistance to radiation therapyby activating mitogenactivated protein kinase mapk pathway in addition cd177 neutrophil ltrates in colorectalcancer patients are associated with adverse outcome in patientsreceiving bevacizumab [antivascular endothelial growth factora vegf a] furthermore lysyl oxidaselike loxl4expressing neutrophils that ltrated colorectal cancer livermetastases were found to identify patients that were resistant toantiangiogenic therapy metabolic programming inneutrophilsrecentinteresthasbeenconceptin thethereofimmunometabolism and the realization that altered cellularmetabolism in ltrating immune cells can have a significantimpact on tumor growth and metastasis neutrophilsare typically viewed as a cell type that is heavily reliant onglycolysis to perform their eï¬ector functions consistentwith this notion neutrophils have very few mitochondria andinhibitors of oxidative phosphorylation oxphos do notalter their rates of oxygen consumption howeverduring tumor progression neutrophils have been shown toundergo a metabolic switch which involves the upregulationof genes associated with oxphos fatty acid metabolism andglycolysis figure neutrophils isolated from lewis lungcarcinoma exhibit increased flux through oxphos glycolysisand increased atp production compared to naïve neutrophilssuggesting that multiple metabolic strategies are engaged intumor ltrating neutrophils recently upregulation offatp2 fatty acid transport protein in neutrophils was shownto increase lipid accumulation in these cells fatp2 regulated theuptake of arachidonic acid which was subsequently convertedto prostaglandin e2 neutrophilderived prostaglandin e2 wasfrontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypefigure metabolic changes in cancerassociated neutrophils neutrophils which possess few mitochondria are reliant on glycolysis to generate atp to fueleffector functions such as phagocytosis generation of reactive oxygen species and netosis in cancer neutrophils upregulate oxidative phosphorylation oxphosand fatty acid transporters to mediate many neutrophil functions including migration and t cell suppression under nutrient limiting conditions such as low glucoseneutrophils can reprogram their metabolism to break down fatty acids or utilize certain amino acids glutamate proline to fuel protumorigenicprometastaticfunctions ppp pentose phosphate pathway glut glucose transporter mct monocarboxylate transporter tca tricarboxylic acid cycle fatp2 fatty acidtransport protein aa arachidonic acid pge2 prostaglandin e2found to be important or neutrophilmediated cd8 t cellsuppression and tumor growth metabolic flexibility refers to the ability of a cell to shiftbetween one metabolic program to another in response tochanging metabolic demands or nutrient supply high metabolicflexibility increases the cell™s ability to survive various andeverchanging metabolic microenvironments neutrophilsubpopulations can also exhibit metabolic flexibility figure in breast cancer splenic neutrophils can engage mitochondrialdependent fatty acid oxidation as a predominate fuel sourceto support ros production and maintain t cell suppression under glucoselimiting conditions similar to certain tumormicroenvironments immature ldns have been shown to utilizeoxphos to generate atp that is required to support theirprotumorigenic functions indeed immature ldns can supportnetosis under nutrient limiting conditions via mitochondrialdependent amino acid catabolism which is importantforefficient breast cancer liver metastasis in addition thelongevity of neutrophils could also be altered due to the enhancedmetabolic flexibility the ex vivo halflife of mouse circulatinghdns and ldns was and h respectively suchobservations raise the intriguing possibility that under certainconditions distinct neutrophil subsets may not be as shortlived as previously thought these studies argue that increasedmetabolic flexibility in distinct neutrophil populations may beimportant for cellular functions that can ‚uence tumor growthand metastatic progressionclinical importance futureperspectives on treatmentin keeping with their protumorigenicmetastatic functions thepresence of neutrophils across diï¬erent cancers was shownto be strongly associated with adverse patient outcomes frontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypeamong certain subtypes of breast cancer er the presence ofa neutrophil ltrate in the primary tumor is also indicativeof worse patient outcomes furthermore in patients withadvanced cancers serum il8 levels and neutrophil ltrationare associated with worse overall survival and diminishedresponse to immune checkpoint inhibitors the mobilization of neutrophils into circulation also hasprognostic significance the neutrophiltolymphocyte rationlr is an important risk stratification and treatment selectiondiagnostic tool for cancer patients a high nlr is associated withpoor prognosis in many solid human cancers “ a highnlr is also associated with decreased overall survival in patientswith tnbc or metastatic breast cancer an important and unanswered question with respect to thenlr is the type of neutrophil that is being detected in thesepatients are they high or lowdensity neutrophils interestinglyldns have been identified in patients with breast cancer lungcancer head and neck cancers urologic cancers and lymphoma “ in patients with advanced lung cancerit wasreported that higher proportion of ldns predictedpoorer survival these observations are in keeping withthe protumorigenic and prometastatic functions associatedwith ldnn2 neutrophils while most studies reveal a negativeprognostic impact of neutrophils in cancer there was one studythat associated the presence of a cd16high cd62dim neutrophilsubset with increased survival of head and neck squamous cellcarcinoma patients these observations highlight the needfor better markers that are capable of discriminating betweenneutrophils that exert antitumor vs those that mediate protumormetastatic eï¬ectsmechanistic insights have greatly advanced our knowledgeoftumorderived factorstumor growth andmetastasis in a neutrophildependent manner additional studiesimpactthatreferences sipsas nv bodey gp kontoyiannis dp perspectives for the management offebrile neutropenic patients with cancer in the 21st century cancer “ 101002cncr20890 kolaczkowska e kubes p neutrophil recruitment and function in healthand ‚ammation nat rev immunol “ 101038nri3399inde visser ke neutrophilssb wellenstein md coï¬eltcancer neutral no more nat rev cancer 101038nrc201652 “ cowland jb borregaard n granulopoiesis and granules of humanneutrophils immunol rev “ 101111imr12440 evrard m kwok iwh chong sz teng kww becht e chenbone marrow neutrophilstraffickinganalysisspecializedpopulationsexpansioninofal developmentaljetrevealsand 101016jimmuni201802002functionseï¬ectorimmunity“79e8 zhu yp padgett l dinh hq marcovecchio p blatchley a wu r identification of an early unipotent neutrophil progenitor with pro tumoralactivity in mouse and human bone marrow cell rep “41e8 101016jcelrep201807097 sagiv jy michaeli j assi s mishalian i kisos h levy l phenotypicdiversity and plasticity in circulating neutrophil subpopulations in cancercell rep “ 101016jcelrep201412039focused on characterizing the phenotypic and functional role ofneutrophils in cancer it may be possible to develop strategies thatspecifically target those neutrophil subsets that actively promotetumor growth and metastasis while sparing those neutrophilsthat possess antitumor and antimicrobial functions finallythe emerging concept of metabolic flexibility that is exhibited bycertain neutrophil subsets may aï¬ord new ways of targeting theseprotumorigenicmetastatic neutrophilsauthor contributionsbh ys and ps wrote the review and prepared the figuresall authors contributed to the and approved thesubmitted versionfundingwork from the authors laboratory cited in this review wassupported by an operating grant to ps from the cancer researchsociety and the terry fox research institute and québecbreast cancer foundation grant bh acknowledgessupport from the charlotte and leo karassik foundation phdfellowship and the rolande and marcel gosselin graduatestudentship ys holds an entrance studentship from thegoodman cancer research centre ps is a mcgill universitywilliam dawson scholarsupplementary materialthe supplementary materialfor this can be foundonline at httpswwwfrontiersins103389fimmu202001778fullsupplementarymaterial coï¬elt sb kersten k doornebal cw weiden j vrijland k hau cs il17producing gammadelta t cells and neutrophils conspireto promote breast“ 101038nature14282cancer metastasis nature hsu be tabaries s johnson rm andrzejewski s senecal j lehuede c immature lowdensity neutrophils exhibit metabolic flexibility thatfacilitates breast cancer liver metastasis cell rep “15e6 101016jcelrep201905091 fridlender zg sun j kim s kapoor v cheng g ling l polarizationof tumorassociated neutrophil phenotype by tgfbeta œn1 versus œn2tan cancer cell “ 101016jccr200906017 ohms m möller s laskay t an attempt to polarize human neutrophilstoward n1 and n2 phenotypes in vitro front immunol 103389fimmu202000532jablonska j leschner s westphal k lienenklaus s weiss s neutrophilsresponsive to endogenous ifnbeta regulate tumor angiogenesis andgrowth in a mouse tumor model j clin invest “ 101172jci37223 finisguerra v di conza g di matteo m serneels j costa s thompsonaa met is required for the recruitment of antitumoural neutrophilsnature “ 101038nature14407 ostuni r kratochvill f murray pj natoli g macrophages and cancer frommechanisms to therapeutic implications trends immunol “ 101016jit201502004frontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotype brandau s moses k lang s the kinship of neutrophils and granulocyticmyeloidderived suppressor cells in cancer cousins siblings or twins semincancer biol “ 101016jsemcancer201302007 vols s sionov rv granot z always look on the bright side antitumor functions of neutrophils curr pharmac design “ granot z henke e comen ea king ta norton l benezra r tumorentrained neutrophils inhibit seeding in the premetastatic lung cancer cell “ 101016jccr201108012 gershkovitz m caspi y fainsodlevi t katz b michaeli j khawaled s trpm2 mediates neutrophil killing of disseminated tumor cells cancer res “ 10115800085472can173614 gershkovitz m fainsodlevi t khawaled s shaul me sionov rvcohendaniel l microenvironmental cues determine tumor cellsusceptibility to neutrophil cytotoxicity cancer res “ 10115800085472can180540 sionov rv fainsodlevi t zelter t polyansky l pham ct granotz neutrophil cathepsin g and tumor cell rage facilitate neutrophilanti8e1624129 1010802162402x20191624129cytotoxicity oncoimmunologytumor mahiddine k blaisdell a ma s crequergrandhomme a lowell caerlebacher a relief of tumor hypoxia unleashes the tumoricidal potentialof neutrophils j clin invest “ 101172jci130952 mensurado s rei m lanca t ioannou m goncalvessousa n kubo h etal tumorassociated neutrophils suppress protumoral il17 gammadeltat cells through induction of oxidative stress plos biol 16e2004990 101371 pbio2004990 singhal s bhojnagarwala ps o™brien s moon ek garfall al rao as etal origin and role of a subset of tumorassociated neutrophils with antigenpresenting cell features in earlystage human lung cancer cancer cell “ 101016jccell201606001et hagerling c gonzalez h salari k wang cy lin c roblescooperationtumor prevents metastatic progressionaliinduced byof breast cancer proc natl acad sci usa 101073pnas1907660116eï¬ector monocyteneutrophilimmunethe primary “ costanzogarvey dl keeley t case aj watson gf alsamraae myu y neutrophils are mediators of metastatic p
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"coronavirus disease COVID19 caused by severe acute respiratory syndrome coronavirus SARSCoV2 emerged in China Currently it is breaking out globally and posing a serious threat to public health The typically clinical characteristics of COVID19 patients were fever and respiratory symptoms and a proportion of patients were accompanied by extrapulmonary symptoms including cardiac injury kidney injury liver injury digestive tract injury and neurological symptoms Angiotensin converting enzyme ACE2 has been proven to be a major receptor for SARSCoV2 and could mediate virus entry into cells And transmembrane protease serine TMPRSS2 could cleave the spike S protein of SARSCoV2 which facilitates the fusion of SARSCoV2 and cellular membranes The mRNA expressions of both ACE2 and TMPRSS2 were observed in the heart digestive tract liver kidney brain and other ans SARSCoV2 may have a capacity to infect extrapulmonary ans due to the expressions of ACE2 and TMPRSS2 in the cells and tissues of these ans It seems that there is a potential involvement of ACE2 and TMPRSS2 expressions in the virus infection of extrapulmonary ans and the manifestation of symptoms related to these ans in patients with COVID19 Here we revealed the expressions of ACE2 and TMPRSS2 in extrapulmonary ans and we also summarized the clinical manifestation and the management of extrapulmonary complications in patients with COVID19 Introduction Since the late a novel coronavirus officially named as severe acute respiratory syndrome coronavirus SARSCoV2 was identified as the pathogen to cause pneumonia [] As a member of the Betacoronavirus genus SARSCoV2 has genomic nucleotides identity with human severe acute respiratory syndrome coronavirus SARSCoV and shares amino acid sequence identity with SARSCoV [] The World Health anization WHO named the disease caused by SARSCoV2 as coronavirus disease COVID19 Until April the virus has swept through countries more than million cases with COVID19 have been confirmed and more than cases died which has been posing significant threats to public health SARSCoV2 can cause respiratory diseases and may lead to acute respiratory distress syndrome ARDS multiple an failure and even death in severe cases [] In addition to typical symptoms such as cough and fever some patients developed the symptoms in multiple systems such as cardiovascular system digestive system and Abbreviations ACE2 Angiotensin converting enzyme AKI Acute kidney injury ALI Acute liver injury ALP Alkaline phosphatase ALS Artificial liver system ALT Alanine aminotransferase AMI Acute myocardial infarction ARDS Acute respiratory distress syndrome AST Aspartate aminotransferase AT2 Alveolar cells BUN Blood urea nitrogen CCLE Cancer Cell Line Encyclopedia CNS Central nervous system COVID19 Coronavirus disease GEO Gene Expression Omnibus GGT Gammaglutamyltransferase GI Gastrointestinal injury GTEx GenotypeTissue Expression ICU Intensive care unit MCS Mechanical circulatory support NP Nucleoprotein PCI Percutaneous coronary intervention SARSCoV2 Severe acute respiratory syndrome coronavirus Scr Serum creatinine ScRNASeq Single cell RNA sequencing STEMI STelevation myocardial infarction TBIL Total bilirubin TEM Transmission electronic microscope TMPRSS2 Transmembrane protease serine VV venousvenous WHO World Health anization Corresponding authors at Department of Infectious Disease and Institute of Hepatology Qingdao Municipal Hospital Qingdao University Digestive Disease Key Laboratory of Qingdao Qingdao China Email addresses xinyongning9812163com Y Xin zlk0823163com L Zhuang 101016jbiopha2020110678 Received June Received in revised form August Accepted August BiomedicinePharmacotherapy1312020110678Availableonline24August2020075333222020TheAuthorsPublishedbyElsevierMassonSASThisisan accessundertheCCBYNCNDlicensehttpcreativecommonslicensesbyncnd40 0cexpressed not only in the cells and tissues of lung but also in extrapulmonary ans [“] Fig In this section the expression levels of ACE2 and TMPRSS2 in extrapulmonary ans including heart kidney liver digestive tract brain and other ans were reviewed Heart M Dong nervous system in the early stages of COVID19 which brings more challenges to the timely diagnosis of patients [] Angiotensin converting enzyme ACE2 as a metalloproteinase is a carboxyterminal dipeptidyl peptidase [] The primary physiological role of ACE2 is involved in the regulation of vasoconstriction and blood pressure [“] Transmembrane protease serine type2 TMPRSS2 belonging to the type II transmembrane serine protease family could cleave the coronavirus spike S protein [“] It was demonstrated that ACE2 and TMPRSS2 were crucial for the entry of SARSCoV and SARSCoV2 into the host cells [] Cell entry of SARSCoV2 depends on binding of the S protein to the specific cellular receptor and S protein priming by host cell proteases As shown in Fig each S protein of SARSCoV2 consists of two subunits a globular S1 domain at the Nterminal region and the membraneproximal S2 domain SARSCoV2 utilizes receptorbinding domain within the S1 domain to bind to the cellular receptor ACE2 which could trigger the effects of TMPRSS2 on the cleavage of protein S at the S1 and S2 sites and priming cell membrane fusion for viral entry [] As receptors and mediators of virus entry are important for determining viral host and an the route of SARSCoV2 infection and the infected an may depend on the expression and distribution of ACE2 and TMPRSS2 [] Studies have shown that ACE2 and TMPRSS2 are expressed not only in lung tissues but also in extrapulmonary ans including heart kidney liver colon esophagus brain gallbladder and testis suggesting that SARSCoV2 may also affect extrapulmonary ans [“] In this review the distributions of ACE2 and TMPRSS2 in extrapulmonary ans and the characteristics and clinical managements of extrapulmonary an injury caused by SARSCoV2 were summarized We believe that this will be important in understanding on the infection of extrapulmonary ans in patients with COVID19 The mRNA expressions of ACE2 and TMPRSS2 in extrapulmonary ans The mRNA expressions of ACE2 in different human ans were analyzed and the results showed that ACE2 was expressed in the heart [] Furthermore Chen analyzed the feature of ACE2 expressions among cardiac cell types and found that ACE2 was specifically expressed in pericyte [] Moreover RNA sequencing from patients with failing hearts and normal donors revealed that myocardial ACE2 expressions were significantly increased in patients with heart failure which was further validated at the protein level by proteomics profiling from heart failure and normal donors [] Another study also showed that the expression of ACE2 in heart tissues of patients with underlying heart disease was higher than that in normal heart tissues [] These two studies suggested that the expression of ACE2 in heart tissue of patients with underlying heart disease was higher than that in normal heart tissue Guo et al analyzed the mRNA expression of TMPRSS2 from the GenotypeTissue Expression GTEx database and the results showed that TMPRSS2 is also expressed in the heart [] By singlecell RNA sequencing scRNASeq to profile the gene expression landscapes of cardiac cells from human embryos Qi revealed that the cardiomyocytes from the heart contain ACE2expressed cells and TMPRSS2expressed cells and the cardiovascular progenitor cells and cells TMPRSS2expressed cells respectively [] These data showed that both ACE2 and TMPRSS2 were expressed in the heart contain ACE2expressed Kidney Studying the viral susceptibility of extrapulmonary ans is important for a deeper understanding for the pathogenesis of SARSCoV infection Studies have shown that ACE2 and TMPRSS2 were Expression analysis from the GTEx database showed that kidney displayed the fifth high expression of ACE2 [] To investigate the expression of ACE2 in kidney Lin analyzed the public singlecell transcriptome dataset of normal kidneys from healthy donors the Fig Entry of SARSCoV2 into host cells SARSCoV2 infected the host cells by the spike protein of the virus and the functions of ACE2 and TMPRSS2 in host cells BiomedicinePharmacotherapy13120201106782 0cM Dong Fig Tissue distributions of ACE2 and TMPRSS2 in human A“B the schematic diagram of the expressions of ACE2 A and TMPRSS2 B in multiple human tissues The colour strength is corresponding to the gene expression level ACE2 and TMPRSS2 were expressed in the brain and heart ACE2 expression is expressed at a relative low level in hepatocytes and mainly located in cholangiocytes while TMPRSS2 is expressed in the hepatocytes and cholangiocytes ACE2 and TMPRSS2 were highly expressed in kidney and intestinal epithelial cells Both ACE2 and TMPRSS2 were also expressed in the esophagus stomach nose testis pancreas breast prostate and thyroid results showed that the ACE2 was distributed across multiple cell types and was mostly enriched in proximal tubule cells [] Fan et al confirmed the specific ACE2 expression in tubular cells from the Gene Expression Omnibus GEO dataset while it was not observed in immune cells and glomerular parietal epithelial cells RNA and protein expression data of ACE2 in different human tissues and cancer cell lines were obtained from three online datasets including the Cancer Cell Line Encyclopedia CCLE GTEx database and the Human Protein Atlas dataset and the results indicated that both mRNA and protein expression levels of ACE2 were relatively high in kidney cells especially in renal tubular cells [] Meanwhile Suryawanshi analyzed the data of kidney tissues in scRNASeq datasets and found that either proximal tubular cells or tubular progenitor cells in the kidney coexpressed ACE2 and TMPRSS2 [] The data of the scRNAseq from GEO dataset GSE134355 showed that ACE2 and TMPRSS2 expression levels were high in nephron epithelial cells epithelial cells endothelial cells and mesangial cells of the kidney [] Recently Pan also found that the TMPRSS2 gene was coexpressed with ACE2 in kidney podocytes [] These data showed that both ACE2 and TMPRSS2 were highly expressed in tissues and cells of kidney Liver Chai et al analyzed the scRNAseq data from GEO database GSE124395 to evaluate ACE2 gene expression in liver the results showed that ACE2 was highly expressed in cholangiocytes which level was about times higher than that in hepatocytes [] The GTEx database also showed that both ACE2 and TMPRSS2 were expressed in the liver [] Zhou identified that TMPRSS2 is highly expressed in hepatocytes from Human Cell Atlas database [] Recently Wen indicated that ACE2 and TMPRSS2 are specifically coexpression in TROP2 liver progenitors of human liver tissue using scRNA sequencing [] These data indicate that ACE2 expression is expressed at a relative low level in hepatocytes and mainly located in cholangiocytes while TMPRSS2 is expressed in hepatocytes Digestive tract A previous study showed that ACE2 could be found in the upper esophagus and it could be detected in stratified epithelial cells and absorptive enterocytes of the ileum and colon [] Quantitative mRNA expression profiling of ACE2 across human tissues by Harmer showed that ACE2 was expressed at a high level in gastrointestinal tissues [] Zhang et al analyzed datasets with singlecell transcriptomes of esophagus gastric ileum colon and lung and the data showed that ACE2 was not only highly expressed in the type II alveolar cells AT2 of lung but also in the stratified epithelial cells ileum absorptive enterocytes cells and colon enterocytes [] Similarly the immunofluorescent staining of esophagus stomach duodenum and rectum showed that ACE2 was stained mainly in the cytoplasm of gastrointestinal epithelial cells [] Besides the scRNAseq data showed that ACE2 was significantly elevated in the proximal and distal enterocytes [] Guo et al suggested that TMPRSS2 was highly expressed in almost all ans of the digestive tract including colon stomach small intestine and esophagus [] Using published scRNAseq data and seven inhouse normal colon samples Lee reported that the coexpressions of ACE2 and TMPRSS2 transcripts were mainly observed in the small intestine and colon [] The highest expressions of TMPRSS2 and ACE2 were found in enterocytes among the intestinal cell types []These data showed that TMPRSS2 and AEC2 are highly expressed in the digestive tract Nervous system Analysis using the GTEx database showed that both TMPRSS2 and ACE2 are expressed at relatively low levels in the brain cortex [] Chen found that ACE2 was relatively highly expressed in some important brain areas such as the substantia nigra and brain ventricles using seven brain transcriptome databases [] ACE2 was expressed at high level in the piriform cortex of human brain and its expression could also be detected in many neurons including both excitatory and inhibitory neurons and some nonneuron cells including astrocytes and oligodendrocytes in human middle temporal gyrus and posterior cingulate cortex [] Qi analyzed the scRNAseq data of substantia nigra BiomedicinePharmacotherapy13120201106783 0cClinical classification of acute cardiac injury NonICUcases ICU cases NonICUcases ICUcases Nonsevere cases Severecases1965 Recoveredcases Died cases NonICUcases ICUcases Survivor cases Nonsurvivor cases Chen [] Hong [] Zhou [] China Korea China M Dong and cortex of brain from GEO database the results showed that both ACE2 and TMPRSS2 were expressed in the oligodendrocyte precursor cells and the astrocytes of the substantia nigra and cortex [] There are limited reports on the expressions of ACE2 and TMPRSS2 in peripheral nervous system Brann analyzed the ACE2 and TMPRSS2 expression in different cell type from human scRNAseq dataset GSE139522 and found that neither olfactory sensory neurons nor olfactory bulb neurons expressed these two genes while ACE2 and TMPRSS2 were expressed in the nonneuronal cells including the sustentacular cells and olfactory bulb pericytes [] These data showed that ACE2 and TMPRSS2 could also be coexpressed in the nervous system Other ans or tissues Table Characteristics of acute cardiac injury after SARScid0 CoV2 infection Study Basic heart disease Acute cardiac injury Country Subject China Wang [] China NA Huang [] Li [] China Moreover ACE2 and TMPRSS2 were also reported to be coexpressed in some other ans [] It has been revealed that both ACE2 and TMPRSS2 are expressed in testis by scRNA sequencing and expression profile analysis indicating that testicular cells might be the potential targets of SARSCoV2 Another report revealed that multiple kinds of cells in the nose including nasal brushing epithelial cells nasal turbinate epithelial cells and nasal airway epithelial cells contained ACE2expressed and TMPRSS2expressed cell clusters [] Moreover ACE2 and TMPRSS2 were also expressed in pancreas breast prostate and thyroid [] and these ans might also be the targets of SARSCov2 Infection of SARSCoV2 and extrapulmonary an injury of patients with COVID19 SARSCoV2 infection and cardiac injury Recently autopsy analysis by Fox revealed that the histopathology of the heart was consistent with the typical pattern of viral myocarditis [] SARSCoV2 RNA was detected in the cardiac tissues of the patients with COVID19 [] These data suggested that SARSCoV2 may directly infect heart The epidemiology of COVID19 reported that cardiac injury was one of the most severe an damages [] The clinical manifestations of cardiac injury in COVID19 patients are complex and could present with heart failure arrhythmias or acute myocardial infarction AMI [ ] Inciardi reported the first case who had the symptom of heart failure at first and later the patient was positive for SARSCoV2 using nucleic acid test [] Cardiac injury is a common symptom in patients with COVID19 Shi reported that patients with COVID19 had cardiac injury [] Moreover there were patients with acute cardiac injury in a cohort including COVID19 patients and of patients with acute cardiac injury in the intensive care unit ICU [] Furthermore a study by Wang showed that there were patients with acute cardiac injury and patients presented with arrhythmia of COVID19 patients while acute cardiac injury was observed in of patients with CIVID19 in the ICU [] These cases suggested that SARSCoV2 may cause serious heart damage which should be widespreadly concerned Furthermore acute cardiac injury is more prevalent in severe cases with COVID19 [“] Table And it has been reported that COVID19 patients with cardiac injury had higher mortality than those without cardiac injury [] In this review we also summarized the possible relationship between basic heart disease and further cardiac injury [“] Table In a cohort of COVID19 patients from Renmin Hospital of Wuhan University China Shi demonstrated that cardiac injury occurred in patients during hospitalization of which had basic heart disease including coronary heart disease and chronic heart failure And only patients with basic heart disease of COVID19 patients without cardiac injury [] Similarly Liu suggested that patients with basic heart disease in COVID19 patients had Table Comorbidity with cardiac injury in COVID19 patients with basic heart disease Subjects with COVID19 Proportion of basic heart disease Patients with Cardiacinjury Study Shi [] Liu [] Xu [] Ma [] Guo [] Patients with basic heart disease With cardiac injury Without cardiac injury cardiac injury compared with patients with basic cardiovascular diseases of COVID19 patients without cardiac injury [] Other studies also indicated that the patients with basic cardiovascular disease are more likely to present heat injury in COVID19 patients [ ] In view of the points above COVID19 patients with underlying cardiac conditions seem to have higher rates of cardiac injury SARSCoV2 infection and kidney injury Recently autopsy analysis on six COVID19 patients showed that varying degrees of acute tubular necrosis were observed in all the renal specimens Nucleoprotein NP antigens and NP positive inclusion body of SARSCoV2 could be seen in kidney tissues from all the samples Moreover viruslike ps were seen in kidney tissues by transmission electronic microscope TEM [] Su analyzed kidney abnormalities in autopsies of patients with COVID19 and found that diffuse proximal tubular damage with the loss of brush border were BiomedicinePharmacotherapy13120201106784 0c SARSCoV2 infection and liver injury M Dong observed Further investigation showed that diffuse necrosis can be seen under the light microscope and electron microscopic examination also showed the clusters of coronavirus ps with distinctive spikes in the tubular epithelium and podocytes [] It was reported that both NP antigens and RNA of SARSCoV2 were detected in urine of COVID19 patients [] These data coincide with the finding of the SARSCoV2 invasion in kidney Collectively SARSCoV2 could directly infect human renal tubules and lead to kidney damage Recent studies have shown that the incidence of acute kidney injury AKI in COVID19 patients ranged from “ and higher frequency of renal function damage with elevated blood urea nitrogen BUN or serum creatinine Scr was observed in COVID19 patients [ ““] Table A study of patients with COVID19 indicated that levels of BUN and Scr were increased in and patients with COVID19 respectively And routine urine tests were performed on patients among which patients were positive for urinary protein and patients were positive for hematuria [] Another study also showed that about patients with COVID19 had abnormal renal function [] Moreover COVID19 patients with more severe disease progression have higher rates of AKI Huang and colleagues reported that of patients with AKI in the ICU were observed and none of the patients who did not require care in the ICU suffered AKI [] Xu found that the fatality rate was obviously higher in COVID19 patients with AKI than those without renal injury [] Furthermore in another study investigating patients with COVID19 at hospital admission more severe patients had higher rates of AKI and the Cox regression analysis also suggested that COVID19 patients who developed AKI had a significantly higher mortality risk [] Therefore AKI is more prevalent in severe cases with COVID19 An autopsy report of a 50yearold patient with COVID19 showed moderate microvesicular steatosis and mild lobular activity in liver tissues [] Moreover Zhao used human liver ductal anoids as a tool to investigate the SARSCoV2 infection and the tissue damage induced by SARSCoV2 ex vivo and the results showed that the expression of SARSCoV2 NP was easily detected in the patchy areas of the hepatic duct indicating that liver ductal anoids were susceptible to SARSCoV2 infection [] In addition SARSCoV2 infection could disrupt the barrier and bile acid transporting functions of cholangiocytes which indicated that SARSCoV2 might directly induce cholangiocyte injury and consequently bile acid accumulation [] In view of the points above liver damage in the COVID19 patients might be directly caused by the viral infection Abnormal liver functions were frequently reported in COVID19 patients [] Epidemiologic studies showed that almost half of the patients had differing degrees of liver damage [“ “] Table Chen reported that out of patients had elevated alanine aminotransferase ALT patients had elevated aspartate aminotransferase AST and had elevated total bilirubin TBIL in Wuhan Jinyintan Hospital Wuhan China [] Similarly a nationwide study involving patients with COVID19 in China showed that more than of patients had elevated ALT and AST and of patients had elevated TBIL [] It was revealed that the levels of direct bilirubin indirect bilirubin ALT alkaline phosphatase ALP and gammaglutamyltransferase GGT were significantly higher in males than that in females with COVID19 and multivariate logistic regression analysis showed that male was an important independent risk factor for predicting acute liver injury ALI in COVID19 patients [] These data indicated that male patients with COVID19 may be more susceptible to liver injury Furthermore Table Characteristics of acute kidney injury after SARScid0 CoV2 infection Study Chen Wang Huang Guan Xu Preexisting kidney conditions NA NA NA [] [] [] [] [] Country China China China China China Subject Li [] Chen [] Hong [] Cheng [] Xiao [] Richardson [] Wan Li Qian Pei [] [] [] [] China China Korea China China America China China China China NA NA NA NA NA NA Scr Serum creatinine BUN Blood urea nitrogen AKI Acute kidney injury Abnormal renal functional indices Scr BUN NA Scr Scr Scr Scr BUN NA Scr BUN Scr NA NA NA Scr BUN Scr NA AKI NA Clinical classification of AKI NA NonICU cases ICU cases ICU cases Nonsevere cases Severe cases Mild cases Severe cases Critical ill cases Nonsevere cases Severe cases Recovered cases Died cases NonICU cases ICU cases NA Nonsevere cases Severe cases Cured cases In hospital cases Died cases Mild cases Severe cases Nonsevere cases Severe cases NA Moderate cases Severe cases Critically ill cases BiomedicinePharmacotherapy13120201106785 0cM Dong Table Characteristics of liver injury after SARScid0 CoV2 infection Study Country Subject China China China China China China China Korea America China China Chen [] Wang Huang Guan [] [] [] Xu Li [] [] Chen Hong [] [] Richardson et Wan Li al [] [] [] Qian [] China NA Patients with preexisting liverconditions NA NA NA Patients with abnormal liver functional indices ALT AST TBIL NA AST AST ALT TBIL ALT AST ALT AST TBIL ALT AST ALT AST TBIL AST ALT AST ALT AST TBIL ALT AST Abnormal liver functional indices in the Nonsevere patients AST ALT NA NA TBIL NA Abnormal liver functional indices in the severe patients ALT NA TBIL NA AST NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Nonsevere patients include patients without ICU care and recovered patients Severe patients include patients with ICU care and death ALT alanine aminotransferase AST aspartate aminotransferase TBIL total bilirubin multiple studies found that AST ALT and TBIL were significantly higher in patients treated in the ICU than that in nonICU patients [] Li suggested that among the patients with abnormal liver function moderate and severe types of patients were more likely to have liver injury and respectively [] Fu analyzed the relationship between ALI and mortality risk in COVID19 patients and the results showed that ALI is more common in the critically ill patients and ALI at the early stage increased death risk of COVID19 patients [] Together abnormal liver functions might be associated with the severity of patients with COVID19 SARSCoV2 infection and digestive tract injury Epithelial cells of the esophagus stomach duodenum and rectum in one COVID19 patient tested positive for SARSCoV2 RNA and the staining of viral NP was also visualized in the cytoplasm of epithelial cells in stomach duodenum and rectum [] Moreover minimally invasive autopsies were performed on three patients died of COVID19 and the results showed that some epithelial cells of the gastrointestinal mucosa were degenerated necrotic and detached [] These studies strongly supported that SARSCoV2 may directly infect the epithelial cells of digestive tract Table SARScid0 CoV2 detection in gastrointestinal specimens Study Subject [] [] Xiao Zhang Tan [] Xing Young Holshue Lescure Tang Wang Xu [] [] [] [] [] [] [] Gastrointestinal samples Stool Anal swabs Rectal swab Stool Stool Stool Stool Stool Stool Rectal swabs Tested positive in gastrointestinal specimens The positive time in gastrointestinal specimens days NA 6cid0 1cid0 5cid0 NA 3cid0 Positive time for Gastrointestinal samples after respiratory samples were negative days NA NA 8cid0 NA NA NA NA NA 2cid0 BiomedicinePharmacotherapy13120201106786 0cM Dong Multiple studies have identified that the SARSCoV2 RNA was detected in anal swabs [] rectal swabs [] and stool specimens [ ] of COVID19 patients It has been demonstrated that SARSCoV2 RNA could be detected in feces from more than half of COVID19 patients [] In another study Xing reported that SARSCoV2 RNA was detected in the feces of three pediatric cases with COVID19 in Qingdao China and the persistence of SARSCoV2 in the digestive tract lasted for 6cid0 days [] The possibility of fecaloral transmission of SARSCoV2 infection needs to be taken into account Furthermore as shown in Table long duration of SARSCoV2 detection in digestive tract by RTPCR has been reported and viral RNA remained detectable in the digestive tract for 2cid0 days after nucleic acid turned negative in respiratory samples [“] The studies suggested that SARSCoV2 could be detected from respiratory tract specimens during the early period to digestive tract specimens during the late period and viral nucleic acid tests in both the respiratory and digestive tract are necessary to confirm the complete clearance of virus Some COVID19 patients presented gastrointestinal symptoms such as diarrhea nausea vomiting and abdominal pain [] Holshue reported the first case of COVID19 patient in the USA which had nausea and vomiting before admission [] Multiple studies found that gastrointestinal symptoms including diarrhea “ nausea and vomiting “ and abdominal pain “ were common at presentation in COVID19 patients [ ““] Table In a cohort of patients with COVID19 in Wuhan China gastrointestinal symptoms were described in up to [] Moreover Sun showed that critically ill patients with COVID19 had gastrointestinal injury GI during hospital stay and the survival curves showed that the mortalities of patients with GI was greater than that of patients without GI [] Jin also found that the rate of the severe type was markedly higher in COVID19 patients with GI symptoms than that in those without GI symptoms [] These data suggested that GI is one of the common extrapulmonary an injuries in COVID19 patients and may be related to the severity of the disease On the other hand many studies showed that patients with COVID19 could present initially with the typical gastrointestinal sympto
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