| Background:Colorectal cancer is one of the most frequently diagnosed cancers worldwide.Recent years,colorectal cancer has been increasing rapidly in China,with the change of lifestyle and aging of population.It has been estimated that 376300 colorectal cancer patients were diagnosed,and 19100 colorectal cancer related deaths were reported in China in 2015.Surgery,radiotherapy,chemotherapy and targeted therapy are the current major therapeutic approaches for colorectal cancer.Although significant progressions have been achieved,there still lacks effective approaches to treat advanced and metastatic diseases.Therefore,it is urgent to develop a novel therapeutic strategy to treat colorectal cancer efficiently.Gene therapy can target to many critical molecules,which promote tumor growth and metastasis at different phases of tumor development and progression.Therefore,gene therapy has emerged as a potential approach for cancer therapy.Oncolytic adenovirus,named as conditionally replicating adenoviruses(CRADs),can selectively replicate in tumor cells and produce cytotoxicity to tumor cells eventually.Lysing of tumor cells,can release viruses to infect tumor cells nearby.Moreover,tumor antigens could be released from host cells to activate local and systematic anti-tumor immunities.Furthermore,oncolytic adenovirus could improve the expression of therapeutic genes.Therefore,combining oncolytic adenoviruses and therapeutic genes might be an effective approach for cancer therapy.Decorin,a prototypic member of the small leucine-rich proteoglycans(SLRPs),is an important regulator of various cellular functions,including adhesion,migration,proliferation,apoptosis,autophagy,differentiation,inflammation and tumorogenesis.Studies have reported that decorin was down-regulated in tumor tissues of various caners.Granulocyte macrophage colony stimulating factor(GM-CSF)can promote the differentiation and maturation of macrophage and dendritic cells(DCs).Researches from clinical researches suggested that GM-CSF is a promising cytokine to therapy cancers.Therefore,we developed an oncolytic adenovirus encoding decorin and GM-CSF,rAd.DCN.GM,and evaluated its anti-tumor effects.And,the mechanisms,including immune activation,were also investigated.Methods:Firstly,tumor samples were obtained from 59 colorectal cancer patients.And,tumor tissues,para-cancerous tissues and distal normal tissues were stored separately according to the results of pathological examination.Patients' data and follow-up data were obtained.The stored tumor tissues and normal tissues were used for RNA isolation.After cDNA was synthesized,the expression of decorin was detected by real-time reverse transcript polymerase chain reaction(RT-PCR)and immunohistochemistry.The target genes of decorin,including Met and VEGFA,were detected by real-time RT-PCR.Secondly,we constructed a replication-defective recombinant adenovirus plasmid encoding decorin,pAd.DCN,by using Adeasy system.Then,the plasmids was cut by PacI and transfected into HEK293 cells to package recombinant adenovirus,Ad.DCN.Then,all of the adenoviruses,including replication-defective adenoviruses(Ad.Null and Ad.DCN),and oncolytic adenoviruses(rAd.Null,rAd.DCN,rAd.GM and rAd.DCN.GM),were growing in HEK293 cells.The adenoviruses were collected and purified by using chlorinated cesium(CsCl)density gradient centrifugation.Finally,the titers and purity were determined on UV spectrophotometry.Thirdly,the characteristics and activities of oncolytic adenoviruses were analyzed in colon cancer cell in vitro.Colon cell lines,SW480,SW620 and CT26.WT were infected by replication-defective adenoviruses and oncolytic adenoviruses.Then,the replication ability was evaluated by amplification of plaques.The expression of decorin and GM-CSF protein in culture media of tumor cells was examined by using ELISA.The cytotoxicity was detected by using sulforhodamine B(SRB)staining.Peripheral blood was collected from colorectal cancer patients,and the mononuclear cells(PBMCs)were isolated.The lysates from oncolytic adenoviruses infected SW480 cells were added to cultured PBMCs for stimulation.The proliferation and cytokines expression in PBMCs were detected by flow cytometry and real-time RT-PCR,respectively.Finally,murine colorectal cancer CT26.WT xenograft models were established in immune competent Balb/c mice.Then,oncolytic adenoviruses were delivered by intratumoral administration.The health status was observed and tumor growth was monitored twice a week after therapy.The adenovirus induced injuries in liver and spleen,and tumor metastasis to lung,were evaluated by pathological examination.The immune phenotypes of immune cells in spleen and peripheral blood were analyzed by flow cytometry.In addition,expression of decorin and its target genes,cytokines and chemokines were detected by real-time RT-PCR in spleen and tumor lesions.Results:In colorectal cancer patients,decorin mRNA expression in tumor tissues was significantly down-regulated,compare to that in normal tissues.And,similar results of decorin protein expression was observed.Moreover,the down-regulation of decorin is associated with tumor recurrence and metastasis.We also found that reduced decorin expression up-regulated its target genes,Met and VEGFA which has been reported to play an important role in tumor recurrence and metastasis.Our lab previously has successfully developed an oncolytic adenovirus encoding decorin and GM-CSF.In this study,we successfully constructed oncolytic adenovirus encoding decorin and GM-CSF,rAd.GM.CSF,and a replication-defective adenovirus expressing decorin,Ad.DCN,by using Adeasy system.Then,adenoviruses,Ad.Null,Ad.DCN,rAd.Null,rAd.DCN,rAd.GM and rAd.DCN.GM were propagated and purified at high quality,which can be used to evaluate its features in vitro and in vivo.In vitro,we evaluated the oncolytic ability,expression of theraputic genes.We showed that:(1)rAd.DCN.GM,rAd.DCN and Ad.DCN could express decorin protein,while rAd.DCN.GM and rAd.GM could express GM-CSF protein efficiently;(2)rAd.DCN.GM replicated in human colon cell lines,SW480 and SW620,but not in mouse colon cell line,CT26.WT;(3)rAd.DCN.GM produced cytotoxicity in both human and mouse colon cancer cells.In addition,Ad.DCN infection down-regulated the expression of decorin target genes,including c-Met,CTNNB1,VEGFA and TGF-?,and also inhibited the cell migration in SW480 cells.Moreover,conditioned media from rAd.DCN.GM infected SW480 cells stimulated the expression of cytokines(INF-?,perforin,granzyme B,IL-12)in PBMCs.In immune competent murine colon cancer CT26.WT xenograft models,oncolytic adenoviruses were delivered to evaluate its anti-tumor effects and anti-tumor immune responses.Our results showed:(1)Oncolytic adenovirus inhibits the tumor growth obviously,while the oncolytic adenovirus encoding therapeutic genes had much stronger inhibition;(2)Oncolytic adenovirus induced cell apoptosis and death,inhibited cell proliferation in the tumor lesions,prevented lung metastasis,with few toxicity in liver and spleen;(3)rAd.DCN.GM expressed decorin and GM-CSF in tumor lesions,which in turn up-regulated E-cadherin,perforin and granzyme B,and down-regulated VEGFA,N-cadherin and Vimentin;(4)rAd.DCN.GM up-regulated the percentage of CD8+ T lymphocytes in peripheral blood,promoted differentiation and maturation of DCs in spleen;rAd.DCN.GM also inhibited the expression of IL-6,TNF-? and TGF-?,and promoted INF-? expression in spleen.Conclusion:Decorin is significantly reduced in colorectal cancer patients,and down-regulation of decorin is closely associated with tumor recurrence and metastasis.Therefore,we developed an oncolytic adenovirus armed with therapeutic genes,decorin and GM-CSF,r Ad.DCN.GM.In vitro,rAd.DCN.GM effectively replicated and produced cytotoxicity in colon cancer cells;produced high level of decorin and GM-CSF protein.In vivo,rAd.DCN.GM inhibited the tumor growth obviously in immune competent murine colon cancer xenograft,and activated the anti-tumor immune responses.Therefore,rAd.DCN.GM might be an effective therapeutic approach to treat advanced and metastatic colon cancer. |
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