Experimental Therapy To Colorectal Cancer Mouse Model With IL-7 Modified Vaccinia Virus

The immunosuppressors in tumor microenvironment lead to the most prominent antitumor cells,effector T cells,decreasion and dysfunction in tumor tissues.How to reverse the immunosuppressive tumor microenvironment,induce and enhance the antitumor immune response is an urgent problem to be solved.Vaccinia virus is an oncolytic virus that can infect tumor cells,promote immunogenic cell death which leads to stimulate antitumor immune response.The cytokine interleukin-7 can enhance the antitumor immune responses through multiple pathways,such as differentiation and survival of naive T cells,function of effector T cells,and generation and maintenance of memory T cells,directly abrogation the Treg-mediated suppression of effector T cells proliferation.IL-7 modified vaccinia virus may induce and enhance the antitumor immune response,promote effector T cells infiltration and cytotoxicity.In this Opinion,the study wants to modify the vaccinia virus with Mus murine Il7 coding sequences to construct IL-7 recombinant vaccinia virus.We investigate the therapeutic efficacy of IL-7 recombinant vaccinia virus in coloretcal cancer mouse model,and expect to provide a new method of cancer immunotherapy.Part ? Construct a mouse colorectal cancer gene transfected cell line stably expressing firefly luciferase and establish a tumor-bearing mouse model by use of itObjective:To construct a mouse colorectal cancer gene transfected cell line stably expressing firefly luciferase,and to establish a tumor-bearing mouse model by use of the gene transfected cell line.Methods:A gene transfected cell line screened by puromycin was constructed with MC38 cells and pLVX-Puro-Luc2P expressing firefly luciferase under the help of lentiviral vectors by liposome transfection.The expression of firefly luciferase in transfectd cells was detected on gene level and protein level by RT-PCR and chemiluminescence,respectively.The transfected cell line tumorigenicity was detected by used the cell line to establish a tumor-bearing mouse model.Meanwhile the tumor formation curve was generated,in addition,the bioluminescence intensity of transfected cell line was detected by in-vivo imaging.Results:It was confirmed that transfected cell line contained firefly luciferase mRNA by RT-PCR.It was confirmed that transfected cell line contained active firefly luciferase by chemiluminescence.Tumor formation rate of transfected cell line was one hundred percent,at the same time the bioluminescence of transfected cell line could be detected by in-vivo imaging.So it was confirmed that transfected cell line tumor-bearing mouse model was successfully constructed,named the cell line as MC38-Luc.Conclusion:A gene transfected cell line stably expressing firefly luciferase was successfully constructed and named as MC38-Luc.A tumor-bearing mouse model was successfully established by use of MC38-Luc.Part ? Study of the therapeutic efficacy of IL-7 recombinant vaccinia virus in colorectal cancer mouse modelObjective:To study the therapeutic efficacy of IL-7 recombinant vaccinia virus in the use of colorectal cancer mouse model.Methods:IL-7 recombinant plasmid was constructed with Mus musculus Il7 coding sequences and vector plasmid pCMS1-IRES.IL-7 recombinant vaccinia virus was constructed with homologous recombination of IL-7 recombinant plasmid into vaccinia viral genome,then the virus was amplified and purified.The expression of IL-7 in IL-7 recombinant vaccinia virus was detected on gene level and protein level by RT-PCR and ELISA,respectively.The replication and cytotoxicity of IL-7 recombinant vaccinia virus were detected.The tumor-bearing mice in colorectal cancer were treated with IL-7 recombinant vaccinia virus,and the tumor growth curve and survival curve were generated.Rechallenge of tumor-free mice with MC38-Luc cells and B16-F10 cells,and the tumor growth of these mice was observed.Results:IL-7 recombinant vaccinia virus was successfully constructed.The virus could inhibit tumor growth and prolong the survival of tumor-bearing mice,moreover,promote a specific and durable memory response upon tumor rechallenge.Conclusion:IL-7 recombinant vaccinia virus could treat colorectal cancer mouse model effectively.