Study On The Mechanism Of Intestinal Flora And Oncolytic Virus Vaccine In The Treatment Of Colorectal Cancer

Research background and purposeColorectal cancer(CRC)is one of the most common malignant tumors in the world,ranking the third in tumor incidence and the second in cancer-related causes of death.More and more evidence shows that intestinal flora plays an important role in the occurrence and development of CRC.In recent years,immunotherapy has become one of the important therapies for some cancers,including colorectal cancer,as emerging therapies such as targeted therapy and immunotherapy have achieved results in many solid tumors.As an important branch of tumor immunotherapy,oncolytic virus has shown its potential and clinical application prospect in the treatment of solid tumors.Studies have shown that changes in intestinal flora structure can affect the efficacy of tumor immunotherapy.The purpose of this study was to analyze the difference of efficacy of OVV-gp33 in the treatment of early-stage and late-stage mouse CRC tumor models,and analyze the characteristics of intestinal microflora of mice before the treatment of early-stage and late-stage mouse tumor models,and influence the microflora of mice through antibiotic cleaning,oral probiotics and fecal coccus transplantation.Based on 16 S r DNA and flow cytometry analysis,To investigate the possible role of intestinal flora in the treatment of mice tumor with OVV-gp33,and to provide a partial basis for further promoting the clinical application of intestinal flora combined with oncolytic virus vaccine.Materials and MethodsMC38 cell lines with stable gp33 expression were obtained by lentivirus transfection combined with purinomycin screening.MC38-gp33 cells were then inoculated intradermal to construct a mouse tumor model,which was defined as an early CRC model group on day 5 of tumor inoculation and a late CRC model group on day 17 of tumor inoculation.Based on the late CRC model,they were further divided into the following three groups: late CRC combined with bifidobacterium and Lactobacillus triple viable tablet group,late CRC combined with FMT group and late CRC antibiotic scavenging bacteria group.Then,the tumor-bearing mice in each group were injected with OVV-GP33(an OVV targeting MC38 neoantigen gp33 of colorectal cancer cells,and named OVV-GP33)through the tail vein.Tumor growth and survival curves of mice were recorded and plotted.Stool of mice was collected,and intestinal flora characteristics of mice in different intervention groups were evaluated by 16 S r DNA sequencing.At the same time,flow cytometry was used to detect the activation of specific CD8+T cells and Treg cells in peripheral blood of mice to evaluate the immune status of mice.To explore the possible interaction mechanism between intestinal flora and OVV-gp33 in the treatment of colorectal cancer.Result1.OVV-gp33 can effectively inhibit tumor growth in early CRC model and prolong the survival time of tumor-bearing mice.However,the advanced CRC model could not achieve the therapeutic purpose.2.Through the analysis of microflora in early and late CRC model mice,we found that with the development of CRC,the abundance and diversity of mice intestinal microflora decreased significantly,and its composition changed significantly.The results showed that the number of Bacteroidetes,verrucobacteria and Proteobacteria increased and the number of Firmicutes decreased,and the ratio of firmicutes to Bacteroidetes(F/B)decreased.3.Improving intestinal flora structure can delay tumor growth and enhance the therapeutic effect of OVV-gp33.Compared with the advanced CRC blank control group,the tumor growth rate of tumor-bearing mice in the combined bifidobacterium Lactobacillus triple viable tablet group and the combined FMT group was slowed down,and the survival time was prolonged after OVV-gp33 treatment,and the mice in the combined FMT model group showed more significant performance.On the contrary,the tumor growth was accelerated and the survival time was shorter after OVV-gp33 treatment in the combined antibiotic cleaning group.4.Through the 16 S r DNA sequencing analysis of the stool of mice in the combined probiotics group and the combined FMT group,it was found that the abundance and diversity of intestinal microorganisms in mice with advanced CRC decreased after the combined probiotics and the combined FMT group,and even lower in the combined FMT group.The results showed that Bacteroidetes and Proteobacteria decreased,Firmicutes and verrucobacteria increased significantly.After antibiotic cleaning,the abundance and diversity of late CRC mice were significantly reduced,including Bacteroides and Firmicutes.Proteobacteria increased significantly.5.KEGG metabolic pathway analysis showed that there were common enrichment pathways in the early CRC model group,the combined probiotics group and the combined FMT group,which were mainly glycolysis/gluconeogenesis,pentose phosphate pathway and lysine biosynthesis pathway.It may be related to T cell differentiation and activation.6.Flow cytometry showed that with the progression of CRC,the activation ratio of specific CD8+T cells decreased,the proportion of Treg cells increased,and the immune response function decreased.After the combination of probiotics and FMT,the activation ratio of specific CD8+T cells in mice was increased and the proportion of Treg cells was decreased after OVV-gp33 treatment,and the immune response function was enhanced,and the combination of FMT was stronger.After further antibiotic cleaning,OVV-gp33 treatment significantly reduced the activation ratio of specific CD8+T cells,increased the proportion of Treg cells,and further decreased the immune response function.ConclusionBased on this study,we reached the following conclusions:1.The tumor formation time of CRC affects the therapeutic effect of OVV-gp33.2.Improving the structure of intestinal flora can enhance the immunotherapeutic effect of OVV-gp33,and the immunotherapeutic effect of OVV-gp33 depends on the presence of intestinal flora.3.Changes in the structure of intestinal flora may affect the differentiation and activation of T cells through metabolic pathways such as glycolysis/gluconeogenesis,pentose phosphate pathway,and lysine biosynthesis pathway.4.Changes in intestinal flora affect the therapeutic efficacy of OVV-gp33 by affecting specific CD8+T cells and the activation ratio of Treg.In conclusion,there is a synergistic effect between intestinal flora and oncolytic virus vaccine in the treatment of advanced colorectal cancer.