The Effect Of Changweiqing On Enhancing PD-1 Inhibitor Against Colon Cancer By Improving Immune Microenvironment

Objective:To investigate the mechanism of the disease tumor immune microenvironment is an important factor affecting the efficacy of immunotherapy.In this study,the effects of Changweiqing combined with PD-1 monoclonal antibody on tumor growth,tumor infiltrating lymphocytes in tumor microenvironment,intestinal mucosal barrier and gut microbiota in mice with subcutaneous transplanted colorectal cancer were detected to explore the specific mechanism of Changweiqing increasing the anti-tumor effect of PD-1 monoclonal antibody.Method:(1)The expression of CD8~+T cells,CD4~+T cells,PD-1 protein and PD-L1 protein in tumor tissues of patients with microsatellite stable and microsatellite unstable colorectal cancer were detected by immunofluorescence.(2)C57BL/6 mice were randomly divided into Control group,Changweiqing group,PD-1 group and Changweiqing combined with PD-1 group after tumor formation.The corresponding drugs were given respectively((1)Control group was given drinking water once a day,0.2ml each time;(2)CWQ group was given Changweiqing solution of2.574g/ml once a day,0.2ml each time;(3)mice in PD-1 m Ab group were given0.1mg/ml of PD-1 by tail vein injection on the 5th,10th and 15th day after administration,0.2ml each time;(4)mice in CWQ+PD-1 group were given 2.574g/ml of Changweiqing solution by gavage,0.2ml each time,and 0.1mg/ml of PD-1 by tail vein injection on the 5th,10th and 15th day after administration,0.2ml each time).The tumor size was measured every day.At the end of administration,the mice were anesthetized and the tumor bodies were stripped,and the tumor weight of each group was measured.The anti-tumor effects of Changweiqing and PD-1 monoclonal antibody were evaluated according to the tumor volume and tumor weight of each group.(3)The contents of CD3~+T cells,CD4~+T cells,CD8~+T cells and PD-1~+CD8~+T cells in tumor tissues were detected by flow cytometry,and the expression of PD-L1 protein in tumor tissues was detected by Western blot.(4)At the end of the experiment,the mice were anesthetized and killed.A section of colon was taken from each mouse,fixed with paraformaldehyde and embedded in sections.The morphological changes of intestinal tissues in each group were detected by HE staining,and the expressions of tight junction proteins claudin,occludin and ZO-1 in each group were detected by immunohistochemistry The effect of the drug on intestinal mucosal barrier in mice.(5)Before the end of the experiment,the feces of mice in each group were collected with sterile tweezers and sterilized cryopreservation tubes.The data of gut microbiota of mice in each group were detected by 16S r RNA macro gene sequencing method.The composition of gut microbiota and different gut microbiota of mice in each group were analyzed by the relevant modules of Meggie cloud platform.At the same time,the relationship between gut microbiota and tumor immune microenvironment was analyzed by RDA/CCA method.Result:(1)The levels of CD8~+T cells,CD4~+T cells,PD-1 protein and PD-L1 protein in patients with microsatellite stable colorectal cancer were significantly lower than those in patients with microsatellite unstable colorectal cancer(all P<0.05).(2)Compared with the Control group,the tumor weight and volume of CWQ group and PD-1 group were decreased,but there was no significant difference(P>0.05).CWQ combined with PD-1 group could significantly reduce the tumor weight(P<0.01)and volume(P<0.01).(3)CWQ and combination group could significantly increase the infiltration of CD3~+T and CD8~+T cells in immune microenvironment(P<0.01),and significantly increase the expression of PD-1 on CD8~+T cells(P<0.05),but had no obvious effect on CD4~+T cells.The combination group could significantly up regulate the expression of PD-L1protein in subcutaneous transplanted tumor tissue,while CWQ and PD-1 had no significant effect on the expression of PD-L1 protein.(4)The results of HE staining showed that PD-1 could induce the sparse arrangement of villi,the rupture and atrophy of villi,the infiltration of inflammatory cells,interstitial edema and hyperemia,and the decrease of goblet cells.CWQ could restore the villi structure changes caused by PD-1 and improve the intestinal microstructure.The results of immunohistochemistry showed that the expressions of occludin,ZO-1 and claudin were decreased in the PD-1 group,while CWQ significantly increased the expressions of occludin,ZO-1 and claudin.(5)Compared with the Control group,the abundances of bacteroidota and desulfobacterota in CWQ group,PD-1 group and combination group were significantly decreased at the portal level,while the abundances of Firmicutes,actinobacteriata and verrucomicrobiota were significantly increased,and the combination group was the most abundant;marvinbryantia,Roseburia,Staphylococcus,Bacteroides and unclassified at the genus level＿c＿＿The abundances of bacilli,A2 and alloprevotella were down regulated,while those of akkermansia and unclassified were down regulated.The abundance of eggerthellaceae was up-regulated.The results of RDA/CCA showed that CD4~+T cells were closely related to Lactobacillus and norank＿F＿＿Muribaculaceae was positively correlated,and negatively correlated with Candida saccharimonas,dubosiella and Staphylococcus.PD-1~+CD8~+T cells,CD8~+T cells and CD3~+T cells were positively correlated with Candida saccharimonas,Staphylococcus and dubosiella,but positively correlated with Lactobacillus,Desulfovibrio and norank＿F＿＿Muribaculaceae was negatively correlated.The correlation results are consistent with the flow detection results.Conclusion:1.There were a large number of CD8~+T cells,CD4~+T cells infiltration and PD-1 and PD-L1 protein high expressions in the tumors of patients with microsatellite unstable colon cancer,and a small number of CD8~+T cells,CD4~+T cells and PD-1 and PD-L1protein low expressions in the tumors of patients with microsatellite stable colorectal cancer,and the immune cells were mainly infiltrated at the edge of the tumor.2.CWQ combined with PD-1 monoclonal antibody can significantly inhibit the growth of tumor,and CWQ can improve the anti-tumor efficacy of PD-1 monoclonal antibody.3.CWQ can promote the infiltration of CD8~+T cells and PD-1~+CD8~+T cells in the tumor tissues of mice,and improve the immune microenvironment in the tumor tissues.4.PD-1 m Ab can destroy the intestinal mucosal barrier,and Changweichuang can restore the disturbance of intestinal mucosal barrier caused by PD-1 m Ab.5.Immune cells are correlated with some specific flora,and CWQ can positively regulate intestinal flora closely related to immune cells.