Anti-tumor Effect Of Tumor Infiltrating Lymphocytes Enhanced By αPD-1 Blockade In Colorectal Cancer

BackgroundAs a malignant neoplasm with high mortality and morbidity,the five-year overall survival rate of CRC remains low despite years of advances in ancestral healing.Recently,cancer immunotherapy has become a hot topic with theαPD-1 blockade approved by FDA.The clinical efficacy ofαPD-1 blockade is desirable in several cancer types,such as melanoma and lung cancer,but colorectal cancer has been shown less effective.Therefore,it is of great significance to explore the immune microenvironment status in colorectal cancer and investigate the anti-tumor mechanism ofαPD-1 blockade.This study will promote the application ofαPD-1 blockade more reasonable and remarkably improve the therapeutic efficacy ofαPD-1 blockade in colorectal cancer in the future.ObjectiveTo investigate the immunosuppressive tumor microenvironment in colorectal cancer and to detect the antitumor effect mechanism ofαPD-1 blockade therapy in the CT26subcutaneous tumor model.Methods1.Clinical sampleThe CD4/CD8 ratio,the percentage of Tregs cells,the expression of PD-1 on TIL,PIL,PBMC from CRC patients,the change of IFN-γinduction on CD8~+T-cells afterαPD-1blockade was analyzed by flow cytometry.The immunosuppressive microenvironment in CRC tissue was investigated.2.Tumor experiments and in vivo blockadeBALB/c mice were inoculated subcutaneously with CT26 tumor cells.The mice were randomized into treatment groups and treated withαPD-1 blockade or PBS,at 7 days post tumor cell inoculation.According to the treatment effect,mice in theαPD-1 blockade group were divided into response and non-response group at 14 days after treatment.Spleens and the tumor volumes were weighed in time and the lymphocyte subsets in spleen and tumor ware analyzed by Flow cytometry.Results1.The percentages of Tregs cell was more significantly enrichment in tumor tissue than matched paraneoplastic tissue and peripheral blood.2.The expression of PD-1 in TIL was significantly higher than that in matched PIL and PBL,and the IFN-γsecretion level of CD8~+PD-1~+TIL cells were less than CD8~+PD-1~-TIL cells.3.In the CT26 tumor cell beared mice,blockade ofαPD-1 inhibited tumor growth and prolonged survival time.4.In the spleen and tumor of the mice inαPD-1 response group,CD3~+T-cells,CD4~+T-cells and CD8~+T-cells were significantly enriched compared withαPD-1 non-response group and PBS control group.5.Distinguished with other subtype cells,the percentages of CD4~+Foxp3~+Tregs in tumor tissue of the mice inαPD-1 response group ware remarkable higher.6.The MDSC percentages of spleen and tumor inαPD-1 response group was lower compared with PBS group andαPD-1 non-response group.The difference between the latter two groupswas not significant.ConclusionsTumor tissue of CRC patients has an immunosuppression in the tumor microenvironment.For tumor model and treatments,blockade ofαPD-1 inhibited CT26tumor growth and prolonged survival time of some mice.Further analysis by flow cytometry showedαPD-1 blockade therapy is associated with immune cell infiltration and MDSC decrease.