The Study On The Mechanism Of MUC1-induced Immunosuppression In Colorectal Cancer

ObjectiveMucin1(MUC1)upregulation in colon cancer has been linked to poor patient outcomes and advanced stage at diagnosis.The aberrant overexpression and glycosylation of MUC1 in various malignancies facilitate oncogenic events from inception to metastasis.Mucin-associated sialyl-Tn(sTn)antigens bind to various receptors present on the dendritic cells(DCs),macrophages,and natural killer(NK)cells,resulting in overall immunosuppression by either receptor masking or inhibition of cytolytic activity.By comparing the growth of MUC1-positive and MUC1-negative colon cancer cells in wild-type and immunodeficient mice,our results found that only wild-type mice showed significant differences in growth rate,indicating that MUC1 is possible to promote tumor growth by inhibiting the T cells anti-tumor response in the tumor microenvironment.The purpose of this study was to address the pro-tumor and immunosuppressive roles of MUC1 in colon cancer.Methods(1)The stable cell lines expressing hMUC1(MUC1/CT26 and MUC1/SW480)were established.The cells were inoculated into the abdomen of wild type and nude mice.The long diameter and short diameter of the tumor were measured twice a week.The tumor-bearing mice were sacrificed to obtain the survival data.(2)The tumor tissues of CT26/MUC1 tumor-bearing mice were collected and homogenized on the indicated time,enzyme-linked immunosorbent assay was used to detect the secretion of cytokines in tumor tissue homogenate.The infilration of CD4+T cells,regulatory T cells(Tregs),CD8+T cells,bone marrow-derived suppressor cells(MDSC)and tumor-associated macrophages(TAM)in tumor tissue were anzlyzed.The function of tumor infiltrated T cell were determined.(3)The tumor-bearing mice were treated by PDL1 antibody.The long diameter and short diameter of the tumor were measured twice a week.The tumor-bearing mice were sacrificed to obtain the survival data.The infiltration of T cells,MDSC,TAM in tumor tissues,the activity of tumor infiltrating lymphocytes and programmed death molecules(PD1),programmed death molecules ligand(PDL1)were analyzed by Flow cytometry.(4)The cell-dependent mechanism of antibody was studied through T cell depletion in animals.(5)The colon cancer specimens were collected from the First Affiliated Hospital of Kunming Medical University.The secretion of cytokines in tumor tissues was determined by enzyme-linked immunosorbent assay(ELISA).The distribution of immune cells in tumor tissues,the activity of tumot infiltrated T cells,and the expression of programmed death molecule PD1 and its ligand PDL1 in tumor tissue were analyzed by Flow cytometry.(6)The mRNA levels of MUC1 in tumor tissues from colon cancer patients was analyzed by searching TCGA database,and the survival time of patients was detected.Results(1)MUC1-positive CT26 cells grew faster than MUC1-negative CT26 cells in murine models with intact immune competence(p<0.05).However,there was no significant difference on tumor growth between MUC 1-positive and MUC 1-negative CT26 tumor cells in nu/nu mice.(2)The inflammatory cytokines were increased in MUC 1-positive tumor tissues.ELISA results suggested that Interleukin-17A(IL-17A)and Interferon-gamma(IFN-y)levels not IL-10,Transforming growth factor-beta(TGF-β)and Tumor necrosis factor-alpha(TNF-α)levels were significantly increased in MUC 1-positive tissues from tumor-bearing mice or in colon tumor patients.Intracellular cytokine staining analysis demonstrated that MUC 1-positive and MUC1high tumor cells promoted the infiltration of IL-17A-producing CD4+T cells and IFN-y-producing CD8+T cells,but not the granzyme B-producing CD8+T cells in tumor tissues.(3)The immune-suppressive cells were accumulated in MUC1-positive tumor tissues.The increased CD4+Foxp3+Tregs were observed in MUC1-positive and MUC1high tumor tissues.The results also indicated that the MDSC and TAM were accumulated in MUC1-positive and MUC1high tumor tissues.PDL1 expression on MDSC,TAMs and tumor cells is greater in MUC1high tumor tissues from mice and patients(p<0.05).We also found that IL-17A and IFN-y stimulation promoted PDL1 expression on CT26 cells,as well as CT26/MUC1 cells,the data also showed that PD1 expression on CD8+T cell in mouse and humans was significantly increased.(4)Targeting PDL1 induces an antitumor immune response in MUC1-positive tumor mouse model.Compared with tumor-bearing mice treated with isotype antibodies,mice treated with PDL1 antibody significantly increased the number of CD8+ T cells in the tumor tissue,decreased the ratio of CD4/CD8 T cells and the ratio of MDSC and TAMs.The ability of CD8+T cells to secrete IFN-y and granzyme B,and the ability of CD4+T cells to produce IFN-y was enhanced(p<0.05).The percentage of PD1+CD8+T cells was also significantly increased in mice treated with the PDL1 antibody.(5)Targeting PDL1 induces antitumor effects.The data suggested that anti-PDL1 antibody treatment could significantly inhibit the growth of tumors on day 18,21,24 and 27 after tumor implantation,and prolong the survival time of tumor-bearing mice compared with the control antibody.However,mice with depleted CD8+cells lost the protective antitumor effects induced by the PDL1 antibody.Notably,MUC1-positive tumor-bearing mice had larger tumors and shorter survival times than MUC1-negative tumor-bearing mice,but MUCl-positive tumor-bearing mice injected with anti-PDLl antibodies had smaller tumors and significantly longer survival times than MUC1-negative tumor-bearing mice injected with anti-PDL1 antibodies.(6)The relationship between the expression of MUC1(copy number)in colon tumor tissues from the TCGA colon cancer database and the patient’s survival was analyzed.Results showed that the survival time of patients with low expression of MUC1 was significantly longer than that of patients with high expression of MUC1(p<0.05).Conclusion:The present study revealed that MUC1,as a tumor-associated antigen,can recruit more tumor-infiltrating lymphocytes into the tumor microenvironment compared with MUC1-negative colon cancer,but that these cells could not serve antitumor roles.Conversely,the present study demonstrated that MUC1-positive colon cancer attracted more Treg cells,MDSC and TAMs to the tumor site than MUC1-negative colon cancer.Furthermore,the data suggested that PD1-PDL1 expression is greater in MUC1-positive colon cancer.Blocking the PD1-PDL1 signaling pathway reduced the percentage of Treg cells,MDSC and TAMs in the tumor microenvironment,enhanced T-cell cytotoxicity and inhibited tumor growth,prolonging the survival time of MUC 1-positive tumor-bearing mice.Therefore,the present study elucidated the role of MUC 1 in tumor immune escape and provides a foundation for the application of PDL1 inhibitors to MUC 1-positive colon cancer.