Therapeutic Effect And Mechanism Of CDK4/6 Inhibitor Combined With Pd-1 Monoclonal Antibody In Mouse Ovarian Cancer

BackgroundImmune checkpoint blockade represented by PD-1/PD-L1 inhibition have shown great promise in treating various kinds of solid tumors.However,their therapeutic effects against ovarian cancer are still limited.Potential mechanisms underlying the limited efficacy of immune checkpoint inhibitors in ovarian cancer involve many aspects,among which limited lymphocyte infiltration and immunosuppressive tumor microenvironment are nonnegligible factors.Increasing evidence showing the immunomodulatory effects of CDK4/6 inhibitors in vivo,including enhanced antigen presentation,increased infiltration of CD8+ T cells and reduced regulatory T cell(Treg)proliferation,which are beyond tumor cell cycle control.CDK4/6 inhibition and Immune checkpoint blockade could be a promising strategy in treating ovarian cancer.However,the effects of CDK4/6 inhibitors on the tumor microenvironment(TME)of ovarian cancer and whether these inhibitors can enhance the efficacy of immunotherapy are still unknown.Aims1.To determine whether CDK4/6 inhibitor has a therapeutic effect on mouse ovarian cancer model;2.To analyze the effects of CDK4/6 inhibition on the immune microenvironment of ovarian cancer and its possible mechanisms;3.To detect the effect of CDK4/6 inhibitor plus PD-1 monoclonal antibody in ovarian cancer;4.To analyze the possible mechanisms of the combined immunotherapy.Methods1.luciferase-tagged ID8 cells and immuno-competent mice were used to establish mouse ovarian cancer model;2.An in vivo image system and survival analysis was used to evaluate the therapeutic effect of abemaciclib in ovarian cancer;3.The effects of abemaciclib on the cell cycle,proliferation and apoptosis of ID8 tumor cells were detected by flow cytometry,immunohistochemistry and TUNEL.4.Immunohistochemistry was used to assess the density of tumor infiltrating lymphocyte after abemaciclib treatment.5.Immunofluorescence was performed to assess the distribution of tumor infiltrating lymphocytes.6.Flow cytometry analysis was performed to evaluate the proportion and activity of CD8+T cells,CD4+T cells,B cells,Dendritic cells,tumor-associated macrophages and MDSC.7.The Mouse Cytokine and Chemokine PCR Array was applied to evaluate transcriptional level changes in genes encoding cytokines and chemokines.8.Mouse cytokine and chemokine array was used to analyze the secretory phenotype of tumor cells.9.Transwell assays were performed to detect the chemotactic ability of the supernatant of tumor cells treated with abemaciclib.10.The effect of chemokine neutralization on tumor progression was evaluated in vivo.11.An in vivo image system and survival analysis was used to evaluate the therapeutic effect of abemaciclib plus anti-PD-1antibody in mouse ovarian cancer.The safety of the combined therapy was also preliminary evaluated.12.To investigate whether the mice that eradicate primary tumors could develop memory response to re-challenge,we reinjected ID8 cells to the mice cured by combination therapy.13.Effect of combined therapy on the activity and function of CD8+ and CD4+T cells was analyzed by flowcytometry.14.An in vivo image system and survival analysis was used to evaluate the effect of CD8+T cell and B cell clearance.15.The significance of CD8+T cells and B cells in the prognosis of human ovarian cancer by KM-plot public database.16.The effect of B cell clearance on the activity and function of CD8+ and CD4+T cells was analyzed by flow cytometry.Results1.Abemaciclib can inhibit the progression of ovarian cancer improve the survival of tumor-bearing mice.2.Abemaciclib induce cell cycle arrest in ID8 cells,inhibit the proliferation of ID8 tumor and promote apoptosis.3.Immunohistochemical analysis showed that abemaciclib promoted lymphocyte infiltration in ID8 tumors.4.The immunofluorescence results showed that the infiltrating CD3+T cells and B cells gathered to form the tertiary lymph node structure in abemaciclib treated group.5.Flow analysis showed that the activity of cytotoxic T cells and B cells increased,the antigen presentation function of dendritic cells enhanced,the expression of M1 phenotype related marker increased and M2 phenotype related marker expression decreased in tumor associated macrophages.6.PCR array analysis showed that abemaciclib increased the transcription level of TH1 cytokines and chemokines.7.Cytokine array showed that ID8 cells treated with abemaciclib secreted more CXCL10 and CXCL13.8.The supernatant of ID8 cells treated with abemaciclib had stronger chemotactic ability to CD8+T cells and B cells,and the chemotactic effect could be blocked by CXCL10 and CXCL13 antibodies.9.The anti-tumor effect of abemaciclib combined with PD-1 monoclonal antibody was better than that of single drug therapy,and the survival of mice in combined treatment group was better.Factorial analysis showed that there was a synergistic effect between abemaciclib and PD-1 monoclonal antibody.10.the mice cured by combined therapy were re-injected with ID8 cells,the tumor growth was significantly smaller than that in the control group.11.Flow analysis showed that the activity and function of CD8+ and CD4+T cells in the combined treatment group were higher than those in the monotherapy group.12.The antitumor activity of abemaciclib plus an anti-PD-1 m Ab is abrogated by CD8+ T cell depletion or B cell depletion.13.Survival analysis showed that high expression of CD8 a and CD19 indicates better prognosis in human ovarian cancer.14.Flow analysis of cytotoxic T cell activity showed that B cell clearance affected the activity of CD4+T cells,and finally led to a decrease in the activity and function of CD8+T cells.Conclusion1.Abemaciclib has anti-tumor effect in mouse ovarian cancer model;2.Abemaciclib can increase immune infiltration promote the formation of tertiary lymph node structure,which provides the basis for the anti-tumor role of PD-1 monoclonal antibody;3.Abemaciclib can increase the activity of anti-tumor immune cells in tumor microenvironment;4.Abemaciclib treated tumor tissues secrete more TH1 cytokines and chemokines and promote the infiltration of immune cells.5.Abemaciclib synergizes with anti-PD-1 therapy in ID8 tumor control,which depends on both CD8+T cells and B cells.