Antitumor Effect Of Radiotherapy Combined With CpG-ODN And PD-1 Blockade In In Murine Tumor Models

Objective:1.To establish a lewis lung cancer model in C57/BL mice.2.To observe the antitumor effect of triple therapy with RT combined with PD-1 inhibitor and CpG-ODN.3.To explore the anti-tumor mechanism of this triple therapy.Method:A tumor-bearing mouse model was established,the right hind limb was subcutaneously implanted as a primary tumor,and the left back was subcutaneously implanted as a secondary tumor.Tumor-bearing mice were randomly divided into 6groups(A.B.C.D.E.F)when the right hindlimb tumor grew to a diameter of about 5 mm.Group A:blank control.Group B:radiotherapy alone RT.Group C:RT combined PD-1blockade.Group D:RT combined with CpG-ODN.Group E:PD-1 blockade combined with CpG-ODN.Group F:triple therapy group.The tumor volume was measured every two days using an electronic vernier caliper as observation.Some mice were used for observation of survival time,observed on the 80th day.Some mice were sacrificed on the24th day to harvest the spleen and the tumor tissue.The single cell suspension was obtained by the grinding method.Tumor infiltrating lymphocytes(TILs)were extracted by enzyme digestion.The frequency and phenotype of CD8~+T/CD4~+T lymphocytes in spleen and TIL were detected by flow cytometry.The levels of IFN-?and TNF-?secreted by CD8~+T cells were detected to analyze the function of CD8~+T lymphocytes.In addition,the maturation and activation of tumor-infiltrating dendritic cells were also examined.What's more,the frequency of CD25~+Foxp3~+Tregs in spleen and TIL was also examined.To observe the effect of triple therapy with CD8~+T cells and CD4~+T cells.Another group G:Triple therapy+CD8~+T cell inhibition antibody group,H group:triple therapy+CD4~+T cell inhibitor antibody group.Tumor-bearing mice with"complete tumor regression"in the triple therapy group were challenged with large doses of tumor cells to verify the immune memory of tumor-bearing mice.Results:Tumor-bearing mice in the triple therapy group showed significant inhibition of tumor growth and even"complete tumor regression",and this anti-tumor effect was systematic and it significantly prolonged the survival time of mice.In the triple therapy group,the proportion of CD8~+T cells was significantly increased.The increased phenotype was mainly effector memory.However,in the CD8~+T cell deletion group,the effect of combination therapy was significantly reduced.Further analysis found that not only the proportion of CD8~+T cell was significantly increased,its ability to activate and kill the tumor also increased significantly,showing the increase of secreted IFN-?and TNF-?.We also detected increased activation of tumor-infiltrating dendritic cells in the triple therapy group.In addition,the triple therapy also reduced the proportion of regulatory T cells which have a negative regulatory effect,further enhancing the antitumor effect of the body.The triple therapy also has an effective immune memory,the impact of inoculation,slightly tumor appeared,then the tumor subsided.Not only that,triple therapy on the blood-borne tumor mice and their lung toxicity is minimal.Conclusion:This triple therapy can effectively inhibit tumor growth,significantly prolong the survival time of mice,enhance the frequency and function of CD8~+T cells,enhance the maturation and activation of tumor infiltrating dendritic cells,and can attenuate tumor local Tregs immunosuppression.In addition,on the basis of effective anti-tumor effect,its safety has also been affirmed,making this new treatment strategy more clinical significance and value.