The Mechanism And Evaluation Of The Combination Therapy Of Cryoablation And PD-1 Blockade In A Murine Model Of Renal Cell Carcinoma

objective Cryoablation,which destroys tumors by inducing an extreme low temperature in the tumor tissue,has been used in the focal treatment of localized RCC.Furthermore,cryoablation can stimulate a specific immune response against tumor.However,the immunological effect is not sufficient to diminish established tumors since tumor cells have evolved efficient mechanisms to escape the detection and destruction from the immune system including checkpoint molecules.The aim of this study is to explore the antitumor immune response induced by cryoablation and the possible relationship between cryoablation and checkpoint PD-L1/PD-1 using a murine model of metastatic renal cell carcinoma.We also evaluate the efficacy of the combination treatment of cryoablation and PD-1 blockade.Methods RENCA cells were inoculated subcutaneously on the bilateral flanks of BALB/C mice at the same time to establish a murine model.The subcutaneous tumor on the left flank was cryo-treated when it reached 50-70mm3 by inserting a cryoprobe into the center of the tumor.The treatment was administered for 30 seconds at a temperature of-100?.To explore the antitumor immune response within the distant tumor microenvironment and the possible relationship with checkpoint PD-L1/PD-1,the percentage of CD8+ and PD-1+ cells in the contralateral tumor were analysed on day 3 and day 7 after cryoablation by flow cytometric analysis.Meanwhile,the m RNA level of IFN-?,GZMB,IL-10 and PD-L1 were analysed by q PCR.To evaluate the efficacy of the combination treatment of cryoablation and PD-1 blockade,mice were divided into four groups and treated with anti-PD-1,cryoablation,cryoablation plus anti-PD-1 or without any treatment.The percentage of CD8+ tumor infiltrating lymphocytes and the m RNA level of cytokine IFN-?,GZMB and IL-10 were examined on day 14 after the initial treatment.Results A significant increase in the percentage of CD8+ tumor infiltrating T lymphocytes was observed on day 3 but decreased on day 7.In the meantime,the m RNA expression of IFN-? and GZMB were upregulated significantly on day 3 and dropped on day 7 after cryoablation.The m RNA expression of IL-10,was not different on day 3 but upregulated on day 7.The proportion of CD8+PD-1+ T cells was significantly increased on day 3 and day 7 after the treatment.Meanwhile,PD-L1 m RNA expression increased on day 3 and decreased on day 7.Cryoablation combined with PD-1 blockade inhibited the outgrowth of the distant tumor with higher percentage of CD8+PD-1+ cells and the m RNA level of IFN-? and GZMB.Conclusion Cryoablation can induce a short-lived antitumor immune response in the distant tumor.Furthermore,the combination therapy of cryoablation and PD-1 blockade can induce a potent and durable immune response and inhibit the outgrowth of distant tumor.