CGAMP Enhances The Antitumor Effect Of Cryoablation In Colon Cancer And Its Mechanism

Cryoablation is a tumor surgery procedure with the advantages of less invasive and precise treatment for local tumors,and has been extensively used in the treatment of solid tumors such as liver cancer,kidney cancer,prostate cancer,lung cancer,and esophageal cancer.At present,the mechanism of tumor cell death and immune response induced by cryoablation is thought to be that freezing causes tumor cells to lyse and die,releasing antigens that are taken up by antigen-presenting cells（such as DC cells）,inducing costimulatory signals to effector T cells,triggering an anti-tumor immune response by effector T cells,and then producing a distant effect.However,the activation of the body’s immune response by cryoablation is not strong enough.Moreover,it is still unclear how cryoablation activates the body’s natural immunity to produce anti-tumor effects.There is an urgent need to enhance the anti-tumor immune effect of cryoablation,by strengthening and consolidating the activation and effect of immune cells in tumor treatment.In recent years,tumor immunotherapy has played an increasingly important role in cancer treatment.How to activate one’s own immune system is the key to treatment success.At the same time,a large number of recent data suggests that under pathological conditions such as tissue injury,self-DNA will accumulate in the cytoplasm,is recognized by cGAS and activates downstream type I interferons to initiate an immune response.Thus,the cGAS-STING regulatory pathway is necessary to generate spontaneous anti-tumor immunity.At present,whether the antitumor effect of cryoablation depends on the cGAS-STING signaling pathway is unknown,and the effect of cryoablation in combination with cGAMP is also to be explored.Therefore,in this thesis,a mouse colon cancer model was constructed using Cgas^-/-and Sting^-/-knockout mice to explore the relationship between cryoablation and the cGAS-STING pathway and the antitumor effect of cryoablation in combination with cGAMP.The research results of the study were as follows:1.Cryoablation mainly induces apoptosis of tumor cells,causes DNA damage and promotes tumor IFNβexpression.The tumor model of colon cancer was constructed in C57BL/6 mice and treated with cryoablation to detect tumor growth.After analysis by immunofluorescence（IF）,western blot（WB）,quantitative real-time polymerase chain reaction（q RT-PCR）method,it was found that cryoablation treatment of colon cancer in mice significantly suppressed tumor volume and body weight.At the same time,cryoablation induced apoptosis and tumor p-H2AX protein expression,indicating that it triggered DNA damage and upregulated the expression of interferon type I（IFNβ）and interferon stimulating genes（ISG56）in tumor tissues.2.Cryoablation-dependent anti-tumor effect of cGAS-STING pathway in mice.To confirm whether the antitumor effect of cryoablation depends on the cGAS-STING pathway,the colon cancer tumor model was constructed inoculating tumor cells in WT,Cgas^-/-and Sting^-/-mice,and the antitumor effect of cryoablation was found to be inhibited in the colon cancer tumor model of Cgas^-/-and Sting^-/-mice.This indicates that the antitumor effect of cryoablation in colon cancer is dependent on the presence of Cgas and Sting genes in the body.3.cGAMP produces anti-tumor effects in mice by activating the cGAS-STING pathway.MC38 cells were stimulated by different concentrations of cGAMP,and the q RT-PCR and Western blot assays showed that cGAMP induced the activation of cGAS-STING signaling pathway in MC38 cells.In addition,by constructing mouse colon cancer tumor model and screening cGAMP treatment concentration,20μg cGAMP was determined as the final treatment concentration and significantly inhibited tumor volume and body weight in mice.It showed that cGAMP produced anti-tumor effects through activating the cGAS-STING pathway in colon cancer.4.cGAMP facilitated cryoablation-induced anti-tumor effects in tumor-bearing mice.By constructing mouses colon cancer tumor model and treated with cryoablation and cGAMP alone or in combination,and it was found that these treatments significantly inhibited tumor growth and prolonged the generation time of tumor-bearing mice,but the combined treatment was more effective.In addition,the combination of cryoablation and cGAMP further increased the expression of IFNβand ISG56 in the tumors by q RT-PCR.The combination of cryoablation and cGAMP significantly enhanced the expression of CD4 T cells and CD8 T cells in tumor tissues after immunohistochemical detection.This indicates that cGAMP can enhance the antitumor effect of cryoablation.In conclusion,we found that cryoablation treatment of mouse colon cancer induced DNA damage in tumor tissues,and its antitumor effects were dependent on the cGAS-STING signaling pathway.The combination of cryoablation and cGAMP showed good therapeutic effects in colon cancer tumor models,increased the expression of IFNβand ISG56 in tumor tissues,and promoted the infiltration of CD4 T cells and CD8 T cells in tumors.These findings reveal the anti-tumor effects between cryoablation and natural immunity and provide a new therapeutic strategy for the clinical treatment of cryoablation.