Blocking Immune Checkpoint PD1 And VISTA In Radiation-treated Mouse CT26 Colon Cancer Model

Objective:T cell activation inhibitor immunoglobulin variable region domain(VISTA)molecules and CD8,CD33,programmed death receptor 1(PD1),forkhead transcription protein 3(FOXP3)in the K-mouse Expression of sarcoma virus oncogene(K-RAS)exon in different colorectal cancer tissues were detected to analyze its clinical significance.Compare the normal mouse group,tumor-bearing control group,CA170 treatment group,high-dose single-irradiation group and high-dose single irradiation combined with CA170 treatment group for different groups of tumors expression of multiple cytokines in tumors and spleens and ratio of immune cells in transplanted tumors after treatment and indicators of tumor shrinkage by construct double tumors model of BALB/C mouse with CT26 colon cancer cells.Then explore the mechanism of combination of high-dose single irradiation and VISTA and PD1 signaling pathway induce the abscopal effect in double tumors model in BALB/C mice.Methods:1.Immunohistochemistry technique was used to detect the expression of VISTA and CD8,CD33,PD1,FOXP3 in colorectal cancer tissues with different mutations of K-RAS gene exons and analyze their clinical significance.2.The mouse colorectal cancer cell line CT26 was cultured to construct a BALB/c mouse double-transplant tumor model.The expression of multiple cytokines in different mouse models before and after treatment was detected by liquid suspension microglobulin assay;The changes of the proportion of immune cells in vivo and in the spleen of transplanted tumors before and after treatment were measured by flow cytometry.Vernier caliper was used to measure the index of tumor regression.Results:1,Colorectal cancer stromal cells express VISTA?CD33 molecules,lymphocytes in colorectal tissue express FOXP3,PD1 and CD8 molecules2.There were significant differences in the expression of VISTA,CD33,CD8 molecules between KRAS gene mutation group and wild group(P<0.05).3.There was a significant difference in the expression of VISTA,CD8 and CD33 molecules between left and right colon cancer(P<0.05).4.High-dose X-ray combined with CA170 treatment significantly reduced the first and second tumors of mice(P<0.05);X-ray high-dose irradiation combined with CA170 significantly increase the expression of cytokines CCL2/MCP-1,IL-? and IFN-? and reduce the expression of VEGF and M-CSF in the first tumors of mice compared to CA170 or irradiation treated group(P<0.05).X-ray high-dose irradiation combined with CA170 significantly increase the expression of cytokines CCL2/MCP-1,IL-1? and IFN-?in the second tumors of mice compared to CA170 treated group(P<0.05).X-ray high-dose irradiation combined with CA170 significantly increase the expression of cytokines CCL2/MCP-1,IL-1 0 and IFN-y and reduce the expression of VEGF in the second tumors of mice compared to irradiation treated group(P<0.05).5.CA170 combined with radiotherapy or monotherapy CA170 treatment increased the infiltration and expansion of TIL in CD3+/CD8+mouse tumors.Conclusion:1.The formation of inhibitory immune microenvironment in colorectal cancer tissues with KRAS mutation may be related to the high expression of VISTA molecules in MDSCs and the inhibition of TIL function by MDSCs.2.High-dose X-irradiation combined with CA170 can significantly amplify the activation of the natural immune system caused by high-dose X-ray irradiation and promote the production of host acquired anti-tumor immunity and significantly inhibit the growth of mouse CT26 cell xenografts tumors.