The Impact Of Radiation Therapy With Different Fractionation Regimen On Immune Microenvironment In Xenograft Carcinoma And Its Mechanism

Objective: To investigate the impact of radiation therapy with different fractionation regimen on immune microenvironment in xenograft carcinoma and its mechanism.Methods: Biweekly inoculation of Lewis cells in the right hindlimb of 6-week-old female C57BL/6J mice established a transplanted tumor model.Mice were randomly divided into blank control group(NC),3Gy*10f group(3Gy),8Gy*3f group(8Gy),20Gy*1f(20Gy)group,4 mice in each group.Different doses of radiation therapy were given on the 9th day,5f/w,and the tumor diameter was measured with a vernier caliper every 3 days.On the 30 th day after the inoculation of the transplanted tumors,the tumor tissue and spleen tissues of the mice were collected and ground to prepare a single cell suspension.The cells were surface-stained,or cytoplasmically stained or nuclear stained,and the regulatory T cells(Treg),myeloid derived suppressor cells(MDSC),CD8+ T cell content and their percentage of lymphocytes.The blood was taken from the eyeballs of the mice after centrifugation,and plasma was obtained.The immune-related cytokines such as IL-2,IL-6,IL-10,IL-13,and IFN-r were detected by ELISA.On this basis,a further mechanism study was conducted to divide the tumor-bearing mice into 5 groups: blank control group(NC),3Gy*10f group(3Gy),8Gy*3f group(8Gy),3Gy*10f+?MDSC(3Gy+)Group,8Gy*3f+?MDSC(8Gy+)groups,3Gy+ and 8Gy+ groups were given intraperitoneal injection of 200 ?g of Gr-1 antibody on the second,sixth and tenth days of radiotherapy.On the 30 th day after transplantation,tumor tissues and spleen tissues of mice were collected.The Treg cells in different treatment groups were detected by FACS method;serum IL-2,IL-6,IL-10,IL-13,IFN-r and other cytokines were detected by ELISA method;the growth of transplanted tumors in each group was recorde.Results: The flow-through results showed that for Treg cells,the percentage of tumor cell infiltrating Treg cells in the 3Gy and 20 Gy groups was 17.5% and 6.8% respectively,there was a statistical difference between the two groups(P<0.05);and the spleen infiltrated There was no significant difference in the percentage of Treg between each group(P>0.05).For MDSC cells,the percentage of MDSC tumor infiltration in mice was 32.6%,19.6%,and 8.3% in the 3Gy,8Gy,and 20 Gy groups, respectively.The MDSC content in the 8Gy and 20 Gy groups was significantly lower than 3Gy group(P<0.05);the spleen infiltration in mice.The MDSC percentages in the 3Gy,8Gy,and 20 Gy groups were 26.7%,12.5%,8.3%,respectively,and the MDSC levels in the 8Gy and 20 Gy groups were significantly lower than 3Gy group(P<0.05).For CD8+ T cells,the percentage of CD8+ T cells infiltrated by mice was 3.3%,2.1%,7.6%,and 9.7% in the NC,3Gy,8Gy,and 20 Gy groups,respectively.The levels of CD8+T in the 8Gy and 20 Gy groups were higher than NC and 3Gy groups(P<0.05).However,there was no significant difference in the percentage of spleen's CD8+T in each group(P>0.05).ELISA results: For IL-6 cytokines,plasma concentrations of NC,3Gy,and 8Gy(pg/ml)were 4.8,12.9,6.4,and 7.8 pg/ml,respectively;3Gy was significantly different from 8Gy and NC groups(P< 0.05);For IFN-r cytokines,the concentrations of NC,3Gy,8Gy,20 Gy were 4.3,6.3,10.4,and 12.6 pg/ml,respectively.The concentrations of IFN-r in plasma of 8Gy and 20 Gy were significantly higher than NC and 3Gy(P<0.05);The three cytokines IL-2,IL-10,and IL-13 were not detected in plasma.Tumor growth curves: NC,3Gy,8Gy,20 Gy,but not statistically significant(P>0.05).On the basis of the appeal test,the MDSC was further removed.The experimental results are as follows.The flow-through results showed that the percentage of tumor infiltrating Treg cells in mice was 23.%,30.1%,18.4%,22.1%,12%% in NC,3Gy,8Gy,respectively.There was a significant difference between the 3Gy and 8Gy groups,3Gy+ and 8Gy+,8Gy and 8Gy+ groups(P<0.05).The percentage of spleen infiltrating Treg cells was not significantly different between the groups(P>0.05).ELISA results: Plasma concentrations of IL-6 were 5.4,18.1,9.7,8.4,4.7 pg/ml in the NC,3Gy,8Gy,3Gy+,and 8Gy+ groups,3Gy and 8Gy groups,3Gy+ and 3Gy+ groups,and 8Gy and 8Gy+.The difference between the three groups was statistically significant(P<0.05);four cytokines such as IL-2,IL-10,IL-13 and IFN-r were not detected in plasma.Tumor growth curves: The tumor volumes(mm3)in the NC,3Gy,8Gy,3Gy+,and 8Gy+ groups were 2289,1609,1377,852,and 690 mm3,and the 3Gy+ and 8Gy+ groups were significantly different from the NC,3Gy,and 8Gy groups,respectively(P<0.05).Conclusion: Comparing with conventional radiotherapy,hypofractionated radiation can reduce the tumor microenvironment of Lewis lung cancer transplanted tumor immunosuppression status,its possible mechanism is by lowering the MDSC inhibitory immune cells and cytokines IL-6.