The Effect Of Aerobic Exercise On The Mobilization Of Anti-tumor Immunity By CD8~+T Cells In Mice

Background:Cancer is currently the second leading cause of death worldwide and its occurrence is associated with immune imbalance.Exercise has great potential in tumor prevention and treatment as an effective way to boost the body’s immune system.Exercise can exert anti-tumor effects by altering the tumor microenvironment,improving metabolism,affecting immune function and thus mobilizing immune cells.This study aims to investigate the effect of aerobic exercise on MC38 colon cancer tumors,to reveal the anti-tumor mechanism of exercise from the perspective of exercise immunity,and provide experimental evidence for exercise against tumors.Methods:Part one:36 male C57BL/6 mice were selected and randomly divided into quiet control MC38 group(MC38-Con,n=10),regular exercise for 8 weeks MC38 group(MC38-8w Exe,n=8),quiet control LLC group(LLC-Con,n=9),and regular exercise for 8 weeks LLC group(LLC-8w Exe,n=9).The exercise group continued to exercise at moderate intensity for 8 weeks on a running platform,and the control mice were kept quietly.After the exercise intervention,mice were inoculated with MC38colon cancer cells or LLC lung cancer cells,and tumors were taken from each group when the mean value of tumor volume reached 800 mm~3in control mice.Tumor volumes were continuously observed after inoculation,tumor weights were weighed,and the infiltration of various types of immune cells in LLC-Con and LLC-8w Exe tumor tissues was detected by flow cytometry.Part two:27 male C57BL/6 mice were selected and randomly divided into quiet control MC38 group(MC38-Con,n=9),regular exercise for 4 weeks MC38 group(MC38-8w Exe,n=9),and regular exercise for 8 weeks MC38 group(MC38-8w Exe,n=9).The exercise group continued to exercise at moderate intensity for 4weeks or 8weeks on a running platform,and the control mice were kept quietly.After the exercise intervention,mice were inoculated with MC38 colon cancer cells,and tumors were taken from each group when the mean value of tumor volume reached 800 mm~3in control mice.Tumor volumes were continuously observed after inoculation,and tumor weights were weighed.Part three:For MC38-Con and MC38-4w Exe tumor tissues,flow cytometry was used to detect the infiltration of various types of immune cells,and immunohistochemistry was used to verify the results of flow cytometry.RNA-seq was used to detect the expression of differential genes,and RT-PCR was used to detect the gene expression of immune cell surface markers.Western Blot was used to detect the expression of key proteins of the JAK1/STAT1 pathway.In addition,18 male C57BL/6 mice were selected and randomly divided into a quiet control group(Con,n=9),and a regular exercise group for 4 weeks(4w Exe,n=9).The exercise group continued to exercise at moderate intensity for 4weeks on a running platform,and the control mice were kept quietly.At the end of the exercise intervention,both groups were sampled.The function of immune cells in peripheral blood was measured by in vitro stimulation.Results:1.Effect of 8-week aerobic exercise on tumors:From day 12 after inoculation of MC38 tumor-bearing mice,tumor volume in the exercise group was significantly smaller than that in the control group(P<0.01),and tumor mass in the exercise group was significantly smaller than that in the control group(P<0.01).In contrast,LLC tumor-bearing mice showed no difference in tumor volume or mass between the exercise group and the control group during the 20 days of tumor inoculation.2.Effect of 4-week aerobic exercise on MC38 colon cancer:From day 12 after inoculation,the tumor volume in the exercise group was significantly smaller than that in the control group(P<0.01),and the tumor mass and tumor mass/body weight ratio in the exercise group were significantly smaller than those in the control group(P<0.05).3.Infiltration of immune cells within MC38 tumor tissues:The flow cytometry results showed that the CD8~+T cells/tumor cells ratio within the tumor tissues of mice in the exercise group was significantly higher than that of the control group(P<0.05),while other immune cells did not show significant differences.Immunohistochemical results of tumor tissues showed positive CD8~+T cells consistent with the flow cytometry results(P<0.05).4.Possible mechanisms of aerobic exercise against MC38 tumors:RNA-seq results showed that the expression of inflammatory cytokines,such as Tgtp2,Zbp1,Gzmc,Gzmd,which associated with interferon regulation and CD8~+T cell effector functions were significantly higher in the exercise group than in the control group(P<0.01).RT-PCR results were consistent with RNA-seq(P<0.01).In vitro stimulation of peripheral blood showed that aerobic exercise enhanced the release of IFN-γfrom CD8~+T cells.Western blot results showed that the phosphorylation levels of JAK1 and STAT1 proteins in tumor cells in the exercise group were significantly higher than those in the control group(P<0.05).Conclusion:Eight weeks of moderate intensity aerobic exercise did not significantly inhibit low immunogenic LLC lung cancer.Four or eight weeks of moderate intensity aerobic exercise can exert tumor suppressive effects on highly immunogenic MC38colon tumor.Exercise induces CD8~+T cell mobilization through activation of the JAK1 and STAT1 protein-mediated signaling pathways,releasing IFN-γand thus exerting a tumor-suppressive effect.