Effect Of Metformin On Immune Function Of G-MDSCs And Its Antitumor Immune Response In Mice

Objective:Granulocytic myeloid-derived suppressor cells(G-MDSCs)were treated with metfotmin in vitro,to observe the changes of immunosuppressive function of G-MDSCs,to detect the expression of the relevant effector molecules of G-MDSCs,and explore possible mechanisms of action;whether or not metformin can affect tumor growth by regulating the function of G-MDSCs in CT-26 tumor-bearing mice.Method:(1)The tumor-bearing mouse model was constructed by subcutaneous injection of CT-26 cells,and the spleen G-MDSCs cells were isolated by immunomagnetic beads sorting(MACS).After treatment with metformin,the phosphorylation of STAT3 was detected by Western-blot;the effect of G-MDSCs on the immunosuppressive function of CD4+ T cells was detected by CFSE;the expression of reactive oxygen specise(ROS)in G-MDSCs was detected by flow cytometry(FCM);the ARG-1 activity was measured using arginase(ARG-1)kit.(2)After treatment with metformin in vitro,the phosphorylation of AMPK was detected by Western-blot.Amplification of AMPK phosphorylation in G-MDSCs was inhibited by AMPK inhibitor Compound C,and then treated with metformin,the phosphorylation of AMPK and STAT3 was detected by Western-blot;the immunosuppressive function of G-MDSCs was detected by CFSE;the expression of ROS in G-MDSCs was detected by flow cytometry(FCM);ARG-1 activity was measured using arginase(ARG-1)kit.(3)Intraperitoneal injection of metformin in tumor-bearing mice.To observe the tumor growth,measure the tumor size and volume;the percentage of total MDSCs,G-MDSCs,CTLs,Thl and Treg cells was measured by flow cytometry(FCM).(4)The isolated G-MDSCs of tumor-bearing mice treated with metformin,the immunosuppressive function of G-MDSCs was detected by CFSE;the expression of ROS in G-MDSCs was detected by flow cytometry(FCM);the ARG-1 activity was measured using arginase(ARG-1)kit;the phosphorylation of STAT3 in G-MDSCs was detected by Western-blot.Results:(1)G-MDSCs sorted from the spleen of tumor-bearing mice were treated with metformin,the results showed that metformin was able to down-regulate STAT3 phosphorylation(P<0.05);metformin significantly reduced the immunosuppressive function of G-MDSCs on CD4+ T cells(P<0.01);the levels of arginase activity and ROS expression were significantly lower than those in the untreated group(P<0.05).(2)After G-MDSCs were treated with metformin,the AMPK phosphorylation levels were significantly increased(P<0.05).In the G-MDSCs culture system,AMPK inhibitor Comound C pretreatment was added,and then treated with metformin,the phosphorylation of AMPK in G-MDSCs was significantly lower than that in the control group(P<0.05),but the phosphorylation level of STAT3 was significantly up-regulated(P<0.05);the immunosuppressive function of G-MDSCs on CD4+ T cells was significantly higher than that of the control group(P<0.001);the expression of ROS was significantly higher than that of the control group(P<0.05);the levels of arginase activity was no significant change.(3)Compared with the saline control group,the tumor growth rate of metformin treatment group was slowed down(P<0.01),the tumor volume decreased,and the weight of the tumor was decreased(P<0.01);the results of FCM showed that the percentage of total MDSCs(P<0.05),G-MDSCs(P<0.01)and Tregs(P<0.001)infiltrated in mice treated with metformin was significantly lower than that in control group;CTLs and Thl were significantly higher than those in the control group(P<0.05).(4)Compared with the saline control group,the immunosuppressive function of spleen G-MDSCs in mice treated with metformin was significantly decreased(P<0.001);the ROS expression level was significantly decreased(P<0.05);the ARG-1 activity was significantly decreased(P<0.05,P<0.01);the phosphorylation of STAT3 in G-MDSCs was significantly down-regulated(P<0.01).Conclusion:(1)Metformin in vitro can enhance the phosphorylation of AMPK,reduce STAT3 phosphorylation levels,thereby reducing the inhibitory function of G-MDSCs.(2)In tumor-bearing mice,metformin can delay tumor growth by down-regulating the immunosuppressive function of G-MDSCs.