Study On The Effect Of Metformin Combined With Radiotherapy On The Polarization Of Tumor-associated Macrophages In Glioblastoma

Objective:To investigate the effects of metformin combined with radiotherapy in vivo and in vitro on the polarization of tumor-associated macrophages(TAMs)in the microenvironment of glioblastoma and its therapeutic effect in the model of gliomablastoma in mice.Methods:1.The effect of metformin(Met)on the proliferation of glioblastoma mouse cell lines GL261-luc and CT-2A was detected by MTS experiment.2.The GL261-luc cells treated with Met and radiotherapy were co-cultured with bone marrow-derived macrophages(BMDM)after induced differentiation,called TAMs.Flow cytometry was used to detect the effect of GL261-luc on TAMs polarization in each group.3.The mouse glioblastoma cell line GL261-luc was transplanted into the frontal lobe of the brain of female C57BL/6 mice fed with SPF for 6 weeks,and the model of glioblastoma was established.After successful tumor implantation was detected(fluorescence value>10~6),56 mice model with similar fluorescence values were randomly divided into 4 groups(n=14):negitive control group(NC group);metformin monotherapy group(Met group,500mg/kg/d);radiotherapy alone group(RT group,8Gy once);metformin combined with radiotherapy group(RT+Met group)was treated with metformin(500mg/kg/d)and whole brain radiotherapy(8Gy once).The polarization of intracranial TAMs in each group was evaluated by flow cytometry,real-time PCR(RT-PCR)and immunohistochemistry,and the results were statistically analyzed.4.The tumor growth in the brain of 4 groups mice(n=8)was detected by using in vivo imaging technology of small animals(IVIS)every week,the tumor growth curve was plotted and the survival period of mice was recorded.Results:1.Met inhibited the proliferation of GL261-luc and CT-2A cells,and the effect increased with the increase of concentration and duration of action.The IC50 was28.38mmol/L and 94.82mmol/L for 1 day,respectively,and the IC50 for 3 days was 15.71mmol/L and 14.71mmol/L,respectively.2.The co-culture of GL261-luc and BMDM in each group had different effects on the proportion of M2-type macrophages(M2)in TAMs,and the proportion of M2decreased after Met treatment compared with that of the NC group,17.33%vs28.13%(p<0.005).After radiotherapy,the M2 proportion of 51.43%increased significantly compared with 28.13%of the NC group(p<0.001),while RT+Met treatment could inhibit the radiation-induced polarization of TAMs to M2,which decreased compared with both the RT group and the NC group,23.02%vs 51.43%vs 28.13%(p<0.005).3.Radiotherapy promoted TAMs recruitment and polarized it to M2-type.The results flow cytometry in vivo tumor tissue showed that TAMs increased from4.38%in NC group to 9.36%in RT group(p<0.05),and the proportion of M2marked by CD206~+after radiotherapy increased from 35.5%to 80%(p<0.001).The proportion of TAMs marked by CD68~+after IHC results was 22.44%higher than NC group 9.1%(p<0.05),and the proportion of M2 marked by CD206~+was16.02%higher than NC group 6.26%(p<0.05).Combination therapy can induce M2,which collected by radiotherapy,to M1.According to RT-PCR results,the expression of M2(IL-10~+/Arg-1~+)after combination therapy was reduced compared with NC group(p<0.05),while the expression of M1(NOS-2~+/i IRF-5~+)was increased(p<0.05).In addition,IHC results showed that the proportion of M2 of CD206~+after combined treatment was 1.4%,significantly lower than that of16.02%in RT group(p<0.001),while the proportion of M1 of i NOS~+after combined treatment was 14.15%,significantly higher than that of 1.56%in RT group(p<0.001).4.Compared with NC group,RT significantly inhibit the growth of glioblastoma in mice and prolong the survival of mice.The median survival of mice in the RT group was 53 days vs 37 days in the control group(p<0.01).Metformin combined with radiotherapy further synergically inhibited tumor growth.After combinedtreatment,the tumor fluorescence values measured weekly in mice were always lower than those of RT group and Met group(p<0.05),and the median survival period of the mice was extended from 53 days of RT group to 62 days(p=0.128).Conclusion:1.Metformin can directly inhibit the proliferation of glioblastoma.Meanwhile,metformin can inhibit M2 polarization of TAMs in glioblastoma,promote M1polarization,and reshape the tumor immune microenvironment.2.Radiotherapy can inhibit the growth of glioblastoma in mice and improve its survival,but at the same time promote the recruitment of TAMs in the local tumor and polarization to M2,resulting in tumor immunosuppressive microenvironment.3.Metformin combined with radiotherapy can induce the transformation of M2collected by radiotherapy to M1 in the tumor microenvironment,enhancing the anti-tumor efficacy.