The use of metformin to enhance radiation effects on M1 macrophage subtype polarization in bone marrow-derived macrophage in glioblastoma tumor environment.

Abstract

e13513 Background: Given the clinical relevance of tumor-associated macrophage (TAM) with its pro-tumor role and as a cell type compromising a large portion in glioblastoma (GBM), reversing the imbalance of TAM polarization in the tumor environment has emerged as a promising novel field for GBM treatment. Radiation therapy (RT) is the standard treatment for GBM patients after surgery, which has been shown to transiently induce M1 polarization of macrophage (M1Ø). Recent studies suggested that metformin could also promote M1Ø in tumor microenvironment. We thus postulate that metformin may enhance and sustain the M1-inducing effect of radiation in GBM. Methods: We first examined the polarization effect of metformin (0.1mM, 1mM and 2mM) on mouse bone marrow-derived macrophage (BMDM) cultured in GBM tumor environment, including media conditioned by GBM cells in monolayer culture or tumor spheres as well as in trans-well co-culturing system. We irradiated GBM cells with different doses (2 Gy, 8 Gy, and 20 Gy) after the treatment of Metformin at various time points; then we used conditioned media to treat BMDM either cultured alone or co-cultured with GBM cells in trans-well system for 24 or 48 hours. A separate set of experiment was conducted by first irradiating GBM cells and then co-culturing them with BMDM at 24 or 48 hours after radiation with metformin added at the start of co-culture. Percentage of various subtypes of BMDM was calculated after flow cytometry. Results: High concentrations of metformin (1mM and 2mM) significantly increased M1Ø and inhibited M2Ø in all culture conditions. Co-culture with irradiated GBM cells or treatment with medium conditioned by irradiated GBM cells could temporally induce M1Ø polarization in BMDM, with the effects being RT dose-dependent. Metformin at high concentrations further promoted M1Ø and suppressed M2Ø polarization in those conditions mimicking tumor microenvironment. This enhancing effect was sustained for at least 48 hours. Conclusions: Metformin at mili-molar concentrations significantly enhances the effects of radiation on M1Ø polarization in BMDM in vitro.