Reshaping Macrophage Polarization Potential Enhances Antitumor Immune Response to Radiation Therapy

Abstract

Although radiation therapy (RT) remains a cornerstone in the treatment of breast cancer, many trials combining RT with immune checkpoint blockade (ICB) have failed to demonstrate benefit in solid tumors including breast cancer. Maximal efficacy of RT relies on the generation of antitumor immunity following treatment which largely consists of cytotoxic T cells and macrophages. Broad depletion of macrophages modestly enhances tumor responses to RT suggesting that they can shape RT-induced antitumor immunity. Although IL4 signaling through GATA-3 is known to polarize T cells into the protumor Th2 phenotype, such central drivers of macrophage polarization are not well established. Given that macrophages abundantly express IL4 receptor, we hypothesized that GATA-3 may direct the transition of macrophages to M2/alternative phase and that genetic ablation of GATA-3 in macrophages can enhance antitumor immunity by arresting macrophage transition to an M2-like pro-tumor state. We generated a macrophage specific GATA-3 KO mouse model (mG3KO) driven by the LysM-Cre promoter. Using a syngeneic orthotopic murine model of breast cancer (EO771), we evaluated the differential effect of RT (16Gy x 1) in WT and mG3KO mice. Multiparametric flow cytometry was performed to investigate the immune changes within the tumor microenvironment on day 3, day 5 and day 10 after RT. T cell depletion was performed using antibodies to CD4 and CD8 by intraperitoneal injections to understand the role of adaptive immunity in the response to RT in WT and mG3KO mice. We found that mG3KO mice bearing advanced EO771 tumors demonstrated significantly improved tumor regression compared to WT mice (p<0.001), which translated to increased overall survival. In vitro characterization of bone-marrow derived macrophages from mG3KO and WT mice suggest that macrophages with ablated GATA-3 expressed increased levels of iNOS and decreased levels of Arginase (Arg-1), consistent with an M1-like phenotype. Immune profiling of the tumors also revealed that mGATA-3 KO animals have significant enrichment of CD8+ T cells in the tumor milieu post RT and these CD8+ T cells express higher amounts of interferon gamma (p<0.001) and Granzyme B (p<0.0015) than their WT counterparts. Using neutralizing antibodies to deplete CD8+ T cells, we show that anti-tumor effects in the mG3KO mice were abolished, suggesting that mG3KO macrophages impact survival, at least, in part by enhancing cytotoxic CD8+T cells. Studies are currently ongoing to reveal the detailed mechanism of GATA-3 ablation in improving the efficacy of RT. Our data indicates that GATA-3 is a central regulator of macrophage polarization in response to RT. Further, directed ablation of GATA-3 appears to drive macrophages towards an M1-like phenotype, which enhances T cell recruitment to irradiated tumors. These data suggest that the antitumor efficacy of RT can be prolonged by targeting GATA-3-dependent signaling within myeloid cells.