Abstract B049: Modulating macrophages to foster antitumor immunity in breast cancer

Abstract Background: Tumor-associated macrophages (TAMs) are critical mediators of immune evasion in cancer and their abundance in the breast tumor microenvironment often correlates with poor prognosis. We sought to define macrophage intrinsic pathways that regulate antitumor immunity. Specifically, we hypothesized that the transcription factor GATA3 promotes the formation of the alternative pro-tumor phenotype and that myeloid-specific GATA3 ablation would enhance RT-induced antitumor immunity by preventing acquisition of this pro-tumor bioactivity. Methods: Triple-negative breast tumors were established by orthotopic implantation of EO771 cells into wild-type (WT) and myeloid-specific GATA3 knockout (mG3KO) C57BL/6 mice. Localized RT (16 Gy) was delivered to tumors, and tumor growth was assessed every two days by caliper measurements. Tumor-infiltrating leukocytes were then isolated for comprehensive profiling of the tumor microenvironment (TME) via multiparametric flow cytometry and single-cell RNA sequencing (scRNA-seq). Immune cell depletion studies were also performed via intraperitoneal injection of neutralizing antibodies against CD4, CD8, and Ly6G to selectively eliminate helper T cells, cytotoxic T cells, and neutrophils, respectively. Results: mG3KO mice demonstrated significantly delayed tumor growth and dramatically enhanced survival following RT (p<0.001) compared to wild type controls. scRNA-seq of post-RT tumor infiltrating leukocytes revealed a sustained pro-inflammatory tumor milieu in mG3KO mice, marked by a reduced immunosuppressive Arg1+ and Chil3+ expressing TAMs and fewer neutrophils. These findings were confirmed by multiparametric flow cytometry, which demonstrated a significant enrichment of CD8+ T cells in mG3KO tumors. Notably, CD8+ T cells from mG3KO mice expressed higher levels of interferon-γ (p < 0.001) and granzyme B (p < 0.0015) versus those from their WT counterparts. Furthermore, depletion of CD8+ T cells, but not neutrophils, with neutralizing antibodies significantly reduced the enhanced tumor control observed in mG3KO mice. Conclusion: In summary, we demonstrate that targeting the ability of macrophages to polarize towards a “pro-tumor” phenotype substantially enhances the response to RT by augmenting anti-tumor immunity. Targeting GATA3-dependent macrophage reprogramming may therefore represent a promising therapeutic strategy to overcome radioresistance in breast cancer. Citation Format: Tahir B. Dar, Harry Xiao, Satchel Stevens, Jolene Viramontes, Anthony T. Nguyen, Julie Jang, David M. Underhill, Stephen L. Shiao. Modulating macrophages to foster antitumor immunity in breast cancer [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr B049.