Abstract B063: Estrogen induces a pro-tumoral phenotype shift in microglia that contributes to E2-unresponsive breast cancer brain metastasis

Abstract Introduction: Younger women with breast cancer (BC) have increased risk to develop brain metastases (BM) irrespective of the tumor subtype. We have shown that pre-menopausal levels of 17-β-Estradiol (E2) promotes BM of estrogen-unresponsive BC cells by modulating astrocyte function. Yet, microglia (the brain resident-macrophages) express estrogen-receptors (ERs), mediate the neuroprotective and homeostatic effects of E2 in the brain, and play pro and anti-tumorigenic roles through brain metastases progression. Thus, we hypothesized that E2 acts on ER+ microglia to regulate its neuroinflammatory properties and suppress anti-tumoral responses in the brain. Results: To assess how E2 modulates brain immune cells, we performed multiparametric flow cytometry across different stages of BM progression in spontaneous and experimental models of BMs. From early to late stages of BM colonization, brains of E2-treated mice showed reduced neutrophils, lymphocyte infiltration, B and CD8+ T cells compared to E2-suppressed mice. scRNAseq of immune cells from BM-bearing mice identified 6 functional microglia subclusters. The migration Mg cluster, marked by microglial homeostatic genes known to promote activation and surveillance movements, the TNF-α cluster, marked by genes associated with promotion of microglial activation, and the Interferon Mg cluster, marked by genes involved in the regulation of type I Interferons were less abundant in E2-treated mice than E2-suppressed mice. Overrepresentation analysis showed microglia from E2-treated mice enriched in translation programs, while microglia from E2-suppresed mice had a significant enrichment of immune leukocyte activation and antigen presentation processes, suggesting E2 suppresses key microglia functions as antigen-presenters and activators of T cells. To determine how microglia from E2-treated or E2-depleted mice differentially influence T cell function, we performed in vitro coculture assays to assess T cell activation, effector-associated markers, and expansion. Microglia from E2-stimulated BM bearing mice showed decreased ability to induce interferon cytotoxic function and expansion of activated T cells, supporting a pro-tumorigenic T cell immune response. Since BMs are usually treated with brain radiotherapy (RT), either as whole brain radiotherapy (WBRT) or stereotactic radiosurgery (SRS), we tested whether E2-suppression impacted radiotherapy response in experimental metastases models. E2-suppression (ovarian suppression in combination with an aromatase inhibitor) significantly increased the effectiveness of radiation in decreasing BM progression. These studies provide a novel mechanism whereby E2 promotes rapid progression of BMs and provides a rationale for the clinical -testing of endocrine therapies in the management of BMs of E2-unresponsive tumors. Citation Format: Karen L. F. Alvarez-Eraso, Maria J. Contreras-Zárate, Andrew Goodspeed, James Costello, Jenny A. Jaramillo-Gómez, Stella Koliavas, R. Alejandro Marquez-Ortiz, Morgan S. Fox, D. Ryan Ormond, Peter Kabos, Mercedes Rincon, Diana M. Cittelly. Estrogen induces a pro-tumoral phenotype shift in microglia that contributes to E2-unresponsive breast cancer brain metastasis abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Brain Cancer; 2026 Mar 23-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86 (6Suppl): Abstract nr B063.