PROJECT SUMMARY Approximately half of triple negative breast cancer (TNBC) patients receive breast conserving therapy, which includes irradiating the primary tumor site. Although this therapy improves prognosis overall, patients continue to experience locoregional recurrences at high rates. The mechanisms controlling recurrence following therapy are poorly understood. Previous research suggests that normal tissue radiation damage may be correlated to recurrence. Pre-clinical studies have shown that tumor cells migrate into irradiated mammary tissue and that macrophages play a critical role in the tumor cell recruitment process. While blockade of macrophage infiltration was shown to eliminate tumor cell migration to irradiated sites, the impact of macrophage phenotype has not been investigated. Macrophages with a wound healing phenotype (M2) are known to promote tumor growth and progression, but whether M2 macrophages influence tumor cell recruitment and proliferation in irradiated tissues is unknown. The central hypothesis of this proposal is that radiation damage in the tissue microenvironment promotes a pre-metastatic niche through immunosuppressive cell infiltration. I will test this hypothesis using pre- clinical orthotopic breast cancer models as well as organoids to study the normal tissue response to radiation and its influence on recurrence. This project is guided by two specific aims: 1) To characterize macrophage phenotypes associated with RT- induced recurrence and 2) to identify targetable secreted factors that contribute to tumor cell recruitment in irradiated tissues. In Aim 1, I will rigorously examine the immune cell infiltrate into irradiated mammary tissue using flow cytometry. To confirm the importance of M2 macrophages in recurrence, I will develop an M2 macrophage-depleted mouse model by blocking polarization with small molecule inhibitors or monoclonal antibodies. Finally, I will determine the impact of M2 macrophage depletion on tumor cell recruitment to irradiated tissues. In Aim 2, I will use an organoid model developed by our laboratory that recapitulates pre-clinical observations. I will analyze secreted pro-tumor factors by co-culturing organoids with macrophages, tumor cells, and CD8+ T cells and characterizing secreted cytokines in conditioned media. I will then evaluate how these factors contribute to tumor cell recruitment in vivo. This study will establish the conditions that lead to recurrence following radiotherapy, including composition of macrophage infiltrate and mechanisms of tumor regrowth, which will have significant implications for TNBC patients vulnerable to recurrence.