Modeling Immune Cell Recruitment and its Impact on Triple Negative Breast Cancer Recurrence in the Irradiated Microenvironment

项目摘要 大约一半的三阴性乳腺癌（TNBC）患者接受保乳治疗， 包括照射原发肿瘤部位。虽然这种治疗总体上改善了预后，但患者继续 局部复发率很高控制治疗后复发的机制 我们对此知之甚少。先前的研究表明，正常组织的辐射损伤可能与 复发临床前研究表明，肿瘤细胞迁移到照射的乳腺组织中， 巨噬细胞在肿瘤细胞募集过程中起关键作用。同时阻断巨噬细胞浸润 显示消除了肿瘤细胞向照射部位的迁移，但对巨噬细胞表型的影响没有 被调查了已知具有伤口愈合表型的巨噬细胞（M2）促进肿瘤生长， 进展，但M2巨噬细胞是否影响照射组织中的肿瘤细胞募集和增殖 不明该建议的中心假设是，组织微环境中的辐射损伤 通过免疫抑制细胞浸润促进转移前小生境。我将使用前- 临床原位乳腺癌模型以及类器官，以研究正常组织对辐射的反应 及其对复发的影响。 该项目由两个具体目标指导：1）表征与RT相关的巨噬细胞表型， 诱导复发和2）鉴定有助于肿瘤细胞募集的靶向分泌因子， 辐照组织。在目标1中，我将严格检查免疫细胞浸润到辐照的乳腺组织中 使用流式细胞术。为了证实M2巨噬细胞在复发中的重要性，我将开发一个M2 通过用小分子抑制剂或单克隆抗体阻断极化的巨噬细胞耗竭小鼠模型 抗体的最后，我将确定M2巨噬细胞耗竭对肿瘤细胞募集到辐射的 组织中在目标2中，我将使用我们实验室开发的类器官模型，该模型概括了临床前 意见。我将通过将类器官与巨噬细胞，肿瘤细胞， 和CD 8 + T细胞，并表征条件培养基中分泌的细胞因子。然后我将评估这些 因子有助于肿瘤细胞在体内募集。这项研究将确定导致复发的条件 包括巨噬细胞浸润的组成和肿瘤再生长的机制， 将对易复发的TNBC患者产生重大影响。

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.