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

项目总结 大约一半的三阴性乳腺癌(TNBC)患者接受保乳治疗，这是 包括照射原发肿瘤部位。尽管这种疗法总体上改善了预后，但患者继续 经历局部区域高复发率。治疗后复发的控制机制 人们对此了解甚少。先前的研究表明，正常的组织辐射损伤可能与 复发。临床前研究表明，肿瘤细胞迁移到受辐射的乳腺组织中， 巨噬细胞在肿瘤细胞募集过程中起着关键作用。在阻断巨噬细胞浸润的同时 被证明可以消除肿瘤细胞向照射部位的迁移，但对巨噬细胞表型的影响却没有 被调查过了。具有伤口愈合表型(M2)的巨噬细胞已知促进肿瘤生长和 进展，但M2巨噬细胞是否影响照射组织中肿瘤细胞的募集和增殖 是未知的。这一建议的中心假设是，组织微环境中的辐射损伤 通过免疫抑制细胞的渗透促进转移前的利基。我将使用Pre--来检验这个假设 临床原位乳腺癌模型以及器官类化合物研究正常组织对辐射的反应 以及它对复发的影响。 该项目由两个具体目标指导：1)鉴定与RT-R相关的巨噬细胞表型。 诱导复发和2)确定有助于肿瘤细胞募集的靶向分泌因子 受辐射的组织。在目标1中，我将严格检查照射后的乳腺组织中的免疫细胞 使用流式细胞术。为了确认M2巨噬细胞在复发中的重要性，我将开发一个M2 小分子抑制剂或单抗阻断极化建立小鼠巨噬细胞耗竭模型 抗体。最后，我将确定M2巨噬细胞耗尽对照射后肿瘤细胞募集的影响 纸巾。在目标2中，我将使用我们实验室开发的一个有机模型，它概括了临床前的情况 观察。我将通过与巨噬细胞，肿瘤细胞， 和CD8+T细胞，并鉴定条件培养液中分泌的细胞因子。然后我会评估这些 肿瘤细胞在体内募集的因素起作用。这项研究将确定导致复发的条件 放射治疗后，包括巨噬细胞渗出的成分和肿瘤再生长的机制，这是 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.