Mechanisms of REST-mediated immunosuppression in cancer

项目摘要 癌细胞中的肿瘤内在因子调节免疫环境，以延长肿瘤的存活和生长 肿瘤。在乳腺癌(BC)中，女性癌症相关死亡的第二大原因，分子亚型 这些因素有效地指导靶向治疗，但不能指导免疫治疗方法。 每种亚型的浸润率差异很大。肿瘤浸润性淋巴细胞(TIL)的存在是高度的 预后与BC的生存和治疗效益有关，但控制它们存在的肿瘤内在因素是 定义不明确。使用TCGA RNA分析，我已经确定了转录抑制物功能的丧失 REST是导致多种BC分子亚型肿瘤中淋巴细胞浸润减少的关键因素。 BC(约15%-20%的肿瘤)失去功能休息与较差的预后有关，然而， 静息功能丧失调节肿瘤免疫微环境的机制尚不清楚 可能为增强治疗反应提供新的靶点。我最近产生了一种小鼠休息基因敲除 BC系(休息不足)和我的体内研究证实，休息不足的肿瘤含有明显较少的淋巴细胞 同时也揭示了亲肿瘤巨噬细胞的显著增加，我们的实验室已经确认的细胞群体是 是BC中淋巴细胞抑制的关键调节器。此外，我的体外研究发现 淋巴管生成的VEGFC和VEGFD是在不休息的BC细胞中差异最大的上调基因。肿瘤 实体瘤中淋巴管生成与T细胞抑制有关，这可能是REST介导的一个原因 抑制淋巴细胞。根据我的初步数据，我假设休息时淋巴细胞抑制- 较少的肿瘤是由肿瘤相关巨噬细胞(TAMs)和淋巴管的极化所协调的 内皮细胞对抑制表型的作用。我提出了两个具体目标来检验我的假设。在《目标1》中，我将 共培养法检测肿瘤内在静息在介导的淋巴细胞抑制中的作用 静息肿瘤TAMs与淋巴细胞联合作用及抗-CSF-1R去除巨噬细胞的实验研究 体内抗原特异性(GFP；绝地)不休息模型的治疗。在目标2中，我将评估VEGFR3、 VEGFC和VEGFD受体，调节静止的肿瘤微环境和对 免疫疗法。为了完成这项任务，我将测试抗VEGFR3阻断在有或没有抗VEGFR3的静息肿瘤上的作用 PD1，并使用RNAseq全面分析肿瘤生长、淋巴管耗竭和免疫反应， 光谱细胞术和循环免疫荧光法。我们提议的研究将提供对 BC中先前未描述的静息介导的免疫抑制方面，并将使用新的小鼠 测试阻断免疫抑制机制的策略的原位模型。最终，我期待着我的 研究结果将揭示(S)抑制BC静息介导的免疫抑制的靶向机制，并提供一种 对治疗躁动的肿瘤有相当大的影响。

Project Abstract Tumor-intrinsic factors in cancer cells modulate the immune milieu to enable prolonged survival and growth of tumors. In breast cancer (BC), the 2nd leading cause of cancer-related deaths in women, molecular subtyping of these factors effectively guides targeted therapies, but does not guide immunotherapy approaches as immune infiltrates vary significantly within each subtype. The presence of tumor-infiltrating lymphocytes (TILs) is highly prognostic for survival and therapeutic benefit in BC, but the tumor-intrinsic factors governing their presence are not well defined. Using TCGA RNA analysis, I have identified loss of function in the transcriptional repressor REST as being a key correlate to reduced lymphocyte infiltration into tumors of multiple BC molecular subtypes. Loss of functional REST in BC (~15-20% of tumors) is associated with poorer prognoses, however, the mechanisms by which loss of REST function modulates the tumor immune microenvironment are not known and may provide novel targets for enhancing therapeutic responses. I recently generated a murine Rest knockout BC line (Rest-less) and my in vivo studies confirmed that Rest-less tumors contain significantly less lymphocytes while also revealing a significant increase in pro-tumor macrophages, a cell population our lab has identified as being key modulators of lymphocyte suppression in BC. Furthermore, my in vitro studies identified lymphangiogenic Vegfc and Vegfd among the most differentially upregulated genes in Rest-less BC cells. Tumor lymphangiogenesis is linked to T cell suppression in solid tumors and may provide a reason for Rest-mediated suppression of lymphocytes. Based on my preliminary data, I hypothesize that lymphocyte suppression in Rest- less tumors is orchestrated by the polarization of tumor-associated macrophages (TAMs) and lymphatic endothelium to a suppressive phenotype. I propose two specific aims to test my hypothesis. In Aim 1, I will determine the role of tumor intrinsic Rest on TAM-mediated lymphocyte suppression using co-culture assays of TAMs from Rest-less tumors together with lymphocytes in vitro and macrophage depletion through anti-CSF-1R treatment in an antigen-specific (GFP; JEDI) Rest-less model in vivo. In Aim 2, I will evaluate how Vegfr3, the receptor for Vegfc and Vegfd, regulates the REST-less tumor microenvironment and responsiveness to immunotherapy. To accomplish this task, I will test anti-Vegfr3 blockade on Rest-less tumors with or without anti- PD1 and comprehensively analyze tumor growth, lymphatic depletion, and immune responses using RNAseq, spectral cytometry, and cyclic immunofluorescence. Our proposed research will provide an understanding of a previously uncharacterized facet of REST-mediated immune suppression in BC and will use novel murine orthotopic models to test strategies that block immune suppressive mechanisms. Ultimately, I anticipate my findings will reveal targetable mechanism(s) to inhibit Rest-mediated immunosuppression in BC and provide a considerable impact on the treatment of REST-less tumors overall.