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使肿瘤细胞能够延长存活和生长。 肿瘤的乳腺癌（BC）是女性癌症相关死亡的第二大原因， 这些因素有效地指导靶向治疗，但不指导免疫治疗方法， 浸润在每个亚型内显著不同。肿瘤浸润淋巴细胞（TIL）的存在是高度相关的。 预后的生存和治疗效益的BC，但肿瘤的内在因素，支配他们的存在， 没有很好的定义。使用TCGA RNA分析，我已经确定了转录抑制因子的功能丧失， REST是减少淋巴细胞浸润到多种BC分子亚型肿瘤中的关键相关因素。 BC（约15-20%的肿瘤）中功能性REST的丧失与较差的肿瘤相关，然而， REST功能丧失调节肿瘤免疫微环境的机制尚不清楚， 可以为增强治疗反应提供新的靶点。我最近做了一个小鼠Rest基因敲除 BC线（Rest-less）和我的体内研究证实，Rest-less肿瘤含有显著较少的淋巴细胞 同时也揭示了促肿瘤巨噬细胞的显着增加，我们的实验室已经确定了一个细胞群， 是BC中淋巴细胞抑制的关键调节剂。此外，我的体外研究发现， 淋巴管生成性Vegfc和Vegfd是静息BC细胞中最差异上调的基因。肿瘤 淋巴管生成与实体瘤中的T细胞抑制有关，并可能为REST介导的T细胞抑制提供原因。 抑制淋巴细胞。根据我的初步数据，我假设淋巴细胞抑制在休息- 更少的肿瘤是由肿瘤相关巨噬细胞（TAM）和淋巴细胞的极化协调的。 抑制性表型。我提出两个具体目标来检验我的假设。在目标1中，我将 使用以下共培养试验确定肿瘤内在Rest对TAM介导的淋巴细胞抑制的作用： 来自静息状态下肿瘤的TAM与体外淋巴细胞和通过抗CSF-1 R清除巨噬细胞 在体内抗原特异性（GFP; JEDI）无静息模型中的治疗。在目标2中，我将评估Vegfr 3， Vegfc和Vegfd的受体，调节无REST的肿瘤微环境和对 免疫疗法为了完成这一任务，我将在有或没有抗-VEGFR 3抗体的情况下测试抗-VEGFR 3抗体对无静息肿瘤的阻断。 PD 1，并使用RNAseq全面分析肿瘤生长、淋巴消耗和免疫反应， 光谱细胞术和循环免疫荧光。我们提出的研究将提供一个了解 以前未表征的方面REST介导的免疫抑制在BC，并将使用新的鼠 原位模型来测试阻断免疫抑制机制的策略。最终，我预计我的 这些发现将揭示抑制BC中REST介导的免疫抑制的靶向机制，并提供一种新的免疫抑制机制。 对REST-较少肿瘤的整体治疗有相当大的影响。

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.