Ethnicity-determined T cell responses and GARP/TGFbeta1 signaling in prostate cancer

项目摘要 前列腺癌（PCa）的发病率和死亡率在非洲裔美国人（AA）男性中最高 与其他种族/民族相比。这些差异即使在考虑了 社会经济因素，表明遗传和未知的生物学因素可能有助于PCa健康 差距。然而，常见的遗传改变，如TMPRRSS 2-ERG基因融合和PTEN缺失， 发现AA PCa的发生频率远低于欧美（EA）PCa。相反地， 在几项研究中报道了AA与EA男性之间肿瘤免疫生物学的显著差异， 其中包括一项癌症疫苗Sipuleucel-T的临床试验，AA男性的总体平均寿命为9个月。 比EA的人更有生存优势 从机制上剖析决定肿瘤细胞敏感性的免疫学和/或生物学因素 和抵抗不同种族的免疫治疗，我们已经开发了AA和EA PCa的原代培养物， 患者来源的肿瘤类器官、正常类器官、癌相关成纤维细胞（CAF）和良性- 相关的成纤维细胞（BAF），并冷冻保存他们的外周血淋巴细胞 （PBL）来自过去几年的同一患者。 我们的初步数据表明，AA CAFs分泌的活性TGF-β 1在培养中的水平增加， 比EA CAFs中等。此外，我们首次证明糖蛋白A重复占主导地位， （GARP）是释放活性TGF-β的对接受体，在邻近基质中过度表达 与EA PCa和AA PCa组织的相邻基质相比，AA PCa的体积。有趣的是， 与EA PCa的相邻基质相比，AA PCa具有增加的细胞毒性CD 8 +T细胞浸润， AA PCa的远距离基质，表明AA基质中的免疫应答较高，但可能不是 由于TGF-β1和GARP的增加而有效。我们与CAFs共培养研究的初步数据 和T细胞进一步支持这种情况，因为我们观察到TGF-β1增加和IFN-γ减少。 在这些共同文化中。 这些结果表明，尽管AA PCa患者可能对免疫疗法更敏感， GARP/TGFβ信号转导代表了AA PCa中的一个脆弱点，并且可以用作发展AA PCa的靶点。 更有效的免疫疗法。因此，我们假设肿瘤和间质之间的相互作用 在AA和EA中，PCa不同地影响T细胞的肿瘤反应性和GARP/TGF-β信号传导， 导致患者之间T细胞肿瘤反应性的差异。 为了验证这一假设，首先我们将确定来自AA和EA PCa患者的T细胞是否表现出 与自体肿瘤类器官和/或CAF共培养中肿瘤反应性的差异。二是我们 初步数据显示，达比加群酯，一种预防中风的抗凝药物， 房颤时，有效阻断GARP/TGF-β 1信号传导， 导致Myc-CaP异种移植肿瘤的持久消退。因此，我们将确定 达比加群可增强抗PD 1/抗CTLA 4在HiMyc和TRAMP转基因小鼠中的抗PCa疗效 PCa小鼠模型。第三，为了进一步增强我们研究的临床相关性，我们将利用现有的 福尔马林固定的石蜡包埋组织块来自141例AA和141例EA匹配的PCa病例， Gleason评分，并确定GARP/TGF-β信号转导与各种浸润性免疫的关系 AA和EA PCa的肿瘤和间质中的细胞。 影响：该提案利用了独特的类器官和细胞资源的开发， 允许解剖因子和机制（即GARP/GARP/TGF-β）信号传导导致免疫差异 AA和EA PCa之间的关系。此外，一种增强PCa免疫检查点治疗的新策略， 一般和AA PCa，特别是通过靶向达比加群酯的再利用， GARP/TGF-β信号在AA PCa的肿瘤微环境中高度活化，如 我们的初步数据达比加群酯显示出与阿司匹林相似的安全性特征或副作用 在人类身上。

Project Summary Prostate cancer (PCa) incidence and mortality rates are the highest in African American (AA) men compared to any other racial/ethnic population. These differences persist even after accounting for socioeconomic factors, suggesting genetics and unknown biological factors may contribute to PCa health disparities. However, common genetic alterations, such as TMPRRSS2-ERG gene fusions and PTEN loss, were found to occur much less frequently in AA PCa than in European American (EA) PCa. Instead, prominent differences in tumor immunobiology between AA vs. EA men were reported in several studies, including a clinical trial with a cancer vaccine, Sipuleucel-T, which AA men had a median nine-month of overall survival advantage over EA men. To mechanistically dissect the immunological and/biological factors that determine tumor cell sensitivity and resistance to immunotherapy of different races, we have developed primary cultures of AA and EA PCa patient-derived tumor organoids, normal organoids, carcinoma associated fibroblasts (CAFs) and benign- associated fibroblasts (BAFs) from many patients and cryopreserved their peripheral blood lymphocytes (PBLs) from the same patient over past years. Our preliminary data show that AA CAFs secrete increased levels of active TGF- in the culture medium than EA CAFs. In addition, we are the first to show that Glycoprotein A repetitions predominant (GARP), the docking receptor for the release of active TGF-β, is over expressed in the adjacent stroma of AA PCa compared to adjacent stroma of EA PCa and AA PCa tissues. Interestingly, the adjacent stroma of AA PCa has increased infiltration of cytotoxic CD8+T cells compared to the adjacent stroma of EA PCa and to the distant stroma of AA PCa, suggesting that immune response is higher in AA stroma but may not be effective due to the increased TGF-β1 and GARP. Our preliminary data of co-culture studies with CAFs and T cells lends further support to the scenario as we observed increased TGF-β1 and reduced IFN-  in these co-cultures. These results suggest that although AA PCa patients may be more responsive to immunotherapies, GARP/TGFβ signaling represents a vulnerable point in AA PCa and may be used as a target for developing more effective immunotherapies. Therefore, we hypothesize that the interaction between tumor and stroma in AA and EA PCa differentially affect the tumor reactivity of T cells and that GARP/TGF-β signaling contribute to the differences in the T cell tumor reactivity among patients. To test the hypothesis, first we will determine whether T cells from AA and EA PCa patients display differences in tumor reactivity in co-cultures with autologous tumor organoids and/or CAFs. Second, our preliminary data have shown that Dabigatran etexilate, an anticoagulant drug for preventing stroke in people with atrial fibrillation, effectively blocks GARP/TGF-1 signaling and that its combination with anti-CTLA4 results in a durable regression of Myc-CaP xenograft tumors. We therefore will determine whether Dabigatran can enhance the anti-PD1/anti-CTLA4’s anti-PCa efficacy in the HiMyc and TRAMP transgenic PCa mouse models. Third, to further enhance the clinical relevance of our study, we will utilize available formalin fixed paraffin embedded tissue blocks from 141 AA and 141 EA matched PCa cases by age and Gleason score and determine the relationship between GARP/TGF-β signaling and various infiltrating immune cells in tumor and stroma of AA and EA PCa. Impact: This proposal capitalizes on the development of unique organoids and cell resources, which allow dissecting factors and mechanisms (i.e. GARP/GARP/TGF-β) signaling leading to immune differences between AA and EA PCa. In addition, a novel strategy for enhancing immune checkpoint therapies in PCa in general and AA PCa in particular may be developed by repurposing Dabigatran etexilate through targeting GARP/TGF-β signaling that is highly activated in the tumor microenvironment of AA PCa, as suggested by our preliminary data. Dabigatran etexilate has shown similar safety profiles or side effects as Aspirin in humans.