Spatial and mechanistic assessment of the role of stromal fibroblasts in driving emergence of aggressive prostate and bladder cancer

摘要 本申请是为了响应被标识为NOT-CA的特别利益通知（NOSI）而提交的- 23-045.前列腺癌和膀胱癌是两种最常见的泌尿生殖系统癌症， 从低到高的侵略性仍然知之甚少。基质-上皮-免疫相互作用，特别是 涉及表达成纤维细胞活化蛋白（FAP）的成纤维细胞，可能有助于肿瘤的侵袭性。 然而，这些细胞在肿瘤微环境（TME）中的确切作用及其分子机制 导致癌症进展的原因尚不清楚。我们假设FAP+成纤维细胞参与细胞间相互作用 和旁分泌信号，支持癌细胞生长和侵袭，导致侵袭性癌症形成。到 为了验证这一假设，我们提出了两个具体目标：（1）整合空间转录组学和多重 人前列腺癌和膀胱癌组织的免疫组织化学/原位杂交（IHC/ISH）分析， 定义FAP+成纤维细胞、癌细胞和其他细胞之间的分子表型和空间关系， TME中的基质细胞区室;和（2）研究FAP+成纤维细胞在侵袭性肿瘤中的功能作用。 使用基因工程小鼠模型（GEMM）和空间转录组学 有和没有FAP基因破坏的改变。我们将进行尖端的空间分辨 使用高级免疫组织化学技术对人前列腺癌和膀胱癌组织进行转录组和多重IHC/ISH分析 平台这些数据将使用AstroPath平台进行整合，该平台将进行扩展，以处理空间数据。 转录组学数据以及多重原位方法。研究FAP+成纤维细胞的功能作用， 在侵袭性癌症的发展中，我们将使用GEMM并评估空间转录组学改变， 没有FAP基因破坏。这些数据将增加表型研究，并允许FAP的调查 在细胞-细胞空间关系和旁分泌信号机制中的作用。将使用以下方法验证结果： 多重免疫组化/原位杂交板。预期结果和影响：我们的研究将提供FAP在前列腺中的作用的见解。 和膀胱癌及其作为治疗和治疗诊断靶点的潜力。空间转录组学的应用 多重原位免疫组织化学将能够识别FAP表达细胞及其空间分布 与其他TME组件的关系。GEMM的使用将有助于调查FAP在以下方面的职能作用： 肿瘤生长、血管生成和转移。在人体组织中验证结果将提供临床相关性。 总之，这项研究将有助于了解前列腺和膀胱的分子机制 癌症，可能导致新的治疗策略，针对FAP。这项试点研究将建立 这些方法和模型在我们的组中的可行性，允许比较FAP+成纤维细胞在两种细胞中的作用。 癌症类型和奠定基础的纵向合作以外的补充奖，以促进 通过我们的U 54 TBEL联盟进行机构间合作。

ABSTRACT This application is being submitted in response to the Notice of Special Interest (NOSI) identified as NOT-CA- 23-045. Prostate and bladder cancers are the two most frequent genitourinary cancers, and their progression from low to high aggressiveness remains poorly understood. Stromal-epithelial-immune interactions, particularly involving fibroblast activation protein (FAP)-expressing fibroblasts, may contribute to tumor aggressiveness. However, the precise role of these cells in the tumor microenvironment (TME) and the molecular mechanisms driving cancer progression remain unclear. We hypothesize that FAP+ fibroblasts engage in cell-cell interactions and paracrine signaling, supporting cancer cell growth and invasion, leading to aggressive cancer formation. To test this hypothesis, we propose two specific aims: (1) Integrate spatial transcriptomics and multiplex immunohistochemistry/in situ hybridization (IHC/ISH) analyses of human prostate and bladder cancer tissues to define the molecular phenotype and spatial relationships between FAP+ fibroblasts, cancer cells, and other stromal cell compartments in the TME; and (2) Investigate the functional role of FAP+ fibroblasts in aggressive prostate and bladder cancer using genetically engineered mouse models (GEMMs) and spatial transcriptomic alterations with and without FAP genetic disruption. We will perform cutting-edge spatially resolved transcriptomic and multiplex IHC/ISH analysis of human prostate and bladder cancer tissues using advanced platforms. These data will be integrated using the AstroPath platform, which will be extended to handle the spatial transcriptomics data alongside the multiplex in situ methods. To investigate the functional role of FAP+ fibroblasts in aggressive cancer development, we will employ GEMMs and evaluate spatial transcriptomic alterations with and without FAP genetic disruption. These data will augment phenotypic studies and allow investigation of FAP's role in cell-cell spatial relationships and paracrine signaling mechanisms. Findings will be validated using multiplex IHC/ISH panels. Expected Results and Impact: Our study will provide insights into FAP's role in prostate and bladder cancer and its potential as a therapeutic and theranostic target. The use of spatial transcriptomics and multiplex in situ immunohistochemistry will enable identification of FAP-expressing cells and their spatial relationship with other TME components. The use of GEMMs will facilitate investigating FAP's functional role in tumor growth, angiogenesis, and metastasis. Validating findings in human tissues will provide clinical relevance. Overall, this study will contribute to understanding the molecular mechanisms underlying prostate and bladder cancer, potentially leading to novel therapeutic strategies targeting FAP. This pilot study will establish the feasibility of these methods and models in our groups, allowing comparison of FAP+ fibroblasts' role in both cancer types and laying the foundation for longitudinal collaboration beyond the Supplement award to facilitate inter-institutional collaboration through our U54 TBEL consortium.