CANCER ASSOCIATED FIBROBLAST CLONALITY, CHROMATIN ACCESSIBILITY AND THERAPEUTIC TARGETING IN PANCREATIC DUCTAL ADENOCARCINOMA STROMA

项目总结 实体瘤，如胰腺导管腺癌(PDAC)，由转化的癌细胞组成 以及一群参与性的基质细胞。这些细胞包括癌症相关的成纤维细胞。 (CAF)，其中 已知可提供支持肿瘤增殖的纤维间质(结缔组织增生症)。我的初步数据 提示实体瘤间质中的成纤维细胞是局部募集和激活的，并以高度克隆性增殖。 举止。这种克隆性提示存在组织驻留的成纤维细胞前体细胞。染色质的变化 可及性被认为在推动成纤维细胞激活方面发挥了关键作用。被激活的特征 成纤维细胞是开发抗癌新基质疗法的关键一步。在这个项目中，我的目标是1) 使用彩虹小鼠，一个随机的， 多色，血统追踪模型。我将使用会徽(表观基因组和线粒体条形码的血统 来自内源性突变)方案，该方案分析单细胞线粒体DNA的体细胞突变 (SC)-转座酶可访问染色质(ATAC)-SEQ数据的分析，以跟踪细胞谱系，以识别和 鉴定可能的成纤维细胞前体细胞。为了进一步探讨成纤维细胞激活的生物学基础 PDAC，我将2)对小鼠和人类PDAC CAF进行sc-RNA-seq以确定功能相关的变化 在成纤维细胞激活时发生的基因表达。我将把CAF基因的表达模式与 基于在我的sc-atac-seq数据集中获得的核读数，染色质可获得性的变化。DNA- 已知可触发粘着斑激酶(FAK)介导的信号转导的结合元件，如Ets1，以及 FAK信号通路在实体瘤中CAF相关的促结缔组织增生症中起重要作用。 我的初步数据显示，PDAC CAF显著上调了CXCL5和CXCL10的表达， 它们是FAK途径的下游趋化因子靶点。因此，我将确定功能效果和 利用体外和体内实验探讨在活化的PDAC CAF中调节这些趋化因子的治疗潜力 活体化验。我将把这些假定的治疗靶点的调节与染色质的具体变化联系起来 使用蛋白质索引(PI)-ATAC的可及性，它允许染色质可及性与蛋白质的关联 在单个细胞级别表达。 这些目标的结果将扩大我们对CAF生物学和 PDAC中的异质性。该项目预计将确定可以利用的新基质目标。 在实体肿瘤的背景下主要或辅助治疗的。

PROJECT SUMMARY Solid tumors such as pancreatic ductal adenocarcinoma (PDAC) are comprised of transformed cancer cells along with a participatory population of stromal cells. These include cancer-associated fibroblasts (CAFs), which are known to provide fibrotic stroma (desmoplasia), which supports tumor proliferation. My preliminary data suggest that fibroblasts in solid tumor stroma are locally-recruited and activated, and proliferate in a highly clonal manner. Such clonality suggests the presence of tissue-resident fibroblast progenitors. Changes in chromatin accessibility are suspected to play a critical role in driving fibroblast activation. Characterization of activated fibroblasts is a key step toward developing novel stromal therapies against cancer. In this project, I aim to 1) quantitate the local, clonal expansion of activated fibroblasts in PDAC using the Rainbow mouse, a stochastic, multi-colored, lineage-tracing model. I will use the EMBLEM (Epigenome and Mitochondrial Barcode of Lineage from Endogenous Mutations) protocol, which analyzes somatic mutations in mitochondrial DNA from single cell (sc)-assay for transposase-accessible chromatin (ATAC)-seq data to track cell lineages, in order to identify and characterize putative fibroblast progenitors. To further explore the biology underlying fibroblast activation in PDAC, I will 2) conduct sc-RNA-seq on mouse and human PDAC CAFs to identify functionally relevant changes in gene expression that occur with fibroblast activation. I will correlate CAF gene expression patterns with changes in chromatin accessibility based on the nuclear reads obtained in my sc-ATAC-seq dataset. DNA- binding elements such as Ets1 which are known to trigger Focal adhesion kinase (FAK)-mediated signaling, and FAK pathway signaling is suggested to play a significant role in CAF-associated desmoplasia in solid tumors. My preliminary data suggest that PDAC CAFs show significant upregulation of CXCL5 and CXCL10 expression, which are downstream chemokine targets of the FAK pathway. As such, I will 3) determine functional effects and explore the therapeutic potential of modulating these chemokines in activated PDAC CAFs using in vitro and in vivo assays. I will correlate modulation of these putative therapeutic targets with specific changes in chromatin accessibility using protein-indexed (Pi)-ATAC, which permits correlation of chromatin accessibility with protein expression at a single cell level. The results of these aims will expand our understanding of CAF biology and heterogeneity in PDAC. This project is expected to identify novel stromal targets that could be exploited therapeutically either primarily or adjunctively in the setting of solid tumors.