Fibroblast Heterogeneity in Pancreatic Cancer

摘要 胰腺导管腺癌(PDA)的特点是广泛的促结缔组织增生性间质，表现为 这是其有效治疗和提高患者存活率的主要挑战。我们试图定义组成 而肿瘤相关成纤维细胞(CAF)是PDA间质中最丰富的细胞群，作为一种 为开发新的治疗策略提供了潜在的途径。尽管CAF在历史上一直是 被认为是促进肿瘤的成分，它们在临床前和临床研究中的消融导致了混合 结果表明，人们对CAF的复杂性知之甚少。为了研究CAF生物学，我们之前 建立胰腺肿瘤类器官/成纤维细胞共培养模型。此外，我们还进行了单细胞RNA- 鼠和人PDA标本测序(scRNA-seq)以确定单细胞的CaF组成 决议。这些分析表明，成纤维细胞是异质的，至少由三个 具有独特转录和功能特征的独特亚型。更重要的是，这种异质性强调了 需要设计有选择性地针对促进肿瘤的CAF人群的治疗方法。尽管我们的工作已经 开始揭示原发性或转移性PDA组织中成纤维细胞的复杂异质性，迭代 在scRNA-seq和分析方法的发展中，已经发现在初级和 转移性动脉导管未闭。为了全面定义原发性和转移性pda的CAF谱系，我们将 利用scRNA-seq，scATAC-seq， 机器学习计算方法，以及检测RNA和蛋白质的原位组织分析方法 物种，如成像质谱仪(AIM 1)。我们假设，对动态的更深入的理解 发生在原发和转移性PDA中的CAF状态将指导特定治疗方案的选择。 为了补充这一分析，我们将确定新的caf亚型，并在一只新的小鼠身上研究它们的动力学。 具有可逆突变的Kras等位基因的模型(目标2)。此外，我们还将通过以下方式研究CaF激活途径 研究了几个与基质激活有关的基因，这些基因是用我们最近开发的 PDA导管内移植模型(目标2)。这些新的媒介似乎在PDA的进展中发挥了作用， 免疫抑制和基质激活，并可能代表新的PDA治疗靶点。最后，我们有 不同的CAF亚型具有不同的免疫调节功能。我们将测试组合 针对PDA微环境中不同的CAF亚型的策略，并研究这些策略的效果 关于小鼠动脉导管未闭模型的肿瘤进展(目标3)。此外，我们还将评估巨噬细胞-CAF的作用。 促进和维护CAF识别中的串扰。总体而言，该项目将阐明PDA CAF的多样性 以及癌细胞在调节CAF亚型中的作用。我们的结果将为CAF目标定位提供新的途径 我们打算为患有这种致命疾病的患者提供更好的治疗方法。

ABSTRACT Pancreatic ductal adenocarcinoma (PDA) is characterized by an extensive desmoplastic stroma that represents a major challenge for its effective treatment and improved survival of patients. We seek to define the composition and roles of cancer-associated fibroblasts (CAFs), the most abundant cell population in the PDA stroma, as a potential avenue for the development of new therapeutic strategies. Although CAFs have been historically considered tumor-promoting components, their ablation in pre-clinical and clinical studies have led to mixed outcomes, indicating the poorly understood complexity of CAFs. To investigate CAF biology, we previously established a pancreatic tumor organoid/fibroblast co-culture model. In addition, we performed single cell RNA- sequencing (scRNA-seq) of murine and human PDA specimens to characterize CAF composition at single-cell resolution. These analyses have revealed that fibroblasts are heterogeneous and comprised of at least three distinct subtypes with unique transcriptional and functional features. More so, this heterogeneity emphasizes the need to design therapies that selectively target the tumor-promoting CAF populations. Although our work has started to reveal the complex heterogeneity of fibroblasts in primary or metastatic PDA tissues, iterative developments in scRNA-seq and analysis methods have revealed four additional CAF subtypes in primary and metastatic PDA. To comprehensively define the CAF repertoire in primary and metastatic PDA, we will systematically differentiate CAF subtypes and their regulatory elements using scRNA-seq, scATAC-seq, machine-learning computational methods, and in situ tissue analytic methods that detect RNA and protein species, such as imaging mass cytometry (Aim 1). We hypothesize that a deeper understanding of the dynamic CAF states that occurs in primary and metastatic PDA will guide the selection of specific therapeutic regimens. To complement this analysis, we will identify new CAF subtypes and study their dynamics in a novel murine model with a reversible mutant Kras allele (Aim 2). Furthermore, we will study CAF-activating pathways by investigating several genes implicated in stromal activation that were revealed using our recently developed intraductal transplantation model of PDA (Aim 2). These new mediators appear to have roles in PDA progression, immunosuppression, and stromal activation, and may represent new PDA therapeutic targets. Finally, we have demonstrated different immunomodulatory functions of distinct CAF subtypes. We will test combinatorial strategies to target distinct CAF subtypes in the PDA microenvironment, and study the effect of these strategies on tumor progression in a murine PDA model (Aim 3). In addition, we will assess the role of macrophage-CAF crosstalk in promoting and maintaining CAF identify. Overall, this project will clarify the diversity of PDA CAFs and the role of cancer cells in regulating CAF subtypes. Our results will provide new avenues for CAF-targeting that we intend to translate towards improved therapies for patients afflicted by this lethal disease.