Analysis of Prrx1-dependent regulation of tumor-associated fibroblasts in pancreatic ductal adenocarcinoma

胰腺导管腺癌中肿瘤相关成纤维细胞的 Prrx1 依赖性调节分析

• 批准号: 424465722
• 负责人: Professor Dr. Maximilian Reichert
• 金额: --
• 依托单位: Klinik und Poliklinik für Innere Medizin II: Gastroenterologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/424465722?language=en
• 关键词: Analysis; Prrx1; dependent; regulation; tumor

Pancreatic cancer is a complex disease in which cellular heterogeneity and plasticity are key characteristics. The tumor stroma, known as desmoplastic reaction, increases tumor complexity and its function in the carcinogenic process is critical. Recently, we identified the transcription factor Prrx1 as a plasticity driver during pancreatitis and carcinogenesis.Cancer-associated fibroblasts (CAFs) display abundant Prrx1 expression compared to cancer cells. Prrx1 orchestrates the transactivation of secreted factors such as tenascin-C, periostin, and HGF. Interestingly, these factors have been suggested to support tumor progression in various cancer types, including pancreatic cancer. To investigate the specific role of Prrx1 in tumor-fibroblast crosstalk, we developed a conditional knockout allele of Prrx1 in order to ablate Prrx1 specifically in fibroblasts in vitro and in vivo. Our in vivo findings demonstrate that Prrx1-ablated CAFs secret a higher amount of extracellular matrix and lead to better differentiated tumors, decreased circulating tumor cells, as well as, fewer metastases. Mechanistically, we discovered that Prrx1 knockout leads to remarkedly increased fibroblast activation as shown by alpha smooth muscle actin (αSMA), and collagen expression, as well as a higher forward migration index. Interestingly, co-culture experiments of tumor cells and CAFs revealed that CAFs render the process of EMT. Furthermore, these cells are also characterized by decreased HGF secretion, which was confirmed in our in vivo model by decreased HGF levels in the serum of these mice. This demonstrates that manipulating Prrx1 expression in the tumor stroma has a significant impact upon tumor development and morphology. To investigate further the role of Prrx1 in the process of tumor initiation, progression and metastasis, we will use the dual recombinase system Pdx1-Flp; FSF-KrasG12D/+; p53frt/+; Sm-22CreERT; Prrx1fl/fl to target specifically tumor cells as well as fibroblasts. Additionally, we are going to perform cytokine analyses from serum samples and RNA sequencing (RNAseq) from tumor cells as well as fibroblasts sorted directly from the pancreatic tumor by genetic lineage labeling. Based on these data we will further characterized the tumor-fibroblast interaction in in vitro assays e.g. using 3D co-culturing experiments, cytokine analyses and evaluating the role of CAF-Prrx1 regarding chemotherapy resistance. Importantly, secreted cytokines and RNAseq data will give us insights in the heterogeneity and plasticity of CAFs. The generated data will provide the fundament for translational research in the human model system, using primary human fibroblasts and organoids for drug screens and functional assays. Taken together, our results indicate that Prrx1 impacts upon several aspects of the crosstalk of tumor cells and CAFs and will pave the way to develop new therapeutic approaches targeting tumor-stroma interactions in PDAC.

胰腺癌是一种复杂的疾病，细胞异质性和可塑性是其关键特征。肿瘤基质，称为促纤维增生反应，增加了肿瘤的复杂性，其在致癌过程中的作用至关重要。最近，我们发现转录因子 Prrx1 是胰腺炎和癌变过程中的可塑性驱动因素。与癌细胞相比，癌症相关成纤维细胞 (CAF) 表现出丰富的 Prrx1 表达。 Prrx1 协调肌腱蛋白-C、骨膜素和 HGF 等分泌因子的反式激活。有趣的是，这些因素被认为支持各种癌症类型（包括胰腺癌）的肿瘤进展。为了研究 Prrx1 在肿瘤-成纤维细胞串扰中的具体作用，我们开发了 Prrx1 的条件敲除等位基因，以便在体外和体内特异性消除成纤维细胞中的 Prrx1。我们的体内研究结果表明，Prrx1 消除的 CAF 分泌更多的细胞外基质，导致肿瘤分化更好、循环肿瘤细胞减少以及转移更少。从机制上讲，我们发现 Prrx1 敲除会导致成纤维细胞活化显着增加，如α平滑肌肌动蛋白（αSMA）和胶原蛋白表达以及更高的前向迁移指数所示。有趣的是，肿瘤细胞和CAF的共培养实验表明，CAF参与了EMT过程。此外，这些细胞的特征还在于 HGF 分泌减少，这在我们的体内模型中通过这些小鼠血清中 HGF 水平降低得到证实。这表明操纵肿瘤基质中的 Prrx1 表达对肿瘤的发育和形态具有显着影响。为了进一步研究Prrx1在肿瘤发生、进展和转移过程中的作用，我们将使用双重组酶系统Pdx1-Flp； FSF-KrasG12D/+； p53frt/+； Sm-22CreERT； Prrx1fl/fl 专门针对肿瘤细胞和成纤维细胞。此外，我们还将对血清样本进行细胞因子分析，对肿瘤细胞以及通过遗传谱系标记直接从胰腺肿瘤中分选的成纤维细胞进行 RNA 测序 (RNAseq)。基于这些数据，我们将进一步表征体外测定中的肿瘤-成纤维细胞相互作用，例如使用 3D 共培养实验、细胞因子分析并评估 CAF-Prrx1 在化疗耐药方面的作用。重要的是，分泌的细胞因子和 RNAseq 数据将使我们深入了解 CAF 的异质性和可塑性。生成的数据将为人类模型系统中的转化研究提供基础，利用原代人类成纤维细胞和类器官进行药物筛选和功能测定。总而言之，我们的结果表明 Prrx1 对肿瘤细胞和 CAF 串扰的多个方面产生影响，并将为开发针对 PDAC 中肿瘤-基质相互作用的新治疗方法铺平道路。