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

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。肿瘤间质被称为促结缔组织反应，它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在胰腺炎和癌症发生中起可塑性驱动作用。与癌细胞相比，肿瘤相关成纤维细胞(CAF)显示出丰富的Prrx1表达。Prrx1协调Tenascin-C、Periostin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的肿瘤进展，包括胰腺癌。为了研究Prrx1在肿瘤-成纤维细胞串扰中的特定作用，我们开发了Prrx1的条件性基因敲除等位基因，以便在体外和体内特异性地去除成纤维细胞中的Prrx1。我们的体内研究结果表明，Prrx1消融的CAF分泌更多的细胞外基质，导致更好的分化肿瘤，减少循环中的肿瘤细胞，以及更少的转移。在机制上，我们发现Prrx1基因敲除导致成纤维细胞活化显著增加，如α-平滑肌肌动蛋白(α-SMA)和胶原表达，以及更高的向前迁移指数。有趣的是，肿瘤细胞和CAF的共培养实验表明，CAF呈现EMT的过程。此外，这些细胞还具有HGF分泌减少的特征，这在我们的活体模型中得到了证实，这些小鼠血清中的HGF水平降低了。这表明操纵Prrx1在肿瘤间质中的表达对肿瘤的发展和形态有重大影响。为了进一步研究Prrx1在肿瘤发生、发展和转移过程中的作用，我们将使用双重重组酶系统Pdx1-FLP；FSF-KrasG12D/+；p53frt/+；Sm-22CreERT；Prrx1fl/fl特异性靶向肿瘤细胞和成纤维细胞。此外，我们将从血清样本中进行细胞因子分析，并从肿瘤细胞和通过遗传谱系标记直接从胰腺肿瘤中分离的成纤维细胞进行RNA测序(RNAseq)。基于这些数据，我们将在体外实验中进一步表征肿瘤-成纤维细胞的相互作用，例如使用3D共培养实验、细胞因子分析和评估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。生成的数据将为人体模型系统中的转译研究提供基础，使用原代人类成纤维细胞和有机化合物进行药物筛选和功能分析。综上所述，我们的结果表明Prrx1对肿瘤细胞和CAF的串扰的几个方面产生了影响，并将为开发针对PDAC中肿瘤-间质相互作用的新的治疗方法铺平道路。

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.