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

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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.