Fibroblast-derived extracellular vesicles promote organ-specific tumor growth and chemoresistance in pancreatic cancer liver metastasis via ptch2 downregulation

Pancreatic ductal adenocarcinoma (PDAC) is the 3 rd leading cause of cancer death in USA with a 5-year survival rate of only 13%. Early diagnosis is very difficult and 53% of patients are diagnosed after metastasis has already occurred, with liver being the most frequently affected site. Both primary tumors and metastases have dense desmoplastic stroma, which contributes significantly to chemoresistance. However, the mechanisms by which primary and metastatic fibroblasts influence tumor behavior remain understudied. This study aims to investigate the contributions of fibroblasts-derived extracellular vesicles (EVs) at the primary versus metastatic sites. To achieve this, we developed tunable 3D biomimetic models that replicate aspects of the tumor microenvironment (TME) of primary PDAC and the liver metastatic niche (LMN). We used matched primary PDAC and liver metastatic (LM) organoids derived from the Kras +/LSL-G12D ; Trp53 +/LSL-R172H ; Pdx1-Cre genetically engineered mouse model of PDAC, combined with either primary PDAC or liver fibroblasts. We hypothesized that primary and metastatic site derived fibroblasts possess distinct properties influenced by their different TMEs, that lead to organ-specific effects on tumor growth and chemoresistance. Supporting this hypothesis, fibroblasts enhanced tumor growth and chemoresistance to gemcitabine, only when paired with the organoids from the same site. Fibroblast-derived EVs were identified as critical drivers of this site-specific tumor support. Mechanistically, we found that expression of Ptch2 , a tumor suppressor in the Sonic Hedgehog (SHH) signaling pathway, is significantly decreased in organoids exposed to site-matched fibroblasts or their EVs, while EV depletion restores Ptch2 expression. Finally, we pharmacologically modulated the SHH pathway via a small molecule Ptch2 agonist, Robotnikinin, which appeared to reduce organoid growth and chemoresistance in our models, indicating a potential role for SHH pathway targeting. These findings suggest that site-specific fibroblast-derived EVs may carry distinct SHH ligands that differentially interact with Ptch2 in tumor cells of the corresponding site, which is currently under investigation. Taken together, our findings underscore the importance of developing models to elucidate the pivotal role of the organ-specific microenvironment in driving tumor progression. Our tunable 3D biomimetic model faithfully recapitulates the tumor-stroma interactions and provides a platform to evaluate therapeutic strategies aimed at disrupting fibroblast-tumor crosstalk. Defining how SHH signaling contributes to tumor growth and chemoresistance driven by fibroblast-derived EVs in an organ-specific manner can potentially uncover novel therapeutic strategies for targeting the metastatic niche in pancreatic cancer.