Abstract 63: Orai1 regulates collagen secretion in pancreatic stellate cells and is enriched in fibrotic cancer-associated fibroblasts in PDAC

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a desmoplastic stroma driven by pancreatic stellate cells (PSCs) and cancer-associated fibroblasts (CAFs). The Ca2+-permeable ion channel ORAI1 is known to influence PSC proliferation and activation, but its role in collagen secretion and tumor fibrosis remains unclear. Experimental Procedures: Using the PSC cell line PS-1, we developed and validated a high-throughput fibrosis assay based on the collagen-binding peptide CNA-35-tdTomato. Collagen secretion was assessed following treatment with transforming growth factor β1 (TGF-β1) and/or vitamin C, with ORAI1 inhibition achieved via siRNA or Synta-66. In vivo, Orai1 expression was examined by immunohistochemistry in the genetically engineered murine PDAC model KPfC (Kraswt/LSL-G12D Tp53fl/+ PDX1Cre+). The intracellular Ca2+ concentration was assessed with the fluorescent Ca2+ indicator Fura-2. Results: TGF-β1 and vitamin C synergistically enhanced collagen deposition in PSCs, a process regulated by ORAI1. Vitamin C induced a significant rise in intracellular Ca2+ in PSCs, which was attenuated by the ORAI inhibitor Synta-66. ORAI1 expression was detected in αSMA+ CAFs in KPfC-derived PDAC tissue, with a higher prevalence in collagen-rich fibrotic tumor regions compared to non-fibrotic areas or non-tumorous tissue. In addition, vessel-lining endothelial cells also express ORAI1. Conclusions: Our findings highlight ORAI1 as a regulator of collagen secretion in PSCs and reveal its enrichment in CAFs within fibrotic PDAC stroma, underscoring its potential role in PDAC-associated desmoplasia. Citation Format: Zoltan Pethö, Ilka Neumann, Andrea Oeckinghaus, Albrecht Schwab, Rieke Schleinhege. Orai1 regulates collagen secretion in pancreatic stellate cells and is enriched in fibrotic cancer-associated fibroblasts in PDAC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 63.