Collagen stiffness shapes the stromal microenvironment architecture in pancreatic cancer in vitro models

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a progressive desmoplastic reaction in which collagen type I accumulation remodels the extracellular matrix (ECM) and profoundly influences tumor-stroma interactions. However, the specific contributions of matrix mechanics and composition to stromal activation and cancer cell behavior remain poorly understood. Here, we developed a mechanically tunable collagen type I-based organotypic platform to investigate how ECM composition and mechanics regulate reciprocal interactions between pancreatic stellate cells (PSCs) and cancer cells during PDAC progression. Hydrogels with increasing collagen content were mechanically characterized (complex modulus ranging from approximately 16 to 154 Pa) and used to establish mono- and co-cultures of PSCs with AsPC-1 and PANC-1 cells. Progressive collagen enrichment promoted coordinated nuclear and cytoskeletal remodeling in PSCs together with increased α-SMA expression. The two pancreatic cancer cell lines displayed distinct responses, reflecting their phenotypic heterogeneity, while co-culture enhanced tumor-stroma crosstalk and promoted a pro-tumorigenic secretory profile. Comparison with mechanically matched Matrigel cultures demonstrated that matrix stiffness alone was insufficient to reproduce the coordinated remodeling observed in collagen hydrogels, revealing that collagen type I-specific biochemical cues drive the structural adaptation of PDAC cells. YAP localization and conditioned medium experiments further distinguished the roles of mechanotransduction and paracrine signaling. Overall, this study establishes a simple, reproducible, and mechanically tunable organotypic platform that recapitulates key features of PDAC desmoplasia and provides a versatile in vitro model for mechanistic studies and therapeutic screening.