Abstract A017: Collagen stiffness shapes the stromal microenvironment architecture in pancreatic cancer in vitro models

Abstract

Pancreatic ductal adenocarcinoma (PDAC) desmoplasia arises from pancreatic stellate cell (PSC) activation and progressive collagen I deposition, driving extracellular matrix (ECM) stiffening that sustains tumor progression, immune evasion, and therapy resistance. Current 2D and organoid models still poorly capture how ECM mechanics and early stroma–tumor crosstalk jointly shape this process. Here, we developed a tunable 3D organotypic platform of collagen type I scaffolds (0.5, 1.5, 2.5 mg/mL), culturing human PSCs alone or in co-culture with AsPC-1 or PANC-1 pancreatic cancer cells (60:40 ratio) and profiling cell morphology, PSC activation, epithelial-to-mesenchymal transition (EMT) markers, and secretome by immunofluorescence, Western blot, and cytokine array. Increasing collagen concentration raised the scaffold's elastic modulus (16–154 Pa) without altering porosity. Embedded PSCs progressively acquired elongated nuclei, reorganized actin architecture, and increased alpha-smooth muscle actin (α-SMA) expression, indicating stiffness-driven stromal activation that was further modulated by cancer cell co-culture. AsPC-1 cells underwent a cadherin switch (E-cadherin loss, N-cadherin gain) upon stromal contact and increasing stiffness, consistent with EMT, whereas PANC-1 cells maintained a stable mesenchymal phenotype reinforced by collagen abundance. Secretome analysis of the stiffest co-culture condition revealed that PSCs dominate the release of angiogenic, pro-inflammatory, and pro-fibrotic factors, shaping an immunosuppressive microenvironment. This tunable collagen-based organotypic platform recapitulates key features of early PDAC desmoplasia, identifying PSCs as primary mechanosensors and major drivers of a tumor-supportive secretome, and offers a tractable system for dissecting stroma–tumor crosstalk and testing stromal-targeted therapeutic strategies.