Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense fibrotic stroma and stiff extracellular matrix (ECM) that together shape the tumor microenvironment (TME) driving disease progression. Mechanical stiffness regulates fibroblast activation and inflammatory signaling through mechanotransduction, yet its contribution to tumor-stroma crosstalk remains incompletely understood. To investigate this, we developed a tunable 3D co-culture organoid model by embedding PDAC cells alone or in combination with stromal cells within soft or stiff collagen-based matrices. Conditioned media were collected one week post-seeding and analyzed via ELISA for TNF-α, IL-1β, and IL-6. Cell viability was assessed using CellTiter-Glo, and fibroblast activation markers (α-SMA, IL-6) were quantified by RT-PCR. Finally, immunostaining was performed to evaluate organoid morphology. Our results show that PDAC cells secreted elevated TNF-α in soft matrices, suggesting early cytokine-driven remodeling. Fibroblasts in stiff matrices exhibited a CAF-like phenotype with strong IL-1β secretion, while both fibroblasts and MSCs produced significantly higher IL-6 under stiff conditions, further amplified in PDAC co-cultures. Stiff ECM enhanced stromal viability and induced α-SMA and IL-6 upregulation, confirming mechanotransductive activation. Morphological analysis revealed that PDAC organoids formed larger, well-organized 3D structures under stiff conditions, indicating matrix-dependent growth and adaptation. We conclude that ECM stiffness acts as a potent regulator of stromal activation and inflammatory cytokine signaling in PDAC, establishing a tumor-promoting feedback loop that sustains fibrosis and inflammation. Future studies will incorporate mechanotransduction inhibitors (e.g., TGF-β or integrin blockers) and extended time courses to further define the dynamics of stiffness-driven inflammation and remodeling in the PDAC microenvironment. Citation Format: Carolyn M. Ruiz-Rivera, Ba Xuan Hoang, Jamel Ali, Thomas D. Schmittgen, Bo Han. A tunable 3D organoid model reveals stiffness-dependent stromal activation and cytokine signaling in pancreatic cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 762.
Ruiz-Rivera et al. (Fri,) studied this question.