Abstract Pancreatic ductal adenocarcinoma (PDAC) develops within a dense, tumor microenvironment (TME) enriched with extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). PDAC tumors also rely heavily on glutamine metabolism. While ECM stiffness and nutrient availability each influence tumor behavior, their combined effects on CAF and PDAC phenotypes in 3D contexts are not well defined. We investigated how matrix stiffness and glutamine supplementation modulate oxidative stress, lipid metabolism, mitochondria, cellular architecture, and PD-L1 expression in CAFs and patient-derived PDAC cells. Soft (3A) and stiff (6A) collagen-based ECM gels were generated in-house. Primary human CAFs or PDAC lines (G43, G46) were embedded in 3D and assessed at 48 hr and 7 days. CAFs were analyzed using CellROX, JC-1, BODIPY, phalloidin, and PD-L1 staining. PDAC cells were cultured in basal DMEM, glutamine-supplemented DMEM, or organoid-like media. Mitochondria were detected using an anti-mitochondrial antibody with AF488 secondary. All samples were imaged in 3D at 20× magnification. ECM stiffness significantly altered CAF oxidative stress responses. At 48 hr, CAFs in soft and stiff ECM showed comparable but detectable CellROX signal, whereas by 7 days, stiff ECM produced a marked increase in oxidative stress with a higher number of strongly CellROX-positive nuclei. Quantification of fluorescence intensity confirmed a time-dependent rise in oxidative stress that was amplified under stiff ECM conditions. This increase aligned with elevated JC-1 J-aggregate formation at 7 days, indicating enhanced mitochondrial polarization. CAFs in stiff ECM also exhibited greater F-actin alignment, cell spreading, and lipid accumulation, suggesting coordinated metabolic and cytoskeletal adaptation to mechanical stress. PD-L1 expression likewise increased over time and remained higher in stiff ECM, supporting a role for mechanosensing in promoting immune-evasive CAF phenotypes. In PDAC cells, ECM stiffness and nutrient environment jointly controlled metabolic outputs. BODIPY staining revealed stiffness-dependent lipid droplet patterns, which were further amplified by organoid-like media. Glutamine supplementation increased mitochondrial signal intensity and reorganized mitochondrial networks in both G43 and G46, consistent with glutamine-driven metabolic activation. ECM stiffness also shaped F-actin architecture and mitochondrial localization, indicating a biomechanical-metabolic interaction. Together, these results show that ECM stiffness and glutamine availability reprogram CAF and PDAC metabolic and immune features. This work underscores the need to incorporate both mechanical and metabolic cues when modeling PDAC and identifies metabolic-immune pathways as potential therapeutic vulnerabilities within the TME. Citation Format: Jonathan Barajas, Carolyn Ruiz, Zhi Yang, Bo Han, Edward Agyare, Xueyou Zhu, Saun-Joo Yoon. ECM stiffness and glutamine availability rewire metabolic and immune phenotypes in 3D Co-culture models of pancreatic cancer and cancer-associated fibroblasts 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 771.
Barajas et al. (Fri,) studied this question.