GelMA Versus Agarose Hydrogels in Pancreatic Cancer 3D Spheroid Modeling: Effects on Morphology, HIF-1α Expression, and Gemcitabine Response

Given its highly aggressive nature and poor clinical outcome, pancreatic ductal adenocarcinoma (PDAC) requires physiologically relevant in vitro models that more accurately reflect tumor biology and drug response. In this study, adhesive and non-adhesive hydrogel microenvironments were comparatively evaluated for pancreatic cancer spheroid modeling using PANC-1 and MIA PaCa-2 cells. Gelatin methacryloyl (GelMA) hydrogels were synthesized, photocrosslinked, and optimized in terms of stability, swelling, degradation, and cytocompatibility, while 3% agarose was used as a non-adhesive counterpart. Although the optimized GelMA formulation showed adequate structural stability and no cytotoxicity, it did not support spheroid formation. In contrast, agarose enabled the formation of compact, viable, and proliferative spheroids in both cell lines. Agarose-derived spheroids exhibited time-dependent growth, positive Ki-67 staining, and increased HIF-1α expression under 3D conditions, indicating the establishment of hypoxia-associated tumor-like microenvironments. Gemcitabine treatment induced a time-dependent reduction in spheroid viability, while viable cell populations persisted throughout exposure, reflecting the heterogeneous therapeutic response typical of 3D tumor models. Overall, these findings provide a comparative, microenvironment-based assessment of pancreatic cancer spheroid modeling, indicating that hydrogel-dependent differences in adhesivity and structural dynamics are important determinants of spheroid assembly, hypoxia-associated molecular adaptation, and chemotherapeutic response. Overall, these findings provide a comparative, microenvironment-based assessment of pancreatic cancer spheroid modeling, indicating that hydrogel-dependent differences in adhesivity and structural dynamics are important determinants of spheroid assembly, hypoxia-associated molecular adaptation, and chemotherapeutic response. Overall, these findings provide a comparative, microenvironment-based assessment of pancreatic cancer spheroid modeling, indicating that hydrogel-dependent differences in adhesivity and structural dynamics are important determinants of spheroid assembly, hypoxia-associated molecular adaptation, and chemotherapeutic response.