The extracellular matrix HA promotes the YAP/hypoxia axis of glioblastoma cells on 3D agar/HA scaffolds

The tumour microenvironment of glioblastoma (GBM) as defined by mechanical heterogeneity, hypoxia, and hyaluronic acid (HA)–rich extracellular matrix (ECM), is a highly dynamic milieu which influences tumour progression and therapeutic resistance. Yet, how these cues converge to regulate mechanosensitive pathways in 3D remains poorly understood. Here, we engineered agar-based porous hydrogels functionalized with HA to independently tune stiffness and ECM composition, creating biomimetic 3D niches for GBM cells. The presence of HA coating showed to increase hydrogel stiffness, promote YAP/TAZ nuclear localisation, and elevate total LATS1/2 expression, consistent with Hippo pathway feedback regulation. Over time, however, hypoxic niches emerged that destabilised this feedback, enabling sustained YAP nuclear activity. HA also modulated OCT4 and Sox2 localisation and attenuated HIF-1α nuclear accumulation, indicating that HA also modulates the spatial distribution and nuclear accumulation of HIF-1α. Also, a cooperative regulation through the HA–CD44–CXCR4 axis, showed integrated biochemical and mechanical signals to reinforce YAP/HIF crosstalk. Together, these results reveal a dynamic interplay between ECM stiffness, HA signalling, and hypoxia in shaping YAP/HIF crosstalk and stem-like phenotypes in GBM and establish our hydrogel platform as a powerful tool to dissect and therapeutically exploit these interactions.