Modeling a hypoxia-integrated glioblastoma microenvironment to mimic tumor heterogeneity and chemoresistance

Glioblastoma (GBM) is a highly aggressive brain tumor in which hypoxia plays a central role in driving tumor progression, cellular plasticity, and resistance to treatment. In order to mimic these pathological features under in vitro conditions, a bioprinted GBM model was developed by integrating a PDMS-based hypoxia chip with a hydrogel composed of hyaluronic acid methacrylate (HAMA) and decellularized extracellular matrix (dECM), aiming to replicate the biochemical, mechanical, and oxygen-deprived conditions of native tumors. Glioblastoma (U87) and microglia (HMC3) cells were bioprinted within the hydrogel in the core and the periphery of the compartmentalized model, respectively. Hypoxic conditions were generated passively by placing a glass barrier and monitored using a fluorescence-based probe. The model was able to reproduce the key GBM features, including pseudopalisading necrosis (central Ki67^-/necrotic and peripheral Ki67⁺/proliferative cells) and a 32% increase in invasion distance under hypoxic conditions. Gene expression analysis revealed that hypoxic conditions induced the upregulation of proliferation (EGFR, Ki67), stemness (SOX2, NES), and invasion (MMP2, CD44, TGFβ) associated markers, while proteomic analysis showed increased glycolysis, HIF1 signaling, and amino acid biosynthesis. Drug testing with temozolomide (TMZ) demonstrated reduced sensitivity under hypoxic conditions, shown by a 56% increase in IC₅₀, reflecting clinically relevant therapy resistance. These findings show the ability of the model to mimic key properties of the GBM microenvironment at both phenotypic and molecular levels and offer a physiologically relevant platform to study GBM biology and evaluate therapeutic responses.