Modeling Hypoxia Integrated Glioblastoma Microenvironment to Mimic Tumor Heterogeneity and Chemoresistance

Abstract

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 with the hydrogel into the core and the periphery of the compartmentalized model, respectively. Hypoxic conditions were generated passively through a barrier and monitored by 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 hypoxic conditions induced the upregulation of proliferation (EGFR, Ki67), stemness (SOX2, NES), and invasion (MMP2) 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, evidenced by a 56% increase in IC50, reflecting clinically relevant therapy resistance. These findings showed the ability of the model to mimic key properties of the GBM microenvironment at both phenotypic and molecular levels and offers a physiologically relevant platform to study GBM biology and evaluate therapeutic responses.