Glutamate Drives Glioblastoma Invasion in Three-Dimensional Hyaluronic Acid Hydrogels

Glioblastoma (GBM) tumors are characterized by an excess of extracellular glutamate, one important source of which is the tumor cells themselves. This abundance of glutamate promotes GBM proliferation, migration, and therapeutic resistance, and causes excitotoxicity in nearby neurons. However, despite glutamate’s clear role in promoting GBM aggression, the exact mechanisms through which excess glutamate drives these phenotypes, particularly three-dimensional (3D) invasion, remain incompletely understood. To address this gap, we used a 3D brain-mimetic hyaluronic acid (HA) hydrogel to investigate the role of glutamate signaling in GBM 3D invasion. We demonstrate that inhibiting the glutamate N -methyl- d -aspartate receptor (NMDAR) reduces invasion from 3D tumorspheres, a result that is reproducible across multiple continuous culture models and a patient-derived xenograft cell line. We then conducted glutamate-driven invasion studies in 3D HA-based devices that can be microdissected to isolate and differentially analyze invasive and noninvasive cells. Transcriptomic analysis of invasive, noninvasive, and drug-treated populations of cells reveals that NMDAR inhibition suppresses several pathways associated with the mechanobiology of invasion, including matrix remodeling and collagen deposition. Correspondingly, supplementation with exogenous collagen VI partially rescued 3D invasion. Our work speaks to the potential value of biomaterial platforms for identifying the autocrine and paracrine mechanisms through which neurotransmitters fuel GBM invasion. Impact Statement Glutamate is an abundant signaling molecule in the glioblastoma (GBM) microenvironment and important driver of disease progression; however, the mechanisms through which glutamate promotes invasion in three-dimensional (3D) environments remain poorly understood. By combining engineered hyaluronic acid hydrogel platforms and transcriptomic analysis, we determine that blocking glutamate signaling through the N -methyl- d -aspartate receptor suppresses invasion in 3D by interfering with extracellular matrix remodeling processes. This work highlights the utility of biomaterial platforms in dissecting neurotransmitter signaling in GBM invasion.