Exogenous matrix‐free biomanufacturing of glioblastoma organoids enables autonomous assembly of neurovascular‐like structures and extracellular matrix

Abstract

Current glioblastoma (GBM) models often rely on chemically undefined exogenous extracellular matrices (ECMs) and serums that limit understanding of autonomous cellular behaviors driving tumor progression. Here, we present an exogenous ECM‐free biomanufacturing process for GBM organoids (GBOs) that recapitulate pathological development relative to size and spatial distribution. These GBOs exhibit a conserved glioma signature established from The Cancer Genome Atlas clinical datasets. Trajectory analyses of neurovascular unit (NVU) zonation, cell‐type differentiation, and basement membrane converged with clinical benchmarks highlighting a critical developmental milestone at the 2 mm diameter stage. Immunofluorescence revealed a matrix‐priming cascade of tenascin‐C (TNC) at 1 mm diameter and fibronectin (FN) at 1.5 mm diameter to provide the necessary biochemical cues and biophysical assembly for endothelial cells at 2 mm diameter. Lastly, the 2 mm diameter GBOs displayed a distinct vascular‐like area characterized by capillary‐scale lumen distributions (5–15 μm) and peak junctional complexity, similar to in vivo capillary beds. However, transcriptional divergences in arterial and proteoglycan markers suggest biochemical cues and morphology are insufficient for full physiological maturation without confirmed internal hemodynamic shear stress. Despite these results, our GBOs serve as a high‐fidelity structural framework for modeling GBM NVU‐like regions that may be used as a translatable platform for TNC‐ and FN‐targeted therapies designed to enhance drug delivery across the NVU.