Characterization of Native Extracellular Matrix of Patient-Derived Glioblastoma Multiforme Organoids

This study presents a scalable method to generate patient-derived glioblastoma organoids (GBOs) without exogenous extracellular matrix (ECM) scaffolds (e.g., Matrigel) by pre-forming glioblastoma spheroids (400–600 µm) and then culturing them in a dimpled bioreactor under sublethal shear stress. Organoids reproducibly reached ≥4 mm diameter within ~6–7 weeks and transitioned from homogeneous spheroids to tissue-like, ECM-rich structures when they exceeded ~1–2 mm. RNA-seq revealed the largest transcriptomic shift between 1 mm and 2 mm GBOs, with ECM-associated gene signatures strongly upregulated at ≥2 mm and collagen VI (COL6A3) emerging as a prominent ECM-related change. Biochemical assays and histology confirmed increased sulfated GAGs and collagen in GBOs relative to spheroids, with spatial localization of HA/sGAGs toward the periphery and altered distribution of collagen IV/VI. Mechanical testing showed spheroids (~300 Pa) and 2 mm GBOs (~600 Pa) exhibited stiffness comparable to diffuse low-grade gliomas and demonstrated cyclic hysteresis behavior. Together, the results support GBOs with natively synthesized ECMs as a more physiologically relevant platform for studying early-stage GBM microenvironment development and ECM-targeted therapeutic strategies.