Understanding Glioblastoma Dynamics Using 3D Organoids and Engineered Extracellular Matrix

ABSTRACT Glioblastoma multiforme (GBM) is an aggressive, angiogenic WHO grade IV glioma marked by rapid progression, therapeutic resistance, and poor prognosis. A defining feature of GBM is the presence of glioma stem‐like cells (GSC), which reside in specialized perivascular niches and drive tumor progression, recurrence, and therapeutic resistance. Current in vitro GBM models inadequately recapitulate the structural and biochemical cues of the native perivascular niche due to the absence of functional vasculature and brain‐mimetic extracellular matrix (ECM), limiting their physiological relevance and predictive power. To address the limitations of existing in vitro GBM models, we developed a patient‐derived GSC derived Matrigel spheroid system that transitions into organoids and enables integration into engineered microenvironments. Our model incorporates GSC spheroids representing proneural and mesenchymal GBM subtypes, a synthetic engineered extracellular matrix (eECM), and endothelial cells (EC) seeded on the matrix surface. Our results show that Matrigel‐derived GSC spheroids progressively differentiate over two weeks, with significantly enhanced expression of cell‐specific markers in the presence of EC. Encapsulation of these organoids within eECM, combined with EC co‐culture, further promoted induction of GBM‐associated genes. Together, this platform provides a modular in vitro system for investigating GBM pathophysiology.