Understanding proneural-mesenchymal transition using patient-derived glioma stem-like cell (GSC) organoids and engineered extracellular matrix.

Glioblastoma multiforme (GBM) is a highly 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 (GSCs), which reside in specialized perivascular niches and drive tumor progression, recurrence, and therapeutic resistance. The blood-brain barrier, coupled with the complex and dynamic tumor microenvironment, poses significant challenges for both treatment and mechanistic investigation. 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 glioma stem cells (GSC) derived Matrigel spheroid system that transitions into organoids and enables integration into engineered microenvironments. Our model incorporates GSC organoids representing proneural and mesenchymal GBM subtypes, a synthetic engineered extracellular matrix (eECM), and endothelial cells (ECs) seeded on the matrix surface. We evaluated the expression of subtype-specific, pro-angiogenic, stemness, and differentiation markers under increasingly complex co-culture conditions. Our results show that Matrigel-derived GSC spheroids progressively differentiate into organoids over two weeks, with significantly enhanced expression of cell-specific markers in the presence of ECs. Encapsulation of these organoids within eECM, combined with EC co-culture, further promoted cellular invasion and induction of GBM associated genes. This in situ encapsulation strategy enables real-time observation of GSC behavior in a tunable microenvironment that mimics key features of the native tumor niche. Together, this platform provides a physiologically relevant and modular in vitro system for investigating GBM pathophysiology. It holds promise for uncovering tumor-specific cellular dependencies, studying GSC-vascular interactions, and conducting high-throughput drug screening under controlled, biomimetic conditions.