Vascularized glioblastoma-on-a-chip reveals microglia-mediated regulation of tumor invasion and stemness.

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor in adults, characterized by rapid progression and poor prognosis. Central to GBM pathobiology are glioma stem cells (GSCs), which preferentially localize within the perivascular niche (PVN) and sustain tumor growth, invasion, and therapeutic resistance through dynamic interactions with the surrounding PVN microenvironment. Notably, microglia (MG), the brain-resident immune cells, represent a substantial fraction of the GBM PVN and play a pivotal role in establishing an immunosuppressive tumor microenvironment (TME). However, the mechanisms by which MG interacts with the brain PVN and how this, in turn, regulates GSC behavior remain poorly understood. To systematically investigate these complex cellular interactions, herein we engineered a compartmentalized human GBM PVN-on-a-Chip (GPoC) model featuring a perfusable brain microvascular network surrounding a tumor core composed of patient-derived GSCs and a stromal region containing brain-resident MG. The presented biomimetic 3D model recapitulates the structural and functional features of the native PVN, enabling well-controlled experimental interrogation and spatial visualization of tumor-immune-vascular crosstalk. Through invasion assay, real-time live imaging, immunofluorescent staining, and secretome profiling, it was evident that the GSC cells exhibited enhanced invasion in the presence of both MG and vasculature. Furthermore, pharmacological intervention studies revealed attenuated GB3 migration under tri-culture conditions, highlighting that the migratory phenotype of the tumor cells is, in part, driven by MG. Overall, the engineered GPoC platform serves as a physiologically relevant human brain tumor model for dissecting MG-mediated mechanisms and advancing therapeutic targeting of the GBM microenvironment.