High-throughput glioblastoma-neurovascular assembloids model blood-tumor barrier disruption, tumor reprogramming and therapeutic efficacy

The blood-tumor barrier (BTB) in glioblastoma is an abnormal neurovascular interface that can be focally leaky yet still restrict effective therapy. We present a rapid human BTB model in which patient derived glioblastoma stem cells (GSCs), human brain endothelial cells, pericytes and astrocytes selfassemble within 72 hours into uniform three-dimensional "BTB assembloids". Thousands can be generated in parallel in standard plates, enabling high-throughput studies and drug testing. These assembloids establish spatial glioma-vascular and glioma-astrocyte niches with focal loss of endothelial tight-junction proteins, astrocyte recruitment and endothelial recruitment toward tumor clusters with localized junctional disruption at the tumor-vascular interface. BTB-conditioned GSCs acquire more invasive, stemness, mesenchymal and angiogenic programs compared with matched GSC neurospheres, and these BTB-conditioned states translate into greater engraftment and infiltrative growth in vivo, accompanied by vascular remodeling and BTB-like barrier disruption. The assembloid platform supports quantitative, "barrier-aware" assessment of compound access and retention in human BTB- (and BBB)-like contexts, revealing that functional P-glycoprotein efflux persists despite increased BTB permeability, thereby dissociating structural barrier leakiness from therapeutic accessibility. It is modular and compatible with iPSC-derived brain cells (for example brain endothelial cells), microglia or other immune components, thereby providing a route toward genotypeand immunity-specific modeling. Together, this establishes BTB assembloids as a scalable human system to interrogate glioblastoma-neurovascular crosstalk and to evaluate brain-tumor therapeutics.