A human iPSC-based neural spheroid platform for modeling glioblastoma infiltration using high-content imaging

Glioblastoma is the most aggressive adult brain tumor, characterized by resistance to therapy and high recurrence due to diffuse infiltration. To mimic glioblastoma migration, we developed a physiologically relevant co-culture model, combining patient-derived glioblastoma cell lines with cortical-like neural spheroids differentiated from human induced pluripotent stem cells. Using high-content imaging, we demonstrate that GBM1 and GBM20 cell lines migrate directionally along axons toward neural spheroids in live imaging assays and infiltrate spheroids extensively in endpoint assays, unlike non-cancerous neural stem cells. A proof-of-principle drug screen identified PF-573228 (FAK inhibitor) and Motixafortide (CXCR4 inhibitor) as potent suppressors of GBM1 and GBM20 infiltration, respectively. Bulk RNA sequencing revealed gene expression profiles correlating with invasive behavior and drug sensitivity. This platform offers a valuable model for studying glioblastoma infiltration along axons and provides proof-of-principle that migration can serve as a measurable and actionable phenotype to screen therapeutic vulnerabilities in glioblastoma.