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

Glioblastoma is the most aggressive adult brain tumour, characterised by resistance to therapy and high recurrence due to diffuse infiltration. 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 GBM20 and GBM1 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 GBM20 and GBM1 infiltration, respectively. Bulk RNA sequencing revealed gene expression profiles correlating with invasive behaviour 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.