Glioblastoma - Neuroblastoma co-cultured multicellular spheroid model for evaluating Temozolomide response

Glioblastoma is the most lethal primary brain tumor, and its resistance to therapy is increasingly understood to arise not from tumor cells alone but also from their integration into the surrounding neural circuitry. However, no existing preclinical spheroid model isolates the direct effect of neuronal cells on glioblastoma's drug response within a rapid, scalable 3D system. We developed a heterotypic 3D co-culture spheroid combining glioblastoma (U87-MG) with a neuronal lineage population (SH-SY5Y) via the agarose liquid overlay method to directly understand this gap. Subsequently, growth, morphology, hypoxia, and temozolomide (TMZ) response over 14 days against glioblastoma only and neuroblastoma only spheroids were characterized. The co-culture adopted a glioblastoma like compact, chemoresistant architecture yet, contrary to what this architecture alone would predict, it showed measurable chemosensitivity at every TMZ concentration tested, including 10M, a dose at which neither monoculture responded at all. This reveals that the neuronal lineage component actively sensitizes glioblastoma to chemotherapy independent of spheroid architecture. Drug-response data from glioblastoma only models may therefore not reflect true tumor behavior within its native neuronal environment, underscoring the need for neuron inclusive platforms in preclinical glioblastoma drug screening.