A method for detecting stemness of glioblastoma: 3D-tumor spheroid assay

Glioblastoma is one of the most common primary malignant brain tumors, characterized by high aggressiveness and chemoresistance. Glioma stem like cells (GSCs) are recognized as critical drivers of tumor initiation, intratumoral heterogeneity, and treatment resistance. This protocol establishes a standardized 3D-tumor spheroid assay to functionally evaluate tumor cell stemness, providing a reproducible platform for investigating glioma malignant phenotypes and screening therapeutic strategies targeting GSCs. This streamlined 96 well assay reduces detection time and resource requirements compared to traditional multi round sphere forming workflows, thereby facilitating high throughput drug screening and detailed mechanistic studies of glioma biology in both research and preclinical settings.