Abstract Background Pediatric brain tumors are the leading cause of cancer-related death in children. Developing novel therapies requires biologically accurate models that reliably predict patient responses. Organoids have emerged as a promising platform for preclinical drug testing, but their development has been limited by low formation rates, scarce tissue availability, and the inability of standard media to meet the distinct growth requirements of tumors from different brain regions. Methodology We developed a series of novel, customized growth media formulations containing a cocktail of growth factors specific to different brain locations during brain development to generate tumor organoids from patient tumors and PDOX models. Tumor tissue was dissociated and cultured to form sphere-like organoids. Organoids were characterized by immunohistochemistry, single-cell RNA sequencing, and whole-exome sequencing. High-throughput screening (HTS) of 90 compounds, pre-selected from prior active hits identified in paired PDOX-derived neurosphere and monolayer cultures, was performed on matched organoids. Results We generated 121 organoids with high success rates: 85.3% from patient tumors (medulloblastoma 11/11, high-grade glioma 11/13, low-grade glioma 27/31, ependymoma 5/6, DIPG 3/5) and 65.5% from PDOX models (medulloblastoma 29/35, high-grade glioma 13/18, low-grade glioma 5/14, ependymoma 1/3, DIPG 7/14). Organoids recapitulated key histological features and expressed lineage and stemness markers (NESTIN, GFAP, NEUN, SOX2, BMI1). Single-cell RNA sequencing of 7 matched tumor–organoid pairs revealed preservation of neoplastic cellular states and transcriptional heterogeneity. Whole-exome sequencing confirmed conservation of somatic variants and copy number alterations. HTS identified 26 drugs with concordant activity in matched organoids, targeting pathways involved in pHGG survival, invasion, and therapeutic resistance. Conclusions Our location-matched, developmentally informed growth media enables robust and reproducible generation of pediatric brain tumor organoids that faithfully mimic parental tumors. This platform provides a powerful tool for preclinical drug screening and holds promise for accelerating the discovery of effective therapies for pediatric brain tumors.
Suárez et al. (Tue,) studied this question.