Patient-derived pediatric brain tumor orthotopic xenografts and tumor organoids faithfully recapitulate primary tumors.

Extensive molecular analyses by many groups have revealed a heterogenous landscape of embryonal brain tumors, but patient-derived tumor organoid (TO) model development has remained limited. Here, we describe the establishment of TO and TO xenografts (TOX) from patient-derived orthotopic xenografts (PDOXs) of medulloblastoma, embryonal tumor with multilayer rosettes, and atypical teratoid rhabdoid tumors. DNA methylation, bulk- and single-cell RNA sequencing, and whole-genome sequencing demonstrated that TOs and TOXs faithfully recapitulate the epigenetic, transcriptomic, and genetic landscape of PDOXs, as well as replicate the intratumor cellular heterogeneity of PDOXs that are often lost in established cell lines. We show that TOs and PDOXs have similar drug responses. The development of embryonal brain TOs will facilitate in vitro functional assays, including high-throughput drug or CRISPR screens, without the need for fresh tumors from tumor-bearing mice. This will accelerate the identification and validation of vulnerabilities and therapeutic strategies for preclinical testing toward clinical trials.