Modeling medulloblastoma pathogenesis and treatment in human cerebellar organoids.

Medulloblastoma (MB) groups 3 and 4 arise from human-specific developmental contexts that remain experimentally inaccessible, limiting mechanistic insight into tumor initiation. We generated cerebellar organoids (CbOs) from expanded potential stem cells (EPSCs) and performed integrated single-cell transcriptomic and DNA methylation analyses, revealing discrete human rhombic lip progenitor populations whose developmental programs align with group 3- and group 4-associated lineages. Using c-MYC overexpression as an initiating oncogenic event, we demonstrated that these lineage-restricted progenitors are susceptible to neoplastic transformation, generating tumors with group 3 MB molecular features in vitro and in vivo. We further established a CbO-MB coculture system that preserves malignant and nonmalignant cellular compartments, hence enabling interrogation of tumor-microenvironment interactions and providing simultaneous readouts of antitumor efficacy and cerebellar toxicity upon drug treatment. Receptor-ligand modeling in this system identifies TGFβ paracrine signaling as a microenvironmental pathway supporting MB growth, which we validated pharmacologically. Further analyses of cocultured MB cells revealed a myogenic differentiation program enriched in MYC-driven MB and recurrent disease, which is associated with poorer prognosis. Together, this work establishes a collection of CbO models for studying group 3/4 MB initiation, growth, and microenvironmental dependencies and provides a tractable platform for further mechanistic and therapeutic investigation of these tumors.