H3K27M drives OPC stemness and intrathecal therapeutic vulnerability in brainstem glioma organoids.

Abstract

Brainstem gliomas, particularly H3K27M-mutant diffuse midline gliomas (DMGs), lack effective therapies owing to anatomic inaccessibility, intact blood-brain barrier, and treatment resistance. Conventional models have low establishment rates and fail to preserve the native tumor microenvironment, limiting translational study for this rare disease. Here, we established a patient-derived brainstem glioma organoid (BSGO) platform with high success, faithfully recapitulating parental tumor features. Multi-omics and functional assays revealed that DMGs maintain oligodendrocyte precursor cell (OPC) stemness by dysregulating ACTIVIN signaling. To overcome delivery barriers and target stemness, we conducted an intrathecal drug screening and identified triple intrathecal therapy (methotrexate + cytarabine + dexamethasone, TIT) as a potent regimen against DMGs. In DMG patients, organoid-avatar guided personalized therapy induced sustained responses, with TIT-associated reductions in OPC markers reliably tracked by cerebrospinal fluid (CSF) proteomic profiling. Together, our study provides an organoid-based platform for mechanistic investigation and personalized intrathecal therapy screening and proposes TIT as a promising translational strategy for DMG.