Transcriptomic profiling of BrO, DMGO and GD2 CAR T cells

Diffuse midline glioma (DMG) is a lethal pediatric brain cancer with limited treatment options. We developed a human brainstem organoid model to study H3.3K27M-driven DMG, enabling formation of patient-like tumors with molecular heterogeneity. To characterize this model, we performed a time-course transcriptomic analysis of healthy organoid development. We then introduced a DMG-inducing plasmid cocktail via electroporation and profiled the resulting tumors using single-cell RNA sequencing. Tumors were treated with GD2 CAR T cells, which were subsequently analyzed by single-cell RNA sequencing to capture treatment responses. Finally, we assessed the role of brain-resident myeloid cells by performing sort-seq on PMP-DMGO cocultures with or without CAR T cell treatment. This platform supports extended in vitro experimentation and reveals immune dynamics relevant for therapy development.