Nested pediatric low-grade glioma cerebral organoid avatars reveal glutamatergic neuron stromal growth dependency.

Pilocytic astrocytomas (PAs), the most common pediatric brain tumor, grow slowly and depend heavily on their local brain microenvironment, hindering accurate humanized model development. To surmount this barrier, we developed mixed assembloids of PAs nested within human iPSC-cerebral organoids (PANCOs) that recapitulate PA histopathology and growth. Consistent with their cell-intrinsic ERK dependence, MEK inhibition reduces PANCO proliferation, whereas clozapine-N-oxide-activatable neural progenitors integrate into PANCOs, preferentially differentiate into glutamatergic neurons, and increase cell-extrinsic PA proliferation, which is blocked by glutamate receptor inhibition. These accurate in vitro pediatric PA avatars provide tractable platforms to discover and validate PA cell-intrinsic and cell-extrinsic growth dependencies.