Nils Bessler, Amber K. L. Wezenaar, Hendrikus C. R. Ariese, Celina Honhoff, Noëlle Dommann, Ellen J. Wehrens, Cristian Ruiz Moreno, Thijs J. M. van den Broek, Raphaël Collot, Daan J. Kloosterman, Farid Keramati, Mieke Roosen, Sam de Blank, Esmée J. van Vliet, Mario Barrera Román, Lucrezia C. D. E. Gatti, Ali Ertürk, Jürgen Kuball, Zsolt Sebestyén, M. Kool, Sara Patrizi, Evelina Miele, Annette Künkele, Mariëtte E.G. Kranendonk, Annelisa M. Cornel, Stefan Nierkens, Christian Mayer, Hendrik G. Stunnenberg, Anna Alemany, María Alieva, Anne C. Rios
Diffuse midline glioma (DMG) is a highly aggressive and untreatable pediatric cancer primarily arising in the pontine brainstem region, necessitating the development of representative models for treatment advance. Here we developed an FGF4-driven human brainstem organoid model, which we used to genetically engineer H3.3K27M-altered DMG. We demonstrated that brainstem pontine glial specification is critical for DMG tumorigenesis, yielding infiltrative tumors that recapitulate patient-representative intratumoral heterogeneity. Prolonged GD2 chimeric antigen receptor (CAR) T cell treatment mirrored clinical outcomes and revealed extensive transcriptional heterogeneity, from which both potent effector and dysfunctional CAR T cell populations could be identified. Furthermore, incorporation of myeloid cells generated DMG-specific microglia that reduced treatment efficacy and revealed CAR T cell functional states most vulnerable to microglia-mediated immunosuppression. Thus, we present a representative DMG model offering a months-long experimental window in vitro, which we leveraged to delineate CAR T cell functionality and microglial impact, aiding therapy development for this devastating disease.