ID #1150 De novo H3.3K27M-altered diffuse midline glioma in human brainstem organoids to dissect GD2 CAR T cell function
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 V U Collot, Daan J Kloosterman, Farid Keramati, Mieke Roosen, Sam de Blank, Esmee van Vliet, Mario Barrera Román, Lucrezia C D E Gatti, Jürgen Kuball, Zsolt Sebestyen, Marcel Kool, Sara Patrizi, Evelina Miele, Annette Kuenkele, Mariëtte E G Kranendonk, Annelisa Cornel, Stefan Nierkens, Christian Mayer, Hendrik G Stunnenberg, Anna Alemany, María Alieva Krasheninnikova, Anne C RiosAbstract
Diffuse midline glioma (DMG) is a highly aggressive and untreatable paediatric cancer primarily arising in the pontine brainstem region, necessitating patient-representative models to advance therapy development. Here, we developed an FGF4-driven human brainstem organoid model and used it to engineer de novo H3.3K27M-altered DMG by introducing H3.3K27M together with partner alterations dnTP53 and PDGFRA-D842V. We demonstrate that brainstem–pontine glial specification is critical for DMG tumorigenesis, yielding diffusely infiltrative tumours that recapitulate patient-like intratumoral heterogeneity and molecular patient-like features. Using a month-long experimental window, prolonged GD2 CAR T-cell treatment mirrored clinically observed response patterns and revealed extensive transcriptional and functional heterogeneity across CAR T populations, including potent effector states as well as dysfunctional or exhausted phenotypes. To incorporate key elements of the tumour–immune microenvironment, we introduced primitive macrophage progenitors that matured into microglia and adopted DMG-associated features. In this immune-enhanced setting, microglia reduced tumour control, amplified suppressive signalling, and exposed CAR T-cell functional states most vulnerable to microglia-mediated immunosuppression.Together, this work establishes a human, brainstem-specific DMG organoid platform that enables mechanistic interrogation of tumour evolution and immune therapy response under sustained treatment pressure. By capturing infiltrative growth, heterogeneity, and microglia–T-cell crosstalk in vitro, the model provides a translationally relevant system to guide optimisation of immunotherapies for DMG.