De novo H3.3K27M-altered diffuse midline glioma in human brainstem organoids to dissect GD2 CAR T cell function

Bessler, N.; Wezenaar, A.K.L.; Ariese, H.C.R.; Honhoff, C.; Dommann, N.; Wehrens, E.J.; Moreno, C.R.; Broek, T.J.M. van den; Collot, R.V.U.; Kloosterman, D.J.; Keramati, F.; Roosen, M.; Blank, S. de; Vliet, E. van; Román, M.B.; Gatti, L.C.D.E.; Ertürk, A.; Kuball, J.; Sebestyén, Z.; Kool, M.; Patrizi, S.; Miele, E.; Künkele, A.; Kranendonk, M.E.G.; Cornel, A.M.; Nierkens, S.; Mayer, C.; Stunnenberg, H.G.; Alemany, A.; Alieva, M.; Rios, A.C.

doi:doi:10.1038/s43018-025-01084-0

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...Show moreDiffuse 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.Show less

Leiden University Scholarly Publications

View statistics

Documents

In Collections

De novo H3.3K27M-altered diffuse midline glioma in human brainstem organoids to dissect GD2 CAR T cell function