TMOD-24. Modeling Pediatric DMG in Human BrOs (Brainstem-regionalized Organoids) Dissects Tumour–Microglia–Immune Crosstalk and CAR T Cell Dysfunction

Abstract Diffuse Midline Glioma (DMG) is a uniformly fatal paediatric brain tumour, most commonly arising in the pons, where access to tissue and effective therapies remain critically limited. To address this, we developed a novel guided human Brainstem-regionalized Organoid (BrO) model using FGF4-based patterning to enrich for pontine glial and neuronal lineages. In situ introduction of H3.3K27M, TP53, and PDGFRA mutations led to the formation of de novoDiffuse Midline Glioma Organoids (DMGOs), which closely recapitulate the infiltrative growth, epigenetic signatures, and single-cell heterogeneity of primary DMG, including all human-specific lineages. Clonal barcoding and lineage tracing revealed that tumour progression in DMGOs is driven by pontine glial progenitors, confirming a developmental window and anatomical specificity for DMG initiation. Leveraging this patient-representative system, we modelled prolonged GD2 CAR T cell treatment and identified functional T cell heterogeneity resembling clinical outcomes, including cytotoxic, exhausted, and metabolically stressed states. Incorporation of stem cell-derived microglia into DMGOs generated an immunosuppressive myeloid compartment that dampened CAR T cell efficacy and induced dysfunctional T cell populations with low effector potential. These microglia adopted tumour-induced phenotypes reflective of the immunosuppressive states observed in pediatric DMG, and their presence led to enhanced CAR T cell exhaustion as well as the emergence of a novel dysfunctional T cell population associated with reduced tumour control and therapy resistance. This study presents BrOs as a clinically relevant and ethically grounded platform to generate human DMGOs, enabling spatially and temporally accurate modelling of pediatric DMG. It supports fundamental and translational applications via de novo tumour formation and prolonged immunotherapy testing within a tumour-relevant niche. By reconstructing tumour–immune–microglia interactions in vitro, our model provides a powerful framework for uncovering resistance mechanisms, refining CAR T cell strategies, and guiding the development of combination therapies for this devastating paediatric disease.

Authors

Institutions

Publication Details

Journal
Neuro-Oncology
Published
2025-11-01
DOI
https://doi.org/10.1093/neuonc/noaf201.1898
Primary Topic
Glioma Diagnosis and Treatment
Type
article
Field-Weighted Citation Impact
0.00
Controls
|||
ALL TIME
JAN
FEB
MAR
APR
MAY
JUN
JUL
AUG
SEP
OCT
article

TMOD-24. Modeling Pediatric DMG in Human BrOs (Brainstem-regionalized Organoids) Dissects Tumour–Microglia–Immune Crosstalk and CAR T Cell Dysfunction

Nils Bessler, Celina Honhoff, Zsolt Sebestyén, Annette Künkele et al.
Neuro-Oncology
Glioma Diagnosis and Treatment
article

TMOD-24. Modeling Pediatric DMG in Human BrOs (Brainstem-regionalized Organoids) Dissects Tumour–Microglia–Immune Crosstalk and CAR T Cell Dysfunction

Nils Bessler, Celina Honhoff, Zsolt Sebestyén, Annette Künkele, Farid Keramati, Thijs van den Broek, Hendrikus C. R. Ariese, Anna Alemany, Ellen J. Wehrens, Hendrik G. Stunnenberg, Mariëtte E.G. Kranendonk, Ali Ertürk, Sam de Blank, Jurgen H. E. Kuball, Annelisa M. Cornel, Mario Barrera Román, Cristian Ruiz Moreno, Noëlle Dommann, Christian Mayer, Lucrezia C D E Gatti, Marcel Kool, María Alieva, Raphaël Collot, Stefan Nierkens, Mieke Roosen, Anne C. Rios, Daan J. Kloosterman, Sara Patrizi, Amber K. L. Wezenaar, E Van Vliet, Evelina Miele
article en

Abstract

Abstract Diffuse Midline Glioma (DMG) is a uniformly fatal paediatric brain tumour, most commonly arising in the pons, where access to tissue and effective therapies remain critically limited. To address this, we developed a novel guided human Brainstem-regionalized Organoid (BrO) model using FGF4-based patterning to enrich for pontine glial and neuronal lineages. In situ introduction of H3.3K27M, TP53, and PDGFRA mutations led to the formation of de novoDiffuse Midline Glioma Organoids (DMGOs), which closely recapitulate the infiltrative growth, epigenetic signatures, and single-cell heterogeneity of primary DMG, including all human-specific lineages. Clonal barcoding and lineage tracing revealed that tumour progression in DMGOs is driven by pontine glial progenitors, confirming a developmental window and anatomical specificity for DMG initiation. Leveraging this patient-representative system, we modelled prolonged GD2 CAR T cell treatment and identified functional T cell heterogeneity resembling clinical outcomes, including cytotoxic, exhausted, and metabolically stressed states. Incorporation of stem cell-derived microglia into DMGOs generated an immunosuppressive myeloid compartment that dampened CAR T cell efficacy and induced dysfunctional T cell populations with low effector potential. These microglia adopted tumour-induced phenotypes reflective of the immunosuppressive states observed in pediatric DMG, and their presence led to enhanced CAR T cell exhaustion as well as the emergence of a novel dysfunctional T cell population associated with reduced tumour control and therapy resistance. This study presents BrOs as a clinically relevant and ethically grounded platform to generate human DMGOs, enabling spatially and temporally accurate modelling of pediatric DMG. It supports fundamental and translational applications via de novo tumour formation and prolonged immunotherapy testing within a tumour-relevant niche. By reconstructing tumour–immune–microglia interactions in vitro, our model provides a powerful framework for uncovering resistance mechanisms, refining CAR T cell strategies, and guiding the development of combination therapies for this devastating paediatric disease.

Neuro-OncologyVol. 27(Supplement_5)
Radboud University Nijmegen (NL), Utrecht University (NL), Novo Nordisk Foundation (DK), Radboud University Medical Center (NL), Helmholtz Zentrum München (DE), University Medical Center Utrecht (NL), Humboldt-Universität zu Berlin (DE), Instituto de Investigaciones Biomédicas Sols-Morreale (ES), Radboud Institute for Molecular Life Sciences (NL), Oncode Institute (NL), Hopp Children's Cancer Center Heidelberg (DE), Department of Embryology (US), Bambino Gesù Children's Hospital (IT), Princess Máxima Center (NL), Center for Translational Molecular Medicine (NL), Max Planck Institute of Neurobiology (DE), Universidad Autónoma de Madrid (ES)
Openalex Percentile: Top 52%
Glioma Diagnosis and Treatment
DOIOpen Access PDFOpenAlex
AI Navigator

Ask Laika to Summarize, Analyze, and Connect papers live on the map.

Summarize Papers & Methodologies

Extract key findings, datasets, and comparative methods across publications.

Benchmark Rankings & Visual Analytics

Rank top research institutions, authors, funders, topics, and journals by Field-Weighted Citation Impact (FWCI) and paper volume with instant charts.

Connect Distant Disciplines

Bridge topological clusters on the map to find hidden collaborative intersections.