Background Diffuse midline gliomas (DMG) are highly malignant incurable pediatric brain tumors. A lack of effective treatment options highlights the need to investigate novel therapeutic strategies. This includes the use of immunotherapy, which has shown promise in other hard-to-treat tumors. To facilitate preclinical immunotherapeutic research, immunocompetent mouse models that accurately reflect the unique genetic, anatomical, and histological features of DMG patients are warranted. Methods We established cell cultures from primary DMG mouse models (C57BL/6) that were generated by brainstem targeted intra-uterine electroporation (IUE). We subsequently created allograft DMG mouse models by orthotopically implanting these tumor cells into syngeneic mice. Immunohistochemistry and -fluorescence, mass cytometry, and cell-viability assays were then used to verify that these murine tumors recapitulated human DMG. Results We generated three genetically distinct allograft models representing histone 3 wildtype (H3 WT) and K27M-mutant DMG (H3.3 K27M and H3.1 K27M). These allograft models recapitulated the histopathologic phenotype of their human counterparts, including their diffuse infiltrative growth and expression of DMG-associated antigens. These murine pontine tumors also exhibited an immune microenvironment similar to human DMG, characterized by considerable myeloid cell infiltration and a paucity of T-lymphocytes and NK cells. Finally, we show that these murine DMG cells display similar sensitivity to histone deacetylase (HDAC) inhibition as patient-derived DMG cells. Conclusions We created and validated an accessible method to generate immunocompetent allograft models reflecting different subtypes of DMG. These models adequately recapitulated the histopathology, immune microenvironment, and therapeutic response of human DMG, providing useful tools for future preclinical studies.

Diffuse midline gliomas (DMG) are highly aggressive pediatric brain tumors with a grim prognosis. A lack of effective treatment options highlights the critical need to investigate new therapeutic strategies. This includes the use of immunotherapy, which has shown promise in other hard-to-treat tumors. To facilitate immunotherapeutic research in this field, and to complement the existing immunodeficient patient-derived DMG models, we developed three distinct immunocompetent mouse models representing different DMG subtypes, i.e., histone 3 wildtype and histone 3 K27M mutant DMG (H3.3K27M or H3.1K27M), that can be used for preclinical testing of new therapies. We first established primary tumor cell cultures from murine DMG tumors that were generated by brainstem-targeted intra-uterine electroporation (IUE). This method enabled the introduction of DMG-associated mutations within the intact developing brainstem, thereby generating DMG tumors in a spatially and temporally defined manner, while maintaining a genetically identical (isogenic) background. We then created allograft DMG mouse models by orthotopically implanting the established primary cell cultures into syngeneic (C57BL/6) mice. Herewith, we provide an allograft tool that is better suitable for large-scale therapeutic studies and more accessible to the scientific community. Importantly, we demonstrated that these allograft models recapitulate the histopathologic phenotype of human DMG, including their diffuse infiltrative growth and expression of DMG-associated antigens. Furthermore, CyTOF mass cytometry analysis indicated that these murine pontine tumors exhibit a tumor immune microenvironment (TIME) similar to human DMG, characterized by considerable myeloid cell infiltration and a paucity of T-lymphocytes and NK cells. As such, we provide a representative model to further delineate the immune landscape in DMG and to preclinically investigate novel (immuno)therapies. Currently, we are using these immunocompetent models to study the interaction between DMG cells and microglia, and we are investigating how we can modify the immune microenvironment to improve checkpoint inhibition in DMG.