MODL-16. Generation of immunocompetent syngeneic allograft mouse models for pediatric diffuse midline glioma Open Access

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.