Generation of immunocompetent syngeneic allograft mouse models for pediatric diffuse midline glioma

Abstract

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.

Keywords: DIPG; DMG; immunotherapy | syngeneic allograft; tumor microenvironment.

Figure 1.

Generation of immunocompetent DMG mouse models. (A) Graphical overview. Figure was made with BioRender. (B) Representative ventral whole brain brightfield and GFP images demonstrating the location of a GFP-positive H3.3^K27M tumor generated by intra-uterine electroporation (IUE). Images of H3^WT and H3.1^K27M models are shown in Supplementary Figure S1.

Figure 2.

Histopathological validation of allograft DMG mouse models. (A) Representative coronal sections (top panels) and high magnification (400×, bottom panels) images of H&E stained DMG allograft tumors across genetic conditions. (B) Representative images (400×) of Ki67 immunohistochemical staining (in brown) of DMG allograft tumor sections showing the tumor core (top panels) and diffuse, infiltrative growth areas (bottom panels). (C) Representative images (400×) of immunohistochemical staining (in brown) for nuclear mutant histone 3 protein (H3K27M, top panels) and histone 3 lysine 27 di- and trimethylation (H3K27me2/3, bottom panels) of DMG allograft tumor sections. Scale bars = 50 μm.

Figure 3.

Characterization of the TIME in IUE DMG mouse models. T-distributed Stochastic Neighbor Embedding (optSNE) clustering of the TIME landscape of whole primary IUE brains across genetic conditions (CyTOF mass cytometry). Indicated cell populations are organized spatially by similarity and distinguished by color.

Figure 4.

Characterization of the TIME in allograft DMG mouse models. (A) Representative images (400×) of CD3 immunohistochemical staining (in brown; marker for T-lymphocytes) of DMG allograft tumors across genetic conditions. Black arrowheads point to the sparsely present CD3-positive cells. (B) Representative immunofluorescent (400×) images of DMG allograft tumor sections co-stained for DAPI (blue) and NKp46 (red; marker for NK cells). White arrowheads point to the sparsely present NKp46-positive cells. (C) Representative images (400×) of Iba1 immunohistochemical staining (in brown; marker for microglia and macrophages) of DMG allograft tumor sections, showing the intra-tumoral heterogeneity with respect to microglia/macrophage density and phenotype. Scale bars = 50 μm.

Figure 5.

Therapeutic sensitivity of murine DMG tumor cells compared to patient-derived DMG cells. Dose–response curves representing cell viability of DMG cell lines generated by brainstem targeted IUE (solid lines) and analogous patient-derived DMG cell lines (dashed lines) across genetic conditions after 96-h treatment with Panobinostat. Data are represented as percentage viability compared to vehicle-treated controls, average ± s.d. (n = 3).