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.

TIM-3 blockade in diffuse intrinsic pontine glioma models promotes tumor regression and antitumor immune memory

“…Interestingly, the highest TIM-3 expression intensity was observed in DC, macrophage, microglia, and Treg populations ( Figures S4 B and S4C). In addition, we confirmed TIM-3 expression in one additional in utero electroporation (IUE) DMG model (PPK 26 ) and in an isogenic orthotopic DMG model (24D-1: H3WT; 26C-7: H3.1K27M, and 26B-7 H3.3K27M) also develop by IUE model 27 ). The expression of TIM-3 in these additional models further supports the notion that, indeed, its expression in DMGs is observed across different models.…”

Microglia in pediatric brain tumors: The missing link to successful immunotherapy

“… 40 , 132 , 133 , 134 , 135 , 136 , 137 Recently, the use of in utero electroporation has allowed for the generation of immunocompetent allograft models of brainstem tumors that closely mimic the immune microenvironment seen in patients, effectively addressing the limitation that has hindered replication of the complexity of the human immune response in traditional mouse models. 138 …”

OV Modulators of the Paediatric Brain TIME: Current Status, Combination Strategies, Limitations and Future Directions

“…Moreover, some immunocompetent models have been developed by introducing driver mutations that were identified in human tumours [28,98]. For example, an immunocompetent mouse model of DMG was successfully generated by Du Chatinier et al [99]. They established tumour cell lines from DMG mouse models that had been induced through intra-uterine electroporation of DMG-specific mutations into the embryonic brainstem [99].…”