Abstract BACKGROUND Diffuse midline glioma (DMG) is an aggressive pediatric brain tumor with no curative treatment. The tumor microenvironment (TME) of DMG, shaped by its unique anatomical location and developmental context, poses significant therapeutic hurdles. While the TME has been characterized by a predominance of tumor-associated microglia and sparse infiltration of blood-derived immune cells, a comprehensive understanding of immune-tumor cell interactions and how to effectively target them with immunotherapies remains challenging. MATERIAL AND METHODS To dissect the DMG TME, we employed a multi-omics approach that integrated single-nucleus RNA sequencing, spatial transcriptomics, and high-dimensional imaging on both patient samples and an immunocompetent in utero electroporation murine DMG model. We characterized the spatial and phenotypic architecture of the tumors and validated a novel immune target using syngeneic mouse models and ex vivo co-culture experiments. RESULTS Our analysis revealed pronounced intratumoral heterogeneity, with two spatially distinct patterns of cancer-immune interaction. A mesenchymal-like niche promoted the recruitment of blood-derived immune cells, whereas another pattern exhibited an immunologically cold phenotype, reminiscent of the immune-privileged healthy brain. Spatially resolved analysis of cell-cell communication within the TME identified the VISTA immune checkpoint pathway as a key regulator of these patterns. Targeting of this axis in a syngeneic mouse model led to significant tumor reduction and 100% survival in mice, highlighting its therapeutic potential. Further validation demonstrated that microglia play a critical role in orchestrating immune cell recruitment and anti-tumor activity, and that modulating the VISTA pathway can reshape the immune response. CONCLUSION Our findings uncover the spatial and cellular complexity of the DMG immune microenvironment and identify the VISTA pathway as a critical modulator of immune suppression. By demonstrating robust therapeutic efficacy upon VISTA blockade, this study lays a strong foundation for developing targeted immunotherapies in DMG and opens new avenues for overcoming immune resistance in pediatric brain tumors.
Collot et al. (Wed,) studied this question.