Transcriptomic and Proteomic Spatial Profiling of Pediatric and Adult Diffuse Midline Glioma H3 K27-Altered, Reveals Region Specific Differences and Limited Overlap between mRNA and Protein.

Diffuse midline glioma, H3 K27-altered (DMG-Alt) are highly aggressive malignancies of the central nervous system (CNS) that primarily affect the pediatric population. Large scale spatial transcriptomic studies have implicated that tumor microenvironmental landscape plays an important role in determining the phenotypic differences in tumor presentation and clinical course, however, data connecting overall transcriptomic changes to the protein level is lacking. The NanoString GeoMx™ Digital Spatial Profiler platform was used to determine the spatial transcriptomic and proteomic landscape in a cohort of both pediatric and adult H3 K27-altered DMG biopsy samples. Three fluorescently labeled antibodies targeting immune cells (CD45), epithelial cells (PanCK), tumor cells (H3 K27M) and a nucleic acid stain (SYTO-13) were used to establish regions of interest (ROI) for genomic and proteomic analysis. We found genetic alterations within the tumor which can be delineated across patient age and spatial location. We show that the H3 K27M mutation itself has a profound impact on tumor cells transcriptomics and interestingly we found limited fidelity between overall transcriptome and proteome. Our data also validate the previously described OPC like precursor signature at the proteomic level and reveal a special shift in the signature based on the local TME composition.

GM-CSF and IL-7 fusion cytokine engineered tumor vaccine generates long-term Th-17 memory cells and increases overall survival in aged syngeneic mouse models of glioblastoma.

Age-related immune dysfunctions, such as decreased T-cell output, are closely related to pathologies like cancers and lack of vaccine efficacy among the elderly. Engineered fusokine, GIFT-7, a fusion of interleukin 7 (IL-7) and GM-CSF, can reverse aging-related lymphoid organ atrophy. We generated a GIFT-7 fusokine tumor vaccine and employed it in aged syngeneic mouse models of glioblastoma and found that peripheral vaccination with GIFT-7TVax resulted in thymic regeneration and generated durable long-term antitumor immunity specifically in aged mice. Global cytokine analysis showed increased pro-inflammatory cytokines including IL-1ß in the vaccinated group that resulted in hyperactivation of dendritic cells. In addition, GIFT-7 vaccination resulted in increased T-cell trafficking to the brain and robust Th-17 long-term effector memory T-cell formation. TCR-seq analysis showed increased productive frequency among detected rearrangements within the vaccinated group. Overall, our data demonstrate that aging immune system can be therapeutically augmented to generate lasting antitumor immunity.