Potentiating Natural Killer Cell-mediated Cytotoxicity in Medulloblastoma 2308814

Abstract Introduction Medulloblastoma (MB) is the most common malignant pediatric brain tumor and comprises four molecular subgroups (WNT, SHH, Group 3, Group 4). Although generally presenting an immunologically “cold” tumor microenvironment, flow cytometric (FC) profiling of primary tumor samples from our laboratory demonstrates the presence of natural killer (NK) infiltrates in the tumor microenvironment, prompting investigation into their functional relevance and therapeutic potential. Methods Healthy human NK cells were expanded under standardized IL-2/IL-15 conditions and co-cultured with MB cell lines (DAOY, D341) and patient-derived cell lines (n = 5, WNT=, SHH=1, Group 3=2, Group 4=1) at graded effector:target ratios and time points. Tumor cell death was quantified by ToxiLight assay and Annexin V/PI by FC analysis. NK activation was assessed by CD45, CD56. And CD107a expression. IFN-γ production was measured by intracellular FC and ELISA. CD226-CD96-TIGIT axis (CD155/PVR,CD112) and PD-1/PD-L1 inhibitory pathways were analyzed by FC and IHC. Multiplex spatial proteomics was performed on FFPE patient samples (n = 26 total; WNT=4, SHH=6, Group 3/4=16) to map NK infiltration and inhibitory signaling across subgroups. Results NK cells induced time- and effector-dependent apoptosis in MB cell lines and primary cultures. Co-culture triggered robust NK activation with IFN-γ production, accompanied by PD-L1 upregulation on tumor cells. NK cytotoxicity varied by molecular subgroup, with SHH tumors showing the greatest sensitivity and Group 3 tumors exhibiting reduced susceptibility. These differences aligned with subgroup-specific inhibitory receptor expression and signaling patterns. Conclusion NK-cell cytotoxicity in medulloblastoma is subtype dependent, with Group 3/4 tumors showing reduced susceptibility. Spatial proteomics revealed distinct subgroup-specific inhibitory architectures that align with differential NK sensitivity, supporting the need for subtype-informed NK-based therapeutic strategies. Funding Source Project to Cure, Olivia Hope Foundation and Hyundai Hope on Wheels Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)