Mentor Name: Ingo Koomoa-Lange

High-risk neuroblastoma remains a leading cause of pediatric cancer mortality, with relapse and multidrug resistance representing major barriers to a durable cure. Unlike many adult cancers, neuroblastoma is driven largely by dysregulated developmental signaling rather than high mutational burden, and affected children are particularly vulnerable to long-term toxicity from intensified cytotoxic therapy. Defining mechanisms that promote treatment resistance is therefore essential for developing more effective and less toxic therapeutic strategies. Calcium signaling regulates proliferation, stress adaptation, and cell fate decisions. Store-operated calcium entry (SOCE) links endoplasmic reticulum calcium depletion to sustained calcium influx through ORAI channels, activating calcineurin and NFAT transcription factors. While SOCE–NFAT signaling is well characterized in T cells, its functional role in pediatric solid tumors remains poorly defined. Our preliminary data using paired patient-derived neuroblastoma cell lines obtained at diagnosis and relapse to demonstrate that drug-resistant cells exhibit enhanced SOCE and elevated basal calcium levels and constitutive NFAT nuclear localization, whereas drug-sensitive cells displayed transient calcium-dependent NFAT activation. In searching for pathophysiological triggers, we found that cytotoxic CD8+ T cells activate the calcium–NFAT pathway in neuroblastoma cells during attempted immune-mediated clearance. These findings suggest that sustained antigen-specific T-cell pressure may serve as a selective force that promotes tumor adaptation rather than complete elimination. Repeated calcium signaling imposed on tumor cells that escape killing may activate NFAT-dependent survival programs, contributing to the emergence of resistant subpopulations. Specific Aim 1 will determine how CD8+ T-cell differentiation state influences calcium signaling responses in neuroblastoma cells. Naïve, effector, and in vitro–activated CD8+ T cells will be isolated from peripheral blood, and their ability to induce calcium mobilization in neuroblastoma cells will be quantified using live-cell confocal imaging. GD2-targeted bispecific T-cell engager (BiTE)–mediated engagement will define antigen-specific calcium responses and tumor cell killing. Specific Aim 2 will determine how repeated CD8+ T-cell challenge shapes tumor survival and adaptation. Neuroblastoma cells will undergo iterative cytotoxic T-cell exposure, and surviving populations will be recovered, expanded, and cryopreserved. Live-cell imaging and caspase-based assays will quantify cell death dynamics. Surviving subpopulations will be evaluated for altered calcium.