Cui Tu, C. C. Terence Tan, J. Monkman, Ana Clara Almeida, Gabrielle Antonio-Carreon, Natacha Omer, Giaan Hull, Kimberly Chung, Ahmed M. Mehdi, Antonietta Salerno, Orazio Vittorio, Aaron T. Mayer, Cleber Machado-Souza, Nanthini Jayabalan, Yi‐Chin Toh, Hui Nee Hon, Fernanda de Almeida Brehm Pinhatti, Selene Elífio-Esposito, Wayne Nicholls, Lúcia de Noronha, Arutha Kulasinghe, Fernando Souza-Fonseca-Guimarães
BACKGROUND: Neuroblastoma is a tumor of the sympathetic nervous system and is the most common extracranial solid malignancy in children. It displays striking clinical heterogeneity, ranging from spontaneous regression to a more aggressive, treatment resistant disease. While previous studies have highlighted the importance of the tumor microenvironment (TME) in shaping disease behavior, how spatial organization and metabolic pathways contribute to high-risk neuroblastoma remains poorly understood. METHODS: Here, we performed spatial transcriptomics profiling using the GeoMx Digital Spatial Profiler (DSP) Whole Transcriptome Atlas (WTA) and a spatial proteomics profile using the Akoya PhenoCycler-Fusion on a cohort of human pediatric neuroblastoma samples to characterize tumor and TME regions. RESULTS: By using the GeoMx WTA panel, high-risk neuroblastoma tumor regions were found to exhibit upregulation of metabolic pathways associated with ferroptosis, including fatty acid metabolism and reactive oxygen species (ROS) signaling. However, these tumors also showed increased glutathione metabolism pathway and elevated GPX4 expression, consistent with a potential compensatory response that may limit ferroptosis-associated cell death. Further in vitro experiments showed that inhibition of GPX4 increased lipid peroxidation and reduced tumor cell viability, consistent with ferroptosis-related processes. Interestingly, spatial proteomic analysis revealed distinct spatial niches in high-risk neuroblastoma, including stroma-secluded immune cells and macrophage enriched areas, both of which were correlated with poor patient survival. CONCLUSIONS: Our integrative spatial multi-omics analysis suggests that high-risk neuroblastoma tumors display ferroptosis-associated metabolic features, with GPX4 inhibition inducing neuroblastoma tumor cell death. We also identify macrophage-tumor interactions that may be linked to ferroptosis sensitivity. Collectively, our study highlights ferroptosis-associated pathways as potential therapeutic avenues in neuroblastoma patients.