Spatial omics resolves adrenergic and mesenchymal cell states in neuroblastoma

Neuroblastoma is a pediatric cancer arising from the developing sympathoadrenal lineage. Although cell lines derived from neuroblastoma patients have features of either adrenergic (ADRN) or mesenchymal (MES) cells, identifying MES cells in patient tumors is challenging and controversial. Here, to characterize intra- and inter-patient cellular heterogeneity, we analyze 54 neuroblastoma tumors spanning broad clinical, genetic, and histologic features using single-cell/single-nucleus RNA sequencing (sc/snRNA-seq), bulk RNA sequencing, spatial transcriptomics, and spatial proteomics. We show that tumor cells occupy distinct cellular neighborhoods separated from nonmalignant immune and stromal cells, yet established cell line-derived gene expression signatures fail to identify MES neuroblastoma cells. We therefore develop ADRN/MES signatures from early-passage orthotopic patient-derived xenografts, enabling the identification of MES tumor cells, validated by transcriptomic, epigenomic, proteomic, ultrastructural, and functional assays. In patients, high MES gene expression correlates with worse event-free and overall survival. Together, this study provides a framework for defining clinically relevant neuroblastoma cell states.