Deciphering Neuroblastomas With Spatial Omics

Neuroblastoma is a pediatric tumour of the developing sympathetic nervous system, characterized by a striking diversity in both clinical presentation and cellular composition. In previous work, we generated a neuroblastoma single-cell RNA-seq reference atlas by integrating data from 61 patients (Bonine et al., Cell Reports, 2024) and identified a remarkable heterogeneity among lymphoid and myeloid cells. We now aim to translate these distinct cellular subpopulations into the spatial context using spatial proteomics and transcriptomics on FFPE samples from high- and low-risk neuroblastomas. Leveraging our neuroblastoma atlas, we selected unique markers or marker combinations for each cell (sub)-population, leading to a 73-plex antibody panel for spatial proteomics with MACSima® Imaging Cyclic Staining, as well as reference-based signatures for cell type annotation in spatially resolved whole-transcriptome profiling (Visium HD). Our spatial proteogenomic mapping revealed a high infiltration of lymphoid cells in low-risk neuroblastoma, in contrast to high-risk neuroblastoma, which exhibited an almost complete absence of lymphocytes. Conversely, myeloid cells dominated the tumor microenvironment in high-risk patients. Notably, specific macrophage subsets formed infiltrative niches within the tumor or along its borders, suggesting a potential role in shaping an immunosuppressive microenvironment in high-risk neuroblastoma. The specifics of this putative role are currently being investigated.