Fig. 3: Intratumoral heterogeneity of childhood neuroblastoma (NB).

4224 single nuclei of human NB tumors were sequenced to an average depth of 676,000 reads per cell. a Cells with high-quality (n = 3212) were grouped into ten different clusters with PAGODA. NB risk groups, INSS stages, and samples are exemplified in (b). c Representation of the hierarchical clustering of cells: undifferentiated (light pink nC2 and pink nC3), mesenchymal stroma (MSC red nC1), endothelial (light blue nC4), T-cells (light green nC10), macrophages (green nC6), and noradrenergic (“NOR”, blue nC5, nC7, nC8, and nC9) and various markers of interest (also in insert d). The bars next to the tSNEs in (d) illustrate the expression measured as the logarithm of the read counts per 10,000. e, f Using a gene enrichment-based approach, the noradrenergic clusters of NB (nC5, nC7, nC8, and nC9) proved to share a significant number of upregulated genes with sympathetic noradrenergic¹⁵ and “ADR” (i.e., adrenergic)¹⁶ transcriptional signatures. Oppositely, the undifferentiated cluster nC3 shared a significant number of upregulated genes with neural-crest cell-like signature¹⁵ and MES (i.e., mesenchymal)¹⁶ signatures. g A comparison of the gene-specific signature of NB clusters with markers defining clusters in eight tumors previously sequenced with 10X¹³, suggests a more significant similarity of the noradrenergic clusters (nC5, nC7, nC8, and nC9) with tumor clusters 1, 2, and 3; and of the undifferentiated nC3 cluster with the tumor cluster 3¹³. Only clusters with the most significant similarities are displayed. h An analysis of the CNVs indicates a significant number of cells with rearrangements (indicated by red, Fisher exact tests) in the undifferentiated cluster nC3 for most samples, while a remarkable number of rearrangements was observed in fewer samples for the NOR (nC5), MSC (nC1), and undifferentiated nC2 clusters. i A subset of the predicted CNVs was validated for each sample, and illustrated in the tSNE for cells with an expected rearrangement (i.e., expected state) overlapping the validated region. Colors displayed in (e), (f), and (g) represent cell clusters by color in (a). An arrow next to the bar indicates a FDR < 1 × 10⁻⁵⁰.