Joint single-cell genetic and transcriptomic analysis reveal embedded premalignant subclones and progressive cell states in human neuroblastoma

Neuroblastoma is a heterogeneous embryonal malignancy and the deadliest tumor of infancy. While its exact origin is still under debate, it is assumed to arise from the multipotent neural crest. To understand the origin and clonal development of various neuroblastoma cell states, we analyzed human tumor samples using single-cell multi-omics, including joint DNA/RNA sequencing of sorted single cells (DNTR-seq). Tumor subclones showed considerable inter- individual variation in terms of copy number profiles and transcriptional phenotype. Despite this heterogeneity, transcriptional plasticity between subclones converges on cellular processes related to axon guidance via the SLIT/ROBO pathway, proliferation, and differentiation. Genomic heterogeneity was also observed for MYCN. Subclones harboring 2p/MYCN gain (< 8 copies) co-exist with amplified subclones (> 20 copies), indicating that MYCN amplification may represent a non-initiating genomic event. Beyond adrenergic heterogeneity, we identify a subpopulation of abnormal cells resembling multipotent Schwann cell precursors, characterized by expression programs of proliferation, apoptosis, and a non- immunomodulatory phenotype. Their genomic profile and phylogeny suggest an ancestral, pre-malignant role in tumorigenesis. While the function of these SCP-like cells in tumor initiation remains to be established, their identification expands the reservoir of tumor cells, considering their multipotency reflecting a central neuroblastoma feature.