Neuroblastoma is a neural crest cell-derived pediatric tumor characterized by high inter- and intra-tumor heterogeneity, and by a poor outcome in advanced stages. Patient-derived xenografts (PDXs) have been shown to be useful models for preserving and expanding original patient biopsies in vivo, and for studying neuroblastoma biology in a more physiological setting. The maintenance of genetic, histologic, and phenotypic characteristics of the original biopsy along serial PDX passages in mice is a major concern regarding this model. Here we analyze consecutive PDX passages in mice, at both transcriptomic and histological levels, in order to identify potential changes or highlight similarities to the primary sample. We studied temporal changes using mRNA and miRNA expression and correlate those with neuroblastoma aggressiveness using patient-derived databases. We observed a shortening of tumor onset and an increase in proliferative potential in the PDXs along serial passages. This behavior correlates with changes in the expression of genes related to cell proliferation and neuronal differentiation, including signaling pathways described as relevant for neuroblastoma malignancy. We also identified new genes and miRNAs that can be used to stratify patients according to survival, and which could be potential new players in neuroblastoma aggressiveness. Our results highlight the usefulness of the PDX neuroblastoma model and reflect phenotypic changes that might be occurring in the mouse environment. These findings could be useful for understanding the progression of tumor aggressiveness in this pathology.

Neuroblastoma is a neural crest-derived pediatric malignancy characterized by marked cellular heterogeneity and variable differentiation status. Undifferentiated tumors are associated with aggressive clinical behavior, treatment resistance and poor outcome, highlighting the need to identify molecular mechanisms that sustain tumor cell plasticity and prevent differentiation. Vaccinia-related kinase 1 (VRK1) is a serine/threonine kinase involved in cell-cycle progression, DNA-damage responses and transcriptional regulation, and has previously been associated with neuroblastoma progression. However, its role in the control of neuroblastoma differentiation remains unclear. Here, we investigated the relationship between VRK1 expression, tumor differentiation and stem-like properties in human neuroblastoma. Analysis of patient tumor datasets and tissue microarrays showed that VRK1 expression is enriched in undifferentiated neuroblastoma and stage 4 tumors, and inversely correlates with established differentiation markers, including DDC, NCAM1 and S100B. This association was maintained in MYCN-non-amplified tumors, indicating that the relationship between VRK1 and differentiation is not dependent on MYCN status. Single-cell transcriptomic analyses further demonstrated elevated VRK1 expression in developmentally immature neural crest progenitor and Schwann cell precursor-like populations. Induction of neuronal or mesenchymal differentiation consistently reduced VRK1 expression in neuroblastoma cell lines and patient-derived cells. Conversely, VRK1 silencing promoted differentiation-marker expression, reduced nestin and Ki67 expression, and produced sustained differentiation-associated changes in xenograft tumors. VRK1 was also enriched in tumorsphere cultures that select for undifferentiated stem-like neuroblastoma cells. VRK1 depletion impaired tumorsphere growth, reduced intratumoral proliferation and altered the balance between undifferentiated cells and differentiated progeny, supporting a role for VRK1 in self-renewal and maintenance of progenitor-like tumor cells. Mechanistically, VRK1 expression positively correlated with the core stemness transcription factor SOX2 in neuroblastoma tumor cells. VRK1 knockdown reduced nuclear SOX2 abundance, whereas VRK1 overexpression increased SOX2 protein levels. In addition, analysis of the VRK1 locus identified an active chromatin configuration and potential SOX2-binding sites, consistent with a regulatory relationship between these factors. Together, these findings identify VRK1 as a regulator of the undifferentiated, proliferative and stem-like state in neuroblastoma. The VRK1-SOX2 axis may contribute to stabilizing tumor-cell immaturity and represents a potential target for differentiation based therapeutic strategies in high-risk neuroblastoma.