Abstract
Neuroblastoma (NBL) is a pediatric cancer responsible for more than 15% of cancer deaths in children, with 800 new cases each year in the United States alone. Genomic amplification of the MYC oncogene family member MYCN characterizes a subset of high-risk pediatric neuroblastomas. Several cellular models have been implemented to study this disease over the years. Two of these, SK-N-BE-2-C (BE2C) and Kelly, are amongst the most used worldwide as models of MYCN-Amplified human NBL. Here, we provide a transcriptome-wide quantitative measurement of gene expression and transcriptional network activity in BE2C and Kelly cell lines at an unprecedented single-cell resolution. We obtained 1105 Kelly and 962 BE2C unsynchronized cells, with an average number of mapped reads/cell of roughly 38,000. The single-cell data recapitulate gene expression signatures previously generated from bulk RNA-Seq. We highlight low variance for commonly used housekeeping genes between different cells (ACTB, B2M and GAPDH), while showing higher than expected variance for metallothionein transcripts in Kelly cells. The high number of samples, despite the relatively low read coverage of single cells, allowed for robust pathway enrichment analysis and master regulator analysis (MRA), both of which highlight the more mesenchymal nature of BE2C cells as compared to Kelly cells, and the upregulation of TWIST1 and DNAJC1 transcriptional networks. We further defined master regulators at the single cell level and showed that MYCN is not constantly active or expressed within Kelly and BE2C cells, independently of cell cycle phase. The dataset, alongside a detailed and commented programming protocol to analyze it, is fully shared and reusable.
Highlights
Cell lines have been widely used in cancer biology as a standard setting to investigate molecular mechanisms and to test the effects of genetic and chemical perturbations
We quantitatively checked the presence of several genes, in terms of average expression across the entire cell population and in terms of number of cells with at least one read mapped on the gene (Figure 1C for BE2C, Figure 1D for Kelly)
The overall analysis highlights that BE2C cells have a markedly higher expression of genes associated to Epithelial-Mesenchymal Transition (EMT) (Figure 5A,B), a pathway generally associated with higher proliferation, chance of metastasis, poor survival, and drug resistance [52]: it can be hypothesized that BE2C cells are a better model for highly aggressive MYCN-amplified NBL than Kelly cells
Summary
Cell lines have been widely used in cancer biology as a standard setting to investigate molecular mechanisms and to test the effects of genetic and chemical perturbations. Recent technological advances have provided the unprecedented opportunity to investigate heterogeneity at single-cell resolution [9], allowing researchers to quantitatively identify cellular subpopulations and to uncover molecular mechanisms underlying phenotypic diversity among cells [10]. Single-cell sequencing has been applied to investigating the early insurgence of drug resistance mechanisms [15,16] and tumor evolution [17,18]. There exist at least three different molecular subtypes in which aggressive NBLs can be categorized, named Mesenchymal, 11q Loss of Heterozygosity and MYCN-Amplified, the last two of which are characterized by specific genomic alterations [23]. There exist several cell lines derived from MYCN-Amplified NBLs, which have been widely characterized both transcriptionally by RNA-Sequencing [25], and epigenetically by ChIP-Sequencing [26,27,28]. The dataset is provided both as raw sequencing reads (provided at the Sequence Read Archive as BAM files aligned on human genome version hg, and containing unaligned reads) and as processed gene counts matrices in the
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