Figure 5. A neural crest stem cell (NCSC) gene signature segregates stage 4/M NB tumors and is associated to tumor aggressiveness.

(A-D) NCSC gene signature defines a subgroup of stage 4/M NB tumors with high probability of relapse. (A) K-means clustering using genes up-regulated in NCSCs (described in Lee et al. [13]), segregating stage 4/M NB tumors into three different groups depending on high, intermediate or low expression levels of the signature (NCH, NCI and NCL tumors respectively). (B) NCH tumors are enriched in NCSC genes related to the mesenchymal phenotype of the neural crest, such as TGFB1, EDNRA and S100A10. By contrast, genes related to neural specification of NCSCs, such as SCG3, POU4F1 and PHOX2B, show lower expression levels within NCH tumors. (C) Transcriptome wide profiling by principal component analysis, revealing a clear segregation between NCH, NCI and NCL tumors. Principal component 2 (PC2) maximized the differences between the three types of tumors. (D) Kaplan-Meier curves showing event-free survival probability of NCH, NCI and NCL tumors, compared to all stage 4/M tumors analyzed together. NCH and NCL tumors present higher probability of relapse compared to NCI neuroblastomas. (E-H) NCH express higher level of mesenchymal related genes. (E) Expression levels of typical mesenchymal genes in NCH tumors compared to NCI and NCL tumors together (NCL+I). All genes analyzed (NT5E, ENG, CD44, PDGFRA, ACTA2, VIM, CSCL12 and SPARC) showed higher expression levels in NCH tumors than in NCL+I tumors. (F) Parametric analysis of gene set enrichment (PAGE) in NCH tumors revealed enrichment in the expression of genes previously described as characteristic of a mesenchymal phenotype. Enrichment z-score for three different gene sets is shown: genes typical of the mesenchymal subtype in glioblastoma (gene set #1, described in Verhaak et al. [48]), genes up-regulated in stromal stem cells (gene set #2, described in Boquest et al. [50]), and genes typical of mesenchymal transition in cancer (gene set #3, described in Anastassiou et al. [49]). The heat map in the upper panel shows the complete gene set map of average z-scores for all genes in every single gene set, calculated for each individual sample. (G) Box-whiskers plots showing the expression of the three different gene sets analyzed in NCH, but now compared to NCL+I tumors. (H) Correlation between gene set enrichment average z-scores in individual tumors over the whole sample of tumors analyzed. NCSC z-scores were correlated to enrichment z-scores for the gene sets analyzed in (F). The results revealed a close correlation between expression of genes up-regulated in NCSCs and genes related to mesenchymal features, both being highly expressed in NCH tumors. (I, J) Genes differentially expressed in NCH tumors are involved in biofunctions and processes related to the stromal component of tumors. (I) Representative biofunctions and processes resulting from gene ontology analysis of genes differentially expressed between NCH and NCL+I tumors. Genes overexpressed in NCH tumors participate preferentially in processes related to the stromal component of tumors, such as “positive regulation of immune response”, “extracellular matrix organization” and “angiogenesis”. (J) Heat map showing the expression levels of genes involved in angiogenesis (GO Path 1525). Tumors are hierarchically clustered and NCSC enrichment status is shown in the upper bar. Whiskers in Box-Whiskers plots indicate maximum and minimum values. (r-value = Pearson's correlation coefficient) (****p < 10^–4, ***p < 0.001, **p < 0.01; Mann-Whitney U-test).