Comprehensive characterization of neuroblastoma cell line subtypes reveals bilineage potential similar to neural crest stem cells.

BACKGROUND: Neuroblastic tumors (NBT) derive from neural crest stem cells (NCSC). Histologically, NBT are composed by neuroblasts and Schwannian cells. In culture, neuroblastic (N-), substrate-adherent (S-) and intermediate phenotype (I-) cell subtypes arise spontaneously. METHODS: Here, neuroblastoma (NB) cell line subtypes were characterized according to embryonic peripheral nervous system development markers (GAP43, Phox2b, Sox10, c-kit, GD2, NF68, vimentin, S100beta, calcyclin and ABCG2), morphological features, gene expression and differentiation potential. I-type cells were investigated as a bipotential (neuronal and glial) differentiation stage. RESULTS: Positive immunostaining of NCSC (GAP43, c-kit, NF68, vimentin and Phox2b) and undifferentiated cell (ABCG2) markers was observed in all NB subtypes. N- and I-type cells displayed cytoplasmic membrane GD2 staining, while nuclear calcyclin was restricted to S-type. N- and I-type cells showed similar phenotype and immunoreactivity pattern. Differential gene expression was associated with each cell subtype. N- and I-type cells displayed similar differentiation capacity towards neuronal and glial lineage fates. S-type cells, upon induction, did not show a neuronal-like phenotype, despite gene expression changes. CONCLUSION: Results suggest that N- and I-type NB cell subtypes represent an immature bilineage stage, able to progress towards neuronal and glial fates upon induction of differentiation. S-type cells appear irreversibly committed to a glial lineage fate.

The calcium-sensing receptor and parathyroid hormone-related protein are expressed in differentiated, favorable neuroblastic tumors.

BACKGROUND: Differentiated histopathology is a favorable prognostic factor in neuroblastic tumors, and molecular pathways underlying neuroblastoma differentiation can be modulated pharmacologically. The calcium-sensing receptor (CaR) and parathyroid hormone-related protein (PTHrP) regulate differentiation processes in some cellular contexts. CaR is up-regulated when neural stem cells are specified to the oligodendrocyte lineage and regulates PTHrP production in astrocytes. The objective of the current study was to assess whether CaR and PTHrP participate in neuroblastoma differentiation pathways. METHODS: CaR and PTHrP messenger RNA (mRNA) and protein expression were analyzed in neuroblastic tumors, and correlation with prognostic factors was assessed. CaR and PTHrP expression levels were analyzed in neuroblastoma cell lines treated with all-trans-retinoic acid or 5-bromo-2'-deoxyuridine (BrdU). RESULTS: CaR expression was correlated with favorable histology, age at diagnosis <1 year, low clinical stage, and low clinical risk. CaR was absent in undifferentiated neuroblasts and was expressed in differentiating neuroblasts. CaR and PTHrP were highly expressed in ganglion and in Schwann-like cells. PTHrP mRNA levels were higher in ganglioneuroblastomas and ganglioneuromas than in neuroblastomas (P < .0001). Both genes were up-regulated in neuroblastomas with treatment-induced maturation features. CaR, but not PTHrP, was up-regulated at early phases of in vitro neuronal differentiation induction. Substrate-adherent, non-neuronal cell lines displayed the highest PTHrP levels among the neuroblastoma cell lines examined. The up-regulation of PTHrP and of 2 glial differentiation markers was observed in 2 cell lines that were treated with BrdU, whereas CaR was induced in only 1 cell line. CONCLUSIONS: CaR and PTHrP were expressed in differentiated, favorable neuroblastic tumors, and both genes were up-regulated by inducing differentiation.

Differential expression of genes mapping to recurrently abnormal chromosomal regions characterize neuroblastic tumours with distinct ploidy status.

BACKGROUND: Neuroblastic tumours (NBTs) represent a heterogeneous spectrum of neoplastic diseases associated with multiple genetic alterations. Structural and numerical chromosomal changes are frequent and are predictive parameters of NBTs outcome. We performed a comparative analysis of the biological entities constituted by NBTs with different ploidy status. METHODS: Gene expression profiling of 49 diagnostic primary NBTs with ploidy data was performed using oligonucleotide microarray. Further analyses using Quantitative Real-Time Polymerase Chain Reaction (Q-PCR); array-Comparative Genomic Hybridization (aCGH); and Fluorescent in situ Hybridization (FISH) were performed to investigate the correlation between aneuploidy, chromosomal changes and gene expression profiles. RESULTS: Gene expression profiling of 49 primary near-triploid and near-diploid/tetraploid NBTs revealed distinct expression profiles associated with each NBT subgroup. A statistically significant portion of genes mapped to 1p36 (P = 0.01) and 17p13-q21 (P < 0.0001), described as recurrently altered in NBTs. Over 90% of these genes showed higher expression in near-triploid NBTs and the majority are involved in cell differentiation pathways. Specific chromosomal abnormalities observed in NBTs, 1p loss, 17q and whole chromosome 17 gains, were reflected in the gene expression profiles. Comparison between gene copy number and expression levels suggests that differential expression might be only partly dependent on gene copy number. Intratumoural clonal heterogeneity was observed in all NBTs, with marked interclonal variability in near-diploid/tetraploid tumours. CONCLUSION: NBTs with different cellular DNA content display distinct transcriptional profiles with a significant portion of differentially expressed genes mapping to specific chromosomal regions known to be associated with outcome. Furthermore, our results demonstrate that these specific genetic abnormalities are highly heterogeneous in all NBTs, and suggest that NBTs with different ploidy status may result from different mechanisms of aneuploidy driving tumourigenesis.