Human fetal neuroblast and neuroblastoma transcriptome analysis confirms neuroblast origin and highlights neuroblastoma candidate genes

Abstract

Background: Neuroblastoma tumor cells are assumed to originate from primitive neuroblasts giving rise to the sympathetic nervous system. Because these precursor cells are not detectable in postnatal life, their transcription profile has remained inaccessible for comparative data mining strategies in neuroblastoma. This study provides the first genome-wide mRNA expression profile of these human fetal sympathetic neuroblasts. To this purpose, small islets of normal neuroblasts were isolated by laser microdissection from human fetal adrenal glands.

Results: Expression of catecholamine metabolism genes, and neuronal and neuroendocrine markers in the neuroblasts indicated that the proper cells were microdissected. The similarities in expression profile between normal neuroblasts and malignant neuroblastomas provided strong evidence for the neuroblast origin hypothesis of neuroblastoma. Next, supervised feature selection was used to identify the genes that are differentially expressed in normal neuroblasts versus neuroblastoma tumors. This approach efficiently sifted out genes previously reported in neuroblastoma expression profiling studies; most importantly, it also highlighted a series of genes and pathways previously not mentioned in neuroblastoma biology but that were assumed to be involved in neuroblastoma pathogenesis.

Conclusion: This unique dataset adds power to ongoing and future gene expression studies in neuroblastoma and will facilitate the identification of molecular targets for novel therapies. In addition, this neuroblast transcriptome resource could prove useful for the further study of human sympathoadrenal biogenesis.

Figure 1

Identification of sympathetic neuroblasts and chromaffin cells in human fetal adrenal glands by immunohistochemical analysis. Sections of a human fetal (19 weeks) adrenal gland, adjacent to those used for laser capture retrieval of cells for mRNA extraction and gene expression profiling, were stained with (a) hematoxylin and eosin or antibodies directed against (b,f) TH, (c,g) CHGA, (d,h) BCL2, and (e,i) HNK1. Whereas the immunoreactivities of BCL2 and HNK1 are specific for neuroblasts, TH and CHGA expression is pronounced in chromaffin cells and weak in neuroblasts [8]. Stars indicate chromaffin cells (TH⁺, CHGA⁺, BCL2^-, and HNK1^-), either solitary or intermingled with neuroblasts. Panels a-e show a cluster of adrenal neuroblasts and panels f-i show cortical area within scattered chromaffin cells adjacent to the neuroblast cluster. Inserts in panels b-e (bars: 10 μm) correspond to the boxed areas in these panels (bars in panels a-i: 100 μm). BCL2, B-cell CLL/lymphoma 2; CHGA, chromogranin A; H&E, hematoxylin and eosin; HNK1, carbohydrate epitope; TH, tyrosine hydroxylase.

Figure 2

Laser capture microdissection of neuroblast clusters. (a) Large cluster of neuroblasts in fetal adrenal glands at 19 weeks' gestational age (mounted hematoxylin and eosin stained cryosections), (b,c) unmounted hematoxylin and eosin stained fetal adrenal cryosections with a neuroblast cluster before and after microdissection (sample 2), and (d) the microdissected neuroblast cluster.

Figure 3

Multidimensional scaling of neuroblast, cortex, and neuroblastoma samples. (a) Multidimensional scaling of neuroblast, cortex, and neuroblastoma samples using all genes (Spearman correlation) and (b) multidimensional scaling of neuroblast, cortex, neuroblastoma, 79 normal tissue samples and other cancer samples (in duplo), and three neural stem cell cultures using the genes that are differentially expressed between fetal adrenal neuroblast and fetal adrenal cortex shows that the neuroblasts cluster very close to the neuroblastomas.

Figure 4

Venn diagram analysis of the genes with detectable expression in neuroblastoma, neuroblast, and cortex samples. (a) All genes, (b) transcription factors (GO:0003700), and (c) neurogenesis genes (GO:0007399). The number of genes that are in common between neuroblast and neuroblastoma is higher than the number of genes that are in common between the neuroblasts and cortex samples (especially for the gene classes transcription and neurogenesis), indicating that neuroblastomas resemble neuroblasts. GO, Gene Ontology.

Figure 5

Positional gene enrichment analysis of genes on chromosome 17. Positional gene enrichment analysis for the genes that are more highly expressed in neuroblastoma compared to normal neuroblasts identified two regions on 17q with significant over-representation (-¹⁰log P values; indicated in grey; the genes in these regions are printed in the boxes). The horizontal red line indicates the multiple testing corrected P value of 0.01, above which the positional gene enrichment value denotes significant over-representation. Vertical lines show the position of the genes on chromosome 17 from the gene list under investigation. The boxplot shows the gene density along the chromosome.

Figure 6

Venn diagram analysis of genes with detectable expression in neuroblast, neuroblastoma, and neural stem cell lines. This analysis shows that neuroblasts have many genes in common with neuroblastoma, but it also demonstrates that neural stem cell lines have more genes in common with the neuroblastomas than with the normal neuroblasts.