Immunomagnetic-Enriched Subpopulations of Melanoma Circulating Tumour Cells (CTCs) Exhibit Distinct Transcriptome Profiles

Abstract

Cutaneous melanoma circulating tumour cells (CTCs) are phenotypically and molecularly heterogeneous. We profiled the gene expression of CTC subpopulations immunomagnetic-captured by targeting either the melanoma-associated marker, MCSP, or the melanoma-initiating marker, ABCB5. Firstly, the expression of a subset of melanoma genes was investigated by RT-PCR in MCSP-enriched and ABCB5-enriched CTCs isolated from a total of 59 blood draws from 39 melanoma cases. Of these, 6 MCSP- and 6 ABCB5-enriched CTC fractions were further analysed using a genome-wide gene expression microarray. The transcriptional programs of both CTC subtypes included cell survival maintenance, cell proliferation, and migration pathways. ABCB5-enriched CTCs were specifically characterised by up-regulation of genes involved in epithelial to mesenchymal transition (EMT), suggesting an invasive phenotype. These findings underscore the presence of at least two distinct melanoma CTC subpopulations with distinct transcriptional programs, which may have distinct roles in disease progression and response to therapy.

Keywords: ABCB5; MCSP; circulating tumour cells; gene expression; melanoma.

Figure 1

Heterogeneity in gene expression of MCSP- and ABCB5-enriched circulating tumour cell (CTC) fractions. Gene expression profiling of five melanoma-specific genes in CTC fractions enriched with MCSP- (M) and ABCB5- (A) coated beads that were positive for any transcript (25 out of 59 samples). Heatmap represents the expression levels of the melanoma-associated genes MLANA, TYR, MAGEA, ABCB5, and PAX3, which were assessed by qRT-PCR. All samples were positive for 18S rRNA (not shown), used as endogenous control. Each heatmap square corresponds to a Ct value for a target gene and a given sample; a higher tone of red indicates a higher gene expression and light yellow indicates no transcripts detected. Unsupervised hierarchical clustering demonstrates the inter- and intra- molecular heterogeneity of CTC subpopulations.

Figure 2

MCSP and ABCB5 CTCs have distinctive gene expression patterns. (A) Unsupervised hierarchical clustering analysis of differentially expressed genes between CTC fractions enriched from samples of healthy donors (N = 6) and melanoma patients (N = 6) using immunomagnetic beads targeting MCSP or ABCB5. Each row is a single gene and each column is a single sample. Red indicated up-regulation and blue indicates down-regulation according to the colour scale at the bottom. The squares indicate the genes that were up-regulated in melanomas (yellow box) and in controls (light green box). Genes that are differentially expressed (with ANOVA p < 0.05 and fold difference 2 vs. respective controls) in either cell type (MSCP or ABCB5 CTCs) were used to perform an unsupervised hierarchical clustering. This analytical approach discriminated the gene expression patterns from MCSP- (dark green box) and ABCB5-enriched (red box) CTC fractions. (B) Comparison of up-regulated genes between MCSP- (green circle) and ABCB5-enriched (red circle) CTC fractions found only 15 genes shared between these two CTC fractions.

Figure 3

MCSP- and ABCB5-enriched CTCs exhibit distinct transcriptional programming, with ABCB5 CTCs displaying an invasive phenotype. Schematic representation of cellular hallmarks enriched by the genes found to be differentially up-regulated in (A) MCSP- and (B) ABCB5-enriched CTCs. Enriched cellular hallmarks in CTCs are shown in green boxes, while genes shown in grey are those that were not found enriched in this study but are known to be important for melanomagenesis. Canonical pathway signalling is indicated by continuous arrows and dotted arrows indicates a previously reported interaction.

Figure 4

Identification of genes specific to single melanoma tumour cells that are also up-regulated in MCSP- and ABCB5-enriched CTC fractions. (A) Pivot plot comparing gene expression profiles of single tumour cells and nonmalignant cells, from publicly available data [31], identified 847 genes that are specifically expressed by single tumour cells (red triangle) and 196 genes specifically expressed by single nonmalignant cells (black triangle). Comparison of these tumour-specific genes against the genes found to be up-regulated in MCSP (B) and ABCB5 CTC fractions (C) identified, respectively, 11 and 10 genes that are commonly expressed by single melanoma tumours as well as by MCSP or ABCB5 CTC fractions.