Single-Cell Gene Expression Signatures Reveal Melanoma Cell Heterogeneity

Oncogene (2015) 34, 3251–3263 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc ORIGINAL ARTICLE Single-cell gene expression signatures reveal melanoma cell heterogeneity M Ennen1, C Keime1, D Kobi1, G Mengus1, D Lipsker1,2, C Thibault-Carpentier1 and I Davidson1 It is well established that tumours are not homogenous, but comprise cells with differing invasive, proliferative and tumour- initiating potential. A major challenge in cancer research is therefore to develop methods to characterize cell heterogeneity. In melanoma, proliferative and invasive cells are characterized by distinct gene expression profiles and accumulating evidence suggests that cells can alternate between these states through a process called phenotype switching. We have used microfluidic technology to isolate single melanoma cells grown in vitro as monolayers or melanospheres or in vivo as xenografted tumours and analyse the expression profiles of 114 genes that discriminate the proliferative and invasive states by quantitative PCR. Single-cell analysis accurately recapitulates the specific gene expression programmes of melanoma cell lines and defines subpopulations with distinct expression profiles. Cell heterogeneity is augmented when cells are grown as spheres and as xenografted tumours. Correlative analysis identifies gene-regulatory networks and changes in gene expression under different growth conditions. In tumours, subpopulations of cells that express specific invasion and drug resistance markers can be identified amongst which is the pluripotency factor POUF51 (OCT4) whose expression correlates with the tumorigenic potential. We therefore show that single-cell analysis can be used to define and quantify tumour heterogeneity based on detection of cells with specific gene expression profiles. Oncogene (2015) 34, 3251–3263; doi:10.1038/onc.2014.262; published online 18 August 2014 INTRODUCTION slow cycling, MITF-low cells have tumour-initiating properties and Tumours are not homogenous, but comprise cells with differing that such cells can arise spontaneously in the cultures of MITF- 10 invasive, proliferative and tumour-initiating potential. Classical expressing cells. anticancer agents target highly proliferative cells; however, the We have used single-cell quantitative (q) PCR to address limited success of this approach with initial tumour regression heterogeneity in melanoma cells in vitro and as xenografted followed by relapse attests the presence of residual, slow-growing, tumours in mice. We show that this method can be used to define therapeutically resistant tumour cells. These nonproliferative, melanoma cell heterogeneity. drug-resistant cells, sometimes termed cancer stem cells or tumour-initiating stem cells, may initiate new tumours when the local environment becomes favourable.1 RESULTS In melanoma, heterogeneity can arise from accumulation of Heterogeneous gene expression in melanoma cells in vitro genetic lesions that promote therapeutic resistance if a small To determine whether it is possible to observe and quantify subpopulation of cells harbour mutations that confer resistance tumour cell heterogeneity from gene expression in single cells, we V600E 2,3 towards drugs specifically targeting oncogenic BRAF . used two melanoma cell lines. MITF-high 501Mel cells proliferate Superimposed on this is phenotype switching where the tumour rapidly in vitro, but are poorly invasive/motile and poorly microenvironment can induce melanoma cells to adopt an tumorigenic when injected subcutaneously in nude mice. MITF- invasive, proliferative or stem-like phenotypes.4,5 Meta-analysis negative 1205Lu cells proliferate slowly in vitro, and are invasive/ of gene expression in more than 500 melanoma samples motile and highly tumorigenic in nude mice.8 RNA-seq identified identified specific signatures involving a group of around 100 3655 genes preferentially expressed in 1205Lu compared with genes whose differential expression correlates strongly with either 501Mel and 2505 genes show the opposite profile (Supplementary the proliferative or invasive cell states.6,7 The strongest marker of Table 1). The Widmer7 proliferative signature genes are preferen- the proliferative phenotype is MIcrophthalmia-associated Tran- tially expressed in 501Mel cells and invasive signature genes scription Factor (MITF) strongly expressed in over 90% of preferentially expressed in 1205Lu cells. We designed primer pairs proliferative cells, but low to undetectable in slow cycling invasive for invasive and proliferative signature genes, for POU3F2/BRN2 cells. MITF is heterogeneously expressed in human tumours and and GLI2, the ‘stemness’ markers POU5F1 (OCT4) and Nanog low-MITF-expressing cells are characterized by high expression of whose expression has been reported to be induced in melanoma the transcription factors POU3F2/BRN28 and/or GLI2,9 that appear cells under conditions that potentiate their tumour-initiating as markers for invasive cells, independent of the BRAF or NRAS potential,11 cell cycle markers such as CCNB1, CCND1 and several oncogenic mutation status. There is also compelling evidence that other genes preferentially or commonly expressed in the two cell 1Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/UDS, Illkirch, France and 2Faculté de Médecine and Service de Dermatologie, Hôpital Civil, Hôpitaux Universitaires de Strasbourg, Strasbourg, France. Correspondence: Dr I Davidson, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/UDS, 1 Rue Laurent Fries, 67404 Illkirch, France. E-mail: [email protected] Received 27 May 2014; revised 8 July 2014; accepted 10 July 2014; published online 18 August 2014 Melanoma heterogeneity M Ennen et al 3252 types, for example the drug resistance marker ABCB512 Increased heterogeneity in cells grown as melanospheres (Supplementary Table 1), to determine whether the differential Melanoma cells can be grown as spheres showing heterogeneous gene expression is represented at the single-cell level. expression of MITF and other markers.13,14 We grew 501Mel and We analysed 501Mel and 1205Lu cells from exponentially 1205Lu cells as spheres for 8 days and assessed the heterogeneity growing monolayer cultures. Heatmaps representing gene expres- of their gene expression profiles. Populations expressing high, low sion generated from qPCR performed on 501Mel cells and or no MITF can be observed in 501Mel-derived spheres (Figure 2a). clustered using the unsupervised Unweighted pair group method Expression of many MITF targets correlates with that of MITF with arithmetic mean identified two major populations expressing although a population expressing high levels of MITF is either high or intermediate levels of MITF expression (Figure 1a). distinguished from the others by lower and more variable High MITF-expressing cells also strongly express many known expression of its target genes. Thus compared with monolayers, MITF target genes (Figure 1a, Supplementary Table 1). In cells with growth as spheres increases cell heterogeneity. Immunostaining intermediate MITF, many 501mel-enriched genes display a more also identified sphere-derived cells with very different MITF levels heterogeneous profile of expression. We also identified a small and many MITF-negative cells (Figure 2b). Similarly, heteroge- number of cells showing strongly reduced MITF where expression neous expression of ITGA4 is also observed both by qPCR and of most of its target genes is also strongly diminished. In contrast, immunostaining. invasive signature and 1205Lu-expressed genes show low to Analysis of 1205Lu cells grown as spheres reveals cells undetectable expression in all cells. with high, low or no detectable ZEB1 expression (Supplementary In analysis of a second set of cells with a smaller number of Figure 3A). In addition, a subpopulation of ZEB1 high cells can be genes, cells with high and intermediate MITF levels are detected, observed that express generally higher levels of many of but are clustered together although separated from the MITF-low the tested genes (*** in Supplementary Figure 3A). Under these population (Supplementary Figure 1A). As seen above, MITF target conditions, the expression of ITGA4 and BIRC3 strongly correlate genes cluster together with MITF separately from invasive with that of ZEB1. Thus, similar to 501Mel cells growth as spheres signature genes. Importantly two cells with undetectable levels leads to increased heterogeneity of the 1205Lu population. of MITF can be seen (** in Supplementary Figure 1A) confirming The cell specificity and heterogeneity of expression are also previous reports that rare MITF-negative cells arise spontaneously underlined by comparisons of the two cell types (Figure 2c) that 10 in 501Mel cultures. Immunostaining confirmed the existence of also highlights a subpopulation of 1205Lu cells showing low 501Mel cells expressing high and lower levels of MITF in expression of many transcripts and thus may correspond to a proportions similar to those seen in the qPCR analysis, whereas different cell state (*** in Figure 2c). no MITF expression was detected in 1205Lu cells (Figure 1b). fi Together these data de ne heterogeneity in the 501mel mono- Correlation of gene expression identifies MITF-regulated genes layer cell population. Overall 1205Lu cell monolayers appear more homogeneous We used the single-cell data to make correlation maps of genes than the 501Mel cells as most 1205lu-expressed genes show showing co-regulated expression. In 501Mel-derived

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