MITF-High and MITF-Low Cells and a Novel Subpopulation Expressing Genes of Both Cell States Contribute to Intra- and Intertumoral Heterogeneity of Primary Melanoma

Abstract

Understanding tumor heterogeneity is an important challenge in current cancer research. Transcription and epigenetic profiling of cultured melanoma cells have defined at least two distinct cell phenotypes characterized by distinctive gene expression signatures associated with high or low/absent expression of microphthalmia-associated transcription factor (MITF). Nevertheless, heterogeneity of cell populations and gene expression in primary human tumors is much less well characterized. We performed single-cell gene expression analyses on 472 cells isolated from needle biopsies of 5 primary human melanomas, 4 superficial spreading, and one acral melanoma. The expression of -high and-low signature genes was assessed and compared to investigate intra- and intertumoral heterogeneity and correlated gene expression profiles. Single-cell gene expression analyses revealed varying degrees of intra- and intertumor heterogeneity conferred by the variable expression of distinct sets of genes in different tumors. Expression of partially correlated with that of its known target genes, while expression correlated best with and Nevertheless, cells simultaneously expressing -high and-low signature genes were observed both by single-cell analyses and RNAscope. Single-cell analyses can be performed on limiting numbers of cells from primary human melanomas revealing their heterogeneity. Although tumors comprised variable proportions of cells with the -high and-low gene expression signatures characteristic of melanoma cultures, primary tumors also comprised cells expressing markers of both signatures defining a novel cell state in tumors .

A PAX3/BRN2 rheostat controls the dynamics of BRAF mediated MITF regulation in MITFhigh/AXLlow melanoma

“…An even more dedifferentiated phenotype reminiscent of a neural crest stem cell (NCSC) like state is driven by RXRG, devoid of both MITF and AXL, but marked by high levels of SOX10; the population with this phenotype gives rise to minimal residual disease (Rambow et al, 2018) Our data support the observed existence of populations of BRN2 high /MITF low cells and vice versa (Goodall et al, 2008;Thurber et al, 2011). However, our model predicts that any intermediate state can exist and indeed in tumours BRN2 are also found significantly co-expressed with MITF in individual melanoma cells; this can be seen in histology (Thurber et al, 2011) as well as by single-cell gene expression analysis (Ennen et al, 2017). Furthermore, because signals from the tumour microenvironment can influence MAPK signalling the dynamic regulation of MITF downstream of BRAF might explain at least in part the significant heterogeneity in MITF expression that is observed in human tumours.…”

Melanoma plasticity and phenotypic diversity: therapeutic barriers and opportunities

“…Taken together this single cell analysis, by and large, confirmed the coexistence of the MITF High and MITF Low phenotypic states, and also revealed that rare drug-resistant cells were found in treatment naïve tumors that would otherwise be classified as MITF High and, therefore, drug-sensitive. However, evidence for any intermediate state, as indicated by the Ennen et al analyses (Ennen et al 2015(Ennen et al , 2017, was not readily apparent. This may be due to the relatively low number of cancer cells analyzed (i.e., only two tumors out of the 20 analyzed exhibited >150 malignant cells) and/or limited number of genes detected per cell due to inherent limitations of the scRNA-seq technique used for this analysis.…”

“…Fifth, single-cell gene expression profiling also points toward an intermediate or poised state in which proliferative and invasive gene expression signatures are present. RT-qPCR profiling of single cells from a series of melanoma biopsies revealed the existence of cells simultaneously expressing genes that belong to both the MITF High and MITF Low gene-expression signatures (Ennen et al 2015(Ennen et al , 2017. Although these studies only examined a limited repertoire of genes, they did highlight substantial heterogeneity in gene expression at the single-cell level; something that may have been missed when analyzing mRNA expression levels from bulk tumor samples or from cell lines.…”

Single-cell gene regulatory network analysis reveals new melanoma cell states and transition trajectories during phenotype switching

“…Recently, scattered evidence illustrates the existence of additional, intermediate state(s) (Haass et al 2014;M. Ennen et al 2015;Marie Ennen et al 2017a;Falletta et al 2017;Tsoi et al 2018;Rambow et al 2018;Tuncer et al 2019).…”