Single-Cell RNA-Seq Reveals Melanoma Transcriptional Heterogeneity

Tumors are composed of multiple types of interacting cells, including malignant tumor, immune, stromal, and endothelial cells. In malignant melanoma, the cellular diversity poses challenges for cancer therapy. To simultaneously identify the major cellular components associated with melanoma and explore the heterogeneities in the malignant and nonmalignant cell types, Tirosh, Izar, and colleagues used single-cell RNA sequencing (RNA-seq) to better understand the complex tumor cellular ecosystem. A total of 4,645 single cells from 19 patients with melanoma were analyzed, including malignant, immune, stromal, and epithelial cells. To distinguish the different cell types, large-scale copy-number variations were identified, and cells were further grouped by their expression profiles. Nonmalignant cells clustered by cell type independent of tumor of origin, whereas malignant cells from each tumor clustered separately, indicating a high degree of intertumor heterogeneity. Malignant cell gene expression analysis also revealed high intratumor heterogeneity in cell cycle genes, indicating low- and high-cycling tumors. Treatment-naïve melanoma cells may harbor subsets of cells that are resistant to targeted therapy. Expression of the transcriptional regulator MITF, a melanoma lineage-survival oncogene, is negatively correlated with expression of the AXL kinase, which is associated with intrinsic resistance to RAF/MEK inhibitors. While bulk tumors could be classified as MITF-high or AXL-high, single-cell analysis revealed the existence of both transcriptional states in every tumor, including treatment-naïve melanoma. Further, RNA-seq of paired BRAF-mutant melanoma biopsies before and after RAF/MEK inhibition indicated that treatment resulted in increased expression of AXL, supporting the initial existence of drug-resistance tumor cell sub-populations. Additionally, enrichment of cancer-associated fibroblasts (CAF) was associated with an AXL-high signature, indicating interactions between melanoma and CAF cells may shape the melanoma cell transcriptome. Taken together, these findings reveal melanoma intratumor and intertumor heterogeneity, identify malignant cell–tumor microenvironment interactions, and detect therapy-resistant tumor subpopulations prior to treatment.

Tirosh I, Izar B, Prakadan SM, Wadsworth MH 2nd, Treacy D, Trombetta JJ, et al. Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq. Science 2016;352:189–96.

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