Melanoma intra-tumor heterogeneity

Tirosh I, Izar B, Prakadan SM, Wadsworth MH 2nd, Treacy D, Trombetta JJ, Rotem A, Rodman C, Lian C, Murphy G, Fallahi-Sichani M, Dutton-Regester K, Lin JR, Cohen O, Shah P, Lu D, Genshaft AS, Hughes TK, Ziegler CG, Kazer SW, Gaillard A, Kolb KE, Villani AC, Johannessen CM, Andreev AY, Van Allen EM, Bertagnolli M, Sorger PK, Sullivan RJ, Flaherty KT, Frederick DT, Jané-Valbuena J, Yoon CH, Rozenblatt-Rosen O, Shalek AK, Regev A, Garraway LA Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq. Science. 2016 Apr 8;352(6282):189-96. doi: 10.1126/science.aad0501
Contact person: Itay Tirosh, tirosh.itay@gmail.com

To explore the distinct genotypic and phenotypic states of melanoma tumors, we applied single-cell RNA sequencing (RNA-seq) to 4645 single cells isolated from 19 patients, profiling malignant, immune, stromal, and endothelial cells. Malignant cells within the same tumor displayed transcriptional heterogeneity associated with the cell cycle, spatial context, and a drug-resistance program. In particular, all tumors harbored malignant cells from two distinct transcriptional cell states, such that tumors characterized by high levels of the MITF transcription factor also contained cells with low MITF and elevated levels of the AXL kinase. Single-cell analyses suggested distinct tumor microenvironmental patterns, including cell-to-cell interactions. Analysis of tumor-infiltrating T cells revealed exhaustion programs, their connection to T cell activation and clonal expansion, and their variability across patients. Overall, we begin to unravel the cellular ecosystem of tumors and how single-cell genomics offers insights with implications for both targeted and immune therapies.

(A) Overview of workflow. (B) Chromosomal landscape of inferred large-scale copy number variations (CNVs) distinguishes malignant from non-malignant cells. The Mel80 tumor is shown with individual cells (y-axis) and chromosomal regions (x-axis). Amplifications (red) or deletions (blue) were inferred by averaging expression over 100-gene stretches on the respective chromosomes. Inferred CNVs are concordant with calls from whole-exome sequencing (WES, bottom). (C,D) Single cell expression profiles distinguish malignant and non-malignant cell types. Shown are t-SNE plots of malignant (C, shown are the six tumors each with >50 malignant cells) and non-malignant (D) cells (as called from inferred CNVs as in B) from 11 tumors with >100 cells per tumor (color code). Clusters of non-malignant cells (called by DBScan, (17)) are marked by dashed ellipses and were annotated as T cells, B cells, macrophages, CAFs and endothelial cells, from preferentially expressed genes.