To explore the distinct genotypic and phenotypic states of melanoma tumors we applied single-cell RNA-seq to 4,645 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 “MITF-high” tumors also contained “AXL-high” tumor cells. Single-cell analyses suggested distinct tumor micro-environmental patterns, including cell-to-cell interactions. Analysis of tumor-infiltrating T cells revealed exhaustion programs, their connection to T cell activation and to 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.

Tumour-initiating cells capable of self-renewal and differentiation, which are responsible for tumour growth, have been identified in human haematological malignancies 1,2 and solid cancers [3][4][5][6] . If such minority populations are associated with tumour progression in human patients, specific targeting of tumour-initiating cells could be a strategy to eradicate cancers currently resistant to systemic therapy. Here we identify a subpopulation enriched for human malignantmelanoma-initiating cells (MMIC) defined by expression of the chemoresistance mediator ABCB5 (refs 7, 8) and show that specific targeting of this tumorigenic minority population inhibits tumour growth. ABCB5 + tumour cells detected in human melanoma patients show a primitive molecular phenotype and correlate with clinical melanoma progression. In serial humanto-mouse xenotransplantation experiments, ABCB5 + melanoma cells possess greater tumorigenic capacity than ABCB5 − bulk populations and re-establish clinical tumour heterogeneity. In vivo genetic lineage tracking demonstrates a specific capacity of ABCB5 + sub-populations for selfrenewal and differentiation, because ABCB5 + cancer cells generate both ABCB5 + and ABCB5 − progeny, whereas ABCB5 − tumour populations give rise, at lower rates, exclusively to ABCB5 − cells. In an initial proof-of-principle analysis, designed to test the hypothesis that MMIC are also

Purpose: To evaluate the effects of BRAF inhibition on the tumor microenvironment in patients with metastatic melanoma.Experimental Design: Thirty-five biopsies were collected from 16 patients with metastatic melanoma pretreatment (day 0) and at 10 to 14 days after initiation of treatment with either BRAF inhibitor alone (vemurafenib) or BRAF þ MEK inhibition (dabrafenib þ trametinib) and were also taken at time of progression. Biopsies were analyzed for melanoma antigens, T-cell markers, and immunomodulatory cytokines.Results: Treatment with either BRAF inhibitor alone or BRAF þ MEK inhibitor was associated with an increased expression of melanoma antigens and an increase in CD8þ T-cell infiltrate. This was also associated with a decrease in immunosuppressive cytokines [interleukin (IL)-6 and IL-8] and an increase in markers of T-cell cytotoxicity. Interestingly, expression of exhaustion markers TIM-3 and PD1 and the immunosuppressive ligand PDL1 was increased on treatment. A decrease in melanoma antigen expression and CD8 T-cell infiltrate was noted at time of progression on BRAF inhibitor alone and was reversed with combined BRAF and MEK inhibition.Conclusions: Together, these data suggest that treatment with BRAF inhibition enhances melanoma antigen expression and facilitates T-cell cytotoxicity and a more favorable tumor microenvironment, providing support for potential synergy of BRAF-targeted therapy and immunotherapy. Interestingly, markers of T-cell exhaustion and the immunosuppressive ligand PDL1 are also increased with BRAF inhibition, further implying that immune checkpoint blockade may be critical in augmenting responses to BRAF-targeted therapy in patients with melanoma.