Analysis single-cell transcriptomics and pseudotime dynamics of patient-derived melanoma cultures
Tobias Gerber,1 Edith Willscher,2 Henry Loeffler-Wirth2, Lydia Hopp2, Dirk Schadendorf3, Manfred Schartl4, Ulf Anderegg,5 Gray Camp1, Barbara Treutlein1, Hans Binder2, Manfred Kunz5,*
1Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology Leipzig, 04103 Leipzig, Germany; 2Interdisciplinary Center for Bioinformatics, University of Leipzig, Härtelstr. 16-18, 04107 Leipzig, Germany; 3Department of Dermatology, Venereology and Allergology, University Hospital Essen, Hufelandstraße 55, 45147 Essen, Germany; 4Department of Physiological Chemistry, University of Würzburg, Biozentrum, Am Hubland, 97074 Würzburg; and Comprehensive Cancer Center Mainfranken, University Clinic Würzburg, 97080 Würzburg, Germany; and Institute for Advanced Study, 3572 Texas A& M University, College Station, Texas 77843-3572, USA; 5Department of Dermatology, Venereology and Allergology, University of Leipzig, Philipp-Rosenthal-Str. 23-25, 04103 Leipzig, Germany
We applied single-cell RNA-seq to measure the transcriptomes of 307 single cells cultured from biopsies of three different metastatic melanoma patients with a BRAF/NRAS wild type, BRAF mutant/NRAS wild type and BRAF wild type/NRAS mutant metastatic melanoma, respectively. Analysis based on self-organizing maps identified sub-populations defined by multiple gene expression modules involved in proliferation, oxidative phosphorylation (oxphos), pigmentation and cellular stroma. Gene expression modules had prognostic relevance when compared with gene expression data from published melanoma samples and patient survival data. We surveyed kinome expression patterns across sub-populations of the BRAF/NRAS wild type sample and found that CDK4 and CDK2 were consistently highly expressed in the majority of cells. Treatment of cells with the CDK4 inhibitor palbociclib restricted cell proliferation to a similar, and in some cases greater, extent than MAPK inhibitors. We further identified a low abundant sub-population in this sample that highly expressed a module containing ABC transporter ABCB5, surface markers CD271 and CD133, and multiple aldehyde dehydrogenases (ALDHs). We further analyzed pseudotime dynamics of the 3 melanoma cultures to study specific and concordant cellular programs with impact on melanoma progression. These analyses provided evidence for common and disparate mechanisms of pseudotime progression in the different melanoma samples. It further unveiled that MITF/AXL expression inversely correlated on single-cell level which appears to be due to opposing regulatory mechanisms. Moreover, oxphos and cellular proliferation signatures were coupled via a switch-like mechanism. Oxphos and glycolysis signatures suggested high growth-promoting and metabolic plasticity in melanoma cells. Taken together, our results describe an intertumor and intratumor heterogeneity in melanoma short-term cultures which might be relevant for patient survival, and suggest promising targets for new treatment approaches in melanoma.
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