title: Mapping heterogeneity in patient-derived melanoma cultures by single-cell RNA-seq.
abstract: Recent technological advances in single-cell genomics make it possible to analyze cellular heterogeneity of tumor samples. Here, we applied single-cell RNA-seq to measure the transcriptomes of 307 single cells cultured from three biopsies of three different patients with a BRAF/NRAS wild type, BRAF mutant/NRAS wild type and BRAF wild type/NRAS mutant melanoma metastasis, respectively. Analysis based on self-organizing maps identified sub-populations defined by multiple gene expression modules involved in proliferation, oxidative phosphorylation, 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, suggesting that these kinases might be involved in melanoma progression. Treatment of cells with the CDK4 inhibitor palbociclib restricted cell proliferation to a similar, and in some cases greater, extent than MAPK inhibitors. Finally, we 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). Patient-derived cultures of the BRAF mutant/NRAS wild type and BRAF wild type/NRAS mutant metastases showed more homogeneous single-cell gene expression patterns with gene expression modules for proliferation and ABC transporters. 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 therapy.
authors.affiliation: Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology Leipzig, 04103 Leipzig, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Gerber
authors.affiliation: Interdisciplinary Center for Bioinformatics, University of Leipzig, 04107 Leipzig, Germany.
authors.forename: Edith
authors.initials: E
authors.lastname: Willscher
authors.affiliation: Interdisciplinary Center for Bioinformatics, University of Leipzig, 04107 Leipzig, Germany.
authors.forename: Henry
authors.initials: H
authors.lastname: Loeffler-Wirth
authors.affiliation: Interdisciplinary Center for Bioinformatics, University of Leipzig, 04107 Leipzig, Germany.
authors.forename: Lydia
authors.initials: L
authors.lastname: Hopp
authors.affiliation: Department of Dermatology, Venereology and Allergology, University Hospital Essen, 45147 Essen, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Schadendorf
authors.affiliation: Department of Physiological Chemistry, University of Würzburg, Biozentrum, Am Hubland, 97074 Würzburg, Germany.|Comprehensive Cancer Center Mainfranken, University Clinic Würzburg, 97080 Würzburg, Germany.|Institute for Advanced Study, 3572 Texas A&M University, College Station, Texas 77843-3572, USA.
authors.forename: Manfred
authors.initials: M
authors.lastname: Schartl
authors.affiliation: Department of Dermatology, Venereology and Allergology, University of Leipzig, 04103 Leipzig, Germany.
authors.forename: Ulf
authors.initials: U
authors.lastname: Anderegg
authors.affiliation: Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology Leipzig, 04103 Leipzig, Germany.
authors.forename: Gray
authors.initials: G
authors.lastname: Camp
authors.affiliation: Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology Leipzig, 04103 Leipzig, Germany.
authors.forename: Barbara
authors.initials: B
authors.lastname: Treutlein
authors.affiliation: Interdisciplinary Center for Bioinformatics, University of Leipzig, 04107 Leipzig, Germany.
authors.forename: Hans
authors.initials: H
authors.lastname: Binder
authors.affiliation: Department of Dermatology, Venereology and Allergology, University of Leipzig, 04103 Leipzig, Germany.
authors.forename: Manfred
authors.initials: M
authors.lastname: Kunz
mesh_terms: D000328:Adult; D000368:Aged; D014408:Biomarkers, Tumor; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002874:Chromosome Mapping; D019295:Computational Biology / Q000379:methods; D051358:Cyclin-Dependent Kinase 4 / Q000037:antagonists & inhibitors; D044127:Epigenesis, Genetic; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D011905:Genes, ras; D018740:Genetic Heterogeneity; D020022:Genetic Predisposition to Disease; D006801:Humans; D008297:Male; D008545:Melanoma / Q000235:genetics* / Q000378:metabolism / Q000401:mortality / Q000473:pathology; D008875:Middle Aged; D009362:Neoplasm Metastasis; D010879:Piperazines / Q000494:pharmacology; D011379:Prognosis; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D011725:Pyridines / Q000494:pharmacology; D017423:Sequence Analysis, RNA / Q000379:methods; D015398:Signal Transduction; D059010:Single-Cell Analysis / Q000379:methods; D059467:Transcriptome
chemical_list: D014408:Biomarkers, Tumor; D010879:Piperazines; D011725:Pyridines; D048493:Proto-Oncogene Proteins B-raf; D051358:Cyclin-Dependent Kinase 4; C500026:palbociclib
keywords: melanoma; single cell transcriptome sequencing; stem cells