Mapping heterogeneity in patient-derived melanoma cultures by single-cell RNA-seq.
Mapping heterogeneity in patient-derived melanoma cultures by single-cell RNA-seq.
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DOI：
10.18632/oncotarget.13666
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发表时间：
2017-01-03
期刊：
影响因子：
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通讯作者：
Kunz M
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文献类型：
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作者：
Gerber T;Willscher E;Loeffler-Wirth H;Hopp L;Schadendorf D;Schartl M;Anderegg U;Camp G;Treutlein B;Binder H;Kunz M
单细胞基因组学的最新技术进步使得分析肿瘤样品的细胞异质性成为可能。在这里，我们应用单细胞RNA-seq来测量分别从具有BRAF/NRAS野生型、BRAF突变体/NRAS野生型和BRAF野生型/NRAS突变体黑色素瘤转移的三个不同患者的三个活检组织培养的307个单细胞的转录组。基于自组织图谱的分析确定了由参与增殖、氧化磷酸化、色素沉着和细胞基质的多个基因表达模块定义的亚群。与已发表的黑色素瘤样本和患者生存数据的基因表达数据相比，基因表达模块具有预后相关性。我们调查了BRAF/NRAS野生型样本亚群中的激酶组表达模式，发现CDK 4和CDK 2在大多数细胞中始终高度表达，表明这些激酶可能参与黑色素瘤进展。用CDK 4抑制剂palbociclib处理细胞，其对细胞增殖的限制程度与MAPK抑制剂相似，在某些情况下更大。最后，我们在该样品中鉴定了低丰度亚群，其高度表达包含ABC转运蛋白ABCB 5、表面标记物CD 271和CD 133以及多种醛脱氢酶（ALDH）的模块。BRAF突变/NRAS野生型和BRAF野生型/NRAS突变转移瘤的患者来源培养物显示出更均一的单细胞基因表达模式，具有增殖和ABC转运蛋白的基因表达模块。总之，我们的研究结果描述了黑色素瘤短期培养中的肿瘤间和肿瘤内异质性，这可能与患者生存有关，并为黑色素瘤治疗中的新治疗方法提供了有希望的靶点。
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.