黑色素瘤转移的分子机制仍然不明确。在本研究中，我们旨在阐明在转移潜力获取过程中基因表达改变的机制，并对原始细胞系中的转移亚克隆进行表征。我们对一个转移能力较差的黑色素瘤细胞系（WM239A）及其具有高肺转移潜力（113/6-4L）和高脑转移潜力（131/4-5B1和131/4-5B2）的亚克隆进行了单细胞RNA测序。无监督聚类分析显示，8173个黑色素瘤细胞可以根据细胞类型（'原生'、'肺'和'脑'簇）划分为三个不同簇，这些簇在MITF和AXL通路的表达以及可能的癌症和细胞周期调控因子上有差异表达；肺簇在基因表达上介于原生簇和脑簇之间。主成分（PC）分析显示，与MITF表达正相关而与AXL通路负相关的PC2（第二主成分）主要区分了不同的细胞类型，而细胞周期通路的PC1也有所区分。伪时轨迹和RNA速度分析显示，在原生簇中存在具有转移潜力的细胞亚群，并且PC2特征变化与转移潜力获取之间存在关联。通过将The Cancer Genome Atlas黑色素瘤样本根据PC2特征表达的四分位数分为PC2高和PC2低簇进行聚类分析，发现PC2高簇与独立的不良预后因素（p值=0.003）以及与PC2低簇相比的基因组和转录组特征有明显差异。总之，我们确定了具有预后意义的黑色素瘤转移特征，并在原始细胞系中发现了可能的促转移亚克隆。© 2023 John Wiley & Sons A/S. 版权所有，由John Wiley & Sons Ltd. 发布。

The molecular mechanisms underlying melanoma metastasis remain poorly understood. In this study, we aimed to delineate the mechanisms underlying gene expression alterations during metastatic potential acquisition and characterize the metastatic subclones within primary cell lines. We performed single-cell RNA sequencing of a poorly metastatic melanoma cell line (WM239A) and its subclones with high metastatic potential to the lung (113/6-4L) and the brain (131/4-5B1 and 131/4-5B2). Unsupervised clustering of 8173 melanoma cells identified three distinct clusters according to cell type ('Primary', 'Lung' and 'Brain' clusters) with differential expression of MITF and AXL pathways and putative cancer and cell cycle drivers, with the lung cluster expressing intermediate but distinct gene profiles between primary and brain clusters. Principal component (PC) analysis revealed that PC2 (the second PC), which was positively associated with MITF expression and negatively with AXL pathways, primarily segregated cell types, in addition to PC1 of the cell cycle pathway. Pseudotime trajectory and RNA velocity analyses suggested the existence of cellular subsets with metastatic potential in the Primary cluster and an association between PC2 signature alteration and metastasis potential acquisition. Analysis of The Cancer Genome Atlas melanoma samples by clustering into PC2-high and -low clusters by quartiles of PC2 signature expression revealed that the PC2-high cluster was an independent significant factor for poor prognosis (p-value = 0.003) with distinct genomic and transcriptomic characteristics, compared to the PC2-low cluster. In conclusion, we identified signatures of melanoma metastasis with prognostic significance and putative pro-metastatic subclones within a primary cell line.© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.