Melanoma cells can switch between a melanocytic and a mesenchymal-like state. Scattered evidence indicates that additional intermediate state(s) may exist. Here, to search for such states and decipher their underlying gene regulatory network (GRN), we studied 10 melanoma cultures using single-cell RNA sequencing (RNA-seq) as well as 26 additional cultures using bulk RNA-seq. Although each culture exhibited a unique transcriptome, we identified shared GRNs that underlie the extreme melanocytic and mesenchymal states and the intermediate state. This intermediate state is corroborated by a distinct chromatin landscape and is governed by the transcription factors SOX6, NFATC2, EGR3, ELF1 and ETV4. Single-cell migration assays confirmed the intermediate migratory phenotype of this state. Using time-series sampling of single cells after knockdown of SOX10, we unravelled the sequential and recurrent arrangement of GRNs during phenotype switching. Taken together, these analyses indicate that an intermediate state exists and is driven by a distinct and stable ‘mixed’ GRN rather than being a symbiotic heterogeneous mix of cells.

黑色素瘤细胞可以在黑色素细胞和间充质样状态之间转换。分散的证据表明可能存在额外的中间状态。在这里，为了寻找这些状态并破译它们潜在的基因调控网络 (GRN)，我们使用单细胞 RNA 测序 (RNA-seq) 研究了 10 种黑色素瘤培养物，以及使用大量 RNA-seq 的另外 26 种培养物。尽管每种文化都表现出独特的转录组，但我们确定了共同的 GRN，它们是极端黑素细胞和间充质状态以及中间状态的基础。这种中间状态得到了独特的染色质景观的证实，并受转录因子 SOX6、NFATC2、EGR3、ELF1 和 ETV4 的控制。单细胞迁移测定证实了这种状态的中间迁移表型。在 SOX10 敲低后使用单细胞的时间序列采样，我们揭示了 GRN 在表型转换过程中的顺序和反复排列。总之，这些分析表明存在一种中间状态，并由一种独特而稳定的“混合”GRN 驱动，而不是细胞的共生异质混合物。