Linking single-cell genomic or transcriptomic profiles to functional cellular characteristics, in particular time-varying phenotypic changes, could help unravel molecular mechanisms driving the growth of tumour-cell subpopulations. Here we show that a custom-built optical microscope with an ultrawide field of view, fast automated image analysis and a dye activatable by visible light enables the screening and selective photolabelling of cells of interest in large heterogeneous cell populations on the basis of specific functional cellular dynamics, such as fast migration, morphological variation, small-molecule uptake or cell division. Combining such functional single-cell selection with single-cell RNA sequencing allowed us to (1) functionally annotate the transcriptomic profiles of fast-migrating and spindle-shaped MCF10A cells, of fast-migrating MDA-MB-231 cells and of patient-derived head-and-neck squamous carcinoma cells, and (2) identify critical genes and pathways driving aggressive migration and mesenchymal-like morphology in these cells. Functional single-cell selection upstream of single-cell sequencing does not depend on molecular biomarkers, allows for the enrichment of sparse subpopulations of cells, and can facilitate the identification and understanding of the molecular mechanisms underlying functional phenotypes.

将单细胞基因组或转录组图谱与功能性细胞特征联系起来，特别是随时间变化的表型变化，可能有助于揭示驱动肿瘤细胞亚群生长的分子机制。在这里，我们展示了具有超宽视野、快速自动图像分析和可被可见光激活的染料的定制光学显微镜，能够在特定功能细胞的基础上对大型异质细胞群中感兴趣的细胞进行筛选和选择性光标记动力学，例如快速迁移、形态变化、小分子摄取或细胞分裂。将这种功能性单细胞选择与单细胞 RNA 测序相结合，使我们能够 (1) 在功能上注释快速迁移和纺锤形 MCF10A 细胞的转录组学概况，快速迁移的 MDA-MB-231 细胞和患者来源的头颈部鳞状癌细胞，以及 (2) 识别关键基因和通路，这些基因和通路驱动这些细胞中的侵袭性迁移和间充质样形态。单细胞测序上游的功能性单细胞选择不依赖于分子生物标志物，允许稀疏细胞亚群的富集，并有助于识别和理解功能表型背后的分子机制。