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.
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The main data supporting the results in this study are available within the paper and its Supplementary Information. Data generated in this study, including source data and the data used to make the figures, are available from Open Science Framework at https://doi.org/10.17605/OSF.IO/HG2NU. The bulk RNA-sequencing data are available at the NCBI BioProject database with the BioProject ID PRJNA751073.
The image-analysis programme used in the study is available at https://sourceforge.net/projects/funseq.
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M.-P.C. acknowledges support from the Oncode Institute, Cancer GenomiCs.nl (CGC), NWO (the Netherlands Organization for Scientific Research) Veni Grant, and Erasmus MC grant. M.-P.C. appreciates Josephine Nefkens Stichting’s support on the UFO microscope. D.B. acknowledges support by an NWO Start-up Grant (740.018.018) and ERC Starting Grant (850818 - MULTIVIsion). P.-R.S. acknowledges support from Ministry of Science and Technology (MOST) in Taiwan (Dragon Gate program: 107-2911-I-002-577 and Columbus Program: 108-2636-M-002-008- &109-2636-M-002-005-). We thank R. Agami and M. Paul for the kind gift of MCF10A-H2B-GFP and U2OS-H2B-mMaple3 cell lines, respectively; A. Theil and T. W. Kan for technical assistance with FACS sorting; J. Kraan and J. Martens for the use of their cell separation machine; the Erasmus MC Center for Biomics for the bulk-cell transcriptomic sequencing; K. T. Chen for the phototagging purification; and P. Keller for discussions about TGMM.
The authors declare no competing interests.
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You, L., Su, PR., Betjes, M. et al. Linking the genotypes and phenotypes of cancer cells in heterogenous populations via real-time optical tagging and image analysis. Nat. Biomed. Eng 6, 667–675 (2022). https://doi.org/10.1038/s41551-022-00853-x