Functional single cell selection and annotated profiling of dynamically changing cancer cells

Abstract

A method connecting single cell genomic or transcriptomic profiles to functional cellular characteristics, in particular time-varying phenotypic changes, would be transformative for single cell and cancer biology. Here, we present fSCS: functional single cell selection. This technology combines a custom-built ultrawide field-of-view optical screening microscope, fast automated image analysis and a new photolabeling method, phototagging, using a newly synthesized visible-light-photoactivatable dye. Using fSCS, we screen, selectively photolabel and isolate cells of interest from large heterogeneous populations based on functional dynamics like fast migration, morphological variation, small molecule uptake or cell division. We combined fSCS with single cell RNA sequencing for functionally annotated transcriptomic profiling of fast migrating and spindle-shaped MCF10A cells with or without TGFβ induction. We identified critical genes and pathways driving aggressive migration as well as mesenchymal-like morphology that could not be detected with state-of-the-art single cell transcriptomic analysis. fSCS provides a crucial upstream selection paradigm for single cell sequencing independent of biomarkers, allows enrichment of rare cells and can facilitate the identification and understanding of molecular mechanisms underlying functional phenotypes.

Spatially Annotated Single Cell Sequencing for Unraveling Intratumor Heterogeneity

“…Next, we imaged the cells using our custom-built Ultrawide Field-of-view Optical (UFO) microscope ( You et al, 2021 ) and identified the outer, middle and inner regions (with a bandwidth of 1,000–1,500 μm) of the patch. Cells were first incubated with photoactivatable Janelia Fluor 646 (JF646) dye, after which we phototagged the outer one-third of the patch ( Figure 1B ; cells emit red fluorescence (λ ex : ∼650 nm, λ em : ∼665 nm) after photoactivation).…”

“…Cell labeling was performed on the Ultrawide Field-of-view Optical (UFO) microscope developed previously ( You et al, 2021 ). Cells were incubated with 40 µM photoactivatable Janelia Fluor 646 (JF646) dye (Tocris) for 20 min and washed with MCF10A culture medium.…”

“…Single-cell sequencing is key to characterizing the complexity of intratumor heterogeneity, but lacks information about functional properties and spatial organization of cells ( Lawson et al, 2018 ). We have recently developed a functionally annotated transcriptomic profiling technique, called functional single cell sequencing (FUNseq), to study heterogeneous populations of tumor cells based on functional features ( You et al, 2021 ). This technology uses live-cell imaging to identify cells with a phenotype of interest (e.g., cell migration or morphology), which can then be phototagged ( via a photopatterned device) with a photoactivatable dye, isolated and subjected to single-cell RNA sequencing (scRNA-seq).…”

Dynamics of Epithelial–Mesenchymal Plasticity: What Have Single-Cell Investigations Elucidated So Far?

“…Tracking the morphological changes upon treatment with EMT-inducing growth factors has helped infer transition rates among different morphological cell states and has been used to develop classifiers to segregate epithelial vs mesenchymal states with 92% accuracy. − Such characterization can be coupled with scRNA-seq data to extract information about E–M heterogeneity. An example would be the development of a functional single-cell selection pipeline that allowed screening based on time-varying cellular dynamics or morphology and selective labeling of cells among a heterogeneous population . Monitoring the migration trajectories of individual cells in the MCF10 cell line, fast-moving cells were identified and phototagged.…”

Light-Seq: light-directed in situ barcoding of biomolecules in fixed cells and tissues for spatially indexed sequencing

“…Recent applications that focus on integrating imaging with transcriptomic measurements of the same individual cells rely largely on physical dissociation and sorting of cells using opto-or microfluidics before, after or during imaging [1][2][3][4] . Alternative methods rely on photoconversion of fluorescent proteins in selected live cells for later sorting [5][6][7] , or photoactivation of messenger RNA capture moieties followed by physical aspiration of target live cells for transcriptomic analysis 8 . These approaches typically require live imaging of cells and extraction of a limited number of visual features before pooling or single-cell sorting of dissociated cells for sequencing.…”