Figure 4: Cancer cells transition to hybrid E/M states and require different EMT TFs during invasion and colony formation.

A) Schema of the longitudinal single-cell RNA sequencing experiment. After isolation, C3(1)-Tag organoids are embedded in collagen I or dissociated into clusters and embedded in Matrigel. After 12h, 3 days and 5 days, organoids are extracted from the matrix and dissociated into single cells. Single tumor cells and 4T1 cells, used as a reference for batch correction, are barcoded per condition before flow sorting for live cells using propidium iodide. Live cells are next used for 10X Genomics barcoding and library preparation. All conditions are then sequenced together.

B) UMAP representation of the sequenced cells from invasion and colony formation assay. Each dot corresponds to single cell and the colors indicate the day the cells were extracted from the matrix.

C) Distribution of the proportion of Ecad⁺ and/or Vim⁺ cells by matrix and day.

D) Schema for shRNA-based gene knock-down in the invasion and colony formation assays. After isolation, organoids are infected with either NT-shRNA or a pool of 3 shRNA sequences against a specific gene. After puromycin selection to eliminate non-infected cells, organoids are either directly lysed to extract mRNA for qPCR analysis, embedded into collagen I, or dissociated into clusters and embedded in Matrigel. After 5 days, the invasion or the number of colonies is measured.

E) mRNA fold change, assayed by qRT-PCR, of Ecad (Cdh1) and Vim gene expression in C3(1)-Tag organoids knocked-down for Grhl2, Zeb2, Foxc2, Zeb1 and Ovol1. Histogram of mean with SEM. r ≥3. * P<0.05, ***P<0.005 (paired T-test, two sided on the deltaCT value between NT-shRNA and shRNA against specific gene).

F) Representative confocal images of maximum intensity projection of whole C3(1)-Tag organoids stained with Vim. GFP indicates infected cells. Scale bars are 100 μm.

G) Quantification of organoid invasion represented in F. Each dot corresponds to one organoid, bars show the medians. **P=0.0016, ***P=0.0003, ****P<0.0001 (Kruskal-Wallis), n=163 organoids, r=3 biological replicates.

H) Representative epifluorescence images of maximum intensity projection of C3(1)-Tag colonies. GFP indicates infected cells. Scale bars are 500 μm.

I) Number of colonies per well. Each dot represents the number of colonies in one well, bars show the medians. *P=0.0267, **P=0.034 (Kruskal-Wallis), n=67 wells, r=3 biological replicates.