Extended Data Fig. 7: FateMap on a triple negative breast cancer cell line reveals between-clone fate type diversity.

a. Nuclei scans (DAPI-stained) of resistant colonies emerging from treatment of the single-cell derived triple negative breast cancer cell line MDA-MB-231-D4 with 1nM paclitaxel. b. For the MDA-MB-231-D4 cell line, we traced representative resistant cells in Adobe Illustrator and created cartoon schematics based on visual inspection of orientation and density. c. Brightfield images of resistant colonies exhibiting different types of morphologies. d. We applied the Uniform Manifold Approximation and Projection (UMAP) algorithm within Seurat to the first 50 principal components to visualize differences in gene expression. 6,535 cells are colored by clusters determined using Seurat’s FindClusters command (“Seurat clusters, resolution = 0.5”). e. We observed silencing of the transcribed barcodes in a subset of colonies, as revealed by epifluorescence imaging of the GFP signal. The colony on the left is strongly expressing a GFP signal while the colony on the right has a very dim GFP signal. f. Cells with assigned barcodes were evenly distributed throughout the UMAP with no clear bias for any specific resistant fate types. g. Four examples from split A (6,535 cells) demonstrate that a clone is constrained largely in specific UMAP regions such that cells within a clone are more transcriptionally similar to each other than cells in other clones. h. Four examples from split B (8,745 cells) demonstrate that a clone is constrained largely in specific UMAP regions such that cells within a clone are more transcriptionally similar to each other than cells in other clones. i. We quantified the preference for a specific cluster across all barcode clones (clone size>4). Specifically, we calculated the fraction of dominant clusters for each clone and found it to be significantly higher (Wilcoxon, unpaired, two-sided) than that for randomly selected cells. The analysis plotted here is for a cluster resolution of 0.5. j. We found that our UMAP had superclusters defined by cell cycle (S, G1, G2M). Of the 3,720 clonal DEGs, 63 are cell cycle genes. We therefore regressed out cell cycle genes and the cell-cycle-genes-regressed data with UMAP. k. We quantified the preference for a specific cluster across all barcode clones after cell cycle regression (clone size>4). Specifically, we calculated the fraction of dominant clusters for each clone and found it to be significantly higher (Wilcoxon, unpaired, two-sided) than that for randomly selected cells. The analysis plotted here is for a cluster resolution of 0.5. l. UMAPs of representative twin clones across the two splits A and B. The twins largely end up with the same transcriptional fate type, invariant of the clone size. This observation suggests that cells are predestined for distinct resistant fate types upon exposure to chemotherapy drug paclitaxel.