Extended Data Fig. 5: FateMap on an NRAS mutant melanoma cell line reveals between-clone fate type diversity.

a. NRAS mutant cell line WM3451 treated with three different doses of trametinib (20 nM, 40 nM, and 50 nM). Representative brightfield images after 2.5 and 5 weeks of drug treatment are shown for each dose. b. (left) UMAP of all barcoded 3451 cells treated with Trametinib. 5,789 cells are colored by clusters determined using Seurat’s FindClusters command at a resolution of 0.6 (i.e. “Seurat clusters, resolution = 0.6”). (right) On the UMAP, we recolored each cell by its expression for a select subset of genes that were identified as differentially expressed in drug resistant cells via the Seurat pipeline. c. Five representative examples demonstrate that a clone (cells sharing the same barcode) is constrained largely in a specific transcriptional cluster such that cells within a clone are more transcriptionally similar to each other than cells in other clones. d. Average pairwise correlation between cells within a clone was estimated based on the expression levels of the top 500 most variable genes. Each point represents the average value for Spearman’s correlation coefficient for all possible pairs of cells within a clone. For each clone, a paired control was created by randomly sampling an equivalent number of cells from the whole population. Higher average correlation coefficient in clones indicates higher transcriptional similarity among cells within a clone, as compared to cells that are not clones. Wilcoxon signed rank test (paired, two-sided) was used to compare the difference in average correlation coefficient. e. UMAP of all barcoded WM3451 P2G7 cells treated with Trametinib. Cells are colored by whether they are a singleton (i.e. clone size = 1). f. UMAPs of representative twin clones (sharing the same barcode) across the two splits A (5,789 cells) and B (7,473 cells). The twins largely end up with the same transcriptional fate type. This observation suggests that drug resistant cells are derived from the same clones having similar transcriptional states and are constrained in the gene expression space.