Extended Data Fig. 4: FateMap on BRAF and NRAS mutant melanoma cell lines reveals between-clone fate type diversity.

a. (left) For another single-cell derived melanoma cell line WM983B E9-C6, we traced representative resistant cells in Adobe Illustrator and created cartoon schematics based on visual inspection of orientation and density. (right) Brightfield images of resistant colonies exhibiting different types of morphologies. b. We applied the Uniform Manifold Approximation and Projection (UMAP) algorithm within Seurat to the first 50 principal components to visualize differences in gene expression. Cells are colored by clusters determined using Seurat’s FindClusters command at a resolution of 0.5 (i.e. “Seurat clusters, resolution = 0.5”) (13,869 and 11,249 total cells respectively for split A and B). c. On the UMAP, we recolored each cell by its expression for a select subset of genes that were identified as differentially expressed via the Seurat pipeline and marked different clusters. MLANA, which marks melanocytes, is found largely in clusters 1,3, and 5; IFIT2, which marks type-1 interferon signaling, is found largely in cluster 4; NGFR, which marks neural crest cells, is found largely in cluster 2 and 4; AXL, which is a canonical resistance marker, is found largely in cluster 5 and 7. d. Six examples to 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. Some clones are larger in size than others, and some exist as singletons, meaning they survive vemurafenib treatment but do not necessarily divide while exposed to the drug. e. 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 test, two-sided, unpaired, p-value = 1.49e-15) than that for randomly selected cells. The analysis plotted here is for a cluster resolution of 0.5. f. Painting of singletons and colonies onto the UMAP demonstrated that singletons and colonies belonged to distinct regions and clusters. g. UMAPs of representative twin clones (sharing the same barcode) 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 vemurafenib. h. NRAS mutant cell line WM3623 treated with three different doses of trametinib (10 nM, 20 nM, and 40 nM). Representative brightfield images after 2.5 and 5 weeks of drug treatment are shown for each dose. i. (left) UMAP of all barcoded 3623 cell line cells treated with Trametinib. 6,397 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. j. 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. k. 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. l. UMAPs of representative twin clones (sharing the same barcode) across the two splits A (6,397 cells) and B (7,538 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. One of the clones appears to be a dominant clone and gives rise to a large fraction of sequenced cells.