Figure 4 from Long-term Multimodal Recording Reveals Epigenetic Adaptation Routes in Dormant Breast Cancer Cells

The adaptive journey of individual lineages at single-cell level. A, Schematic cartoon of TRADITIOM LSC experimental design. A low-complexity (100 barcodes for MCF7 and 200 barcodes for T47D) barcoded population was seeded in T75 flask format and exposed to ET (TAM and –E2 for MCF7 and –E2 for T47D) in a nonperturbed system (no serial passaging). Cells were collected at the indicated time points (duplicates of T0, 1 month, 2 months, and 4 awakening samples for MCF7 and duplicates of T0 and 1 month for T47D) and analyzed by scRNA-seq. Cells were imaged once a week till awakening with 108 scanning windows covering each flask. B, Proliferation dynamics of TRADITIOM LSC for a representative sample [AI (−E2) aw1] determined by weekly imaging along with the local confluency heat map at the time of collection (awakening). Magenta lines follow the growth dynamics of main awakening areas from time zero (T0) to overt expansion that represents the collection point for scRNA-seq analysis. Violin plot shows the confluency of each scanning area at T0 (onset of estrogen deprivation). The T0 confluency of the awakening area is highlighted in purple. C, Heat map of winner barcodes’ frequency for TRADITIOM LSC carbon copies for –E2 (AI) arm at the time of awakening, in the POT population (pretreatment), at the start of the experiment (T₀, time zero) and at early [1 month (1 mo)] and late dormancy [2 months (2 mo)] stages derived from either genomic barcode sequencing (g) or scRNA-seq (sc). D, UMAP projections of MCF7 TRADITIOM LSC –E2 (AI) arm of T0, early [1 month (1 mo)] and late dormancy [2 months (2 mo)] and awakening samples. E, Adaptive journey of winner and non-winner lineages (others) of each TRADITIOM LSC –E2 (AI) awakening sample from T0 to awakening (early progression). Pie charts depict the occupancy of miscellaneous UMAP clusters for each lineage. The approximate awakening time of each carbon copy (replicate) is shown with arrows. F, UMAP projections of T47D TRADITIOM LSC samples at T0 and dormancy [1 month (1 mo)]. G, Dot plot indicates similarity of transcriptional space occupied by MCF7 cells under −E2 treatment (AI) with those under TAM (tamoxifen) and with T47D counterpart under −E2 treatment. Marker genes for clusters in T0, dormancy, transition, and awakening from MCF7 TAM and T47D −E2 UMAPs were checked for enrichment in cluster marker genes from MCF7 −E2 (AI) UMAP. Size of the dot represents the significance of enrichment in context to –log₁₀(P value; correction method: FDR, P ≤ 0.05). The color of the dots corresponds to the respective color of clusters at T0, dormancy, and awakening annotated by colored bars next to cluster names on the x and y axes (T0: gray, dormancy: yellow, awakening: magenta).