Figure 5 from Long-term Multimodal Recording Reveals Epigenetic Adaptation Routes in Dormant Breast Cancer Cells

Failed awakenings. A, TRADITIOM Live awakening topography analysis depicting the awakening dynamics. Twelve carbon copies (cc; replicates) were seeded in a 48-well plate and imaged two times a week for 150 days using IncuCyte Zoom (9 scanning windows per well) to monitor awakening dynamics. Awakening was defined as wells reaching a confluency of 50% (FA: failed awakening; D: dormant; AW: awakening; GA: global awakening; LA: localized awakening; 1LA: 1 localized awakening; >1LA: more than 1 localized awakening). B, IncuCyte time-lapse images from a failed and a bona fide awakening are shown as an example. C, Proliferation dynamics of a representative MCF7 [AI (−E2) awakening 2] and T47D TRADITIOM LSC sample determined by weekly IncuCyte time-lapse imaging until awakening. Pink lines indicate failed awakenings. Magenta lines follow the growth dynamics of main awakening areas. D, MCF7 Geminin-mCherry NLS-GFP cells were treated with estrogen deprivation (−E2) for 3 months to establish a detailed understanding of long-term dormancy–awakening dynamics. Image sets were analyzed using Essenbio Sartorius software from daily imaging. 35% of replicates (n = 60) had dormant persister cells/small colonies until day 88. The proportion of Geminin-mCherry-positive (S/G₂–M; pink lines) is indicated normalized to total count which was quantified by NLS-GFP (gray lines). E, Distribution of Ki-67 expression levels in winner and non-winner (others) lineages associated with either G₁ or S/G₂–M states across T0, dormancy, and awakening samples of MCF7 TRADITIOM LSC cell-cycle regressed data set. F, Cell-cycle regressed UMAPs of the subset of cells in S/G₂–M state of TRADITIOM MCF7 LSC data set. G, Pathway enrichment analysis comparing failed awakenings (S/G₂–M cells in dormancy: cluster 0, 3) to bona fide awakenings (S/G₂–M cells of awakening winner lineages: cluster 4).