Extended Data Fig. 5: In vivo 4D live imaging of LGR5⁺p27⁺ and LGR5⁺p27⁻ cells.

a. Representative images from LGR5 lineage tracing in LGR5-CreER/LGR5-tdTomato/p27-mVenus/tdiRFP-BFP CCO32 xenogfrafts. Both LGR5⁺p27⁺ and LGR5⁺p27^– cells stochastically convert their colour from tdiRFP to TagBFP2 (BFP (tr), blue) by day 4. Nuclear counterstaining (DRAQ5) is shown in white. b. Percentage of p27⁺ cells (mean ± s.e.m.) in BFP⁺ clones on day 5 post-tamoxifen treatment (left), and in LGR5⁺ cells in organoids without tamoxifen treatment (right). Each dot indicates one mouse. Statistics, two-sided Welch's t-test. c. Optical sections of LGR5⁺p27⁺BFP⁺ cells in untreated CCO20 xenografts (from Fig. 3c, bottom) that remained as single cells over the observation period (left). Representative clone expansion from an LGR5⁺p27⁺ cell-derived BFP⁺ clone (from Fig. 3f, bottom) following IRI treatment (right). The clones of interest are framed with white dotted line. d. The size transition of clones traced from individual BFP⁺ single LGR5⁺p27⁺ (green) or LGR5⁺p27^– (grey) cells in CCO20 or CCO32 xenografts. Each line represents one clone. e. The survival of the clones traced from single LGR5⁺p27⁺ (green line) or LGR5⁺p27^– (black dotted line) CCO32 cells with (bottom) or without (top) IRI treatment. p = 2.7 x 10^–10, log-rank test. f. Cleaved caspase-3 (green, left) and TUNEL (green, right) staining, and LGR5-tdTomato expression in untreated CCO32 xenografts. Nuclear counterstaining (white). Insets show higher magnification. g. The proportion of apoptotic cells (mean ± s.e.m.) marked by cleaved caspase-3 or TUNEL in LGR5⁺ cells (red) or LGR5^- cells (grey) in untreated CCO32 xenografts. Each dot represents one image. Statistics, two-sided Welch’s t-test. p = 4.5 x 10^–06 (TUNEL). h. The transition of the proportion of LGR5⁺ cells in traced clones analysed by in vivo live imaging. Clones that expanded over time are shown in red, and those that diminished are shown in blue. Regression lines were made using local polynomial regression, and the blue or red area shows the 95% confidence interval. Scale bars: 100 μm (a: left, g), 20 μm (a: right, c, d)

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