Extended Data Fig. 2: Visualization and characterization of LGR5⁺p27⁺ CRC cells.

a. Ki67 immunostaining (green) and LGR5 expression in CCO7 xenograft tumours (top) and the parental patient tissue (bottom). The expression of LGR5 is shown by the LGR5 reporter (LGR5-tdT; red) in xenografts and by LGR5 ISH (red) in clinical sections. Nuclear counterstaining (white). Higher magnification images of the regions bounded by white box are shown on the right. Yellow arrowheads and white arrowheads indicate LGR5⁺Ki67⁺ cells and LGR5⁺Ki67^– cells, respectively. b. The expression of cyclin-dependent kinase CDK inhibitors (CDKis; p21, p57 and p27, green) and Ki67 (red) in clinical samples. Nuclear counterstaining, white. The proportion of CDKi-positive cells in Ki67⁺ or Ki67^– cells (mean ± s.e.m.) in CCO12 clinical samples (bottom right). Each dot represents one image. p27 marks nearly all Ki67^– cells and does not overlap with Ki67. c. Immunostaining of p27 (green) and Ki67 (red) in CCO7 and CCO20 clinical samples. Nuclear counterstaining, white. The proportions of the indicated cell populations in clinical samples (right). d. The expression of LGR5-tdTomato (red) and p27-mVenus (green) reporters in CCO20 and CCO32 organoids. e. The expression of LGR5-tdTomato (red) and p27-mVeuns (green) reporters in CCO7 organoids cultured in the indicated conditions for 6 days. The percentage of the LGR5⁺p27⁺ area per LGR5⁺ area (mean ± s.e.m.). Each dot represents one organoid. Statistics, two-sided Welch’s t-test. From the left, n = 9, 10, 10 and 10 organoids. f. Flow cytometry dot plots of CCO32 organoids with the indicated reporter(s), which shows appropriate compensation. Representative confocal images of sorted LGR5⁺p27⁺ and LGR5⁺p27^– cells are shown on the right. g, h. The number (g) and size (h) of organoid colonies formed from sorted LGR5⁺p27⁺ (green) and LGR5⁺p27^– cells (grey). 1,000 cells were sorted from each population and the images on day 9 (CCO20, CCO7), day 7 (CCO12) or day 8 (CCO32) were analysed. Data are shown as mean ± s.e.m. Each dot indicates one well. Statistics, two-sided Welch’s t-test. The p-value for CCO20 in (h) is p = 4.7 x 10^–07. i. Time-lapse live imaging of LGR5-tdTomato (red) and p27-mVenus (green) reporter organoid derived from sorted CCO32 LGR5⁺p27⁺ or LGR5⁺p27^– single cells. j. Proportion of LGR5⁺p27⁺ or LGR5⁺p27⁻ cells per all cells (mean ± s.e.m.) in organoids derived from sorted CCO32 LGR5⁺p27⁺ or LGR5⁺p27^– single cells at day 10 after sorting. Statistics, two-sided Welch’s t-test. Scale bars: 25 μm (i), 50 μm (d, e), 100 μm (a, b, c)

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