Extended Data Fig. 11: Metastatic relapse in different mouse CRC models arises from HRCs.

a, Inducible ablation and surgery schedule of mice with AKP Emp1-iCT primary tumours. Panels A and A’ show immunostaining of TOM and E-CADHERIN demonstrating effective ablation of Emp1-high cells in primary CRCs. Dashed lines delimitate the caecum edge. Scale bars, 500 µm. b, Primary tumour area (mean ± SD) measured after resection. Each dot is a mouse, n = 12 (control) and 6 (DIM) mice. P-value for linear model after boxcox transformation. c, Liver metastases (mean ± SD) generated by MTO AKP Emp1-iCT up to one month after primary tumour resection. Each dot is a mouse, n = 12 (control) and 6 (DIM) mice. P-value for generalized linear model with negative binomial family. Bottom panel indicates the percentage of mice that developed liver metastases in the same experiment. Analysed with a two-sided fisher test. d, Inducible ablation and surgery schedule of mice with AKPS Emp1-iCT primary tumours. Panels D and D’ show immunostaining of TOM demonstrating effective ablation of Emp1-TOM^high cells in DIM-treated primary tumours. Dashed lines delimitate the caecum edge. Scale bars, 250 µm. e, Primary tumour area (mean ± SD) measured after resection. Each dot is a mouse, n = 17 (control) and 19 (DIM) mice. P-value for linear model after boxcox transformation. f, Liver metastases (mean ± SD) generated by MTO AKPS Emp1-iCT up to one month after primary tumour resection. Each dot is a mouse, n as in panel e. P-value for generalized linear model with negative binomial family. Bottom panel indicate the percentage of mice that developed liver metastases in the same experiment. Analysed with a two-sided fisher test. g, Inducible ablation schedule of mice implanted with AKTP Emp1-iCT MTOs in the rectum. h, Longitudinal intravital BLI quantification of AKTP MTOs implanted in the rectum. i, Representative TOM and E-CADHERIN immunostainings of lungs from mice bearing AKTP rectal tumours. Lung metastases of increasing size are shown. Note that TOM expression is higher in micrometastases and progressively reduced. Scale bars, 50 µm. j, Primary rectal tumour area (mean ± SD) measured at sacrifice. Each dot is a mouse, n = 9 (control) and 10 (DIM) mice. P-value for linear model after boxcox transformation. k, Lung (left panel) and liver (middle panel) metastases (mean ± SD) generated by MTO Emp1-iCT injected in the rectum. Each dot is a mouse, n as in panel j. P-value for generalized linear model with Poisson family. Right panel shows the percentage of mice that developed metastases in the same experiment. Analysed with a two-sided fisher test. l, CRISPR-Cas9 targeting strategy to introduce an DTR-GFP cassette into the Lgr5 locus of MTOs. Confocal imaging of immunostaining for EGFP and EPCAM in Lgr5-DTR-EGFP organoids. Scale bar, 30 µm. Right panel shows a representative flow cytometry plot of EGFP expression in wild-type and Lgr5-EGFP organoids. m, Relative Lgr5 mRNA expression (mean ± SD) of Lgr5-EGFP^high versus -low cells isolated from Lgr5-DTR-EGFP subcutaneous tumours. n = 3 biological replicates. Two-sided t-test normalizing to B2M. n, Immunofluorescence showing EGFP and E-CADHERIN in primary tumours. Insets (N’ and N’’) correspond to invasion fronts and tumour buds lacking EGFP expression at higher magnification. Scale bars, 500 µm (D) and 100 µm (D’ and D’’). o, Quantification of Lgr5-EGFP^high cells (defined as cells in percentile 90 for EGFP expression) in the tumour core, invasion fronts and tumour buds. Boxes represent the first, second (median) and third quartiles. Whiskers indicate maximum and minimum values. Paired two-sided Wilcoxon test on percentages. n = 11 mice. p, Representative images of Lgr5-EGFP staining in micro (P) and small (P’) metastases. Dashed lines and the yellow arrow surround a micrometastasis. Scale bars: (F) 50 µm; (F’) 250 µm. q, Percentage of tumour area containing Lgr5-EGFP^high and Lgr5-EGFP^low cells versus metastases size. Each dot represents an individual metastasis. r, CRISPR-Cas9 targeting strategy to introduce an iCaspase-9-TOM cassette into the LGR5 locus of AKTP MTOs. s, Representative flow cytometry plot of TOM expression in Lgr5-iCasp9-tdTomato organoids. t, Quantification of Lgr5 mRNA (mean ± SD) by RT-qPCR in Lgr5-TOM^high and Lgr5-TOM^low cells dissociated from primary tumours grown for 4 weeks. n = 3 primary tumours. Analysed with a mixed effects linear model. u, Timing of inducible ablation and surgery in mice implanted with AKTP Lgr5-iCasp9-TOM primary tumours. v, Representative flow cytometry plot of Lgr5-TOM fluorescence in controls versus dimerizer-treated mice. DAPI-/EPCAM+ cells are shown. w, Percentage (mean ± SD) of Lgr5^high tumour cells (defined as the top 10% of the TOM+ population) in control and treated mice. n = 4 mice each group. Two-sided Wilcoxon test. x, Primary tumour area measured after resection. n = 15 mice each group. Mean with SD, p-value of linear model after boxcox transformation. y, Liver metastases counted at experimental endpoints after primary tumour resection. n = 16 (control) and 21 (Lgr5-ablation) mice. Mean ± SD. Analysed with a linear model with negative binomial family. Left panel show the percentage of mice that developed liver metastases in control and Lgr5-ablated tumours in the same experiment. Two-sided Fisher test.

Source data