Extended Data Fig. 5: Mex3a+ cells regenerate organoids after chemotherapy in A, AP and AKP MTOs.
From: Mex3a marks drug-tolerant persister colorectal cancer cells that mediate relapse after chemotherapy
a, Schematic representation of CRISPR–Cas9-based generation of AKP advanced colorectal cancer organoids from Mex3a-creERT2 AP organoids. b, Representative image and flow cytometry plot of an AKP organoid 16 hours after addition of 4-OH-Tamoxifen. Values indicate mean ± s.e.m. n = 3 independent experiments. c, Representative images of AKP organoids 16 h after addition of 4-OH-Tamoxifen in complete stem cell medium or in medium supplemented with TGF-β1 for 7 days. Scale bar 50 µm. d, AKP and APS bearing Mex3a-creERT2 alleles were treated with Tamoxifen in complete stem cell media. After 16 h, organoids were cultured in absence of EGF (APS) or presence of TGF-β1 (AKP) for 4 days. Percentage of tdTomato+ cells was quantified by flow cytometry (n = 3 independent experiments), Linear model no adjustment for multiple comparisons. e, tdTomato-positive cells cultured in Stem Cell Media were recovered by flow cytometry at 1d, 5d or 10 days post recombination. mRNA abundance was measured by RT-qPCR (n = 3 independent experiments, paired two-tailed t-test) f, Recombination in Mex3a+ cells present in AKP organoids was induced with Tamoxifen 16 h before addition of chemotherapy. Organoids were treated during 3 days and then allowed to recover for 30 days. Representative pictures of organoids at different time points are shown. Scale bar 50 µm. g, Lineage-tracing of Mex3a+ cells in organoids derived from APCMin/+ mice. Recombination was induced with Tamoxifen 16 h before addition of chemotherapy. Organoids were treated during 3 days and then allowed to recover for 20 days. h, Representative pictures of Adenoma organoids at different time points are shown. Scale bar 50 µm i, Flow cytometry quantification of tdTomato+ cells in adenomas referred to the number of viable cells over time (n = 4 independent organoids). Scale bar 50 µm. Linear model with no adjustment for multiple comparisons. j, Schematic representation of introduction of p53 null alleles in Mex3acreERT2/+ adenomas organoids. k, p53-/- APC-/- organoids were induced with Tamoxifen 16 h before addition of chemotherapy. Proportion of tdTomato+ cells over live cells was calculated by Flow cytometry. n = 2 independent organoids, linear model with no adjustment for multiple comparisons. Data in panels d, e, i and k are presented as mean values +/− SEM.
