Figure 6 from A Plastic EMP1+ to LGR5+ Cell State Conversion as a Bypass to KRASG12D Pharmacologic Inhibition in Metastatic Colorectal Cancer

RMC-9945 increases β-catenin/TCF transcriptional activity. A, Schematic representation of seven TCF optimal promoter (TOP) luciferase reporter lentiviral vector introduced into AKTP MTOs. TOP-Luciferase quantification of AKTP TOP-Luciferase MTOs, normalized to CellTiter-Glo parallel wells treated with vehicle or RMC-9945 for 1, 2, and 3 days (n = 6). Linear mixed-effect model followed by EMMs with pairwise comparisons. B, Schematic of in vitro AKTP MTOs treated with vehicle or RMC-9945 for 48 hours, with media refresh at 12 hours. C, Immunofluorescence image quantification of the percentage of nuclear (4′,6-Diamidino-2-phenylindole, DAPI-positive) β-catenin–positive cells within AKTP MTO vehicle-treated or RMC-9945–treated organoids. D, Representative images of the quantification displayed in C. Dashed circles illustrate nuclear β-catenin–positive cells. Scale bars: 20 µm. P value is derived from unpaired t test. E, Experimental setup of RMC-9945 treatment of liver metastases. Treatment started at day 21 after inoculation of liver metastases implanted in the spleen of C57BL/6J mice and continued for 11 days. D and F, Immunofluorescence representative images (F), and quantification (G) of the percentage of nuclear (DAPI-positive) β-catenin–positive cells within liver metastasis derived from AKTP MTOs, treated with vehicle or RMC-9945. Scale bar, 100 µm top, 20 µm bottom. White arrows highlight examples of nuclear β-catenin–positive cells. P value is derived from unpaired t test. H, Heatmap showing expression of TFs in AKTP MTO organoids treated with vehicle or RMC-9945. I, Violin plots showing single-cell expression levels of WNT-related TFs in liver metastases treated with vehicle or RMC-9945. J, Schematic representation of the nTCF4-ERT2 construct which translocates into the nucleus and conditionally induces a WNT-OFF state upon treatment with 4-OH-tamoxifen. K, Schematic representation of the experiments displayed in L and M, consisting on nTCF4-ERT2 infected AKTP MTOs treated with 4-OH-tamoxifen in the corresponding conditions since 1 day after being seeded (day -2) and with RMC-9945 or vehicle for 7 days. L, Relative mRNA expression of indicated marker genes in vehicle-treated and RMC-9945–treated (with or without 4OHT) AKTP MTOs for 48 hours. Mean ± SEM. P values are derived from ANOVA followed by Šídák multiple comparisons test. M, AKTP MTO organoid growth (normalized bioluminescence) treated with vehicle, RMC-9945, 4-OH-tamoxifen + vehicle, or 4-OH-tamoxifen + RMC-9945. 4-OH-tamoxifen conditions are normalized to the mean luminescence of 4-OH-tamoxifen treatment at experimental day 0 and non–4-OH-tamoxifen–treated conditions are normalized to the mean luminescence of non–4-OH-tamoxifen treatment experimental day 0. Mean ± SEM; P values are derived from ANOVA followed by Šídák multiple comparisons test.