Figure 1 from A Plastic EMP1+ to LGR5+ Cell State Conversion as a Bypass to KRASG12D Pharmacologic Inhibition in Metastatic Colorectal Cancer

KRASG12D inhibition enforces a Lgr5+ ISC-like transcriptional program in liver metastases. A, Experimental setup of RMC-9945 treatment of liver metastases. Treatment started at day 15, 21, or 25 after inoculation of liver metastases implanted in the spleen (IS) of C57BL/6J mice and continued for 11 days. B, Longitudinal intravital bioluminescence quantification of AKTP MTOs treated with vehicle or RMC-9945. C, Treatment from day 15, day 21, and day 25 of established liver metastases (LiM) generated by injecting AKTP MTOs in the spleen of C57BL/6J mice. Number of liver metastases. Vehicle n = 5 mice, D15 n = 7, D21 n = 8, and D25 = 8. Mean ± SEM; P values are derived from ANOVA followed by two-sided Dunnett tests. D, UMAP visualization of scRNA-seq (n = 11,410) showing vehicle-treated (gray) and RMC-9945–treated (green) cells. Mice were treated from day 21 to day 26 (6 days), and liver metastasis dissected. E, UMAP projection with data points colored by Louvain clusters (resolution 0.6), in which each cluster is represented in a unique color to emphasize the dataset’s underlying structure. F, Bar plot showing the frequency distribution of cells in the vehicle and RMC-9945 groups within each cluster, ordered by their association with CSC, intermediate, or HRC states. Percentages represent the proportion of cells in each group per cluster. G and H, UMAP visualization and violin plot showing HRC (EpiHR+) cells (G) and the expression (MAGIC values) of the Emp1 gene (H). I and J, UMAP visualization and violin plot showing CSCs (Lgr5+ ISC-like) and the expression (MAGIC values) of the Lgr5 gene (J). MAGIC, Markov Affinity-based Graph Imputation of Cells. K, UMAP visualization and violin plot showing proliferative cells. Gene signatures used for the analysis are detailed in Supplementary Table S1. In the UMAP visualization, black indicates the top 20% (quantile 80) of cells based on feature expression (true) and gray indicates the remaining cells (false). The gene expression UMAP is split by the vehicle and RMC-9945 groups. L, Treatment from day 21 of established liver metastases (LiM) generated by injecting AKTP Emp1-Tom MTOs in the spleen of C57BL/6J mice. M, Percentage of Lgr5 mRNA spots detected by RNAscope on the total cell number detected in each metastasis. Each dot represents one metastasis. Vehicle n = 56, RMC-9945 n = 71. Mean ± SEM; P values are derived from unpaired t test. N, Representative example of Lgr5 mRNA FISH combined with tdTomato immunofluorescence on liver metastases sections. Scale bar, 400 μm. O, Treatment from day 15 of established liver metastases (LiM) generated by injecting AKTP MTOs Emp1-Tom in the spleen of C57BL/6J mice. P, Percentage of Lgr5 mRNA spots detected by RNAscope on the total cell number detected in each metastasis. Each dot represents one metastasis. Vehicle n = 95, RMC-9945 n = 87. Mean ± SEM; P values are derived from unpaired t test. Q, Representative example of Lgr5 mRNA FISH combined with tdTomato immunofluorescence on liver metastases sections. Scale bar, 100 μm.