Figure 4 from A Plastic EMP1+ to LGR5+ Cell State Conversion as a Bypass to KRASG12D Pharmacologic Inhibition in Metastatic Colorectal Cancer

Dynamics of cell state conversion by KRAS inhibition. A, Emp1-iCasp9-tdTomato and Lgr5-EGFP alleles introduced in AKTP MTOs. Confocal imaging of TOM, EGFP, and E-cadherin immunostaining in CRISPR knock-in MTOs. Single z-plane. Scale bar, 30 µm. B, Schematic of in vitro AKTP MTOs treated with vehicle or RMC-9945 for 48 hours, with media refresh at 12 hours, during live dOPM imaging started at day 3. C, Representative 3D orthogonal planes from vehicle-treated (top) and RMC-9945–treated (bottom) organoids. Images show Lgr5-EGFP (green)- and Emp1-TOM (red)-expressing organoids at 16-hour time intervals over a 48-hour period. Arrows pointing at TOM-expressing cells becoming GFP-high. Scale bar, 50 µm. D, Graph showing the median and IQR of the median fluorescence intensity of Lgr5-EGFP in vehicle- and RMC-9945–treated AKTP MTOs over a 48-hour period. Vehicle n = 20, RMC-9945 n = 40. E, Graph showing the median and IQR of EGFP/TOM median intensity ratio over a period of 48 hours, measured at 1-hour time intervals upon vehicle and RMC-9945 treatment. EGFP/TOM pixel ratio was normalized at organoid level at time 0. Vehicle n = 20, RMC-9945 n = 40. Bottom in D and E show –log10 P value derived from Welch t test (D) and Welch t test followed by Wilcoxon signed-rank test (E). F, Experimental setup of Slam-seq experiments. Dashed line indicates MTO harvesting. G, Z-scored and smoothed normalized gene expression of the EpiHR (Cañellas-Socias et al; ref. 13), fetal [Mustata and colleagues (28)], and Lgr5+ ISCs [Muñoz and colleagues (29)] gene signatures across different time points. P values are derived from Dunnett tests. H, Barplots showing the Log2 fold change (FC) of nascent RNAs upon RMC-9945 treatment at different time points, compared with the vehicle for Lgr5+ ISC and EpiHR. Wald test–derived significant P values are shown in red.