Adrià-Jaume Roura

Supplementary Table S2. Hallmark, GOCC, KEGG and custom gene signatures of AKTP MTO, AKP CTO, PDO131, and PDO C31M. Selection related with Figure 2 (B, G, J, M)

Supplementary Table S4 from A Plastic EMP1+ to LGR5+ Cell State Conversion as a Bypass to KRASG12D Pharmacologic Inhibition in Metastatic Colorectal Cancer

Supplementary Figures S1-S14 related to the manuscript "A plastic EMP1+ to LGR5+ cell state conversion as a bypass to KRAS-G12D pharmacological inhibition in metastatic colorectal cancer"

Supplementary Table S5 from A Plastic EMP1+ to LGR5+ Cell State Conversion as a Bypass to KRASG12D Pharmacologic Inhibition in Metastatic Colorectal Cancer

Time-lapse imaging of representative AKTP MTO organoids from vehicle and RMC-9945 treatment conditions. Initial imaging (time 0h) was performed prior to vehicle or RMC-9945 treatment. Upon treatment, organoids were imaged every hour over a 48-hour period. Top (vehicle) and bottom (RMC-9945) panels show XY planes positioned at three z-depths relative to the centre of the organoids volume (-10 µm, 0 µm and +10 µm). Lgr5-EGFP is shown in green and Emp1-TOM in red. Scale bar, 50 µm.

Gene signatures used in Figure 1 (G, I, K), in Supplementary Figure S2, for the GSEA analysis in Figure 2 (B, G, J, M), in Supplementary Figure S5, in Figure 3 (A, B, C).

Supplementary Table S3. Gene expression related to Supplementary Figure S6B, C, G, H, I, J

Dependency on Lgr5+ CSCs in RMC-9945–treated metastases. A, Relative mRNA expression of Gfp and Lgr5 genes in sorted GFP-high, -medium, -low, and -negative population from AKTP MTO-derived liver metastases. Mean ± SEM. B, Schematics of in vivo RMC-9945 treatment (7 days, starting at day 21 after implantation), Diphtheria toxin (DT) treatment (one injection any other day), or combination of RMC-9945 and DT, of liver metastases implanted in the spleen of C57BL/6J mice. C, Representative histograms of Lgr5-EGFP expression of the EPCAM+/CD45− population in mice treated with vehicle, RMC-9945, DT, and RMC-9945 + DT obtained by flow cytometry. D, Representative pictures of livers after 7 days of treatment with vehicle, RMC-9945, DT, and RMC-9945 + DT. Arrows pointing at liver metastases. E and F, Quantifications of total number of metastases per mouse in each condition described (E) and of the proportion of metastases per size (higher diameter; F). Vehicle n = 5 mice, RMC-9945 n = 10, DT n = 8, and RMC-9945 + DT = 7. Mean ± SEM. For E, P values are derived from ANOVA followed by Tukey tests. For F, P values are derived from the β-binomial regression model. G, Schematics of in vivo RMC-9945 treatment (10 days, starting at day 15 after implantation), DT treatment (one injection every day), or combination of RMC-9945 and DT, of liver metastases implanted in the spleen of C57BL/6J mice. H and I, Quantifications of total number of metastases per mouse in each condition described (H) and of the proportion of metastases per size (higher diameter; I). Vehicle n = 8 mice, RMC-9945 n = 12, DT n = 8, and RMC-9945 + DT = 12. Mean ± SEM. For H, P values are derived from ANOVA followed by Dunnett tests. For I, P values are derived from the β-binomial regression model. J, Representative pictures of livers after 10 days of treatment with vehicle, RMC-9945, DT, and RMC-9945 + DT. Arrows pointing at liver metastases. K, Kaplan–Meier survival plot of mice treated with the indicated treatments. P values are derived from Gehan–Breslow–Wilcoxon test.

Gene signatures used in Figure 1 (G, I, K), in Supplementary Figure S2, for the GSEA analysis in Figure 2 (B, G, J, M), in Supplementary Figure S5, in Figure 3 (A, B, C).

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.