Journal: bioRxiv

Article Title: Leveraging WNT Hyperactivation to Kill Colorectal Cancer While Rejuvenating Healthy Intestine

doi: 10.1101/2025.10.05.680591

Figure Lengend Snippet: (A) Dose-response curves of GSK3i (LY2090314) treatment across a panel of patient-derived human colorectal cancer organoids. Growth measured by resazurin metabolic assay after 72 hours, normalized to vehicle control. n=10 individual patient samples; demographic and clinical data provided in Supplementary Table 1. Data shown as mean ± SD. (B) Assessment of GSK3i effects on nascent tumor formation. Apc fl/fl ;Kras LSL-G12D ;Trp53 fl/fl ;Rosa26 LSL- tdTomato ;Lgr5-CreERT2 organoids were treated with 4-hydroxytamoxifen (500 ng/ml) for 12 hours to induce Cre-mediated recombination, then cultured ± GSK3i (100 nM) for 4 days. Red fluorescence indicates successful Cre recombination and tumor allele activation. DAPI (blue) marks all cell nuclei; DAPI-positive/tdTomato-negative cells represent untransformed organoids that escaped recombination. Representative images from n=3 independent experiments. (C) Apoptosis detection in AKP tumor organoids. Representative fluorescence images and quantification using Apotracker dye after 24 hours of GSK3i treatment (100 nM). n=4 biological replicates. **p<0.01, unpaired t-test. (D) In vivo tumorigenicity of Wnt3a-KPT organoids. Left: Gross brightfield and fluorescence images of mouse liver 5 weeks after orthotopic implantation of Kras G12D ;Trp53 -/- ;tdTomato organoids expressing EF1A-driven Wnt3a transgene (Wnt3a-KPT). Right: Hematoxylin and eosin staining confirming tumor formation. Representative images from n=5 mice. (E) Normalized red fluorescence of TOP/tdTomato reporter in human CRC organoids, normalized by cell number, primary data found in U,V. demonstrating WNT reporter activity inversely correlating with cell number across all concentrations. (F,G,H) WNT pathway reporter analysis in mouse colorectal cancer organoids. (F) Representative brightfield (top) and tdTomato fluorescence (bottom) images of Apc -/- ;Kras G12D ;Trp53 -/- (AKP) organoids transduced with 13xTCF-tdTomato (TOP/tdTomato) reporter, cultured with GSK3i dose response for 72 hours. (G) Quantification of tdTomato fluorescence intensity per well from (F). (H) Corresponding growth measurements of AKP organoids by resazurin assay. n=5 biological replicates for (G) and (H). Data shown as mean ± SD. GSK3i = LY2090314 for all experiments. Scale bars: 500 μm (B,C,F); 5 mm (D, gross images); 200 μm (D, histology).

Article Snippet: For the Regional Basic Diet induced environmental enteropathy model, mice were weaned onto the formulated diet (Research Diets D09081701), which has significantly decreased protein, moderately decreased fat and minerals, compared to the isocaloric OpenStandard diet control, with ad libitum access to food., For induction of Cre-mediated recombination of Apc fl/fl; Villin CreERT2; Rosa26 LSL-tdTomato mice, 100 μl of 1 mg/ml tamoxifen (MedChem Express, HY-13757A) in corn oil were injected intraperitoneally once.

Techniques: Derivative Assay, Metabolic Assay, Control, Cell Culture, Fluorescence, Activation Assay, In Vivo, Expressing, Staining, Activity Assay, Transduction, Resazurin Assay