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) GSK3 inhibitor screen for wildtype mouse colon organoid growth measured by resazurin metabolic activity assay after 4 days in culture. Eleven small molecule GSK3 inhibitors were tested for their capacity to support wildtype mouse colon organoid growth in media lacking WNT3A or R-spondin. Growth is expressed as fold-change relative to untreated control organoids. The decrease at higher concentrations reflects compound toxicity at supraphysiological doses. n=3 biological replicates per condition. (B) Representative brightfield images of wildtype mouse colon organoids grown with LY2090314 (1 μM) without additional WNT agonists, showing cystic morphology characteristic of WNT-activated organoids similar to APC-mutant organoids. Images taken after 4 days in culture. (C) Schematic of CRISPR-Cas9 dual-guide vector construct used to generate GSK3A/B double knockout organoids, and representative brightfield image demonstrating their growth in WNT agonist-free media, confirming genetic WNT independence. (D) GSK3 inhibitor screen for off-target growth effects using GSK3A/B knockout organoids with the same compounds tested in (A). Growth measured by resazurin assay demonstrates compound specificity. n=3 biological replicates per condition. (E,F) On-target versus off-target validation comparing effects of CHIR99021 (E) and LY2090314 (F) on wildtype organoids (top graphs, demonstrating WNT activation) versus GSK3A/B knockout organoids (bottom graphs, revealing off-target effects). LY2090314 shows superior on-target selectivity with minimal off-target toxicity. (G) Hematoxylin and eosin histology of small intestine from mice fed control diet or regional basic diet (RBD) to induce environmental enteropathy, with or without GSK3 inhibitor nanoparticle treatment (daily enemas for 2 weeks). GSK3 inhibitor treatment restores villus architecture and crypt depth in enteropathy model. n=5 mice per group, representative of two independent experiments. (H,I) Quantification of villus length (H) and crypt depth (I) from histology in (G), demonstrating GSK3 inhibitor-mediated intestinal regeneration in the enteropathy model. (J) Immunohistochemistry for GFP in LGR5-EGFP-CreERT2 mice after 2 weeks of enema treatment with blank nanoparticles or GSK3 inhibitor nanoparticles (3 times weekly). GSK3 inhibitor treatment increases LGR5+ stem cell numbers and crypt depth. n=5 mice per group. (K,L) Quantification of LGR5-GFP positive cells per crypt (K) and crypt depth (L) from immunohistochemistry in (J), confirming enhanced stem cell function and tissue regeneration. (M) Ex vivo organoid growth potential of isolated colon crypts from mice in (J), cultured in dose-response of GSK3 inhibitor. Crypts from GSK3 inhibitor-treated mice show enhanced growth capacity, demonstrating improved stem cell function. Crypts isolated 24 hours after final treatment. Growth measured by resazurin assay after 4 days. Statistical Analysis: Data presented as mean ± SD. Statistical comparisons performed using unpaired two-tailed t-tests for (H, I, K, L) and one-way ANOVA with Tukey’s post-hoc test for dose-response curves (A, D, M). ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. Scale Bars: 500 μm (B), 250 μm (C), 100 μm (G, J). GSK3i refers to LY2090314 throughout. See also .

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: Metabolic Assay, Control, Mutagenesis, CRISPR, Plasmid Preparation, Construct, Double Knockout, Knock-Out, Resazurin Assay, Biomarker Discovery, Activation Assay, Immunohistochemistry, Cell Function Assay, Ex Vivo, Isolation, Cell Culture, Two Tailed Test