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: The following GSK3 inhibitors were obtained and prepared according to manufacturer’s specifications: LY2090314 (Cayman Chemical, 22211), CHIR99021 (LC Laboratories, C-556), A1070722 (Sigma-Aldrich, SML0863), QS11 (Cayman Chemical, 21247) 9-ING-41 ((MedChem Express, HY-113914), SB216763 (Cayman Chemical, 10010246) BIM I (Cayman Chemical 13298), BIM IX (Cayman Chemical,13334), Tideglusib (Cayman Chemical, 16727), BIO (Cayman Chemical, 13123) CHIR98014 (Cayman Chemical, 15578) and in general were dissolved in DMSO.

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

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 mouse colon organoid growth with GSK3i (LY2090314) treatment. Genotypes tested: wildtype (WT), Apc -/- (A), Apc -/- ;Kras G12D ;Trp53 -/- (AKP), and Apc -/- ;Kras G12D ;Trp53 -/- ; Smad4 -/- (AKPS). Growth measured by resazurin assay after 72 hours and normalized to vehicle control for each genotype. n=3 biological replicates. (B) Dose-response curves of human colon organoid growth with GSK3i treatment. Normal colon organoids and patient-derived colorectal cancer organoids (CRC lines: MGH1, PDM2, PDM7) were cultured for 72 hours. Growth measured by resazurin assay and normalized to vehicle control. n=3 biological replicates. (C) Representative histological images of AKP mouse colon cancer organoids showing the effect of GSK3 inhibitor treatment. Top row: untreated control organoids (0 nM). Bottom row: organoids treated with 1000 nM GSK3 inhibitor (LY2090314) after 36 hours. Left panels show hematoxylin and eosin (H&E) staining demonstrating normal organoid architecture in control conditions versus disrupted morphology with cellular debris following GSK3 inhibitor treatment. Right panels show immunohistochemical staining for cleaved caspase-3 (CC3), a marker of apoptosis. Brown staining indicates CC3-positive apoptotic cells, which are markedly increased in the GSK3 inhibitor-treated organoids compared to untreated controls. Scale bars = 50 μm. Right: Quantification of cleaved caspase 3 intensity per cell (control: n = 3,797 cells from 13 fields; treated: n = 4,677 cells from 14 fields; 3 biological replicates each; ****p < 0.0001, unpaired t-test). (D,E) Competition assays between fluorescently labeled organoids cultured in media lacking WNT agonists. (D) Wildtype organoids (cyan) co-cultured with Apc -/- organoids (magenta) ± GSK3i (111 nM). (E) Wildtype organoids (cyan) co-cultured with AKP organoids (magenta) ± GSK3i (125 nM). Images taken after 5 days of co-culture. (F-I) Validation of autocrine WNT3A expression effects. (F) Immunofluorescence of Kras G12D ;Trp53 -/- ; tdTomato organoids with EF1A-driven Wnt3a transgene (Wnt3a-KPT). WNT3A (green), Hoechst 33342 (blue), tdTomato (red). (G) Growth curve of Wnt3a-KPT organoids with GSK3i dose response. (H) Control KPT organoids lacking Wnt3a transgene show no WNT3A staining. (I) Growth curve of control KPT organoids with GSK3i dose response. n=3 biological replicates for (G) and (I). (J-M) shRNA-mediated Apc knockdown experiments in WNT agonist-free media. Growth measured after 72 hours ± doxycycline (2 μg/ml) to induce shApc. (J,K) Wildtype organoids without (J) or with (K) shApc induction. (L,M) GSK3A/B double knockout organoids without (L) or with (M) shApc induction. n=3 biological replicates. (N) Dose-response curves of wildtype and AKP organoids cultured with concentrated WNT3A-conditioned media (WRN supernatant). Growth measured by resazurin assay after 72 hours. n=3 biological replicates. (O) Immunohistochemical staining for beta-catenin in mouse colon cancer organoids. Top panel: untreated control organoids (0 nM) showing baseline beta-catenin expression localized to cell membranes and partially to nuclei. Bottom panel: organoids treated with 1000 nM GSK3 inhibitor (LY2090314), 36 hours of treatment, demonstrating marked accumulation of beta-catenin with intense nuclear and cytoplasmic staining, consistent with hyperactivation of canonical WNT signaling. The increased beta-catenin levels correlate with the induction of apoptosis observed in these "over-WNTed" tumor organoids. Scale bars = 50 μm. Right: Quantification of nuclear beta-catenin intensity (control: n = 805 cells from 10 fields; treated: n = 1,608 cells from 13 fields; 3 biological replicates each; ****p < 0.0001, unpaired t-test). (P-V) WNT pathway activity reporter analysis. (P) Schematic of 13xTCF-tdTomato (TOP/tdTomato) reporter construct. (Q,R,S) Wildtype mouse organoids transduced with TOP/tdTomato reporter: (Q) representative brightfield and fluorescence images, (R) quantification of tdTomato fluorescence intensity per organoid, (S) corresponding growth measurements. (T,U,V) Human CRC organoids (PDM7) transfected with TOP/tdTomato reporter: (T) representative images, (U) quantification of tdTomato fluorescence intensity per organoid, (V) corresponding growth measurements. All measurements after 72 hours of GSK3i treatment. n=4 biological replicates. Scale bars: 500 μm (D,E,P,S); 100 μm (F,H) 50 μm (C). All growth assays measured by resazurin metabolic activity. GSK3i = LY2090314. Data shown as mean ± SD. Statistical analysis by unpaired two-tailed t-test (C) or one-way ANOVA with Tukey’s post-hoc test (other panels). ***p<0.001, **p<0.01.

Article Snippet: The following GSK3 inhibitors were obtained and prepared according to manufacturer’s specifications: LY2090314 (Cayman Chemical, 22211), CHIR99021 (LC Laboratories, C-556), A1070722 (Sigma-Aldrich, SML0863), QS11 (Cayman Chemical, 21247) 9-ING-41 ((MedChem Express, HY-113914), SB216763 (Cayman Chemical, 10010246) BIM I (Cayman Chemical 13298), BIM IX (Cayman Chemical,13334), Tideglusib (Cayman Chemical, 16727), BIO (Cayman Chemical, 13123) CHIR98014 (Cayman Chemical, 15578) and in general were dissolved in DMSO.

Techniques: Resazurin Assay, Control, Derivative Assay, Cell Culture, Staining, Immunohistochemical staining, Marker, Labeling, Co-Culture Assay, Biomarker Discovery, Expressing, Immunofluorescence, shRNA, Knockdown, Double Knockout, Activity Assay, Construct, Transduction, Fluorescence, Transfection, Two Tailed Test

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) Principal component analysis of bulk RNA-sequencing from AKPT organoids cultured with GSK3i dose response (0, 10, 100, 1000 nM LY2090314) for 72 hours. Representative brightfield images of organoids at each concentration shown below PCA plot. n=3 biological replicates per condition. Scale bar: 200 μm. (B) Unsupervised hierarchical clustering heatmap of differentially expressed genes from RNA-seq dataset in (A). Red box highlights Rhoc expression pattern across GSK3i concentrations. Color scale represents z-score of log2-transformed expression values. (C) Rhoc gene expression quantification across GSK3i doses. Data shown as counts per million mapped reads (CPM) from RNA-seq analysis. n=3 biological replicates, mean ± SD. One-way ANOVA with Tukey’s post-hoc test. (D) Immunofluorescence staining for RHOC protein (green) with DAPI nuclear counterstain (blue). Top: AKPT mouse organoids. Bottom: Patient-derived human colorectal cancer organoids (PDM7). Left: vehicle control. Right: 1000 nM GSK3i treatment for 72 hours. Representative images from n=3 independent experiments. Scale bar: 20 μm. (E) Representative histological images of AKP mouse colon cancer organoids following 36-hour treatment with GSK3 inhibitor after 2 days of growth. Top panels: Hematoxylin and eosin (H&E) staining. Bottom panels: RHOC immunohistochemical staining. Left column: untreated control organoids (0 nM GSK3 i) showing normal organoid architecture with minimal RHOC expression. Right column: organoids treated with 1000 nM GSK3 inhibitor (LY2090314) displaying disrupted morphology with cellular debris in H&E sections and marked upregulation of RHOC protein (intense brown staining) throughout the organoid epithelium. The dramatic RHOC induction correlates with activation of the non-canonical WNT/planar cell polarity pathway that executes apoptosis in hyperactivated WNT ("over-WNTed") tumor cells. Scale bars = 50 μm. (F,G) Rescue of GSK3i-induced growth inhibition by ROCK inhibition. (F) Representative brightfield images of AKP organoids treated with GSK3i (100 nM) ± ROCK1/2 inhibitor (Y-27632, 10 μM) for 72 hours. Scale bar: 100 μm. (G) Quantification of organoid growth by resazurin assay. n=4 biological replicates, mean ± SD. **p<0.01, one-way ANOVA. (H,I) Effect of ROCK inhibition on metastatic potential. (H) Representative gross brightfield (top) and tdTomato fluorescence (bottom) images of liver metastases 5 weeks after splenic injection. Left: AKPST organoids. Right: GSK3A/B knockout KPST organoids. Both organoid lines were cultured continuously with ROCK inhibitor (Y-27632, 10 μM) prior to injection. Scale bar: 5 mm. (I) Quantification of liver tumor burden calculated as Σ(mean fluorescence intensity × tumor area). n=5 mice per group, representative of 2 independent experiments. *p<0.05, unpaired t-test. (J) Genetic validation of RHOC-ROCK pathway. Growth curves of organoids with GSK3i dose response: AKPVT (control), AKPVT Rhoc -/- (RHOC knockout), and AKPVT Rock1 -/- / Rock2 -/- (ROCK1/2 double knockout). Growth measured by resazurin assay after 72 hours, normalized to untreated control for each genotype. n=3 biological replicates, mean ± SD. GSK3i = LY2090314 for all experiments. Data shown as mean ± SD. *p<0.05, **p<0.01.

Article Snippet: The following GSK3 inhibitors were obtained and prepared according to manufacturer’s specifications: LY2090314 (Cayman Chemical, 22211), CHIR99021 (LC Laboratories, C-556), A1070722 (Sigma-Aldrich, SML0863), QS11 (Cayman Chemical, 21247) 9-ING-41 ((MedChem Express, HY-113914), SB216763 (Cayman Chemical, 10010246) BIM I (Cayman Chemical 13298), BIM IX (Cayman Chemical,13334), Tideglusib (Cayman Chemical, 16727), BIO (Cayman Chemical, 13123) CHIR98014 (Cayman Chemical, 15578) and in general were dissolved in DMSO.

Techniques: RNA Sequencing, Cell Culture, Concentration Assay, Expressing, Transformation Assay, Gene Expression, Immunofluorescence, Staining, Derivative Assay, Control, Immunohistochemical staining, Activation Assay, Inhibition, Resazurin Assay, Fluorescence, Injection, Knock-Out, Biomarker Discovery, Double Knockout