P0075Human intestinal organoids recapitulate fistula-associated hybrid epithelial-to-mesenchymal transition (EMT): A model of Crohn’s disease fistula formation

Background Fistulas are a debilitating complication of Crohn’s disease (CD), characterized by abnormal invasive tracts lined with transitional cells undergoing hybrid epithelial-to-mesenchymal transition (EMT)1,2. These cells retain partial epithelial identity while acquiring migratory properties3. The mechanisms driving this hybrid EMT and associated tissue disruption remain poorly defined, due to a lack of physiologically relevant models. We aimed to model fistula-associated EMT using human intestinal organoids (HIOs) derived from both ileal and colonic tissue. Methods HIOs were derived from ileal (healthy n = 4; CD n = 2) and colon (healthy n = 4) biopsies. HIOs were cultured in 50% Matrigel with 8-day CD fistula-enriched cytokine treatment of TGFβ, TNFα, IL-13 & IL-22. Some HIOs were treated daily with candidate anti-fistula compound Upadacitinib (JAK1 inhibitor). HIO morphology was monitored via live cell imaging, gene and protein expression was quantified by RT-qPCR and Western blot, and cytoskeletal organization was assessed by F-actin staining. Results Cytokine stimulation induced hybrid EMT in ileal and colon HIOs, with transition from 3D organoids to flattened, collectively migrating sheets. Morphometric analysis of treated HIOs revealed increased diameter and loss of circularity and solidity. Upadacitinib co-treatment reduced EMT morphology in colon HIOs (D2), with increased circularity and solidity and decreased diameter. F-actin staining showed cytoskeletal remodeling with filopodia, lamellipodia, and stress fiber formation but retained cell-cell contacts. Ileal HIOs had strong gene upregulation of SNAI2 (11-fold), ZEB1 (2.5-fold), ETS1 (2-fold), and ITGB6 (6-fold), while SNAI1 and ACTA2 were suppressed (10-fold and 2-fold, respectively), distinguishing this from complete EMT. Markers for Paneth cells (LYZ) and goblet cell (MUC2) were reduced (8-fold and 2.5-fold, respectively). Colon HIOs showed similar gene expression profiles to ileum with stronger upregulation of SNAI2 (15-fold) and ETS1 (6-fold). Both ileal and colonic HIOs had ITGB6 upregulation (with unchanged ITGB5), identifying integrin αvβ6 as the primary integrin involved in this EMT process. MMP9 and FN1 upregulation (9-fold and 5-fold, respectively) in colonic HIOs provides further evidence of EMT. Western blot analysis revealed CTNNB1 (β-catenin) degradation following cytokine stimulation. Conclusion CD fistula-enriched cytokines induce a SNAI2-driven hybrid EMT program in ileal and colonic HIOs, providing a physiologically relevant model of the transitional cell phenotype within CD fistula tracts. Upadacitinib attenuates early EMT features, highlighting its potential as a candidate therapy for preventing fistula progression in CD by targeting epithelial populations. References: 1.Bataille F, Rohrmeier C, Bates R, et al. Evidence for a role of epithelial mesenchymal transition during pathogenesis of fistulae in Crohnʼs disease. Inflammatory Bowel Diseases. 2008;14(11):1514-1527. https://doi.org/10.1002/ibd.20590. 2.Scharl M, Frei S, Pesch T, et al. Interleukin-13 and transforming growth factor β synergise in the pathogenesis of human intestinal fistulae. Gut. 2012;62(1):63-72. https://doi.org/10.1136/gutjnl-2011-300498 3.Georgette C, Tandon R, Simmons A. Pathogenesis of Fistulating Crohn’s Disease: A Review. Cellular and Molecular Gastroenterology and Hepatology. 2022;15(1):1-11. https://doi.org/10.1016/j.jcmgh.2022.09.011 Conflict of interest: Ms. Kola-Ilesanmi, Darasimi: No conflict of interest Stephens, Matthew: No conflict of interest Thorne, Andrew: No conflict of interest Mackenzie, Colin: No conflict of interest Mcdonald, Braedon: No conflict of interest Visser, Frank: No conflict of interest Hirota, Simon: Consultant with Access Nutrients