P207 - EMT and Perianal Crohn’s Disease: A Model for Understanding Fistula Development

Purpose/Background: Background: Perianal fistulizing Crohn’s disease (CD+PAF) is difficult to treat and can lead to significant morbidity for patients. Evidence suggests that epithelial-to-mesenchymal transition (EMT) may play a role in the development of fistulas by promoting cellular migration and altering tissue architecture. EMT markers, such as the loss of E-cadherin (epithelial marker) and the gain of Vimentin (mesenchymal marker), are associated with the EMT, and have been seen on histology from surgically excised fistula. Our study aims to develop a model for studying EMT in CD+PAF and investigate the effects of immune-modulating treatments on this process.

Methods/Interventions:

Methods: We used two epithelial models to study EMT in the context of fistula development. SW1463 immortalized rectal epithelial cells, and IBD patient-derived organoid epithelial cells. Organoids were generated from both inflamed and uninflamed IBD-derived tissues. Cells were grown in a monolayer culture on slides and a gap in the monolayer created via a scratch-assay (SW1463) or culture well insert (organoid-derived cultures). Cells were treated with proinflammatory cytokines (IL-2+IL-6) or EMT-inducing TGF-b, co-cultured with or without healthy patient-derived peripheral blood mononuclear cells (PBMCs) and treated with or without upadacitinib (a JAK inhibitor clinically used to treat IBD). Cell migration to close the gap, as a model of wound healing, was measured over 3-5 days, and then slides were stained with immunofluorescence for E-cadherin and Vimentin expression.

Results/Outcomes:

Results: Using both SW1463 and IBD-organoid-derived epithelial cells, we built a model system to evaluate a role for EMT in fistula. Using SW1463 rectal epithelial cells, IL-2+IL-6 proinflammatory cytokines did not affect wound healing compared to no treatment. However, the EMT-promoting cytokine TGF-b enhanced wound closure over no treatment. Co-culture with PBMCs or upadacitinib enhanced wound closure under all conditions. Organoid-derived cells from inflamed IBD tissue showed a delayed wound healing compared to organoid-derived cells from non-inflamed tissue, additional treatment and co-culture experiments ongoing. Immunofluorescence staining for E-Cadherin and Vimentin revealed a dynamic shift consistent with EMT during wound healing.

Conclusion/Discussion:

Discussion: We have developed a model to study the potential role for EMT in perianal fistula formation in Crohn’s disease, using cultured rectal epithelial cells and IBD-patient derived organoid epithelial cells. The system can be modulated with cytokines, immune cell co-culture and drug treatment to assess effects on wound healing. Our model systems demonstrate that epithelial cells undergo EMT during migration, which suggests a potential target for treating fistulae. The use of patient-derived organoids offers a reliable model system for patient-specific testing of therapeutics.

Conclusion: Our findings suggest EMT as a modifiable factor in the perianal fistulas in Crohn’s disease. We have set up model systems using patient-derived samples to lay the groundwork for testing future therapeutic approaches aimed at modulating EMT for treating perianal fistula. We are currently running further experiments to develop innovative immuno-modulating models to ameliorate the burden of patients with CD+PAF.

Immunofluorescence staining patient-derived organoid cells
Patient-derived organoid cells from a patient with inflammatory bowel disease in which migration assay was carried out (Ibidi culture-insert 2 chamber 24-well plate - Ref: 80242). The wound was done by removing a silicon insert that is represented horizontally in the photomicrograph. We performed staining for E-cadherin (488 green rabbit Ab) as an epithelial marker and Vimentin (594 red mouse Ab) as an Epithelial-to-mesenchymal Transition marker. Images were taken using confocal microscopy. The photomicrograph shows how epithelial cells at the wound healing edge express Vimentin as a marker of EMT during migration.