TFF2, CXCR4 and EGF‐R mediated gastric wound repair in vitro in gastric organoids

Abstract

BackgroundTrefoil factor 2 (TFF2), a secreted peptide found in the stomach mucosa, is known to play an important role in promoting gastric epithelial repair following damage. We previously demonstrated in vivo that TFF2 acts via the chemokine C‐X‐C receptor 4 (CXCR4). Due to the complexity of dissecting molecular pathways in vivo, the mechanisms of TFF2 action remain unclear. Epidermal growth factor receptor (EGF‐R) is implicated in repair and has been shown to be involved in the TFF2 pathway outside of the gastrointestinal tract. Utilizing the 3D primary culture of gastric epithelial cells known as gastric organoids, which mimic in vivo gastric epithelium, we tested the role of EGF‐R and TFF2‐driven restitution. We tested the hypothesis that TFF2 activates CXCR4 and transactivates EGF‐R to promote gastric restitution.MethodsGastric organoids were generated from isolated corpus tissue of wild‐type (WT) and TFF2 knockout (TFF2−/−) mice. Gastric organoids were cultured for 4–5 days prior to experiments. Damage was induced in gastric organoids by 5s exposure of single epithelial cells to high intensity two‐photon 720 nm light (photodamage), resulting in damage to the targeted cell and subsequent cell death. Progression of damage and repair was evaluated using confocal/2‐photon microscopy, imaging cell nuclei (10 μg/ml Hoechst33342 vital DNA stain) and measuring the intensity of Lucifer yellow ([LY] 20 μM added to culture medium) in the luminal side and extracellular space outside of the organoid.ResultsUnperturbed organoids maintain the integrity of the epithelial barrier and show no sign of basal cell migration. Photodamage injury in WT organoids resulted in the simultaneous events of dead cell exfoliation and migration of undamaged neighboring cells to restore a continuous epithelium in the damaged site. To assess repair of damage, we measured the rate of cell exfoliation (μm/min) as determined by nuclear (Hoechst) and fluorescence (LY) confocal images. Photodamaged TFF2−/− gastric organoids showed slowed repair of damage (0.09 ± 0.02 μm/min, n=5) compared to WT organoids (0.5 ± 0.045 μm/min, n=5). Addition of 1 μg/mL AMD3100 (CXCR4 inhibitor) to WT organoids similarly slowed repair (0.08 ± 0.01 μm/min, n=6), suggesting the presence of CXCR4 receptor in the gastric organoid model. Moreover, photodamage in WT organoids exposed to 200 nM AG1478 (EGF‐R inhibitor) resulted in a slower repair at 0.14 ± 0.03 μm/min (n=3).ConclusionResults demonstrate that TFF2, CXCR4 and EGF‐R are required for efficient epithelial restitution in gastric organoids. Outcomes suggest that gastric organoids can be a useful tool to elucidate cellular signaling mechanisms involved in epithelial repair of gastric damage.Support or Funding InformationSupported by NIH R01 DK102551