542 Multi Layered Columnar Epithelium (MLCE) Induced by Bile At the Squamo-Columnar Junction in Mice Originates From Squamous and Columnar Cells

Abstract

Barrett's esophagus (BE), a premalignant condition of esophageal adenocarcinoma (EAC) is defined as the replacement of normal stratified squamous epithelium of the lower esophagus by metaplastic columnar epithelium (Spechler 2014). The observation of multi-layered columnar epithelium regularly observed at the squamo-columnar junction (SCJ) in BE patients suggests a transitional stage between these two types of epithelia. Studies, in both human and rats, have shown that especially the combination of acid and biles can induce multilayered columnar epithelium (MLCE) and metaplasia at the SCJ (Vaezi 1995, Chen 2008, Glickman 2009). In this study we investigated if the diverse layers as observed in MLCE is a transitional stage between epithelia originating from one progenitor cell, or is a mix of different types of epithelia with diverse progenitors. 0.5% deoxycholic acid (DCA) was given to mice in drinking water for a maximum of 30 weeks. Animals were sacrificed and studied by histology and immunohistochemistry (IHC) at different time points. The combination of bile and acid induced gland formation at the SCJ starting from week 10. The MLCE glands were positive in the outer layer for squamous markers K14, p63 and K5 and in the inner layer for columnar markers K19, TFF2 and Alcian Blue. In one of our previous studies we also observed Lgr5 positive cells (RNA in situ) in these multi-layered glands (Mari et al. 2014). To investigate if the multi-layered epithelium originates from a squamous or columnar stem cell, we performed lineage tracing experiments. A Cytokeratin 5 (K5)-cre (n=20) mouse specific for tracing squamous progenitor lineages, and a LeucineG-coupled receptor (Lgr5)-cre mouse (n=20) were crossed with Rosa-lacZ mice. Lineage tracing was induced by tamoxifen injection prior to bile treatment at the age of 8 weeks. Mice were sacrificed 15 weeks after induction. K5-lacZ positive cells were present in the outer layer of the gland demonstrating that the origin of these cells is from squamous progenitors, which is in concordance with the IHC results. Surprisingly, the columnar inner layer of the gland was devoid of K5and Lgr5-lacZ positive cells, suggesting that the origin cell for this layer is neither K5 or Lgr5. Negative K5 lineage tracing in columnar cells within the MLCE in mice excludes the transdifferentiation hypothesis in which squamous cells change into columnar cells, which has been a prevailing theory for many years. The negative lineage experiments for LGR5 suggests a columnar cell of origin, other than Lgr5 for the inner layer. Continuous acid and bile reflux changes the environment at the SCJ in mice and disrupts the natural homeostatic environment of squamous and columnar cells. It appears that MLCE is the result of competitive interactions between cell lineages driven by environmental changes.