RA10.03: REGENERATING ESOPHAGEAL SQUAMOUS EPITHELIUM FROM SQUAMOUS PROGENITOR CELLS THROUGH BMP2/4 SIGNAL INHIBITION IN MODELS OF BARRETT's ESOPHAGUS: A PROMISING CHEMOPREVENTIVE TREATMENT FOR ESOPHAGEAL ADENOCARCINOMA
Authors: Krishnadath Kausilia 
Published: March 2019

Abstract

Background
Barrett's esophagus is a metaplastic condition in which the esophageal normal squamous mucosa is replaced by metaplastic columnar type of epithelium, which is a the result of chronic duodeno-gastric reflux, which causes an inflammatory response. One way to divert the risk of malignant progression of Barrett's esophagus would be by re-establishing squamous epithelium through inhibiting Barrett cells. In earlier studies the SHH-BMP4 pathway has been identified as one of the critical pathways involved in the generation of columnar metaplasia. BMPs are secreted stromal factors for instance involved in development and homeostasis of columnar epithelia. Therefore targeting BMPs to modulate signaling and inhibit columnar cell growth and proliferation could be an attractive approach for diverting cancer risk of metaplastic columnar epithelia such as Barrett's esophagus.

Methods
We first set out to study BMP signaling through analysis of RNA sequencing to identify which BMPs are involved in Barrett's esophagus and in three different models we investigated the effects of specific BMP inhibition and neo-squamous regeration. We finally investigated the origin of the neo-squamous cells by lineage tracing in one of our models.

Results
We found that In a transgenic mouse model, conditional knock out of the BMP inhibitor Noggin increased BMP signaling and development of columnar metaplasia arising from multilayered glands glands at the squamo-columnar junction in the stomach (figure…). We demonstrate that inhibiting BMP2 and 4, drives development of squamous epithelium in these glands. In for its effect of epithelial healing in an cryo-ablation model. Additionally, to perform lineage tracing using K5-cre-Tomato-GFP lineage tracing mice to investigate the site of origin of the neo-squamous epithelium.
Method: Cryoablation of the stomach epithelium in wild-type and CK5 lineage tracing mice was performed just distal of the squamo-columnar junction and mice were treated with saline or intraperitoneal injections of the BMP inhibitor for 0, 7, 14 and 21 days. The healing process of the ablated area was investigated by histology and immunohistochemistry (IHC) using a panel of both squamous (CK5 and p63) and columnar markers (Villin, CK19).
Results: At 21 days post ablation, we observed that the inhibition of BMP4 promoted the re-generation of neo-sqaumous epithelium in the ablated area, while the control group showed re-generation of normal stomach epithelium. IHC showed that the neo-squamous epithelium was positive for the CK5 and p63 squamous markers. Lineage tracing indicated that the squamous cells originated for multilayered glands at the squamo-columnar junction and from the adjacent squamous epithelium (Figure 1).

Conclusion
Our results demonstrate that inhibition of BMP2/4 after ablation of columnar epithelium at the squamo-columnar junction, promotes development of neo-squamous epithelium from submucosal multi-layered glands residing at the SCJ and adjacent squamous epithelium, at the same time the regeneration of columnar epithelium is inhibited. These pre-clinical findings can be translated to a clinical setting in order to optimize ablative therapies for treatment of BE patients.

Disclosure
All authors have declared no conflicts of interest.