In standard conditions designed to grow keratinocytes in 3D culture (Okawa et al., 2007
), Barrett’s Esophagus cells express both squamous and columnar cytokeratins and exhibit a more squamous-like, multilayered growth morphology. Previous studies demonstrated that ATRA was observed to be important for columnar differentiation of esophageal tissue in vivo
(Chang et al., 2007
). Chang et al. demonstrated that squamous biopsies grown as explant cultures differentiate to a columnar phenotype when treated with ATRA and that this change was caused by altered differentiation, and not enhanced proliferation of a particular cell type within the explant tissue. Vitamin A was previously demonstated to be critical to the development of chick esophageal epithelium where it strongly inhibited the development of a thick, stratified esophageal epithelium in favor of a psuedostratified esophageal epithelium consisting of columnar, ciliated and mucosal cells (Aydelotte, 1963
). Based on these studies, we used ATRA as a differentiation factor for BE cells.
Our 3D model of Barrett’s Esophagus established all-trans retinoic acid as a differentiation factor for the BE cell line CP-A, leading to a reduction in squamous cytokeratin expression and an increase in columnar cytokeratin expression after three weeks. Among the other BE cell lines, ATRA did not alter cytokeratin expression, but it did alter morphology leading to more single-layer BE-like growth. Further analysis of biological differences between these four BE cell lines will help elucidate the differences observed with ATRA treatment.
The development of BE in vivo
is a response to the constant reflux of acid and bile salts from the stomach into the esophagus in patients with gastro-esophageal reflux disease (GERD). It is hypothesized that BE may actually be a protective mechanism against esophageal tissue damage during reflux (Reid et al., 2010
). As the esophagus is constantly wounded with this milieu of irritants, the lining responds by producing a cell similar to the intestines, which can withstand low pH, and which produces mucous for protection (Reid et al. 2010
). Over time, the squamous lining is replaced with a more protective columnar lining in some GERD patients.
Acid exposure on ex vivo
biopsies from BE patients resulted in enhanced survival of BE cells over squamous cells (Dvorakova et al., 2005
). Feagins et al. however demonstrated that acid exposure on BE cells in 2D culture had anti-proliferative effects (Feagins et al., 2007
). Other studies however demonstrated enhanced proliferation and decreased apoptosis in vitro
and ex vivo
with acid (Fitzgerald et al., 1996
), (Shirvani et al., 2000
). Acid pulsing of a non-neoplastic BE cell line (BAR-T) demonstrated a change towards a colonic phenotype, indicated by an increase in CK8/18 expression (Bajpai et al., 2008
; Das et al. 2011
), and increased colony formation and tumorigenicity (Das et al. 2011
). Based on the potential effects of acid on various BE tissue and cell culture models, we demonstrated here that application of acid on non-dysplastic metaplasia CP-A cells for 3 weeks did not affect cytokeratin profiles nor growth patterns. The lack of altered expression in squamous and columnar cytokeratins with acid suggests that these cells may have an upregulated protective pathway to retain normal levels of cytokeratin expression under stress conditions and protect against changes induced by the toxic effects of acid. Alternatively, the use of bile salts along with acid, and more frequent pulses could reveal more effects on BE growth in 3D culture.
In basic growth conditions without ATRA or acid, CP-A cells, derived from non-dysplastic metaplasia, demonstrated a highly invasive phenotype, unlike in vivo
growth of BE metaplasia. This suggests that researchers should be cautious in interpreting CP-A cells as a model of metaplasia and note that unlike the other cell lines tested here, they have LOH at chromosome 5q, which contains the APC tumor suppressor gene (Palanca-Wessels et al., 1998
). APC blocks cell proliferation and induces differentiation (Jankowski and Odze 2009
). Tests for the relationship between mutation of APC and disruption to cellular growth in 3D culture are beyond the scope of this study but warrant further investigation.
The model developed in this study opens up many potential applications to study Barrett’s esophagus in vitro. Most importantly, a 3D model of BE, with modifications, can be used to test potential therapeutics in a more physiologically realistic (though still artificial) culture environment. In addition, the model can be used to study factors necessary in vivo for malignant transformation of esophageal adenocarcinoma derived from benign BE. By understanding this transition, potential agents can be developed that interfere with this conversion. These cell lines may provide a platform to test the generality of potential therapies or treatments in a 3D culture environment as a stepping stone between standard 2D cell culture and other models. The heterogeneity in cell line responses ensures that the diversity present between patients and within a single patient can be captured experimentally.