Development and characterization of an organotypic model of Barrett's esophagus

Abstract

Understanding the molecular and cellular processes underlying the development, maintenance, and progression of Barrett's esophagus (BE) presents an empirical challenge because there are no simple animal models and standard 2D cell culture can distort cellular processes. Here we describe a three-dimensional (3D) cell culture system to study BE. BE cell lines (CP-A, CP-B, CP-C, and CP-D) and esophageal squamous keratinocytes (EPC2) were cultured on a matrix consisting of esophageal fibroblasts and collagen. Comparison of growth and cytokeratin expression in the presence of all-trans retinoic acid or hydrochloric acid was made by immunohistochemistry and Alcian Blue staining to determine which treatments produced a BE phenotype of columnar cytokeratin expression in 3D culture. All-trans retinoic acid differentially affected the growth of BE cell lines in 3D culture. Notably, the non-dyplastic metaplasia-derived cell line (CP-A) expressed reduced squamous cytokeratins and enhanced columnar cytokeratins upon ATRA treatment. ATRA altered the EPC2 squamous cytokeratin profile towards a more columnar expression pattern. Cell lines derived from patients with high-grade dysplasia already expressed columnar cytokeratins and therefore did not show a systematic shift toward a more columnar phenotype with ATRA treatment. ATRA treatment, however, did reduce the squamoid-like multilayer stratification observed in all cell lines. As the first study to demonstrate long-term 3D growth of BE cell lines, we have determined that BE cells can be cultured for at least 3 weeks on a fibroblast/collagen matrix and that the use of ATRA causes a general reduction in squamous-like multilayered growth and an increase in columnar phenotype with the specific effects cell-line dependent.

Fig 1

H&E staining of organotypic reconstructs demonstrate ATRA generally reduces epithelial thickness in BE and squamous cells at 3 weeks (see brackets). A. EPC2 cell line, B. CP-A cell line, showing invasion (arrows) and goblet cells, C. CP-B cell line, D. CP-C cell line, E. CP-D cell line. Scale bar = 75 μm.

Fig 2

Alcian Blue staining of CP-A organotypic reconstructs at 3 weeks with or without ATRA, demonstrates presence of goblet cells preferentially in control conditions. Scale bar = 100 μm. arrows=goblet cells

Fig 3

Squamous cytokeratin expression is reduced with ATRA treatment in squamous and CP-A cells, but not in high-grade dysplasia. A. EPC2 CK13 B. CP-A CK13 C. CP-A CK14 D. CP-B CK14 E. CP-D CK14. Scale bar = 100 μm. arrows=cytokeratin positive cells.

Fig 4

Columnar CK19 expression in ATRA-treated CP-D cells and CK8 in CP-A cells were diminished, without measurable differences in other BE cells. A. CP-D week 3 CK19, B. EPC2 week 3 CK19, C. EPC2 week 3 CK8, D. CP-A Week 1 CK8, E. CP-A Week 3 CK8. Scale bar = 100 μm. Arrows=cytokeratin positive cells

Fig 5

CP-A organotypic reconstructs demonstrated no effect of acid pulsing over 3 weeks on squamous and columnar cytokeratin expression. A. CP-A Week 3 H&E, B. CP-A Week 1 CK14, C. CP-A Week 3 CK14 Scale bar = 100 μm. arrows=cytokeratin positive cells