An Immunofluorescent Method for Characterization of Barrett’s Esophagus Cells

Summary

There is a discernible need for improved information on molecular drivers of Barrett’s Esophagus. Immunofluorescent staining is a useful technique for understanding the effects of cell signaling on cell morphology. We present a simple, effective protocol for the use of immunofluorescent staining to assess therapeutic treatment in Barrett’s Esophagus cells.

Abstract

Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One of the primary risk factors of EAC is Barrett’s esophagus (BE), a metaplastic change of the normal squamous esophagus in response to chronic heartburn. Despite the well-established connection between EAC and BE, interrogation of the molecular events, particularly altered signaling pathways involving progression of BE to EAC, are poorly understood. Much of this is due to the lack of suitable in vitro models available to study these diseases. Recently, immortalized BE cell lines have become commercially available allowing for in vitro studies of BE. Here, we present a method for immunofluorescent staining of immortalized BE cell lines, allowing in vitro characterization of cell signaling and structure after exposure to therapeutic compounds. Application of these techniques will help develop insight into the mechanisms involved in BE to EAC progression and provide potential avenues for treatment and prevention of EAC.

Introduction

Barrett’s esophagus (BE) is a metaplastic change in the normal squamous epithelium of the esophagus and a consequence of chronic exposure to the gastric contents resulting from gastroesophageal reflux disease (GERD)¹. BE is thought to be a protective mechanism in response to GERD, however the presence of BE imparts an increased risk of esophageal adenocarcinoma (EAC), a disease which carries a significantly poor survival¹. Current estimates suggest that up to 5.6% of the American population have BE, however as BE is often asymptomatic, it is thought that the majority of BE remains undiagnosed². As incidence rates of both GERD and....

Protocol

1. Cell Line Maintenance

1. Immortalized Human BE high-grade dysplastic (CP-B, CP-C, CP-D) and metaplastic (CP-A) cell lines by stable transfection of the human telomerase reverse transcriptase (TERT) protein¹¹.
2. Maintain BE cell lines in keratinocyte media supplemented with bovine pituitary extract (BPE), epidermal growth factor (EGF), 5% fetal bovine serum (FBS), nonessential amino acids (NEAA), penicillin-streptomycin-neomycin (PSN), and standard tissue culture conditions (37 °C, 5% CO₂, 98% humidity).

2. BE Cell Plating

1. Preparation of 12-well plate/coverslips. Place 18 mm c....

Results

An example of the results obtained from application of the described procedures is illustrated in Figures 1A-D. Coverslips with CP-D and CP-C BE cells were treated for 24 hr with 1 μM of the Src family inhibitor, SKI-606, or vehicle (DMSO) and stained using the above procedures for the Adherens Junction and Wnt signaling protein, β-catenin. Visualization of β-catenin was achieved by labeling with an anti-rabbit IgG coupled to a green fluorophore, while labeling of the cell nucleus was accomplished by lab.......

Discussion

We have outlined a method for application of IF of BE cells towards elucidating the physiological effects of targeted therapies upon these cells. While we have described the use of these procedures towards BE cells, we have found that these methods are also applicable to a variety of different cell types^13,17,18. Further, these procedures can be altered in several ways to optimize staining for specific targets.

We find one parameter that has a direct effect upon proper IF is the cho.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CP-A (Metaplastic Cell Line)	ATCC	CRL-4027
CP-B (High-grade Dysplastic Cell Line)	ATCC	CRL-4028
CP-C (High-grade Dysplastic Cell Line)	ATCC	CRL-4029
CP-D (High-grade Dysplastic Cell Line)	ATCC	CRL-4030
Keratinocyte-SFM (1x), Liquid	Life Technologies	17005-042
0.25% Trypsin-EDTA (1x), Phenol Red	Life Technologies	25200056
Fetal Bovine Serum, Qualified, HI	Life Technologies	10438026
PSN antibiotic mixture	Life Technologies	15640
PBS - Phosphate-Buffered Saline	Life Technologies	10010049
Pro-long Gold Antifade Reagent with DAPI	Life Technologies	P36931
Circular Glass Coverslip 18 mm	Fisher Scientific	15-183-86
β-catenin Primary Antibody (Rabbit)	Cell Signaling	9562S
Alexa Fluor 488 (Goat Anti-Rabbit)	Life Technologies	A11008
10 cm TC treated PS dish, sterile	USA Scientific	CC7682-3394
12-well TC treated PS plate, sterile	USA Scientific	5666-5180
DMSO	Sigma-Aldrich	276855
Paraformaldehyde	Sigma-Aldrich	P6148
Ammonium chloride	Sigma-Aldrich	A9434
Saponin	Sigma-Aldrich	47036
Bovine Serum Albumin - Fraction V	Sigma-Aldrich	85040C
SKI-606 (Bosutinib)	Selleck Chemicals	S1014
Square Bioassay Dish	Thermo Scientific	240835
Parafilm	VWR	82024-546
Disposable Pasteur Pipettes, Flint Glass	VWR	14672-380
Nexcelom Mini Cell Counter	Nexcelom
Cellometer Counting Chambers	Nexcelom	CHT4-SD100-014
Zeiss Apotome microscope	Zeiss