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.
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.
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 EAC have seen continued growth3, it has become important to understand the molecular mechanisms involved in progression of BE to EAC, particularly as this information could potentially provide therapeutic approaches towards preventing progression of BE to EAC.
Patient directed studies have resulted in the current paradigm of BE-EAC pathogenesis1. Chronic inflammation, injury, and genotoxic damage resulting from prolonged exposure to gastric contents exert selective pressure on the BE lesion promoting neoplastic progression to EAC. A number of genetic alterations have been identified during BE to EAC progression. However, despite this there is a distinct lack of understanding about the exact changes in cell signaling and subsequent effects upon cell structure and function.
Immortalized in vitro cell lines are useful tools for studying the effects of cell signaling, particularly in investigating the effects of targeted therapeutic compounds. The recent commercial availability of immortalized BE cell lines allows for such studies. Although high-throughput assays, such as the widely used viability assays, can be valuable in assessing the effects of targeted therapies upon cell proliferation and survival4-6, these assays are not useful for interrogating the effects of cell signaling upon cell morphology. Immunofluorescent staining (IF) is a useful technique for investigating the effects of targeted therapies upon the morphological, growth, and survival characteristics of cells and the proteins that are involved. Our laboratory has adapted these methods towards immortalized BE cell lines, using IF to evaluate the effects of a clinically available drug upon BE cell lines in hopes of delineating a possible chemopreventative treatment and biomarker for drug treatment7. Similarly, application of these techniques in EAC, BE and immortalized esophageal cell lines has delineated critical findings regarding BE to EAC progression8-10. We find IF analysis of BE cells treated with targeted therapies has value, allowing for characterization of changes in cell structure and protein localization. Here, we present our methods for IF of immortalized BE cells to characterize drug treatment.
1. Cell Line Maintenance
2. BE Cell Plating
3. Drug Treatment of BE Cells
4. Immunofluorescent Staining
5. Microscopic Visualization of Coverslips
Stained cells can be imaged via either confocal microscopy or an IF capable upright microscope. Although confocal microscopy provides high-resolution images at discrete depths, an upright microscopic is capable of producing useful images faster. For brevity, a method of imaging stained BE cells using standard microscopy is presented. In general, to image fluorescein isothiocyanate (FITC) or similar fluorophores (i.e. green fluorescent protein), Texas Red and DAPI, requires a microscope with filters capable of discriminating these fluorophores. Check the microscope before beginning protocol to determine compatible fluorophores.
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 labeling with DAPI (blue). Coverslips were mounted to microscope slides using hard set mounting and cells were imaged using laser scanning confocal microscope using a 63X/1.4N plan-apochromat objective. The chosen green fluorophore was excited at 488 nm and visualized using an emission filter at 505-525 nm. Similarly, DAPI was excited at 405 nm and emission collected using a filter at 410-460 nm. Activity of the tyrosine kinase, Src is increased in BE, particularly in high grade dysplasia12. In concordance with observations in colorectal and prostate cancer cells13-15, inhibition of Src results in relocalization of β-catenin from the cytoplasm/nucleus (arrows, Figures 1A, 1C, and 1E) to the cell membrane (arrowheads, Figures 1B, 1D, and 1F). Using these techniques, we and others have previously shown that inhibition of Src also alters the expression and localization of the tumor suppressor, p27kip17,16 and these methods are currently used within our laboratory.
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Figure 1. IF of immortalized BE cells following treatment with an inhibitor of Src kinase. CP-C (A-D) and CP-D (E, F) were treated with vehicle (DMSO, left panels) or 1 μM of the Src inhibitor, SKI-606, for 24 hr (right panels). BE cells were fixed and stained according to the described protocol. β-catenin (green) was visualized using a specific antibody and a secondary antibody coupled to Alexa Fluro 488, while the nucleus was stained with DAPI (blue). Note the change in localization for β-catenin from the nucleus (arrows) to the cell membrane (arrowheads) following treatment with SKI-606. C,D) Magnified images of white boxes in A and B, further highlight the changes in β-catenin localization. These images represent ideal staining with minimal background and distinct staining for the chosen target. Bars, 7 μm (A, B, D, F), 0.5 μm (C, D).
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 types13,17,18. 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 choice of fixation. We have described the use of 4% PFA/PBS for fixation in the above procedures but alternative fixatives are also applicable. Fixation with cold methanol (-20 °C) for 20 min after washing with PBS is suitable for certain proteins/antibodies13. Use of cold methanol for fixation negates the need to permeabilize cells. However, the choice of fixative (4% PFA/PBS or cold methanol) must be determined empirically.
The procedures outlined above describe visualization of a single protein; however this method is easily adaptable to identification of an additional target13. For optimal IF of two proteins, primary antibodies raised in two different species (e.g., one raised in rabbit and one in mouse), and secondary antibodies specifically recognizing each species, coupled to different fluorophores, are necessary. Secondary antibodies coupled to green and red fluorophores, combined with DAPI provide an excellent contrast between targets, in addition to generally avoiding interference from the multiple fluorophores. For IF of two targets in BE cells, perform sequential incubation of two primary and their respective secondary antibodies following fixation. Steps for washing and mounting of slides remain the same.
In summary, we provide a simple and useful protocol for IF of BE cells. Application of these procedures can yield information regarding the effects of drug treatment, introduction of ectopic genes of interest, or RNAi downregulation of genes of interest in BE cells.
The authors have nothing to disclose.
This work was supported by grants from the St, Joseph’s Foundation (AJF, LJI) and American Lung Association, RG-224607-N (LJI).
Name of the Material/Equipment | Company | Catalog Number | Comments/ Description (optional) |
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 18mm | Fisher Scientific | 15-183-86 | |
β-catenin Primary Antibody (Rabbit) | Cell Signaling | 9562S | |
Alexafluor 488 (Goat Anti-Rabbit) | Life Technologies | A11008 | |
10cm 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 Pipets, Flint Glass | VWR | 14672-380 | |
Nexcelom Mini Cell Counter | Nexcelom | ||
Cellometer Counting Chambers | Nexcelom | CHT4-SD100-014 | |
Zeiss Apotome microscope | Zeiss |