Human biopsy specimens
For expression analysis of the Wnt-pathway components, endoscopic biopsies were taken from Barrett’s mucosa with intestinal metaplasia, high-grade intraepithelial neoplasia, esophageal adenocarcinoma. Normal mucosa was taken from patients with Barrett’s mucosa using a safety distance of at least 3 cm to the Barrett’s mucosa. Biopsies were placed directly to RNAlater® (Sigma-Aldrich, Taufkirchen, Germany) for a maximum of 24 h and stored at - 80 °C until further usage. Seven specimens of squamous epithelium, Barrett’s mucosa, HGIN, and EAC were investigated. The diagnoses of Barrett’s mucosa, HGIN, or EAC were confirmed by an expertized GI pathologist.
Reagents and antibodies
Recombinant human Wnt3a (rhWnt3a, 200 ng/mL) was purchased from R&D Systems (Minneapolis, USA). The following antibodies phospho-GSK3β-Ser9 (D85E12), GSK3β (D5C5Z), phospho-Akt-Ser473 (D9E), Akt (C67E7), phospho-β-catenin-Ser552 (D8E11), and β-catenin (D10A8) were all purchased from Cell Signaling Technology (Danvers, USA) and the β-actin antibody from Sigma-Aldrich (Taufkirchen, Germany). The peroxidase-conjugated goat anti-rabbit and goat anti-mouse antibodies were purchased from Jackson ImmunoResearch (Suffolk, UK).
Cell culture
In total, six different cell lines, representing Barrett’s sequence, were cultivated. EPC1-hTERT (EPC-1) and EPC2-hTERT (EPC-2) were a generous gift from Dr. Hiroshi Nakagawa [18]. They are immortalized human squamous esophageal cells and were cultivated as previously described [19]. The metaplastic cell line CP-A (CRL-4027) and the dysplastic cell line CP-B (CRL-4028) were purchased from LGC Standards (Wesel, Germany) and cultivated in MCDB 153 growth medium (Biochrom, Berlin, Germany) with supplements according to the manufacturer’s protocol. Originating from esophageal adenocarcinomas, OE33 (ECACC-96070808) and OE19 (ECACC-96071721), cells were acquired from Sigma-Aldrich (Taufkirchen, Germany). They were cultivated in RPMI 1640 growth medium (Gibco, Waltham, USA), supplemented with 10% FBS (Biochrom, Berlin, Germany). All cell lines were incubated at 37 °C with 5% CO2.
Wnt3a treatment
EPC-1, EPC-2, CP-A, CP-B, OE33 and OE19 cells with a confluence of about 80% were seeded with 250,000 cells per well in a 6-well plate and incubated at 37 °C with 5% CO2. After one day, the medium was changed and one day later, cells were incubated with FCS free medium for three hours. The cells were stimulated with 200 ng rhWnt3a at 37 °C, 5% CO2 for one hour. Removing medium and washing two times with cold PBS, the stimulation with Wnt3a was stopped.
RNA isolation and cDNA synthesis
For RNA isolation from cell cultures, the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) was used. RNA isolation from biopsies was done with TRI Reagent® (Sigma-Aldrich, Taufkirchen, Germany), followed by DNase (NEB, Frankfurt a.M., Germany) treatment. CDNAs were synthesized from 250 ng to 1000 ng of total RNA using the RevertAid RT synthesis kit (Thermo Scientific, Darmstadt, Germany) according to the manufacturer’s protocol.
RNA isolation from FFPE specimens
For isolating RNA from paraffin embedded biopsies, we used the AllPrep DNA/RNA FFPE Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. 125 ng of total RNA were used to synthesize cDNA using SuperScript IV Reverse Transcriptase (Invitrogen, Darmstadt, Germany).
Quantitative RT-PCR
Quantitative RT-PCR was conducted by using Takyon No ROX SYBR®Core Kit dTTP Blue (Eurogentec, Lüttich, Belgium) with 1 μl of cDNA and 14 μl of reaction mixture. Primers used for quantitative RT PCR are shown in Table 1. Expression of Wnt3a was analyzed using a TaqMan Assay (Hs00263977_m1, ThermoFisher, Darmstadt, Germany) in combination with Blue Probe qPCR Kit (Biozym, Hessisch Oldendorf, Germany). Normalization was done for β-actin expression.
Qualitative RT- PCR
Using DreamTaq Green PCR Master Mix (Thermo Scientific, Darmstadt, Germany), polymerase chain reaction was performed with 1 μl of cDNA per 20 μl reaction mix. The following protocol was used for 40 cycles: denaturation 98 °C for 2 min, annealing 94 °C for 10 s, elongation 60 °C for 20 s. 10 μl of cycled PCR products were applied to 2% ethidiumbromid stained agarose gel and visualized with UV light. Primers used for qualitative RT PCR are shown in Table 1.
Westernblot analysis
Harvested cells were lysed in RIPA buffer. Using the method of Bradford, protein concentrations were detected. 20 μg of protein were separated on 8% or 10% sodium dodecyl sulphate (SDS)-polyacrylamide gels and blotted on nitrocellulose membranes. After blocking the membranes with 5% low fat milk for one hour, they were incubated with a specific primary antibody over night at 4 °C. Detecting bound antibody, a peroxidase conjugated secondary antibody goat anti-mouse or goat anti-rabbit was used incubating for 1 h at room temperature. Protein bands were visualized with ECL chemiluminescence detection (Millipore, Billerica, USA). The dilutions of the different antibodies are shown in Table 2. Westernblots were analyzed densitometrically using ImageJ.
Immunohistochemistry
Paraffin embedded biopsies from squamous epithelium, Barrett’s metaplasia, HGIN and EAC were cut in 3 μm sections and deparaffinized using a series of different concentrated alcohols and xylol. Endogenous peroxidase was blocked by 3% H2O2 for 20 min at 4 °C. The slices were cooked in citrate buffer with a pressure cooker for 10 min. Blocking unspecific bindings was performed in several steps, first with 5% NormalGoatSerum (Jackson Immuno Research, Suffolk, UK) for 20 min at room temperature and second with Reagents from the Streptavidin/Biotin blocking kit (GeneTex, Irvine, CA), as described in the manufacturer’s protocol. Diluted 1:50 in 1% BSA in PBS, the monoclonal primary antibody against WNT3A (AM09053PU-N, OriGene Technologies GmbH, Herford, Germany) was incubated over night at 4 °C. Detecting bound antibody, a biotin-SP conjugated goat anti-mouse (Jackson Immuno Research, Suffolk, UK) and a peroxidase conjugated Streptavidin (Jackson Immuno Research, Suffolk, UK), both diluted 1:1000 in 1% BSA in PBS, were utilized for 1 h at room temperature. Results were visualized with diaminobenzidine and counterstained with hematoxylin. Breast cancer tissue was used as positive control. Evaluation was done using a staining index (1 < 30%, 2 < 60%, 3 < 100% stained) with categorization for analyzing the stroma, the squamous epithelium, and the metaplastic or EAC tissue.
Statistical analysis
Analyses were performed using GraphPad Prism 6 (GraphPad Software Inc., San Diego, USA). Results are presented as mean ± standard error (S.E.M.) and a p-value of less than 0.05 was considered statistically significant.