E-Cadherin

Gingival tissues from healthy volunteers were obtained at the NIDCR clinic (Clinical Protocol # 06-D-0144). After surgery, fresh tissues derived from the retro molar area of the oral cavity were incubated overnight in trypsin 0.25% (Sigma Aldrich, CA) at 4°C. Next day, the epithelial compartment was mechanically dissociated from the connective tissue and minced to fine fragments. Keratinocytes were then filtered through a 100 µm cell strainer (BD falcon), pellet at 125 g for 5 min at 4°C, resuspended in keratinocyte serum-free culture medium (Invitrogen, CA), and plated on 60 mm dishes kept in a controlled humidity, temperature, and CO₂ environment [25] . Using this procedure, we observed that most human oral epithelial cells can be expanded for a finite number of passages, achieving a replicative senescent state after approximately 55 days, but few cultures escaped cell senescence, and instead became immortal (G.L. Sanchez, K.L, R.C., J.S.G et al., manuscript in preparation). To date, these cells, termed spontaneously immortalized normal oral keratinocytes, NOK-SI, have been cultured for more than 2 years, retaining epithelial morphology, proliferative capacity, and the expression of typical markers such as cytokeratins and E-cadherin. NOK-SI cell line is routinely cultured in Keratinocyte-SFM medium (Gibco, USA) supplemented with BPE and EGF, penicillin, streptomycin, and maintained at 37°C in a 5% CO2-humidified incubator.

Immunohistochemical staining for p-STAT3, p-AKT, MET, E-cadherin, and BIM was performed using the streptavidin-peroxidase method. Briefly, 4-μm-thick sections were deparaffinized, rehydrated, and treated with 0.3% H₂O₂ to block endogenous peroxidase activity. Following rehydration through graded concentrations of ethanol and autoclave, nonspecific binding sites were blocked with 10% normal goat serum. The sections were then incubated at 4 °C overnight with the following antibodies: rabbit antibodies against human p-STAT3 (Cell Signaling Technology, #4060, dilution 1:100), p-AKT (Cell Signaling Technology, catalog #9145, dilution 1:100), MET (Abcam, catalog #Ab227637, dilution 1:30), E-cadherin (Cell Signaling Technology, catalog #3195, dilution 1:50), and BIM (Cell Signaling Technology, catalog #2933, dilution 1:100). The sections were then incubated with biotinylated peroxidase-labeled anti-rabbit antibody (DakoCytomation, catalog #K4003) for 30 min, followed by incubation with streptavidin–biotin peroxidase complex solution. The chromogen 3,3′-diaminobenzidine tetrahydrochloride was used. The expression of p-STAT3, p-AKT, MET, and BIM was defined in samples as any intensity of antibody staining and ≥ 1% of the tumor^{33 (link)–36 (link)}. Samples with no E-cadherin membranous staining in any percentage of the tumor were categorized as a loss of expression, while those with E-cadherin membranous staining were categorized as having preserved expression^{37 (link)}. Finally, the sections were lightly counterstained with hematoxylin. The stained tissue sections were independently scored by Dr. Chang-Yao Chu at Chi-Mei Medical Center and other pathologists at NCKUH who were blinded to the patients’ clinical characteristics and outcomes.

Whole-mount immunofluorescent staining was performed using a modification of a previously reported method^{61 (link)}. The ileal tissue was fixed in 4% paraformaldehyde (Sigma) for 2 h at room temperature. Fixed tissue was permeabilized with 0.5% Triton X-100 (Sigma) overnight at room temperature, and then blocked with 10% goat serum (Sigma) and 0.5% Triton X-100 overnight at 4 °C. For SD and CDAHFD group, antibody reaction was performed with FITC labeled mouse anti-Crp1 antibody (50 μg/mL, clone 77-R63, produced in our laboratory) and Alexa Fluor 647-labeled anti-mouse/human CD324 (E-cadherin) antibody (1:100, clone DECMA-1, BioLegend). For CDAHFD + PBS and CDAHFD + R-Spo1 group, the primary antibody reaction was performed with rabbit anti-Olfactomedin 4 (Olfm4) antibody (1:80, clone D6Y5A, Cell Signaling) for 1 day at 4 °C, and then the secondary antibody reaction was performed with Alexa Fluor 555 conjugated F(ab′)2-goat anti-rabbit IgG (dilution 1:500, Thermo Fisher Scientific) and FITC labeled mouse anti-Crp1 antibody overnight at 4 °C. After washing, nuclei were stained with DAPI (Thermo Fisher Scientific). Samples were immersed in the optical-clearing solution (RapiClear 1.52, Sunjin Lab).
For quantification of Crp1 fluorescence intensity and counting numbers of Paneth cells and stem cells, Z-stack images were obtained using a confocal microscope (A1, Nikon) equipped with CFI Apo LWD 20X WI λS (Nikon). The number of Paneth cells was quantified by counting Crp1 immunostaining positive cells on 3 fields (150 × 150 μm²) per tissue. The number of stem cells was quantified counting Olfm4 positive cells on 3 fields (150 × 150 μm²) per tissue. Crp1 fluorescence intensity per Paneth cell was measured by creating a region of interest using image analysis software, NIS-Elements AR ver. 5.11 (Nikon), on 3 fields (150 × 150 μm²) per tissue, and the mean intensity per field was calculated. For quantification of the number and diameter of Paneth cell granules, Z-stack images were obtained using A1 with CFI Apo TIRF 60X Oil (Nikon). The number and diameter of Paneth cell granules were measured on 3 fields (33 × 33 μm², 2 Paneth cells/field) per tissue.