Tissue samples
Mammary gland samples were collected from 74 female domestic dogs who had been seen at a veterinary clinic due to a mass in the mammary gland. The canine mammary tissues were surgically removed from these animals at a mean age of 12 years (7–15 years) and were submitted to the University of Ulsan College of Medicine between June 2014 and May 2015. After microscopic examination, 5 samples were diagnosed as non-neoplastic lesions and 69 specimens were diagnosed as CMTs.
Histological evaluation
Slides were evaluated for growth pattern (ductular, papillary, or solid), invasion pattern (expansile, local, regional, nodal, or vascular), mitotic index, degree of necrosis, anaplasia, and inflammation. The morphologic diagnosis of CMT was based on the classification of Goldschidt et al. [36] Using this approach, the classification of benign mammary tumors includes adenoma simple, intraductal papillary adenoma, ductal adenoma, adenoma complex, benign mixed, fibroadenoma, and myoepithelioma. Malignant classifications include carcinoma simple, carcinoma complex, carcinoma mixed, anaplastic carcinoma, lipid-rich carcinoma, inflammatory carcinoma, mucinous carcinoma, and adenosquamous carcinoma. The carcinoma simple class has tubular, tubulopapillary, cystic-papillary, and cribriform subclasses. Tumors that were too poorly differentiated to be morphologically diagnosed were classified as solid carcinomas. Malignancy was evaluated using the following criteria: tumor type, tumor size, tubular formation pattern, significant nuclear and cellular pleomorphism, mitotic index (number of cells with mitotic figures per 10 high-power fields from the neoplastic area with mitotic activity), and presence of areas of necrosis [22]. All samples were also classified according to their morphologic origin. All microscopic evaluations were performed by two veterinary pathologists.
Immunohistochemistry
Sections (3 μm) from paraffin-embedded tissue blocks of canine mammary gland tumor tissues were mounted on glass slides. Immunohistochemistry was performed using an automated slide preparation system (Benchmark XT; Ventana Medical Systems Inc., Tucson, AZ). Deparaffinization, epitope retrieval, and immunostaining were performed according to the manufacturer’s instructions with cell conditioning solutions (standard, for 60 min) and the BMK ultraVIEW diaminobenzidine detection system (Ventana Medical Systems). Tumor sections were stained with EZH2 (1:100, ab109398, Abcam, Cambridge, MA) for 36 min at 37 °C, followed by Ultraview HRP universal Multimer for 8 min at 37 °C. Positive signals were amplified using ultraVIEW copper, and sections were counterstained with hematoxylin and bluing reagent for 4 min respectively.
Immunohistochemical Evaluation of EZH2
EZH2 expression was evaluated on the slides using a semiquantitative scoring system described previously with some modifications [10]. Samples were evaluated for staining intensity (0, none; 1, weakly positive; 2, moderately positive; and 3, strongly positive).
SDS-PAGE and western blot
Approximately 10 mg of CMT tissue were prepared by TissueLyser II (Qiagen, Valencia, CA) and suspended in sample buffer (62 mmol/liter Tris-Cl, pH 6.8, 2 % SDS, 10 % glycerol, and 0.01 % bromophenol blue with 5 % 2-mercaptoethanol), incubated for 5 min at 100 °C, and then electrophoretically separated in a 12 % polyacrylamide mini-gel. Electrophoresis was performed in Tris-buffered saline (TBS) at a constant current of 60 mA for 2 h. Molecular weight standards (P8502-050; GenDEPOT) were run simultaneously. The gel was stained with Coomassie Blue. A parallel SDS-PAGE gel was run as described above, and the separated proteins were transferred directly by tank blotting onto a polyvinyl difluoride transfer membrane (Bio-Rad Corp, Hercules, CA) for 90 min at a constant current of 80 mA. After saturation of the nonspecific sites with 5 % nonfat milk/TBS overnight at 4 °C, the proteins were probed with a 1:500 dilution of rabbit anti-EZH2 antibody (ab186006; Abcam, Cambridge, MA) overnight at 4 °C. The blot was then washed in 20 mM Tris-HCl, pH 7.5, and 0.14 mM NaCl containing 0.5 % Tween 20 (TBS-Tween) and then incubated for 2 h in an anti-rabbit HRP-conjugated IgG antibody (SC-2004; Santa Cruz, Santa Cruz, CA) diluted 1:1000 in TBS-Tween at room temperature. The immunoblot was exposed to an enhanced chemiluminescence immunoassay substrate reagent (DG-WP250; DoGen, Seoul, Korea) for 1 min to detect signals and the membrane was exposed to X-ray film for 5 min. Band intensity on exposed film was semi-quantified using ImageJ software (National Institutes of Health, Bethesda, MD).
Statistical analysis
Data are expressed as the mean ± standard deviation of the mean. Since our data were not normally distributed in the Kolmogorov-Smirnov test, we compared the data with the Kruskal-Wallis test, which is a non-parametric method using the SPSS version 21 (IBM Corp., Armonk, NY). If significant, paired comparisons were done with the Mann Whitney test. A Bonferronic correction was applied to correct for multiple comparisons of the primary end point.
