Activated EphA2 Processing by MT1-MMP Is Involved in Malignant Transformation of Ovarian Tumours In Vivo

Materials and Methods

Tissue samples. This study included ovarian tumour tissue samples from 206 patients, which were obtained from ovarian tumour files at the Department of Pathology, Fukuoka University Hospital. The patients were clinically treated at the Department of Gynecologic Oncology, Fukuoka University Hospital between January 2000 and December 2014. Samples of ovarian epithelial tumours included 45 benign ovarian tumours (15 serous cyst adenomas, 17 mucinous cyst adenomas, and 13 endometrial cysts), 54 ovarian borderline tumours (OBTs; 12 serous borderline tumours, and 42 mucinous borderline tumours), and 107 ovarian carcinomas (47 serous adenocarcinomas, 24 endometrioid carcinomas, 16 mucinous carcinomas, and 20 clear cell carcinomas). Tumor tissue specimens for western blot analyses were frozen in liquid nitrogen and kept at −80°C until use.

Immunohistochemistry. Surgically resected specimens were fixed in 10% formalin and processed into paraffin blocks. Tissues were sectioned (3-μm thickness), and the sections were deparaffinized and immersed in 0.3% hydrogen peroxide in methanol for 10 min at room temperature (RT) to block endogenous peroxidase activity and then heated in 10 mM citrated buffer (pH 6.0) in a microwave oven (700 W) for 10 min to retrieve epitopes. After non-specific sites were blocked with 5% non-fat dry milk for 1 h at RT, these sections were incubated overnight at 4°C with polyclonal antibodies against the C-terminal of EphA2 (C-EphA2) (1:200; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) (9), the N-terminal of EphA2 (N-EphA2) (1:800; Abgent, San Diego, CA, USA) (10), or monoclonal antibodies against MT1-MMP (1:50; 2D12) (11). Subsequently, these sections were washed and incubated with ChemMate EnVision (Dako, Carpinteria, CA, USA). Immunoreacitve proteins were visualized with 3, 3’-diaminobenzidine (DAB; Dako), followed by counterstaining with haematoxylin. In all cases skin sections were stained to represent the normal control on the same glass slide in order to elucidate the staining pattern and intensity in normal epidermis (3).

Immunohistochemical results were assessed as described in previous reports (3, 9). Similar expression levels of N-EphA2 and C-EphA2 indicated that the EphA2 likely retained the N-terminal ligand-binding domain. In contrast, reduced expression levels of N-EphA2 compared with the C-terminal indicated that N-EphA2 was likely to have been cleaved off.

Visual- and computer-supported evaluation of immunohistochemical staining. Immunohistochemical expression of C-EphA2, N-EphA2, and MT1-MMP in all samples was assessed using Tissue Studio v.2.0 software (Definiens AG, Munich, Germany). For image analysis, each immunohistochemically stained slide was scanned and converted to a whole-slide image (WSI, also known as a virtual slide) with NanoZoomer 2.0-RS (Hamamatsu Photonics, Hamamatsu, Japan) at 20× magnification. On each WSI, the tumour area was selected by a hand-drawing tool, and tumour cells exhibiting cytoplasmic expression of EphA2 and MT1-MMP were identified using Tissue Studio v.2.0, installed on the server for NanoZoomer 2.0-R.S. The DAB colour intensity of positive tumour cells in every unit area was measured, and the average value per unit area (/μm²) was calculated using Tissue Studio v.2.0. The intensity of EphA2 and MT1-MMP staining was measured in epithelial cells of normal skin, which had been added as a positive control to each glass slide alongside tumour tissues. Furthermore, the ratio of the average staining intensity of tumour cells to that of the control epithelial cells was calculated.

Protein extraction and western blot analysis. Proteins were extracted from frozen tissues in RIPA lysis buffer (50 mM Tris-HCl, ph7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40; Millipore, Bedford, MA, USA), containing protease inhibitors (Complete Mini; Roche Applied Sciences, Penzberg, Germany) using a homogenizer on ice. The extracts were clarified by centrifugation (14,000 rpm for 20 min at 4°C) and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred electrophoretically to Immobilon membranes (Millipore). Nonspecific sites were blocked with 5% dry non-fat milk in TBS at 37°C for 1 h, and then the membrane was incubated overnight at 4°C with anti-EphA2 (Santa Cruz Biotechnology) (9) and anti-MT1-MMP (2D12) (11) antibodies. After three washes with TBS-T (TBS containing 0.05% Tween-20), the membranes were incubated for 1 h with peroxidase-conjugated anti-rabbit IgG; immunoreactive proteins were detected with chemiluminescence reagents according to the manufacturer's instructions (PerkinElmer, Waltham, MA, USA).

In situ Proximity Ligation Assay (PLA). Duolink PLA (Olink, Uppsala, Sweden), was used to detect protein-protein interactions. This technique utilizes one pair of oligonucleotide labelled antibodies binding in close proximity (30-40 nm apart) to two different proteins in a complex. PLAs were performed according to the manufacturer's instructions. Briefly, sections were pretreated, and primary antibodies against C-EphA2 (1:200; Santa Cruz Biotechnology) (9), N-EphA2 (1:800; Abgent) (10), and MT1-MMP (1:50; 2D12) (11) were applied for 1 h at 37°C. For isotype controls, the primary antibody was substituted with either rabbit (C-EphA2 or N-EphA2) or mouse (MT1-MMP) IgG. Sections were then washed twice for 5 min each in Duolink wash buffer A (Olink) before PLA PLUS and MINUS probes (Olink) were applied for 1 h at 37°C. Following washing (as previously), ligation-ligase solution (Olink) was applied to each sample for 30 min at 37°C. Sections were washed again, and amplification-polymerase solution (Olink) was applied for 120 min at 37°C. In paraffin sections of formalin-fixed tissues, dot signals were visualized with DAB. The number of in situ PLA signals per cell was determined using Tissue Studio v.2.0.

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Figure 1.

Representative immunohistochemical expression of C-EphA2, N-EphA2, and MT1-MMP in ovarian epithelial carcinomas (OECs; SC: Serous carcinoma; EC: endometrioid carcinoma; MC: mucinous carcinoma; CC: clear cell carcinoma).

Compliance with ethical standards. This study was approved by the Ethics Committee of Fukuoka University School of Medicine (No.15-3-14). Use of anonymous and redundant tissue is part of the standard treatment agreement with patients in our hospitals when no objection has been expressed.\

Statistical analyses. Quantitative data are presented as mean±standard deviation (SD) and were analysed using the Student's t-test. A p-value <0.05 was considered indicative of statistical significance. All data analyses were conducted using the Excel statistical software package (Ekuseru-Toukei 2015; Social Survey Research Information Co., Ltd., Tokyo, Japan).