MMP7 Expression in Colorectal Tumours of Different Stages

Patients and Methods

Patients. A prospective study was undertaken of patients referred for elective colorectal cancer treatment to the Pietro Valdoni Department of Surgery, Sapienza University of Rome. Exclusion criteria were neoadjuvant radiotherapy, chemoradiotherapy, language problems and consent withdrawal. Twenty-eight patients were recruited prospectively according to guidelines for treatment of colorectal disease after routine clinical assessment. The study was approved by the Human Ethics Committee at the Sapienza University of Rome and registered at Clinical Trials, ID NCT 01570452. The study was carried-out over a period of three years (September 2005-September 2008). Twenty-eight patients completed the study (eight with benign and 20 with malignant colorectal tumours) after acceptance of informed consent. Among benign tumours, only polyps not suitable for endoscopic resection were included. Serum controls were taken from 10 healthy volunteers after acceptance of informed consent. Staging was performed after colonoscopy and biopsy, with abdominal computed tomography (CT) and chest radiography. Clinical variables were included in a database. Patients with severe dysplasia or large symptomatic low-to-moderate dysplastic adenomas and adenocarcinoma underwent surgery. Intraoperative blood samples were collected to determine baseline parameters, specific oncomarkers (carcinoembryogenic antigen, CEA; cancer antigen 19-9, CA19-9, cancer antigen 50, CA 50) and MMP7. Intraoperative specimens were collected from cancer tissues and from normal surrounding mucosa (about 2-2.5 cm from the tumour edge). Lymph nodes from the colonic mesentery close to the tumour were collected. The specimens were fixed in 4% formaldehyde before histopathological examination.

Samples and serum preparation. Tissue samples from tumoural and mucosal tissue, as well as from lymph nodes, were kept in sterile tubes at −80°C. They were then cut obtaining aliquots weighing between 50 and 100 mg. These were treated with 300 μl of lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.2% NP-40, 1% CHAPS, 2 mM EDTA dissolved in tetra-distilled water). A mixture of protease inhibitors (Complete-Mini Protease Inhibitor Cocktail Tablets; Roche, Mannheim, Germany) was added just before use. Samples were first homogenized by Ultra-Turrax® (T10 basic; IKA®, Staufen, Germany), then sonicated for 20 sec and centrifuged at 14,000 rpm for 20 min (19). The supernatants were then collected. Venous blood samples were drawn into sterile vacuum tubes and left at room temperature for 30 min, then centrifuged at 4,000 rpm for 15 min, to divide serum from pellet, as standard laboratory protocol. Serum was immediately aliquoted and stored at −80°C until assayed. The protein content of supernatants and serum samples was determined by using the Bradford assay.

Histology. Samples were fixed in 4% phosphate-buffered formaldehyde and later embedded in paraffin. Sliced specimens stained with hematoxylin and eosin were analyzed under light microscopy. At least three slides were studied from each specimen by a blinded observer. Stage definition was stated according to 2002 UICC classification (20).

Immunohistochemistry. For immunohistochemistry, standard avidin-biotin procedures for human MMP7 were used. After deparaffinization and washing in phosphate-buffered solution (PBS), endogenous peroxidase activity was blocked by incubating the sliced sections in 3% hydrogen peroxide in PBS for 10 min. Analysis for MMP7 was performed using anti-MMP7 (MAB-10756; Immunological Sciences, Rome, Italy) following the manufacturer's instructions. Biotin-conjugated secondary antibody and streptavidin-conjugated horseradish peroxidase (Dako North America, Inc., CA, USA) was applied to sections for 45 min at room temperature, and developed using 3,3’-diaminobenzidine (DAB) as substrate. Finally, counterstaining with haematoxylin was performed. Sections were mounted and the grade of staining was determined on randomly selected areas counter-checked for intensity by a blinded observer.

MMP7 determination. In supernatants and serum samples, total human MMP7 levels were determined using an enzyme-linked immunosorbent assay (ELISA) kit (Quantikine®, R&D Systems, Minneapolis/USA). Diluted samples (150 μl) were added to a 96-well microtiter plate, pre-coated with a monoclonal antibody to human MMP7 and incubated at room temperature for a further 2 h on a microplate shaker. After washing, 200 μl of the secondary antibody solution were added, and the plate was incubated for 2 h at room temperature on the shaker. After washing, the substrate solution was added and incubated at room temperature in the dark. A 50 μl stop solution was added after 30 min and the optical density was measured using a microtiter plate reader (Opsys MR™; Dinex Technologies, Inc.; Chantilly, VA, USA) at 450 nm, with correction wavelength set at 570 nm.

Western blot analysis. For western blot analysis, supernatants obtained from lymph node specimens were separated on a sodium dodecyl sulphate-polyacrylamide electrophoresis gel with a concentration of acrylamide specific for MMP7 and β-actin. Proteins were blotted onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA) and probed with the following antibodies: anti-MMP7 (MAB-10756 Immunological Sciences) and anti-β-actin (A 5060; Sigma Chemical Co., St. Louis, MO, USA). Antigens were detected with an enhanced chemoluminescence (ECL) kit from Amersham (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK). All western blotting images were acquired and analyzed through an Imaging Fluor S densitometer (Bio-Rad Laboratories, Hercules). The optical density (O.D.) of each condition was correlated to the signal of the β-actin internal control.

Statistical methods. Data are expressed as the mean±standard deviation (SD). The statistical comparisons between groups were performed by using the analysis of variance (ANOVA) followed by the Bonferroni post hoc test. Differences were considered significant at p<0.05. Analysis was performed by using a statistical software (GraphPad Software, Inc., San Diego, CA, USA).