MKN-1 (adenosquamous carcinoma), MKN-7 (well-differentiated adenocarcinoma), MKN-74 (moderately differentiated adenocarcinoma) and NUGC-4 (signet ring cell carcinoma) cells, which are all derived from human gastric cancer, and Huh7 cells, which lack endogenous ERC/mesothelin expression and derived from human hepatocellular carcinoma, were provided by the RIKEN BRC Cell Bank (Tsukuba-shi, Ibaraki, Japan). TMK-1 (poorly differentiated adenocarcinoma) cells, which are derived from human gastric cancer, were provided by Hiroshima University (Hiroshima-shi, Hiroshima, Japan). NUGC-3 (poorly differentiated adenocarcinoma) cells, which are derived from human gastric cancer, were provided by the Health Science Research Resources Bank (Sennan-shi, Osaka, Japan). NCI-H226 cells, which were used as a positive-control and are derived from human mesothelioma, were provided by the American Type Culture Collection (Manassas, VA, USA). All of the cells, except the Huh7 cells, were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS). The Huh7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS. The culture supernatants and cells were harvested after being cultured for 48 h at 37°C under a 5% CO₂ atmosphere, at which point they had reached >80% confluence.

The mRNA levels of ERC/mesothelin in the cultured cells (MKN-1, MKN-7, MKN-74, NUGC-3, NUGC-4 and TMK-1) were analyzed by RT-PCR. RT-PCR was carried out using the Titan RT-PCR system (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions. Cells in Petri dishes were lysed with TRIzol reagent according to the acid guanidinium thiocyanate-phenol-chloroform extraction method (20) (Invitrogen Life Technologies, Carlsbad, CA, USA). Total RNA was extracted from these lysates according to the manufacturer's instructions. Total RNA (1 μg) was reverse transcribed for 30 min at 50°C and subjected to PCR amplification. The primers used to amplify ERC/mesothelin mRNA were as follows: sense, 5′-CAAGAAGTGGGAGCTGGAAG-3′ and antisense, 5′-GTCTCCAGGGACGTCACATT-3′. As a control for the RT-PCR, β-actin mRNA was amplified using the following β-actin-specific primers: sense, 5′-CCGCGAGAAG ATGACCCAGA-3′ and antisense, 5′-CAGGAGGAGCAATG ATCTTG-3′. All primers were purchased from Operon (Tokyo, Japan). After an initial denaturation step of 2 min at 94°C, each sample was subjected to 30 cycles of amplification (denaturation, 30 sec at 94°C; annealing, 30 sec at 55°C and elongation, 1 min at 68°C) followed by a final elongation step of 2 min at 68°C. The PCR products (20 μl) were analyzed on a 2% agarose gel containing 1 μg/ml ethidium bromide.

Cell surface C-ERC/mesothelin expression was analyzed by flow cytometry, as described previously (21). Briefly, 5×10⁵ cells were incubated for 30 min at 4°C with 1 μg/ml of the C-ERC/mesothelin-specific mouse monoclonal antibody 22A31, as described previously (21), or with 1 μg/ml of normal mouse IgG1κ diluted in 2% FCS (100 μl). After being washed with 2% FCS in medium, the cells were resuspended in 100 μl of 2% FCS in medium containing 2 μg/ml of Alexa Fluor 488-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR, USA) to detect the primary antibodies, before being incubated at 4°C for 30 min. After being washed with phosphate-buffered saline (PBS), the stained cells were analyzed with an LSRFortessa™ cell analyzer (BD Biosciences, San Jose, CA, USA).

The culture supernatants of the MKN-1, MKN-7, MKN-74, NUGC-3, NUGC-4, and TMK-1 cells were harvested and adjusted with a solution containing 2% sodium dodecyl sulfate (SDS), 10% glycerol, 50 mM Tris-HCl (pH 6.8), and 100 mM dithiothreitol (DTT), before being boiled for 3 min. The culture supernatants were then electrophoresed on 10% Laemmli gels and transferred onto nitrocellulose membranes. The membranes were then blocked in a mixture of 1% skimmed milk and PBS supplemented with 0.1% Tween-20 (PBS-T) for 1 h at room temperature. Next, the membranes were incubated with the N-ERC/mesothelin-specific mouse monoclonal antibody (MoAb) 7E7, as described previously (7), or a rabbit anti-actin polyclonal antibody (sc-1616-R) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; 1:500 dilution) in a mixture of PBS-T and 1% skimmed milk for 1 h at room temperature. The goat anti-mouse or anti-rabbit Ig secondary antibodies, which were conjugated with peroxidase labeled-dextran polymers (EnVision K4001 or K4003; DakoCytomation, Glostrup, Denmark) at a dilution of 1:50 in a mixture of PBS-T and 1% skimmed milk, were allowed to react with the membranes at room temperature for 1 h. The ECL detection system (GE Healthcare, Buckinghamshire, UK) was used to visualize ERC/mesothelin on the membranes.

The present study included 50 patients with gastric cancer (40 men and 10 women; age range, 33–89 years; mean age, 71.5) who were diagnosed and provided blood samples between January 2009 and September 2011 at Juntendo Shizuoka Hospital, Juntendo University School of Medicine. All clinical diagnoses of gastric cancer were confirmed by microscopic examinations of the material obtained during surgery or endoscopic resection. The present study was approved by the Institutional Review Board of Juntendo Shizuoka Hospital, Juntendo University School of Medicine. All patients provided informed consent. Blood samples were collected from each patient before surgery or endoscopic resection. Thirty-seven healthy controls (age range, 53–79 years; mean age, 66.8) were sampled at random from a database, as described previously (9).

Tissue sections (3 μm) were prepared from archival formalin-fixed, paraffin-embedded specimens. After being deparaffinized, the tissue sections were heated in target retrieval solution (DakoCytomation) for antigen retrieval and were then treated with 3% hydrogen peroxide. Non-specific binding sites were blocked by incubating the samples with 5% normal goat serum in PBS for 20 min at room temperature. Next, the sections were incubated overnight with primary antibody solutions diluted in PBS-T at 4°C. We used mouse monoclonal anti-human C-ERC/mesothelin antibody 22A31 (1:40 dilution) as the primary antibody and the EnVision+ system labeled with horseradish peroxidase (HRP) polymers (DakoCytomation) as the secondary antibody. Diaminobenzidine was used as the peroxidase substrate. The C-ERC/mesothelin immunostaining was analyzed using the scoring system described by Einama et al(18). For the immunostained slides, the proportion of stained cancer cells was scored as +1 for 1–10, +2 for >10–50 and +3 for >50%. The intensity of the staining was scored as +1 for weak intensity and +2 for moderate to strong intensity, and the location of the staining was also recorded; i.e., in the luminal membrane or the cytoplasm. The final evaluation of C-ERC/mesothelin expression was based on the following scoring system: ‘positive-staining’ was defined as a proportion score of ≥+3 and/or an intensity score of +2, while ‘negative staining’ was defined as a total score of <+3, except in cases involving a proportion score of +1 and an intensity score of +2.

The N-ERC/mesothelin concentrations in the patient sera and the cell culture supernatants of the MKN-1, MKN-7, MKN-74, NUGC-3, NUGC-4 and TMK-1 cells were analyzed using the sandwich ELISA method (7), which was performed as described previously (7,9) using the 7E7 MoAb and the HRP-conjugated polyclonal antibody-282. The absorbance of the solution was measured at 450 nm in an ELISA reader (EMax; Molecular Devices, Sunnyvale, CA, USA).

The data were analyzed with GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). The measurement data were analyzed using the Mann-Whitney and Kruskal-Wallis tests, while the categorical data were analyzed using Fisher's exact test. P<0.05 was considered to indicate a statistically significant result.

In 5 of the 6 investigated gastric cancer cell lines (MKN-1, MKN-7, MKN-74, NUGC-4 and TMK-1), ERC/mesothelin mRNA and C-ERC protein were detected by RT-PCR (Fig. 1), flow cytometry (Fig. 2A) and immunohistochemistry (Fig. 2B). The C-ERC/mesothelin was localized in the cytoplasm and/or on the cell membrane (Fig. 2). ELISA demonstrated that 3 of these 5 cell lines (MKN-1, NUGC-4 and TMK-1) excreted N-ERC into the culture medium (Fig. 3A). However, western blotting only detected N-ERC in the media from 2 cell lines (NUGC-4 and TMK-1) (Fig. 3B).

Immunohistochemically, positive C-ERC/mesothelin staining was observed on the cancer cell membrane and in the cytoplasm (Fig. 4). Among the 50 tumors examined, 29 (58%) were positive for C-ERC/mesothelin. Luminal membrane expression of C-ERC/mesothelin was observed in 15 cases (30%).

The correlations between the clinicopathological features and C-ERC/mesothelin expression in the primary tumors are summarized in Table I. C-ERC/mesothelin positivity was not associated with any of the parameters. However, the incidence of C-ERC/mesothelin luminal membrane expression was correlated with lymphatic invasion (P=0.0286).

The N-ERC/mesothelin levels in the sera of the patients were evaluated. We selected 38 of the 50 patients for age-matching with the control group. The sera of the 38 age-matched gastric cancer patients (26 men and 10 women, age range, 33–78 years; mean age, 65.0) were compared with those of the 37 healthy controls (24 men and 13 women, age range, 53–79 years; mean age, 66.8) as described previously (9). There was no significant difference between the serum N-ERC/mesothelin concentrations of the cancer patients and healthy controls (P=0.3986) (Fig. 5A). When TNM stage was analyzed, a significant correlation with the serum N-ERC/mesothelin concentration was not detected (P=0.7948) (Fig. 5B).