Mesothelin Expression in Human Lung Cancer

Materials and Methods

Tissues and cell lines. Frozen and fixed lung tumor samples were acquired from the Cooperative Human Tissue Network (Charlottesville, VA), which is funded by the National Cancer Institute. Nine human lung cancer cell lines in the NCI-60 cell line panel were obtained from the National Cancer Institute Anticancer Drug Screen Program. They are A549 (adenocarcinoma), Hop-62 (adenocarcinoma), Hop-92 (large cell), EKVX (adenocarcinoma), NCI-H226 (mesothelioma), NCI-H322M (adenocarcinoma), NCI-H460 (large cell), NCI-H522 (adenocarcinoma), and NCI-H23 (adenocarcinoma).

Reverse transcription-PCR analysis. Primers used in this study are Meso64 (mesothelin, sense), 5′-CGAGATAGACGAGAGCCT-3′; and Meso65 (mesothelin, antisense). Total RNAs from different cancer cell lines and cancer tissues were isolated using TRIzol reagents (Invitrogen, Carlsbad, CA). First-strand cDNA was prepared from the isolated RNA using a first-strand cDNA synthesis kit (Amersham Biosciences, Piscataway, NJ). PCR was done on cDNA using the Tgo DNA polymerase with 3′-5′ exonuclease proofreading activity (Roche Molecular Biochemicals, Indianapolis, IN). The PCR conditions used are initial denaturation at 94°C for 2 min, 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and elongation at 72°C for 2 min. The expected size of the PCR products of mesothelin is shown in Fig. 1 .

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Fig. 1.

Mesothelin mRNA and protein expression in frozen tissue specimens of lung cancer patients. A, reverse transcription-PCR detection of mesothelin mRNA in frozen tissue specimens. Water, a template-free negative control; Ov, Ovcar-3 cells as a positive control; Ad, lung adenocarcinoma tissues; Sq, squamous cell carcinoma; Sm, small cell lung cancer. B, Western blot detection of mesothelin proteins in frozen tissue specimens. MSLN, mesothelin. H9 is the A431.MSLN⁺ (H9) stable transfected cell line used as a positive control (18).

Immunohistochemistry. Tissue sections obtained from archival paraffin-embedded tumor blocks from the original biopsies of patient or surgical resection were evaluated for mesothelin expression using anti-mesothelin mAbs MB and 5B2 with peroxidase immunohistochemistry and with citraconic anhydride antigen retrieval (27).

Measurement of mesothelin expression by flow cytometry. Flow cytometric analysis was done by following an established protocol (18). Briefly, 5 × 10⁵ cells were incubated with 1 μg/mL of mAb (MN or MB) in 500 μL of PBS containing 5% bovine serum albumin and 0.1% sodium azide. After incubation for 1 h at 4°C, the cells were washed once with the same buffer and incubated with 1:200 dilution of phycoerythrin-labeled goat F(ab′)₂ anti-mouse IgG (Invitrogen) for 1 h. After washing twice, the cells were suspended in 0.5 mL of PBS, and the fluorescence associated with the live cells was measured using a FACSCalibur flow cytometer (BD Biosciences, Palo Alto, CA). To quantitatively measure the mesothelin sites on the cell surface, the mean fluorescence intensity of cells was converted to the molecular number of fluorochromes per cell using QuantiBrite PE (BD Biosciences) calibration beads.

Western blot analysis. Immunoprecipitation assay buffer [50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA (pH 8), 0.1% Triton X-100, 1 mmol/L phenylmethylsulfonyl fluoride, 1 μg/mL aprotinin, 1 μg/mL leupeptin, and 1 μg/mL pepstatin] containing 2% SDS was used to solubilize cells on the dish or the cell pellet in the tube. Protein concentration was determined by the bicinchoninic acid protein assay (Pierce, Rockford, IL) according to the manufacturer's protocol. Equivalent amounts (40 μg per lane) of whole-cell lysates were separated by a 4% to 20% Tris-glycine SDS-PAGE and transferred onto nitrocellulose filters. Western blots were done using a standard method. Each blot was treated with 1 μg/mL anti-mesothelin mAb MN. Primary antibodies were detected by secondary goat anti-mouse antibodies conjugated with horseradish peroxidase. Signals were visualized by the Enhanced Chemiluminescence kit (Amersham Biosciences).

Cytotoxicity assays. The cytotoxicity of immunotoxin SS1P was determined on NSCLC cell lines by a WST cell death assay as described previously (28).

Results and Discussion

Mesothelin expression in human lung cancers. To determine how frequently mesothelin is expressed in human lung cancer, we characterized the mRNA and protein expression of mesothelin in lung cancer samples. We used reverse transcription-PCR to examine if mesothelin mRNA was expressed in 16 lung cancer patient specimens. The reverse transcription-PCR data in Fig. 1A show that mesothelin is expressed in 10 of 12 (83%) lung adenocarcinomas, 2 of 2 squamous cell carcinomas, and 1 of 2 small cell lung cancers. In Fig. 2B , we did Western blot experiments on 11 lung adenocarcinomas. The mesothelin precursor protein with a molecular weight of 71 kDa was detected in 9 of 11 (82%) of adenocarcinomas, and its mature form (molecular weight, 40 kDa) was detected in 55% (6 of 11; Fig. 1B). The reverse transcription-PCR and immunoblotting results show that mesothelin is expressed in most lung adenocarcinoma specimens.

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Fig. 2.

Summary of immunoreactivity patterns in cancer patient samples. Immunohistochemical evaluation using two different mAbs, 5B2 (A) and MB (B), shows strong and diffuse expression of mesothelin in two of four representative lung adenocarcinoma (Adeno) samples. Ca, carcinoma.

We also examined mesothelin protein expression in human lung cancer specimens by immunohistochemistry. Two of four lung adenocarcinoma tissue samples had strong mesothelin immunoreactivity using two different mAbs: 5B2 (Fig. 2A) and MB (Fig. 2B). With both mAbs, the immunoreactivity was mainly distributed in the plasma membrane and the Golgi region of cells, with some signal present diffusely in the cytoplasm. Lower mesothelin immunoreactivity was observed in the other two adenocarcinomas. Some of the cytoplasmic mesothelin signals observed in lung adenocarcinoma specimens by immunohistochemistry may be due to the unprocessed 71-kDa precursor form of mesothelin detected by immunoblotting (Fig. 1B). In both squamous cell carcinomas, the immunoreactivity was greatly diminished. As previously reported in mesothelioma, there was a strong signal at the cell membrane (2). Mesothelin immunoreactivity was also seen on the membrane of normal mesothelial cells.

Characterization of mesothelin expression in lung cancer cell lines. To further characterize the expression of mesothelin in human lung cancer cells, we examined all nine lung cancer cell lines from the NCI-60 cell line panel (29): eight NSCLC lines and one mesothelioma line (NCI-H226). Among the NSCLC lines, there are six adenocarcinoma lines (A549, Hop-62, EKVX, NCI-H322M, NCI-H522, and NCI-H23) and two large cell lines (Hop-92 and NCI-H460). As shown in Fig. 3A , mesothelin mRNA was expressed in 7 of 9 (78%) lung cancer cell lines: A549, Hop-92, EKVX, NCI-H226, NCI-H322M, NCI-H460, and NCI-H522. Both mesothelin mRNA (Fig. 3A) and proteins (Fig. 3B) were expressed at a high level in EKVX, NCI-H460, NCI-H322M, NCI-H522, and NCI-H226 cells. As shown in Fig. 3B, there are two major bands representing the 71-kDa precursor and 40-kDa mature forms of mesothelin in all the NSCLC lines. Some minor and low–molecular weight bands were detected probably due to the degradation of mesothelin. In the mesothelioma (NCI-H226) and ovarian cancer (Ovcar-3) lines, the 40-kDa mature form is the dominant form present.

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Fig. 3.

Mesothelin mRNA and protein expression in the NCI-60 lung cancer cell lines. A, reverse transcription-PCR detection of mesothelin mRNA. Water, a template-free negative control for PCR contamination; Ovcar-3, a positive control. B, Western blot detection of mesothelin proteins. H9 is the A431.MSLN⁺ (H9) stable transfected cell line used as a positive control (18).

To determine how much mesothelin is present on the surface of NSCLC cells, we did flow cytometric analysis of A549, EKVX, NCI-H322M, NCI-H460, and NCI-H522 cells. As shown in Fig. 4 , adenocarcinoma NCI-H322M and EKVX cell lines had the highest surface expression (∼100,000 sites per cell) of mesothelin proteins, and A549, NCI-H460, and NCI-H522 cell lines had modest surface expression (6,000-10,000 sites per cell) of mesothelin.

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Fig. 4.

Flow cytometric analysis of mesothelin protein expression in NCI-60 lung cancer cell lines. NCI-H322M (H322M), NCI-H522 (H522), A549, NCI-H460 (H460), and EKVX cells were probed with a mesothelin-specific mAb (solid dark line) or an irrelevant isotype mAb control (gray line). Ovcar-3 was used as a positive control.

Cytotoxicity of mesothelin-specific immunotoxin SS1P on human lung cancer cell lines. To determine if mesothelin could function as a new therapeutic target in lung cancer, we tested an anti-mesothelin recombinant immunotoxin, SS1(dsFv)PE38 or SS1P, that is composed of the Fv portion of antibody SS1 and a truncated form of Pseudomonas exotoxin. SS1P has been evaluated in phase I studies for mesothelioma and ovarian cancer patients (6). The cytotoxicity of immunotoxin SS1P was determined on five NSCLC cell lines (A549, EKVX, NCI-H322M, NCI-H460, and NCI-H522) by a WST cell death assay (Fig. 5 ). In NCI-H322M and EKVX, the two adenocarcinoma cell lines with the highest mesothelin expression on the cell surface (Fig. 4), SS1P was very active with IC₅₀ values ranging from 2 ng/mL (NCI-H322M) to 5 ng/mL (EKVX). In the A549 and NCI-H522 cell lines with low mesothelin expression, lower but significant cytotoxic activity (IC₅₀ = 200 ng/mL) was observed.

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Fig. 5.

Inhibition of cell viability on NCI-60 lung cancer cells by immunotoxins. Cancer cells (10,000 per well) incubated with various concentrations of anti-mesothelin immunotoxins (SS1P) for 72 h. Cell viability was determined by a WST assay as described in Materials and Methods. Points, means of triplicate determinations; bars, SD. The dashed line indicates 50% inhibition of cell viability, which is halfway between the level of viability in the absence of toxin and that in the presence of 10 μg/mL of cycloheximide.

In the present study, we found that mesothelin mRNA and protein are present in a substantial number of lung adenocarcinomas. Expression was confirmed using cell lines where mesothelin mRNA was detected in seven of nine lines of the NCI-60 panel. Furthermore, flow cytometric analysis showed high surface expression of mesothelin in the NCI-H322M and EKVX lung adenocarcinoma cell lines.

One unusual feature of the lung cancer samples is that there is more intracellular mesothelin reactivity than in mesotheliomas, ovarian cancers, or normal mesothelial cells. In addition, there seems to be substantial amounts of the high–molecular weight mesothelin precursor present in lung cancers, possibly accounting for its intracellular location.

Two phase I clinical trials of immunotoxin SS1P were recently completed at the National Cancer Institute in mesothelioma and ovarian cancer patients. Several minor but significant antitumor responses were observed.³ Phase II studies of SS1P will begin in 2007. In the current study, we showed that immunotoxin SS1P, specific for mesothelin, has a strong cytotoxic activity on NCI-H322M and EKVX cell lines with IC₅₀ values ranging from 2 to 5 ng/mL. This work suggests that SS1P also merits evaluation for the treatment of NSCLC.