Establishment and characterization of a cell line (NOMH-1) originating from a human endometrioid adenocarcinoma of the ovary

On June 30, 1993, we performed an abdominal simple total hysterectomy, bilateral salpingo-oophorectomy, and omentectomy on a 44-year-old woman with ovarian cancer FIGO stage IIc (Figure 1). The patient gave informed consent for performance of this study. Histology was performed on the resected tissue, and the tumor was diagnosed as an endometrioid adenocarcinoma of the ovary by intraoperative diagnosis of frozen section. Assessment of preoperative serum tumor marker levels showed that CA 125 was 639 U/ml (normal level <35 U/ml), CA 19–9 was 108 U/ml (<37 U/ml), and CEA was 2.4 ng/ml (<2.5 ng/ml). The patient was treated twice with 100 mg cisplatin (CDDP) and 400 mg etoposide (VP-16) intraperitoneally, and 5 times with 450 mg carboplatin (CBDCA), 60 mg pirarubicin hydrochloride (THP), and 400 mg cyclophosphamide (CPA) intravenously. However, a follow up examination on March 25, 1994 showed that her serum tumor markers were increased: CA 125 was 158 U/ml, CA 19–9 was 438 U/ml, and CEA was 6.3 ng/ml. Her clinical condition deteriorated progressively because of a recurrent abdominal tumor, and she died on October 11, 1994.

Tissue from the resected ovarian tumor was finely minced with a pair of sharp blades in a dish including serum-free Ham’s F-12 medium (Flow Laboratories Inc., McLean, VA), stirred slowly with a magnetic stirrer in a 0.25% trypsin solution (Flow Laboratories Inc.), centrifuged at 70 g for 5 min, and placed in culture medium at 37°C in humidified 5% CO₂ and 95% air. Cells were cultured in Ham’s F-12 medium plus 20% precolostrum newborn calf serum (Mitsubishi Chemical Industries Ltd., Tokyo, Japan) with kanamycin. Then, subcul-tures were passaged with 0.1% trypsin and 0.02% ethylenediamine-tetraacetic acid (EDTA) solution every 4 weeks. Six months after the primary culture, the concentration of precolostrum newborn calf serum in the culture medium was reduced from 20% to 10%.

Living cells grown in culture flasks were examined using a phase contrast microscope. For histology, the original tumor was fixed in 10% formalin, embedded in paraffin, and 4 μm-sections were stained with hematoxylin-eosin (HE). Monolayer cells cultured on slides were fixed in 90% ethanol and stained by Papanicolaou’s procedure [4]. For electron microscopy, the original tumor was fixed by immersion in a mixture of 1.25% glutaraldehyde and 1% paraformaldehyde buffered with phosphate saline, pH 7.4, at 4°C for 3 hr. After washing with phosphate buffered saline (PBS), the tumor was postfixed with 1% osmium tetroxide at 4°C for 1 hr, then washed in PBS, dehydrated in graded concentrations of ethanol, and embedded in Epon 812. Sections 0.5 μm thick were cut with a 6000 ultramicrotome (Sorvall, Du Pont, CT) and stained with toluidine blue. Ultrathin sections exhibiting light gold interference color were cut from the corresponding areas in the toluidine blue stained sections, double stained with uranyl acetate and lead citrate, and observed under a JEM-100SX electron microscope (JEOL, Tokyo, Japan) at 80 kV [5].

Cell characteristics were examined in passages 5–10. Suspensions of 1 × 10⁵ cells were plated in 35 mm plastic dishes and incubated for 44 days. Then, cells from two dishes were counted every other day by an automatic cell counter (Coulter Counter^R, Coulter Electronics, Luton, England). The population doubling time and saturation density were calculated from the growth curve. For studies of plating efficiency, 1×10² and 2×10² single-suspension cells were placed into five 60 mm plastic dishes each and cultured for 21 days. Plating efficiency was determined by the ratio of the number of colonies (more than 10 cells) to the total number of inoculated cells. For the mitotic index, monolayer cells were cultured for 5 days and treated with 1×10^-7 M colcemid (Demecolcine Solution, Wako Pure Chemical Industries, Osaka, Japan) for 4 hr, placed in a 0.2% KCl solution for 15 min, and then fixed step-by-step in a methanol: acetic acid solution (3:1). After air-drying, the cells were stained with Giemsa and the number of mitotic cells in 1,000 cells were counted.

Histograms of chromosome number distribution were determined using 39 metaphase plates. Their karyotypes were analyzed in 10 cells in accordance with the International System for Human Cytogenetic Nomenclature (ISCN 1995).

Ten million cells (passage 10) were inoculated subcutaneously into the dorsal region of 6-week-old nude mice (BALB/cAJcl-nu; Clea Japan, Inc., Tokyo, Japan). When the tumors had grown to 5–10 mm in diameter after 4 weeks, they were removed and processed for morphological examinations. For histology, the excised tumors were fixed in 10% formalin, embedded in paraffin, and stained with HE. For electron microscopy, the tumors were cut into pieces with a pair of sharp blades fixed, stained, and observed as described above.

Culture medium in which 2×10⁶ cells / 5 ml were cultured for 7 days was examined for α fetoprotein (AFP), CA 125, CA 19–9, CA 72–4, CEA, human chorionic gonadotropin (HCG), squamous cell carcinoma (SCC) antigen, and tissue polypeptide antigen (TPA) by radioimmunoassay or chemiluminescent immunoassay.

Deparaffinized sections of the original and heterotransplanted tumors on glass slides were immunohistochemically stained using the universal Immuno-enzyme Polymer (UIP) method (Envision kit; DAKO, Glostrup, Denmark). Slides were immersed in 0.03% hydrogen peroxidase and absolute methanol to block endogenous peroxidase, and then washed with phosphate buffered saline and heated in an autoclave for 20 min. After cooling, the slides were incubated with a primary antibody at room temperature for 40 min and then reacted with Envision (DAKO) at room temperature for 30 min, followed by incubation with diaminobenzidine (DAB) for 5–10 min. Antibodies against CA 125 (DAKO), CA 19–9 (DAKO), CEA (DAKO), and P53 (DAKO) were used to detect tumor markers, and against human estrogen receptor α (Nichirei Bioscience Inc., Tokyo, Japan) and human progesterone receptor (Nichirei) to detect hormone receptors.

The effects of actinomycin D (ACD, MSD KK, Tokyo, Japan), doxorubicin (ADM, Kyowa Hakko Kirin Co., Ltd., Tokyo, Japan), 5-fluorouracil (5-FU, Kyowa Hakko Kirin Co.), mitomycin C (MMC, Kyowa Hakko Kirin Co.), bleomycin (BLM, Nippon Kayaku Co., Ltd., Tokyo, Japan), vinblastine (VLB, Nippon Kayaku Co.), vincristine (VCR, Nippon Kayaku Co.), carboplatin (CBDCA, Bristol-Myers K.K., Tokyo, Japan), cisplatin (CDDP, Bristol-Myers), etoposide (VP-16, Bristol-Myers), paclitaxel (PTX, Bristol-Myers) [6], 4-hydroperoxy-cyclophosphamide (CPA, Shionogi & Co., Ltd., Osaka, Japan), irinotecan SN-38 (CPT-11, Yakult Honsha Co., Ltd., Tokyo, Japan) [7], and methotrexate (MTX, Pfizer Japan Inc., Tokyo, Japan), which are often used to treat gynecological cancers [8], on the cultured cells were investigated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay. The drugs were dissolved in culture medium and used immediately. For MTT assay, 5 x 10³ cells in 100 μl medium were seeded in tetraplicate into each well of 96-well microwell plates. For continuous drug exposure experiments, various diluted drugs in 50 μl were added after 48 hr of incubation. The wells were incubated for 72 hr after the addition of drugs. MTT (50 μl of 2 mg/ml; Wako Pure Chemical Industries, Ltd, Osaka, Japan) was added to each well, and the plates were incubated for 4 hr. The medium was then removed, 150 μl of dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO) was added to each well, and the plates were agitated for 5 min. The optical density was then read at 570 nm on a microplate reader (Bio-Rad Laboratories, Hercules, CA) and the effective concentration of the median lethal dose was calculated from the dose response curve (EC50: dose of drug required to reduce final cell number or optical density in MTT assay to 50% of control) [9].

Light microscopy revealed the original tumor to be an endometrioid adenocarcinoma, which showed tubular glands lined by stratified non-mucin containing epithelium (Figure 2).

Tissue fragments from the original tumor were cultured and after a 2-month stationary period, definite out-growths developed. Initially, contamination by spindle-shaped fibroblasts was observed, but they disappeared from the cultures upon passaging the cells, which were named NOMH-1. The NOMH-1 cells grew well, without interruption, for more than 232 months, and more than 50 serial passages were successively carried out. They continue to exhibit stable growth.

The cultured cells grew in monolayer and appeared to be epithelial, showing a pavement-like arrangement. Multilayering of cells was easily observed even before they reached confluency (Figure 3). The cells were polygonal and showed neoplastic features such as bizarre aggregation of chromatin granules, thickened nuclear membrane, and multiple large nucleoli. Multinucleated giant cells were also seen (Figure 4). Electron microscopy revealed that adjacent cells showed desmosome-like junctions (Figure 5a). The cells had indented nuclei (figure not shown), abundant mitochondria in the cytoplasm, and numerous microvilli on the cell surface (Figure 5b). These features suggested that the cells were epithelial in origin.

The growth curve was examined in passage 5 of the NOMH-1 cell line. Three days after culturing, the cells grew logarithmically (Figure 6). The population doubling time, saturation density, plating efficiency, and mitotic index were 273 hr, 3.7 × 10⁴ cells/cm², 3.6%, and 4.9%, respectively.

This cell line had a modal chromosome number that was in the hypertriploid range (73–80) (Figure 7). Chromosomal analysis showed that all of the cells had 6q- and most of the cells had 8p+ (Figure 8).

Histopathologically, the transplanted tumors were endometrioid adenocarcinomas that showed tubular glands lined by stratified non-mucin containing epithelium, which closely resembled the original tumor (Figure 9). Electron microscopy showed desmosome-like junctions and numerous microvilli as observed in the original tumor.

This cell line was positive for the following tumor markers: CEA, 1,000 ng/ml; CA 19–9, 305 U/ml; CA 72–4, 5.0 U/ml; and TPA, >1,500 U/L. On the other hand, they were negative for: AFP, <1 ng/ml; CA 125, 20 U/ml; HCG, <1 mIU/ml; and SCC antigen, <0.5 ng/ml. Expression of CEA and CA 19–9 was demonstrated immunohistochemically in the cancer cells of the original and heterotransplanted tumors (Figure 10). In contrast, CA125 and P53 were negative immunohistochemically.

Neither estrogen receptor (ER) nor progesterone receptor (PgR) were detected immunohistochemically in the original and heterotransplanted tumors.

The EC50 values of anti-cancer drugs for NOMH-1 cells are listed in Table 1 (Figure 11).