Establishment and characterization of a cell line (HCH-1) originating from a human clear cell carcinoma of the ovary

We performed an abdominal simple total hysterectomy, bilateral salpingo-oophorectomy, and omentectomy on a 67-year-old woman with ovarian cancer FIGO stage IIc 19 years ago. The right ovarian tumor was cystic with some parts of solid lesions (Fig. 1). The histology was clear cell carcinoma of the ovary. Preoperative serum tumor markers were CA 125; 340.9 U/ml (normal level <35 U/ml), CA 19–9; 75.4 U/ml (<37 U/ml), α-feto-protein (AFP); 8.2 ng/ml (<20 ng/ml), carcinoembryonic antigen (CEA); 1.3 ng/ml (<5.0 ng/ml), and squamous cell carcinoma (SCC) antigen; 0.6 ng/ml (<1.5 ng/ml). The patient was treated with intraperitoneal 100 mg cisplatin and 400 mg etoposide twice, and intravenous 450 mg carboplatin, 60 mg pirarubicin hydrochloride, 400 mg cyclophosphamide four times. However her clinical condition deteriorated progressively because of the brain metastasis with increasing CA 125. Although the metastatic tumor was resected by surgery, showing adenocarcinoma, she died 23 months after the first surgery. Unfortunately we had not an opportunity to culture the metastatic tumor. The patient gave written informed consent before surgery according to institutional guideline.

The material from the 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, USA), 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 subcultures were made with 0.1 % trypsin and 0.02 % ethylenediamine-tetraacetic acid (EDTA) solution every 4 weeks. Three months after 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 light microscopy, 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 [6].

Cell characteristics were examined chronologically. Suspensions of 1×10⁵ cells each were placed on 35 mm plastic dishes. Cells were incubated for 15 days, and cells in two dishes were counted by an automatic cell counter (Coulter Counter^R, Coulter Electronics, Luton, England) every other day. 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 onto five-60 mm plastic dishes each and cultured for 14 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, the 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 h, 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 mitoses in 1000 cells were counted.

Histograms of chromosome number distribution were determined on 50 metaphase plates. Their karyotypes were analyzed in 9 cells in accordance with the International System for Human Cytogenetic Nomenclature (ISCN 1995).

Ten million cells (passage 10) were inoculated subcutaneously into the backs of 6-week-old SCID mice (SCID/Os; Shionogi, Osaka, Japan). When the tumors had grown to 5–10 mm in diameter after 4 weeks, they were removed and processed for morphological examinations. For light microscopy, the excised tumors were fixed in 10 % formalin, embedded in paraffin, and stained with HE. 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 h. After washing with phosphate buffered saline (PBS), the tumor was postfixed with 1 % osmium tetroxide at 4 °C for 1 h. The tumor was 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, USA) and stained with toluidine blue. Ultrathin sections exhibiting a light gold interference color were cut from appropriate areas chosen from 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 [7].

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

The deparaffinized sections of the original tumor and heterotransplanted tumor on glass slides were stained immunohistochemically using the universal Immuno-enzyme Polymer (UIP) method (HISTOFINE simple stain MAX PO; Nichirei Bioscience Inc., Tokyo, Japan). They were immersed in 0.03 % hydrogen peroxidase and absolute methanol to block endogenous peroxidase. The samples were then washed with phosphate buffered saline and heated in a microwave oven for 10 min at a power of 700 W. After cooling, the slides were incubated with a primary antibody at 4 °C for 12 h and then added and reacted with Histfine Simple stain MAX PO at room temperature for 30 min, followed by diaminobenzidine (DAB) for sections for 5–10 min. Antibodies used as tumor markers were CA 125 (DAKO, Glostrup, Denmark), CA 19–9 (DAKO) and, as hormone receptor markers, were anti-human estrogen receptor α (Nichirei) and anti-human progesterone receptor (Nichirei). Hepatocyte factor-1β (HNF-1β) [8] (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and Annexin IV [9] (Santa Cruz Biotech) which commonly express in ovarian clear cell carcinoma were also used.

A comparison was made between 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) [10], 4-hydroperoxy-cyclophosphamide (CPA, Shionogi & Co., Ltd., Osaka, Japan), irinotecan SN-38 (CPT-11, Yakult Honsha Co., Ltd., Tokyo, Japan) [11] and methotrexate (MTX, Pfizer Japan Inc., Tokyo, Japan) which are often used to treat gynecological cancers [12]. They were dissolved in culture medium and used immediately. For 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay, 96-well micro well plates were set up in quadruplicate, each well containing 5×10³ cells in 100 μl medium. For continuous drug exposure experiments, various diluted drugs in 50 μl were added after 48 h of incubation. The wells were incubated for 72 h after the addition of drugs. MTT (50 μl of 2 mg/ml; Wako Pure Chemical Industries) was added to each well, and the plates were incubated for 4 h. The medium was then removed, 150 μl of dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, USA) 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, USA) and the effective concentration for 50 % kill 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) [13].

DNA was extracted from cultured cells using DNA extraction Kit (PureLink™ Genomic DNA Mini Kit, Invitrogen, Life Technologies, Carlsbad, CA, USA). Somatic mutations (substitutions, insertions or deletions) were assessed using the Ion AmpliSeq™ Cancer Hotspot Panel v2, designed to amplify 207 amplicons covering ~2800 COSMIC mutations from the 50 most commonly reported oncogenes and tumor suppressor genes: ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53 and VHL (Ion Torrent, Life Technologies) [14, 15] (Additional file 1: Table S1).

Light microscopy revealed the original tumor to be clear cell carcinoma, with a clear cytoplasm (Fig. 2a) and a partial hobnail appearance (Fig. 2b).

After a 2-month stationary period, the fragments developed distinct out-growths. Initially, contamination of spindle shaped fibroblasts was observed, but they disappeared from the cultures upon passaging the cells. The HCH-1 cells grew well, without interruption, for more than 230 months, and more than 50 serial passages were successively carried out. They continue to exhibit stable growth.

The monolayer cultured cells appeared to be epithelial, showing a pavement-like arrangement. Multilayering was observed and confluency could not be achieved (Fig. 3). The cells were polygonal and showed such neoplastic features as bizarre aggregation of chromatin granules, thickened nuclear membrane, and multiple large nucleoli. Multinucleated giant cells were also seen (Fig. 4).

The growth curve was examined in passages 6 and 25 of the HCH-1 cell line. Three days after culturing, the cells grew logarithmically (Fig. 5). The population doubling time, the saturation density, the plating efficiency and the mitotic index were 66.4 h, 5.4×10⁴ cells/cm², 18.8 %, 2.7 % in the 6th generation and 48.7 h, 8.2×10⁴ cells/cm², 12.9 %, 8.5 % in the 25th generation respectively.

A modal chromosome number was in the hypodiploid range (39–44) (Fig. 6). Chromosomal analysis showed the following abnormalities; 40–42, X, der(X)t(X;1)(p22;q21-23) [7], −1 [9], −4 [9], −5 [7], −8 [3], −9 [6], −10 [3], −11 [9],-12 [3], add (12)(p13) [7], −13 [9], −15 [9], −16 [9], −17 [5], −18 [7], add (18)(q23) [3], +19 [2], −19 [4], +20 [3], −21 [4], −22 [3], +5-9mar (Fig. 7).

Histopathologically, the transplanted tumors were clear cell carcinoma i.e. with clear cytoplasm, closely resembling the original tumor (Fig. 8). Electron microscopy revealed that adjacent cells possessed desmosome-like junctions (Fig. 9a). The cells had indented nuclei and abundant mitochondria in the cytoplasm, and numerous microvilli on the surfaces (Fig. 9b). These features suggest that the cells were epithelial in origin.

Positive levels were observed for: CA 125, 1400 U/ml; CA 19–9, 355 U/ml; and TPA, >1500 U/L. Negative markers were: AFP, 1 ng/ml; CEA, 0.7 ng/ml; CA 72–4, 8.8 U/ml; HCG, <1 mIU/ml; and SCC antigen, <0.5 ng/ml.

CA 125, CA 19–9, HNF-1β, and Annexin IV were demonstrated immunohistochemically in cancer cells from the original tumor and heterotransplanted tumors (Figs. 10 and 11). Estrogen receptor and progesterone receptor were not detected using immunocytochemistry.

The EC50 values of anti-cancer drugs for HCH-1 cells are listed in Table 1 (Additional file 2: Figure S1).

The amount of 8.1 μg DNA was extracted from 2×10⁶ cells. No variant was found in hotspot locations of 50 cancer genes but 12 of called variants outside of hotspot locations were found (Table 2).