GYNECOLOGIC ONCOLOGY 38, 37-45
(1990)
Establishment and Characterization of a New Human Cell Line Derived from Ovarian Clear Cell Carcinoma W. S. FELIX WONG, Y. F. WONG, Y. T. ANGELA NG,* P.
DOLLY
HuANG,*
E.
C.
CHEw,t
T.
H.
Ho,?
AND M. Z. ALLAN CHANG Departments of Obstetrics and Gynaecology, * Morbid Anatomy, and tilnatomy, Chinese University Received
November
A new cell line, designatedOCCl, was establishedfrom the ascitic fluid of a patient with a clear cell carcinomaof the ovary. The cell line grew well without interruption for over 12 months and over 80 passages.The doubling times of OCCl were 36 and 38 hr at the 10th and 40th passages,respectively.Chromosomal analysisof the cell line showedhypertriploidy with modal number around 70-77. Several structural chromosomalabnormalities wereconsistentlyfound. Electron microscopyrevealedthat OCCl produceda basementmembrane-likestructure in vitro. Histological evaluation of xenografts from OCCl in the 33th passage implanted and grown in nude (athymic) mice revealeda morphologyidentical to that of the original tumor. o 1990 Academic PUSS, Inc.
INTRODUCTION
In recent years, a number of cell lines from various malignant ovarian tumors have been established. They provide investigators with opportunities to study their histogenesis, carcinogenesis [ 11, and cellular sensitivity to cytotoxic agents [2,3]. Furthermore, neoplastic cells in culture may also provide material for the production of tumor type-specific monoclonal antibodies [4-61 which would be useful in immunodiagnostic studies and immunotherapeutic manipulations. We report the establishment and characterization of a cell line (OCCl), which has retained its tumorigenic potential in vitro, obtained from the ascitic fluid of a patient with ovarian clear cell carcinoma. MATERIALS
AND METHODS
Materials. A 47-year-old woman underwent total abdominal hysterectomy and bilateral salpingo-oophorectomy for ovarian cyst on 18 January 1988. Histology of the ovarian cyst showed clear cell carcinoma. The tumor was composed of papillary or glandular structures con-
of Hong Kong, Shatin, N.T., Hong Kong
8, 1989
sisting of sheets of tumor cells with large pleomorphic nuclei and clear eosinophilic cytoplasm (Fig. 1). After operation the patient developed ascites rapidly before she received any chemotherapy. A sample of ascitic fluid was taken by abdominal paracentesis and subjected to primary culture on August 20, 1988. Culture technique and media. The ascitic fluid was centrifuged at 1OOgfor 5 min. The sediments were resuspended in the growth medium of RPM1 1640 (GIBCO Laboratories, Grand Island, NY) supplemented with 10% fetal bovine serum (Flow Laboratories, UK), 100 IU/ml penicillin, and 100 pg/ml streptomycin (Flow Laboratories, UK). Stationary cell cultures were set up in Corning flasks (25 cm*) and incubated in 5% CO2 and 95% air at 37°C. The initial two cell colonies contained both epithelial tumor cells and fibroblasts. Tumor cells were separated from fibroblasts by the double trypsinization technique with 0.05% trypsin and 0.02% EDTA (Flow Laboratories, UK) based on the observation that fibroblasts detach earlier from the flask after trypsinization. After four subcultures, fibroblasts was found to have completely disappeared from the tumor cell population in the culture. Morphology. The cultured cells grown on coverslips were fixed with 10% formalin and stained with hematoxylin and eosin, and the morphology was observed under light microscope. Another sample of tumor cells growing on collagen matrix in culture flasks was rinsed three times with phosphate-buffered saline at 37°C and fixed in situ with 2.5% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.4, at 4°C. They were then postfixed for 1 hr in 1% osmium tetroxide in the same buffer. For transmission electron microscopy (TEM), specimens were rinsed several times followed by routine procedures for dehydration and embedding in Spurr resin. Ultrathin sections were stained with uranyl acetate and lead citrate
37 0090-8258/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
38
WONG ET AL.
FIG. 1. Histophotographs of an ovarian clear cell carcinoma. The tumor is composed of papillary or glandular structures (a) and sheets of tumor cells (b) with large pleomorphic nuclei and clear or eosinophilic cytoplasm. H&E x 400.
and examined with a ieol IOOCXII electron microscope at 80 kV. For scanning electron microscopy @EM), the specimens were rinsed, dehydrated, critical point dried, coated with gold-paliadi~m, and examined with a Jeol JSM-35CF scanning electron microscope at 1.5kV. Growth characreristics. The growth curve, population doubling time, saturation density, and plating efficiency were studied at the 10th and 40th passages. Cells at a concentration of 5 x 104/ml were seeded into 35mm-
diameter petrie dishes and incubated at 37°C under humidified 5% CO2and 95% air. A cell count in three dishes was taken daily after trypsinization and the averagenumbers of cells were plotted to obtain the growth curve. The population doubling time and saturation density were determined by studying the growth curve. Plating efficiency was determined by plating 250 single suspended cells into a plastic 60-mm-diameter dish and culturing for 14days. The cell colonies formed were stained
NEW HUMAN CELL LINE FROM OVARIAN
with Giemsa. Plating efficiency was determined as the ratio of the number of visible colonies (more than 10 cells) to the number of inoculated cells. Slides were prepared by cytocentrifuge of OCCl tumor cells after harvest from the culture and were fixed in acetone before incubation with monoclonal antibody Ki-67 to study immunocytochemically the growth fraction of the cell line. Chromosome analysis. Cytogenetic study of the OCCl line was done on passage 3 1. Logarithmic-phase growing tumor cells, which had been maintained in RPMI-1640 medium with 10% fetal bovine serum, were fed fresh medium 24 hr before harvest. Colcemid at a final concentration of 0.05 pg/ml was used for metaphase arrest. Culture was trypsinized 5 hr after the addition of colcemid. The cell pellet was resuspended in 45 mM KCI at 37°C for a half-hour. After the hypotonic treatment, cells were fixed in acetic acid and methanol (1:3 v/v). Metaphase was spread by the dropping method on dry slides. Chromosomes were analyzed after the trypsin Giemsa banding technique. Heterotransplantation. Approximately 1.6 x IO’ cells (at passage 33) were transplanted into the flanks of Sweek-old female nude mice (BALB-Nu/Nu). The tumors were examined histologically by light microscopy 5 months after transplantation. CA-125 levels in culture medium. Approximately 5 x lo4 cells in triplicate at passage 20 were cultured for 2 days and CA-125 levels in the growth media was measured by a CA-125 radioimmunoassay (Abbott Laboratory, USA).
CLEAR CELL CARCINOMA
39
RESULTS Morphological Characteristics OCCI cells maintained on glass coverslips grew as a monolayer of closely opposed epithelial-like cells. The individual cells varied somewhat in size and shape. The nuclei, which occupied about 50% of the cell profile, were irregular with prominent nucleoli (Fig. 2). Under the transmission electron microscope, the tumor cells were observed to contain abundant polyribosomes and scanty free ribosomes. Isolated granular endoplasmic reticulum and mitochondria were also observed. The mitochondria appeared rather dense. Most of the tumor cells were shown to contain vacuoles of different size (Fig. 3). In tumor aggregates, glandular-like structures were often seen. Microvilli and tight junctions were also present (Fig. 4). When tumor cells were growing on collagen gel, a basement membrane was always sandwiched between tumor cells and the collagen matrix (Fig. 5). Under the scanning electron microscope, the OCCl tumor cells growing on collagen were observed to form a continuous sheet. Focal areas of cell accumumlation were always observed (Fig. 6). At a higher-power view, some of the tumor cells were shown to be covered with long and interwoven filapodia (Fig. 7). Growth Characteristics Analysis of the growth curve of the OCCI line (Fig. 8) shows that at the 10th and 40th passages, the popu-
FIG. 2. Monolayer cultured cells of the OCCl line at the 31th passage. Intracytoplasmic vacuoles, nuclei of varying sizes, and fine granular chromatin with distinct nucleoli can be observed. H&E, x400.
40
WONG ET AL.
FIG. 3. Part of an OCCl cell showing abundant polyribosomes and vacuoles (V) of different size in the cytoplasm.
lation doubling times are 38 and 36 hr, the plating efficiencies are 20 and 22%, and the saturation densities are 2.22 and 2.25 x lo6 tells/35-mm-diameter dish, respectively. The growth fraction of OCCl tumor cells in the logarithmic phase of growth, maintained in RPMI-1640 cmture medium and incubated in 5% CO;?at 37°C had been studied with monoclonal antibody Ki-67 [7]. This antibody labeled all cycling cells in the culture and produced
positive nuclear staining. The labeling index by Ki-67 is around 85-90% in Day 2 cultures. Chromosome Analysis Fifty metaphases were counted. Over 90% of the mitotic cells at passage 31 showed hypertriploidy. The in vitro modal number is around 70-77. On the basis of 30 metaphases analyzed, structural
FIG. 4. A glandular-like structure with a lumen (L) is often seen in tumor aggregates. Note that microvilli (arrow) and tight junctions (TJ are present.
NEW HUMAN CELL LINE FROM OVARIAN
FIG. 5. OCCl cells growing on collagen gel. A basement membrane-like
abnormalities of chromosomes 1, 7, 8, 9, 12, 16, and 17 were found to be consistent in all cells. A typical karyotype of this cell line (Fig. 9) is 74, XX, -1, +t(l;l4), +t(l;l4), +del(l), +del(l), +2, +3, +4, +5, +5, t-5, +6, +7, +7, +t(7;?), +8p+, -9, +de1(9), +de1(9), +lO, + 11, +12, +de1(12), +13, +15, +16p+, del(l7), +19, +20, +21, fmarl, + mar2.
CLEAR CELL CARCINOMA
structure
41
(arrow) is found between tumor cells and matrix.
somes have always been observed to appear in pairs, suggestive of a missing chromosome X. Nude Mouse Studies
After subcutaneous inoculation with OCCl cells, four of four mice in the series developed a solid tumor in the flank, with a latent period of 2 to 4 months. Tumors were described by light microscopy as clear cell carcinoma. The histopathology of these tumors resembles that of the
FIG. 6. Survey scanning electron micrograph of a sheet of OCCl cells growing on collagen gel. Note that cells are accumulating at different spots (A).
42
WONG ET AL.
FIG. 7. Higher-power view of OCCI cells growing on collagen gel. Note that some of the tumor cells possess long and interwoven fils (arrows).
original tumor from which the OCCl cell line was derived (Fig. 10). CA-125 Levels in the Culture Medium When medium from culture of OCCl cells was analyzed for CA-125 by radioimmunoassay, it was shown to contain 13 U of CA-125 per milliliter. Control medium, i.e., medium not exposed to OCCl cells, did not contain a detectable quantity of CA-125 (< 1 U/ml). Ill0
1
FIG. 8. The growth curve of the 10th (0) and 40th (0) cultures. Each point represents the mean value of triplicate cultures. The SD did not exceed 10%.
DISCUSSION About 40 established cell lines have been characterized from ovarian malignancies [81. Most of them were obtained from ascitic cells, while a minor percentage of the continuously growing ovarian tumor cell lines were derived from solid primary tumors. In our study we established a new cell line derived from an uncommon ovarian clear cell carcinoma. The establishment of the OCC 1 line is thus potentially useful for developing model systems to study this type of malignancy. It also provides the opportunity to investigate differences between other epithelial and metastatic ovarian cancers. The cytogenetic characterization of OCCl reveals that it is a hypertriploid cell line, with over 90% of cells showing triploid chromsomes. In addition, at least one specific marker chromosome is observed. The malignant character also emerges from its ability to grow stably in vitro for a long time. The development of xenografts in immunodeficient nude mice shows the continued tumorigenic potential of this cell line, and suggests that these tumor cells, which have maintained their malignant characteristics in vitro, are either unmodified or minimally modified descendents of the original ovarian clear cell carcinoma. The antigenic determinant CA-125 is common to many forms of ovarian cancer, and can be identified using the monoclonal antibody OC-125 [91. This antibody was obtained from mice immunized with the ovarian cystadenocarcinoma cell line OVCA433 [9,10] and stains almost all ovarian carcinoma tissues [51. The cells of the OCCl
NEW HUMAN CELL LINE FROM OVARIAN
CLEAR CELL CARCINOMA
43
1 13
19
mar 1
14
20
21
22
mar 2
FIG. 9. Typical karvotype of OCCI cell line after G-band in passage 31. +5, +5:‘+6, +71 ii, +t(7;?). +Sp+, -9, +del(9), +del(9): tI0. +21, +marl, +mar2.
+5,
line produced and shed CA-125 into the medium, thus supporting the ovarian carcinoma origin. A basement membrane-like structure was produced by OCCl in culture as revealed by the electron microscopy. Basement membranes are sheets of extracellular matrix found in every organ where parenchymal cells interface with interstitial connective tissue [l I]. They are also of particular importance in the development of epithelial malignancies since they are the first barrier encountered in the invasive process of carcinomas. Thus, attention has been focused on basement membranes for determining the biologic behavior of malignant tumors and in particular the progression from in situ lesion to invasive neoplasm [12]. An endodermal cell subline (PF HR-9) was established which is both tumorigenic and basement membrane forming [13]. Another example is the Engelbreth-Holm Swarm (EHS) sarcoma. This transplantable mouse tumor contains large amounts of basement membrane matrix [14]. In the study of the formation of base-
74, XX,
+ll,
-1,
+l2,
+t(l;lA), +t(l;ld), +del(t), +del(l), +2, +3, +4, +del(l2), +13, +l5, +16p+, del(l7). +l9, +20,
ment membrane by a malignant mouse keratinocyte cell line in organotypic culture and transplants, Hornung et al. [ 121found that irrespective of the amount of basement membrane components synthesized, none of the cell lines formed a structured basement membrane in organatypical cultures in vitro. To the best of our knowledge, the OCCl line established in the present study seems to be the only cell line established from an adenocarcinoma which produces a basement membrane-like structure in vitro. The biochemical nature of this structure is currently under investigation. Once the basement membrane has been characterized, it will be able to be produced in large quantity for the study of interactions between basement membrane and normal and tumor cells. The role of basement membrane in the growth of normal and tumor cells can also be explored. For developing immunodiagnostic techniques for ovarian carcinoma, and possible immunotherapeutic manipulations in other gynecologic malignancies, it is neces-
44
WONG ET AL.
FIG. 10. Histophotograph of sections of a tumor nodule obtained from an athymic nude mouse. Large nests and sheets of carcinoma cells with abundant vacuolated or clear cytoplasm are separated by delicate fibrous septa. H&E, x400.
sary to identify, isolate, and purify tumor-specific antigens. The tumor biopsy specimen or tumor removed at the operation is always contaminated by stromal cells, blood cells, and a variety of tissue antigens. However, tumor cell lines in culture like OCCl may provide an excellent source of pure tumor-specific antigens for the above-mentioned purposes. In summary, we have developed a stable cell line, OCC 1, from the ascitic fluid produced by an ovarian clear cell carcinoma. This cell line is able to produce CA-125 and to grow in nude mice, forming xenografts of an adenocarcinomatous character. ACKNOWLEDGMENTS We thank Miss Rita, Department of Anatomy, Chinese University of Hong Kong, for technical assistance with the electron microscopic study, and Mr. Stephen Ho, Department of Oncology, Prince of Wales Hospital, Hong Kong, for testing CA-125. We are also grateful to the Gynaecologic Cancer Research Laboratory, Chinese University of Hong Kong, for providing the culture reagents and equipment for development of this cell line.
REFERENCES 1. Filmus, .I., Trent, J. M., Pullano, R., and Buick, R. N. A cell line from a human ovarian carcinoma with amplification of the K-ras gene, Cancer Res. 46, 5179-5182 (1986). 2. Hill, B. T., Whelan, R. D. H., Gibby, E. M., Sheer, D., Hosking, L. K., Shellard, S. H., and Rupniak, H. T. Establishment and characterization of three new human ovarian carcinoma cell lines and initial evaluation of their potential in experimental chemotherapy studies, Znf. J. Cancer 39, 219-225 (1987).
3. Wolf, C. R., Hayward, I. P., Lawrie, S. S., Buckton, K., McIntyre, M. A., Adams, D. J., Ewis, A. D., Scott, A. R. R., and Smith, J. F. Cellular heterogeneity and drug resistance in two ovarian adenocarcinoma cell lines derived from a single patient, Znt. .Z. Cancer 139, 695-702 (1987). 4. Poels, L. G., Peters, D., van Megen, Y., Vooijs, G. P., Verheyen, R. N. M., Willemen, A.. van Niekerk, C. C., Jap, P. H. K., Mungyer, G., and Kenemans, P. Monoclonal antibodies against human ovarian tumour-associated antigens, .Z.Nat/. Cancer Inst. 76, 781791 (1986). 5. Kabawat, S. E., Bast, R. C., Welch, W. R., Knapp, R. C., and Calvin, R. B. Immunopathologic characterization of a monoclonal antibody that recognizes common surface antigens of human ovarian tumours of serous, endometrioid, and clear cell types, Amer. J. Clin. Puthol. 79, 98-104 (1983). 6. Miotti, S., Canevari, S., Menard, D. M., Mezzanzanica, D., Porro, G., Pupa, S. M., Regazzoni, M., Tagliabue, E., and Colnaghi, M. I. Characterization of human ovarian carcinoma-associated antigens defined by novel monoclonal antibodies with tumour-restricted specificity, ht. J. Cancer 39, 297-303 (1987). 7. Pileri, S., Gerdes, J., Rivano, M., Tazzari, P. L., and Magnani, M. Immunohistochemical determination of growth fractions in human permanent cell lines and lymphoid tumours: A critical comparison of the monoclonal antibodies OKT9 and Ki-67, Bit. J. Haematol. 65, 271-276 (1987). 8. Poels, L. G., Jap, P. H. K., Ramaekers, F. F. C. S., Scheres, C. M. G., Thomas C. M. G., Vooijs, P. G., Croes, H. J. E., and Mungyer, G. Characterization of a hormone-producing ovarian carcinoma cell line, Gynecol. Oncol. 32, 203-214 (1989). 9. Bast, R. C., Feeney, M., Iazarus, H., Nadler, L. M., Colvin, P. B., and Knapp, R. C. Reactivity of a monoclonal antibody with ovarian carcinoma, J. C/in. Invest. 68, 1331-1337 (1981). 10. Bast, R. C., Klug, T. L., John, E. T., Jenison, E., Niloff, J. M. L., Berkowitz, R. S., Leavitt, T., Griffiths, C. T., Parker, L., Zurawski, V. R., and Knapp, R. C. A radioimmunoassay using a
NEW HUMAN CELL LINE FROM OVARIAN monoclonal antibody to monitor the course of epithehal ovarian cancer, N. Engl. J. Med. 309, 883-887 (1983). 1I. Vracko, R. Anatomy of basal lamian scaffold and its role in maintenance of tissue stucture, in Biology and chemistry of basement membranes (N. A. Kefalides, Ed.), Academic Press, New York, pp. 165-176 (1978). 12. Hornung, J., Bohnert, A., Phan-Than, L., Krieg, T., and Fusenig, N. E. Basement membrane formation by malignant mouse kera-
CLEAR CELL CARCINOMA
45
tinocyte cell lines in organotypic culture and transplants: Correlation with degree of morphologic differentiation, J. Cancer Res. C/in. Oncol. 113, 325-341 (1987).
13. Engvall, E., and Rouslahti, E. Binding of soluble form of fibroblast surface proteins, fibronectin to collagen, Int. J. Cancer 20, I (1977). 14. Orkin, R. W., Gehron, P., McCoodwin, B., Martin, G. R., Valentine, T., and Swarm, R. A murine tumour producing a matrix or basement membrane, J. Exp. Med. 145, 204-220 (1977).
(1990)
Establishment and Characterization of a New Human Cell Line Derived from Ovarian Clear Cell Carcinoma W. S. FELIX WONG, Y. F. WONG, Y. T. ANGELA NG,* P.
DOLLY
HuANG,*
E.
C.
CHEw,t
T.
H.
Ho,?
AND M. Z. ALLAN CHANG Departments of Obstetrics and Gynaecology, * Morbid Anatomy, and tilnatomy, Chinese University Received
November
A new cell line, designatedOCCl, was establishedfrom the ascitic fluid of a patient with a clear cell carcinomaof the ovary. The cell line grew well without interruption for over 12 months and over 80 passages.The doubling times of OCCl were 36 and 38 hr at the 10th and 40th passages,respectively.Chromosomal analysisof the cell line showedhypertriploidy with modal number around 70-77. Several structural chromosomalabnormalities wereconsistentlyfound. Electron microscopyrevealedthat OCCl produceda basementmembrane-likestructure in vitro. Histological evaluation of xenografts from OCCl in the 33th passage implanted and grown in nude (athymic) mice revealeda morphologyidentical to that of the original tumor. o 1990 Academic PUSS, Inc.
INTRODUCTION
In recent years, a number of cell lines from various malignant ovarian tumors have been established. They provide investigators with opportunities to study their histogenesis, carcinogenesis [ 11, and cellular sensitivity to cytotoxic agents [2,3]. Furthermore, neoplastic cells in culture may also provide material for the production of tumor type-specific monoclonal antibodies [4-61 which would be useful in immunodiagnostic studies and immunotherapeutic manipulations. We report the establishment and characterization of a cell line (OCCl), which has retained its tumorigenic potential in vitro, obtained from the ascitic fluid of a patient with ovarian clear cell carcinoma. MATERIALS
AND METHODS
Materials. A 47-year-old woman underwent total abdominal hysterectomy and bilateral salpingo-oophorectomy for ovarian cyst on 18 January 1988. Histology of the ovarian cyst showed clear cell carcinoma. The tumor was composed of papillary or glandular structures con-
of Hong Kong, Shatin, N.T., Hong Kong
8, 1989
sisting of sheets of tumor cells with large pleomorphic nuclei and clear eosinophilic cytoplasm (Fig. 1). After operation the patient developed ascites rapidly before she received any chemotherapy. A sample of ascitic fluid was taken by abdominal paracentesis and subjected to primary culture on August 20, 1988. Culture technique and media. The ascitic fluid was centrifuged at 1OOgfor 5 min. The sediments were resuspended in the growth medium of RPM1 1640 (GIBCO Laboratories, Grand Island, NY) supplemented with 10% fetal bovine serum (Flow Laboratories, UK), 100 IU/ml penicillin, and 100 pg/ml streptomycin (Flow Laboratories, UK). Stationary cell cultures were set up in Corning flasks (25 cm*) and incubated in 5% CO2 and 95% air at 37°C. The initial two cell colonies contained both epithelial tumor cells and fibroblasts. Tumor cells were separated from fibroblasts by the double trypsinization technique with 0.05% trypsin and 0.02% EDTA (Flow Laboratories, UK) based on the observation that fibroblasts detach earlier from the flask after trypsinization. After four subcultures, fibroblasts was found to have completely disappeared from the tumor cell population in the culture. Morphology. The cultured cells grown on coverslips were fixed with 10% formalin and stained with hematoxylin and eosin, and the morphology was observed under light microscope. Another sample of tumor cells growing on collagen matrix in culture flasks was rinsed three times with phosphate-buffered saline at 37°C and fixed in situ with 2.5% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.4, at 4°C. They were then postfixed for 1 hr in 1% osmium tetroxide in the same buffer. For transmission electron microscopy (TEM), specimens were rinsed several times followed by routine procedures for dehydration and embedding in Spurr resin. Ultrathin sections were stained with uranyl acetate and lead citrate
37 0090-8258/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
38
WONG ET AL.
FIG. 1. Histophotographs of an ovarian clear cell carcinoma. The tumor is composed of papillary or glandular structures (a) and sheets of tumor cells (b) with large pleomorphic nuclei and clear or eosinophilic cytoplasm. H&E x 400.
and examined with a ieol IOOCXII electron microscope at 80 kV. For scanning electron microscopy @EM), the specimens were rinsed, dehydrated, critical point dried, coated with gold-paliadi~m, and examined with a Jeol JSM-35CF scanning electron microscope at 1.5kV. Growth characreristics. The growth curve, population doubling time, saturation density, and plating efficiency were studied at the 10th and 40th passages. Cells at a concentration of 5 x 104/ml were seeded into 35mm-
diameter petrie dishes and incubated at 37°C under humidified 5% CO2and 95% air. A cell count in three dishes was taken daily after trypsinization and the averagenumbers of cells were plotted to obtain the growth curve. The population doubling time and saturation density were determined by studying the growth curve. Plating efficiency was determined by plating 250 single suspended cells into a plastic 60-mm-diameter dish and culturing for 14days. The cell colonies formed were stained
NEW HUMAN CELL LINE FROM OVARIAN
with Giemsa. Plating efficiency was determined as the ratio of the number of visible colonies (more than 10 cells) to the number of inoculated cells. Slides were prepared by cytocentrifuge of OCCl tumor cells after harvest from the culture and were fixed in acetone before incubation with monoclonal antibody Ki-67 to study immunocytochemically the growth fraction of the cell line. Chromosome analysis. Cytogenetic study of the OCCl line was done on passage 3 1. Logarithmic-phase growing tumor cells, which had been maintained in RPMI-1640 medium with 10% fetal bovine serum, were fed fresh medium 24 hr before harvest. Colcemid at a final concentration of 0.05 pg/ml was used for metaphase arrest. Culture was trypsinized 5 hr after the addition of colcemid. The cell pellet was resuspended in 45 mM KCI at 37°C for a half-hour. After the hypotonic treatment, cells were fixed in acetic acid and methanol (1:3 v/v). Metaphase was spread by the dropping method on dry slides. Chromosomes were analyzed after the trypsin Giemsa banding technique. Heterotransplantation. Approximately 1.6 x IO’ cells (at passage 33) were transplanted into the flanks of Sweek-old female nude mice (BALB-Nu/Nu). The tumors were examined histologically by light microscopy 5 months after transplantation. CA-125 levels in culture medium. Approximately 5 x lo4 cells in triplicate at passage 20 were cultured for 2 days and CA-125 levels in the growth media was measured by a CA-125 radioimmunoassay (Abbott Laboratory, USA).
CLEAR CELL CARCINOMA
39
RESULTS Morphological Characteristics OCCI cells maintained on glass coverslips grew as a monolayer of closely opposed epithelial-like cells. The individual cells varied somewhat in size and shape. The nuclei, which occupied about 50% of the cell profile, were irregular with prominent nucleoli (Fig. 2). Under the transmission electron microscope, the tumor cells were observed to contain abundant polyribosomes and scanty free ribosomes. Isolated granular endoplasmic reticulum and mitochondria were also observed. The mitochondria appeared rather dense. Most of the tumor cells were shown to contain vacuoles of different size (Fig. 3). In tumor aggregates, glandular-like structures were often seen. Microvilli and tight junctions were also present (Fig. 4). When tumor cells were growing on collagen gel, a basement membrane was always sandwiched between tumor cells and the collagen matrix (Fig. 5). Under the scanning electron microscope, the OCCl tumor cells growing on collagen were observed to form a continuous sheet. Focal areas of cell accumumlation were always observed (Fig. 6). At a higher-power view, some of the tumor cells were shown to be covered with long and interwoven filapodia (Fig. 7). Growth Characteristics Analysis of the growth curve of the OCCI line (Fig. 8) shows that at the 10th and 40th passages, the popu-
FIG. 2. Monolayer cultured cells of the OCCl line at the 31th passage. Intracytoplasmic vacuoles, nuclei of varying sizes, and fine granular chromatin with distinct nucleoli can be observed. H&E, x400.
40
WONG ET AL.
FIG. 3. Part of an OCCl cell showing abundant polyribosomes and vacuoles (V) of different size in the cytoplasm.
lation doubling times are 38 and 36 hr, the plating efficiencies are 20 and 22%, and the saturation densities are 2.22 and 2.25 x lo6 tells/35-mm-diameter dish, respectively. The growth fraction of OCCl tumor cells in the logarithmic phase of growth, maintained in RPMI-1640 cmture medium and incubated in 5% CO;?at 37°C had been studied with monoclonal antibody Ki-67 [7]. This antibody labeled all cycling cells in the culture and produced
positive nuclear staining. The labeling index by Ki-67 is around 85-90% in Day 2 cultures. Chromosome Analysis Fifty metaphases were counted. Over 90% of the mitotic cells at passage 31 showed hypertriploidy. The in vitro modal number is around 70-77. On the basis of 30 metaphases analyzed, structural
FIG. 4. A glandular-like structure with a lumen (L) is often seen in tumor aggregates. Note that microvilli (arrow) and tight junctions (TJ are present.
NEW HUMAN CELL LINE FROM OVARIAN
FIG. 5. OCCl cells growing on collagen gel. A basement membrane-like
abnormalities of chromosomes 1, 7, 8, 9, 12, 16, and 17 were found to be consistent in all cells. A typical karyotype of this cell line (Fig. 9) is 74, XX, -1, +t(l;l4), +t(l;l4), +del(l), +del(l), +2, +3, +4, +5, +5, t-5, +6, +7, +7, +t(7;?), +8p+, -9, +de1(9), +de1(9), +lO, + 11, +12, +de1(12), +13, +15, +16p+, del(l7), +19, +20, +21, fmarl, + mar2.
CLEAR CELL CARCINOMA
structure
41
(arrow) is found between tumor cells and matrix.
somes have always been observed to appear in pairs, suggestive of a missing chromosome X. Nude Mouse Studies
After subcutaneous inoculation with OCCl cells, four of four mice in the series developed a solid tumor in the flank, with a latent period of 2 to 4 months. Tumors were described by light microscopy as clear cell carcinoma. The histopathology of these tumors resembles that of the
FIG. 6. Survey scanning electron micrograph of a sheet of OCCl cells growing on collagen gel. Note that cells are accumulating at different spots (A).
42
WONG ET AL.
FIG. 7. Higher-power view of OCCI cells growing on collagen gel. Note that some of the tumor cells possess long and interwoven fils (arrows).
original tumor from which the OCCl cell line was derived (Fig. 10). CA-125 Levels in the Culture Medium When medium from culture of OCCl cells was analyzed for CA-125 by radioimmunoassay, it was shown to contain 13 U of CA-125 per milliliter. Control medium, i.e., medium not exposed to OCCl cells, did not contain a detectable quantity of CA-125 (< 1 U/ml). Ill0
1
FIG. 8. The growth curve of the 10th (0) and 40th (0) cultures. Each point represents the mean value of triplicate cultures. The SD did not exceed 10%.
DISCUSSION About 40 established cell lines have been characterized from ovarian malignancies [81. Most of them were obtained from ascitic cells, while a minor percentage of the continuously growing ovarian tumor cell lines were derived from solid primary tumors. In our study we established a new cell line derived from an uncommon ovarian clear cell carcinoma. The establishment of the OCC 1 line is thus potentially useful for developing model systems to study this type of malignancy. It also provides the opportunity to investigate differences between other epithelial and metastatic ovarian cancers. The cytogenetic characterization of OCCl reveals that it is a hypertriploid cell line, with over 90% of cells showing triploid chromsomes. In addition, at least one specific marker chromosome is observed. The malignant character also emerges from its ability to grow stably in vitro for a long time. The development of xenografts in immunodeficient nude mice shows the continued tumorigenic potential of this cell line, and suggests that these tumor cells, which have maintained their malignant characteristics in vitro, are either unmodified or minimally modified descendents of the original ovarian clear cell carcinoma. The antigenic determinant CA-125 is common to many forms of ovarian cancer, and can be identified using the monoclonal antibody OC-125 [91. This antibody was obtained from mice immunized with the ovarian cystadenocarcinoma cell line OVCA433 [9,10] and stains almost all ovarian carcinoma tissues [51. The cells of the OCCl
NEW HUMAN CELL LINE FROM OVARIAN
CLEAR CELL CARCINOMA
43
1 13
19
mar 1
14
20
21
22
mar 2
FIG. 9. Typical karvotype of OCCI cell line after G-band in passage 31. +5, +5:‘+6, +71 ii, +t(7;?). +Sp+, -9, +del(9), +del(9): tI0. +21, +marl, +mar2.
+5,
line produced and shed CA-125 into the medium, thus supporting the ovarian carcinoma origin. A basement membrane-like structure was produced by OCCl in culture as revealed by the electron microscopy. Basement membranes are sheets of extracellular matrix found in every organ where parenchymal cells interface with interstitial connective tissue [l I]. They are also of particular importance in the development of epithelial malignancies since they are the first barrier encountered in the invasive process of carcinomas. Thus, attention has been focused on basement membranes for determining the biologic behavior of malignant tumors and in particular the progression from in situ lesion to invasive neoplasm [12]. An endodermal cell subline (PF HR-9) was established which is both tumorigenic and basement membrane forming [13]. Another example is the Engelbreth-Holm Swarm (EHS) sarcoma. This transplantable mouse tumor contains large amounts of basement membrane matrix [14]. In the study of the formation of base-
74, XX,
+ll,
-1,
+l2,
+t(l;lA), +t(l;ld), +del(t), +del(l), +2, +3, +4, +del(l2), +13, +l5, +16p+, del(l7). +l9, +20,
ment membrane by a malignant mouse keratinocyte cell line in organotypic culture and transplants, Hornung et al. [ 121found that irrespective of the amount of basement membrane components synthesized, none of the cell lines formed a structured basement membrane in organatypical cultures in vitro. To the best of our knowledge, the OCCl line established in the present study seems to be the only cell line established from an adenocarcinoma which produces a basement membrane-like structure in vitro. The biochemical nature of this structure is currently under investigation. Once the basement membrane has been characterized, it will be able to be produced in large quantity for the study of interactions between basement membrane and normal and tumor cells. The role of basement membrane in the growth of normal and tumor cells can also be explored. For developing immunodiagnostic techniques for ovarian carcinoma, and possible immunotherapeutic manipulations in other gynecologic malignancies, it is neces-
44
WONG ET AL.
FIG. 10. Histophotograph of sections of a tumor nodule obtained from an athymic nude mouse. Large nests and sheets of carcinoma cells with abundant vacuolated or clear cytoplasm are separated by delicate fibrous septa. H&E, x400.
sary to identify, isolate, and purify tumor-specific antigens. The tumor biopsy specimen or tumor removed at the operation is always contaminated by stromal cells, blood cells, and a variety of tissue antigens. However, tumor cell lines in culture like OCCl may provide an excellent source of pure tumor-specific antigens for the above-mentioned purposes. In summary, we have developed a stable cell line, OCC 1, from the ascitic fluid produced by an ovarian clear cell carcinoma. This cell line is able to produce CA-125 and to grow in nude mice, forming xenografts of an adenocarcinomatous character. ACKNOWLEDGMENTS We thank Miss Rita, Department of Anatomy, Chinese University of Hong Kong, for technical assistance with the electron microscopic study, and Mr. Stephen Ho, Department of Oncology, Prince of Wales Hospital, Hong Kong, for testing CA-125. We are also grateful to the Gynaecologic Cancer Research Laboratory, Chinese University of Hong Kong, for providing the culture reagents and equipment for development of this cell line.
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