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Human Peritoneal Mesothelial Cells Are More Potent Than Ovarian Cancer Cells in Producing Tumor Marker CA-125
GYNECOLOGIC ONCOLOGY ARTICLE NO.
62, 384–389 (1996)
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Human Peritoneal Mesothelial Cells Are More Potent Than Ovarian Cancer Cells in Producing Tumor Marker CA-1251 ALAIN G. ZEIMET,*,2 CHRISTIAN MARTH,* FELIX A. OFFNER,† PETER OBRIST,† MICHAEL UHL-STEIDL,* HANS FEICHTINGER,† SYLVIA STADLMANN,† GU¨NTER DAXENBICHLER,* AND OTTO DAPUNT* *Department of Obstetrics and Gynecology, University Hospital, and †Department of Pathology, University of Innsbruck, Innsbruck, Austria Received January 31, 1996
The aim of the present study was to investigate the extent to which human peritoneal mesothelial cells (HPMCs) are able to participate in the release of tumor marker CA-125 in ovarian cancer and other conditions associated with an involvement of the peritoneum. For this purpose CA-125 shedding was measured in the supernatant culture medium of HPMCs obtained from various donors and seven well-established ovarian cancer cell lines (OVCAR-3, 2780, 2774, SKOV-6, SKOV-8, HOC-7, HTB-77). Furthermore, the influence of inflammatory cytokines [interleukin-1b (IL-1b), tumor necrosis factor a (TNF-a), interferon g (IFN-g)] on CA-125 release in normal and malignant cells was also studied. Constitutive CA-125 shedding was found to be about five times higher in HPMCs as compared with the investigated ovarian cancer cell lines. IL-1b and TNF-a treatment of HPMCs resulted in a significant reduction in CA-125 release; however, no consistent pattern in CA-125 secretion was found during incubation with either IL-1b or TNF-a in the various malignant cell lines. IFNg, on the other hand, induced a highly significant increase in CA125 secretion in ovarian cancer cells, but did not influence the shedding of CA-125 in HPMCs. q 1996 Academic Press, Inc.
INTRODUCTION
CA-125, the antigenic determinant defined by the murine monoclonal antibody OC-125 [1] is associated with highmolecular-weight glycoproteins [2]. This tumor-associated antigen is expressed by most epithelial ovarian cancers [1, 3] and endometrial adenocarcinomas [4]. Synthesis of CA125, however, is not restricted to malignant transformed cells. It is also expressed in benign ovarian tumors [5, 6], and in normal tissues such as the Fallopian tube, the endometrium [7], and the amniotic cells of the placenta and the umbilical cord [8]. The OC-125 determinant is also shed 1 This work was supported in part by the Fondation Lions ‘‘Vaincre le Cancer,’’ Luxembourg. 2 To whom requests for reprints should be addressed at the Department of Obstetrics and Gynecology, University Hospital, Anichstrasse 35, A6020 Innsbruck, Austria. Fax: (0512) 504-3055.
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MATERIAL AND METHODS
Isolation and Culture of Human Peritoneal Mesothelial Cells Human peritoneal mesothelial cells (HPMCs) were obtained from omental tissue of seven different consenting patients undergoing elective abdominal hysterectomy which was performed for severe dysplasia of the uterine cervix
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0090-8258/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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from the cell surface, and elevations of serum CA-125 levels are associated with untreated ovarian and advanced endometrial cancers [9, 10]. Over the last decade a growing body of literature has emphasized the role of CA-125 as the leading tumor marker in ovarian cancer because of its clinical value in preoperative diagnosis and monitoring of the disease [11, 12]. Modest elevations in CA-125 serum concentrations, however, are also observed in benign conditions like menstruation [7], endometriosis [6], pelvic inflammatory disease [13, 14], liver cirrhosis [15], and peritoneal trauma [16]. In addition, elevated CA-125 serum levels are commonly found in nonmalignant disorders associated with ascites and pleural effusions [17, 18]. Kabawat et al. demonstrated CA-125 immunohistochemically in mesothelial cells lining the adult pleura, pericardium, and peritoneum [19]. Expression of mesothelial CA-125 was found to be particularly high in areas of inflammation and adhesions. Since very high concentrations of tumor-associated antigen CA-125 are directly detectable in malignant as well as benign ascitic and pleural fluid [20], the aim of the present study was to assess the role of peritoneal mesothelial cells in CA-125 production. CA-125 release in primary cultured human peritoneal mesothelial monolayers was compared with CA-125 shedding in seven well-characterized ovarian cancer cell lines: OVCAR3, SKOV-6, SKOV-8, HOC-7, 2774, 2780, HTB-77. Moreover, the influence of the inflammatory cytokines, tumor necrosis factor a (TNF-a), interleukin-1b (IL-1b), and interferon-g (IFN-g), on cell growth and CA-125 release was investigated in human mesothelial and ovarian cancer cells.
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(n Å 1) or intramural fibroids not exceeding a diameter of 3 cm (n Å 6). None of the donors had CA-125 serum levels above the normal range at the time of surgery or had a history of previous conditions related to elevated CA-125 serum levels. Isolation of cells was carried out by a modified method previously described elsewhere [21]. Briefly, small biopsies (approximately 3 cm3) of omental tissue were rinsed in phosphate-buffered saline (PBS), transferred to a 0.05% solution of collagenase I (Worthington, Boehringer-Mannheim) and allowed to float for 1.5–2 hr at 377C. After removal of the fat tissue, the solution containing mesothelial cells was filtered through a 200-mesh steel sieve. Thereafter, cells were collected by centrifugation (10 min, 400g), suspended in Eagle’s Minimum Essential Medium (EMEM, Gibco, Paisley Scotland, Great Britain), supplemented with 10% fetal calf serum, penicillin/streptomycin (100 IU/ml, 100 mg/ml, Seromed), and grown to confluence in 25-cm2 culture flasks (Falcon, Oxnard, CA) in a humidified atmosphere of 10% CO2 in air at 377C. Cells used for experiments were either passage 2 or 3. Characterization of Mesothelial Cells HPMCs were identified as being mesothelial in origin by their uniform cobblestone appearance after confluence, by the presence of desmosomes and surface microvilli, and by their coexpression of vimentin and the cytokeratin types 8 and 18 [22]. Culture purity was confirmed by flow cytometry using an indirect immunofluorescence method [23]. Cells were detached with collagenase I/Versene (0.05% w/v, 0.02% w/v in PBS), washed twice in PBS, suspended at a concentration of 0.5 1 106/ml, and incubated with monoclonal antibodies (MAbs) against CD34 and CD68 (Becton & Dickinson). MAb CAM 5.2 (Becton & Dickinson) and MAb V9 (Dakopatts, Glostrup, Denmark) were used for detection of cytokeratins 8 and 18 and vimentin, respectively. For this purpose cells were fixed in ethanol and washed three times in PBS before application of the primary MAbs at appropriate dilutions. After rinsing in PBS an FITC-conjugated (Fab)2 fragment (goat anti-mouse, Dakopatts) was used as secondary reagent. Omission of primary MAb was used as control. Samples of 10,000 cells were analyzed using a fluorescence-activated cell sorter (FACScan, Becton & Dickinson) at a wavelength of 488 nm. Repeated analyses of different passage 2 cultures did not reveal any cells positive for CD68 or CD34, thus excluding the presence of contaminating macrophages or endothelial cells. In all samples analyzed mesothelial identity was confirmed by uniform positive staining of the cells for cytokeratins 8 and 18 as well as vimentin. Ovarian Carcinoma Cell Lines The established human ovarian carcinoma cell lines HTB 77, SKOV-6, SKOV-8, OVCAR-3, 2774, 2780, and HOC-
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7 were kindly provided by Dr. T. Hamilton, NIH, Bethesda, Dr. G. Gastl, Memorial Sloan Kettering Cancer Center, New York, and Dr. C. Dittrich, Vienna, Austria. They were all maintained in 75-cm2 culture flasks (Falcon) in the same medium as the mesothelial cells (see above). Modulation of CA-125 Secretion in HPMC and Ovarian Cancer Cells For experiments, HPMCs and ovarian cancer cells were first detached with trypsin/Versene (0.05%/0.02%) and then washed once in culture medium. Each treatment or control group consisted of 12 independently grown cultures of every ovarian cancer cell line or HPMC specimen. For this purpose, cells were seeded into 24-well tissue culture dishes in a volume of 1 ml medium. HPMCs were used at a concentration of 1 1 105 cells/ml, ovarian cancer cell lines at a concentration of 2.5 to 4 1 104 cells/ml. Twenty-four hours after seeding, exponentially growing cells were washed twice with fresh medium and subsequently incubated for 48 hr in the absence or presence of the following cytokines: recombinant IL-1b (provided by Dr. P. Lomedico, Hoffman LaRoche, Nutley, NJ), human recombinant TNF-a and recombinant IFN-g (kindly provided by Dr. G. Adolf, E. Boehringer Institute, Vienna, Austria), each at a final concentration of 10 ng/mL. At the end of the incubation period supernatants were removed, centrifuged, and stored at 0707C until assayed. The remaining cells were detached enzymatically and the cell number of the samples determined using a Coulter Counter (Luton, U.K.). Incubation of the cells with cytokines for up to 48 hr did not have any significant effect on cell viability as shown by trypan blue exclusion. Cell numbers were calculated as a percentage of the respective values under control conditions. Values were plotted relative to the control values, which were set at 100%. Measurement of CA-125 CA-125 concentrations obtained under the various experimental conditions were determined in the cell culture supernatants by sandwich solid-phase radioimmunoassay (Centocor, Malvern, PA). All CA-125 measurements were done in duplicate and performed according to the instructions of the manufacturer. Briefly, polystyrene beads coated with mouse monoclonal anti-CA 125 (OC 125) were incubated with the samples along with 125I-labeled mouse monoclonal anti-CA 125. After incubation for 20 hr at room temperature, the beads were washed three times with 5 ml distilled water and counted in a gamma scintillation counter. The interassay and intraassay coefficients of variation were 5.3 and 4.2%, respectively. The lower limit of detection as defined by 2 SD of the zero standards was 1.2 U/ml supernatant culture medium. The highest standard was 500 U/ml. All data for secreted CA-125 are expressed as arbitrary units related to 1 1 105 cells (U/105 cells).
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TABLE 1 CA-125 Secretion in Human Peritoneal Mesothelial Cellsa Control HPMC-10 HPMC-20 HPMC-21 HPMC-27 HPMC-28 HPMC-31 HPMC-32
54 64 65 10 13 39 15
[45; 72] [51; 69] [49; 72] [8; 26] [11; 29] [30; 52] [12; 26]
Median valued
39 [17; 65]
IL-1b (10 ng/ml)
TNF-a (10 ng/ml)
IFN-g (10 ng/ml)
37 43 26 1.5 3.5 16 5
[22; 49] [31; 61] [12; 33]b [1.2; 2.5]c [2; 8.5]b [3.5; 23]b [1.2; 10]b
28 42 36 1.5 5 20 7
[12; 36]b [29; 56] [19; 42]b [1.2; 2.5]c [1.2; 7]b [17; 29] [1.2; 10]b
56 55 62 7 4 32 12
16
[4; 33]
20
[5; 37]
32 [12; 55]
[40; 66] [43; 65] [55; 72] [4; 19] [1.2; 8]b [25; 40] [10; 18]
a
Median values (U/105 cells) with their respective first and third quartiles [Q1 ; Q3] obtained from 12 wells examined P õ 0.05 vs control. c P õ 0.01 vs control. d Median values (U/105 cells) with their respective first and third quartiles [Q1 ; Q3] of all HPMC samples evaluated (n Å 84/treatment group). b
Statistical Analyses Data were analyzed by nonparametric procedures. A Mann– Whitney U test was used to analyze differences between measured values of CA-125 for ovarian cancer cell lines and HPMCs. Cytokine-treated cultures were compared with control cultures using the Wilcoxon signed rank test. Correlation analyses were performed by the Spearman rank test. Probability values less than 0.05 were considered significant. RESULTS
Constitutive CA-125 synthesis and shedding were demonstrable in HPMC monolayer cultures of all seven donors investigated. Median values obtained from CA-125 concentrations measured in 12 supernatants of each of the seven primary HPMC cultures ranged from 10 to 65 U/105 cells. The median concentration of all HPMCs investigated for CA-125 release was 39 U/105 cells (Table 1). With the exception of the ovarian cancer cell line 2774, a constitutive CA-125 synthesis was found in all other cell lines studied. While unstimulated HTB 77 cells secreted only traces of measurable CA-125 (2 U/105 cells), highest CA125 shedding was found in the supernatant of untreated OVCAR-3 cells (13 U/105 cells). The median value from the six CA-125-producing ovarian cancer cell lines was 8.5 U/105 cells (Table 2). Constitutive CA-125 secretion was found to be significantly higher in exponentially growing HPMCs as compared with the various ovarian cancer cell lines cultured under the same conditions (P õ 0.01). As shown in Fig. 1, IFN-g treatment resulted in significant (P õ 0.05) suppression of the proliferation of both HPMCs [median inhibition, 20%; (Q1 , 16%; Q3 , 31%); range, 5–47%] and the ovarian cancer cell lines [median inhibition, 63%; (Q1 , 46%; Q3 , 89%); range, 30–94%]. In HPMCs treated with IFN-g, either no change in CA-125 shedding or a significant reduction in
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CA-125 release into the supernatant culture medium was observed (Table 1, Fig. 2). This was contrasted by an IFNg-induced increase in CA-125 release in the ovarian cancer cell lines (Fig. 3). As listed in Table 2, this increase in antigen shedding was statistically significant in cell lines HOC-7 and HTB-77 (P õ 0.01), OVCAR-3, and SKOV-6 (P õ 0.05). The most prominent inductive effect was observed in the HTB-77 cell line, with a more than 30-fold increase in CA-125 shedding. IL-1b had no significant effect on the cell growth rate of either the various ovarian cancer cell lines or the HPMCs (Fig. 1). While CA-125 release was significantly (P õ 0.05) reduced in HPMCs (median value, 16 U/105 cells) by IL-1b treatment (Fig. 2), no consistent pattern of CA-125 shedding could be pointed out in the malignant cell lines investigated (Fig. 3). As listed in Table 2, CA-125 release was either enhanced (OVCAR-3), reduced (SKOV-6, SKOV-8 and HOC-7), or found not to be influenced (2780, HTB-77) by IL-1b. TNF-a proved to increase growth in HPMCs [median stimulation, 15%; (Q1 , 12%; Q3 , 22%); range, 7–59%], but did not affect proliferation of any of the ovarian cancer cell lines studied (Fig. 1). While in HPMCs, TNF-a treatment inhibited CA-125 shedding into the culture medium (median concentration of 20 U/105 cells, P õ 0.05), antigen release was either not influenced (SKOV-8, HOC-7, HTB-77), increased (OVCAR-3, 2780) or even reduced (SKOV-6) in malignant cell lines (Table 2, Fig. 3). In cell line 2774 neither IFN-g, IL-1b, nor TNF-a showed an inductive effect on CA-125 release, which would have resulted in antigen detection by the antibody OC-125 in our standard procedures. Finally, no relationship between CA125 release and proliferation rate was evidenced either in HPMCs or in ovarian cancer cells in the present study. DISCUSSION
In epithelial ovarian cancer, the clinical utility of serum marker CA-125 in preoperative diagnosis and monitoring of
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TABLE 2 CA-125 Secretion in Human Ovarian Cancer Cell Linesa Control OVCAR-3 2780 HTB-77 SKOV-6 SKOV-8 HOC-7
13 5 2 12 9 8
Median valued
[8; 15.5] [1.2; 8] [1.2; 7] [6; 14] [4; 12] [6; 19]
8.5 [5; 12]
IL-1b (10 ng/ml) 20.5 6 2.5 6 5 3.5
TNF-a (10 ng/ml)
[18; 23]b [3; 11] [2; 8.5] [2.5; 8.5]b [3; 12] [1.2; 6]b
5.5 [3; 8.5]
IFN-g (10 ng/ml)
36 11 6 6 11 10.5
[32; 45]c [9.5; 11]b [3.5; 12] [4; 9]b [6; 15] [7.5; 15]
25 7 75 19 13 92
[20; 27]b [4; 11] [62; 102]c [15; 28]b [5; 17] [78; 105]c
11
[4.5; 13]
22 [16; 72]
a
Median values (U/105 cells) with their respective first and third quartiles [Q1 ; Q3] obtained from 12 wells examined. P õ 0.05 vs control. c P õ 0.01 vs control. d Median values (U/105 cells) with their respective first and third quartiles [Q1 ; Q3] of all ovarian cancer cell samples evaluated (n Å 60/treatment group). b
the disease has been extensively documented in recent years. Elevations in CA-125 serum levels were, however, also observed in benign disease, especially in conditions associated with pleural or peritoneal involvement. Abundant amounts of the tumor marker CA-125 were also directly detected in malignant as well as benign ascitic and pleural fluids [24]. Hunter et al. were unable to demonstrate a correlation between serum and peritoneal CA-125 levels in patients with benign gynecologic conditions or ovarian cancer [20]. Conflicting, moreover, were the reports dealing with the relation-
FIG. 1. Proliferation of ovarian cancer cells and human peritoneal mesothelial cells (HPMC) under cytokine treatment. Data are expressed as a percentage of the cell number obtained in controls; solid lines and bars represent median values and interquartile ranges, respectively.
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ship between CA-125 serum levels and the actual tumor load. While some investigators have shown that CA-125 serum levels are closely related to tumor mass [12, 25, 26], others have found these levels to be more strongly associated with the presence of ascites than with the surgically proven tumor load [21]. The data of Buller et al. indicate that paracentesis may result in a substantial decrease in CA-125 serum content, thus mimicking surgical debulking [24]. The present in vitro study provides evidence of a constitutive and abundant synthesis and shedding of the CA-125 antigen by HPMCs. This is in agreement with the observations recently reported by others [27, 28]. Constitutive CA-125 secretion by HPMCs was found to be about fivefold that determined in the supernatant culture medium of various ovarian cancer cell lines grown under the same conditions. To our knowledge, no direct comparison between healthy mesothelial cells and ovarian cancer cells addresssing the potency of CA-125 synthesis has been undertaken so far. We con-
FIG. 2.
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CA-125 secretion by human peritoneal mesothelial cells.
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FIG. 3.
CA-125 secretion by ovarian cancer cells.
fined our investigation to measuring the fraction of shedded CA-125, accumulated over 48 hr in the supernatant culture medium, because in previous studies we learned that CA125 turnover is very rapid and that within 24 hr the total amount produced in each cell was released into the culture medium [29]. Our data suggest that mesothelial cells are a major source of elevated circulating CA-125 levels in benign disease associated with peritoneal damage. Moreover, it seems justified to speculate that peritoneal mesothelial cells also contribute significantly to CA-125 release into the ascitic fluid and the serum in ovarian cancer patients. To what extent these cells are involved in the origin of physiological CA-125 serum levels and the reported cyclic changes in CA-125 serum levels in normally menstruating women [7] remains to be established. When monolayer cultures were treated with inflammatory cytokines the most prominent differences between HPMCs and malignant cells with respect to CA-125 shedding were observed after treatment with IFN-g. In HPMCs, CA-125 shedding either was not affected by IFN-g or showed a significant decrease, whereas CA-125-producing ovarian cancer cells unanimously showed an increase in CA-125 release. In HTB-77 cells a similar induction of CA-125 release by IFN-g had already been observed in earlier investigations [30]. Given that these differences between normal and malignant cells can be confirmed in studies including freshly isolated ovarian carcinoma cells, tumor marker CA125 can be considered a further suitable parameter in an IFN-g-based dynamic in vivo test, with the aim of predicting subclinical disease status before second-look procedures in ovarian cancer. Such a dynamic stimulatory test was recently proposed by Scambia et al. for antigen 90K, another tumorassociated protein [31]. In contrast, TNF-a and IL-1b treatment of the various ovarian cancer cell lines did not result in a coherent pattern for CA-125 expression.
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In HPMCs, treatment with TNF-a and IL-1b resulted in a significant reduction of CA-125 shedding into the culture medium. These results, however, are in disagreement with those recently reported by Zeillemaker et al., who found on the contrary that IL-1b and TNF-a enhance CA-125 secretion in HPMCs [27]. This imparity may be due to the fact that these authors determined CA-125 shedding after a duration of cytokine treatment as short as 6 hr. As the induction of CA-125 is, however, dependent on intact protein biosynthesis [29] and as the cellular turnover of antigen CA-125 was found to take at least 24 hr [30], it seemed more appropriate to choose a treatment time of 48 hr to study specific modulatory effects of cytokines on CA-125 synthesis and shedding. In accordance with the results of Zeillemaker et al. [27] it may be possible that IL-1b and TNF-a treatment leads to early depletion of cellular CA-125, followed by prolonged inhibition of CA-125 synthesis. Further in vitro studies are needed to elucidate the time-dependent effects had by inflammatory cytokines alone or in various combinations on the government of CA-125 expression. The results of the present study clearly indicate the important role of HPMCs in CA-125 shedding as well as in benign and malignant disease. This fact should be included in diagnostic considerations and in the exact interpretation of elevations of circulating levels of this tumor marker. ACKNOWLEDGMENTS The authors thank Mrs. Elisabeth Perkmann and Mrs. Julia Ro¨ssler for their excellent technical assistance.
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Human Peritoneal Mesothelial Cells Are More Potent Than Ovarian Cancer Cells in Producing Tumor Marker CA-1251 ALAIN G. ZEIMET,*,2 CHRISTIAN MARTH,* FELIX A. OFFNER,† PETER OBRIST,† MICHAEL UHL-STEIDL,* HANS FEICHTINGER,† SYLVIA STADLMANN,† GU¨NTER DAXENBICHLER,* AND OTTO DAPUNT* *Department of Obstetrics and Gynecology, University Hospital, and †Department of Pathology, University of Innsbruck, Innsbruck, Austria Received January 31, 1996
The aim of the present study was to investigate the extent to which human peritoneal mesothelial cells (HPMCs) are able to participate in the release of tumor marker CA-125 in ovarian cancer and other conditions associated with an involvement of the peritoneum. For this purpose CA-125 shedding was measured in the supernatant culture medium of HPMCs obtained from various donors and seven well-established ovarian cancer cell lines (OVCAR-3, 2780, 2774, SKOV-6, SKOV-8, HOC-7, HTB-77). Furthermore, the influence of inflammatory cytokines [interleukin-1b (IL-1b), tumor necrosis factor a (TNF-a), interferon g (IFN-g)] on CA-125 release in normal and malignant cells was also studied. Constitutive CA-125 shedding was found to be about five times higher in HPMCs as compared with the investigated ovarian cancer cell lines. IL-1b and TNF-a treatment of HPMCs resulted in a significant reduction in CA-125 release; however, no consistent pattern in CA-125 secretion was found during incubation with either IL-1b or TNF-a in the various malignant cell lines. IFNg, on the other hand, induced a highly significant increase in CA125 secretion in ovarian cancer cells, but did not influence the shedding of CA-125 in HPMCs. q 1996 Academic Press, Inc.
INTRODUCTION
CA-125, the antigenic determinant defined by the murine monoclonal antibody OC-125 [1] is associated with highmolecular-weight glycoproteins [2]. This tumor-associated antigen is expressed by most epithelial ovarian cancers [1, 3] and endometrial adenocarcinomas [4]. Synthesis of CA125, however, is not restricted to malignant transformed cells. It is also expressed in benign ovarian tumors [5, 6], and in normal tissues such as the Fallopian tube, the endometrium [7], and the amniotic cells of the placenta and the umbilical cord [8]. The OC-125 determinant is also shed 1 This work was supported in part by the Fondation Lions ‘‘Vaincre le Cancer,’’ Luxembourg. 2 To whom requests for reprints should be addressed at the Department of Obstetrics and Gynecology, University Hospital, Anichstrasse 35, A6020 Innsbruck, Austria. Fax: (0512) 504-3055.
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MATERIAL AND METHODS
Isolation and Culture of Human Peritoneal Mesothelial Cells Human peritoneal mesothelial cells (HPMCs) were obtained from omental tissue of seven different consenting patients undergoing elective abdominal hysterectomy which was performed for severe dysplasia of the uterine cervix
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from the cell surface, and elevations of serum CA-125 levels are associated with untreated ovarian and advanced endometrial cancers [9, 10]. Over the last decade a growing body of literature has emphasized the role of CA-125 as the leading tumor marker in ovarian cancer because of its clinical value in preoperative diagnosis and monitoring of the disease [11, 12]. Modest elevations in CA-125 serum concentrations, however, are also observed in benign conditions like menstruation [7], endometriosis [6], pelvic inflammatory disease [13, 14], liver cirrhosis [15], and peritoneal trauma [16]. In addition, elevated CA-125 serum levels are commonly found in nonmalignant disorders associated with ascites and pleural effusions [17, 18]. Kabawat et al. demonstrated CA-125 immunohistochemically in mesothelial cells lining the adult pleura, pericardium, and peritoneum [19]. Expression of mesothelial CA-125 was found to be particularly high in areas of inflammation and adhesions. Since very high concentrations of tumor-associated antigen CA-125 are directly detectable in malignant as well as benign ascitic and pleural fluid [20], the aim of the present study was to assess the role of peritoneal mesothelial cells in CA-125 production. CA-125 release in primary cultured human peritoneal mesothelial monolayers was compared with CA-125 shedding in seven well-characterized ovarian cancer cell lines: OVCAR3, SKOV-6, SKOV-8, HOC-7, 2774, 2780, HTB-77. Moreover, the influence of the inflammatory cytokines, tumor necrosis factor a (TNF-a), interleukin-1b (IL-1b), and interferon-g (IFN-g), on cell growth and CA-125 release was investigated in human mesothelial and ovarian cancer cells.
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(n Å 1) or intramural fibroids not exceeding a diameter of 3 cm (n Å 6). None of the donors had CA-125 serum levels above the normal range at the time of surgery or had a history of previous conditions related to elevated CA-125 serum levels. Isolation of cells was carried out by a modified method previously described elsewhere [21]. Briefly, small biopsies (approximately 3 cm3) of omental tissue were rinsed in phosphate-buffered saline (PBS), transferred to a 0.05% solution of collagenase I (Worthington, Boehringer-Mannheim) and allowed to float for 1.5–2 hr at 377C. After removal of the fat tissue, the solution containing mesothelial cells was filtered through a 200-mesh steel sieve. Thereafter, cells were collected by centrifugation (10 min, 400g), suspended in Eagle’s Minimum Essential Medium (EMEM, Gibco, Paisley Scotland, Great Britain), supplemented with 10% fetal calf serum, penicillin/streptomycin (100 IU/ml, 100 mg/ml, Seromed), and grown to confluence in 25-cm2 culture flasks (Falcon, Oxnard, CA) in a humidified atmosphere of 10% CO2 in air at 377C. Cells used for experiments were either passage 2 or 3. Characterization of Mesothelial Cells HPMCs were identified as being mesothelial in origin by their uniform cobblestone appearance after confluence, by the presence of desmosomes and surface microvilli, and by their coexpression of vimentin and the cytokeratin types 8 and 18 [22]. Culture purity was confirmed by flow cytometry using an indirect immunofluorescence method [23]. Cells were detached with collagenase I/Versene (0.05% w/v, 0.02% w/v in PBS), washed twice in PBS, suspended at a concentration of 0.5 1 106/ml, and incubated with monoclonal antibodies (MAbs) against CD34 and CD68 (Becton & Dickinson). MAb CAM 5.2 (Becton & Dickinson) and MAb V9 (Dakopatts, Glostrup, Denmark) were used for detection of cytokeratins 8 and 18 and vimentin, respectively. For this purpose cells were fixed in ethanol and washed three times in PBS before application of the primary MAbs at appropriate dilutions. After rinsing in PBS an FITC-conjugated (Fab)2 fragment (goat anti-mouse, Dakopatts) was used as secondary reagent. Omission of primary MAb was used as control. Samples of 10,000 cells were analyzed using a fluorescence-activated cell sorter (FACScan, Becton & Dickinson) at a wavelength of 488 nm. Repeated analyses of different passage 2 cultures did not reveal any cells positive for CD68 or CD34, thus excluding the presence of contaminating macrophages or endothelial cells. In all samples analyzed mesothelial identity was confirmed by uniform positive staining of the cells for cytokeratins 8 and 18 as well as vimentin. Ovarian Carcinoma Cell Lines The established human ovarian carcinoma cell lines HTB 77, SKOV-6, SKOV-8, OVCAR-3, 2774, 2780, and HOC-
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7 were kindly provided by Dr. T. Hamilton, NIH, Bethesda, Dr. G. Gastl, Memorial Sloan Kettering Cancer Center, New York, and Dr. C. Dittrich, Vienna, Austria. They were all maintained in 75-cm2 culture flasks (Falcon) in the same medium as the mesothelial cells (see above). Modulation of CA-125 Secretion in HPMC and Ovarian Cancer Cells For experiments, HPMCs and ovarian cancer cells were first detached with trypsin/Versene (0.05%/0.02%) and then washed once in culture medium. Each treatment or control group consisted of 12 independently grown cultures of every ovarian cancer cell line or HPMC specimen. For this purpose, cells were seeded into 24-well tissue culture dishes in a volume of 1 ml medium. HPMCs were used at a concentration of 1 1 105 cells/ml, ovarian cancer cell lines at a concentration of 2.5 to 4 1 104 cells/ml. Twenty-four hours after seeding, exponentially growing cells were washed twice with fresh medium and subsequently incubated for 48 hr in the absence or presence of the following cytokines: recombinant IL-1b (provided by Dr. P. Lomedico, Hoffman LaRoche, Nutley, NJ), human recombinant TNF-a and recombinant IFN-g (kindly provided by Dr. G. Adolf, E. Boehringer Institute, Vienna, Austria), each at a final concentration of 10 ng/mL. At the end of the incubation period supernatants were removed, centrifuged, and stored at 0707C until assayed. The remaining cells were detached enzymatically and the cell number of the samples determined using a Coulter Counter (Luton, U.K.). Incubation of the cells with cytokines for up to 48 hr did not have any significant effect on cell viability as shown by trypan blue exclusion. Cell numbers were calculated as a percentage of the respective values under control conditions. Values were plotted relative to the control values, which were set at 100%. Measurement of CA-125 CA-125 concentrations obtained under the various experimental conditions were determined in the cell culture supernatants by sandwich solid-phase radioimmunoassay (Centocor, Malvern, PA). All CA-125 measurements were done in duplicate and performed according to the instructions of the manufacturer. Briefly, polystyrene beads coated with mouse monoclonal anti-CA 125 (OC 125) were incubated with the samples along with 125I-labeled mouse monoclonal anti-CA 125. After incubation for 20 hr at room temperature, the beads were washed three times with 5 ml distilled water and counted in a gamma scintillation counter. The interassay and intraassay coefficients of variation were 5.3 and 4.2%, respectively. The lower limit of detection as defined by 2 SD of the zero standards was 1.2 U/ml supernatant culture medium. The highest standard was 500 U/ml. All data for secreted CA-125 are expressed as arbitrary units related to 1 1 105 cells (U/105 cells).
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TABLE 1 CA-125 Secretion in Human Peritoneal Mesothelial Cellsa Control HPMC-10 HPMC-20 HPMC-21 HPMC-27 HPMC-28 HPMC-31 HPMC-32
54 64 65 10 13 39 15
[45; 72] [51; 69] [49; 72] [8; 26] [11; 29] [30; 52] [12; 26]
Median valued
39 [17; 65]
IL-1b (10 ng/ml)
TNF-a (10 ng/ml)
IFN-g (10 ng/ml)
37 43 26 1.5 3.5 16 5
[22; 49] [31; 61] [12; 33]b [1.2; 2.5]c [2; 8.5]b [3.5; 23]b [1.2; 10]b
28 42 36 1.5 5 20 7
[12; 36]b [29; 56] [19; 42]b [1.2; 2.5]c [1.2; 7]b [17; 29] [1.2; 10]b
56 55 62 7 4 32 12
16
[4; 33]
20
[5; 37]
32 [12; 55]
[40; 66] [43; 65] [55; 72] [4; 19] [1.2; 8]b [25; 40] [10; 18]
a
Median values (U/105 cells) with their respective first and third quartiles [Q1 ; Q3] obtained from 12 wells examined P õ 0.05 vs control. c P õ 0.01 vs control. d Median values (U/105 cells) with their respective first and third quartiles [Q1 ; Q3] of all HPMC samples evaluated (n Å 84/treatment group). b
Statistical Analyses Data were analyzed by nonparametric procedures. A Mann– Whitney U test was used to analyze differences between measured values of CA-125 for ovarian cancer cell lines and HPMCs. Cytokine-treated cultures were compared with control cultures using the Wilcoxon signed rank test. Correlation analyses were performed by the Spearman rank test. Probability values less than 0.05 were considered significant. RESULTS
Constitutive CA-125 synthesis and shedding were demonstrable in HPMC monolayer cultures of all seven donors investigated. Median values obtained from CA-125 concentrations measured in 12 supernatants of each of the seven primary HPMC cultures ranged from 10 to 65 U/105 cells. The median concentration of all HPMCs investigated for CA-125 release was 39 U/105 cells (Table 1). With the exception of the ovarian cancer cell line 2774, a constitutive CA-125 synthesis was found in all other cell lines studied. While unstimulated HTB 77 cells secreted only traces of measurable CA-125 (2 U/105 cells), highest CA125 shedding was found in the supernatant of untreated OVCAR-3 cells (13 U/105 cells). The median value from the six CA-125-producing ovarian cancer cell lines was 8.5 U/105 cells (Table 2). Constitutive CA-125 secretion was found to be significantly higher in exponentially growing HPMCs as compared with the various ovarian cancer cell lines cultured under the same conditions (P õ 0.01). As shown in Fig. 1, IFN-g treatment resulted in significant (P õ 0.05) suppression of the proliferation of both HPMCs [median inhibition, 20%; (Q1 , 16%; Q3 , 31%); range, 5–47%] and the ovarian cancer cell lines [median inhibition, 63%; (Q1 , 46%; Q3 , 89%); range, 30–94%]. In HPMCs treated with IFN-g, either no change in CA-125 shedding or a significant reduction in
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CA-125 release into the supernatant culture medium was observed (Table 1, Fig. 2). This was contrasted by an IFNg-induced increase in CA-125 release in the ovarian cancer cell lines (Fig. 3). As listed in Table 2, this increase in antigen shedding was statistically significant in cell lines HOC-7 and HTB-77 (P õ 0.01), OVCAR-3, and SKOV-6 (P õ 0.05). The most prominent inductive effect was observed in the HTB-77 cell line, with a more than 30-fold increase in CA-125 shedding. IL-1b had no significant effect on the cell growth rate of either the various ovarian cancer cell lines or the HPMCs (Fig. 1). While CA-125 release was significantly (P õ 0.05) reduced in HPMCs (median value, 16 U/105 cells) by IL-1b treatment (Fig. 2), no consistent pattern of CA-125 shedding could be pointed out in the malignant cell lines investigated (Fig. 3). As listed in Table 2, CA-125 release was either enhanced (OVCAR-3), reduced (SKOV-6, SKOV-8 and HOC-7), or found not to be influenced (2780, HTB-77) by IL-1b. TNF-a proved to increase growth in HPMCs [median stimulation, 15%; (Q1 , 12%; Q3 , 22%); range, 7–59%], but did not affect proliferation of any of the ovarian cancer cell lines studied (Fig. 1). While in HPMCs, TNF-a treatment inhibited CA-125 shedding into the culture medium (median concentration of 20 U/105 cells, P õ 0.05), antigen release was either not influenced (SKOV-8, HOC-7, HTB-77), increased (OVCAR-3, 2780) or even reduced (SKOV-6) in malignant cell lines (Table 2, Fig. 3). In cell line 2774 neither IFN-g, IL-1b, nor TNF-a showed an inductive effect on CA-125 release, which would have resulted in antigen detection by the antibody OC-125 in our standard procedures. Finally, no relationship between CA125 release and proliferation rate was evidenced either in HPMCs or in ovarian cancer cells in the present study. DISCUSSION
In epithelial ovarian cancer, the clinical utility of serum marker CA-125 in preoperative diagnosis and monitoring of
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TABLE 2 CA-125 Secretion in Human Ovarian Cancer Cell Linesa Control OVCAR-3 2780 HTB-77 SKOV-6 SKOV-8 HOC-7
13 5 2 12 9 8
Median valued
[8; 15.5] [1.2; 8] [1.2; 7] [6; 14] [4; 12] [6; 19]
8.5 [5; 12]
IL-1b (10 ng/ml) 20.5 6 2.5 6 5 3.5
TNF-a (10 ng/ml)
[18; 23]b [3; 11] [2; 8.5] [2.5; 8.5]b [3; 12] [1.2; 6]b
5.5 [3; 8.5]
IFN-g (10 ng/ml)
36 11 6 6 11 10.5
[32; 45]c [9.5; 11]b [3.5; 12] [4; 9]b [6; 15] [7.5; 15]
25 7 75 19 13 92
[20; 27]b [4; 11] [62; 102]c [15; 28]b [5; 17] [78; 105]c
11
[4.5; 13]
22 [16; 72]
a
Median values (U/105 cells) with their respective first and third quartiles [Q1 ; Q3] obtained from 12 wells examined. P õ 0.05 vs control. c P õ 0.01 vs control. d Median values (U/105 cells) with their respective first and third quartiles [Q1 ; Q3] of all ovarian cancer cell samples evaluated (n Å 60/treatment group). b
the disease has been extensively documented in recent years. Elevations in CA-125 serum levels were, however, also observed in benign disease, especially in conditions associated with pleural or peritoneal involvement. Abundant amounts of the tumor marker CA-125 were also directly detected in malignant as well as benign ascitic and pleural fluids [24]. Hunter et al. were unable to demonstrate a correlation between serum and peritoneal CA-125 levels in patients with benign gynecologic conditions or ovarian cancer [20]. Conflicting, moreover, were the reports dealing with the relation-
FIG. 1. Proliferation of ovarian cancer cells and human peritoneal mesothelial cells (HPMC) under cytokine treatment. Data are expressed as a percentage of the cell number obtained in controls; solid lines and bars represent median values and interquartile ranges, respectively.
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ship between CA-125 serum levels and the actual tumor load. While some investigators have shown that CA-125 serum levels are closely related to tumor mass [12, 25, 26], others have found these levels to be more strongly associated with the presence of ascites than with the surgically proven tumor load [21]. The data of Buller et al. indicate that paracentesis may result in a substantial decrease in CA-125 serum content, thus mimicking surgical debulking [24]. The present in vitro study provides evidence of a constitutive and abundant synthesis and shedding of the CA-125 antigen by HPMCs. This is in agreement with the observations recently reported by others [27, 28]. Constitutive CA-125 secretion by HPMCs was found to be about fivefold that determined in the supernatant culture medium of various ovarian cancer cell lines grown under the same conditions. To our knowledge, no direct comparison between healthy mesothelial cells and ovarian cancer cells addresssing the potency of CA-125 synthesis has been undertaken so far. We con-
FIG. 2.
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CA-125 secretion by human peritoneal mesothelial cells.
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FIG. 3.
CA-125 secretion by ovarian cancer cells.
fined our investigation to measuring the fraction of shedded CA-125, accumulated over 48 hr in the supernatant culture medium, because in previous studies we learned that CA125 turnover is very rapid and that within 24 hr the total amount produced in each cell was released into the culture medium [29]. Our data suggest that mesothelial cells are a major source of elevated circulating CA-125 levels in benign disease associated with peritoneal damage. Moreover, it seems justified to speculate that peritoneal mesothelial cells also contribute significantly to CA-125 release into the ascitic fluid and the serum in ovarian cancer patients. To what extent these cells are involved in the origin of physiological CA-125 serum levels and the reported cyclic changes in CA-125 serum levels in normally menstruating women [7] remains to be established. When monolayer cultures were treated with inflammatory cytokines the most prominent differences between HPMCs and malignant cells with respect to CA-125 shedding were observed after treatment with IFN-g. In HPMCs, CA-125 shedding either was not affected by IFN-g or showed a significant decrease, whereas CA-125-producing ovarian cancer cells unanimously showed an increase in CA-125 release. In HTB-77 cells a similar induction of CA-125 release by IFN-g had already been observed in earlier investigations [30]. Given that these differences between normal and malignant cells can be confirmed in studies including freshly isolated ovarian carcinoma cells, tumor marker CA125 can be considered a further suitable parameter in an IFN-g-based dynamic in vivo test, with the aim of predicting subclinical disease status before second-look procedures in ovarian cancer. Such a dynamic stimulatory test was recently proposed by Scambia et al. for antigen 90K, another tumorassociated protein [31]. In contrast, TNF-a and IL-1b treatment of the various ovarian cancer cell lines did not result in a coherent pattern for CA-125 expression.
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In HPMCs, treatment with TNF-a and IL-1b resulted in a significant reduction of CA-125 shedding into the culture medium. These results, however, are in disagreement with those recently reported by Zeillemaker et al., who found on the contrary that IL-1b and TNF-a enhance CA-125 secretion in HPMCs [27]. This imparity may be due to the fact that these authors determined CA-125 shedding after a duration of cytokine treatment as short as 6 hr. As the induction of CA-125 is, however, dependent on intact protein biosynthesis [29] and as the cellular turnover of antigen CA-125 was found to take at least 24 hr [30], it seemed more appropriate to choose a treatment time of 48 hr to study specific modulatory effects of cytokines on CA-125 synthesis and shedding. In accordance with the results of Zeillemaker et al. [27] it may be possible that IL-1b and TNF-a treatment leads to early depletion of cellular CA-125, followed by prolonged inhibition of CA-125 synthesis. Further in vitro studies are needed to elucidate the time-dependent effects had by inflammatory cytokines alone or in various combinations on the government of CA-125 expression. The results of the present study clearly indicate the important role of HPMCs in CA-125 shedding as well as in benign and malignant disease. This fact should be included in diagnostic considerations and in the exact interpretation of elevations of circulating levels of this tumor marker. ACKNOWLEDGMENTS The authors thank Mrs. Elisabeth Perkmann and Mrs. Julia Ro¨ssler for their excellent technical assistance.
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