Human ovarian surface epithelium in primary culture

Summary The ovarian surface epithelium (OSE) represents a minute fraction of the cell mass of the ovary but gives rise to over 80% of human ovarian carcinomas. No experimental models for the study of human OSE exist. To characterize OSE cells in culture, explants of ovarian surface from normal ovary of premenopausal women were grown on plastic, glass, and collagen gel in 25% fetal bovine serum/Waymouth's medium 752/1. About 25% of explants produced epithelial outgrowths. Morphologically, these outgrowths resembled OSE in vivo and endothelial and mesothelial cells in culture, but they differed from cultured ovarian stromal, granulosa, and luteal cells. Only OSE among ovarian cell types were intensely keratin positive by immunofluorescence. Keratin also distinguished OSE cells from the keratin-negative endothelial cells. Most but not all OSE colonies tested showed 17β-hydroxysteroid dehydrogenase (HSD) activity, which was absent in peritoneal mesothelial cells. Colonies from most patients were limited to a few millimetres and became stationary within a few weeks. Changes that accompanied cessation of growth included senescence, increased keratin content, or the formation of multicellular papillary aggregates. With time, OSE cells tended to assume a fibroblast-like morphology but remained keratin positive and continued to resemble OSE by scanning electron microscopy (SEM). Subcultured OSE cells persisted in a stationary keratin-positive form for many weeks. Throughout this study, all pavementlike epithelial outgrowths that were contiguous with an explant stained for keratin; thus, such colonies can be assumed to be OSE. Conversely, fibroblast-shaped cells may represent OSE as indicated by keratin content and SEM appearance. The methods presented here permit culture of normal human OSE under conditions in which the cells exhibit morphologic plasticity, variable 17β-HSD activity, and presence of keratin. Supported by a grant and a research associateship to N. A. by the National Cancer Institute of Canada.     

Serial propagation of human ovarian surface epithelium in tissue culture

Most human ovarian cancers are thought to arise in the ovarian surface epithelium (OSE). The precise role of OSE in carcinogenesis has not been defined because no appropriate animal models for the study of this tissue exist and culture of human OSE has been limited to primary outgrowths. In this report, we describe conditions for serial cultivation of normal human OSE. Premenopausal ovarian tissue was obtained at surgery. OSE growth was compared in media MCDB 202, 199 and Waymouth's 752/1 (WM) supplemented with 5, 15, or 25% fetal bovine serum (FBS), with/without 20 ng/ml epidermal growth factor (EGF) and 0.4 micrograms/ml hydrocortisone (HC). The rate and extent of OSE outgrowths from explants in primary culture were greatest in either WM or 199/202 (1:1), supplemented with 15% FBS/EGF/HC. In early passage cultures, cell proliferation was most rapid and extensive in 199/202 with 15% FBS, EGF, and HC. In this medium, OSE cells were subcultured up to 10 times and underwent 20-25 population doublings over 5 weeks. The population doubling time during rapid growth was approximately 48 h. Seeding efficiencies of up to 53% and cloning efficiencies of up to 13% were obtained. Early passage OSE cells reversibly modulated from a slow growing, epithelial, intensely keratin-positive form in 199/202 medium lacking EGF/HC, to a rapidly proliferating, elongate, less keratin-positive form in medium with EGF/HC. OSE cells grown in WM/5-15% FBS were epithelial and near-stationary. Thus, culture conditions have been defined for ovarian carcinogen assays requiring either proliferating or stationary cell populations, and for further studies of the role of OSE in ovarian biology.     

Differentiation and growth potential of human ovarian surface epithelial cells expressing temperature-sensitive SV40 T antigen

The epithelial ovarian carcinomas arise in the ovarian surface epithelium (OSE) which is the mesothelial covering of the ovary. Studies of human USE have been hampered by the small amounts and limited lifespan of this epithelium in culture. OSE cells expressing SV40 large T antigen (Tag) or the HPV genes E6 and E7 have increased growth potentials but lack some of the normal characteristics of OSE. In this study, we used conditional SV40 Tag expression to produce OSE cells with increased proliferative potentials but relatively normal phenotypes. Primary OSE cultures from three women, one of whom had a BRCA1 mutation, were infected with a temperature-sensitive Tag construct (tsTag), and from these, 28 monoclonal and four polyclonal lines were isolated. The effects of temperature changes were examined in two monoclonal and two polyclonal lines. At the permissive temperature (34 degrees C), these cell lines underwent 52-71 population doublings (PD) compared to 15-20 PD for normal OSE. Nuclear SV40-Tag and p53 expression, demonstrated by immunofluorescence, showed that tsTag was uniformly present and biologically active in all lines. At 34 degrees C, culture morphologies ranged from epithelial to mesenchymal. The mean percentage of cells expressing the epithelial differentiation marker, keratin. varied between lines from 20 to 97%. Collagen type III, a mesenchymal marker expressed by OSE in response to explantation into culture, was present in 24-43% of cells. At 39 degrees C, tsTag was inactivated by 2 d while nuclear p53 staining diminished to control levels over 2 wk. Over 3 d. the cells assumed more epithelial morphologies, keratin expression reached 85-100% in all lines and collagen expression increased significantly in two lines. The cultures with the BRCA1 mutation expressed the most keratin and the least collage n III at both temperatures. As indicated by beta-galactosidase staining at pH 6.0, changes leading to senescence were initiated at 39 degrees C by 6 h and were present in all cells after 24 h. However, the cells underwent 1-3 population doublings over up to 1 wk before growth arrest and widespread cell death, thus providing an experimental system where large numbers of OSE cells with different genetic backgrounds and growth potentials can be studied without the concurrent influence of Tag.     

Formation and barrier function of tight junctions in human ovarian surface epithelium

The normal ovarian surface epithelium (OSE) is a primitive epithelium made up by a single layer of mesothelial-type epithelial cells. When these cells get trapped in the ovarian stroma, expression of epithelial specific markers, such as E-cadherin, are induced. Most epithelial cells are also characterized by the ability to form tight junctions (TJ). Incomplete TJ have earlier been demonstrated in the OSE by electron microscopy studies. We have investigated expression and localization of the TJ proteins ZO-1, occludin, and claudin-1 in tissue biopsies from normal human ovaries and OSE in culture. The dynamics of TJ formation were studied in human OSE cultured on porous filters in culture inserts by measuring trans epithelial resistance (TER) including Ca(2+) switch experiments. Confluent OSE cells were also analyzed by electron microscopy. The results show that normal human OSE has expression of all three TJ proteins investigated. These proteins, ZO-1, occludin, and claudin-1, were localized to OSE cell borders both in ovarian biopsies and in cultured OSE. There was no difference in this regard between fertile and postmenopausal women. Cells in culture were polarized and presented junctional complexes seen by electron microscopy. In the Ca(2+) switch experiments, removing free Ca(2+) transiently, TER decreased significantly (P < 0.05) in the Ca(2+)-free group compared with nontreated OSE. TER was fully restored after 24 h. N-cadherin but not E-cadherin was expressed in the OSE and localized to the cell borders. We conclude that normal human OSE express and form functional TJ both in vivo and vitro. This report also describes a method to study the influence of ovarian-derived mediators on TJ in cultured OSE.     

Conditionally immortal ovarian cell lines for investigating the influence of ovarian stroma on the estrogen sensitivity and tumorigenicity of ovarian surface epithelial cells

The tendency of the ovarian surface epithelium (OSE) to undergo metaplastic and morphogenetic changes during the life cycle, at variance with the adjacent peritoneal mesothelial cells, suggests that its biology may be regulated by underlying ovarian stromal cues. However, little is known about the role that the ovarian stroma plays in the pathobiology of the OSE, largely because of the lack of a suitable in vitro model. Here, we describe the establishment and characterization of conditionally immortalized ovarian stromal and surface epithelial cell lines from H-2K(b)-tsA58 transgenic mice that carry the thermolabile mutant of SV-40 large T antigen under the control of an interferon-gamma (IFN-gamma)-inducible promoter. These cells express functional T antigens, grow continuously under permissive conditions at 33 degrees C in the presence of IFN-gamma, and stop dividing when the activity and expression of the tumor antigen is suppressed by restrictive conditions without IFN-gamma at 39 degrees C. Morphological, immunohistochemical, and ultrastructural analyses show that conditionally immortal OSE cells form cobblestone-like monolayers, express cytokeratin and vimentin, contain several microvilli, and develop tight junctions, whereas stromal cells are spindle-like, express vimentin but not cytokeratin, and contain rare microvilli, thus exhibiting epithelial and stromal phenotypes, respectively. At variance with the reported behavior of rat epithelial cells, conditionally immortal mouse epithelial cells are not spontaneously transformed after continuous culture in vitro. More importantly, conditioned media from stromal cells cultured under permissive conditions increase the specific activity of the endogenous estrogen receptor in BG-1 human ovarian epithelial cancer cells and promote these cells' anchorage-independent growth, suggesting the paracrine influence of a stromal factor. In addition, stromal cells cultured under restrictive conditions retain this growth-stimulatory activity, which, therefore, appears to be independent of T antigen expression. These established cell lines should provide a useful in vitro model system for studying the role of cellular interactions in OSE cell growth and tumorigenesis.     

Expression and function of HOXA genes in normal and neoplastic ovarian epithelial cells

We studied the roles of three HOXA genes in cultured normal ovarian surface epithelial (OSE) cells and ovarian cancer cells. They included HOXA4 and HOXA7 because, by cDNA microarray analysis, these were more highly expressed in invasive ovarian carcinomas than in benign or borderline (noninvasive) ovarian tumors, and HOXA9 because it characterizes normal oviductal epithelium, which resembles ovarian serous adenocarcinomas. The three HOXA genes were more highly expressed when OSE cells were dividing and motile than when they were confluent and stationary, and also when they dispersed in response to EGF treatment or to reduced calcium concentrations in culture media. The expression of the HOXA genes varied among ovarian cancer cell lines, but was highest in lines with compact epithelial morphologies. We focused on HOXA4 as the most highly expressed in the ovarian carcinoma array. HOXA4 expression did not parallel proliferative activities of either OSE or ovarian cancer lines. Moreover, modifying HOXA4 expression in ovarian cancer cell lines did not alter either E-cadherin expression or CA125 secretion. However, HOXA4 downregulation enhanced EGFR phosphorylation and migration in serum-starved OSE and ovarian cancer cells in response to EGF, and enhanced migration of all ovarian cancer lines in 5% serum even without EGF treatment. Thus, HOXA4 expression does not correlate with proliferation or with epithelial differentiation, but it increases in response to OSE cell dispersion and negatively regulates EGFR activation and the motility of OSE and of ovarian cancer cells. HOXA4 expression was highest in cancer lines with compact epithelial growth patterns, suggesting, again, an anti-dispersion function. In summary, increased HOXA4 expression in ovarian cancer appears to constitute a tumor-suppressive, homeostatic response to aberrant cell behavior, and, in particular, to cell dispersion and migration.     

Ovarian surface epithelium: autonomous production of connective tissue-type extracellular matrix.

The ovarian surface epithelium (OSE) is known to contribute to postovulatory repair of the ovarian cortex by proliferation and migration over the site of follicular rupture, and by deposition of a basement membrane. We examined the production of other extracellular matrix components in culture by OSE cells of the rat (ROSE), using immunofluorescence microscopy, electron microscopy, and proline incorporation. We compared recently explanted cells in low passage, the immortal line ROSE 239, whose growth pattern resembles low passage cultures, and the immortal line ROSE 199, which forms ridges and papillae. The epithelial nature of all three cell types was confirmed by the presence of keratin and laminin. All three cell types secreted collagen types I and III and at least one (ROSE 199) produced highly polymerized banded fibrils, which are characteristic for stromal or interstitial extracellular matrix. Simultaneous production of collagen types I and III, keratin, and laminin by cloned subpopulations ruled out an origin of the lines in mixed epithelial/fibroblast populations. The results demonstrate that OSE has the capacity to synthesize major components of connective tissue stroma. They suggest that this epithelium, in addition to its postulated proteolytic role, may also express synthetic activity in the remodelling of the ovarian cortical stroma. A capacity of OSE cells to produce stromal components autonomously might be an important factor in the formation of ovarian surface papillae and in neoplastic progression of OSE-derived ovarian carcinomas.     

Isolation and characterization of rabbit ovarian surface epithelium,granulosa cells,and peritoneal mesothelium in primary culture

Summary Mammalian ovarian surface epithelial (OSE) cells and peritoneal mesothelial (PM) cells have a common embryologic origin, yet certain morphologic and histochemical characteristics are different in the adult. In this study, a two-step culture method was developed to examine the characteristics of these two cell types in vitro. OSE, PM, and ovarian granulosa (GC) cells were isolated from estrous rabbits and cultured for 6 d in 5% serum-supplementedd-valine medium (to inhibit fibroblast growth), then incubated for a further 2 d in serum-free McCoy's 5A medium. This study showed that rabbit OSE and PM cells in vitro maintained certain in vivo morphologic characteristics; OSE cells exhibited distinct cell borders and abundant microvilli of homogeneous size and shape, whereas PM cells were characterized by obscure cell borders and abundant microvilli of heterogeneous form. GC in vitro exhibited overlapping cell borders and sparse microvilli of homogeneous structure. This study showed for the first time that cultured rabbit OSE and PM cells, but not GC, contain distinct filaments of cytokeratin 18. In addition, rabbit OSE cells and GC, but not PM cells, contained 17β-hydroxysteroid dehydrogenase. However, only GC contained delta 5-3β hydroxysteroid dehydrogenase. OSE, PM, and GC maintained their ultrastructural and histochemical characteristics in serum-free medium. These results suggest that rabbit OSE cells in vitro could be distinguished from PM cells by histochemical and ultrastructural differences. Furthermore, because these characteristics were not altered in serum-free medium, the two-step culture method will be valuable in further hormonal studies of these cells in vitro. This work was supported in part by Grant No. 202-3192 from the University of South Dakota Parsons Fund     

Close similarity between cultured human omental mesothelial cells and endothelial cells in cytochemical markers and plasminogen activator production

Summary The mesothelial cells obtained from human omental adipose tissue showed a typical cobblestone monolayer and reacted strongly with keratin, but did not have Von Willebrand factor. Ultrastructurally these cells revealed the existence of desmosome-like cell junctions as well as intracellular canaliculi, tubular structures surrounded by microvilli, and tonofilament-like filaments. The mesothelial cells grew much faster in the medium containing epidermal growth factor, actively took up acetylated-low density lipoprotein into their cytoplasm, and released angiotensin-converting enzyme. They also released urokinase-type plasminogen activator, but only half as much as do human umbilical vein endothelial cells; release of tissue-type plasminogen activator was not observed. Inasmuch as the mesothelial cells also released plasminogen activator inhibitor-1, as do human umbilical vein endothelial cells, we could not detect u-PA activity in culture medium. u-PA may play a role in the protection against adhesion among visceral organs. These observations indicate that cultured human mesothelial cells have characteristics closely related to those found in human endothelial cells.     

Establishment of an outgrowth culture system to study growth regulation of normal human epithelium

Summary To study the growth regulation of epithelial cells as a sheet, I developed an outgrowth culture system for normal human ectocervical epithelial (NHCE) cells, whereby outgrowths from tissue explants increase their radius in a constant rate over time. Cinematographic observation revealed that throughout the outgrowths the cells coordinately migrate and proliferate. To date, all 59 specimens examined have shown similar growth characteristics, with explant size not causing any difference in the growth rate; 10⁸ cells/specimen can easily be obtained in 3 wk. Cell densities of outgrowths also remain constant. Moreover, there is no fibroblast contamination, and removal of explants does not affect growth rate. Therefore, pure epithelial outgrowth in uniform growth condition can be prepared for further experiments. The results demonstrate that the outgrowth culture system is an attractive model for analysis of growth control mechanisms in normal human epithelium in vitro.     

Intermediate filaments in rat ovarian surface epithelial cells: changes with neoplastic progression in culture.

Interrelationships between neoplastic progression and the expression of intermediate filaments were examined in primary cultures, immortal lines, and Kirsten murine sarcoma virus (KiMSV) transformed lines of rat ovarian surface epithelial (ROSE) cells. Immunofluorescence microscopy revealed abundant keratin filaments in all cells of primary cultures. In immortal, nontumorigenic lines, keratin filaments were detected in fewer cells, in smaller numbers, and in microscopically altered forms. The percentage of keratin-positive cells ranged from 4 to 54%. Its expression was inversely proportional to cell density. Keratin expression was similar in the two immortal lines, although one had retained a monolayered epithelial growth pattern resembling primary cultures, while in the other the growth pattern of the cells was more atypical. The two KiMSV-transformed lines were previously shown to produce tumors in vivo that resemble human ovarian endometrioid stromal sarcomas. In spite of this histologic appearance, the proportion of keratin-positive cells in these cells was increased over the immortal lines. Keratin expression was unrelated to cell density, and keratin in most virally transformed cells was limited to few, fine filaments. In thymidine-labelled immortal and virus-transformed cultures stained for keratin, no correlation was found between keratin expression and proliferative activity. The keratin profiles of primary and immortal cultures were identical on Western blots, with subtypes ranging from 52 to 66 kDa. The two virally transformed lines lacked some of the subtypes. Vimentin networks were faint or absent in primary cultures. In the immortal and the virus-transformed lines, neoplastic progression was associated with increasing vimentin expression but with no changes in filament morphology and distribution. The results show that the abnormalities in intermediate filament expression that accompany immortalization do not preclude the retention of a normal epithelial morphology and growth pattern in this cell type. Furthermore, the number of intermediate filaments and their intracellular distribution appear to be altered at an earlier stage in neoplastic progression than those mechanisms that select for specific keratin subtypes, or those that respond to regulation by cell density. Finally, the presence of keratin in the KiMSV-transformed lines examined in this study supports the hypothesis that human ovarian stromal sarcomas can arise in the OSE.     

Primary culture of ovarian surface epithelial cells and ascites-derived ovarian cancer cells from patients

Our laboratory has refined the technique for isolating primary cultures of normal human ovarian surface epithelial (OSE) cells by combining two different protocols involving the enzymatic and mechanical removal of OSE cells from ovarian biopsies. A simple protocol of obtaining primary epithelial ovarian cancer (EOC) cells from the ascites fluid removed from patients with high-grade ovarian cancer is also described. These methods allow for the direct application of many molecular and cellular analyses of normal versus cancer cells isolated freshly from patients, with the added potential for retrospective analyses of archived cells and tissues. Thus, we have included optional steps for the immediate preparation of ascites-derived EOC cells to be used for subsequent cytological analyses. Initial isolation of OSE or EOC cells can be completed in 1 h, and primary cells are further expanded in culture for several weeks.     

Gonadotropin and steroid hormones stimulate proliferation of the rat ovarian surface epithelium

The ovarian surface epithelium (OSE) is a single layer of flattened or cuboidal cells covering the ovary. Ninety percent of all human ovarian malignancies arise from this layer of cells. Incessant ovulation, hyperovulation induced by infertility treatment, and hormone replacement therapy have been suggested as risk factors for ovarian cancer. In this study, two groups of rats, with and without surgically induced injury to the ovary, were treated with 17beta-estradiol, pregnant mare's serum gonadotropin (PMSG), human chorionic gonadotropin (hCG), or the combination PMSG/hCG, and the proliferative response of the OSE cells was measured using bromodeoxyuridne (BrdU) and (3)H-thymidine. All hormones, alone or in combination with ovarian surgery, were found to increase significantly the rate of proliferation of the rat OSE. These data demonstrate that hormones associated with infertility treatments and hormone replacement therapy, as well as injury- or ovulation-induced rupture of the ovarian surface, stimulate the rat OSE, and hence could have a role in the development of ovarian cancer via proliferation-associated mutagenesis, or alternatively, by promoting the rapid selection of OSE cells with accumulated mutations.     

Estrogen stimulation of ovarian surface epithelial cell proliferation

Summary Ovarian cancer is the leading cause of gynecological cancer mortality, and 85–90% of this malignancy originates from the ovarian surface epithelium (OSE). The etiology of ovarian epithelial cancer is unknown but a role for estrogens has been suspected. However, the effect of estrogens on OSE cell proliferation remains to be determined. Using the rabbit model, our studies have demonstrated that 17β-estradiol stimulates OSE cell proliferation and the formation of a papillary ovarian surface morphology similar to that seen in human ovarian serous neoplasms of low malignant potential. Immunohistochemical staining of ovarian tissue sections with an antibody to the estrogen receptor α demonstrates its expression in both OSE cells and stromal interstitial cells. In primary ovarian cell cultures, the proliferative response of the epithelial cells to 17β-estradiol depends on the expression of the estrogen receptor α in the epithelial cells. However, when the epithelial cells are grown together with ovarian stromal cells, their proliferative response to this hormone is greatly enhanced, suggesting the involvement of stromal-epithelial interactions. These studies suggest a role for estrogens and the estrogen receptor α in OSE growth.     

In vitro three-dimensional modelling of human ovarian surface epithelial cells

Objectives:  Ninety percent of malignant ovarian cancers are epithelial and thought to arise from the ovarian surface epithelium (OSE). We hypothesized that biological characteristics of primary OSE cells would more closely resemble OSE in vivo if established as three-dimensional (3D) cultures.
Materials and methods:  OSE cells were cultured as multicellular spheroids (MCS) (i) in a rotary cell culture system (RCCS) and (ii) on polyHEMA-coated plastics. The MCSs were examined by electron microscopy and compared to OSE from primary tissues and cells grown in 2D. Annexin V FACS analysis was used to evaluate apoptosis and expression of extracellular matrix (ECM) proteins was analysed by immunohistochemical staining.
Results:  On polyHEMA-coated plates, OSE spheroids had defined internal architecture. RCCS MCSs had disorganized structure and higher proportion of apoptotic cells than polyHEMA MCSs and the same cells grown in 2D culture. In 2D, widespread expression of AE1/AE3, laminin and vimentin were undetectable by immunohistochemistry, whereas strong expression of these proteins was observed in the same cells grown in 3D culture and in OSE on primary tissues.
Conclusions:  Physiological and biological features of OSE cells grown in 3D culture more closely resemble characteristics of OSE cells in vivo than when grown by classical 2D approaches. It is likely that establishing in vitro 3D OSE models will lead to greater understanding of the mechanisms of neoplastic transformation in epithelial ovarian cancers.     

Explant-cell culture of primary mammary tumors from MMTV-c-Myc transgenic mice

We have established an explant-cell culture system for mammary gland tumors from c-myc oncogene-expressing transgenic mice and potentially other transgenic strains. By coating culture dish surfaces with fetal bovine serum and using culture media supplemented with low serum and growth factors, the mammary tumor specimens could be maintained in culture for over 3 mo. Throughout the culture period, the explants produced abundant outgrowths of epithelial cells. As the outgrowths of epithelial cells filled the dishes, the explants were serially transferred from one dish to another-a process that could be repeated at least six times, thus providing a continuous supply of primary tumor cells. This culture system provides a useful tool for studying the biology of mouse mammary gland tumors and possibly tumors from other organ sites.