Ovarian surface epithelial cells are positionally organized as a single cell layer by a sheet of basement membrane. It is believed that the contact of the ovarian surface epithelial cells with the basement membrane regulates cell growth and ensures the organization of the epithelium. Disabled-2 (Dab2), a signal transduction protein and a candidate tumor suppressor of ovarian carcinoma, functions in positional organization of ovarian surface epithelial cells. In ovarian carcinomas, genetic and epigenetic changes enable the tumor cells to escape positional control and proliferate in a disorganized fashion. Alterations in the extracellular environment may also be critical for tumor initiation and progression.
We analyzed and compared the presence of collagen IV and laminin, the scaffold proteins of the basement membrane, and Dab2 in 50 ovarian tumors that are restricted to the ovaries and in 50 metastases of ovarian tumors by immunohistochemistry. Expression of collagen IV, laminin, and Dab2 was also analyzed by Northern blotting in a panel of human ovarian surface epithelial and cancer cell lines.
The basement membrane is often absent in morphologically benign ovarian surface and cyst epithelium and low-grade tumors and collagen IV and laminin are absent in the extracellular matrix of most of the primary tumors tested. Of the 50 ovarian tumors confined to the ovaries, 6% (3 of 50) were collagen IV positive and 24% (12 of 50) were laminin positive tumors. Of the 50 metastatic tumors, 16% (8 of 50) are collagen IV positive and 86% (43 of 50) are laminin positive. In addition, even in the metastatic ovarian tumors that are largely collagen IV negative, there are pockets of local areas in which the tumor cells are surrounded by collagen IV-positive staining. Dab2 is absent in the majority of ovarian tumors found in both ovaries and metastatic sites. In both nontumorigenic human ovarian surface epithelial and cancer cell lines, collagen IV, laminin, and Dab2 are expressed aberrantly.
Basement membranes are thin sheets of extracellular matrix that surround epithelial and endothelial cells, nerves, and muscles, and separate the cells from the interstitial stroma.1–3 The major components of basement membranes, laminin, entactin, heparin sulfate proteoglycans, and collagen IV, form a dense meshwork.1–6 Other minor components may include fibronectin, tenascin, SPARC (osteonectin), and fibulin-1.3, 5, 7
In the ovary, the surface epithelial cells grow as a single layer in contact with the basement membrane,6, 8–10 which acts as both a mechanical barrier and an organizer of the tissue structure. Laterally, the epithelial cells are interconnected through specialized adherent areas, including adhesive junctions, desmosomes, and tight junctions.3, 6, 10 The basement membrane of the ovarian surface epithelium is a well defined layer and the epithelial cells are organized by attaching to the basement membrane.
The ovarian surface epithelial cells play a role in ovulation, contributing to postovulatory repair by proliferation and migration over the site of follicular rupture.8–11 In ovulation, proteases and hydrolyases are produced and secreted to degrade the basement membranes of both the follicles and surface epithelium, allowing the release of the ovules.12–17 Prostaglandins are believed to be the main mediators for the activation of preovulatory proteolysis.18–20 Following ovulation, the surface epithelial cells proliferate and deposit a basement membrane as part of the repair process.5 It has been suggested that the risk of ovarian cancer increases with the number of ovulatory cycles. According to the incessant ovulation hypothesis,21, 22 the increased cancer risk is due to the genetic mutations that might occur during proliferation of the surface epithelial cells in the process of postovulatory repair. This idea has been tested in the laboratory by culturing rat ovarian surface epithelial cells and is supported by in vitro experimental results.23, 24 A competing gonadotropin stimulation hypothesis suggests that the surges of pituitary gonadotropins associated with each ovulation may directly act on the ovarian surface epithelium to increase the risk of tumorigenicity.22, 25, 26
In addition to its biomechanical role, the basement membrane acts as a cellular signal regulator of growth, differentiation, polarity, and gene expression.27–30 In neoplastic transformation, the epithelium of the ovarian surface or inclusion cysts first becomes multicell layered and forms adenomas.31–35 Subsequently, the epithelial cells may puncture the basement membrane and invade the stroma or the tumor cells may become detached from the primary tumors and spread in the peritoneal cavity as ascites. The detached tumor cells often reattach and establish secondary metastatic tumors in the surrounding tissues of the peritoneal cavity, mainly in the omentum.36–39 Distant metastasis of ovarian tumor cells through blood vessels and the lymphatic system in later stages is also common40 The hallmark of malignancy is the disorganization of the epithelial-derived tumor cells. The cells are often no longer arranged as a single cell layer and lose their basal-apical polarity. Presumably, the tumor cells now reside in a much different extracellular environment and the genetic and epigenetic alterations in the tumor cells allow these cells to survive and proliferate, resisting the positioning rules imposed on normal epithelial cells.41–42
The basement membrane status of ovarian tumors has been investigated.42–53 It is generally believed that low-grade and benign tumors associate with the presence of the basement membrane at the interface between the epithelial tumor cells and the adjacent stroma and that loss of the basement membrane is associated with tumor invasion and metastasis.45–48 In addition, the absence of collagen IV and laminin basement membrane associates with dedifferentiation of tumor cells.49, 50 In some cases, the presence or absence of collagen IV or laminin was associated with particular histologic subtypes of ovarian carcinomas.51, 53 and may even correlate with growth and tumor grades.48, 50 Additional studies have underlined the importance of collagenase and other matrix-degrading proteases in tumor invasion and metastasis.54–50 Overexpression or activation of these proteolytic enzymes may account for the degradation of collagen IV and laminin produced by the epithelial tumor cells. However, the intrinsic ability of the ovarian surface epithelial cells to express collagen IV and laminin may also be regulated and altered in tumors.61 Most of these immunohistochemical studies of collagen IV and laminin have focused on the basement membrane at the interface between tumor cells and the stroma.
In the current study, we examined the expression and deposition of collagen IV and laminin around epithelial ovarian tumor cells (instead of at the interface between tumor cells and the stroma). We found that the expression and the presence of collagen IV and laminin are regulated dynamically in the process of ovarian tumor development and spreading. We conclude that in ovarian tumor development, the epithelial cells initially lose expression of collagen IV and laminin. However, in metastases, the expression is often restored and the collagen IV and laminin proteins are deposited around the tumor cells.
MATERIALS AND METHODS
Ovarian Tumor Tissues
Representative sets of 50 ovarian tumors confined to the ovaries and 50 ovarian-derived tumors removed from metastatic sites were selected for analysis. These are paraffin-embedded, archived tumor tissue samples obtained from the Emory University Hospital. The pathology characterizations were provided by Dr. Cohen (Department of Pathology, Emory University) and grades and histologic subtypes were confirmed in sections stained with hematoxylin and eosin. All the tumors were identified as being of ovarian surface epithelial origin. The use of human tissues was examined and approved by the Human Investigation Committee. Safety and ethical guidelines were followed in using the human tumor tissues according to the requirements of the institution.
Of the 50 tumors confined to the ovary, there are 2 endometrioid tumors, 5 mucinous tumors, 7 poorly differentiated adenocarcinomas, and 36 serous tumors. These tumors were used in a previous investigation.53 Of the 50 metastatic tumors, the sites of metastasis included 1 right obturator lymph node, 1 left ovary (primary in right ovary), 1 colon, 1 left pelvic wall, and 46 omentum. All extraovarian tumors were considered to be peritoneal spread, instead of distant metastases. Due to concern about patient privacy, information on the pairing of primary and metastatic tumors was not obtained. Because of the few tumors at metastatic sites other than the omentum and because we did not find a specific pattern, no effort was made to differentiate between specific metastatic sites in this study.
The immunostaining of Disabled-2 (Dab2), collagen IV, and laminin was performed and analyzed as previously reported.42, 53, 62, 63 Some of the slides were stained with periodic acid–Schiff (PAS) according to standard methods by immersing the slides in PAS solution (Sigma, St. Louis, MO) for 20 minutes and counterstaining with hematoxylin. The slides were scored independently by three persons including a pathologist. For collagen IV and laminin staining, only staining within the epithelial/tumor compartment (likely tumor derived) but not the well defined basement membrane separating epithelial and stromal compartments was scored. Positive scoring was given when the epithelial staining of collagen IV and laminin was higher than 10%. If tumor cytosolic Dab2 staining was positive in more than 10% of the cells, the tumor was scored as Dab2 positive. The results from three independent determinations were compared. Any differences in scoring results were discussed and the slides were further examined to reach a common conclusion.
Human Ovarian Surface Epithelial Cell Lines (HIO)
Human ovarian surface epithelial cell lines were derived and characterized as described previously.64, 65 Briefly, ovarian specimens were obtained from women undergoing prophylactic oophorectomies. The ovarian surface epithelium was scraped from the ovarian surface and the cells were cultured in medium 199 and MCDB-105 (1:1) supplemented with 4% fetal bovine serum (FBS) and 0.2 U/mL of insulin (Novagen, Madison, WI). After primary cultures were established (1–2 months), the cells were transfected with an SV40 large T antigen (SV40Tag) expression vector. These cells are referred to as human “immortalized” ovarian surface epithelial (HIO)-cells. In contrast to primary human ovarian surface epithelial cells, which undergo less than 10 population doublings before entering replicative senescence, HIO clones derived following introduction of SV40Tag undergo an additional 20–30 population doublings before ceasing proliferation.64, 65 RNA used in the Northern blots was isolated from HIO cells at the following passages (p): HIO-103, p11; 105, p10; 107, p7; 114, p11; and 118, p34.
Cell Culture and Northern Blot Analysis
Ovarian tumor cell lines were previously established66 or obtained from ATCC (Rockville, MD). The tumor cells were cultured in Dulbecco's modified Eagle medium with 10% FBS. Total RNA was isolated from 100-mm plates of 80% confluent monolayers using the guanidinium isothiocyanate/phenol/chloroform extraction procedure as described.67 Northern blot analysis was performed using [32P]-labeled cDNA fragments with a random prime labeling kit, Prime-It II (Stratagene, La Jolla, CA). Human Dab2 cDNA was reported previously.68 Plasmids containing partial cDNA of collagen IV alpha 1 and alpha 2 and laminin beta-1 were from the ATCC. The gel-purified cDNA fragments from restriction digestion were used as probes. All cDNA fragments were sequenced to verify the identity.
Alteration of the Basement Membrane of Ovarian Surface Epithelium
The ovarian surface epithelium is normally a single cell layer of flat or cuboidal epithelial cells organized by a sheet of basement membrane. Columnar morphology of the surface epithelial cells is observed occasionally in the nontumor parts of the ovarian tissues obtained during surgery. Collagen IV and laminin are two of the major constituents of the basement membrane lining the epithelium, separating epithelial from stromal components. We characterized the basement membrane by PAS staining to visualize glycoproteins and by immunostaining of collagen IV and laminin (Fig. 1). Collagen IV staining locates discretely within the basement membrane under the epithelium (arrow) separating the stroma. Laminin staining is more promiscuous and appears around epithelial cells in addition to the basement membrane (arrow). The glycoprotein-rich basement membrane is indicated by the PAS staining of a layer structure lining the epithelium (Fig. 1, arrow). In normal ovaries, there is no collagen IV and laminin staining in the stroma, although a noncollagen IV matrix is present as depicted here by PAS staining.
Consistent with a previous study,53 we observed that the preneoplastic surface epithelium immediately adjacent to tumor areas often lacks an intact basement membrane as indicated by the absence of collagen IV and laminin staining. For example, in the nontumor part of the ovary adjoined to the tumor, an intact collagen IV-positive basement membrane is evident in the surface epithelium (Fig. 2A, arrow), even though the epithelial cells are columnar. In the same section, a morphologically preneoplastic epithelial cyst lacks a collagen IV-containing basement membrane (Fig. 2A, double arrows), concurring with the observation that the ovarian tumor precursor lesions are often found in inclusion cysts.31, 33 In another borderline low-grade ovarian tumor of endometrioid histologic subtype (Fig. 2B), no collagen IV and laminin-positive basement membrane fibers are evident between the epithelial and stromal components. In confirmation of our previous observation,63 the alteration of the basement membrane is associated with early transformation of ovarian surface epithelial cells before malignant morphologic transformation and invasion of the stroma.
Extracellular Microenvironment in Ovarian Carcinomas Confined to Ovaries
Fifty cases of ovarian cancer were selected for analysis. We separated the tumor specimens of each case into primary tumors confined to the ovary and metastatic tumors in the peritoneal tissues. First, we examined the 50 ovarian carcinomas confined to the ovary tissues by immunostaining collagen IV and laminin to investigate the presence of the basement membrane components around the tumor cells. In an ovarian tumor section, the morphologically normal surface epithelium exhibits an intact basement membrane (Fig. 3, arrow) and invasive tumor cells in the stroma lack collagen IV staining (Fig. 3, double arrows). Often, the ovarian tumor is free of collagen IV staining but laminin staining may be positive. In a few tumors, collagen IV and laminin are present as basement membrane-like fibers around tumor cells. However, the majority of the primary ovarian carcinomas are devoid of collagen IV (94% [47 of 50 tumors]) and laminin (76% [38 of 50 tumors]) around the tumor cells (Table 1). The data in Table 1 are replotted to indicate staining of collagen IV relating to staining of laminin in tumor or stromal areas (Table 2). The expression of Dab2, a candidate tumor suppressor42, 69, 70 that is often lost in preneoplasia of ovarian epithelial cells,53, 63 was also lost in the majority (96%) of tumors (Table 1).
Table 1. Alteration of Extracellular Matrix around Tumor Cells Confined to Ovarian Tissues or of Metastasesa
Nonmetastatic (+)/Total (%)
Metastatic (+)/Total (%)
Human ovarian carcinomas excised from ovaries (50 nonmetastic samples) or metastatic sites (50 metastatic samples) were formalin fixed and paraffin embedded. Sections (5 μm) were cut and immunostained with an anti–collagen IV, anti-laminin, and an affinity-purified anti-Dab2 antibody. Only staining within (for Dab2) or surrounding (for collagen IV and laminin) tumor cells was scored as positive.
Collagen IV (+)
Table 2. Correlations of Laminin and Collagen IV Staining in Ovarian Tumorsa
The results of the staining in Table 1 are reformatted to reveal the relationship among expression of Dab2, collagen IV, and laminin in individual tumors, in tumor (T) or stroma (S) areas. For example, there are 38 tumors in which both the tumor and stroma areas are negative for both collagen IV (T−, S−) and laminin (T−, S−).
Figure 4 shows examples of ovarian-confined carcinomas immunostained with collagen IV and laminin. The staining of blood vessels serves as an internal positive control. In the majority of these tumors (Fig. 4A, B), there is little or no collagen IV and laminin staining around tumor cells or in the stroma adjacent to the tumor cells. In a small percentage (24% [12 of 50]) of tumors (Fig. 4C), the tumor cells are slightly laminin positive, but collagen IV staining is absent.
Extracellular Microenvironment in Metastatic Ovarian Carcinomas
We also analyzed the 50 ovarian metastases by immunostaining laminin, collagen IV, and Dab2. The majority of these specimens are secondary tumors established in the omentum. Upon inspection, one obvious difference between these metastatic lesions and primary tumors is the elevated deposition of collagen IV and laminin around tumor cells within the metastatic tumors (Table 1). In the secondary metastatic tumors, 16% (8 of 50) exhibit collagen IV staining, 86% (43 of 50) show laminin staining around or in contact with tumor cells, and Dab2 expression is lost in 88% of the tumors. There is also an increase in collagen IV and laminin staining in the stroma around the metastatic tumor cells (Table 2). Examples of collagen IV and laminin staining in these metastatic ovarian tumors are shown in Figure 5. The metastatic tumor cells (Fig. 5A) are negative for collagen IV and positive for laminin staining. Other tumors (Fig. 5B) are laminin positive and the stroma is positive for collagen IV staining. Some tumors in Figure 5C,D have collagen IV and laminin- positive materials around the tumor cells and one tumor (Fig. 5E) is collagen IV positive but laminin negative. Metastatic ovarian carcinoma has an enhanced deposition of collagen IV and laminin around tumor cells and in the stroma.
Although increased compared with tumors confined to the ovaries, metastases that are collagen IV positive are still a relative minor category (16% [8 of 50 tumors]). However, all metastatic tumors that are collagen IV negative (in which >90% of the tumor cells are collagen IV negative) exhibit pockets of tumor cells that are stained positive (Fig. 6). In two examples (Fig. 6A,B), pockets of tumor cells with collagen IV-positive material around the tumor cells (arrow) are present in a tumor that is largely free of collagen IV staining. In addition, part of a tumor with collagen IV and laminin-positive basement membrane-like material surrounding the cells resides next to another area of a collagen IV and laminin-negative tumor on the same section of a metastatic ovarian tumor (Fig. 6C). The heterogeneous nature of collagen IV staining is not common in ovary-confined tumors. Therefore, tumors confined to ovaries tend to be negative for collagen IV and laminin and secondary (metastatic) ovarian tumors are often collagen IV and laminin positive either in the entire tumor mass or in small pockets within the largely collagen IV-negative tumors.
Correlation of Collagen IV Deposition around Tumor Cells and Dab2 Expression
The different basement membrane components in the extracellular environment of the ovarian tumors may require a different signaling strategy for the tumor cells adapting to the various cell contact signaling. One would predict a different set of genetic and epigenetic changes in tumor cells between the collagen IV and laminin-positive and negative tumors. A correlation was found between the expression of Dab2 and the presence of collagen IV around the tumor cells, further confirming a previous observation.42
In the majority tumors within or outside the ovaries, Dab2 expression was absent, consistent with previous studies.42, 53, 63, 69, 70 The absence of Dab2 expression is correlated with the absence of collagen IV, but not laminin, staining of an ovarian-confined tumor (Fig. 7A) and a tumor found in the omentum (Fig. 7C). A fraction of ovarian tumors are Dab2 positive. Two (4%) tumors are confined to ovaries and six (12%) tumors spread to secondary sites. These tumors are positive for collagen IV deposition around tumor cells (Fig. 7B) for a tumor confined to ovary and for a metastasized ovarian tumor found in the omentum (Fig. 7D). All Dab2-positive ovarian tumors are also positive for deposition of collagen IV around tumor cells, but some collagen IV-positive tumors may be Dab2 negative.
Expression of Collagen IV and Laminin in Human Ovarian Surface Epithelial Cells and Tumor Cell Lines
It is possible that the fluctuation of expression of collagen IV and laminin by ovarian surface epithelial and tumor cells contributes to the change in the presence of collagen IV and laminin in the extracellular matrix of the tumors.61 We investigated the expression of collagen IV and laminin in a panel of ovarian surface epithelial cell lines (Fig. 8). Five HIO surface epithelial cell lines were examined (Fig. 8A). These cell lines were derived independently from human primary ovarian surface epithelial cells following introduction of SV40Tag to prolong life in culture. Even in these early passage cell lines, collagen IV and laminin are aberrantly expressed. Laminin mRNA is absent in the HIO-103 and HIO-107 lines and collagen IV expression is also absent (or very low) in HIO-103. In the seven tumor cell lines tested (Fig. 8A), two (A2780 and OVCAR-10) lack laminin expression and three (A2780, OVCAR-5, OVCAR-10) lack collagen IV expression. Collagen IV and laminin are expressed aberrantly in both HIO surface epithelial cells and tumor cell lines.
The expression of collagen IV varies quantitatively between tumor and nontumor cells, whereas the expression of laminin is constant (Fig. 8A). The expression of collagen IV alpha I and alpha II genes is very similar. However, the expression of collagen IV and laminin is disassociated. The lack of collagen IV and laminin staining in some tumors may be due to variable expression in the tumor cells. The expression of Dab2 was lost in most of the ovarian tumor cell lines.42, 53, 63, 69, 70 Consistently, Dab2 expression is present in the ovarian surface epithelial cell lines HIO-105, HIO-114, and HIO-118, but is low in the HIO-103 and HIO-107 lines, and is absent in all the tumor lines (Fig. 8A). ES2 is one of the few ovarian tumor lines that express Dab2.63 ES2 cells are also positive for collagen IV and laminin expression (Fig. 8B). One of the ovarian tumor lines, OV1016, lacks both collagen IV and laminin expression (Fig. 8B) and SKOV3, a metastatic line, exhibits strong collagen IV but no laminin expression (Fig. 8B). Collagen IV and laminin are expressed aberrantly in ovarian tumor cell lines. In tumor lines, expression of collagen IV correlates with the ability of the cells to metastasize (Bruening and Godwin, unpublished data).
In the current study, we surveyed the presence of basement membrane components, collagen IV and laminin, in ovarian tumors and derived secondary tumors. We observed that basement membranes are often absent in the morphologically nonneoplastic epithelium, consistent with the previous observation53 that the basement membrane of the ovarian surface epithelium is often absent transiently before morphologic transformation of the epithelium. We found that collagen IV and laminin staining is often absent in primary ovarian tumors confined to the ovary, due to either degradation or lack of synthesis. We speculate that the loss of cellular expression contributes to the absence of collagen IV and laminin in the extracellular matrix because aberrant expression of collagen IV and laminin was observed in tumor cell lines by Northern blot. It is equally possible that the produced collagen IV and laminin are degraded by increased proteolytic activity. The emergence of collagen IV and laminin-positive materials around tumor cells, and the presence of pockets of collagen IV and laminin-positive tumor cells in ovarian tumors spread to secondary sites, suggests that reexpression of collagen IV and laminin may be prometastatic.
Loss of Basement Membrane Structure in Premalignant Transformation of Ovarian Surface Epithelium
It is well recognized that the basement membrane of the epithelium is degraded during the invasion of carcinoma cells.55–58 However, a previous study53 suggested that in the ovarian surface epithelium, the degradation or loss of the basement membrane is an early event before the morphologic transformation of epithelial cells. Our result is consistent with that study,53 indicating that collagen IV and laminin are lost early. Morphologically normal surface epithelium or cysts are often devoid of an intact basement membrane and collagen IV and laminin are lost in both low-grade and invasive tumors. The loss of collagen IV and laminin is an early event not solely associated with tumor invasion. In a physiologic situation, the intactness of the basement membrane of the ovarian surface epithelium may be regulated because the basement membrane must be degraded before the release of ovules during ovulation.11–20 The basement membrane is degraded by prostaglandin-mediated expression of proteinases,18–20 because the degradation of the surface epithelium and ovulation can be blocked by indomethacin or collagenase inhibitors and an increase in protease secretion was observed during ovulation. However, it is also possible that the expression of collagen IV and laminin by ovarian surface epithelial cells is regulated during ovulation.11, 18
The frequent absence or aberrant expression of collagen IV and laminin in cultured human ovarian surface epithelial cells as determined by Northern blot suggests that the loss of collagen IV and laminin expression may occur in premalignant ovarian surface epithelial cells. We believe that both an increase in protease production and suppression of collagen IV and laminin expression may lead to the loss of the basement membrane in the premalignant transformation of the ovarian surface epithelium.
A Subtype of Ovarian Tumors Defined by Collagen IV-Positive Extracellular Matrix
Ovarian surface epithelial cells in culture autonomously synthesize and secrete basement membrane components such as collagen IV and laminin.5 The extracellular basement membrane components in ovarian tumors have been investigated using immunohistochemistry.42–53 It is unclear whether the epithelial-derived tumor cells continue to produce collagen IV and laminin and we do not know the fate of these basement membrane components. The information concerning the extracellular environment of the tumor cells is of importance because the major results of the intrinsic genetic and epigenetic mutations of the tumor cells are for the tumor cells to survive and proliferate in the altered extracellular environment of the tumors.
Upon analysis of the tumors, we classified the ovarian tumors into several subclasses based on the patterns of collagen IV and laminin staining. Although they may be present in the basement membranes at the interface of tumor and stroma, collagen IV and laminin staining is low or absent around the tumor cells in most of the tumors.
A smaller fraction of both ovarian-localized and metastatic tumors is positive for collagen IV, whereas a much higher percentage are laminin positive, in either the matrix around the tumor cells or stroma. The collagen IV and laminin deposited in the extracellular matrix of the tumor cells are produced and secreted by the tumor cells. A unique subtype of ovarian tumors is identified in this study. These are characterized by the presence of a collagen IV and laminin-positive basement membrane-like structure (referred to in this study as a pseudo basement membrane) around the tumor cells. The pseudo basement membranes are arranged in two ways. The basement membrane content may be deposited on one side of each tumor cell. Therefore, the tumor cells are polarized or exhibit an asymmetric extracellular environment (Figs. 5D, 7B,D). In the second arrangement, the pseudo basement membrane surrounds the outline of the tumor cells, making the tumor cells nonpolarized (Figs. 5E, 6A–C).
The correlation between cellular Dab2 expression and the presence of collagen IV deposition around tumor cells suggests that Dab2 may be involved in the signaling pathway for epithelial cells to contact the collagen IV-positive basement membrane. Ovarian surface epithelial cells undergo oncogenic transformation by two distinct pathways (Fig. 9A). The cells either lose Dab2 expression and became collagen IV independent or the cells retain Dab2 expression and acquire the ability to deposit a collagen IV-positive pseudo basement membrane around the cells. These two types of ovarian cancers (Fig. 9A), i.e., Dab2-negative and collagen-negative and Dab2-positive and pseudo basement membrane-positive, may behave differently in regard to proliferation, invasion, and metastasis. However, a close correlation was not found between Dab2 expression and the Mib-l proliferative index in ovarian tumors.53 We found that a higher percentage of metastatic than primary ovarian tumors are of the pseudo basement membrane-positive type. An obvious speculation is that pseudo basement membrane-positive ovarian tumors are more metastatic. The relative small number of tumors analyzed and the current design of the research have not addressed this correlation adequately. Further investigation is needed to examine critically the correlation between pseudo basement membrane-positive ovarian tumors and the tendency to metastasize or spread.
Absence of Collagen IV and Laminin in Tumors Confined to Ovaries and Restoration of Collagen IV and Laminin Deposition in Tumor Metastasis
Tumor metastasis is generally accepted as the end result of a multistep process, including tumor initiation and development, escape of cells from the primary tumor, entry into the bloodstream and lymphatic system, evasion of host defense mechanisms, and extravasation and colonization of a specific target tissue.40 Ovarian tumors, however, can spread by a unique mechanism. Tumor cells can “shed” from the primary tumors and spread, adhere, and establish secondary tumors in peritoneal tissues by direct contact and transfer.31, 36–39 In the late stages of ovarian carcinoma, malignant cells are often found both in ovaries and in peritoneal tissues, mainly the omentum.
In this study, we selected tumor tissue confined to the ovary and secondary tumors excised from peritoneal metastatic sites for analysis. By comparing primary and metastatic ovarian tumors for collagen IV and laminin staining (Table 1), an obvious observation is that the fractions of collagen IV and laminin-positive tumors are increased greatly in the metastases. We speculate that although collagen IV and laminin in the basement membrane are lost in the premalignant ovarian surface epithelium, and deposition around tumor cells is absent in most of the primary tumors, reexpression and the presence of a pseudo basement membrane may facilitate the spreading of ovarian tumor cells.
Collagen IV and laminin staining are more heterogeneous in metastatic ovarian tumors. For metastatic ovarian tumors, even in collagen IV-negative tumors, pockets of tumor cells stained positive for collagen IV. Because the expression of collagen IV and laminin may be regulated dynamically in premalignant and neoplastic ovarian surface epithelial cells, we speculate that a metastatic tumor could be derived from a clone of collagen IV-positive tumor cells. Upon establishment in the secondary site, tumor cells may gradually lose the ability to produce collagen IV. Supporting the idea of a dynamic regulation of collagen IV and laminin during tumor malignant transformation, some studies71, 72 have revealed that the synthesis of laminin and collagen IV is regulated by signaling pathways such as Akt/PKB and TGF-beta. To include our current observation, a previous model53 on the transformation of ovarian surface epithelium may be modified to include the reexpression of collagen IV and laminin in the spread of tumor cells (Fig. 9B).
We conclude that the expression of collagen IV and laminin in ovarian surface epithelial cells is regulated dynamically. The loss of collagen IV and laminin occurs in premalignant cells and during the expansion of tumor mass. However, it is likely that reexpression of collagen IV and laminin is advantageous for the spread of ovarian tumor cells to metastatic sites in the peritoneum. These conclusions were made based on primarily histologic observations and should be tested in experimental models to verify if alteration of the extracellular matrix impacts ovarian epithelial cell transformation, tumor development, and metastasis.
The authors thank Drs. Cathy Bingham, Zhe-Sheng Chen, and Andrey Frolov for reading and commenting during the process of preparing the manuscript. They thank Jennifer Smedberg and Malgorzata Rula for their excellent technical assistance, Ms. Diane Lawson for her technical assistance in immunostaining, and Ms. Patricia Bateman for her secretarial assistance.