Affiliation of authors: Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston.
Correspondence to: Isaiah J. Fidler, D.V.M., Ph.D., Department of Cancer Biology, Box 173, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030 (e-mail: ijfidler{at}mdanderson.org).
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To produce new vasculature during angiogenesis, endothelial cells must migrate, divide, and form tubes (4). Proteolysis of components of the extracellular matrix (8,9) allows endothelial cells to migrate and releases stored angiogenic signaling molecules from the extracellular matrix (10,11). High levels of matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9, also called type IV collagenases or gelatinases) in tissues have been associated with active neovascularization (811). Indeed, inhibitors of MMPs, such as tissue inhibitor of MMP-2 (TIMP-2), have been shown to inhibit in vitro proliferation and tube formation by endothelial cells (12,13). TIMP-2 as well as tissue inhibitor of MMP-1 (TIMP-1) have been shown to suppress in vivo angiogenesis by mouse B16 melanoma cells (14) and Burkitt's lymphoma cells (15). Studies in mice that were genetically modified to lack MMP-9 expression (10,11,16,17) have shown that MMP-9 contributes to the angiogenic switch that occurs during carcinogenesis (10,11). Furthermore, these studies suggested that MMP-9 is expressed by inflammatory cells and not by neoplastic cells, at least in these animal models.
Human ovarian cancer cells produce both MMP-2 and MMP-9 (1820), and increased expression of these MMPs in human ovarian cancer cells is associated with their invasive and metastatic potentials (2125). However, in situ hybridization studies have demonstrated that MMP-9 mRNA expression is detectable not only within neoplastic epithelial areas of the tumors (23) but also in stromal areas, which raises the possibility that the MMP-9 expressed by stromal cells, in addition to that expressed by tumor cells, may contribute to the malignant behavior of ovarian cancers.
To examine the contribution of mouse stromal MMP-9 to the progressive growth of human ovarian cancer cells, we generated a strain of nude mice that have a homozygous null mutation in the MMP-9 gene (i.e., MMP-9-/- nude mice). We implanted human ovarian cancer cells SKOV3.ip1 and HEY-A8 into the peritoneal cavities of MMP-9-/- nude mice and into those of nude mice with intact MMP-9 genes (MMP-9+/+ nude mice) and measured tumor incidence, angiogenesis, and progressive growth.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The SKOV3 cell line was originally obtained from the American Type Culture Collection (Manassas, VA). The SKOV3.ip1 variant cell line was isolated from ascites fluid that had accumulated in a nude mouse that was injected intraperitoneally with SKOV3 cells (26). The HEY-A8 cell line was obtained from Dr. Gordon B. Mills (The University of Texas M. D. Anderson Cancer Center). Human ovarian cancer cells were maintained in culture (5% CO2 and 95% air at 37 °C) in minimal essential medium (MEM; Life Technologies, Rockville, MD) supplemented with 10% fetal bovine serum, sodium pyruvate, nonessential amino acids, L-glutamine, and vitamin solution (Life Technologies).
Mice
Athymic BALB/c nude mice with wild-type MMP-9 genes (MMP-9+/+ nude mice) were purchased from the Animal Production Area of the National Cancer Institute, Frederick Cancer Research Facility (Frederick, MD). Mice that lacked an intact MMP-9 gene were originally developed via homologous recombination in mice with a 129/CD1 genetic background (17). We generated nude mice that lacked an intact MMP-9 gene (MMP-9-/- nude mice) in our animal facility by interbreeding MMP-9-/- 129/CD1 mice with MMP-9+/+ nude mice for eight generations. The genotypes of the resulting mice were determined by using polymerase chain reaction analysis (17). The nude mice were housed in laminar flow cabinets under pathogen-free conditions and were used for all studies when they were 8 weeks old. Animals were maintained according to institutional regulations in facilities approved by the American Association for Accreditation of Laboratory Animal Care, in accordance with current regulations and standards of the United States Department of Agriculture, Department of Health and Human Services, and the National Institutes of Health.
Collagenase Activity Assay
SKOV3.ip1 cells, HEY-A8 cells, and macrophages (2 x 105) were seeded in six-well plates and incubated overnight at 37 °C. The cells were washed twice with Hanks' balanced salt solution (HBSS) and cultured for an additional 24 hours in serum-free medium. Macrophages were treated with lipopolysaccharide (at 0 µg/mL, 0.1 µg/mL, or 1.0 µg/mL; Sigma Chemical Co., St. Louis, MO) for an additional 30 minutes. Culture supernatants were collected for assays of collagenase activity; cells attached to the plate were stained for viability with trypan blue, and the viable cells were counted. Culture supernatants (40 µL) were resolved on a 7.5% sodium dodecyl sulfate polyacrylamide gel that contained 1 mg/mL gelatin (Sigma Chemical Co., St. Louis, MO). The gel was washed for 30 minutes at room temperature in wash buffer (50.0 mM TrisHCl [pH 7.5], 15.0 mM CaCl2, 1.0 µM ZnCl2, 2.5% Triton X-100) and then incubated for 24 hours at 37 °C in the same buffer that contained Triton X-100 at a final concentration of 1%. The gel was then stained with 0.1% Coomassie Brilliant Blue R-250; clear zones against the blue background indicated the presence of gelatinolytic (i.e., collagenase) activity.
Collection of Mouse Peritoneal Exudate Macrophages
We collected peritoneal exudate macrophages from MMP-9+/+ and MMP-9-/- nude mice by peritoneal lavage with HBSS 4 days after the mice were given an intraperitoneal injection with 2 mL of thioglycollate broth (Baltimore Biological Laboratories, Cockeysville, MD) (27). The macrophages were concentrated by centrifugation for 5 minutes at 895g and resuspended in MEM supplemented with 5% fetal bovine serum, and 3 x 107 cells were plated into each tissue culture flask and incubated at 37 °C for 2 hours. The flasks were washed with MEM to remove nonadherent cells. The adherent cells were greater than 98% pure macrophages.
Matrigel Invasion Assay
The in vitro invasion assay was performed as previously described (28) with minor modifications. Invasion chambers containing polycarbonate filters (8-µm pore size; BD Biosciences, Franklin Lakes, NJ) were coated with growth-factor-reduced Matrigel matrix (50 µg/filter; BD Biosciences, Bedford, MA). Macrophages were harvested from cultures by tapping the flask sharply to dislodge cells, counted, and seeded (2 x 105 cells per chamber) in the upper compartment of each invasion chamber in MEM containing 0.1% bovine serum albumin (BSA) for 24 hours at 37 °C. Either conditioned medium from 1 x 106 SKOV3.ip1 or HEY-A8 cells cultured for 48 hours or MEM was placed in the lower compartment and served as a chemoattractant. After 24 hours at 37 °C, cells were harvested from the lower compartment and from the undersurface of the filter. Each assay was performed on duplicate filters, and the experiments were repeated twice. The harvested cells were counted, and the mean number of cells per chamber was calculated and recorded.
Macrophage Chemotaxis Assay
Macrophage chemotaxis was examined by using 24-well transwell migration chambers (8-µm pore size; BD Biosciences) as described previously (29). Macrophages (1 x 106) in MEM containing 0.1% BSA were seeded in the upper compartment of each chamber. MEM containing recombinant mouse monocyte chemoattractant peptide-1 (JE/MCP-1) at 1100 ng/mL (R&D Systems, Minneapolis, MN) was added to the lower compartment. The cells were incubated at 37 °C and then harvested after 3 hours or 24 hours by scraping from the lower compartment and the undersurface of the filter and counted. All assays were performed in triplicate, and the experiments were repeated twice. The mean number of cells per chamber was calculated and recorded.
Tumor Growth In Vivo
Cultured human tumor cells were harvested by a brief treatment with 0.25% trypsin and 0.02% EDTA. Single-cell suspensions of 1 x 106 cells that had a viability of greater than 95% by trypan blue dye exclusion were injected into the peritoneal cavities of female MMP-9+/+ and MMP-9-/- nude mice (10 mice in each group) (21,30). The mice were monitored daily for evidence of disease (e.g., abdominal swelling, hunched posture, listlessness) and killed when they became moribund or at day 30 (HEY-A8 cells) or day 45 (SKOV3.ip1 cells) after the intraperitoneal injection, whichever came first. All of the mice were necropsied, and the size and volume of ascites fluid for each were recorded.
Immunohistochemistry and Quantitation of Microvessel Density
Peritoneal tumors of similar size were harvested at autopsy and processed for immunostaining as previously described (30) using the rat polyclonal antibody F4/80, which recognizes macrophage-specific antigen F4/80 (Serotec, Inc., Raleigh, NC) (27), anti-MMP-9 monoclonal antibody (human specific; Oncogene Research Products, Cambridge, MA), anti-vascular endothelial growth factor (VEGF) polyclonal antibody (mouse and human cross-reactive; Santa Cruz Biotechnology, Santa Cruz, CA), anti-basic fibroblast growth factor (anti-bFGF) monoclonal antibody (mouse and human cross-reactive; Sigma Chemical Co.), and anti-CD31/PECAM-1 monoclonal antibody, which recognizes plateletendothelial cell adhesion molecule-1 (PECAM-1) on endothelial cells (mouse specific; BD PharMingen, San Diego, CA), and the appropriate horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Negative controls were stained with nonspecific immunoglobulin G (IgG) and the appropriate horseradish peroxidase-conjugated secondary antibody. All sections were counterstained with Gill's hematoxylin. The immunostained tumor sections were examined by bright field microscopy, and the macrophages and blood vessels in 10 random 0.159-mm2 fields of each sample were counted (21,30). Microvessel density was defined as the mean number of blood vessels per field of tumor at x100 magnification (1 field = 0.159 mm2). Images were digitized by using a Sony 3CD color video camera (Sony Corp., Tokyo, Japan) and a personal computer equipped with Optimas image analysis software (Optimas Corp., Bothell, WA).
Immunofluorescence Double Staining for F4/80 and MMP-9
Frozen peritoneal tumors were cut into 8-µm sections and fixed in cold acetone. Sections were incubated with 4% fish gelatin in phosphate-buffered saline for 10 minutes to block nonspecific binding and then incubated with a 1 : 50 dilution of a goat monoclonal anti-mouse MMP-9 antibody (R&D Systems) for 18 hours at 4 °C. Bound antibody was detected by using biotinylated mouse anti-goat IgG (1 : 200 dilution; Biocare Medical, Walnut Creek, CA) and streptavidin-conjugated Alexa 594 (1 : 400 dilution; Molecular Probes, Eugene, OR). The sections were incubated for 10 minutes with 4% fish gelatin to block nonspecific binding and then overnight with F4/80 (1 : 10 dilution). Bound F4/80 was detected by using anti-rat Alexa 488 (1 : 200 dilution; Molecular Probes). Sections were mounted in medium containing 0.1 M propyl gallate to minimize photobleaching (Sigma Chemical Co.). Immunofluorescence microscopy was conducted on a Zeiss Axioplan fluorescence microscope (Carl Zeiss, Inc., Thornwood, NY). Images were captured with a cooled C5810 camera (Hamamatsu Photonics KK, Bridgewater, NJ) using Optimas software (Media Cybernetics, Silver Spring, MD) run on a Dell personal computer. MMP-9 staining was identified by red fluorescence, and F4/80 staining was detected by green fluorescence. Colocalization of MMP-9 and F4/80 was detected by yellow fluorescence.
Spleen Cell Reconstitution Experiment
Spleens were harvested from 4-week-old MMP-9+/+ and MMP-9-/- nude mice, placed in MEM, and forced through a wire mesh to disaggregate spleen cells. The spleen cells were washed with HBSS, treated for 60 seconds with ammonium chloride (140 meq/L), then washed again with HBSS. We determined spleen cell viability by trypan blue exclusion; 1 x 107 viable spleen cells were injected intravenously into each mouse. Spleen cells were injected on days 1, 3, and 5. Tumor cells (1 x 106) were injected intraperitoneally on day 6. Mice with SKOV3.ip1-derived tumors were killed on day 50. Mice with HEY-A8-derived tumors were killed on day 35. Tumors were excised and weighed, and the volume of ascites fluid for mice injected with SKOV3.ip1 cells was measured.
Statistical Analysis
The statistical significance of the in vitro results was determined by using Student's t test (two-tailed). The in vivo data were analyzed by the MannWhitney U test. All statistical tests were two-sided.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the first set of experiments, we injected SKOV3.ip1 and HEY-A8 cells into the peritoneal cavities of MMP-9+/+ and MMP-9-/- nude mice (10 mice of each genotype for each cell line). All of the MMP-9+/+ nude mice injected with either SKOV3.ip1 or HEY-A8 cells developed peritoneal tumors. By contrast, only four of the MMP-9-/- nude mice injected with HEY-A8 cells and six of the MMP-9-/- nude mice injected with SKOV3.ip1 cells developed peritoneal tumors. Moreover, the peritoneal tumors in the MMP-9-/- nude mice were statistically significantly smaller (P = .006 and .042 for HEY-A8 and SKOV3.ip1 cells, respectively) and produced statistically significantly less ascites fluid (P = .005 for SKOV3.ip1 cells) than those that developed in the MMP-9+/+ nude mice (Table 1). For example, the median weight of tumors in MMP-9+/+ nude mice injected with HEY-A8 and SKOV3.ip1 cells was 3.8 g (interquartile [IQ] range = 1.74.4 g) and 2.3 g (IQ range = 1.12.9 g), respectively, whereas in MMP-9-/- nude mice, it was 0 g (IQ range = 01.7 g) and 0.9 g (IQ range = 01.3 g), respectively. Thus, disruption of the MMP-9 gene in the recipient mice decreased tumorigenicity and progressive growth of human ovarian cancer cells in those animals.
|
Because both the human ovarian cancer cells and cells from the MMP-9+/+ mice were potential sources of MMP-9, we next determined whether the difference in tumor incidence between the MMP-9+/+ and MMP-9-/- mice was dependent on MMP-9 expressed by mouse cells or on MMP-9 expressed by the human cancer cells. Using gelatin zymographic analysis to detect collagenase activity, we found that SKOV3.ip1 cells expressed a low level of MMP-9 activity, whereas HEY-A8 cells expressed a high level of MMP-9 activity (data not shown). The two cell lines expressed similar levels of MMP-2 activity in vitro (data not shown). We also determined the in vivo expression level of MMP-9 in tumor cells growing in mice by immunohistochemistry with an anti-human MMP-9 antibody. As shown in Fig. 1, the level of MMP-9 in SKOV3.ip1 and HEY-A8 tumor cells in MMP-9-/- nude mice was similar to that in tumor cells growing in MMP-9+/+ nude mice. These data suggested that expression of MMP-9 by human ovarian cancer cells was not sufficient to promote tumorigenicity in this mouse model.
|
MMP-9 is expressed in macrophages, which are a major component of the lymphoreticular cells that infiltrate ovarian tumors that grow in humans as well as in nude mice (21,31). We used immunohistochemistry to characterize the tumor-infiltrating macrophages in SKOV3.ip1 cell- and HEY-A8 cell-derived tumors of similar size. Specific staining for macrophages with the F4/80 antibody (27) revealed the presence of macrophages throughout the HEY-A8- and SKOV3.ip1-derived tumors in MMP-9+/+ nude mice (the mean number was 269 macrophages (95% confidence interval [CI] = 231 to 307 macrophages) and 144 macrophages [95% CI = 129 to 159 macrophages], respectively) (Fig. 1 and Table 2
). By contrast, in MMP-9-/- mice, tumors derived from HEY-A8 and SKOV3.ip1 cells contained fewer macrophages (the mean number was 104 macrophages [95% CI = 87 to 121 macrophages] and 51 macrophages [95% CI = 42 to 60 macrophages], respectively) (Table 2
).
|
To determine the role of MMP-9 in macrophage function, we collected and analyzed macrophages from peritoneal exudates from 10 MMP-9+/+ and 10 MMP-9-/- nude mice that had not been injected with either human tumor cell line. The mean number of peritoneal exudate macrophages recovered from the MMP-9+/+ mice was 30.1 x 106 macrophages per mouse (95% CI = 21.6 x 106 to 38.5 x 106 macrophages), which was statistically significantly more than the mean number of peritoneal exudate macrophages recovered from the MMP-9-/- mice (16.9 x 106 macrophages per mouse, 95% CI = 9.8 x 106 to 23.4 x 106 macrophages) (P = .035, MannWhitney U test). Gelatin zymographic analysis revealed that peritoneal exudate macrophages from MMP-9-/- nude mice had no detectable MMP-9 activity, whereas those from MMP-9+/+ nude mice had substantial activity (Fig. 2, A). Moreover, the MMP-9 activity in peritoneal exudate macrophages from MMP-9+/+ nude mice could be induced by lipopolysaccharide in a dose-dependent manner (Fig. 2, A
).
|
To determine whether peritoneal exudate macrophages from MMP-9-/- nude mice also had a lower response to chemotactic signals in the absence of a Matrigel matrix, we measured the migration of the macrophages through an uncoated filter insert using JE/MCP-1 as a chemoattractant (32). We detected no statistically significant differences in chemotactic migration between peritoneal exudate macrophages from MMP-9+/+ nude mice and those from MMP-9-/- nude mice (data not shown).
Angiogenesis in Human Ovarian Tumors in MMP-9-/- and MMP-9+/+ Nude Mice
Because tumor-infiltrating macrophages have been shown to augment neoplastic angiogenesis (33), we examined whether macrophage infiltration into the human ovarian tumors was associated with the formation of blood vessels. Tumors produced by SKOV3.ip1 or HEY-A8 cells in MMP-9-/- and MMP-9+/+ nude mice were resected, and tumor sections (not necessarily adjacent) were processed for immunohistochemistry. The blood vessels in the tumors were identified in different sections by staining the sections with an anti-CD31 antibody, and macrophages were identified by using an anti-F4/80 antibody (Fig. 1). In MMP-9+/+ nude mice, human ovarian tumors derived from SKOV3.ip1 and HEY-A8 cells were highly vascularized and had a microvessel density of 70 vessels/field (95% CI = 54 to 88 vessels/field) and 118 vessels/field (95% CI = 93 to 144 vessels/field), respectively, whereas in MMP-9-/- nude mice, the mean vessel densities were 32 vessels/field (95% CI = 21 to 44 vessels/field) and 44 vessels/field (95% CI = 31 to 57 vessels/field), respectively (P = .003).
We also used immunohistochemistry to evaluate the expression levels of the proangiogenic molecules VEGF and bFGF in the peritoneal tumors. VEGF was expressed at higher levels in both SKOV3.ip1 and HEY-A8 tumors growing in MMP-9+/+ nude mice than in tumors growing in MMP-9-/- nude mice (Fig. 1). No substantial differences were found in the levels of bFGF among the four groups of mice (data not shown).
Effect on Tumorigenicity of Reconstitution of MMP-9-/- Nude Mice With Spleen Cells From MMP-9+/+ Nude Mice
The spleens of young mice are a rich source of monocytes and macrophages (34). We tested whether injection of nucleated spleen cells from young MMP-9+/+ nude mice into MMP-9-/- nude mice would stimulate the growth of SKOV3.ip1 and HEY-A8 cells injected into the peritonea of the MMP-9-/- nude mice. The presence of MMP-9-expressing macrophages in the peritonea of MMP-9-/- nude mice was confirmed by MMP-9 collagenase activity assays (data not shown). The tumorigenicity of SKOV3.ip1 cells was determined at 6 weeks after tumor cell injection. As shown in Table 2, reconstitution of MMP-9-/- nude mice with splenocytes from MMP-9+/+ nude mice was associated with a statistically significant enhancement of the growth of peritoneal tumors derived from SKOV3.ip1 cells (P = .016) and the formation of ascites fluid (P = .011). Moreover, the microvessel density of peritoneal tumors was statistically significantly higher in MMP-9-/- mice that were injected with splenocytes from MMP-9+/+ mice than that in MMP-9-/- mice injected with splenocytes from MMP-9-/- mice (P = .003), a finding that was consistent with relative levels of macrophage infiltration into the tumors (Table 2
). The growth of HEY-A8 cells in the peritoneal cavities of MMP-9-/- nude mice was statistically significantly lower than that in MMP-9+/+ nude mice (the median tumor weights were 0 g and 3.4 g, respectively, P = .041). Reconstitution of MMP-9-/- nude mice with spleen cells from MMP-9+/+ nude mice (but not with spleen cells from MMP-9-/- nude mice) was associated with a statistically significant increase in the growth of these tumors (the median tumor weights were 2.2 g and 0 g, respectively, P = .043) (Table 2
).
MMP Production by Tumor-Infiltrating Macrophages
In the final set of experiments, we examined whether MMP-9 expression in the tumor-infiltrating macrophages was associated with progressive growth of human ovarian cancers in nude mice. Tumors produced by the injection of SKOV3.ip1 or HEY-A8 cells into MMP-9+/+ or MMP-9-/- nude mice were resected and processed for immunohistochemistry. MMP-9-expressing mouse cells were detected with an anti-MMP-9 antibody that was specific for murine MMP-9 and were visualized by red fluorescent signals. Tumor-infiltrating macrophages were detected with an antibody against the macrophage-specific marker F4/80 and were visualized by green fluorescent signals. Colocalization of the two antibodies yielded a yellow fluorescent signal. Fig. 3 shows that human ovarian tumors in MMP-9+/+ nude mice contained F4/80-positive macrophages that expressed MMP-9, whereas tumors in MMP-9-/- nude mice contained F4/80-positive macrophages that did not express MMP-9. In fact, the majority of the tumor-infiltrating mouse cells in human ovarian tumors from MMP-9+/+ mice were positive for both MMP-9 and F4/80.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
MMP-9 promotes the migration and invasion of cancer cells into and out of blood vessels by mediating the proteolytic degradation of type IV collagen in the basement membrane (8,9). Experimental metastasis of murine tumor cells has been shown to be suppressed in MMP-9-deficient mice (35). MMP-9 and one of its indirect activators, urokinase-type plasminogen activator, were recently reported to be required for the intravasation of tumor cells in a chick embryo metastasis model (36). MMP-9 also contributes to carcinogenesis in pancreatic islets and in skin epithelium by triggering the angiogenic switch (10,11). In a mouse model of skin carcinogenesis, MMP-9 was predominantly expressed in inflammatory cells rather than in oncogene-positive neoplastic cells, suggesting that inflammatory cells are the critical suppliers of MMP-9 in this pathway of carcinogenesis (10). Specifically, Coussens et al. (37) have demonstrated that the mast cells that are prevalent in hyperplasia, dysplasia, and invading cancer fronts, play an important role in the angiogenic switch. In our study, we observed that mouse macrophages that expressed MMP-9 were the predominant cell type that infiltrated the human ovarian tumors. In the MMP-9-/- nude mice, reduced tumorigenicity and angiogenesis were associated with inhibition of macrophage infiltration into the lesions. Moreover, reconstitution of MMP-9-/- nude mice with spleen cells from MMP-9+/+ nude mice was associated with the infiltration of tumors by MMP-9-expressing macrophages, enhanced angiogenesis, and tumor growth.
The extent of angiogenesis is determined by the balance between positive and negative regulatory molecules that are produced by tumor cells as well as by stromal (i.e., nontumor) cells (3,4). Activated macrophages influence the angiogenic process by secreting enzymes that can break down the extracellular matrix and by secreting angiogenic molecules and growth factors, such as bFGF, transforming growth factor-alpha and -beta (TGF- and -
), insulin-like growth factor-I, platelet-derived growth factor, and VEGF/vascular permeable factor (VEGF/VPF) (3840). These factors induce endothelial cells to migrate and proliferate. The number of macrophages that infiltrate human ovarian cancers has been shown to directly correlate with microvessel density (31), and macrophages isolated from ascitic fluid aspirated from women with advanced ovarian cancer has been shown to produce angiogenic effects in vitro and in vivo (41). In clinical samples of human ovarian tumors, MMP-9 is expressed in both epithelial and stromal cells (18).
Our findingthat decreased angiogenesis of ovarian tumors in MMP-9-/- nude mice was associated with a decrease in macrophage infiltration into the tumorssupports the conclusion that macrophages positively influence the vascularization of human ovarian tumors. Moreover, we also observed that tumors from MMP-9-/- nude mice had lower levels of VEGF than did tumors from MMP-9+/+ nude mice, and that this decrease was associated with a decrease in macrophage infiltration. Thus, our data suggest that one mechanism by which macrophages could promote angiogenesis is through the proangiogenic molecule, VEGF.
To infiltrate a tissue, macrophages must penetrate the extracellular matrix. Our data provide direct evidence that MMP-9 is involved in this process. Consistent with the decrease in macrophage infiltration into tumors growing in MMP-9-/- nude mice, peritoneal exudate macrophages from MMP-9-/- nude mice were less able to penetrate a reconstituted extracellular matrix than were those from MMP-9+/+ nude mice. Whether these data are applicable to other infiltrating cells, e.g., neutrophils, is controversial (42,43).
In summary, we have demonstrated that host-derived MMP-9 contributes to the angiogenesis, growth, and formation of ascites fluid by human ovarian cancers in nude mice. In this tumor model, a major source of MMP-9 is the macrophage. Our data do not exclude the possibility that other host cells, such as endothelial cells, mast cells, and neutrophils, could have contributed MMP-9 (10,4446). In any event, we found that deficiency of MMP-9 in host cells (but not in tumor cells) inhibited neoplastic angiogenesis and, hence, the carcinomatosis of two human ovarian cancer cell lines in this animal model. Targeting expression of MMP-9 in tumor cells, and more so in nontumor cells, may therefore be an effective approach to control angiogenesis and carcinomatosis of human ovarian tumors.
![]() |
NOTES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We thank Steven D. Shapiro (Washington University School of Medicine, St. Louis, MO) for providing the MMP-9-/- 129/CD1 mice, Walter Pagel for critical editorial review, and Lola López for expert assistance in the preparation of this manuscript.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1 American Cancer Society. Cancer Facts and Figures, 2002, p. 4. Available at: http://www.cancer.org/eprise/main/docroot/STT/content/STT_1x_Cancer_Facts_Figures_2002.
2 Ozols RF, Schwartz PE, Eifel PJ. Ovarian cancer, fallopian tube carcinoma and peritoneal carcinoma. In: DeVita VT, Hellman S, Rosenberg SA, editors. Principles and practice of oncology. 5th ed. Philadelphia (PA): Lippincott-Raven; 1997. p. 150239.
3 Folkman J. The role of angiogenesis in tumor growth. Semin Cancer Biol 1992;3:6571.[Medline]
4 Fidler IJ, Ellis LM. The implications of angiogenesis for the biology and therapy of cancer metastasis. Cell 1994;79:1858.[Medline]
5 Hollingsworth HC, Kohn EC, Steinberg SM, Rothenberg ML, Merino MJ. Tumor angiogenesis in advanced stage ovarian carcinoma. Am J Pathol 1995;147:3341.[Abstract]
6 Kohn EC. Angiogenesis in ovarian carcinoma: a formidable biomarker. Cancer 1997;80:221921.[Medline]
7 Abulafia O, Triest WE, Sherer DM. Angiogenesis in primary and metastatic epithelial ovarian carcinoma. Am J Obstet Gynecol 1997;177:5417.[Medline]
8 Liotta LA, Steeg PS, Stetler-Stevenson WG. Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation. Cell 1991;64:32736.[Medline]
9 Aznavoorian S, Murphy AN, Stetler-Stevenson WG, Liotta LA. Molecular aspects of tumor cell invasion and metastasis. Cancer 1993;71:136883.[Medline]
10 Coussens LM, Tinkle CL, Hanahan D, Werb Z. MMP-9 supplied by bone marrow-derived cells contributes to skin carcinogenesis. Cell 2000;103:48190.[Medline]
11 Bergers G, Brekken R, McMahon G, Vu TH, Itoh T, Tamaki K, et al. Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nat Cell Biol 2000;2:73744.[Medline]
12 Murphy AN, Unsworth EJ, Stetler-Stevenson WG. Tissue inhibitor of metalloproteinase-2 inhibits bFGF-induced human microvascular endothelial cell proliferation. J Cell Physiol 1993;157:3518.[Medline]
13 Schnaper HW, Grant DS, Stetler-Stevenson WG, Fridman R, D'Orazi G, Murphy AN, et al. Type IV collagenase(s) and TIMPs modulate endothelial cell morphogenesis in vitro. J Cell Physiol 1993;156:23546.[Medline]
14 Valente P, Fassina G, Melchiori A, Masiello L, Cilli M, Vacca A, et al. TIMP-2 over-expression reduces invasion and angiogenesis and protects B16F10 melanoma cells from apoptosis. Int J Cancer 1998;75:24653.[Medline]
15 Guedez L, McMarlin AJ, Kingma DW, Bennett TA, Stetler-Stevenson M, Stetler-Stevenson WG. Tissue inhibitor of metalloproteinase-1 alters the tumorigenicity of Burkitt's lymphoma via divergent effects on tumor growth and angiogenesis. Am J Pathol 2001;158:120715.
16 Itoh T, Tanioka M, Yoshida H, Yoshioka T, Nishimoto H, Itohara S. Reduced angiogenesis and tumor progression in gelatinase A-deficient mice. Cancer Res 1998;58:104851.[Abstract]
17 Vu TH, Shipley JM, Bergers G, Berger JE, Helms JA, Hanahan D, et al. MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 1998;93:4112.[Medline]
18 Naylor MS, Stamp GW, Davies BD, Balkwill FR. Expression and activity of MMPS and their regulators in ovarian cancer. Int J Cancer 1994;58:506.[Medline]
19 Fishman DA, Bafetti LM, Banionis S, Kearns AS, Chilukuri K, Stack MS. Production of extracellular matrix-degrading proteinases by primary cultures of human epithelial ovarian carcinoma cells. Cancer 1997;80:145763.[Medline]
20 Moore DH, Allison B, Look KY, Sutton GP, Bigsby RM. Collagenase expression in ovarian cancer cell lines. Gynecol Oncol 1997;65:7882.[Medline]
21 Yoneda J, Kuniyasu H, Crispens MA, Price JE, Bucana CD, Fidler IJ. Expression of angiogenesis-related genes and progression of human ovarian carcinomas in nude mice. J Natl Cancer Inst 1998;90:44754.
22 Stack MS, Ellerbroek SM, Fishman DA. The role of proteolytic enzymes in the pathology of epithelial ovarian carcinoma. Int J Oncol 1998;12:569 76.[Medline]
23 Huang LW, Garrett AP, Bell DA, Welch WR, Berkowitz RS, Mok SC. Differential expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 protein and mRNA in epithelial ovarian tumors. Gynecol Oncol 2000;77:36976.[Medline]
24 Lengyel E, Schmalfeldt B, Konik E, Spathe K, Harting K, Fenn A, et al. Expression of latent matrix metalloproteinase-9 (MMP-9) predicts survival in advanced ovarian cancer. Gynecol Oncol 2001;82:2918.[Medline]
25 Davidson B, Goldberg I, Gotlieb WH, Kopolovic J, Ben-Baruch G, Nesland JM, et al. High levels of MMP-2, MMP-9, MT1-MMP, and TIMP2 mRNA correlate with poor survival in ovarian carcinoma. Clin Exp Metastasis 1999;17:799808.[Medline]
26 Yu D, Wolf JK, Scanlon M, Price JE, Hung MC. Enhanced c-erbB-2/neu expression in human ovarian cancer cells correlates with more severe malignancy that can be suppressed by E1A. Cancer Res 1993;53:8918.[Abstract]
27 Huang S, Xie K, Bucana CD, Ullrich SE, Bar-Eli M. Interleukin 10 suppresses tumor growth and metastasis of human melanoma cells: potential inhibition of angiogenesis. Clin Cancer Res 1996;2:196979.[Abstract]
28 Xie H, Turner T, Wang MH, Singh RK, Siegal GP, Wells A. In vitro invasiveness of DU-145 human prostate carcinoma cells is modulated by EGF receptor-mediated signals. Clin Exp Metastasis 1995;13:40719.[Medline]
29 Skobe M, Hamberg LM, Hawighorst T, Schirner M, Wolf GL, Alitalo K, et al. Concurrent induction of lymphangiogenesis, angiogenesis, and macrophage recruitment by vascular endothelial growth factor-C in melanoma. Am J Pathol 2001;159:893903.
30 Huang S, Robinson JB, DeGuzman A, Bucana CD, Fidler IJ. Blockade of nuclear factor-B signaling inhibits angiogenesis and tumorigenicity of human ovarian cancer cells by suppressing expression of vascular endothelial growth factor and interleukin-8. Cancer Res 2000;60:53349.
31 Orre M, Rogers PA. Macrophages and microvessel density in tumors of the ovary. Gynecol Oncol 1999;73:4750.[Medline]
32 Luini W, Sozzani S, Van Damme J, Mantovani A. Species-specificity of monocyte chemotactic protein-1 and -3. Cytokine 1994;6:2831.[Medline]
33 Polverini PJ, Leibovich JS. Induction of neovascularization in vivo and endothelial proliferation in vitro by tumor-associated macrophages. Lab Invest 1984;51:63542.[Medline]
34 van Furth R, Diesselhoff-den Dulk MM. Dual origin of mouse spleen macrophages. J Exp Med 1984;160:127383.[Abstract]
35 Itoh T, Tanioka M, Matsuda H, Nishimoto H, Yoshioka T, Suzuki R, et al. Experimental metastasis is suppressed in MMP-9-deficient mice. Clin Exp Metastasis 1999;17:17781.[Medline]
36 Kim J, Yu W, Kovalski K, Ossowski L. Requirement for specific proteases in cancer cell intravasation as revealed by a novel semiquantitative PCR-based assay. Cell 1998;94:35362.[Medline]
37 Coussens LM, Raymond WW, Bergers G, Laig-Webster M, Behrendtsen O, Werb Z, et al. Inflammatory mast cells upregulate angiogenesis during squamous epithelial carcinogenesis. Genes Dev 1999;13:138297.
38 Mantovani A, Bottazzi B, Colotta F, Sozzani S, Ruco L. The origin and function of tumor-associated macrophages. Immunol Today 1992;13:26570.[Medline]
39 Sunderkotter C, Steinbrink K, Goebeler M, Bhardwaj R, Sorg C. Macrophages and angiogenesis. J Leukoc Biol 1994;55:41022.[Abstract]
40 Koch AE, Polverini PJ, Kunkel SL, Harlow LA, DiPietro LA, Elner VM, et al. Interleukin-8 as a macrophage-derived mediator of angiogenesis. Science 1992;258:17981801.[Medline]
41 Sheid B. Angiogenic effects of macrophages isolated from ascitic fluid aspirated from women with advanced ovarian cancer. Cancer Lett 1992;62:1538.[Medline]
42 Betsuyaku T, Shipley JM, Liu Z, Senior RM. Neutrophil emigration in the lungs, peritoneum, and skin does not require gelatinase B. Am J Respir Cell Mol Biol 1999;20:13039.
43 Liu Z, Shipley JM, Vu TH, Zhou X, Diaz LA, Werb Z, et al. Gelatinase B-deficient mice are resistant to experimental bullous pemphigoid. J Exp Med 1998;188:47582.
44 Fidler IJ. Critical factors in the biology of human cancer metastasis: twenty-eighth G.H.A. Clowes memorial award lecture. Cancer Res 1990;50:61308.[Abstract]
45 Fidler IJ. Modulation of the organ microenvironment for treatment of cancer metastasis. J Natl Cancer Inst 1995;87:158892.[Medline]
46 Coussens LM, Werb Z. Inflammatory cells and cancer: think different! J Exp Med 2001;193:F236.
Manuscript received November 20, 2001; revised May 27, 2002; accepted June 10, 2002.
This article has been cited by other articles in HighWire Press-hosted journals:
![]() |
||||
|
Oxford University Press Privacy Policy and Legal Statement |