Cancer Research Meeting Calendar Jordan
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chantrain, C. F.
Right arrow Articles by DeClerck, Y. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chantrain, C. F.
Right arrow Articles by DeClerck, Y. A.
[Cancer Research 64, 1675-1686, March 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Stromal Matrix Metalloproteinase-9 Regulates the Vascular Architecture in Neuroblastoma by Promoting Pericyte Recruitment

Christophe F. Chantrain1, Hiroyuki Shimada2, Sonata Jodele1, Susan Groshen3, Wei Ye3, David R. Shalinsky4, Zena Werb5, Lisa M. Coussens6 and Yves A. DeClerck1

Departments of 1 Pediatrics and Biochemistry and Molecular Biology and 2 Pathology, Keck School of Medicine, University of Southern California and The Saban Research Institute, Childrens Hospital Los Angeles, Los Angeles, California; 3 Department of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California; 4 Department of Pharmacology, Agouron Pharmaceuticals, Inc., a Pfizer Company, La Jolla, California; and 5 Department of Anatomy and 6 Cancer Research Institute and Department of Pathology, University of California San Francisco, San Francisco, California


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Advanced stages of neuroblastoma show increased expression of matrix metalloproteinases MMP-2 and MMP-9 (Y. Sugiura et al., Cancer Res., 58: 2209–2216, 1998) that have been implicated in many steps of tumor progression, suggesting that they play a contributory role. Using pharmacological and genetic approaches, we have examined the role of these MMPs in progression of SK-N-BE (2).10 human neuroblastoma tumors orthotopically xenotransplanted into immunodeficient mice. Mice treated with Prinomastat, a synthetic inhibitor of MMPs, showed an inhibition of tumor cell proliferation in implanted tumors and a prolonged survival (50 versus 39 days in control group, P < 0.035). Treatment with Prinomastat did not affect formation of liver metastases (P = 0.52) but inhibited intravascular colonization by the tumor cells in the lung by 73.8% (P = 0.03) and angiogenesis in both primary tumors and experimental liver metastases. The primary tumors from Prinomastat-treated mice showed a 39.3% reduction in endothelial area detected by PECAM/CD31 staining in tumor sections (P < 0.001), primarily due to the presence of smaller vessels (P = 0.004). MMP-2 is expressed by neuroblastoma tumor cells and stromal cells, whereas MMP-9 is exclusively expressed by stromal cells, particularly vascular cells. To examine the contribution of MMP-9 to tumor angiogenesis, we generated RAG1/MMP-9 double-deficient mice. We observed a significant inhibition of angiogenesis in the immunodeficient RAG1/MMP-9 double-deficient mice orthotopically implanted with tumor cells (P = 0.043) or implanted s.c. with a mixture of tumor cells and Matrigel (P < 0.001). Using an FITC-labeled lectin, we demonstrated an inhibition in the architecture of the tumor vasculature in MMP-9-deficient mice, resulting in fewer and smaller blood vessels. These changes were associated with a 48% decrease in pericytes present along microvessels. Taken together, the data demonstrate that in neuroblastoma, stromally derived MMP-9 contributes to angiogenesis by promoting blood vessel morphogenesis and pericyte recruitment.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Matrix metalloproteinases (MMPs) are a family of at least 26 endopeptidases that have been associated with a number of invasive and metastatic human malignancies such as prostate, lung, breast, and colon cancer and are involved at multiple levels of tumor progression (1) . These proteinases, including MMP-2 and MMP-9 (gelatinases A and B, respectively), can degrade the extracellular matrix and basement membrane as well as growth factors, cytokines, and growth factor binding proteins (2 , 3) , suggesting that they play an active role not only in promoting tumor cell invasion and metastasis but also in regulating the tumor microenvironment (4 , 5) . In many cancers, stromal cells rather than neoplastic cells are the source of MMP production like MMP-9 (6 , 7) and MMP-13 (8) . The expression of these MMPs in stromal cells is in some cases stimulated by factors expressed by neoplastic cells (9 , 10) . In addition, MMPs contribute to the creation of a vascular stroma that presumably facilitates tumor cell proliferation. In particular, MMP-2 and MMP-9 play a positive role in activating angiogenesis by promoting the invasion of the extracellular matrix by microvascular endothelial cells and by increasing the bioavailability of vascular endothelial cell growth factor (11, 12, 13) .

Neuroblastoma is the most common extracranial solid childhood tumor. It arises from the neural crest and typically forms tumors in the adrenal medulla or paraspinal sympathetic ganglia of the abdomen, chest, or neck (14) . Although localized and well-differentiated tumors can be successfully treated by surgical resection eventually associated with adjuvant chemotherapy, the prognosis of regionally invasive (stage III) or metastatic tumors (stage IV) remains poor (15) . The most commonly involved sites of metastasis in neuroblastoma are the bone marrow, the bone, and the liver. We and others (16 , 17) have previously reported the expression of two MMPs, MMP-2 and MMP-9, in human neuroblastoma primary tumors. In these analyses, we observed that MMP-2 was produced by both neoplastic cells and stromal cells but that MMP-9 was only expressed by stromal cells. The expression of these proteases and, in particular, MMP-9 was significantly higher in stage III and IV tumors, suggesting a positive role for these MMPs in neuroblastoma progression (17) .

Taking advantage of an orthotopic metastatic human tumor xenograft model in immunodeficient mice recently developed in our laboratory (18) , we have taken a pharmacological approach using a synthetic MMP inhibitor and a genetic approach using MMP-9-deficient mice to investigate the role of MMPs in neuroblastoma progression. Our data demonstrate a critical role for MMP-9 in the formation of mature blood vessels and in the recruitment of pericytes along endothelial cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
The human neuroblastoma cell line SK-N-BE(2) was obtained from Dr. June Biedler (Memorial Sloan-Kettering Cancer Center, New York, NY). Cells were transfected with an enhanced green fluorescent protein (EGFP) cDNA as described previously (18) . One clone SK-N-BE(2) .10, stably expressing EGFP, was selected for the experiments. In experiments using fluorescent conjugated lectin, untransfected SK-N-BE(2) cells were used. EGFP-transfected and -untransfected cell lines were equally tumorigenic in immunodeficient mice. Cells were routinely grown in RPMI medium supplemented with 10% FBS, 2% penicillin-streptomycin, and 2 mM L-glutamine. Cells were split and maintained in subconfluent culture for experiments.

Animal Models.
For the pharmacological approach, 6–12-week-old female B17 Scid mice were obtained from Harlan-Sprague Dawley (St. Louis, MO). For the genetic approach, mice carrying homozygous null mutations in the MMP-9 gene (19) and RAG1 gene (20) were backcrossed into FVB/n mouse strain for five generations and maintained in the homozygous null (ko/ko) state. Immunodeficient MMP-9ko/ko mice were generated by successive intercrossing of MMP-9ko/ko mice with RAG1ko/ko mice to generate RAG1/MMP-9 double-deficient mice. All animals were maintained in a germ-free barrier facility according to the guidelines of the Childrens Hospital Los Angeles Animal Care Facility. Animal experiments were reviewed and approved by the Institution Animal Care Utilization Committee at Childrens Hospital Los Angeles. In the orthotopic metastatic model, mice were placed under general anesthesia and implanted with a 1-mm3 SK-N-BE(2) or SK-N-BE(2) .10 tumor fragment sown on the left adrenal gland as described previously (18) . Animals were observed for signs of tumor growth (abdominal distension), local tumor invasion (leg palsy), weight loss, and distress. Animals developing signs of acute distress were sacrificed by CO2/O2 euthanasia. At necropsy, cells from bone marrow, liver, and lung tissues were harvested and snap frozen for extraction of total cellular RNA. Fragments of liver and lung were obtained, fixed in 4% paraformaldehyde, and embedded in paraffin. Adrenal tumors were dissected, removed with the entire kidney, weighed, and sectioned in three pieces. One piece was fixed in 4% paraformaldehyde and paraffin embedded. Another piece was embedded in OCT, snap frozen in liquid nitrogen, and stored at -70°C. A third piece was snap frozen and stored at -70°C to obtain tissue lysates used for zymographic analysis. For the experimental liver metastasis model, 100 µl of a SK-N-BE(2) .10 cell suspension (107 cells/ml) were injected into the tail vein of 6–12 week-old female B17 Scid mice. After 28 days animals were sacrificed, the liver was carefully dissected and macroscopic metastatic nodules were counted at the surface. Selected macroscopic nodules were also dissected, snap frozen in OCT, and stored at -70°C for additional analysis.

Matrigel Plug Assay.
A total of 0.5 ml of Matrigel (10 mg/ml; Becton Dickinson Biosciences, Bedford, MA) mixed with 107 SK-N-BE(2) .10 cells was injected s.c. in the abdominal midline of 8–10-week-old male RAG1ko/ko mice and RAG1/MMP-9 double-deficient mice. After 10 days, animals were sacrificed, and the Matrigel plug was harvested. One part of the plug was weighed and dissolved in Matrisperse (Becton Dickinson Biosciences) overnight at 4°C. After centrifugation at 15,000 rpm for 15 min at room temperature, the hemoglobin content in the supernatant was quantified using the Drabkin’s procedure (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instructions. A second part of the plug was fixed in 10% formalin, paraffin embedded, stained with H&E, and examined under a light microscope.

Prinomastat Preparation and Administration.
Prinomastat (AG3340) was provided by Agouron Pharmaceuticals, Inc., a Pfizer Company (La Jolla, CA). Prinomastat was solubilized in acidified water (pH 2.3) at a final concentration of 20 mg/ml. The solution was filtered under sterile condition, stored at 4°C, and freshly made every 2 weeks. Prinomastat was given by gastric gavage using 22G feeding needles at a dose of 100 mg/kg twice a day, 6 days a week until sacrificed. Control animals received an equal volume of vehicle solution (acidified water).

Immunohistochemistry.
The presence of endothelial cells in tumor tissue was determined by immunohistochemical detection of PECAM/CD31 and quantified on whole sections by using a computerized method recently described by us (21) . Briefly, frozen 7-µm sections were incubated in the presence of a rat antimouse PECAM/CD31 antibody (Product 01951D; PharMingen, San Diego, CA). A biotinylated goat antirat antibody (Product 31831; Pierce, Rockford, IL) was used as secondary antibody. The sections were then incubated with an avidin-biotin-peroxidase complex (Vectastain ABC kit; Vector Laboratories, Inc., Burlingame, CA), revealed in the presence of 3,3'-diaminobenzidine tetrahydrochloride (Sigma-Aldrich, St. Louis, MO), and counterstained in 1% methylgreen. Digital pictures of the whole tumor tissue sections were acquired using a high-resolution Polaroid SprintScan 4000 35-mm film slide scanner (Polaroid Corp., Cambridge, MA) and a PathScan Enabler 4000 microscope slide holder (Meyer Instrument Co., Houston, TX). Using MetaMorph 4.6 software, the endothelial area (EA), the microvessel density (MVD), and the mean vessel size (MVS) were determined on whole tumor tissue sections. For each tumor, a minimum of three serial sections were analyzed. The detection of tyrosine hydroxylase (TH), an enzyme specifically expressed by neuroblastoma cells (22) , was similarly performed by immunohistochemistry. Four-µm sections from paraffin-embedded tissues were pretreated with Antigen Retrieval Citra (Bio Genex, San Ramon, CA) for 20 min in steam water. Slides were incubated with an antihuman TH monoclonal antibody (Pel-Freez Biologicals, Rogers, AR) at a 1:1000 dilution at 37°C for 30 min. The sections were then processed with Ventana Enhanced DAB Detection kit according to the manufacturer’s instructions. Nuclear counterstaining was done with Mayer’s hematoxylin. The presence of neoplastic cells in mouse tissues was quantified by calculating the number of TH-positive cells in 10 randomly chosen microscopic fields under x400 magnification. For bromodeoxyuridine (BrdUrd) staining, mice were injected with BrdUrd (100 mg/kg) i.v. 1 h before being sacrificed to optimize incorporation in rapidly proliferating tumor cells compared with normal cells. The detection of BrdUrd was performed on 4-µm paraffin-embedded tumor sections pretreated with 0.1% trypsin for 10 min and blocked with 0.5% rabbit serum and 0.1% Tween. The slides were then incubated in the presence of an anti-BrdUrd mouse monoclonal antibody (Product RPN202; Amersham Pharmacia Biotech, Inc., Piscataway, NJ) at a 1:50 dilution for 1 h at room temperature. After washing in PBS, the slides were incubated in the presence of a biotinylated goat antimouse antibody (Vector Laboratories, Inc.) at a 1:100 dilution for 30 min at room temperature. The sections were then incubated with an avidin-biotin-peroxidase complex, revealed in the presence of 3,3'-diaminobenzidine tetrahydrochloride, and counterstained with Mayer’s hematoxylin as previously indicated. The number of BrdUrd-positive nuclei in tumor cells was counted in five randomly chosen microscopic fields under x200 magnification and calculated as a percentage of the total number of tumor cell nuclei in the same field. Sections were also examined for apoptosis and Ki67 expression. For apoptosis, we used the terminal deoxynucleotidyl transferase-mediated nick end labeling assay according to the instructions of the manufacturer (Apoptag Apoptosis Detection Systems; Serologicals Corp., Norcross, GA). For Ki67 expression, we used a monoclonal mouse antihuman Ki67 antibody as primary antibody (dilution 1:50; DAKO Corp., Carpinteria, CA) and a biotinylated multiswine antigoat, mouse, and rabbit immunoglobulin as secondary antibody (dilution 1:100; DAKO Corp.). The expression of MMP-9 was examined by immunohistochemistry on 4-µm paraffin-embedded tumor sections pretreated with proteinase K (Roche Diagnostics Corp., Indianapolis, IN) at a concentration of 10 µg/ml at 37°C for 5 min. After washing, the slides were incubated overnight at 4°C in the presence of 1:1000 diluted rabbit antimurine MMP-9 antibody as described previously (19) . A biotinylated goat antirabbit antibody (Vector Laboratories, Inc.) at a dilution of 1:100 was used as a secondary antibody. Slides were then treated with a horseradish peroxidase-conjugated streptavidin (Product RPN1231; Amersham Pharmacia Biotech, Inc.) diluted at 1:200 for 1 h at room temperature, revealed with 3,3'-diaminobenzidine tetrahydrochloride, and counterstained with 1% methylgreen. The detection of smooth muscle actin (SMA) was performed on 5-µm paraffin-embedded sections, using a monoclonal mouse anti-SMA antibody (dilution 1:50; DAKO Corp.) as primary antibody and a biotinylated swine antigoat, mouse, and rabbit immunoglobulin as secondary antibody. To detect the presence of endothelial cells and pericytes in the same histological sections, we used paraffin-embedded tumor sections boiled in unmasking solution (Vector H3300) by three cycles of 4 min in a microwave (1300 W, power 5). We used a goat polyclonal anti-PECAM/CD31 antibody (Santa Cruz Biotechnology) and a mouse monoclonal antihuman SMA antibody as primary antibodies. As secondary antibodies we used a Cy3-conjugated donkey antigoat IgG (Jackson Immunoresearch) at a dilution of 1:200 and a FITC horse antimouse IgG (Vector Laboratories, Inc.) at a dilution of 1:200. Slides were examined at x16 under a Leica fluorescent microscope and analyzed for red (PECAM/CD31) and green (SMA) fluorescence. Quantification was done using Metamorph 4.6 software.

Real-Time Reverse Transcriptase-PCR for the Detection of EGFP-Expressing Cells.
Quantification of mRNA coding for EGFP in total cellular RNA obtained from bone marrow, lung, and liver tissues was achieved by real-time reverse transcriptase-PCR. cDNA was obtained from total cellular RNA using the Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD) in the presence of random primers. Values for the amount of EGFP RNA were normalized for the amount of 18 sRNA present in each sample. For the detection of EGFP mRNA, we used the following primers: sense primer: 5'-GAACTCCAGCAGGACCATGTG-3'; antisense primer: 5'-CTGCTGCCCGACAA CCA-3'; and fluorescent-labeled probe 5'-6FAM-CCCTGAGCAAAGACCCCAACGAG A-TAMRA-3'. For the detection of 18 sRNA, the sense primer was 5'-CGGCTACCACATCCAAGGAA-3', the antisense primer was 5'-GGGCCTCGAAA GAGTCCTGT-3', and the probe was 5'-TET-CAGCAGGCGCGCAAATTACCC-TAMRA-3'. The amplification reaction was performed in the presence of 1 µg of cDNA, 200 nmol of each primer, 200 nmol of probe, and 25 µl of Taqman Universal Master Mix (Applied Biosystems, Foster City, CA) for a total volume of 50 µl. EGFP and 18 sRNA were amplified in separate wells. The amplification reaction included 40 cycles with a denaturation step at 95°C for 15 s and a primer annealing extension step at 60°C for 1 min. The analysis was performed using ABI Prism 7700 sequence detector (Applied Biosystems). For each plate, a standard curve of the cycle threshold (Ct) versus the relative amount of EGFP RNA was obtained by measuring the Ct value for SK-N-BE(2) .10 total cDNA serially diluted in the presence of murine liver cDNA. The Ct of each sample was then converted in arbitrary units based on the standard curve and was normalized for the amount of 18 sRNA. Each sample was analyzed in duplicate, and the standard curve was performed using triplicate samples of each serially diluted mixture.

Transmission Electron Microscopy.
Tumor samples were fixed with 2% glutaraldehyde in 0.1 M PBS (pH 7.4) for 3 h and postfixed with 2% OsO4 for 1 h. The samples were then dehydrated through graded ethanol solutions and embedded in Epon. Ultra-thin sections were cut, mounted on collodion one-hole grids, stained with uranyl acetate and lead citrate, and examined with a Philips CM12 transmission microscope.

SDS-Polyacrylamide Gelatin Zymography.
Frozen tumor tissues were homogenized in lysis buffer [10 mM Tris HCl (pH7.4), 150 mM NaCl, 10% glycerol, and 1% Triton X-100]. Aliquots of tumor lysates containing 10 µg of proteins were loaded on a 1% gelatin, 0.1% SDS, 8% polyacrylamide gel, and electrophoresed at 120 V for 2 h at 4°C. After electrophoresis, gels were incubated overnight at 37°C in substrate buffer [50 mM Tris, HCl (pH 7.4), and 10 mM CaCl2], stained with Coomassie Brilliant Blue, and destained in methanol:acetic acid:water (50:10:40). Aliquots of serum-free conditioned media from human HT1080 cells and murine NIH3T3 cells were used as markers for human and murine MMP-2 and MMP-9.

Fluorescent Angiography.
Tumor vasculature was visualized by fluorescent angiography using a FITC-labeled Lycopersicon esculentum lectin (Vector Laboratories, Inc.) injected i.v. (100 µg in 100 µl of 0.9% NaCl) in mice. Two min after lectin injection, mice were anesthetized with 2% avertin, the chest was opened, the right atrium was incised to drain the blood, and the vasculature was washed free of blood by slow perfusion of 20 ml of 0.9% NaCl into the left ventricle. Tumors were then harvested and examined unfixed under fluorescent confocal microscopy. We used a Leica SP confocal DM IRBE-inverted microscope with a 488-nm argon ion laser and a RSP500 beam splitter. Detection of the Lycopersicon esculentum lectin was achieved with a 503–537-nm emission band, and nonconfocal visualization of the tumor, suture, and fat tissue was done with a 488-nm transmitted light channel. Datasets typically represented 1000 x 1000 x 120 µm (512 x 512 pixels x 61 planes). The analysis used the entire z-series of all three channels.

Statistical Analysis.
Kaplan-Meier plots and the log-rank test were used to compare survival time between the Prinomastat-treated and the control mice. Survival time was calculated as the days from the date of tumor implantation to the date of death. The effect of Prinomastat on macroscopic tumor growth was tested by using a two-sample t test comparing the tumor weights of mice in the treated group and in the control group at day 28. The analysis for cell proliferation was based on arcsine-transformed data. ANOVAs were performed to test the difference in EGFP expression in tumor cells in the lung and the liver, comparing the control and the Prinomastat-treated mice and adjusting for the survival time. The main effects of treatment and survival and the interaction between treatment and survival were tested. Animals were grouped based on the calendar time they were studied, and these groupings were included in the analysis. For the analysis of EGFP mRNA detection by reverse transcriptase-PCR, the mean percent change in EGFP expression from the control to the treated group was determined and the associated 95% confidence interval (CI) was calculated using the SD obtained by the delta method. The treatment effect on TH immunohistochemistry was tested using two-sample t test. EA, MVD, and MVS measurements on PECAM/CD31-stained sections were compared between the control and the Prinomastat-treated mice, using ANOVA. To compare the detection of macroscopic liver nodules in mice injected i.v. with neuroblastoma cells between the treated and the untreated group, we used the Kruskal-Wallis test. The percent change in EA and its 95% CI were calculated using the method similar to that for EGFP expression described above. The analyses for MVD and MVS were based on log-transformed data to reduce the heteroscedasticity, in particular, the association between the means and the SDs. Means and 95% CIs were calculated based on the log-transformed data and then transformed back to the original scale. EA was also compared among the control, the mice receiving early treatment of Prinomastat, and the mice receiving late treatment of Prinomastat using ANOVA based on the log-transformed data. The least significant difference method was used for the pairwise comparisons once the overall F test was significant at the 0.05 level. ANOVA was also used to compare EA, MVD, and MVS between the MMP-9-deficient mice and the control mice and to compare hemoglobin concentrations among control, MMP-9-deficient, and Prinomastat-treated control mice. The least significant difference method was used for the pairwise comparisons of hemoglobin concentrations once the overall F test was significant at the 0.05 level. Correlation analyses were done by examining the scatter plots and by calculating the Spearman correlation coefficient.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Tumor Progression in Mice Orthotopically Implanted with Neuroblastoma Tumors.
We first evaluated the sequence of tumor progression, including growth of primary tumor, angiogenesis, and formation of distant micrometastases in an orthotopic model of neuroblastoma development. This was achieved by growing tumor fragments derived from EGFP expressing SK-N-BE(2) .10 neuroblastoma cells near the left adrenal gland of 15 Scid mice, which were sacrificed at specific time intervals. The systematic histological analysis of primary tumors harvested at days 7, 14, and 21 after implantation identified the contribution of angiogenesis to the growth of the primary tumor (Fig. 1, A and B)Citation . At day 7, a small tumor of the size of the original fragment (mean diameter, 1.0 ± 0.2 mm) was found. This tumor tissue consisted primarily of tumor cells with no evidence of vascularization. At day 14, tumors were the same size (mean diameter, 1.3 ± 0.4 mm), but there was histological evidence of vascularization as indicated by the presence of RBC in capillary structures. At day 21, the tumors were larger (mean diameter, 4.1 ± 0.7 mm) and consisted of islands of neoplastic cells surrounded by stromal septa rich in PECAM/CD31-positive endothelial cells. The appearance of tumor cells in distant organs, including the lung, the liver, and the bone marrow, was detected by EGFP mRNA measurement using real-time reverse transcriptase-PCR (Fig. 1C–E)Citation . On days 14 and 21, the levels of EGFP-expressing tumor cells in these organs were below the detection limit (10-6). On day 28, four of six specimens examined were positive for tumor cells, and by day 35, six of six specimens were positive. The presence of tumor cells in the lung and the liver was confirmed by TH immunohistochemistry on day 35. This analysis revealed an important difference in the localization of the tumor cells between the lung and the liver (Fig. 1, F and G)Citation . In the lung, tumor cells were confined to the inside of blood vessels without evidence of extravasation. In the liver, the cells were located in the parenchyma and showed signs of local invasion.



View larger version (76K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Tumor progression in mice orthotopically implanted with human neuroblastoma tumors. A, H&E-stained sections of orthotopic tumors harvested at days 7, 14, and 21. Neuroblastoma cells (N) are seen around the surgical suture (S) used for implantation. The tumor tissue was progressively infiltrated by capillaries (Cap). Immunohistochemistry for PECAM/CD31 on a tumor obtained at day 21 is shown as inset. B, the data represent the mean tumor diameter (±SD) of three tumors harvested at each time point. C–E, the presence of tumor cells in the lung (C), the liver (D), and the bone marrow (E) was detected by real-time reverse transcriptase-PCR on mRNA extracted from these organs at indicated time. The data represent the amount of enhanced green fluorescent protein (EGFP) mRNA in arbitrary units (A.U.) in organs obtained from two mice at each time point. F and G, presence of tumor cells detected by tyrosine hydroxylase immunohistochemistry on day 35 in the lung vasculature (F) and the liver parenchyma (G). The basement membrane was identified by staining with periodic acid Schiff. Arrows indicate invasion of the liver parenchyma by tumor cells. Bars are 100 µm (A) and 50 µm (F and G).

 
Neuroblastoma Tumors Express MMP-2 and MMP-9.
The presence of MMPs in primary tumors was examined by gelatin zymography (Fig. 2A)Citation . In all samples, a gelatinolytic band comigrating with murine pro-MMP-9 from NIH3T3 cells was detected. The electrophoretic mobility of this MMP in the polyacrylamide gel indicated its murine origin compared with the faster migrating human pro-MMP-9 derived from HT1080 cells. In some specimens (samples 1 and 2), a slightly faster migrating band was also detected. Evidence that this band represents the active form of MMP-9 was obtained by treating the tumor sample with the pro-MMP activator, p-amino phenylmercuric acetate (1 µM at 37°C for 30 min). Thus, although MMP-9 was mainly found in a proform, in some tumor samples the activated form was detected. Pro-MMP-2 and MMP-2 were also detected in all tumor samples. Neither pro nor mature MMP-9 was detected in the conditioned medium of the SK-N-BE(2) .10 cell line or in an extract from a murine adrenal gland. This observation is consistent with our previously reported finding that in human neuroblastomas the expression of MMP-9 is restricted to stromal cells (17) . Pro-MMP-2 and pro-MMP-9 were also present in the lysate of metastatic liver nodules obtained after i.v. injection of tumor cells (experimental metastatic model, see below). In these specimens, the murine origin of pro-MMP-9 was similarly demonstrated by its slower mobility (Fig. 2B)Citation . Complete inhibition of the gelatinolytic activity of these proteases in the gels was achieved by incubation overnight in the presence of Prinomastat at a concentration of 10 ng/ml or higher, confirming that these gelatinolytic activities were because of MMPs (Fig. 2C)Citation .



View larger version (58K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Expression of matrix metalloproteinases (MMPs) in tumor lysates and inhibition by Prinomastat. A, the presence of MMPs in lysates of three primary tumors incubated or not with the pro-MMP activator p-amino phenylmercuric acetate (APMA) and one adrenal gland was examined by SDS-polyacrylamide gelatin zymography as described in "Materials and Methods." Samples of serum-free conditioned media of SK-N-BE(2)(2).10, NIH3T3, and HT1080 cells were used as controls. m indicates murine origin, and h indicates human origin. When indicated, samples were incubated with APMA (1 µM) for 30 min at 37°C. B, the presence of pro-MMP-2 and pro-MMP-9 in four liver metastases and a sample of normal liver was detected by zymography. C, inhibition of MMP activity in the lysates of a primary tumor and a liver metastasis was documented by incubating the gels overnight in the presence of increased concentrations of Prinomastat. For each experiment shown in A to C, 10 µg of tumor lysate were loaded into each lane of the gels.

 
Effect of MMP Inhibition on Mouse Survival and Tumor Proliferation.
To assess the role of MMP-2 and MMP-9 on neuroblastoma progression, we initiated a series of experiments in which mice orthotopically implanted with neuroblastoma tumors were treated from day 1 after implantation with Prinomastat and examined for primary tumor development. We selected a dose of 100 mg/kg as previously used and documented a mean plasma level of Prinomastat of 2163 ng/ml 1 h after oral administration (n = 5, data not shown). Although we did not examine plasma levels at other times, published pharmacokinetic studies (23) indicate that this selected dose and schedule provide a plasma level constantly >1 ng/ml, a concentration that is well above the Ki for MMP-2 (0.02 ng/ml) and MMP-9 (0.11 ng/ml). Two experiments involving a total of 38 mice were done, and the data of one experiment, with 17 mice, are presented in Fig. 3ACitation . They indicate a positive effect of Prinomastat on survival that was statistically significant with a median survival of 50 days for the treated group versus 39 days for the control group (P = 0.035). To evaluate the effect of Prinomastat on tumor growth, we performed an additional experiment in 18 mice. These mice (9 treated and 9 untreated) were sacrificed 28 days after implantation, and BrdUrd was injected i.v. before sacrifice to measure tumor cell proliferation. We observed a mean wet tumor weight of 0.22 g (95% CI = 0.06–0.37) in the treated group compared with 0.47 g (95% CI = 0.09–0.86) in the untreated group (Fig. 3B)Citation . The range of tumor weights in the control group was large, and the difference between the two groups was not statistically significant (P = 0.17). However a more precise measurement of tumor growth such as the analysis of BrdUrd incorporation in tumor cells (Fig. 3C to E)Citation indicated a decrease in BrdUrd-positive tumor cells with a mean of 60.9% positive tumor cells in treated mice versus 79.4% in the control group. This difference was small but statistically significant (P = 0.007). The data are consistent with Prinomastat having an inhibitory effect on tumor cell proliferation, although not sufficient to influence tumor mass.



View larger version (55K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Effect of Prinomastat treatment on tumor-bearing mouse survival and tumor growth. A, Kaplan-Meier plot of survival time between Prinomastat-treated (n = 9) and control (n = 8) mice. The data shown are from one of two experiments. B, the data represent the wet weight of each tumor (in g) obtained from mice treated with Prinomastat or untreated and sacrificed at day 28 (n = 9 in each group). The mean values are shown by a bar. C, the data represent the mean percent (±SD) of bromodeoxyuridine (BrdUrd)-positive nuclei of tumor cells counted in five microscopic fields (x200) in histological sections of tumors obtained from control (n = 6) and treated (n = 5) mice. D and E, representative histological sections of tumors immunostained for BrdUrd. Bars are 50 µm./

 
Effect of MMP Inhibition on Lung and Liver Colonization.
Considering the well-known involvement of MMPs in tumor metastasis (1 , 2 , 4) , we examined the effect of Prinomastat treatment on the development of spontaneous micrometastases. This analysis was performed by real-time reverse transcriptase-PCR quantification of EGFP mRNA in samples of lung and liver harvested from mice treated and not treated with Prinomastat. In the lung, there was a significant inhibition in the tumor burden detected after Prinomastat treatment (decrease by 73.8%, 95% CI = 40.9–100, P = 0.03) compared with untreated mice, after adjusting for the survival time. Furthermore, there was a slower rate of increase of EGFP detection with time in Prinomastat treated mice compared with the control group (P = 0.022 for the interaction term; Fig. 4ACitation ). In the liver, Prinomastat had no effect on tumor burden (P = 0.52; Fig. 4BCitation ). Immunohistochemistry of TH in these organs consistently indicated an inhibitory effect of Prinomastat treatment on the presence of neoplastic cells in the lung but not in the liver (Fig. 4, C and DCitation , P < 0.001 for the lung, P = 0.10 for the liver). As previously observed, tumor cells were confined to the inside of blood vessels in the lungs, whereas in the liver, they were present in the parenchyma (Fig. 4E–H)Citation . To confirm the absence of effect of Prinomastat treatment on liver colonization, we tested its effect on the formation of macroscopic experimental metastases in the liver after i.v. injection of tumor cells. For this experiment, three groups of 15 mice each, injected i.v. with SK-N-BE(2) .10 cells, were examined 28 days after injection for the presence of macroscopic metastatic tumor nodules on the surface of the liver. Treatment with Prinomastat was initiated either 48 h before tumor cell injection (early treatment) or 48 h after tumor cell injection (late treatment), and control mice were treated with the vehicle solution. The data (Table 1)Citation revealed no difference in the number of mice developing macroscopic liver metastases and in the number of macroscopic liver nodules among the three experimental groups (P = 0.66). These data confirm an absence of involvement of MMPs in neuroblastoma liver colonization.



View larger version (64K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Effect of Prinomastat on tumor burden in lung and liver. The data represent an analysis performed in two experiments in which SK-N-BE (2).10 tumor-bearing mice were treated and not treated with Prinomastat. A and B, the detection of tumor cells in the lung (A) and the liver (B) was achieved by real-time reverse transcriptase-PCR analysis of enhanced green fluorescent protein (EGFP) mRNA. The data represent the amount of EGFP mRNA [in arbitrary units (A.U.)] for each sample of organ harvested at the indicated time (days after implantation). C and D, the data represent the mean number of tyrosine hydroxylase (TH)-positive cells/10 microscopic field (x400) in sections of each organ (C = lung, D = liver) obtained at the time of death (n = 6 in each group). E–H, representative histological sections stained for TH. E and G = lung; F and H = liver. The inset in E shows the staining of the basement membrane by periodic acid Schiff. Bars are 50 µm (E and G), 20 µm (F), and 40 µm (H). Closed symbols = control; open symbols = treated.

 

View this table:
[in this window]
[in a new window]

  		Table 1 Effect of Prinomastat treatment on experimental neuroblastoma metastasis

 
MMPs Contribute to Neuroblastoma Angiogenesis.
We also examined the effect of Prinomastat on angiogenesis in primary orthotopic tumors by immunodetection of cells expressing the endothelial cell marker PECAM/CD31. We performed three independent measurements on whole tumor sections stained for PECAM/CD31 to calculate the EA, the MVS, and the MVD using a recently reported computerized method developed in our laboratory (Fig. 5ACitation ; Ref. 21 ). Prinomastat had no effect on the MVD (P = 0.95) but had a significant inhibitory effect on the EA (average, 39.3% reduction, 95% CI = 9.9–48.7, P < 0.001) and on the MVS (average, 48.8% reduction, 95% CI = 6.4–58.9, P = 0.004). There was a weak correlation between EA and MVS in this experiment (r = 0.41). However, this association did not reach statistical significance (P > 0.10) because the number of animals for this analysis (n = 18) was limited. Thus, the data suggest that a primary effect of MMP inhibition on angiogenesis is on the size of the blood vessels rather than on their number. This was confirmed by routine microscopic examination of PECAM/CD31-stained sections (Fig. 5, B and C)Citation . When this analysis was performed on experimental liver metastasis in mice injected i.v. with SK-N-BE(2) .10 cells, a similar inhibitory effect of Prinomastat on EA was observed (P = 0.001, data not shown).



View larger version (40K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Effect of matrix metalloproteinase (MMP) inhibition on tumor angiogenesis. A, microvessel density (MVD), endothelial cell area (EA), and microvessel size (MVS) were quantified on whole sections of primary tumors immunostained for PECAM/CD31. The data represent the values for each parameter from 8 control tumors and 9 treated tumors. The mean values are indicated by a bar. B and C are representative histological tumor sections stained for PECAM/CD31 expression (bars are 100 µm).

 
Stromal MMP-9 Contributes to Neuroblastoma Angiogenesis and is Expressed by Vascular Cells.
Because both MMP-2 and MMP-9 are present in this model of neuroblastoma progression and both proteases are effectively inhibited by Prinomastat, the above described experiments do not discriminate between the specific contributions of each MMP to angiogenesis. We exploited the observation that in neuroblastoma, MMP-9 is exclusively expressed by stromal cells and generated MMP-9 null mice in a RAG1-deficient background. Control RAG1ko/ko mice (n = 13) and RAG1ko/ko/MMP-9ko/ko mice (n = 12) were implanted with SK-N-BE(2) .10 tumor fragments obtained from s.c. tumors grown in RAG1ko/ko/MMP-9ko/ko mice to ensure an absence of MMP-9 in the implant. The tumors were harvested 2 and 4 weeks after implantation. As anticipated, pro-MMP-9 was not detectable by zymographic analysis in tumor extracts derived from MMP-9ko/ko mice but was present in tumors obtained from control mice. In contrast, tumors from both types of mice contained a similar amount of pro-MMP-2, indicating that MMP-2 expression did not compensate for a lack of MMP-9 expression in tumor tissue (Fig. 6A)Citation .



View larger version (57K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 6. Inhibition of angiogenesis in tumor-bearing MMP-9ko/ko mice. A, The expression of matrix metalloproteinase (MMP)-2 and MMP-9 in primary tumors derived from MMP-9ko/ko and control mice was detected by SDS-polyacrylamide gelatin zymography. In each lane, 10 µg of tumor lysate were loaded. The data represent two tumor samples in each group and are representative of 10 samples similarly analyzed. B, microvessel density (MVD), endothelial cell area (EA), and microvessel size (MVS) were determined in PECAM/CD31-immunostained sections of tumors implanted in control and MMP-9ko/ko. The data show the measurements obtained for each parameter with the mean indicated by a bar. Closed symbols = control (n = 13); open symbols = MMP-9ko/ko (n = 12). C and D, representative histological sections stained for PECAM/CD31 in control and MMP-9ko/ko mice. Bars are 100 µm.

 
Similar to our previous studies with Prinomastat, we observed no significant difference in MVD in PECAM/CD31-stained sections of primary tumors among both groups (mean MVD, 12.03 vessels/10,000 pixels in MMP-9ko/ko mice versus 12.79 in control mice, P = 0.41; Fig. 6BCitation ). However, the mean values for EA and MVS in tumors derived from MMP-9ko/ko mice were significantly lower compared with the mean values obtained in tumors in the control group (mean EA = 4.46%, 95% CI = 4.15–4.77 in MMP-9ko/ko mice and 6.17%, 95% CI = 5.90–6.44 in control, P = 0.043; mean MVS = 36.14 pixels, 95% CI = 33.24–39.04 pixels in MMP-9ko/ko; and 49.01 pixels, 95% CI = 46.96–51.53 pixels in control, P = 0.037). Consistently, we observed smaller microvessels in MMP-9-deficient mice by histological examination (Fig. 6, C and D)Citation . There was a statistically significant correlation between EA and MVS in all tumors (n = 25) with larger vessel sizes leading to greater EA (r = 0.82; P < 0.001).

In previous other tumor models, e.g., skin carcinogenesis (7) and ovarian cancers (24) , MMP-9 expressed by inflammatory cells like mast cells and macrophages has been found to contribute to angiogenesis. Accordingly we determined the origin of MMP-9 by immunohistochemistry. We observed the presence of the protein in the tumor stroma and in connective tissue septa surrounding nodules of tumor cells where it was predominantly present around blood vessels and in vascular cells. Consistently, no protein was detected in tumors derived from MMP-9ko/ko mice (Fig. 7)Citation . Taken together, the observation that MMP-9 is predominantly present in vessels and the observation that smaller vessels are present in tumors derived from MMP-9ko/ko mice suggest that MMP-9 has a direct role in regulating vasculature architecture.



View larger version (151K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 7. Matrix metalloproteinase (MMP)-9 expression in neuroblastoma tumors. MMP-9 expression was detected by immunohistochemistry in tumors derived from control (A and C) and MMP-9ko/ko mice (B and D). Bars are 100 µm in (A) and (B) and 25 µm in (C) and (D). Open arrowheads: vascularized connective tissue septum. Closed arrowheads: microvascular endothelial cells; asterisk: intravascular RBCs; T = tumor nodule.

 
Effect of MMP-9 on Tumor Vascular Architecture.
To confirm the effect of MMP-9 on blood vessel size and determine whether it is affecting the formation of mature perfused (carrying red cells) blood vessels, we used a plug of polymerized Matrigel mixed with SK-N-BE(2) .10 cells and implanted into RAG1ko/ko/MMP-9ko/ko (n = 5) and RAG1ko/ko control mice untreated (n = 6) and treated (n = 6) with Prinomastat. Ten days after implantation, the plugs were collected and examined for hemoglobin concentration and for the presence of blood vessels on routine H&E-stained sections. In contrast to PECAM/CD31 immunohistochemistry, this assay, which measures hemoglobin concentration, is a better index of vascularization. Plugs implanted in MMP-9ko/ko mice and in Prinomastat-treated control mice were macroscopically less vascularized than plugs implanted in untreated control mice (Fig. 8A)Citation . Consistently, we observed a significant decrease in hemoglobin concentration in plugs derived from MMP-9ko/ko mice (mean hemoglobin concentration = 0.74 g/dl, 95% CI = 0.18–1.29, P < 0.001) and from Prinomastat-treated control mice (mean hemoglobin concentration = 1.34 g/dl, 95% CI = 0.83–1.85, P < 0.001) when compared with untreated control mice (mean hemoglobin concentration = 3.32 g/dl, 95% CI = 2.81–3.82, the overall P for comparing the three groups in terms of hemoglobin concentration was <0.001; Fig. 8BCitation ). Interestingly, whereas Matrigel plugs obtained from untreated control mice contained numerous microvascular structures filled with RBC located around clusters of tumor cells, plugs obtained from MMP-9ko/ko mice or treated control mice contained fewer vascular structures that were empty of RBC, suggesting an effect of MMP-9 on the formation of an organized vascular network (Fig. 8C–E)Citation .



View larger version (84K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 8. Inhibition of vascularization of Matrigel plugs in MMP-9ko/ko and Prinomastat-treated mice. A, photograph of Matrigel plugs harvested 10 days after implantation. B, the data represent the values of hemoglobin concentration in g/dl in each plug analyzed, with the mean values indicated by a bar (control and treated control n = 6, MMP-9ko/ko n = 5). C–E, paraffin-embedded sections of Matrigel plugs stained with H&E. Open arrowheads indicate RBCs, and closed arrows indicate the presence of endothelial cells. Clusters of tumor cells are indicated by T. Bars are 50 µm.

 
We next used fluorescent angiography in vivo in orthotopic tumors derived from SK-N-BE(2) cells that do not express EGFP and compared the vascular architecture between MMP-9ko/ko and control mice (Fig. 9)Citation . This analysis revealed a marked difference in vascular organization between tumors derived from MMP-9ko/ko mice and tumors derived from control mice. In small (<1.5 mm in diameter) tumors obtained 15 days after implantation, some blood vessels stained with FITC-lectin were seen in control mice, whereas in MMP-9ko/ko mice, only rare fluorescent microvessels were detected, and areas of extravasated FITC-lectin were seen. In larger tumors obtained at days 23 and 31 after implantation, a significant network of fluorescent microvessels was detected in tumors from control mice, whereas in tumors from MMP-9ko/ko mice, only a few blood vessels were observed, and there was little evidence of vascular organization.



View larger version (133K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 9. Effect of matrix metalloproteinase (MMP)-9 on vascular architecture. Control mice (A–C) and MMP-9ko/ko mice (D–F) implanted with SK-N-BE(2) tumors were injected with FITC-labeled lectin before sacrifice as described in "Materials and Methods." Tumors were harvested at indicated times and examined unfixed under confocal microscope. The figures are a reconstruction of the maximum intensity obtained in the entire Z-series acquired at 2-µm intervals over a thickness of 120 µm. The space occupied by the suture, the retroperitoneal fat, and the tumor tissue was delineated by examination of the specimen under transmitted light and is represented by white dotted lines. The data are representative of two experiments with 6 mice each. Bars are 100 µm. Open arrowhead: extravasated FITC lectin.

 
To obtain additional insight into the role of MMP-9 on the tumor vasculature and, in particular, to evaluate the ability of newly formed microvessels to recruit pericytes, sections of primary orthotopic tumors derived from MMP-9ko/ko mice and control mice were immunostained for the presence of SMA. This analysis revealed a significant difference between MMP-9ko/ko and control. In tumors from control mice, SMA-positive pericytes formed a uniform layer around vascular structures containing RBCs. In contrast, in tumors derived from MMP-9ko/ko, the layer of SMA-positive cells was discontinuous, suggesting a lack of pericyte recruitment along blood vessels in MMP-9ko/ko mice (Fig. 10, A and B)Citation . This observation was confirmed by transmission electron microscopy analysis of these tumor samples. In tumors derived from control mice, a uniform and continuous layer of pericytes was detected around the endothelial cells. In tumors derived from MMP-9ko/ko mice, few pericytes were detected, and they only partially surrounded the endothelial layer (Fig. 10, C and D)Citation . To quantify this lack of pericyte recruitment, tumor sections were then double stained for the presence of endothelial cells using an anti-PECAM/CD31 antibody and pericytes using an anti-SMA antibody. A Cy3 (red)-labeled secondary antibody for PECAM/CD31 and a FITC (green) secondary antibody for SMA was then used to analyze sections for the presence of endothelial cells (red) and pericytes (green) by double fluorescence (Fig. 10, E and F)Citation . The presence of endothelial cells and pericytes in tumor sections was then quantified by calculating the red (endothelial cells) or green (pericytes) fluorescent area in six fields for each tissue section examined using Metamorph 4.6 software. The recruitment of pericytes along endothelial cells was quantified by calculating the ratio green fluorescence:red fluorescence. The data show a statistically significant decrease in pericytes (P = 0.0003) and to a lesser degree in endothelial cells (P = 0.041) in tumors derived from MMP-9ko/ko mice when compared with controls (Fig. 10G)Citation . The mean ratio pericyte:endothelial cell in tumors from MMP-9ko/ko mice was 2.3, which represent a 48% decrease compared with a mean ratio of 4.87 in tumors derived from control mice (P = 0.023; Fig. 10HCitation ). Thus, the data point to a specific role for MMP-9 in the formation of mature blood vessels by promoting the recruitment of pericytes along endothelial cells.



View larger version (70K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 10. Effect of matrix metalloproteinase (MMP)-9 on pericyte recruitment. Sections of neuroblastoma tumors derived from control mice (A and C) and MMP-9ko/ko mice (B and D) were examined for the presence of pericytes by immunohistochemistry for smooth muscle actin (A and B) and by transmission electron microscopy (C and D). Paraffin-embedded tumor sections double stained for endothelial cells (red) and pericytes (green) examined by fluorescence microscopy (E and F). The amount of endothelial cells (EC) and pericytes in tumor sections was quantified by calculating the fluorescent area/field (216,000 µm2). The data represent the mean area of six fields examined each in three tissue sections/tumor. We analyzed three separate tumors from each group (G). The recruitment of pericytes along endothelial cells was then quantified by calculating the ratio pericyte:endothelial cells (H). E = endothelial cell, P = pericyte, r = RBC.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The orthotopic tumor model used in this study was particularly suitable to examine the role of MMPs and more specifically the contribution of stromal MMP-9 in neuroblastoma for two reasons. First, as indicated in our time course analysis, it reproduced the natural sequence in tumor progression with an initial phase of vascularization of the implant followed by stimulation of tumor growth and ultimately metastatic colonization. Second, this model reproduced a pattern of MMP-9 expression restricted to stromal cells as previously reported in human neuroblastoma by us and seen in other cancers (7 , 17 , 24) . This model was therefore selected to examine the role of MMP in neuroblastoma progression using pharmacological and genetic approaches. Prinomastat was selected for the pharmacological approach because of its higher inhibitory activity for MMP-2 and MMP-9 compared with other MMPs such as MMP-1 and MMP-7 and its documented activity in several human tumor xenograft models (25, 26, 27) .

There is a large body of literature supporting an important and positive contributory role of MMPs in cancer metastasis, including skin, colon, breast, and prostate. Some of this evidence comes from observations showing a positive correlation between the presence and expression of these MMPs and the metastatic stage of a cancer (28 , 29) . Other evidence comes from studies demonstrating that enhanced expression of MMPs in tumor cells increases their invasive and metastatic potential (30 , 31) and, vice versa, that down-regulation of these MMPs by antisense strategies or by overexpression of natural inhibitors inhibits metastasis (32 , 33) . The antimetastatic activity of natural and synthetic inhibitors of MMPs has also been well demonstrated in preclinical models (26 , 34, 35, 36) . It has been generally assumed that the major role of MMPs in metastasis is to promote the invasion of tumor cells and their ability to extravasate and colonize distant organs. However, some investigators have shown that tumor cells overexpressing a natural inhibitor of MMPs such as TIMP-1 can extravasate (37 , 38) , and we have previously demonstrated that forced expression of TIMP-2 in melanoma cells does not inhibit spontaneous metastasis (39) . Studies of cellular invasion in tumor-prone mouse models harboring homozygous deletions in the MMP-9 gene or in cancer patients treated with MMP inhibitors also do not reveal impaired migratory or invasive behavior of neoplastic cells (11 , 40) . Consistently, our data demonstrate that MMP-2 and MMP-9 do not contribute significantly to neuroblastoma extravasation and liver colonization in our orthotopic and experimental metastatic models. Accordingly, we observed that Prinomastat did not inhibit the invasion of neuroblastoma cells through a Matrigel-coated filter (data not shown).

The inhibitory effect of Prinomastat on the presence of tumor cells in the lung vasculature is an intriguing and not entirely understood observation. Neuroblastoma rarely metastasizes to the lung, and in our model, although tumor cells were found in the lung vasculature, they never truly metastasized and never formed macroscopic nodules as observed in the liver. It is conceivable that lung colonization reflects the hematogenous spread of the tumor cells as a consequence of the degree of angiogenesis in the primary tumor. However, in this case, one would anticipate less liver metastasis in Prinomastat-treated mice, which was not the case. We also did not find a correlation between MVS and EA in the primary tumor and metastases in lung, liver, and bone marrow (data not shown). Alternatively, Prinomastat may affect tumor cell arrest, proliferation, and survival in the lung vasculature. For example, investigators have shown that B16 melanoma cells and breast carcinoma cells arrest in the lung vasculature where they attach to the pulmonary endothelium and proliferate without extravasating (41 , 42) . In our studies, we found a decrease in the percentage of Ki67-positive (proliferating) tumor cells in the lung of Prinomastat-treated mice and no difference in terminal deoxynucleotidyl transferase-mediated nick end labeling-positive apoptotic cells (data not shown), suggesting that the inhibitory effect of Prinomastat on lung colonization may involve inhibition of proliferation of tumor cells inside the lung vasculature. The exact mechanism by which Prinomastat inhibits lung colonization will require additional investigation. Consistent with our observation is the observation of Hiratsuka et al. (43) who recently reported in a mouse model that MMP-9 is induced in premetastatic lung endothelial cells and macrophages by a distant primary tumor and that suppression of MMP-9 markedly reduces lung metastases.

In the tumor microenvironment, MMP-9 is expressed by a variety of stromal cells. Our studies demonstrate that vascular cells are an important source of MMP-9 and that MMP-9 positively contributes to neuroblastoma angiogenesis. However, in squamous epithelial carcinomas, MMP-9 is predominantly expressed by inflammatory cells such as mast cells, neutrophils, and macrophages, and its expression and activation coincides with angiogenic switch in premalignant lesions (7) . In a murine model of pancreatic cancer, MMP-9 expression by inflammatory cells similarly coincides with the formation of angiogenic islands, and this process is delayed in the presence of an MMP inhibitor (11) . Other investigators have shown that MMP-9 expressed by macrophages that colonize ovarian tumors in mice contributes to angiogenesis and ascites formation (24) . Thus, depending on the type of tumor, both inflammatory cells and vascular cells can contribute to MMP-9 production.

Our data with Prinomastat treatment and MMP-9-deficient mice both point to a contribution of MMPs to angiogenesis in neuroblastoma. Whereas experiments with Prinomastat did not allow differentiation between a role for MMP-2 or MMP-9, experiments in MMP-9-deficient mice indicate that genetic ablation of this single MMP was at least as effective as the MMP inhibitor, pointing therefore to an important role for MMP-9 in angiogenesis. The contribution of MMP-9 to tumor vascularization is complex and involves both stimulatory and inhibitory effects. MMP-9 promotes vascular endothelial cell invasion upon stimulation by protein kinase C activation or thrombospondin-1 (44 , 45) and promotes endothelial cell morphogenesis (13) . It also increases the bioavailability of vascular endothelial cell growth factor (11) . MMP-9 can also inhibit angiogenesis because of its proteolytic effect on plasminogen to generate the angiogenesis inhibitory peptide, angiostatin (46) , and on collagen type IV {alpha}3 chain to generate tumstatin (47) . Interestingly, MMP-9-deficient mice have a normal estrous cycle, breed normally, and undergo normal pregnancies, suggesting an absence of necessary involvement for MMP-9 in the physiological vascular response associated with these processes. However, MMP-9-deficient mice have an abnormal pattern of skeletal growth plate vascularization and ossification, resulting in a transient lengthening of the growth plate to about eight times normal. This effect involves a delayed release of angiogenic activators (19) . There are, however, significant differences between these physiological processes and tumor angiogenesis (48) .

The exact mechanism by which MMP-9 contributes to angiogenesis in neuroblastoma has thus far not been explored. It is unlikely related to changes in vascular endothelial cell growth factor bioavailability because we did not observe changes in vascular endothelial cell growth factor solubility in tumors derived from Prinomastat-treated mice compared with controls (data not shown). The analysis of angiogenesis in our orthotopic neuroblastoma model indicated a significant inhibitory effect of MMP inhibition and genetic ablation of MMP-9 on microvessel size and EA and an insignificant effect on MVD. Three lines of evidence supporting the hypothesis that MMP-9 is important to the architecture of the tumor vasculature and the formation of mature blood vessels were then obtained. The Matrigel plug implantation experiment indicated a marked decrease in hemoglobin concentration, an indicator of the perfusion of the tumor vasculature, in plugs derived from Prinomastat-treated mice and MMP-9-deficient mice compared with plugs derived from control mice. Fluorescent angiography then provided more definitive evidence by demonstrating a significant difference in tumor vascular architecture between MMP-9-deficient and control mice, with a marked inhibition in the formation of perfused microvessels in the absence of MMP-9. We then discovered that the basis for this difference in vascular architecture was in the organization of pericytes along endothelial cells. We observed a 48% decrease in pericyte recruitment along microvessels in the absence of MMP-9, indicating, for the first time, that MMP-9 may play a critical role in recruiting pericytes in the vasculature. Pericytes are cells of smooth muscle cell lineage that become associated with endothelial cells as soon as they form a primitive blood vessel network (49 , 50) . In tumor angiogenesis, the role of pericytes has recently been recognized as promoting the maturation of newly formed blood vessels and preventing their regression (51) . The longitudinal spreading of pericytes along blood vessels depends on PDGF-B produced by endothelial cells and PDGF receptor-ß expressed by pericytes (52) . Whether MMP-9 has a direct effect on pericyte or affects PDGF-B/PDGF-ß interaction is unknown but presently investigated. Interestingly, in a recent study, Spurbeck et al. (53) demonstrated that forced expression of tissue inhibitor of metalloproteinase-3 in murine neuroblastoma and melanoma tumors resulted in an unanticipated increase in the number of PECAM/CD31-positive endothelial cells but a decreased pericyte recruitment. In additional support for a role of MMP-9 in pericyte recruitment is the observation that MMP-9 expression is localized to the perivascular smooth muscle cells and pericytes at the proliferating borders of human glioma (54) and in pericytes of venules in ulcerative bowel disease (55) . Accordingly, we observed a strong expression of MMP-9 in vascular and perivascular cells in neuroblastoma tumors.

In summary our work points out that different types of tumors use MMP-9 in different and distinctive ways and that MMP-9, although derived from stromal cells in most cases, can be vascular or inflammatory in origin. By demonstrating the contribution of MMP-9 to the formation of a capillary network in tumors and the recruitment of pericytes, our data provide a new insight into the multiple contributions of stromal MMP-9 to tumor angiogenesis and progression.


   ACKNOWLEDGMENTS
 
We thank Katie Lacina and Morgan Woo for their excellent technical assistance. We also thank Drs. George McNamara, Hongjun Peng, and Khalid Bajou for their help in generating the images in confocal microscopy, and Jackie Rosenberg for typing the manuscript.


   FOOTNOTES
 
Grant support: NIH Grants PO1 CA81403 (to Y. A. DeClerck, H. Shimada, S. Groshen), RO1 NS39278 (to Z. Werb), and PO1 CA72006 (to Z. Werb, L. M. Coussens). C. F. Chantrain was the recipient of a Research Career Development Fellowship from the Saban Research Institute of Childrens Hospital Los Angeles (July 1, 2001 to Dec 30, 2002) and of a grant from the Salus Sanguinis Foundation, Catholic University of Louvain, Brussels, Belgium.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: C. F. Chantrain is currently at the Department of Pediatrics, Division of Hematology-Oncology, Catholic University of Louvain, School of Medicine, Brussels, Belgium.

Requests for reprints: Yves A. DeClerck, Childrens Hospital Los Angeles, 4650 Sunset Boulevard, MS #54, Los Angeles, CA 90027. Phone: (323) 669-2150; Fax: (323) 664-9455; E-mail: declerck{at}hsc.usc.edu

Received 1/24/03. Revised 12/11/03. Accepted 12/18/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Nelson A. R., Fingleton B., Rothenberg M. L., Matrisian L. M. Matrix metalloproteinases: biologic activity and clinical implications. J. Clin. Oncol., 18: 1135-1149, 2000.[Abstract/Free Full Text]
  2. Chang C., Werb Z. The many faces of metalloproteases: cell growth, invasion, angiogenesis and metastasis. Trends Cell Biol., 11: S37-S43, 2001.[Medline]
  3. McCawley L. J., Matrisian L. M. Matrix Metalloproteinases: they’re not just for matrix anymore. Curr. Opin. Cell Biol., 13: 534-540, 2001.[CrossRef][Medline]
  4. Stetler Stevenson W. G., Liotta L. A., Kleiner D. E., Jr. Extracellular matrix: role of matrix metalloproteinases in tumor invasion and metastasis. FASEB J., 7: 1434-1441, 1993.[Abstract]
  5. 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, 94: 353-362, 1998.[CrossRef][Medline]
  6. Himelstein B. P., Canete Soler R., Bernhard E. J., Dilks D. W., Muschel R. J. Metalloproteinases in tumor progression: the contribution of MMP-9. Invasion Metastasis, 14: 246-258, 1994.[Medline]
  7. Coussens L. M., Tinkle C. L., Hanahan D., Werb Z. MMP-9 supplied by bone marrow-derived cells contributes to skin carcinogenesis. Cell, 103: 481-490, 2000.[CrossRef][Medline]
  8. Basset P., Okada A., Chenard M. P., Kannan R., Stoll I., Anglard P., Bellocq J. P., Rio M. C. Matrix metalloproteinases as stromal effectors of human carcinoma progression: therapeutic implications. Matrix Biol., 15: 535-541, 1997.[CrossRef][Medline]
  9. Himelstein B. P., Muschel R. J. Induction of matrix metalloproteinase 9 expression in breast carcinoma cells by a soluble factor from fibroblasts. Clin. Exp. Metastasis, 14: 197-208, 1996.[Medline]
  10. Guo H., Zucker S., Gordon M. K., Toole B. P., Biswas C. Stimulation of matrix metalloproteinase production by recombinant extracellular matrix metalloproteinase inducer from transfected Chinese hamster ovary cells. J. Biol. Chem., 272: 24-27, 1997.[Abstract/Free Full Text]
  11. Bergers G., Brekken R., McMahon G., Vu T. H., Itoh T., Tamaki K., Tanzawa K., Thorpe P., Itohara S., Werb Z., Hanahan D. Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nat. Cell Biol., 2: 737-744, 2000.[CrossRef][Medline]
  12. Itoh T., Tanioka M., Yoshida H., Yoshioka T., Nishimoto H., Itohara S. Reduced angiogenesis and tumor progression in gelatinase A-deficient mice. Cancer Res., 58: 1048-1051, 1998.[Abstract/Free Full Text]
  13. Chandrasekar N., Jasti S., Alfred-Yung W. K., Ali-Osman F., Dinh D. H., Olivero W. C., Gujrati M., Kyritsis A. P., Nicolson G. L., Rao J. S., Mohanam S. Modulation of endothelial cell morphogenesis in vitro by MMP-9 during glial-endothelial cell interactions. Clin. Exp. Metastasis, 18: 337-342, 2000.[CrossRef][Medline]
  14. Reynolds C. P., Seeger R. C. Neuroblastoma Haskell C. eds. . Cancer Treatment, Ed. 4 860-871, W. B. Saunders Philadelphia 1994.
  15. Matthay K. K., Villablanca J. G., Seeger R. C., Stram D. O., Harris R. E., Ramsay N. K., Swift P., Shimada H., Black C. T., Brodeur G. M., Gerbing R. B., Reynolds C. P. Treatment of high-risk neuroblastoma with intensive chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13-cis-retinoic acid. Children’s Cancer Group. N. Engl. J. Med., 341: 1165-1173, 1999.[Abstract/Free Full Text]
  16. Ara T., Kusafuka T., Inoue M., Kuroda S., Fukuzawa M., Okada A. Determination of imbalance between MMP-2 and TIMP-2 in human neuroblastoma by reverse-transcription polymerase chain reaction and its correlation with tumor progression. J. Pediatr. Surg., 35: 432-437, 2000.[CrossRef][Medline]
  17. Sugiura Y., Shimada H., Seeger R. C., Laug W. E., DeClerck Y. A. Matrix metalloproteinases-2 and -9 are expressed in human neuroblastoma: contribution of stromal cells to their production and correlation with metastasis. Cancer Res., 58: 2209-2216, 1998.[Abstract/Free Full Text]
  18. Moats R., Ma L. Q., Wajed R., Sugiura Y., Lazaryev A., Tyska M., Jacobs R., Fraser S. E., Nelson M. D., Jr., DeClerck Y. A. Magnetic resonance imaging for the evaluation of a novel metastatic orthotopic model of human neuroblastoma in immunodeficient mice. Clin. Exp. Metastasis, 18: 455-461, 2001.
  19. Vu T. H., Shipley J. M., Bergers G., Berger J. E., Helms J. A., Hanahan D., Shapiro S. D., Senior R. M., Werb Z. MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell, 93: 411-422, 1998.[CrossRef][Medline]
  20. Chen J., Shinkai Y., Young F., Alt F. W. Probing immune functions in RAG-deficient mice. Curr. Opin. Immunol., 6: 313-319, 1994.[CrossRef][Medline]
  21. Chantrain C. F., De Clerck Y. A., Groshen S., McNamara G. Computerized quantification of tissue vascularization using high resolution slide scanning of whole tumor sections. J. Histochem. Cytochem., 51: 151-158, 2003.[Abstract/Free Full Text]
  22. Wirnsberger G. H., Becker H., Ziervogel K., Hofler H. Diagnostic immunohistochemistry of neuroblastic tumors. Am. J. Surg. Pathol., 16: 49-57, 1992.[Medline]
  23. Shalinsky D. R., Brekken J., Zou H., Kolis S., Wood A., Webber S., Appelt K. Antitumor efficacy of AG3340 associated with maintenance of minimum effective plasma concentrations and not total daily dose, exposure or peak plasma concentrations. Investig. New Drugs, 16: 303-313, 1998.[CrossRef][Medline]
  24. Huang S., Van Arsdall M., Tedjarati S., McCarty M., Wu W., Langley R., Fidler I. J. Contributions of stromal metalloproteinase-9 to angiogenesis and growth of human ovarian carcinoma in mice. J. Natl. Cancer Inst. (Bethesda), 94: 1134-1142, 2002.[Abstract/Free Full Text]
  25. Shalinsky D. R., Brekken J., Zou H., Bloom L. A., McDermott C. D., Zook S., Varki N. M., Appelt K. Marked antiangiogenic and antitumor efficacy of AG3340 in chemoresistant human non-small cell lung cancer tumors: single agent and combination chemotherapy studies. Clin. Cancer Res., 5: 1905-1917, 1999.[Abstract/Free Full Text]
  26. Shalinsky D. R., Brekken J., Zou H., McDermott C. D., Forsyth P., Edwards D., Margosiak S., Bender S., Truitt G., Wood A., Varki N. M., Appelt K. Broad antitumor and antiangiogenic activities of AG3340, a potent and selective MMP inhibitor undergoing advanced oncology clinical trials. Ann. N. Y. Acad. Sci., 878: 236-270, 1999.[CrossRef][Medline]
  27. Price A., Shi Q., Morris D., Wilcox M. E., Brasher P. M., Rewcastle N. B., Shalinsky D., Zou H., Appelt K., Johnston R. N., Yong V. W., Edwards D., Forsyth P. Marked inhibition of tumor growth in a malignant glioma tumor model by a novel synthetic matrix metalloproteinase inhibitor AG3340. Clin. Cancer Res., 5: 845-854, 1999.[Abstract/Free Full Text]
  28. Kleiner D. E., Stetler-Stevenson W. G. Matrix metalloproteinases and metastasis. Cancer Chemother. Pharmacol., 43: S42-S51, 1999.
  29. Coussens L. M., Fingleton B., Matrisian L. M. Matrix metalloproteinase inhibitors and cancer: trials and tribulations. Science (Wash. DC), 295: 2387-2392, 2002.[Abstract/Free Full Text]
  30. Tsunezuka Y., Kinoh H., Takino T., Watanabe Y., Okada Y., Shinagawa A., Sato H., Seiki M. Expression of membrane-type matrix metalloproteinase 1 (MT1-MMP) in tumor cells enhances pulmonary metastasis in an experimental metastasis assay. Cancer Res., 56: 5678-5683, 1996.[Abstract/Free Full Text]
  31. Noel A., Lefebvre O., Maquoi E., VanHoorde L., Chenard M. P., Mareel M., Foidart J. M. Stromelysin-3 expression promotes tumor take in nude mice. J. Clin. Investig., 97: 1924-1930, 1996.[Medline]
  32. Khokha R. Suppression of the tumorigenic and metastatic abilities of murine B16–F10 melanoma cells in vivo by the overexpression of the tissue inhibitor of the metalloproteinases-1. J. Natl. Cancer Inst. (Bethesda), 86: 299-304, 1994.[Abstract/Free Full Text]
  33. Hua J., Muschel R. J. Inhibition of matrix metalloproteinase 9 expression by a ribozyme blocks metastasis in a rat sarcoma model system. Cancer Res., 56: 5279-5284, 1996.[Abstract/Free Full Text]
  34. Alvarez O. A., Carmichael D. F., DeClerck Y. A. Inhibition of collagenolytic activity and metastasis of tumor cells by a recombinant human tissue inhibitor of metalloproteinases. J. Natl. Cancer Inst. (Bethesda), 82: 589-595, 1990.[Abstract/Free Full Text]
  35. Khokha R., Zimmer M. J., Wilson S. M., Chambers A. F. Up-regulation of TIMP-1 expression in B16–F10 melanoma cells suppresses their metastatic ability in chick embryo. Clin. Exp. Metastasis, 10: 365-370, 1992.[CrossRef][Medline]
  36. Eccles S. A., Box G. M., Court W. J., Bone E. A., Thomas W., Brown P. D. Control of lymphatic and hematogenous metastasis of a rat mammary carcinoma by the matrix metalloproteinase inhibitor batimastat (BB-94). Cancer Res., 56: 2815-2822, 1996.[Abstract/Free Full Text]
  37. Koop S., Khokha R., Schmidt E. E., MacDonald I. C., Morris V. L., Chambers A. F., Groom A. C. Overexpression of metalloproteinase inhibitor in B16F10 cells does not affect extravasation but reduces tumor growth. Cancer Res., 54: 4791-4797, 1994.[Abstract/Free Full Text]
  38. Chambers A. F., MacDonald I. C., Schmidt E. E., Morris V. L., Groom A. C. Preclinical assessment of anti-cancer therapeutic strategies using in vivo videomicroscopy. Cancer Metastasis Rev., 17: 263-269, 1998.[CrossRef][Medline]
  39. Montgomery A. M., Mueller B. M., Reisfeld R. A., Taylor S. M., DeClerck Y. A. Effect of tissue inhibitor of the matrix metalloproteinases-2 expression on the growth and spontaneous metastasis of a human melanoma cell line. Cancer Res., 54: 5467-5473, 1994.[Abstract/Free Full Text]
  40. van Kempen L. C., Coussens L. M. MMP9 potentiates pulmonary metastasis formation. Cancer Cell, 2: 251-252, 2002.[CrossRef][Medline]
  41. Al Mehdi A. B., Tozawa K., Fisher A. B., Shientag L., Lee A., Muschel R. J. Intravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: a new model for metastasis. Nat. Med., 6: 100-102, 2000.[CrossRef][Medline]
  42. Wong C. W., Song C., Grimes M. M., Fu W., Dewhirst M. W., Muschel R. J., Al Mehdi A. B. Intravascular location of breast cancer cells after spontaneous metastasis to the lung. Am. J. Pathol., 161: 749-753, 2002.[Abstract/Free Full Text]
  43. Hiratsuka S., Nakamura K., Iwai S., Murakami M., Itoh T., Kijima H., Shipley J. M., Senior R. M., Shibuya M. MMP9 induction by vascular endothelial growth factor receptor-1 is involved in lung-specific metastasis. Cancer Cell, 2: 289-300, 2002.[CrossRef][Medline]
  44. Park M. J., Park I. C., Lee H. C., Woo S. H., Lee J. Y., Hong Y. J., Rhee C. H., Lee Y. S., Lee S. H., Shim B. S., Kuroki T., Hong S. I. Protein kinase C {alpha} activation by phorbol ester induces secretion of gelatinase B/MMP-9 through ERK 1/2 pathway in capillary endothelial cells. Int. J. Oncol., 22: 137-143, 2003.[Medline]
  45. Qian X., Wang T. N., Rothman V. L., Nicosia R. F., Tuszynski G. P. Thrombospondin-1 modulates angiogenesis in vitro by up-regulation of matrix metalloproteinase-9 in endothelial cells. Exp. Cell Res., 235: 403-412, 1997.[CrossRef][Medline]
  46. Pozzi A., LeVine W. F., Gardner H. A. Low plasma levels of matrix metalloproteinase 9 permit increased tumor angiogenesis. Oncogene, 21: 272-281, 2002.[CrossRef][Medline]
  47. Maeshima Y., Sudhakar A., Lively J. C., Ueki K., Kharbanda S., Kahn C. R., Sonenberg N., Hynes R. O., Kalluri R. Tumstatin, an endothelial cell-specific inhibitor of protein synthesis. Science (Wash. DC), 295: 140-143, 2002.[Abstract/Free Full Text]
  48. Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat. Med., 6: 389-395, 2000.[CrossRef][Medline]
  49. Gerhardt H., Betsholtz C. Endothelial-pericyte interactions in angiogenesis. Cell Tissue Res., 314: 15-23, 2003.[CrossRef][Medline]
  50. Hellstrom M., Gerhardt H., Kalen M., Li X., Eriksson U., Wolburg H., Betsholtz C. Lack of pericytes leads to endothelial hyperplasia and abnormal vascular morphogenesis. J. Cell Biol., 153: 543-553, 2001.[Abstract/Free Full Text]
  51. Carmeliet P. Angiogenesis in health and disease. Nat. Med., 9: 653-660, 2003.[CrossRef][Medline]
  52. Hellstrom M., Kalen M., Lindahl P., Abramsson A., Betsholtz C. Role of PDGF-B and PDGFR-ß in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse. Development (Camb.), 126: 3047-3055, 1999.[Abstract]
  53. Spurbeck W. W., Ng C. Y., Strom T. S., Vanin E. F., Davidoff A. M. Enforced expression of tissue inhibitor of matrix metalloproteinase-3 affects functional capillary morphogenesis and inhibits tumor growth in a murine tumor model. Blood, 100: 3361-3368, 2002.[Abstract/Free Full Text]
  54. Forsyth P. A., Wong H., Laing T. D., Rewcastle N. B., Morris D. G., Muzik H., Leco K. J., Johnston R. N., Brasher P. M. A., Sutherland G., Edwards D. R. Gelatinase-A (MMP-2), gelatinase-B (MMP-9) and membrane type matrix metalloproteinase-1 (MT1-MMP) are involved in different aspects of the pathophysiology of malignant gliomas. Br. J. Cancer, 79: 1828-1835, 1999.[CrossRef][Medline]
  55. Arihiro S., Ohtani H., Hiwatashi N., Torii A., Sorsa T., Nagura H. Vascular smooth muscle cells and pericytes express MMP-1, MMP-9, TIMP-1 and type I procollagen in inflammatory bowel disease. Histopathology, 39: 50-59, 2001.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
V. C. Ardi, P. E. Van den Steen, G. Opdenakker, B. Schweighofer, E. I. Deryugina, and J. P. Quigley
Neutrophil MMP-9 Proenzyme, Unencumbered by TIMP-1, Undergoes Efficient Activation in Vivo and Catalytically Induces Angiogenesis via a Basic Fibroblast Growth Factor (FGF-2)/FGFR-2 Pathway
J. Biol. Chem., September 18, 2009; 284(38): 25854 - 25866.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. D. Martin, K. J. Carter, S. R. Jean-Philippe, M. Chang, S. Mobashery, S. Thiolloy, C. C. Lynch, L. M. Matrisian, and B. Fingleton
Effect of Ablation or Inhibition of Stromal Matrix Metalloproteinase-9 on Lung Metastasis in a Breast Cancer Model Is Dependent on Genetic Background
Cancer Res., August 1, 2008; 68(15): 6251 - 6259.
[Abstract] [Full Text] [PDF]

Home page
 Exp. Biol. Med.Home page
S. Arnold, E. Mira, S. Muneer, G. Korpanty, A. W. Beck, S. E. Holloway, S. Manes, and R. A. Brekken
Forced Expression of MMP9 Rescues the Loss of Angiogenesis and Abrogates Metastasis of Pancreatic Tumors Triggered by the Absence of Host SPARC
Experimental Biology and Medicine, July 1, 2008; 233(7): 860 - 873.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
V. W.M. van Hinsbergh and P. Koolwijk
Endothelial sprouting and angiogenesis: matrix metalloproteinases in the lead
Cardiovasc Res, May 1, 2008; 78(2): 203 - 212.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
M. Gerg, C. Kopitz, S. Schaten, A. Tschukes, C. Kahlert, M. Stangl, C. W. Hann von Weyhern, B. L.D.M. Brucher, D. R. Edwards, K. Brand, et al.
Distinct Functionality of Tumor Cell-Derived Gelatinases during Formation of Liver Metastases
Mol. Cancer Res., March 1, 2008; 6(3): 341 - 351.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
Y. Y. Mahller, S. S. Vaikunth, M. C. Ripberger, W. H. Baird, Y. Saeki, J. A. Cancelas, T. M. Crombleholme, and T. P. Cripe
Tissue Inhibitor of Metalloproteinase-3 via Oncolytic Herpesvirus Inhibits Tumor Growth and Vascular Progenitors
Cancer Res., February 15, 2008; 68(4): 1170 - 1179.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
P. Nangia-Makker, T. Raz, L. Tait, V. Hogan, R. Fridman, and A. Raz
Galectin-3 Cleavage: A Novel Surrogate Marker for Matrix Metalloproteinase Activity in Growing Breast Cancers
Cancer Res., December 15, 2007; 67(24): 11760 - 11768.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
L. Ivanciu, R. D. Gerard, H. Tang, F. Lupu, and C. Lupu
Adenovirus-Mediated Expression of Tissue Factor Pathway Inhibitor-2 Inhibits Endothelial Cell Migration and Angiogenesis
Arterioscler Thromb Vasc Biol, February 1, 2007; 27(2): 310 - 316.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
A. E. M. Dirkx, M. G. A. oude Egbrink, J. Wagstaff, and A. W. Griffioen
Monocyte/macrophage infiltration in tumors: modulators of angiogenesis
J. Leukoc. Biol., December 1, 2006; 80(6): 1183 - 1196.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. Jost, A. R. Folgueras, F. Frerart, A. M. Pendas, S. Blacher, X. Houard, S. Berndt, C. Munaut, D. Cataldo, J. Alvarez, et al.
Earlier onset of tumoral angiogenesis in matrix metalloproteinase-19-deficient mice.
Cancer Res., May 15, 2006; 66(10): 5234 - 5241.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
V. W.M. van Hinsbergh, M. A. Engelse, and P. H.A. Quax
Pericellular Proteases in Angiogenesis and Vasculogenesis
Arterioscler Thromb Vasc Biol, April 1, 2006; 26(4): 716 - 728.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
L. Blavier, A. Lazaryev, F. Dorey, G. M. Shackleford, and Y. A. DeClerck
Matrix metalloproteinases play an active role in wnt1-induced mammary tumorigenesis.
Cancer Res., March 1, 2006; 66(5): 2691 - 2699.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
A. Zijlstra, M. Seandel, T. A. Kupriyanova, J. J. Partridge, M. A. Madsen, E. A. Hahn-Dantona, J. P. Quigley, and E. I. Deryugina
Proangiogenic role of neutrophil-like inflammatory heterophils during neovascularization induced by growth factors and human tumor cells
Blood, January 1, 2006; 107(1): 317 - 327.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
E. I. Deryugina, A. Zijlstra, J. J. Partridge, T. A. Kupriyanova, M. A. Madsen, T. Papagiannakopoulos, and J. P. Quigley
Unexpected Effect of Matrix Metalloproteinase Down-Regulation on Vascular Intravasation and Metastasis of Human Fibrosarcoma Cells Selected In vivo for High Rates of Dissemination
Cancer Res., December 1, 2005; 65(23): 10959 - 10969.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
M. Hecht, J. H. Schulte, A. Eggert, J. Wilting, and L. Schweigerer
The neurotrophin receptor TrkB cooperates with c-Met in enhancing neuroblastoma invasiveness
Carcinogenesis, December 1, 2005; 26(12): 2105 - 2115.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
S. Jodele, C. F. Chantrain, L. Blavier, C. Lutzko, G. M. Crooks, H. Shimada, L. M. Coussens, and Y. A. DeClerck
The Contribution of Bone Marrow-Derived Cells to the Tumor Vasculature in Neuroblastoma Is Matrix Metalloproteinase-9 Dependent
Cancer Res., April 15, 2005; 65(8): 3200 - 3208.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
Y. Sohara, H. Shimada, C. Minkin, A. Erdreich-Epstein, J. A. Nolta, and Y. A. DeClerck
Bone Marrow Mesenchymal Stem Cells Provide an Alternate Pathway of Osteoclast Activation and Bone Destruction by Cancer Cells
Cancer Res., February 15, 2005; 65(4): 1129 - 1135.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
P. B. Cornet, C. Galant, Y. Eeckhout, P. J. Courtoy, E. Marbaix, and P. Henriet
Regulation of Matrix Metalloproteinase-9/Gelatinase B Expression and Activation by Ovarian Steroids and LEFTY-A/Endometrial Bleeding-Associated Factor in the Human Endometrium
J. Clin. Endocrinol. Metab., February 1, 2005; 90(2): 1001 - 1011.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
A. Ferrario, C. F. Chantrain, K. von Tiehl, S. Buckley, N. Rucker, D. R. Shalinsky, H. Shimada, Y. A. DeClerck, and C. J. Gomer
The Matrix Metalloproteinase Inhibitor Prinomastat Enhances Photodynamic Therapy Responsiveness in a Mouse Tumor Model
Cancer Res., April 1, 2004; 64(7): 2328 - 2332.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chantrain, C. F.
Right arrow Articles by DeClerck, Y. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chantrain, C. F.
Right arrow Articles by DeClerck, Y. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online