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Basic and Translational Investigations |
Departments of Neurological Surgery (R.D., C.P., K.L., P.L., A.H., H.S., S.V., G.B.) and Pathology (S.V.) and Brain Tumor Research Center and Comprehensive Cancer Center (C.P., G.B.), University of California San Francisco, San Francisco, CA, USA; Department of Neurological Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA (R.D.); Western Australian Institute for Medical Research, Perth, Australia (R.G.)
Address correspondence to Gabriele Bergers, University of California San Francisco, Department of Neurological Surgery, 513 Parnassus Ave., Box 0520, San Francisco, CA 94143-0520, USA (gabriele.bergers{at}ucsf.edu).
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Key Words: angiogenesis • glioblastoma multiforme • invasion • matrix metalloproteinase-2 • survival
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously generated genetically engineered wild-type (WT)-GBMs by isolating astrocytes from the hippocampus of neonatal mice and transforming them with the oncogenes SV40 large T-antigen and V12–H-ras.14 When WT-GBM cells were injected intracranially into the striatum of mice, they developed into aggressive GBMs that exhibited all the major hallmarks of human GBMs. WT-GBM cells grew as invasive, highly angiogenic tumors with a leaky, tortuous vasculature and with hypoxic and pallisading necrotic centers within the tumor mass.14 Notably, WT-GBM, like human GBM, infiltrated as single cells into the brain parenchyma and moved along basement membranes, preferentially along blood vessels. We found that MMP-2 was expressed in endothelial cells and tumor cells, reflecting its expression pattern in human GBMs. Because MMP-2ko mice are viable and fertile, we were able to generate MMP-2ko GBM cells using the same technique as described above in order to study the impact of MMP-2 loss in tumor cells and/or host cells by comparing propagation of WT-GBM and MMP-2ko GBM in both MMP-2–proficient and MMP-2ko hosts. Our studies reveal that MMP-2 regulates vascular patterning and branching, thereby affecting tumor cell survival and tumor invasion in a dose-dependent manner in GBM.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Soft Agar Assay
Cells were plated in triplicate in a six-well plate at 5,000 cells/well, mixed in 0.35% low-melting-temperature agarose (Cambrex Bioscience, Rockland, ME, USA). After culturing cells for 14 days, the wells were stained with 0.005% crystal violet for 2 h and analyzed for colony number and size.
Intracranial Implantation of Astrocytes
Thirty-two Ragko and MMP-2ko Ragko mice, 6–8 weeks of age, were implanted intracranially with 2.5 µl of 0.7 x 106 WT or MMP-2ko/ko-transformed astrocytes as previously described.14 Mice were anesthetized and heart-perfused with 4% paraformaldehyde (PFA) and/or phosphate-buffered saline (PBS). All implantation experiments were repeated up to three times for a total of 6–12 mice per group.
Tissue Preparation
For fixed sections, animals were heart-perfused with 4% PFA. The brains were fixed in formalin overnight and then immersed in 70% ethanol and embedded into paraffin, or immersed in 30% sucrose/PBS overnight, embedded in OCT freezing medium, and stored at –80°C. For unfixed sections, brains were embedded in OCT medium after heart perfusion with PBS.
Substrate Zymography
Tumors were removed from the brain, weighed, and then homogenized (1:3 wt/vol) in RIPA lysis buffer containing 20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), and 1x Roche complete protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA). Equivalent amounts of soluble extracts were analyzed by gelatin zymography on 10% SDS-polyacrylamide gels copolymerized with 1 mg/ml gelatin in sample buffer (5% SDS, 0.25 M Tris-HCl, 25% glycerol, 0.1% bromophenol blue, pH 6.8). After electrophoresis, gels were washed for 40 min in 2.5% Triton X-100, rinsed in water, and incubated for 16 h at 37°C in gelatinase buffer (50 mM Tris-HCl, pH 7.5, 5 mM CaCl2, 1 µM ZnCl2). Gels were then stained in Coomassie Brilliant Blue R-250 staining solution (Bio-Rad, Hercules, CA, USA) and destained in a solution of 30% ethanol and 10% acetic acid. Negative staining denotes the locations of active proteinase bands. Ethylenediaminetetraacetic acid (EDTA; 20 mM), which chelates magnesium and thereby inactivates MMPs, was added to the gelatinase buffer overnight to confirm that the proteases were metalloproteinases.
Histopathological Analysis
Hematoxylin and eosin staining was done in three whole-brain tumor samples for each tumor/host combination and analyzed by a neuropathologist who was blinded to the tumor/host types. Tumors were graded for mitoses, nuclear pleomorphism, necrosis, microperivascular spread, subarachnoid spread, white matter spread, and single cell/perineuronal spread.
Proliferation rate was determined by calculating the ratio of Ki-67–positive cells to all tumor cells per high-power field (x40) in three to eight tumor samples, and three to five high-power fields per sample for each tumor/host combination. Apoptotic indices were obtained by calculating the ratio of cells identified as positive by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) to all tumor cells per high-power field (x40) in three to seven tumor samples, and three to six high-power fields per sample for each tumor/host combination.
Invasiveness of tumors was determined by staining tumor cells with an antibody for SV40 large T-antigen on whole-brain sections. Tumors were graded from 1 to 3, where 1 indicates minimal distant spread of tumor cells and 3 indicates substantial and marked distant spread. Five to eight tumor samples per tumor/host combination were analyzed.
Infiltration was quantified by counting the number of infiltrating cells at the invasive edge (x20 field) in 50-µm frozen sections that were double-stained with large T-antigen and CD31. Infiltrating cells were defined as single cells at the invasive edge that were not associated with a blood vessel. Two to four different tumor samples and 3–10 sections per sample were analyzed per group.
Visualization of the vasculature was revealed by injecting mice intravenously with 0.05 mg fluorescein- or rhodamine-conjugated Lycopersicon esculentum (tomato) lectin (Vector Laboratories, Burlingame, CA, USA) and subsequent heart perfusion with 4% PFA and/or PBS. Brains were frozen in OCT and sectioned at 15 µm and 50 µm thicknesses. Vessel density was determined by calculating the area of CD31 staining using an ImageJ v1.34 software program (NIH) in x20 fields of two to five different tumor samples per group and three to seven different sections per tumor sample.
Quantification of pericyte coverage was performed by collecting fluorescent images of tumor sections on a Zeiss Axioskop 2 with x20 Plan Neofluar lenses and a Zeiss Axiocam color charge-coupled device. Red, green, and blue staining was quantitatively evaluated using ImageJ v1.34 software. The total area of CD31, desmin, or 
-smooth muscle actin (-SMA) staining was obtained. The fraction of pericyte coverage was calculated as the ratio of the area of desmin or -SMA staining (red) to the area of CD31 staining (green). For desmin staining, 8–17 tumor sections per group were evaluated. For -SMA staining, four to nine tumor sections per group were evaluated.
Immunohistochemical Analyses
Frozen (15 µm and 50 µm thickness) and paraffin (6 µm thickness) sections were used for immunohistochemical analysis. Fixed frozen sections were postfixed with 4% PFA, unfixed frozen sections were fixed with 100% methanol at –20°C, and paraffin sections were deparaffinized and subjected to graded rehydration. Astrocytoma cells were identified with a rabbit anti-SV40 T-antigen antibody (1:500; a gift from Dr. Douglas Hanahan, University of California San Francisco). Endothelial cells were visualized with a rat antimouse CD31 antibody (1:100; BD Biosciences Pharmingen, San Jose, CA, USA) in frozen sections and an antimouse endoglin antibody (R&D Systems, Minneapolis, MN, USA) in paraffin sections. Vascular endothelial growth factor receptor 2 (VEGFR2) staining was carried out on paraffin-embedded sections with a goat antimouse VEGFR2 antibody (1:50, R&D Systems). VEGF-VEGFR2 complex was visualized in frozen sections with mouse monoclonal antibody Gv39M (1:50; EastCoast Bio, North Berwick, ME, USA). Apoptotic cells were assessed on both paraffin and frozen sections by TUNEL staining as previously described.15 Proliferating cells were detected on both paraffin and frozen sections with a rat antimouse Ki-67 antibody (1:100; DAKO Corp., Carpinteria, CA, USA). Pericytes were identified with a mouse antihuman desmin (1:100; DAKO Corp.) and mouse antihuman SMA (1:500; DAKO Corp.). Primary antibody reaction products were visualized with respective biotinylated secondary antibodies (1:200; Vector) and then incubated with an ABC kit and 3,3-diaminobenzidine chromophore (Vector). For fluorescent visualization of antibody reactions, secondary antibodies were labeled with fluorochrome AlexaFluor350, Alexa Fluor488, or AlexaFluor594 (1:200; Molecular Probes, Eugene, OR, USA). Photomicrographs were taken with a Zeiss Axiovert 2 microscope, using Openlab 3 software (Improvision, Lexington, MA, USA). Levels in images were adjusted in Adobe Photoshop 7.
Fluorescence-Activated Cell Sorting Analysis
After the mice were euthanized, tumors were removed from the brain. Tumors were minced with a razor blade and digested at 37°C for 30 min with 20 ml enzyme mixture containing minimal essential medium with Earle's balanced salt solution, 1 mM L-cysteine, 0.5 mM EDTA, 1 µg/ml DNAse I (Worthington Biochemical Corp., Lakewood, NJ, USA), and 20 U/ml papain (Worthington). Cells from the digested tumors were passed through a 70-µm cell strainer and washed with Dulbecco's modified Eagle's medium. Red blood cells were lysed with PharmLyse (BD Biosciences Pharmingen) and washed. Cell pellets were resuspended in PBS containing 1% bovine serum albumin. Cells were incubated with primary antibodies on ice. The following primary antibodies were used (all from BD Biosciences Pharmingen): phycoerythrin-CD31 (1:50), rat antimouse CD11b (1:50), rat antimouse Ly-6G (GR1 antigen) (1:60), and rabbit antimouse CD45 (1:50). If the primary antibody was unlabeled, secondary antibodies fluorescently labeled with the fluorochrome Alexa Fluor647 (1:100; Molecular Probes) were added to the cell suspension.
RNA Isolation, Reverse Transcriptase PCR, and Real-Time PCR Analysis
RNA isolation was performed on cells sorted via fluorescence-activated cell sorting (FACS), on cell cultures, and on whole tissue. FACS-sorted cells and cells obtained from culture were placed in a cell lysis solution containing β-mercaptoethanol (Qiagen Inc., Valencia, CA, USA). RNA was isolated following RNeasy Mini Kit protocols (Qiagen Inc.). Whole tissues were flash frozen in liquid nitrogen and stored at –80°C until required. After thawing tissue on ice, 1 ml of TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was added, and the tissue was homogenized immediately using a rotor-stator homogenizer (Fisher-Scientific, Pittsburgh, PA, USA). Total RNA was harvested per the manufacturer's instructions. cDNA was used for qualitative PCR (Roche Diagnostic Corp., Indianapolis, IN, USA). The following primer sequences were used: For MMP-2, (F) 5'-CAGTGACACCACGTGACAAGC-3'; (R) 5'-GGCAGCATCTAGTTGCTGGAC-3'; 60°C. For the ribosomal protein L19, (F) 5'-CTGAAGGTCAAAGGGAATGTG-3'; (R) 5'-GGACAGAGTCTTGATGATCTC-3'; 58°C.
Quantitative real-time PCR analysis of VEGFR2 expression was performed using the iQ SYBR Green Supermix and iCycler thermocycler (Bio-Rad Laboratories), according to the manufacturer's instructions. To control for variations in input cDNA between samples, L19 amplifications were performed in parallel for normalization. All measurements were collected in triplicate and confirmed by independent experiments. Primers for real-time PCR were, for VEGFR2, (F) 5'-GCGGGCTCCTGACTACAC-3'; (R) 5'-CCAAATGCTCCACCAACTCTG-3'; 60°C.
VEGF and VEGFR2 Western Blot Analysis
For VEGF Western blot analysis, proteins from 45-µg WT-GBM and MMP-2ko GBM tumor extracts, 15-µg WT-GBM and MMP-2ko GBM astrocyte supernatants, and 20-µg WT-GBM and MMP-2ko GBM astrocyte cell extracts were separated by SDS-polyacrylamide gel electrophoresis in 15% Tris-tricine gels and transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA, USA) using 20% methanol. Blots were incubated with epitope-specific rabbit anti-VEGF antibody (provided by Donald Senger, Beth Israel Deaconess Medical Center).16 VEGF protein was detected as previously described.17 Anti--tubulin antibody was used to control for equal protein loading (1:5,000; Calbiochem Brand, EMD Biosciences, San Diego, CA, USA) in tumor and cell extracts. Equal amounts of proteins were loaded for astrocyte supernatants. For VEGFR2 Western blot analysis, 100 µg protein from WT-GBM and MMP-2ko GBM tumor extracts were loaded and separated on 15% Tris-glycine gels. Proteins were transferred to Immobilon-P using 5% methanol to ensure transfer of proteins with higher molecular weight. VEGFR2 was detected using an anti-VEGFR2 antibody (1:1,000; Cell Signaling Technology, Danvers, MA, USA).
Nonradioactive In Situ Hybridization
Nonradioactive in situ hybridization of RGS-5 was performed as previously described.17
Supplemental Material and Methods
For the soft agar assay, cells were plated in triplicate in a six-well plate at 5 x 105 cells/well, mixed in 0.35% low-melting-point agarose (Invitrogen). After culturing the cells for 21 days, the wells were stained with 0.005% crystal violet for 4 h and analyzed for colony number and size. Insulin-like growth factor binding protein-2 (IGFBP-2) Western blots were performed as described for VEGF Western blot analysis using an anti-IGFBP-2 antibody (1:500; AF 797, R&D Systems) and an anti--tubulin antibody.
Statistical Analysis
All experiments were repeated two to four times. Statistical analyses were performed over all groups with the Kruskal-Wallis H-test to determine statistical significance using p < 0.05 followed by a Mann-Whitney U-test for pairwise comparison. A p value (exact significance) of <0.05 was considered statistically significant. Kaplan-Meier curves and the log-rank test were used to compare survival times among various groups of mice. All calculations were performed using SPSS version 11.0 (SPSS Inc., Chicago, IL, USA).
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Results |
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Generation of MMP-2ko GBMs
Since MMP-2 is expressed in both normal host and tumor cells, we intended to generate a system in which MMP-2 is deleted in all cell types. We generated a GBM cell line that lacks MMP-2 activity by isolating astrocytes from MMP-2ko neonatal mice and transforming the astrocytes with the oncogenes SV40 large T-antigen and V12–H-ras (Fig. 1B). For controls, we employed WT-GBM cells that were generated in the same way, but with astrocytes isolated from WT, MMP-2–proficient mice.14 We confirmed that both WT-GBM and MMP-2ko GBM cell lines had a similar transformation status by performing soft agar assays, which showed that both cell lines gave rise to similar numbers of soft agar colonies (see supplementary data, Fig. S1). Furthermore, both cell lines exhibited comparable levels of SV40 large T-antigen and H-ras oncogene expression (see supplementary data, Fig. S1).
GBM-Bearing Mice Have a Survival Benefit in the Complete Absence of MMP-2
We next assessed whether MMP-2 affects survival of GBM-bearing mice. We first injected MMP-2ko GBM cells into an MMP-2ko host (MMP-2koT/MMP-2koH). In the complete absence of MMP-2, GBM-bearing mice had a three- to fourfold increase in survival benefit when compared to the WT situation (p = 0.001) in which WT-GBM cells were injected into MMP-2–proficient WT mice (WTT/WTH) (Fig. 1C, a and b). We then generated heterozygous scenarios (Fig. 1B) in which we either injected WT-GBM cells into an MMP-2ko host (WTT/MMP-2koH), or MMP-2ko GBM cells into an MMP-2–proficient WT host (MMP-2koT/WTH). While the survival rate of the WTT/MMP-2koH group was comparable to that of the WTT/WTH group, the mean survival of the MMP-2koT/WTH group was twice as long as the mean survival of the WTT/WTH group (p = 0.007, Fig. 1C, a,b). Thus, removing MMP-2 from the tumor cells alone had a significant effect on survival. As illustrated in Fig. 1C (b), Kaplan-Meier survival curves confirmed that the MMP-2koT/MMP-2koH and MMP-2koT/WTH groups had higher survival rates than the WTT/WTH group (p = 0.001).
Neuropathological analysis showed that all tumors in each of the four groups exhibited typical features of high-grade astrocytomas, including high numbers of mitotic figures and cellular pleomorphism/atypia. Surprisingly, pallisading necrosis was observed in tumors of the WT and heterozygous groups but not in tumors that were completely deficient in MMP-2 (MMP-2koT/MMP-2koH group) (data not shown). The absence of necrosis may be partly related to the absence of infiltration by tumor cells into the brain parenchyma.19 We then assessed the proliferative rate of the tumors from each of the four groups, but did not find any statistically significant differences between the growth rate of tumors that lost MMP-2 activity (either in the tumor or in host cells) and the growth rate of those that maintained MMP-2 activity (Fig. 1C, c). We did detect, however, a 2.4-fold increase in the apoptotic rate of GBMs that were completely deficient in MMP-2 (p = 0.056), which most likely contributed to the slower growth of the tumors in the MMP-2koT/MMP-2koH group (Fig. 1C, d). The elevated numbers of apoptotic cells were observed only in the complete absence of MMP-2, implying that MMP-2 deficiency in either tumor cells or host cells alone was not sufficient to increase apoptosis in GBM. The increased apoptotic ratio in tumors when MMP-2 activity is completely abolished might suggest that MMP-2 is linked to a tumor survival factor. Indeed, MMP-2 and other metalloproteinases have been shown to degrade IGFBP-2, unmasking insulin-like growth factors that act as cell survival factors.20,21 We investigated IGFBP-2 levels in the presence and absence of MMP-2 by Western blot analyses and found that IGFBP-2 in tumors is degraded to a similar extent independent of MMP-2 (see supplementary data, Fig. S2).
MMP-2 Affects Vascular Density by Regulating Blood Vessel Branching
MMP-2 can potentially mediate tumor cell survival by regulating the activity of survival factors or by supporting new blood vessel formation during tumor propagation. To elicit whether MMP-2 affects GBM neovascularization, we sought to visualize and compare the vascular network in GBMs in the presence and absence of MMP-2 (Fig. 2A–D). We found that the vasculature in WT-GBM tumors, independent of the MMP-2 status of the host, had undergone vascular remodeling as in human GBMs. Tumor vessels of GBMs in the WTT/MMP-2koH group (Fig. 2B) were hyperdilated and irregularly shaped, as were tumor vessels of the WTT/WTH group (Fig. 2A). A subtle difference could be observed in tumor vessels of GBM from the MMP-2koT/WTH cohort that looked somewhat irregular and leaky, but also exhibited a mild increase in vessel density (p = 0.023), as well as some normalization of vessel morphology to slightly thinner and less tortuous vessels (Fig. 2C). The complete absence of MMP-2 in both tumor and host cells, however, led to a dramatic and unexpected change in both vessel morphology and density. GBM blood vessels from the MMP-2koT/MMP-2koH cohort were three- to fourfold denser than vessels from all other groups (p = 0.001) and formed a highly branched and slim vascular network (Fig. 2D). Thus, there appears to be an MMP-2 dose response, but the complete absence of MMP-2 is required to completely alter the vascular phenotype. It was puzzling that tumor cells in such a vascular-rich environment were more prone to apoptosis, raising the question of whether these blood vessels are functionally impaired.
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MMP-2 Regulates Pericyte Activation and Recruitment
In addition to the lower levels of VEGFR2 in tumors totally devoid of MMP-2, we found that pericytes did not become activated in GBMs of the MMP-2koT/MMP-2koH group as assessed by the lack of RGS-5 expression (Fig. 3A). RGS-5 is a member of the RGS family of GTPase-activating proteins for G-proteins and is induced in developing and angiogenic pericytes in the brain and pancreas.17,24,25 Furthermore, MMP-2ko GBM had approximately 30% to 40% less pericyte coverage of blood vessels than did WT-GBM (desmin, p = 0.031; -SMA, p = 0.001), further supporting the notion that pericytes were not activated and recruited to the vascular site (Fig. 4A–F). Interestingly, although WT-GBM tumors contained more pericytes, their blood vessels were more dilated than those of MMP-2ko tumors, which contained fewer pericytes.
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MMP-2 Affects Tumor Cell Infiltration and Perivascular Tumor Cell Invasion
Finally, we observed that MMP-2 affected the invasive behavior of GBMs. GBMs are highly invasive tumors that can infiltrate as single cells into the brain parenchyma and can invade along basement membranes, including the leptomeninges, ventricles, and vascular basement membranes. WT-GBM exhibited all of these invasive features (Fig. 5A), but in the complete absence of MMP-2, tumors surprisingly grew even more diffusely (Fig. 5B). We quantified the invasive pattern by grading and counting invading tumor cells, and by distinguishing between infiltrative tumor behavior, in which tumor cells infiltrate into the brain parenchyma without being in contact with blood vessels (Fig. 5A, a, yellow arrows), and perivascular invasion, in which tumor cells move into the normal tissue by moving along and being closely associated with blood vessels (Fig. 5B, b, white arrowheads). We found that GBMs from the WTT/WTH and WTT/MMP-2koH groups were equally infiltrative, whereas tumors from the MMP-2koT/WTH and MMP-2koT/MMP-2koH cohorts were both much less infiltrative (p = 0.004 and p = 0.015; Fig. 5C). On the other hand, MMP-2ko tumors increased their perivascular invasion mode compared with WT controls (p = 0.065 and p = 0.030; Fig. 5D). In summary, loss of MMP-2 did not inhibit invasion, but altered the mode of diffuse tumor growth by causing tumor cells to preferentially move along blood vessels.
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Discussion |
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Finally, our observations that GBMs without MMP-2 activity grew slower and exhibited a significant increase in mean survival, although tumor cells still invaded predominantly along blood vessels, might make MMP-2 an attractive target for GBM therapy.
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Acknowledgments |
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Received for publication April 30, 2007. Accepted for publication October 24, 2007.
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References |
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