Generation of MMP-2ko–Transformed Mouse Astrocytes
Primary astrocytes were isolated from 1- to 2-day-old WT and MMP-2ko/ko FvBN Ragko mice as described previously.14
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 aga-rose (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 × 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.
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.
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 1× 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.
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 (×40) 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 (×40) 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 (×20 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 × 20 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 ×20 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 α-smooth muscle actin (α-SMA) staining was obtained. The fraction of pericyte coverage was calculated as the ratio of the area of desmin or α-SMA staining (red) to the area of CD31 staining (green). For desmin staining, 8–17 tumor sections per group were evaluated. For α-SMA staining, four to nine tumor sections per group were evaluated.
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 Alex-aFluor350, AlexaFluor488, 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 AlexaFluor647 (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′-CAGTGACAC-CACGTGACAAGC-3′; (R) 5′-GGCAGCATCTAGTT-GCTGGAC-3′; 60°C. For the ribosomal protein L19, (F) 5′-CTGAAGGTCAAAGGGAATGTG-3′; (R) 5′-GGA-CAGAGTCTTGATGATCTC-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′-GCGGGCTC-CTGACTACAC-3′; (R) 5′-CCAAATGCTCCACCA-ACTCTG-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-α-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 × 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-α-tubulin antibody.
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).