Patients and tissue specimens
The Institutional Review Board of Kyushu University School of Medicine, Fukuoka, Japan approved the protocol to obtain and examine surgical GCT specimens. Twenty-one GCT patients were surgically treated in the Department of Orthopaedic Surgery, Kyushu University. All tumor specimens were formalin-fixed and paraffin-embedded, and 5-mm sections were cut from one representative block for molecular analyses.
Sprague-Dawley rats were purchased from KBT Oriental (Saga, Japan). Recombinant human VEGF was obtained from Genzyme/Techne (Minneapolis, MN). Anti-VEGF, -Flt-1 and -Flk-1 Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-FAK Ab was obtained from Upstate Biotechnology (Lake Placid, NY). Antibodies specific for the phosphotyrosine residue at position 397 in FAK (pY-FAK Ab) and anti-tyrosine phosphorylated Flt-1 (pY-Flt-1) were purchased from Invitrogen (Carlsbad, CA) and Oncogene (San Diego, CA), respectively. The VEGF receptor tyrosine kinase (RTK) inhibitor (ZD4190) was purchased from Calbiochem (San Diego, CA).
Rat osteoclast precursor cells (rOPCs) were harvested using by the modified method as previously described (Takeshita S et al. 2000). Briefly, the femurs and tibias of 1-day-old Sprague-Dawley rats were aseptically resected. The bone ends were cut and the marrow cavity was flushed with α-MEM. The marrow cells were collected, washed and cultured in α-MEM containing 10% FCS and rhM-CSF (100 ng/mL) supplemented with 100 U/mL penicillin and 100 mg/mL streptomycin. After three days of culture, the cells were vigorously washed to remove the nonadherent cells, detached by pipetting and subcultured. After culturing for an additional three days, the cells were harvested and used as rOPCs.
Immunohistochemistry and immunofluorescence
Immunohistochemistry was performed as previously described . Surgical specimens were initially decalcified for two weeks in an EDTA-containing buffer and embedded in paraffin. The endogenous peroxidase activity was quenched by incubating the sections for an additional 30 min in absolute methanol and 3% hydrogen peroxide. The slides were then incubated with the appropriate primary Abs, followed by biotinylated secondary Abs and peroxidase-conjugated streptavidin. The signals were detected using 3-amino-9-ethylcarbazole in N,N-dimethylformamide. To examine the pY-Flt-1 and pY-FAK levels in GCT samples, the staining intensity of each specimen was scored as follows: 1 (weak staining; less than 10% of cells were positive), 2 (intermediate staining; 10-50% positive) and 3 (strong staining; >50% positive). All molecular variables were scored by one investigator, who was blinded to the clinical stages of the patients.
For immunofluorescence, the samples were incubated with the primary Abs overnight at 4°C. The samples were washed in PBS and then incubated with FITC or TRITC-conjugated secondary Abs. Then, the sections were mounted and examined by confocal laser scanning microscopy.
Tissue culture of giant cell tumors of bone
Primary cultures of GCTs were obtained from surgical samples of lytic bone lesions. As previously described , fresh tumor tissues were minced in DMEM containing 10% FBS supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin. The cell suspension containing small tissue pieces was plated in a 10 cm-culture dish and incubated at 37°C in a humidified atmosphere with 5% CO2 and 95% air. Half of the culture medium was replaced every three days with fresh DMEM containing 10% FBS. When the cells reached confluency, the primary cultures were scraped and subcultured. After several passages, the multinucleated giant cells and monocyte/macrophage-like round cells progressively disappeared from the cultures and only the proliferating spindle-shaped cells remained. At passage eight, the cells were cultured with serum-free DMEM for 24 h and the conditioned medium was collected, filtered through 2.5 μm filters, and used as GCT-conditioned medium (GCT-CM).
When the cells reached approximately 70% confluency, they were harvested and solubilized in lysis buffer [20 mM Tris (pH 7.4), 250 mM NaCl, 1.0% NP40, 1 mM EDTA, 50 mg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride]. The protein quantity was determined with a Bradford protein assay (Bio-Rad, Hercules, CA). The samples were separated on 4-12% gradient pre-cast MOPS-polyacrylamide gels (Novex, San Diego, CA) and blotted onto nitrocellulose filters. After transfer, the filters were pre-treated with TBS containing 5% dry milk and 0.05% Triton X for 2 h at room temperature and then incubated with the indicated primary antibodies for 2 h at room temperature. After several washes, the membranes were probed with the appropriate horseradish peroxidase-conjugated secondary Abs at room temperature for 1 h. After the final wash, the immunoreactivity of the blots was detected using an enhanced chemiluminescence system (Amersham, Arlington Heights, IL).
Enzyme-linked immunosorbent assay (ELISA) for VEGF
The VEGF levels in GCT-CM were determined using an ELISA kit from R&D Systems (Minneapolis, MN).
Cell proliferation assay
rOPCs cells seeded in culture plates were incubated in serum-free media with various reagents (GCT-CM, VEGF and ZD4190) for 24 h. The cell growth rate was determined using a Celltiter-Glo Luminescent Cell Viability Assay Kit (Promega, Madison, WI) according to the manufacturer's protocol.
The chemotaxis assay was performed using transwell chambers (Costar, Cambridge, MA) as previously described [13–15]. Briefly, rOPCs were suspended in serum-free α-MEM containing 1% bovine serum albumin and seeded in the upper chamber. The lower chamber was filled with serum-free α-MEM supplemented with or without various cytokines. Polyvinylpyrrolidone-free polycarbonate filters with 8.0-μm pores were coated with type IV collagen and inserted between the two chambers. Then, the cells were allowed to migrate for 6 h at 37°C. After this incubation period, the cells that had migrated to the lower side of the filter were fixed, stained and counted using five fields/filter under a microscope.
The results obtained from the chemotaxis and cell proliferation assays are expressed as the means ± SD and were statistically analyzed by the Student's t-test. The association between the expression levels of various molecular factors (pY-FAK and pY-Flt-1) and the clinical stages were analyzed using the Mann-Whitney U test.