Cancer Cell Biology
Human chondrosarcoma secretes vascular endothelial growth factor to induce tumor angiogenesis and stores basic fibroblast growth factor for regulation of its own growth
Article first published online: 10 OCT 2001
Copyright © 2002 Wiley-Liss, Inc.
International Journal of Cancer
Volume 97, Issue 3, pages 313–322, 20 January 2002
How to Cite
Furumatsu, T., Nishida, K., Kawai, A., Namba, M., Inoue, H. and Ninomiya, Y. (2002), Human chondrosarcoma secretes vascular endothelial growth factor to induce tumor angiogenesis and stores basic fibroblast growth factor for regulation of its own growth. Int. J. Cancer, 97: 313–322. doi: 10.1002/ijc.1607
- Issue published online: 27 DEC 2001
- Article first published online: 10 OCT 2001
- Manuscript Accepted: 30 JUL 2001
- Manuscript Revised: 11 JUN 2001
- Manuscript Received: 7 MAR 2001
- tumor angiogenesis;
- vascular endothelial growth factor;
- basic fibroblast growth factor;
- transforming growth factor;
- endothelial cell
Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are well-known factors that induce neovascularization in many tumors. The molecular mechanisms that regulate tumor angiogenesis in human chondrosarcoma are not clear. We assessed in this work the angiogenic activities of a human chondrosarcoma cell line (OUMS-27) in vivo and determined the efficacies of angiogenic factors derived from OUMS-27 cells on human umbilical vein endothelial cells (HUVECs) in vitro. Tumor xenografts induced an increase in the formation of neovessels, but the distributions of Ki-67 antigen, VEGF and bFGF were unaffected. We also demonstrated that OUMS-27 cells secreted VEGF165 into the culture medium and that it was the maximal angiogenic factor to stimulate endothelial proliferation and migration in chondrosarcoma. Anti-VEGF antibodies induced an approximately 70% inhibition of these responses of HUVECs, but did not have any effect on OUMS-27 cells. Anti-bFGF antibodies suppressed not only the activities of HUVECs but also the growth of tumor cells in vitro. We indicate that angiogenesis is principally elicited by VEGF165 and that tumorigenesis is mainly regulated by bFGF stored in the extracellular matrix of OUMS-27 cells. The present study may offer the availability of combination therapies for inhibition of VEGF and bFGF action on vascular endothelial cells and chondrosarcoma cells, respectively. © 2001 Wiley-Liss, Inc.
Chondrocytes are packed within specialized extracellular matrix (ECM) comprising macromolecules such as proteoglycan and type II collagen that are not usually expressed in other tissues and organs.1 Cartilaginous tissues such as articular cartilage and growth plate have been described to be rarely invaded by tumors and to have a tolerance against neovascularization.2, 3 Langer et al.4, 5 and Kuettner et al.6 reported that some proteins extracted from cartilage had anti-angiogenic activity and inhibited tumor growth. Several proteins have been identified as anti-angiogenic factors derived from cartilage, such as cartilage-derived inhibitor,7 transforming growth factor β1 (TGFβ1),8 chondromodulin-I9 and chondrocyte-derived inhibitor of angiogenesis and metalloproteinase activity.10 Several reports have stated that angiogenic proteins such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), are expressed by various chondrocytes in different developmental stages.11–15 These findings suggest that the balance between angiogenic and anti-angiogenic factors in cartilaginous tissues would be shifted toward the latter.16
Chondrosarcomas are usually derived from cartilage, however, they induce tumor angiogenesis in many cases. Hayami et al.17 reported that human chondrosarcoma cell line OUMS-2718 shows a marked reduction in chondromodulin-I expression, which may explain the lack of the anti-angiogenic property of OUMS-27 cells. Takigawa et al.19 cloned from another human chondrosarcoma cell line HCS-2/8 cells that produced not only anti-angiogenic factors such as chondrocyte-derived inhibitor of angiogenesis and metalloproteinase activity10 but also an angiogenic factor, connective tissue growth factor.20, 21 Tumor angiogenesis is thus thought to be dependent on the balance between these molecules.
Angiogenesis is induced in general by not only a decrease in the level of anti-angiogenic factors but also an increase in that of angiogenic factors. In fact, the upregulation of VEGF and bFGF in many tumors has been observed in recent years.22 Ayala et al.23 demonstrated, by immunohistochemical analysis, that VEGF expression was to a greater extent detected in higher-grade chondrosarcomas and associated with the number of intratumoral microvessels. The efficacy of anti-bFGF immunotherapy in rat chondrosarcoma was reported by Coppola et al.24 The mechanism or molecular events of angiogenesis in chondrosarcoma, however, have not been clarified yet. In addition, tumor resection with removal of a wide margin including normal tissue has been the best therapy, which elongates survival time and postoperative chemotherapy or radiation is no better than an adjunct method in the treatment of chondrosarcoma.25
We hypothesize that OUMS-27 cells secrete both VEGF and bFGF to maintain their own growth. To confirm the efficiency of anti-angiogenic therapy targeting these molecules in chondrosarcoma, we performed the following experiments: first, we investigated whether these angiogenic factors were expressed in OUMS-27 cells. Second, we assessed the angiogenic activity of OUMS-27 cells in vivo. Finally, we examined the validity of the use of antibodies against VEGF and bFGF for anti-angiogenic therapy in vitro. We found that VEGF165 protein was secreted and that 70% of the angiogenic activity of OUMS-27 cells was dependent on VEGF. In contrast, bFGF mainly acted as a protein stored in ECM for tumor growth. Our study suggests that anti-angiogenic therapies might be also useful against human chondrosarcoma.
MATERIAL AND METHODS
Cells and cell culture
The OUMS-27 human chondrosarcoma cell line was a kind gift from Dr. Toshiyuki Kunisada, who established it in 1998.18 OUMS-27 cells were maintained and sub-cultured as described previously.18 Human umbilical vein endothelial cells (HUVECs) were obtained from Kurabo (Osaka, Japan) and were cultured in Medium 200S (M200S) containing 2% low serum growth supplement (LSGS) (both obtained from Cascade Biologics, Portland, OR) on gelatin-coat dishes (Becton Dickinson, Franklin Lakes, NJ). Cells between passage 2 and 6 were used for experiments. HUVECs were incubated at 37°C in 5% CO2 in a humidified atmosphere and sub-cultured at a density of 2,500 cells/cm2 every 5–7 days with 0.25% trypsin, 1 mM EDTA solution (Life Technologies, Rockville, MD) and trypsin neutralizing solution (Kurabo). The medium was changed every 2 days.
RNA isolation and Northern blot analyses
Total cellular RNAs from confluent cells were extracted with 4 M guanidine thiocyanate lysis buffer and centrifuged for 20 hr through a 5.7 M cesium-chloride solution at 180,000g, 20°C. After denaturation, RNA samples (10 μg) underwent electrophoresis in 1% agarose-2.2 M formaldehyde gels and were then transferred to nylon membranes (Hybond N+, Amersham Pharmacia Biotech, Buckinghamshire, UK). The membranes were prehybridized for 1 hr with Church buffers at 65°C and were hybridized overnight at 65°C with the 32P-labeled specific probes (see below). The cDNA probes were labeled with [α-32P]-dCTP (Amersham Pharmacia Biotech) by using multiprime DNA labeling system RPN1601Z (Amersham Pharmacia Biotech). The membranes were washed for 10 min at 65°C in 2× SSC and then incubated twice for 10 min each time at 65°C in 0.1× SSC. Autoradiography was performed for 24–72 hr at −70°C by using Kodak BioMax MS film (Kodak, Tokyo, Japan) with an intensifying screen. The VEGF, fms-like tyrosine kinase-1 (FLT-1, VEGFR-1), kinase insert domain-containing receptor (KDR, VEGFR-2), bFGF and fibroblast growth factor receptor-1 (FGFR-1) signals were then normalized to the levels of GAPDH signals.
RT-PCR and Southern blot analyses
RNA samples (5 μg) were reverse-transcribed for 50 min at 42°C with reverse transcriptase (ReverTra Ace, Toyobo, Osaka, Japan). The cDNAs underwent PCR amplification in the presence of 10 pmol of each specific primer, 200 μM deoxyribonucleotide triphosphates and 0.5 U of Taq DNA polymerase (Qiagen K.K., Tokyo, Japan). For all the RT-PCR fragments, the reaction was allowed to proceed for 30 cycles, each cycle consisting of 30 sec at 94°C, 30 sec at 58°C and 40 sec at 72°C, in an automated thermal cycler (Perkin Elmer Applied Biosystems, Foster, CA). The reaction was initiated by 2 min of incubation at 94°C and ended after 4 min of extension at 72°C. PCR products were electrophoresed on 1% agarose gels stained with ethidium bromide except for products of VEGF isoforms, which were separated on 1.5% gels. For Southern blotting analysis, PCR products were transferred to Hybond N+ membranes by alkali blotting and hybridized overnight with the 32P-labeled cDNA probes. The membranes were washed twice for 10 min each time at 65°C in 2× SSC and then twice for 10 min each time at 65°C in 0.1× SSC. The membranes were exposed against Kodak MXJB-1 film with an intensifying screen for 5–60 min at −70°C. To identify the VEGF transcript isoforms and common parts, we used the following sense and antisense oligonucleotides: 5′-CGCGGATCCCTTTCTGCTGTCTTGGGTGC-3′ and 5′-CGGAATTCTCACCGCCTCGGCTTGTCAC-3′ for spanning exons 1–8 and 5′-CGCGGATCCCTTTCTGCTGTCTTGGGTGC-3′ and 5′-CGGAATTCCTGTAGGAAGCTCATCTCTC-3p for spanning exons 1–5. The cDNA fragments corresponding to FLT-1, KDR, bFGF and FGFR-1 were amplified with the following sets of primers: 5′-GAGATCAGGAAGCACCATAC-3′ and 5′-GAAGAGAGTCGCAGCCACAC-3′ for FLT-1, 5′-GATTCCTACCAGTACGGCAC-3′ and 5′-CAAACAGGTGTGGGCAACTC-3′ for KDR, 5′-GGAGTGTGTGCTAACCGTTA-3′ and 5′-TAGCTTTCTGCCCAGGTCCT-3′ for bFGF and 5′-CATCAACCACACATACCAGC-3′ and 5′-GAGTCCGATAGAGTTACCCG-3′ for FGFR-1. The 452-bp fragment of GAPDH was amplified by using GAPDH 0.45-kb control amplimer set (Clontech Laboratories, Palo Alto, CA). PCR product for VEGF (exons 1–5) was ligated to pBluescript II SK+ (Stratagene, La Jolla, CA). PCR fragments for FLT-1, KDR, bFGF, FGFR-1 and GAPDH were ligated to pCRII vectors by using a TA cloning kit (both obtained from Invitrogen, San Diego, CA). The sequencing reactions of these fragments were performed with a BigDye Terminator Cycle Sequencing Ready Reaction Kit and AmpliTaq FS (Perkin Elmer). The sequencing was performed on an ABI Prism 310 Genetic Analyzer (Perkin Elmer). Registered deviations from the published sequences were confirmed by repeating the sequence analysis. These confirmed fragments were used as cDNA probes for Southern and Northern blot analyses.
ELISA and Western blot analyses
Confluent OUMS-27 cells in 75-cm2 tissue culture flasks (Greiner, Frickenhausen, Germany) were starved for 48 hr in serum-free Dulbecco's modified Eagle's medium (DMEM, Life Technologies) or M200S and then the conditioned medium (CM) was collected and centrifuged to discard all cellular fragments. VEGF and bFGF proteins were quantified by using Quantikine human VEGF and bFGF ELISA kits (both obtained from R&D Systems, Minneapolis, MN), respectively, according to the manufacturer's protocol. After collection of culture supernatants, cells were trypsinized and counted with a hemocytometer. Levels of VEGF and bFGF proteins were calculated as nanograms of VEGF or picograms of bFGF/106 cells as described.26, 27 These CM were used in proliferation assays and migration assays. Preliminary experiments confirmed that the VEGF ELISA kit detected both VEGF121 and VEGF165.
In addition, we performed Western blot analyses for VEGF and bFGF. The confluent OUMS-27 cells in 75-cm2 tissue culture flasks were starved for 72 hr in serum-free DMEM. The CM was collected and centrifuged to discard all cellular fragments and then the cells were washed twice with ice-cold phosphate buffered saline (PBS) and scraped from the culture plates with 500 mM NH3OH. These samples were precipitated with trichloroacetic acid solution, resuspended in a minimal volume of 1 M NaOH and lysed in standard SDS-PAGE sample buffer under non-reducing conditions. Fifty micrograms of protein were electrophoresed through a 12% polyacrylamide gel and the separated proteins were transferred onto Immune-Blot PVDF membranes (Bio-Rad, Hercules, CA). After blocking with 1% bovine serum albumin (BSA, Sigma, St. Louis, MO) in PBS for 1 hr at room temperature, the membranes were incubated with goat anti-human VEGF polyclonal antibody (anti-VEGF antibody, R&D Systems) or rabbit anti-human bFGF polyclonal antibody (anti-bFGF antibody, Pepro Tech, London, UK) for 1 hr. After the standard washes in PBS-T (PBS containing 0.05% Triton X-100), the bands were visualized by using a Vectastain ABC-AP kit and Vector blue alkaline phosphatase substrate kit III (Vector Laboratories, Burlingame, CA) according to the procedures prescribed in their manuals. Biotinylated antibodies against goat IgG and rabbit IgG in the kits were used for the detection of VEGF and bFGF, respectively. Recombinant human VEGF121 (rh-VEGF121, R&D Systems), recombinant human VEGF165 (rh-VEGF165, R&D Systems) and recombinant human bFGF (rh-bFGF, Pepro tech) were detected as a 28-, 42- and 17.2-kDa proteins, respectively, under non-reducing conditions. The cross reactivity to VEGF121 and VEGF165 of anti-VEGF antibody was tested in a preliminary experiment.
In vivo tumor models
Male BALB/c nu/nu mice (Clea Japan, Tokyo, Japan), 6 weeks of age, received in their right flank subcutaneous injections of cultured OUMS-27 cells (1 × 107 cells) suspended in a 200-μl volume of DMEM. Tumors appeared ∼1 week after implantation. Tumor size was measured with a caliper and tumor volume (TV) was calculated by using a standard formula, TV = width2 × length × 0.52.17 After ∼3 weeks, animals were randomized, into groups each of 3 mice, with comparable tumor size within and among the groups (the TV ranged from 210∼260 mm3). The animals were killed at various time points (3, 6 and 16 weeks after inoculation). Tumors from each mouse were removed for histological and immunohistochemical analyses.
Histology and immunohistochemistry
Half of the removed tumor xenograft was fixed with 4% paraformaldehyde-buffered solution. Tissue samples were embedded in paraffin for hematoxylin and eosin (H& E) staining, safranin O staining and immunohistochemistry of Ki-67. From paraffin blocks, 4.5-μm thick serial sections were cut, dewaxed and rehydrated in water for the staining procedures. The localization of glycosaminoglycans was visualized by using safranin O staining.28, 29 Sections were stained in 0.1% safranin O solution (pH 7.4) for 3 min at room temperature. The synthesis or depletion of proteoglycans was assessed by the uptake of safranin O dye (Chroma, Köngen, Germany).30 Proliferative activity was evaluated by using mouse anti-human Ki-67 monoclonal antibody (anti-Ki-67 antibody) and HistoMouse-Plus kit as per manual instructions (both obtained from Zymed Laboratories, San Francisco, CA). Tumor sections were pretreated with autoclave heating for antigen retrieval before staining for Ki-67. PBS without primary antibody was used as the negative control. Several fields were assessed randomly except for necrotic areas, under high-power magnification (×400) and the Ki-67-positive index was determined by scoring the positive cells per total cells in 4 fields, as described.31, 32
The other half of each tumor tissue sample was frozen in liquid nitrogen and stored at −80°C. Seven-μm thick serial sections were cut at −20°C, treated for 5 min in cold acetone, air-dried and blocked with 1% BSA for 30 min at room temperature. Two types of antibody, anti-VEGF and anti-bFGF antibody, were used for immunohistochemical staining. Staining with each antibody diluted to a final concentration of 10 μg/ml in PBS was performed for 1 hr at room temperature in a humid atmosphere. PBS without primary antibody was used for the negative control. Sections were washed 3 times in PBS and stained with Vectastain ABC-AP kit according to instructions in the manual. Subsequent staining was performed with a Vector blue alkaline phosphatase substrate kit III and then the sections were stained with Vector nuclear fast red for counterstaining.
To determine the degree of tumor-induced angiogenesis, 7-μm cryostat sections of tumor xenografts were fixed in acetone for 5 min, air-dried, blocked with 1% BSA for 30 min at room temperature and stained with rat anti-mouse CD31 monoclonal antibody (anti-CD31 antibody, Pharmingen, San Diego, CA) (1:50 dilution in PBS) for 1 hr. PBS without primary antibody was used for the negative control. The tissue localization of CD31 was visualized by incubating the sections for 45 min at room temperature with fluorescein-conjugated affinity-purified goat antibody against rat IgG (ICN Pharmaceuticals, Aurora, OH) and was followed by a 5 min incubation in Hoechst 33258 (Wako, Osaka, Japan) as a counterstain.
Two CD31-immunostained sections were analyzed per tumor. The entire slide was scanned at a magnification of ×100 to determine 4 areas at the periphery of the tumor showing the greatest vascularization. A countable microvessel was defined as any stained endothelial cell or cell cluster that was separated from the adjacent microvessels. Neither the presence of vessel lumens nor of red blood cells were necessary to define a microvessel. Branching structures were counted as a single vessel. CD31-immunostained plasma cells were eliminated from the count. Microvessels in normal subcutaneous tissue adjacent to the tumor were taken as an internal control. The final quantification was carried out on the number of microvessels per mm2 obtained from the 4 fields at a magnification of ×200, as described.33, 34
Cell proliferation assays
Cultured HUVECs were washed with PBS and dispersed in a 0.25% trypsin, 1 mM EDTA solution. A cell suspension was made in M200S (LSGS+), plated as 1 × 104 cells/well onto 96-well plastic tissue culture plates (Coster, Cambridge, MA) and incubated (37°C, 5% CO2) for 24 hr. After the cultures had been washed with PBS, the media were replaced with LSGS-free M200S containing 0.1% BSA. The medium containing rh-VEGF165 (5 ng/ml) or rh-bFGF (10 ng/ml) and the CM of OUMS-27 cells cultured in M200S (see above) were used for proliferation assays.
Cultured OUMS-27 cells were washed with PBS and dispersed in a 0.1% trypsin, 5 mM EDTA solution. A cell suspension was made with DMEM (containing fetal bovine serum, FBS, Sigma), plated as 1 × 104 cells/well onto 96-well plates and incubated (37°C, 5% CO2) for 24 hr. After the cells had been washed with PBS, the media were replaced with serum-free DMEM containing 0.1% BSA. The medium containing rh-VEGF165 (5 ng/ml), rh-bFGF (10 ng/ml) or rh-TGFβ1 (1 ng/ml, R&D Systems) and the CM of OUMS-27 cells cultured in DMEM (see above) were used for proliferation assays. In some cases, neutralizing antibodies (anti-VEGF, anti-bFGF or mouse anti-human TGFβ1 antibody, R&D Systems) were added to the medium at a final concentration of 10 μg/ml and the cells were incubated for 1 hr at room temperature before the proliferation assays. These plates were incubated (37°C, 5% CO2) for 24 hr for proliferation and then the cells were incubated with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide, Chemicon, Temecula, CA) according to the manufacturer's protocol. Each of the test samples was added to 4 wells (n = 4). The optical density was measured by using a Model 550 microplate reader (Bio-Rad) with a test wavelength of 595 nm and a reference wavelength of 655 nm. The data obtained by substraction of readings at 655 nm from those at 595 nm were used for evaluation. The assays were run in quadruplicate and similar results were obtained.
Cell migration assays
Cell migration assays were performed in a modified Boyden chamber (48-well chemotaxis chamber AP48, Neuro Probe, Gaithersburg, MD). Polycarbonate membranes (8-μm pore, 25 × 80 mm size, polyvinylpyrrolidone free, Neuro Probe) were coated with 100 μg/ml of rat tail type I collagen in 0.2 N acetic acid (Collaborative Biomedical Products, Bedford, MA) for 2 days and air-dried. The membrane was placed over the bottom chamber filled with the desired medium (n = 4): rh-VEGF165 (5 ng/ml) or rh-bFGF (10 ng/ml) in M200S (DMEM) containing 0.1% BSA or CM of OUMS-27 cells. BSA was added to the CM at a final concentration of 1 mg/ml before use. Neutralizing antibodies were added to the medium at a final concentration of 10 μg/ml, 1 hr before the migration assays, with this pretreatment at room temperature. HUVECs were suspended in M200S and 1 × 104 cells in 50 μl were added to the upper chamber of each well. OUMS-27 cells were suspended in DMEM. The assembled chemotaxis chamber was incubated for 6 hr at 37°C with 5% CO2 to allow cells to migrate through the collagen-coated polycarbonate membrane. Non-migrating cells on the upper surface of the membrane were removed by scraping with a wiper tool (Neuro Probe) and the membrane was stained with Diff-Quik (VWR Scientific Products, Bridgeport, NJ). The total number of cells with nuclei that migrated per well was counted as described.35 The assays were performed in quadruplicate.
Frozen sections were exposed to heparitinase, which cleaves heparan sulfate proteoglycans, to elucidate the stored bFGF in the ECM synthesized by OUMS-27 cells. Heparitinase (Seikagaku, Tokyo, Japan) was diluted in an indicated buffer and incubated at 37°C for 2 hr according to the manufacturer's protocol. Immunostainings with an anti-bFGF antibody were performed in the same method (see above).
All experiments were repeated at least twice and similar results were obtained. Data were expressed as means ± SD. Differences among groups were compared by using the Mann-Whitney U-test. Statistical significance was established at p < 0.05.
OUMS-27 cells secrete VEGF165 protein and hold bFGF protein
We investigated the expressions of VEGF, bFGF and their receptors in OUMS-27 cells. First, we performed RT-PCR analyses using first-strand cDNAs reverse-transcribed from RNAs isolated from cultured OUMS-27 cells. The RT-PCR fragments of VEGF, FLT-1, KDR, bFGF and FGFR-1 were detected in significant amounts (Fig. 1a). The amplified fragment of KDR, which mainly transduces the signals of VEGF, was in relatively low level. The results of Northern blot analyses indicated that the cells expressed VEGF165 and FGFR-1 mRNA at a fairly high level (Fig. 1b). We also performed RT-PCR using exon-1 and exon-8 primers to determine the proportion of VEGF isoforms. Four isoforms (VEGF121, VEGF145, VEGF165 and VEGF189) were obtained; however, VEGF206 was not detected (Fig. 1c). We also examined the expression of these mRNAs in HUVECs by Northern blot analysis and observed VEGF, FLT-1 (the 7.5-kb mRNA for the full-length receptor was detected, but the shorter 2.7-kb transcript for its soluble variant was not seen), KDR (7.5-kb), bFGF (7.4- and 4.4-kb) and FGFR-1 mRNAs (data not shown). Thus, it appeared that OUMS-27 cells expressed VEGF165 mRNA at a significant level, whereas bFGF expression remained.
We next tried to detect VEGF and bFGF proteins in the CM and the cell lysate (CL) of OUMS-27 cells. VEGF165 protein was identified in both CM and CL, but VEGF121 protein was not detected in either (Fig. 1d). The signal of bFGF protein of 17.2-kDa was only seen in the CL (Fig. 1d). We then performed ELISA to measure the concentrations of VEGF and bFGF protein in the CM of OUMS-27 cells as described in Material and Methods. The levels of VEGF and bFGF proteins in the M200S was determined as 4.8 ng/106 cells/48 hr and 113 pg/106 cells/48 hr, respectively. The DMEM contained VEGF (or bFGF) protein at a concentration of 4.3 ng (155 pg)/106 cells/48 hr. These data suggested that VEGF165 protein was the major VEGF isoform secreted from OUMS-27 cells and bFGF protein was conceivably low and mostly trapped in the cell surface or cytoplasm.
Inoculated OUMS-27 cells induce angiogenesis in an in vivo model
To demonstrate whether tumor angiogenesis could be induced in vivo by OUMS-27 cells, we injected these cells into nude mice. The inoculated OUMS-27 cells retained their proliferative activities in the subcutaneous space of the mice. After 3 weeks, the mean TV reached 237 mm3 (ranged from 219–252 mm3). Tumor specimens showed that 1 lobule had formed, containing a mass of OUMS-27 cells, but there were no intratumoral blood vessels and no necrotic areas when the sections were observed in H&E staining (Fig. 2a). TV reached 555 (522∼587) mm3 by 6 weeks after the injection. We observed 2 or 3 lobules surrounded and divided by fibrous tissues that had originated from mouse cells. The section showed that a few microvessels had penetrated into the septa (Fig. 2b). Several necrotic zones were observed nearby the center of the tumor lobules. TV was calculated to be 8,300 (7,480∼9,120) mm3 after 16 weeks. By this phase, the lobules had become enveloped by fibrous tissues, the necrotic area had increased and the microvessels had invaded into tumors through the involucre (Fig. 2c). To determine the grade of angiogenesis in this in vivo model, we stained the serial sections using anti-CD31 antibodies (Fig. 2d–g). The number of CD31-positive microvessels per mm3 among the 4 indicated groups was augmented as the tumor grew (number of microvessels/mm3: internal control, 3 weeks, 6 weeks and 16 weeks after inoculation was 99.3 ± 2.52, 116.7 ± 9.29, 169.7 ± 8.02 and 204.3 ± 8.51, respectively; p < 0.05; Fig. 3). These results suggest that the injected human OUMS-27 cells occupied the subcutaneous space of the nude mice and induced angiogenesis from the surrounding host tissues, which induction was probably essential for the tumors to survive.
OUMS-27 cells retain their proliferative and angiogenic activities for long duration in the in vivo tumor model
Because tumor angiogenesis was expected to involve the expression of angiogenic factors and the proliferation of OUMS-27 cells, we subsequently performed the following histochemical analyses. First, we assessed the production of proteoglycans in OUMS-27 cells by safranin O staining. The staining pattern was almost the same among the 3 groups (Fig. 2h–j). Second, the assessment of proliferation by anti-Ki-67 antibodies showed no significant difference among the groups (Fig. 2k–n). The rate of Ki-67-positive cells was 54.8 ± 8.1%, 57.7 ± 9.5% and 52.6 ± 8.3% in 3 weeks, 6 weeks and 16 weeks, respectively, after the inoculation (p > 0.05). The Ki-67-positive cells were rarely observed around necrotic areas. Finally, we immunostained the tumor tissues with anti-VEGF or anti-bFGF antibodies. The staining pattern of VEGF and bFGF proteins was not much different among the sections (Fig. 2o–v). It was not possible for us to evaluate which of the 2 growth factors was predominantly contributing to the tumor angiogenesis. These findings may be due to the invariance of inherent activity of OUMS-27 cells in vivo at any time point. It appears that tumor angiogenesis is induced by the increase in the total amount of VEGF in this in vivo model, not by the increment of it per cell.
VEGF secreted from OUMS-27 cells stimulates the proliferation of HUVECs and bFGF is the regulator for the proliferation of OUMS-27 cells
We next analyzed, by proliferation assays, the influences of angiogenic factors from OUMS-27 cells to endothelial cells in vitro. Neutralizing antibodies against VEGF and bFGF were simultaneously used for the evaluation of their angiogenic effects. Figure 4a and b demonstrates that the CM of OUMS-27 cells stimulate the proliferation of both HUVECs and OUMS-27 cells. The increase in HUVEC proliferation stimulated with the CM was inhibited to the 28% of the maximum proliferation by anti-VEGF antibody and anti-bFGF antibody also displayed an inhibitory effect (Fig. 4c). OUMS-27 cells were not affected by rh-VEGF165, but responded to rh-bFGF with 37% increase in proliferation (Fig. 4b). The upregulation of OUMS-27 proliferation in the CM was reduced to 34% of the maximum by anti-bFGF antibody (Fig. 4d, right columns). Control experiments were performed using antibodies without stimulus (0.1% BSA) and confirmed the lack of an effect on the cell proliferations (Fig. 4c,d, left columns). These data demonstrate that the proliferation of endothelial cells is mainly affected by VEGF165 protein secreted from OUMS-27 cells, which proliferation was inhibited significantly by the specific antibody.
Conditioned medium of OUMS-27 cells activates the migration of HUVECs and OUMS-27 cells in different manners
We performed cell migration assays to assess the effect of the angiogenic factors on the migration activity of HUVECs and OUMS-27 cells. The migration of HUVECs was stimulated by rh-VEGF165, rh-bFGF and the CM with significant differences (p < 0.05; Fig. 5a). Figure 5c shows the individual effects with or without antibodies, on HUVECs. No accessory migration of OUMS-27 cells was induced by rh-VEGF165, in line with the results of OUMS-27 proliferation assay; however, their migration was strongly stimulated by rh-bFGF and CM (Fig. 5b). These data clearly demonstrate that bFGF is one of the major stimulant for the migration of OUMS-27 cells and that VEGF has no biological effect on them (Fig. 5d). From these results, it appears that VEGF was also necessary for the migration of endothelial cells and that approximately 50% of the invasive activities of OUMS-27 cells depended on bFGF.
Basic FGF is stored in the ECM of chondrosarcoma and TGFβ1 also stimulates OUMS-27 proliferation
We performed heparitinase digestion of tumor sections to assess the stored bFGF binding to cell surface heparan sulfate proteoglycans. The stainability by using anti-bFGF antibody was decreased or fully diminished by heparitinase treatments (Fig. 6a,b). The immunostaining with anti-TGFβ1 antibody was not affected by heparitinase treatments (data not shown). In addition, the mRNAs of type I, type II TGFβ receptor and TGFβ1 were detected in OUMS-27 cells by RT-PCR (data not shown) so that we further analyzed the effect of TGFβ1 on OUMS-27 proliferation. Rh-TGFβ1 induced the 20% increase of OUMS-27 proliferation and its effect was inhibited to basal level by anti-TGFβ1 antibodies (Fig. 6c). The increase of OUMS-27 proliferation cultured in the CM was prevented to 34 and 58% level of control by anti-bFGF antibody and anti-TGFβ1 antibody, respectively. These results demonstrate that OUMS-27 growth is regulated by synergistic effects of bFGF and TGFβ1.
Our results illustrate the production of VEGF and bFGF proteins in a human chondrosarcoma cell line. VEGF and bFGF have been shown to induce angiogenesis by activating endothelial cells in many cases of tumors and other inflammatory diseases such as rheumatoid arthritis and proliferative retinopathies.36–38 The effects of these angiogenic factors on endothelial cells have been investigated in detail,11, 39, 40 however, the interactions between these molecules and human chondrosarcomas are still unclear. In our study, VEGF165 was shown to be the major regulator of tumor angiogenesis in human chondrosarcoma, acting in a paracrine fashion. The effect of bFGF derived from chondrosarcoma cells on endothelial cells was little, however, the tumor growth was mainly dependent on the bFGF stored in the ECM and cell surface of chondrosarcoma.
We recognized in our study that 4 human VEGF isoforms encoding 121, 145, 165 and 189 amino acid residues, respectively, were present in OUMS-27 cells, produced by alternative splicing of the VEGF mRNA.41 Many articles have described that tumor cells mainly express VEGF121 and VEGF165 isoforms.36, 42 Our results support these previous reports. High expression of VEGF165 mRNA was shown in Figure 1b and only VEGF165 protein of 40-kDa was detected in the CM of OUMS-27 cells (Fig. 1d). Therefore, we suggest that the estimated VEGF in CM represented the concentration of VEGF165. These results suggest that OUMS-27 cells secreted VEGF165 protein as a main VEGF isoform.
Charnock-Jones et al.43 described that VEGF145 was specific isoform in the female gynecological tract. The VEGF145 isoform was detected by RT-PCR (failed to be detected by Northern blot analysis) in endometrial adenocarcinoma cells stimulated with insulin-like growth factor-1.44 Although previous studies36, 42 could not detect VEGF145 except in female inherent tissue, we obtained the first finding that the VEGF145 isoform is also exhibited in a male chondrosarcoma. VEGF145 has been reported as a specific protein that binds to ECM produced in corneal endothelial cells by a different mechanism.45 The present findings suggest that chondrosarcoma may be characterized by (i) distinct ECM containing VEGF145, (ii) an another pathway for angiogenesis mediated by VEGF145 and (iii) atypical tumor growth stimulated by a specialized population of endothelial cells. Further studies to analyze the effect of the VEGF145 isoform on chondrosarcoma cells are required.
Basic FGF is a multipotential factor that stimulates proliferation, migration and differentiation of many types of cells.46 This molecule is not secreted, consistent with the lack of a signal peptide.46 We could measure its low concentration in the CM of OUMS-27 cells by ELISA. And also, we detected bFGF protein in the CL (Fig. 1d) and demonstrated that it was stored in the ECM of chondrosarcoma by binding heparan sulfate proteoglycans (Fig. 6a,b). Sasaki et al.47 reported that the concentration of bFGF released from rabbit articular cartilage into CM was under 100 pg/ml and that this level was elevated by interleukin-1. Our results support previous studies and suggest the dependence of tumorigenesis on bFGF in human chondrosarcoma.
The avascular feature of cartilage is regulated by the differentiation of chondrocytes and the ECM condition. Horner et al.12 demonstrated that an increase in VEGF expression on human neonatal growth plate cartilage occurred with the maturation of the chondrocytes. Transferrin (iron transporter) has been identified as the major angiogenic molecule released from hypertrophic chondrocytes and shown to promoted the migration of endothelial cells.48 Alini et al.49 purified a novel angiogenic chemoattractant from bovine hypertrophic chondrocytes. The regulation, however, is disorganized in chondrosarcoma because of its oncogenicity. Ayala et al.23 suggested that upregulation of VEGF expression and neovascularization led to a high grade of human chondrosarcoma. The present study demonstrated that the VEGF165 protein is the active angiogenic stimulator in human chondrosarcoma. The proliferation and migration of endothelial cells mostly depended on a paracrine factor, VEGF; and bFGF had a minor effect in the in vitro assays. In contrast, the reactions of tumor cells in these assays support the results of Northern blot analyses demonstrating that OUMS-27 cells expressed FGFR-1 but not FLT-1 or KDR (Fig. 1b) and suggest that chondrosarcoma growth is mainly dependent on bFGF produced by the cell itself.46, 50 Furthermore, we analyzed the other factor derived from OUMS-27 cells. TGFβ1 has been shown to stimulate the proliferation of rat chondrosarcoma cells.51, 52 OUMS-27 cells expressed the mRNAs of TGFβ1, type I and II receptor of TGFβ and platelet-derived growth factor receptor in RT-PCR but not detected hepatocyte growth factor receptor (data not shown). Moreover, the OUMS-27 proliferation in their CM was inhibited to basal levels in the presence of anti-TGFβ1 and anti-bFGF antibodies (Fig. 6c). These results suggest that TGFβ1 is also an important factor to regulate human chondrosarcoma growth.
Formation of tumor vessels has attracted much interest in recent decades.38, 40 Figure 7 is a schema that summarizes tumor angiogenesis in chondrosarcoma cells revealed by our study. The animal model we employed was useful to investigate anti-angiogenic effects, because injected OUMS-27 cells developed the systematic angiogenesis and we could easily assess the grade of neovascularization. Although angiogenic activity increased in the tumor, the immunostaining pattern of the VEGF, bFGF for that for Ki-67 did not show any significant time related difference. We suppose that the activity of cell proliferation and potential for secreting the growth factors per cell may not change despite the enlargement of the tumor. Uria et al.53 described that the expression of collagenase-3 (matrix metalloproteinase-13) was induced by bFGF in HCS-2/8 human chondrosarcoma cells, so that the stored bFGF in the ECM might support the penetration of newborn microvessels in this tumor model. In addition, the OUMS-27 cells used in our study did not show any calcification but continued producing cartilaginous tissues; therefore, reactivity to several differentiation factors might be missing in chondrosarcoma cells.
Wide margin tumor resection has been considered to be the best therapy for high-grade chondrosarcomas.25 At the experimental level, Baird et al.54 successfully inhibited the growth of mouse transplantable chondrosarcoma by using antibody against immunoreactive FGF extracted from the tumor itself. Coppola et al.24 also suppressed rat chondrosarcoma by intralesional injection of anti-bFGF antibody and suggested that the anti-tumor force of the antibody was due to an inhibitory effect on the proliferation of endothelial cells. In our study, we found that anti-bFGF antibody inhibited not only the endothelial growth but also the proliferation of OUMS-27 cells in vitro and that the latter effect was stronger than the former one. Two opposite results have been reported: an anti-bFGF antibody inhibited tumor growth,24, 54, 55 but on the other hand, it did not prevent tumorigenesis.56, 57 In the therapy of chondrosarcomas, the system of delivery of anti-bFGF antibody to tumor cells is considered to be the most important point, because bFGF is trapped around tumor cells. We propose that anti-bFGF therapy requires a peritumoral or intratumoral approach to chondrosarcoma, because anti-bFGF antibody hardly infiltrates into cartilage-specific ECM as described.14 Although no inhibitory effect of anti-VEGF antibody on chondrosarcoma has been reported yet, Gerber et al.58 demonstrated that soluble VEGF receptor prevented endochondral ossification in growth plates. These results suggest that the following combinations might be useful in chondrosarcoma therapy: (i) anti-VEGF antibody therapy to inhibit neovascularization and (ii) anti-bFGF antibody therapy to suppress tumor growth.
In conclusion, we demonstrate that VEGF165 protein secreted from human chondrosarcoma cell line (OUMS-27) stimulates the proliferation and migration of endothelial cells. In addition, bFGF is stored in the ECM of chondrosarcoma and cooperatively regulates the proliferation and migration of OUMS-27 cells with TGFβ1.
We would like to thank Ms. N. Shimazaki for her useful technical advice on the in vivo studies. We are grateful to Miss A. Yoshida for the histological analyses. Dr. N. Yamaguchi is acknowledged for her technical support regarding the migration assay. We thank lab members for comments and discussion. Finally, we greatly appreciate Dr. Toshiyuki Kunisada for generously providing us with the human chondrosarcoma cell line (OUMS-27).
- 25Chondrosarcoma of bone: an assessment of outcome. J Bone Joint Surg Am 1999; 81-A: 326–38., , , et al.
- 28Chemical basis for the histological use of safranin O in the study of articular cartilage. J Bone Joint Surg Am 1971; 53-A: 69–82..