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Human Cancer Biology |
Department of Oncological Sciences and Laboratories of 1 Clinical Oncology and 2 Molecular Angiogenesis, University of Turin Medical School, IRCC Institute for Cancer Research and Treatment, Turin, Italy
Requests for reprints: Giuliana Cavalloni, Department of Oncological Sciences, Institute for Cancer Research and Treatment, Strada Provinciale 142, 10060 Candiolo, Turih, Italy. Phone: 39-011-9933523; Fax: 39-011-9933524; E-mail: giuliana.cavalloni{at}ircc.it.
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: osteosarcoma • chemokines • chemokine receptors • tumor progression
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Metastasis development is a complex mechanism in which many factors, such as properties of tumor cells as well as the microenvironment in which they localize, can potentially influence tumor dissemination (5). Recent studies indicate that the metastatic process shares many similarities with leukocyte trafficking (6).
Chemokines are a superfamily of cytokine-like proteins that are grouped into CXC and CC chemokines that selectively attract and activate different cell types. These secreted proteins induce, through their interaction with G-protein-coupled receptor, cytoskeleton rearrangement, firm adhesion to endothelial cells and directional migration (7, 8).
The CXC chemokine stromal cell–derived factor 1 (SDF-1 or CXCL12; ref. 7) is expressed on the surface of vascular endothelial cells and is secreted by stromal cells from a variety of tissues such as bone marrow, lung, and liver (9). Its chemotactic effect is mediated by interaction with the chemokine receptor 4 (CXCR4 or CD184; refs. 10–12). It has been shown that endothelial cells expressing the CXCR4 receptor are strongly chemoattracted by SDF-1 (13). Hematopoietic stem cells are selective in their migratory response to SDF-1, which regulates their specific homing in marrow microenvironment (14–16).
Involvement of the CXCR4/SDF-1 axis has been shown during tumor progression of breast, prostate, and pancreatic cancer as well as in rhabdomyosarcoma and lymphoma (17–20). Owing to the importance of the CXCR4/SDF-1 axis in homing and in promoting cell migration in different kinds of tumor, it is possible that also osteosarcoma uses this system to produce metastases.
Recent studies show that CXCR4 inhibition can affect tumor cell dissemination in non-Hodgkin lymphoma, melanoma, and breast cancer (21, 22). The polyphemusin II peptide T22 and its derived T134 have shown in vitro the ability to prevent HIV type 1 (HIV-1) infection by blocking HIV-1 binding to its coreceptor CXCR4. T134 peptide exhibits higher activity and significantly less cytotoxicity in comparison with T22; it competes with SDF-1 at much lower concentration and shows a higher affinity than SDF-1 for the binding to CXCR4 (23–27).
Here we analyzed CXCR4 expression on some osteosarcoma cell lines and evaluated in vivo the potency of T134 peptide to modulate SDF-1-mediated chemotaxis and migration of osteosarcoma ells. In this study, we showed the bility of T134 to block CXCR4/SDF-1 interaction and to prevent osteosarcoma cell dissemination in the metastatic progression.
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MATERIALS AND METHODS |
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Bone marrow stromal cells from normal donors were obtained, after informed consent was given, by culturing 107 bone marrow mononuclear cells as previously described (28).
Human umbilical vein endothelial cells (HUVEC) were isolated from umbilical cord, after informed consent was given, by collagenase treatment and cultured in dishes precoated with 0.04% gelatin according to Jaffe et al. (29). Briefly, the cells were maintained in M199 medium supplemented with 100 units/mL penicillin/streptomycin, 2 mmol/L L-glutamine (all from Biochrom, Berlin, Germany), 20% FCS, 40 µg/mL endothelial cell growth factor (Boehringer Mannheim, Mannheim, Germany), and 50 µg/mL heparin (Sigma-Aldrich, Deisenhofen, Germany). Cells were subcultured after trypsinization and used throughout passages 2 to 4. Bone marrow stromal cells and HUVEC proved to be a constant and reproducible source of SDF-1.
Flow Cytometric Analysis
Cells were washed in 1x PBS containing 0.1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO) and 0.01% sodium azide. The cells were labeled for 45 minutes at 4°C with mouse monoclonal antibody (mAb) R-phycoerythrin-conjugated anti-human CXCR4 (clone 12G5, R&D Systems, Minneapolis, MN). For intracellular CXCR4 staining we blocked cell surface expression by incubating with 10 µg/mL of nonconjugated anti-human CXCR4 mAb for 1 hour at 4°C. The cells were than washed, fixed, and permeabilized to stain the intracellular CXCR4 with anti-human CXCR4-PE mAb. After an additional wash, the receptor expression on 10,000 cells was determined using a FACS Vantage (Becton Dickinson, San Jose, CA). Cell acquisition and analysis were done using CellQuest software program (Becton Dickinson).
Within the range of cell concentrations used [(1 to 5) x 105] the cell density did not influence the result.
Western Blot Analysis
Five to 10 million cells were washed with 1x PBS and lysed with lysis buffer (50 mmol/L Tris-HCl pH 7.5, 250 mmol/L NaCl, 2 mmol/L EDTA, 50 mmol/L NaF, 0.1 mmol/L Na3VO4, 0.5% Nonidet-P40, 1 mmol/L DTT) and a mixture of protease inhibitors (Sigma-Aldrich) for 15 minutes at 4°C and centrifuged at 14,000 rpm for 15 minutes. The protein concentration of cell lysates was measured using the Bio-Rad DC protein assay kit (Hercules, CA) and 20 µg of proteins were resolved on 10% SDS-PAGE and electrotransferred to 0.45-µm polyvinylidene difluoride (Amersham Pharmacia Biotech, Piscataway, NJ) at 180 mA for 90 minutes at 4°C. Nonspecific sites were blocked by incubating for 1 hour with 5% nonfat dry milk (Bio-Rad Laboratories) in 1x TBST 20 mmol/L Tris-HCl pH 7.5, 500 mmol/L NaCl, 0.1% Tween 20. The membrane was first incubated overnight with 1 µg/mL of mouse mAb anti-CXCR4 (clone 12G5, BD PharMingen, San Diego, CA) and then with 1 µg/mL horseradish peroxidase–conjugated secondary anti-mouse antibody (Amersham Pharmacia Biotech) in 1x TBST with 1% nonfat dry milk. After each incubation step, the membrane was washed for 30 minutes with 1x TBST, revealed with a chemiluminescence reagent (ECL, Amersham Pharmacia Biotech) and exposed to autoradiography film.
Determination of SDF-1 Production by Reverse Transcription–PCR
Total RNA was isolated from 5 x 106 to 10 x 106 cells using Trizol reagent (Invitrogen SRL) according to the manufacturer's instructions; 2.5 µg of total RNA were reverse transcribed for 45 minutes at 42°C with 100 pmol oligo(dT) primers (Roche Diagnostics, Milan, Italy) and 200 units of cloned Moloney murine leukemia virus reverse transcriptase in 1x reverse transcription buffer supplemented with 0.01 mol/L DTT, 1 mmol/L of each deoxynucleotide triphosphate and 200 units of RNase inhibitor (all from Invitrogen SRL) in a final volume of 25 µL. The resulting cDNA was subjected to a 50-µL PCR reaction containing 1x PCR buffer (Invitrogen SRL) supplemented with 1.5 mmol/L MgCl2, 200 µmol/L deoxynucleotide triphosphates, and 20 pmol of each primer.The primers for human SDF-1 were 5'-GGGGGAATTCCATGAACGCCAAGGTCGTGGTC-3' (forward) and 5'-GGGGTCTAGAGGGCATGGATGAATATAAGCTGC-3' (reverse).Thermal cycle conditions were 94°C for 1 minute, 57°C for 1 minute, and 72°C for 1 minute for two cycles followed by 41 cycles of 94°C for 1 minute, 65°C for 1 minute, and 72°C for 1 minute. All PCR products were analyzed on a 2% agarose gel with ethidium bromide staining.
SDF-1 ELISA
To determine SDF-1 secretion in conditioned medium, primary human osteosarcoma cell lines were plated in medium containing 10% FCS. The medium was changed on days 3 and 5. After the cells had reached confluence, the supernatants were collected following the manufacturer's instructions. The media were then analyzed by antibody sandwich ELISA (R&D Systems) with a detection range of 156 to 10,000 pg/mL SDF-1.
Modulation of CXCR4 Expression on Osteosarcoma Cells by Exposure to Recombinant Human SDF-1
Modulation of CXCR4 was done on SJSA cells; 5 x 105 cells were incubated in a 24-well plate with serum-free medium for 1 hour and were then cultured with or without 100 ng/mL of recombinant human SDF-1 (rhSDF-1, R&D Systems) diluted in bovine serum albumin, and incubated at 37°C for 1 and 3 hours. They were then washed in 1x PBS and stained to analyze CXCR4 expression by FACS analysis as described above.
Zymographic Analysis
Gelatinolytic activities of matrix metalloproteinases (MMPs) were assessed under nonreducing conditions using a modified SDS-PAGE. The supernatants of osteosarcoma cell lines were collected after 48 hours of standard culture condition. Aliquots of supernatants for each cell line were mixed with Laemmli buffer and applied onto an 8% polyacrylamide gel copolymerized with 1 mg/mL gelatin (Sigma-Aldrich). Electrophoresis was done under constant voltage (150 V) for 2 hours. The gel was washed 3 x 20 minutes each with 2.5% Triton X-100 (Sigma-Aldrich) to remove SDS and to allow the electrophoresed enzymes to renature before incubation in zymography buffer (50 mmol/L Tris-HCl, 200 mmol/L NaCl, 10 mmol/L CaCl2, 0.06% Brij 35 solution 30% pH 7.5) for 24 hours at 37°C. The gel was then stained with 0.5% Coomassie brilliant blue G-250 (Sigma-Aldrich) in 1:3:6 acetic acid/methanol/water for 15 minutes and destained with 1:4.5:4.5 acetic acid/methanol/water. Prestained standard high-range protein marker (Amersham Pharmacia Biotech) was used to determine the molecular weight of the gelatinase. The clear bands represented gelatinase activity. To determine whether zones of lysis detected by zymography were produced by MMPs, one gel was incubated with zymography buffer in the presence of 5 mmol/L EDTA. To evaluate the SDF-1 effect on MMPs modulation, we cultured the MG-63 and SJSA cell lines for 24 hours with and without rhSDF-1 (100 ng/mL) and did the zymographic assay on the supernatants as described above.
Migration Assay
The invasion potential was evaluated using Transwell chambers (1 cm2 per well, Costar, Bodenheim Germany). The upper and lower culture were separated by an 8-µm pore-size polyvinylpyrrolidone-free polycarbonate filters (Nucleopore, Corning Costar Corp., Cambridge, MA). Briefly, the lower compartment of the chamber was loaded with aliquots of serum-free medium plus rhSDF-1 (100 ng/mL). Before the invasion assay, filters were coated with 100 µg/well Matrigel (Becton Dickinson) diluted in culture medium. Tumor cells were seeded onto the reconstituted basement for 48 hours (5 x 104 cells per well) at 37°C in humidified atmosphere with 5% CO2. Cells that passed the synthetic basement were fixed with ethanol and stained with Giemsa solution (Diff-Quik kit, Baxter Diagnostics, Milan, Italy). Each experiment was done in triplicate. Cells migrated to the underside of the filter were counted in five fields (magnification x100) in each well by light microscopy. To evaluate random migration, in one set of experiments rhSDF-1 was added to both upper and lower compartments.
Adhesion Assay
To evaluate the SDF-1 contribution on adhesion, bone marrow stromal and HUVEC cells (5 x 104 per well) were plated for 24 hours in a 24-well plate at a density compatible with monolayer confluence and were treated with 100 ng/mL rhSDF-1 for 1 hour at 37°C to produce the active form of SDF-1. SJSA cells (1 x 106 cells/mL), labeled before assay with the fluorescent dye calcein-AM (Calbiochem, Darmstadt, Germany) for 5 minutes at 37°C, were detached and subsequently added to bone marrow stromal or HUVEC cell monolayer for 30 and 60 minutes at 37°C in humidified atmosphere. Unattached cells were removed, and the remaining cells were washed with PBS and harvested to quantify the adherent cell fluorescence in a 96-well fluorescent plate reader (HTS 7000 Bio Assay Reader, Perkin-Elmer). To show CXCR4-dependent adhesion we pretreated SJSA cells with 10 ng/mL neutralizing anti-CXCR4 antibody for 1 hour at 4°C before the adhesion to SDF-1-unstimulated bone marrow stromal and HUVEC cells.
Blockade of CXCR4 Receptor
For CXCR4 neutralization in in vitro studies, the interaction between CXCR4 and SDF-1 was blocked by treating SJSA cells for 1 hour at 4°C with a nonconjugated anti-human CXCR4 mAb (clone 12G5, 10 µg/mL, R&D Systems) before migration and adhesion assays.
For in vivo neutralization, CXCR4 receptor was blocked by using T134 (150 µg/kg), a small analogue of T22 peptide (14 amino acid residues, Sigma-Genosys, The Woodlands, Texas), for 1 hour at 4°C before in vivo assays.
3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide Assay
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was done according to the manufacturer's recommendation (Sigma-Aldrich). Briefly, 1 x 104 SJSA cells were seeded in 96-well plate in 150 µL DMEM containing 10% of FCS with or without rhSDF-1 stimulation (100 ng/mL). After 24 and 48 hours, MTT solution was added to each well and plates were incubated for 3 to 4 hours; subsequently, DMSO (Sigma-Aldrich) was added to the wells for 5 minutes. The plates were then read at 595 nm using an automate plate reader (Perkin-Elmer).
In vivo Metastasis Studies
Female BALB/c mice (6 to 8 weeks old) were supplied by The Jackson Laboratory (Bar Harbor, ME), maintained at the animal facilities of CIOS (Turin, Italy), and handled according to institutional regulations under sterile conditions in cage microisolators.
To investigate the involvement of the CXCR4/SDF-1 system during osteosarcoma metastatic process in vivo, one group of mice was given an injection of 106 SJSA cells alone into the tail vein and another group of mice was given an injection of SJSA cells and T134 (150 µg/kg) together. The animals were then given i.v. injections of T134 peptide thrice per week at the same concentration. No toxicity for the anti-human CXCR4 peptide was observed during preliminary in vivo experiments.
All mice were sacrificed 5 weeks after injection and lungs were examined macroscopically and microscopically for the presence of metastases. For histology and immunohistochemistry evaluation, lungs were collected and fixed in 10% formalin and embedded in paraffin. Sections 4 µm thick were stained with H&E for conventional histology.
Statistical Analysis
Each chemoinvasion and adhesion experiment was done in triplicate. The results were analyzed using Student's t test. All P values are two tailed with a significance of P < 0.05. For in vivo experiments, pulmonary metastasis development was compared in different groups of treatment by using Student's t test.
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RESULTS |
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Flow cytometric analysis showed that cell surface receptor expression was detectable on all tested cell lines, at different levels and in each cell line with high variability of expression, as shown in Fig. 1A. We observed that the CXCR4 level expression was influenced by the culture condition of the cells. In confluent cells the CXCR4 staining decreased compared with growing cells. Because SJSA cells expressed the highest level of CXCR4, we have chosen this cell line for our study.
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The expression of the SDF-1 mRNA on the four osteosarcoma cell lines was evaluated by reverse transcription–PCR. SDF-1 transcript was detected in all cell lines tested with different levels of expression. High level of SDF-1 transcript was found in MG-63 and U2-OS (Fig. 2A).
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SDF-1 Induces Chemoinvasion and Adhesion in Osteosarcoma Cells
SDF-1 stimulation induces a down-regulation of CXCR4 on SJSA cells; in a representative experiment shown in Fig. 3A, the expression of CXCR4 significantly decreased from 86% (untreated cells) to 16% and 8% after 1 and 3 hours of SDF-1 treatment, respectively. In separated experiments we analyzed modulation of CXCR4 also in confluent SJSA cells (about 20% of CXCR4+ cells) and we obtained a significant down-regulation (to 6%; data not shown). Down-regulation of the receptor is the result of CXCR4 internalization as shown by the increase of intracellular CXCR4 expression (Fig. 3B).
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To evaluate whether SDF-1 acts on MMPs modulation, with consequent increase of the metastatic potential, MG-63 and SJSA cell lines were stimulated for 24 hours with rhSDF-1 and then checked for the change of gelatinolytic activity. We found an increase on pro-MMP-9 secretion on MG-63 cell line supernatant but no modulation in SJSA cell line. Moreover, after rhSDF-1 treatment we found an increased expression of the lower band (83 kDa) in both cell lines (Fig. 4B). By contrast, SDF-1 treatment did not induce MMP-2 secretion on supernatants (data not shown).
To verify the involvement of CXCR4/SDF-1 complex during osteosarcoma progression, we did a Matrigel migration assay with osteosarcoma cells toward medium with or without rhSDF-1 (100 ng/mL). As shown in Fig. 5B, we observed a massive migration of SJSA cells in response to rhSDF-1. On the contrary, MG-63 cell migration in response to rhSDF-1 was lower as compared to SJSA cell locomotion (Fig. 5D).
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Moreover, we analyzed the role of SDF-1 in osteosarcoma cell adhesion. The rhSDF-1 pretreatment of the monolayer of endothelial and bone marrow stromal cells was done to enhance the biological activities of endogenous SDF-1 as described by Amara et al. (35). Figure 6A shows that the adhesion of SJSA cells to bone marrow stromal and endothelial cells significantly increase in the presence of rhSDF-1. To show that this phenomenon was CXCR4/SDF-1 system dependent, we blocked the receptor on SJSA cells and showed that the adhesion to HUVEC and to bone marrow stromal cells decreased at about 25% (Fig. 6B).
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T134 Inhibits Osteosarcoma Cell Metastases to the Lung
To further investigate whether CXCR4 is involved in in vivo osteosarcoma progression, we blocked the chemokine receptor with T134, a 14-amino-acid peptide, which has been shown to specifically block CXCR4 receptor (24, 25).
Injection of SJSA cells into a tail vein of the mice determined lung metastasis formation in 5 of 5 mice. Figure 7B shows a representative lung section of a mouse given an injection of SJSA cells alone in which we can observe an extensive metastasis of osteosarcoma cells. On the contrary, coinjection of T134 with SJSA cells and the following peptide treatment completely prevent lung metastasis formation, both macroscopically and microscopically, in 6 of 6 mice (Fig. 7C; P < 0.001).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, we analyzed the possibility that the metastatic process in osteosarcoma involves the CXCR4/SDF-1 axis.
In vitro studies on osteosarcoma cell lines showed an inverse correlation between ligand and receptor expression, suggesting that CXCR4 expression is regulated by autocrine production of SDF-1. The exposure of osteosarcoma CXCR4+ cells to exogenous rhSDF-1 caused a down-regulation of the receptor. This down-regulation induced by SDF-1 is the result of CXCR4 internalization, as occurs in hematopoietic cells (36).
MMPs have extracellular matrix–degrading activity and they are involved during cancer progression; the increase of MMPs activity contributes to acquisition of malignant properties (30, 31). It was previously shown that endothelial, epithelial, and hematopoietic cells express MMPs and that growth factors and cytokines could induce MMP-2 and MMP-9 (gelatinase A and B) secretion (33, 34). We detected pro-MMP-9 (92 kDa) and MMP-9 (83 kDa), but not MMP-2 in osteosarcoma cell line supernatants. We observed that rhSDF-1 treatment increased the pro-MMP-9 release in the supernatant of the MG-63 cell line but not in the SJSA cell line. Nevertheless, rhSDF-1 treatment increased the gelatinolytic activity of MMP-9 (83 kDa) corresponding to an active form of MMP-9, as described by other authors (32), but had no effects on the MMP-2 secretion.
Chemoinvasion of SJSA cell line, which expresses high levels of CXCR4, occurs following SDF-1 ligand gradient. On the contrary, MG-63 cell migration toward rhSDF-1 was significantly lower according to the level of CXCR4 expression. We showed that migration is CXCR4 dependent: by blocking CXCR4/SDF-1 interactions with a monoclonal neutralizing antibody 
-CXCR4 before assay, cell migration was significantly impaired.
Different levels of CXCR4 on osteosarcoma cells might explain the different behavior of tumor cells. Autocrine regulation of CXCR4 by endogenous SDF-1, as occurs on MG-63, might limit their metastatic potential but support local invasion (30, 31), as shown by low migration capacity and high MMP-9 production.
SDF-1 production might be involved in the localization of CXCR4-positive cells to tissue compartments. It has been shown that SDF-1 binding on cell membrane heparin and glycosaminoglycans produces a biological active form of SDF-1 (37). Adhesion of SJSA cells on a monolayer of endothelial or bone marrow stromal cells was increased after rhSDF-1 treatment. The blockade of the receptor with a mAb -CXCR4 caused a reduction of cell adhesion. The SDF-1 ligand promotes migration and adhesion processes of osteosarcoma cells, but it does not influence their proliferation and survival as described in rhabdomyosarcoma and in lymphohematopoietic cells (19, 38); however, SDF-1 might synergize in vivo with other growth factors as previously described (39).
Osteosarcoma is characterized by a high local aggressiveness with a tendency to metastasize to the lung, one of the preferential sites for SDF-1 production, so a specific regulation of CXCR4 can occur at this level. These in vitro experiments with stromal and endothelial cells cannot address the question of tissue specificity; therefore we set up an in vivo model with T134 peptide that mimics the region of SDF-1 involved in binding to CXCR4 and prevents receptor activation. It was shown that daily treatment with T22 reduces pulmonary metastases in mice after inoculation of CXCR4-transduced melanoma cells (22).
Here we reported that coinjection of osteosarcoma cells and T134 peptide into BALB/c nu/nu mice strongly prevents lung metastasis formation. Because T134 is CXCR4 specific and other receptors might be involved in osteosarcoma progression, such as CCR7 or CCR5 (40, 41), blockage of the CXCR4/SDF-1 pathway could not completely prevent metastasis formation. In our model, T134 inhibits the osteosarcoma cell adhesion to the pulmonary epithelium because at this level there is a stronger interaction of T134 with CXCR4 than with SDF-1; this peptide competes with SDF-1 at much lower concentration and its smaller size confers higher affinity for CXCR4.
In conclusion, our results strongly suggest that the CXCR4/SDF-1 pathway might have a role in osteosarcoma tumor progression, supporting some of the sequential events that are involved in metastasis formation. Small CXCR4 antagonists such as T134 could represent the starting point for innovative therapeutic strategies in osteosarcoma treatment.
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FOOTNOTES |
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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.
Received 3/23/04; revised 10/ 6/04; accepted 10/29/04.
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associates with heparan sulfates through the first ß-strand of the chemokine. J Biol Chem 1999;274:23916–25.. Glycobiology 2000;10:21–9.This article has been cited by other articles:
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