Zoledronic acid protects against osteosarcoma-induced bone destruction but lacks efficacy against pulmonary metastases in a syngeneic rat model

Authors

  • Agatha Labrinidis,

    1. Discipline of Orthopaedics and Trauma, University of Adelaide, The Royal Adelaide Hospital and The Hanson Institute, Adelaide, South Australia, Australia
    Search for more papers by this author
  • Shelley Hay,

    1. Discipline of Orthopaedics and Trauma, University of Adelaide, The Royal Adelaide Hospital and The Hanson Institute, Adelaide, South Australia, Australia
    Search for more papers by this author
  • Vasilios Liapis,

    1. Discipline of Orthopaedics and Trauma, University of Adelaide, The Royal Adelaide Hospital and The Hanson Institute, Adelaide, South Australia, Australia
    Search for more papers by this author
  • David M. Findlay,

    1. Discipline of Orthopaedics and Trauma, University of Adelaide, The Royal Adelaide Hospital and The Hanson Institute, Adelaide, South Australia, Australia
    Search for more papers by this author
  • Andreas Evdokiou

    Corresponding author
    1. Discipline of Orthopaedics and Trauma, University of Adelaide, The Royal Adelaide Hospital and The Hanson Institute, Adelaide, South Australia, Australia
    • Discipline of Orthopaedics and Trauma, University of Adelaide, Level 4, Bice Building, Royal Adelaide Hospital, North Terrace, Adelaide 5000, South Australia, Australia
    Search for more papers by this author
    • Fax: 618-8232-3065


Abstract

Osteosarcoma (OS) is the most common primary malignant tumor of bone in children and adolescents. In spite of successful control of the primary tumor, death from lung metastasis occurs in more than a third of patients. To investigate the efficacy of zoledronic acid (ZOL) on the development, progression and metastatic spread of OS, we used a rat model of OS, with features of the disease similar to human patients, including spontaneous metastasis to lungs. Rat OS cells were inoculated into the tibial marrow cavity of syngeneic rats. OS development was associated with osteolysis mixed with new bone formation, adjacent to the periosteum and extended into the surrounding soft tissue. Metastatic foci in the lungs formed 3–4 weeks postcancer cell transplantation. Treatment with a clinically relevant dose of ZOL was initiated 1 week after tumors were established and continued once weekly or as a single dose. ZOL preserved the integrity of both trabecular and cortical bone and reduced tumor-induced bone formation. However, the overall tumor burden at the primary site was not reduced because of the persistent growth of cancer cells in the extramedullary space, which was not affected by ZOL treatment. ZOL treatment failed to prevent the metastatic spread of OS to the lungs. These findings suggest that ZOL as a single agent protects against OS-induced bone destruction but lacks efficacy against pulmonary metastases in this rat model. ZOL may have potential value as an adjuvant therapy in patients with established OS.

Osteosarcoma (OS) is defined as an osteoid-producing malignant sarcoma. It is the most frequent primary malignancy of the skeleton in children and adolescents, developing mainly before the age of 30.1, 2 Current standard treatment consists of surgery and various combinations of chemotherapy.3 Treatment of OS has undergone considerable changes over the past 20 years, with more efficacious chemotherapy significantly improving long-term survival. However, response to chemotherapy depends on the type and combination of drugs used, the doses given and the sensitivity/resistance of the tumor cells. Further, despite the recent advances, the development of drug resistance to chemotherapy remains a problem in OS therapy.4 Metastatic spread, preferentially to the lungs compared with other sites, is seen in more than a third of presenting patients, and 90% of recurrent patients, and is correlated with extremely poor survival statistics.5–9 Therefore, therapies that could inhibit metastatic OS disease would have considerable potential to reduce mortality in OS.

Bone lesions caused by OS are characterized on the basis of their radiologic appearance and appear as osteolytic, osteoblastic (osteosclerotic) or a combination of both.10 Osteolysis is a common manifestation of OS, even within predominantly osteoblastic lesions, and is mediated primarily by osteoclasts and their bone resorbing activity.1, 2 Factors released from the bone are believed to stimulate tumor growth and tumor cells are in turn able to produce factors that stimulate osteoclast differentiation and activity, resulting in the establishment of a mutually beneficial relationship, often termed “the vicious cycle” because of its progressively destructive nature.11 Conversely, tumor cells associated with osteoblastic lesions may stimulate osteogenesis.12, 13

Bisphosphonates (BPs) are commonly used for the prevention and treatment of various bone diseases characterized by increased bone resorption, such as Paget's disease and osteoporosis.14 The highly selective localization and retention of BPs in bone is the basis for their use in skeletal disorders. Nitrogen-containing BPs, such as zoledronic acid (ZOL), inhibit bone resorption by preventing prenylation of GTPases, such as Ras and Rho, which are required for many cellular processes and ultimately induce cell death in osteoclasts.15 In addition to their antiresorptive activity, there is growing evidence supporting the direct effects of BPs on cancer cells, at least in vitro. In this respect, ZOL exhibits the highest potency of its class. A wide variety of tumor cell types have been used to demonstrate the antitumor activity of BPs, including leukemia,16 breast cancer,17 prostate cancer18 and OS.19–23 These studies have shown that BPs can dose dependently inhibit proliferation and induce apoptosis in tumor cells. A reduction in tumor cell adhesion, invasion and angiogenesis has also been reported, potentially making BPs attractive agents in the treatment of metastatic malignancies.24, 25

Preclinical animal models of metastatic cancer have demonstrated a reduction in tumor-induced osteolysis with ZOL treatment.25, 26 Reports of animal models of prostate cancer have all shown reduced osteolysis with ZOL treatment, with conflicting results in osteoblastic lesions.27 In the clinic, BP treatment is the current standard practice for palliative treatment of bone metastases.14 ZOL has proven to be effective in large nonrandomized clinical trials and is the first BP to show significant clinical benefit in patients with bone metastases from various primary tumors.25 Prolonged treatment with ZOL seems to be safe and well tolerated and this combination of potency and safety makes it a useful adjuvant therapy in bone metastasis,28 although prolonged treatment with nitrogen-containing BPs in cancer patients appears to be associated with the risk of developing osteonecrosis of the jaws.29

The efficacy of ZOL against mouse and rat OS has been evaluated previously by 2 separate studies using animal models, in which tumor cells were injected either intravenously or were in contact with the bone surface.30, 31 OS originates in the bone so that animal models with ectopic tumor implantation, such as intravenous injections or subcutaneous implantation, lack relevance for patients with OS. We have recently demonstrated the ability of ZOL to inhibit the development and progression not only of the osteolytic but also the osteoblastic component of primary OS lesions in an immunocompromised murine model. However, in the mouse model, lung metastases were not reduced and may even have been promoted with ZOL treatment, highlighting the need for further investigation before clinical application of ZOL is considered for OS therapy.23 An important limitation of the immunocompromised moue model was that it did not take into account the role of the immune system in the establishment and metastasis of OS. Therefore, in our study, we established a syngeneic animal model, in which rat OS cells were inoculated directly into the tibial marrow cavity of immune-competent rats. We report here that treatment with clinically relevant doses of ZOL following tumor establishment had a significant protective effect on OS-induced bone destruction but lacked efficacy in reducing the overall tumor burden at the primary site or in preventing pulmonary metastases in this rat model that closely mimics the clinical features of patients with OS.

Material and Methods

Cells and reagents

The rat OS cell line MSK-8G32 was kindly provided by Dr. Paul Reynolds (Hanson Institute, Adelaide, Australia), and the cells were cultured in Dulbecco's modified Eagle's medium, supplemented with glutamine (2 mM), penicillin (100 IU/ml), streptomycin (100 μg/ml), gentamicin (160 μg/ml) and 10% fetal bovine serum (Biosciences, Sydney, Australia), in a humidified atmosphere containing 5% CO2.

ZOL was generously provided by Novartis Pharma AG (Basel, Switzerland).

Measurement of cell viability

For determination of ZOL effects on cell growth, 1 × 104 cells per well were seeded into 96-well microtiter plates and allowed to adhere overnight. Cells were then incubated with fresh media containing increasing concentrations of ZOL (1–100 μM) for 72 hr. Cell viability was determined by staining with crystal violet and measuring optical density at 570 nm wavelength. The experiments were performed in quadruplicate and were repeated at least twice. The results of representative experiments are given as the mean ± SD.

Apoptosis analysis

DAPI staining of nuclei.

Cells were seeded on plastic chamber slides and treated with 40 μM ZOL for 48 hr. After 2 washes with PBS, cells were fixed in methanol for 5 min, washed again with PBS and incubated with 0.8 mg/ml of 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI, Roche Diagnostics, Castle Hill, NSW, Australia) in PBS for 15 min at 37°C. After several washes in PBS, the coverslips were mounted on PBS/glycerol. DAPI staining was visualized by fluorescence microscopy.

Measurement of DEVD-caspase activity.

DEVD-caspase was assayed by cleavage of zDEVD-AFC (z-asp-glu-val-asp-7-amino-4-trifluoro-methyl-coumarin), a fluorogenic substrate based on the peptide sequence at the caspase-3 cleavage site of poly(ADP-ribose) polymerase. Cells (5 × 105) grown in 24-well plates were treated as indicated, washed once with HBSS and resuspended in 200 μl of NP-40 lysis buffer containing 5 mM Tris-HCl, 5 mM EDTA and 0.5% NP40, pH 7.5. After 15 min in lysis buffer at 4°C, insoluble material was pelleted at 15,000g and an aliquot of the lysate was tested for protease activity. To each assay tube containing 8 μM of substrate in 1 ml of protease buffer (50 mM Hepes, 10% sucrose, 10 mM DTT and 0.1% CHAPS, pH 7.4) was added 20 μl of cell lysate. After 4 hr at room temperature, fluorescence was quantified (Exc 400, Emis 505) in a Perkin Elmer LS50 spectrofluorimeter. Optimal amounts of added lysate and duration of assay were taken from linear portions of curves as determined in preliminary experiments. One unit of caspase activity was taken as 1 fluorescence unit (at slit widths of 10 nm) per 4 hr incubation with substrate.

Detection of unprenylated and total Rap1A

To determine the effect of ZOL on the prenylation of small GTPases in the OS cells, lysates from ZOL-treated cells were analyzed by Western blotting for the presence of the unprenylated form of Rap 1A. Cells were seeded into 25 cm2 flasks and were incubated for 72 hr with media containing 40 μM ZOL. Cells were lysed in buffer containing 10 mM Tris HCl, pH 7.6, 150 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 2 mM sodium vanadate and a cocktail of protease inhibitors (Roche Diagnostics, NSW AUS) and stored at −70°C until ready to use. Cell extracts were mixed with an equal volume of sample buffer containing 12 mM Tris HCl, pH 6.8, 6% SDS, 10% β-mercaptoethanol, 20% glycerol and 0.03% bromophenol blue. Protein samples were boiled for 5 min and electrophoresed under reducing conditions in 4–20% polyacrylamide gels. Separated proteins were electrophoretically transferred to PVDF transfer membrane (Novex, San Diego, CA) and blocked in PBS containing 5% blocking reagent (Amersham, Castle Hill, NSW, Australia) for 1 hr at room temperature. Immunodetection was performed overnight at 4°C in PBS/blocking reagent containing 0.1% Tween 20, using anti-Rap 1A antibody, which is specific for the unprenylated form of Rap 1A, or an anti-Rap 1A antibody, which detects total Rap 1A (both antibodies from Santa Cruz Technology, CA), diluted according to the manufacturer's instructions. Filters were rinsed several times with PBS containing 0.1% Tween 20 and incubated with 1:5,000 dilution of anti-goat alkaline phosphatase-conjugate (Amersham Biosciences, Castle Hill, NSW, Australia) for 1 hr. Bound proteins were detected and quantitated using the Vistra ECF substrate reagent kit (Amersham) using a FluorImager (Molecular Dynamics, Sunnyvale, CA).

Rat model of osteosarcoma

Male Fischer 344 (F344) rats, 5–6 weeks old, were housed in accordance with the guidelines approved by the Institute of Medical and Veterinary Science animal ethics research committee. Animals were acclimatized for 1 week before commencement of procedures. The rats were anesthetized by i.p. injection with 80 mg ketamine/kg body weight and 10 mg xylazine/kg body weight. The left tibia was shaved free of hair, wiped with 70% ethanol and a 23-gauge needle was inserted through the tibial plateau with the knee flexed, and 1 × 105 MSK-8G cells resuspended in 50 μl of PBS were injected into the marrow space. All animals were injected with PBS in the contralateral tibia, as a control. After tibial injection, rats were randomly assigned to 3 groups of 6 animals each. Rats were weighed regularly and radiographs were taken every 2 weeks to determine the extent of osteolysis. At sacrifice, all the major organs and both hind limbs were collected for micro-CT and histological analysis.

Treatment with zoledronic acid

ZOL was dissolved in sterile water and prepared in sterile 1× PBS. ZOL at 100 μg/kg body weight was administered by s.c. injection at weekly intervals for 5 weeks, starting 1 week after cancer cell implantation. On a mg/kg basis, this dose of ZOL is approximately equivalent to the approved human dose of 4 mg i.v., but the weekly dosing frequency is higher than the once monthly regimen used clinically in oncology patients with bone metastases. Therefore, in a second cohort of animals, ZOL was administered as a single dose only of 100 μg/kg, equivalent to the clinical dose of 4 mg given to patients once monthly.33

Radiography

Animals were anesthetized by i.p. injection with 80 mg ketamine/kg body weight and 10 mg xylazine/kg body weight and were laid onto X-ray film with hind limbs spread to enable clear imaging of the tibiae. Radiographs were taken using the HP cabinet X-Ray System-Faxitron Series. Exposure time and intensity was optimized with final settings of 18 sec at 60 kVp. Kodak min R-2000 film was used (Kodak, Australasia Pty, Melbourne, Australia).

Microcomputer tomography analysis

For micro-CT imaging, hind limbs were dissected and placed in 100% ethanol. Both the right and left tibiae of each animal were mounted in the CT specimen tube and placed securely into a SkyScan-1072 X-ray micro-CT Scanner (Aartselaar, Belgium). The program was commenced with magnification set to give scan slices of 18 μm. Three-dimensional (3D) images were generated using Cone-Beam reconstruction and 3D visualization (Skyscan). Using the 2D images obtained from the micro-CT scan, the growth plate was identified and 500 sections, starting from the growth plate/tibial interface and moving down the tibia, were selected. Histograms representing trabecular bone volume (TBV, mm3) were generated and compared to the control tibia.

Histology

Tibiae were fixed in 10% buffered formalin, followed by EDTA decalcification in 10% EDTA solution and 7% nitric acid at room temperature. Decalcification was confirmed by radiography before sectioning. Samples were paraffin embedded, sectioned longitudinally at 6 μM and stained with hematoxylin and eosin (H&E). Analysis was performed on a Nikon Eclipse TE300 inverted microscope (Nikon Corporation, Tokyo, Japan). Tumor area was calculated in a blinded fashion from digitized images of histological slides obtained from a standard 5 megapixel resolution camera coupled to a microscope and using Scion Imaging software (Scion Corporation, MA). Tumor size, defined as the tumor area, was calculated from the section of the tibia that best represents the center of the tumor mass and expressed as an average tumor area per group in absolute units (mm2). For pulmonary metastases, sections were stained with H&E, and the number and size of each lung metastasis were measured with Scion image analysis software. Representative lung samples were resin embedded, sectioned longitudinally at 6 μM and stained with Von Kossa or Alizarin Red. Slides were fixed in 10% formalin and washed twice with dH2O. One percent silver nitrate was added for 30 min in direct sunlight for Von Kossa staining, or 2% Alizarin Red for 5 min at room temp, then washed twice with dH2O. Von Kossa stains were finally rinsed with 2.5% sodium thiosulfate for 5 sec and allowed to dry. Tartrate-resistant acid phosphatase (TRAP) staining of osteoclasts was performed using the leukocyte acid phosphatase (TRAP) kit, as per the manufacturer's instructions (Sigma-Aldrich, St. Louis, MO).

Statistical analysis

The continuous outcome bone volume was analyzed using mixed model ANOVA to allow for clustering of rats (i.e., more than 1 observation per rat). Post hoc pair-wise comparisons were undertaken with no adjustment made for multiple comparisons. In all cases, p < 0.05 was considered statistically significant.

Results

Effect of ZOL on viability of MSK-8G cells in vitro

The effect of ZOL on the viability of the MSK-8G rat OS cells was tested in vitro. Treatment with ZOL for 72 hr resulted in a dose-dependent inhibition of cellular proliferation of MSK-8G cells in monolayer cultures (Fig. 1a). Exposure to greater than 10 μM ZOL resulted in cells detaching from the substratum within 24–48 hr, similar to effects we reported previously for human OS cells.20 Loss of attachment with ZOL treatment was concomitant with a dose-related increase in caspase-3-like activity (Fig. 1a). We have previously published data showing that caspase-3 activation is secondary to the apoptotic activity of ZOL in OS.20 Morphological evidence characteristic of apoptosis, including chromatin condensation, nuclear fragmentation and the formation of dense rounded apoptotic bodies, was seen at doses greater than the half maximal effective dose of 40 μM ZOL, as assessed by DAPI staining of nuclei (Fig. 1b). Lysates from untreated cells and cells treated with 40 μM ZOL were collected after 48 hr and analyzed by Western blotting. Concomitant with apoptosis induction was cleavage of the apoptosis target protein PARP with ZOL treatment (Fig. 1c). Also, consistent with the importance of the mevalonate pathway as an intracellular target for the effects of ZOL, the untreated cells expressed only the prenylated form of Rap1A, whereas in the ZOL-treated cells both prenylated and unprenylated RAP 1A were present (Fig. 1c).

Figure 1.

Activity of Zol against MSK-8G OS cells in vitro. (a) The rat MSK-8G OS cells line were seeded in 96-well plates at 1 × 104 cells per well and treated with increasing doses of ZOL, as indicated. Cell viability (▪) was assessed by crystal violet staining 72 hr after treatment. Data are presented as the mean ± SD of quadruplicate wells and are expressed as a percentage of the number of control cells. Cell lysates were collected and used to determine caspase-3-like activity (▴), using the caspase-3-specific fluorogenic substrate, zDEVD-AFC, as described in the Material and Methods. Data points show means of quadruplicate results from a representative experiment, repeated at least twice. (b) Cells were treated with 40 μM ZOL and left for 48 hr before cells were fixed with methanol and incubated with DAPI, before washing in PBS and mounting on PBS/glycerin. DAPI staining was visualized by fluorescence microscopy. (c) Western blot analysis revealed the presence of unprenylated Rap1A protein and cleavage of PARP in cell lysates treated with ZOL but not in the untreated control cells. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Rat model of OS development, progression and metastatic spread

The rat model chosen for these studies recapitulates the features of development of OS and the spontaneous metastasis to lungs seen in the human disease, in the context of an intact immune system. MSK-8G rat OS cells were injected directly into the tibial marrow cavity of F344 rats, using the method we have previously described.23 All animals injected with OS cells developed tumors, which were visible by 3 weeks after injection. Visible and palpable tumors confirmed successful implantation and indicated that tumors had penetrated the tibial cortex and extended into the surrounding soft tissue. X-ray images of tumor-bearing tibiae taken just before sacrifice confirmed the presence of osteolysis in all animals within the vehicle (PBS)-treated group (Fig. 2a). A characteristic radiodense “sunburst” configuration, consistent with new bone formation, was also evident, extending perpendicular to the cortex and into the surrounding soft tissue mass (Fig. 2a). Micro-CT images of the tibiae were reconstructed to produce 3D images. These revealed extensive bone remodeling, characterized by areas of osteolysis mixed with areas of osteosclerosis in the tumor-bearing tibiae (Fig. 2b). Cross sections of the micro-CT images of the normal nontumor-bearing tibiae demonstrated significant cortical bone destruction and loss of trabecular bone in all tumor-bearing tibiae, representing the osteolytic component of OS lesions. In addition, marked spicular new bone formation, extending from the cortex, was clearly evident in all tumor-bearing tibiae, confirming the plain radiographic data (Figs. 2b2d). Histological analysis demonstrated that the spicular new bone was mineralized woven bone, and although it was mainly present as an extension from the cortex, it was also present within the extramedullary tumor mass highlighting the bone-forming ability of these tumors (Fig. 2e).

Figure 2.

OS-induced bone destruction in an immune-competent rat model. (a) Radiographs showing areas of osteolysis and areas of new bone formation extending from the cortex. (b) Ex vivo representative 3D reconstructed micro-CT images. (c, d) Serial cross section m-CT views with progressive loss of trabecular and cortical bone. Also shown is an extensive network of mineralized new bone extending perpendicular from the cortex. (e) Histological sections at low and high magnification showing that new bone formation is mineralized woven bone predominantly extending from the periosteum but also present within the extramedullary tumor mass (arrow).

Effect of ZOL on OS-induced bone destruction

Eighteen animals were inoculated with cancer cells on Day 0 and animals were randomly assigned into 3 groups of 6 rats each group: (i) the control vehicle group, (ii) the ZOL single dose only group and (iii) the ZOL once weekly treated group. ZOL treatment commenced 7 days after cancer cell transplantation, to allow establishment of tumor cells within the bone environment, and was administered at 100 μg/kg subcutaneously (s.c.)

Radiographic images of bones from both the ZOL-treated group of animals were highly radiodense when compared with the vehicle-treated group, reflecting increased bone density due to inhibition of bone resorption. This effect of ZOL was more pronounced in areas of increased bone turnover, such as the distal femurs and proximal tibiae, and was not restricted to the tumor site, as the contralateral nontumor-bearing tibiae also showed this effect (Figs. 3a and 3b). Qualitative micro-CT assessment of bone architecture showed no evidence of osteolysis in both the ZOL treatment groups when compared with vehicle treatment. In addition, the amount of spicular new bone formation extending from the cortex was also significantly reduced with ZOL treatment (Fig. 3a). Longitudinal and cross-sectional micro-CT images confirmed the increase in trabecular bone density in both tumor- and nontumor-bearing tibiae that were treated with ZOL (Figs. 3a and 3b). To quantify the TBV, we compared the left tumor-bearing tibiae of the ZOL-treated animals with that of the vehicle-treated animals at a selected region beginning at the growth plate and extending downward 500 μm × 18 μm slices, which encompassed all of the OS lesions. The TBV of both the ZOL-treated groups was significantly higher than the vehicle alone-treated group (Fig. 4a), thus confirming the qualitative assessment which showed an increase in trabecular bone density with ZOL treatment. The protective effect of ZOL on OS-induced bone destruction was due to a decrease in osteoclastic bone resorption as evident by the significant decrease in the number of TRAP-positive osteoclasts lining the bone surface (Fig. 4b). This effect was most pronounced in the ZOL weekly treatment group.

Figure 3.

Radiographic and ex vivo micro-CT images. (a) Tumor-bearing and (b) contralateral nontumor-bearing tibiae from vehicle-treated and ZOL-treated rats. Transverse and cross section views of representative tibiae show loss of cortical and trabecular bone in tumor-bearing tibia with spicular new bone extending from the cortex in the vehicle group. ZOL treatment given once only or weekly inhibits OS-induced bone destruction, reduced spiculation and increased trabecular density in both tumor- and nontumor-bearing tibiae. ZOL treatment increased radiodensity in areas of higher bone turnover, including proximal tibia and distal femur in both tumor- and nontumor-bearing tibiae. Depicted are representative images that best illustrate the effect of treatment. White arrows indicate increased radiodensity.

Figure 4.

Effect of ZOL on bone volume. Trabecular bone volume was measured from 500 micro-CT slices of the proximal tibia starting from the growth plate, using the program CTan. (a) Graph represents the trabecular bone volume in mm3 in the tumor-bearing tibiae of each group, **represents p = 0.001. Data shown in each case are the average bone volume from all animals in that group: points are means ± SEM. (b) Histological sections (n = 4 per group) showing TRAP staining of osteoclasts in the tibia of untreated and ZOL single and weekly treated rats, respectively.

Effect of ZOL on primary tumor growth and on pulmonary metastases

The intra- and extraosseous tumor burden in the tibiae following ZOL treatment was calculated from histological sections and expressed as an average tumor area per group (Fig. 5a). Treatment with ZOL either as a single dose or as a weekly dose did not decrease the extramedullary tumor growth in the tibiae as assessed by histomorphologic analysis at the end of the experiment. In contrast, from histological inspection, the intraosseous tumor burden was significantly decreased with both of the ZOL treatment regimens and cancer cells were barely detectable within the bone marrow space, which had now been significantly reduced and spatially constrained because of the increase in trabecular bone density (Fig. 5b).

Figure 5.

Quantification of the effect of ZOL on primary tumor growth. (a) Total tumor burden was measured using the histology images and the area expressed as an average per group is shown in the graph as mm2 (data are expressed as mean ± SEM). (b) Histological sections from representative tibiae stained with H&E, showing intraosseous (IO) tumor burden in the vehicle animal only and extramedullary tumor burden (EM).

Pulmonary metastases were present in all animals, irrespective of treatment. The metastatic tumor nodules in the lungs were highly mineralized and stained positive for Von Kossa and Alizarin Red, confirming the presence of phosphate deposits within the tumor mass (Fig. 6a). Tumor burden was quantified ex vivo using histology and calculated as a percent of total lung area. The tumor burden in the lungs was similar between the 2 ZOL treated groups and was not significantly different from that of the vehicle-treated animals (Fig. 6b). Similarly, the number of metastatic nodules was no different between the groups (Fig. 6c). Histological analysis of kidney, liver, heart and spleen tissue sections obtained at autopsy from animals transplanted with OS cells showed no evidence of metastatic spread in these organs (data not shown).

Figure 6.

Effects of ZOL treatment on pulmonary metastases. Histological sections of the lungs were photographed and used to measure tumor area and total lung area. (a) Tumor within lung was confirmed using histological slides stained with H&E and compared to normal lung histology. The mineralization status of the lung metastases was confirmed using histological slides stained with Von Kossa and Alizarin Red. (b) Graph represents the tumor area as a percent of total lung area and is expressed as an average per group. (c) Graph represents the average number of metastatic loci per group. Data shown in each case are an average from a representative section of each animal: points are means ± SEM.

Discussion

OS has a variable bone-forming ability but is destructive by virtue of its ability to expand in bone by inducing osteoclast-mediated bone resorption. The effect of BPs on OS cells, specifically alendronate and pamidronate, has been reported as having proapoptotic effects on canine21 and human34, 35 OS cells in vitro. Three recent studies have reported on the effect of ZOL in animal models of mouse and rat OS. Ory et al. investigated a mouse model, in which mouse OS cells were injected intravenously and showed that ZOL treatment suppressed lung metastases and prolonged the overall survival of OS-bearing mice.31 Heymann et al. also showed that ZOL completely prevented lung metastases and caused reduction in osteolytic lesions in a rat model, in which intact rat OS tissue was implanted subcutaneously and in contact with the tibial bone surface.30 Using a subcutaneous implanted mouse OS model, Koto et al. showed that clinically relevant doses of ZOL administered i.p inhibited lung metastases but had no effect on the growth of OS at the primary site.36 Our experimental model involves the direct transplantation of OS cells into the tibial marrow cavity, attempting to simulate the normal development, progression and metastatic spread of OS and to enable the evaluation of ZOL treatment in the bone environment. We previously demonstrated that ZOL is able to inhibit the development and progression not only of the osteolytic but also the osteoblastic component of primary OS lesions in a nude mouse model using human OS cells. However, we showed in the same study that lung metastases were not reduced and may even have been promoted, with ZOL treatment, indicating that caution is required before any clinical application of ZOL is considered for OS therapy.23 More recently, using a similar animal model, Dass and Choong37 have shown that ZOL administration to SAOS-2 tumor-bearing mice resulted in primary tumor growth inhibition, reduction in lung metastases and a dramatic decrease in osteolysis. The reasons for these different observations are not clear but may relate to cell type, dose of ZOL and scheduling of treatment protocols. For example, the dosing schedule of the Dass study equates to 8 times the current clinical dose.

Our previous mouse study had 1 important limitation, in that it did not take into account the role of the immune system in tumor growth and metastasis. Therefore, in our study, we established a syngeneic animal model, in which rat OS cells were inoculated directly into the tibial marrow cavity of immune-competent rats. We selected the rat MSK-8G OS cell line, a derivative of the MSK cell line.32 MSK-8G cells when implanted directly into the tibial marrow cavity develop locally, growing tumors of mixed osteolytic/osteosclerotic lesions when analyzed radiographically and histologically. One added advantage is that, as in the human disease, MSK-8G cells injected intratibially form pulmonary metastases that are easily quantifiable upon gross and histological inspection of the lungs 3–4 weeks after tumor cell inoculation.

Although ZOL treatment offered considerable protection against OS-induced bone destruction, there was no significant effect on the overall tumor burden (intraosseus and extramedullary) at the primary site. However, the intraosseus tumor burden diminished considerably such that tumor cells were barely detectable within the bone marrow, as shown in histologic sections of the tibiae. It should be noted that the trabecular bone density was considerably increased with ZOL treatment, an effect that was more pronounced with the weekly ZOL treatment regimen. This effect of ZOL translated to a significant reduction in bone marrow volume, contributing to the spatial constraints preventing tumor cell growth within the marrow space but not in the surrounding soft tissue. The effects of ZOL on trabecular bone density were due to the actions on osteoclast formation and activity as ZOL treatment decreased the number of TRAP+ve osteoclasts lining the bone surfaces. Consistent with our findings, Buijs et al.38 have recently shown that ZOL or Fc-OPG treatment following intratibial injection of highly osteolytic breast cancer cells protected against cancer-induced osteolysis and although the intraosseus tumor burden was diminished, the overall tumor burden was also not significantly affected because of the persistent growth in the extramedullary space.

In the context of the effects of ZOL on OS-induced bone destruction and on primary tumor growth, the results presented here are in complete agreement with those we have recently published.23 However, although our previous study demonstrated an increase in pulmonary metastases with ZOL treatment in immune-compromised animals, this effect was not seen in this syngeneic rat model of OS, where the immune system is intact. ZOL was shown previously to directly stimulate gamma-delta T cells to secrete high levels of proinflammatory cytokines leading tumor cell death in vitro.39–42 A previous study in severe combined immunodeficiency mice demonstrated that mice reconstituted with human peripheral blood lymphocytes or purified gamma-delta T cells were able to kill tumor cells in vivo.39 In a clinical trial, Wilhelm et al.43 reported that BP therapy led to a strong correlation between gamma-delta T cell expansion and patient response, highlighting the potential contribution of the immune system in mediating the anticancer actions of ZOL in vivo. It should be noted, however, that in our study, ZOL treatment failed to restrain pulmonary metastases, indicating that the immune system is unlikely to provide significant therapeutic benefit in the treatment of OS.

In conclusion, we demonstrate that ZOL as a single agent protects against OS-induced bone destruction but lacks efficacy against pulmonary metastases in this syngeneic immune-competent rat model. The important question that needs to be addressed is whether a combinatorial approach of ZOL with clinically relevant chemotherapeutic drugs may be a useful therapeutic option for the treatment of OS patients in the future. This issue will need to be addressed in properly designed preclinical and clinical studies.

Acknowledgements

A. Evdokiou is a research fellow of the National Health and Medical Research Council of Australia (NHMRC) and A Labrinidis is a postdoctoral fellow supported by the National Breast Cancer Foundation (NBCF). The authors thank Dr. Jonathan Green (Novartis, Switzerland), Prof. Peter Choong (St Vincent's Hospital, Vic, Australia), Dr. Paul Reynolds (IMVS, SA, Australia), Dr. Masakazo Kogawa (IMVS, SA, Australia) and Dr. Kikuo Takahashi (Chiga University Hospital, Japan) for help and advice.

Ancillary