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Chondrosarcoma, identified by Lichtenstein and Jaffe as a malignant bone tumor clearly distinct from osteosarcoma, is currently defined as malignant cartilage tumor arising de novo or within a preexisting benign cartilage tumor.1 Human chondrosarcomas, which represent about 25% of all bone sarcoma,2, 3 are classified as low, intermediate or high grade on the basis of histological and cytological features.4 Overall local recurrence and 10-year survival are 24–33% and 46–70%, respectively.5, 7 Risk factors for the former include inadequate surgical margins5, 8 and tumor size, and for the later include local recurrence, extracompartmental spread and high histological grade.5 No effective adjuvant treatment (radiation therapy, chemotherapy) is available.6
As evidenced for bone metastases, a vicious cycle between osteoclasts, bone stromal cells/osteoblasts and cancer cells has been hypothesized during the progression of primary bone tumors.9 Accordingly, suppression of osteoclasts would be a primary approach to inhibit local cancer growth. Among the potential drugs available, bisphosphonates (BPs) are an important class of molecules for the treatment of bone diseases with different molecular mechanisms of action. Nitrogen-containing BPs act by inhibiting the recruitment, proliferation and differentiation of preosteoclasts, or by impeding the resorptive activity of mature osteoclasts.10, 11, 12, 13 They also shorten the life span of osteoclasts by inducing their apoptosis.14 Previous studies revealed that BPs have the ability to reduce the osteolytic bone resorption associated with multiple myeloma and breast cancer15, 16 and also show efficacy in cancer metastases to bone due to prostate cancer and other solid tumors, demonstrating that this BPs can reduce skeletal morbidity in both osteolytic and osteoblastic diseases.17, 18 A clear direct antitumor activity on breast cancer cells has been demonstrated in vitro.19 In primary malignant bone tumor, inhibition of human osteosarcoma cell growth by pamidronate and clodronate20, 21 and zoledronic acid (ZOL)22 have been reported in vitro. These results are consistent with our in vitro and in vivo studies demonstrated efficacy of ZOL on osteosarcoma tumor progression and metastatic spreading.23, 24
Hence, the purpose of the present study was to determine the efficacy of ZOL on chondrosarcoma in vitro and in vivo, in term of local tumor growth, animal survival after and before intralesional curettage using a rat transplantable model of chondrosarcoma.
Material and methods
Dulbecco's modified eagle's medium (DMEM), L-glutamine and trypsin were obtained from Invitrogen (Eragny, France), fetal bovine serum (FBS) from Hyclone (Perbio, France). Rat swarm chondrosarcoma (RCS) was a generous gift from Dr. P.A. Guerne (Geneva, Switzerland).25, 26 The subline used in the present study expresses collagen II. Although RCS cells are not able to form mineralized nodules in vitro, they decrease the capacity of bone marrow to mineralize and do not modify the osteoclastic differentiation.26 After subcutaneous or intramuscular development, RCS was well delimited, poorly vascularized, lobular in organisation and soft (unlike normal cartilage). Histological examination revealed chondroid tumor proliferation consisting of lobules of variable size containing chondroid stroma delimited by fine septa. This chondrosarcoma displayed the histological feature of grade II chondrosarcoma.26 Zoledronic acid (ZOL) was kindly provided as the research grade disodium salt by Novartis Pharma AG (Basel, Switzerland).
In vivo assessment of ZOL treatment
Male Sprague-Dawley rats were purchased from the “Centre d'Elevage Janvier” (Le Genest Saint Isle, France). Four-week-old rats were housed under pathogen free conditions at the Experimental Therapy Unit (Medicine Faculty of Nantes, France), in accordance with the institutional guidelines of the French Ethical Committee and under the supervision of authorized investigators. For the implantation, the rats were anaesthetized by inhalation of a combination isoflurane (Abbott, Rungis, France)/air (1.5%, 1 L/min) associated with an intramuscular injection of Imalgene (100 mg/kg, Merial Lab., Lyon, France). Allograft transplantations of tumor fragments were performed as follows: using a lateral approach, the cortical surface of the diaphysis was scarified laterally on 10 mm, a 10 mm3 fragment of RCS was placed contiguous to the scarified surface, and the cutaneous and muscular wounds were sutured. Tumors appeared at the graft site 7–11 days later.
Two protocols were applied: (i) curative treatment: rats were individually identified and assigned to control or treatment group 4 days after implantation (6 animals/group). Each rat in treated group received s.c. 100 μg/kg ZOL twice a week, started at day 4 after implantation until euthanasia; (ii) in other series, rats bearing growing tumors with a volume >1200 mm3, which were considered as progressive tumors, were individually identified at day 20 after implantation and assigned to the control or treatment group (6 animals/group). Treatment group received 100 μg/kg ZOL s.c. the same day, and twice a week until euthanasia. All animals were blindly operated at day 24 by intralesional curettage.
In all series (n = 3), control groups received the same volume of PBS s.c. at the same schedule as treated animals. The animals were weighed twice a week, at the same time the tumor volume was calculated from the measurement of 2 perpendicular diameters using a caliper. Each tumor volume (V) was calculated according to the following formula : 0.5 × L × (S)2, where L and S are the largest and smallest perpendicular tumor diameters, respectively. The animals were sacrificed, except spontaneous death, when tumor became too bulky and when life of the animal was threatened.
In vitro analyses
The RCS cells was derived from the Swarm tumor used in the present study. Briefly, RCS were isolated from small tumor fragments, treated 2 hr with 1 mg/mL collagenase A (Boehringer Manheim). The cell suspension was then washed 3 times with PBS, cultured in DMEM supplemented with 10% FCS, 1% glutamine and maintained at 37°C in a humidified atmosphere with 5% CO2. The medium was replaced twice a week and the adherent RCS were replated using a 0.05% trypsine–0.02% EDTA solution. Replicate subconfluent cell cultures in 96-multiwell plates were treated for 1 to 3 days with increasing concentrations of ZOL (10−7–10−4 M, diluted in PBS). Cell proliferation was determined by a cell proliferation reagent assay kit using sodium 3′[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate (XTT) (Roche Molecular Biomedicals, Mannheim, Germany). Cell viability was also assessed by trypan blue exclusion and manual counting.
Induction of apoptosis
Programmed cell death was monitored microscopically following Hoechst staining and cell viability by trypan blue exclusion. RCS cells were seeded at 104 cells/well in a 24-multiwell plate and cultured for 24 hr as described above, before being incubated with ZOL at indicated concentrations during 24, 48 and 72 hr. Trypsinized cells were then resuspended in the presence of Hoechst n°33258 staining (10 μg/mL; Sigma) for 30 min at 37°C. Cells were then observed by UV microscopy (Leica, Wetzlar, Germany). Induction of apoptosis was also investigated by cleavage of caspase-1 and -3 substrates, in cell lysates with or without ZOL treatment. RCS cells were seeded at 15 × 103 cells/well (in a 24-multiwell plate), then incubated with ZOL (1 and 10 μM) for 24, 48 and 72 hr. Cells treated with UV for 30 sec, 24 hr before cell extraction, were used as positive controls. At the end of the incubation period, the cells (adherent and non adherent) were lysed with 50 μL of RIPA buffer for 30 min. The cells were then scraped off and protein content was quantified in parallel samples using the BCA (bicinchominique acid + Copper II sulfate) assay. Caspase 1 and 3 activities were assessed on 10 μL of the cell lysate with the kit CaspACE™ Assay System (Fluorometric, Promega, Madison, USA) following the manufacturer's instructions.
Cell cycle analysis
Confluent cultures of RCS cells were incubated for 24, 48 or 72 hr with or without 10 μg/mL ZOL, trypsinized, washed twice and lysed in PBS containing 0.12% Triton X-100, 0.12 mM EDTA and 100 μg/mL ribonuclease A. Then 50 μg/mL propidium iodide were added for each sample for 20 min at 4°C in the dark. The intensity of propidium iodide labelling was measured by flow cytometry (FACScan, BD Biosciences) using the CellQuest software.
For in vivo experimentations, the nonparametric Willcoxon test was used to compare the tumor volume (quantitative data) between controls and ZOL-treated animals. The cumulative rate of overall survival was calculated according to actuarial method and the end point considered was either death of animals or tumor volume superior to 20,000 cm3. The differences of actuarial survival were determined by the K2 test. Statistical evaluation of the in vitro proliferation data was performed by Student's t-test. Results are given as mean + SD and results with p < 0.05 were considered significant.
ZOL slows down the chondrosarcoma tumor progression and significantly enhances rat survival in curative treatment
In curative treatments, rats received s.c. 100 μg/kg ZOL twice a week, from day 4 after tumor implantation until death. The mean tumor volume was significantly lower in the ZOL-treated group than in control group at day 25 (4318 + 2278 and 10355 + 7414 mm3 respectively, p < 0.05, Fig. 1a) and at day 27 (5253 + 4133 and 15092 + 10781 respectively, p < 0.05). The volume tumor progression calculated between day 14 and 27 was significantly higher in the control group than in treated animals (13986 + 10986 versus 4418 + 4059 mm3, p < 0.05, Fig. 1b). As a consequence, ZOL significantly prolonged overall survival of chondrosarcoma-bearing rats, as the probability of survival at day 40 was 0.3 + 0.197 for the control group compared to 0.667 + 0.33 for treated animals (p < 0.05) with tumor volume superior to 20,000 mm3 as the end point (Fig. 1c).
Recurrent tumors after intralesional curettage grow slower in ZOL-treated rats
In a second set of experiments, the effects of ZOL were examined on tumor growth that occurred after intralesional curettage. In these series, the rats were operated at day 24 after tumor implantation. The treatment group received s.c. 100 μg/kg ZOL, 4 days before intralesional curettage, then twice a week until euthanasia. In this protocol, even if ZOL treatment failed to prevent local growth, it occurred later in all cases. The mean tumor volume was smaller for ZOL-treated animals from day 32 to 54. For example, mean tumor volume was 2.6-fold higher in control group than in the treated group at day 54, 31204 + 10781 and 12066 + 5492 mm3, respectively (p < 0.05, Fig. 2a). At all time points, the control value is always superior to ZOL-treated animals. In the same way, volume tumor progression between day 39 and 49 was significantly higher in control group than in the treated animals (15691 + 3173 versus 7396 + 5621 mm3, p < 0.03, Fig. 2b). In another independent series, the mean tumor volumes were always much higher in controls than in ZOL-treated rats, even if the differences were not found significant (21821 + 22913 and 7171 + 11232 at day 46, and 12253 + 5479 versus 5547 + 9218 at day 42, respectively) (data not shown).
ZOL inhibits RCS cell proliferation and increases cell death
To determine whether the antitumor activity of ZOL observed in vivo could be mediated by a direct antitumor effect on cell proliferation, the effect of ZOL (0.1–100 μM for 72 hr) was assessed in vitro on the RCS cell proliferation (Fig. 3a). The XTT viability test showed that ZOL significantly decreased chondrosarcoma cell proliferation with an IC50 value of 1.7 μM. Thus, ZOL inhibited cell proliferation by 38% at 1 μM and by 100% at 10 and 100 μM (p < 0.001) (Fig. 3a). To determine whether these effects were due to inhibition of cell proliferation and/or induction of cell death, the effects of ZOL were assessed by counting viable cells based on trypan blue exclusion. The results confirmed the low proliferation rate of RCS cells in the control group (30% increase after 72 hr of culture, Fig. 3b) and a decrease of alive cell number over 72 hr in the presence of 1 μM ZOL compared to the control group (38% decrease, p < 0.01). During the same period of culture, a marked increase of cell death was observed in the presence of ZOL (20% compared to 4.7% in the control group, p < 0.01), thus suggesting that no proliferation takes place in the ZOL-treated cell groups (Fig. 3b). All the RCS cells died after 72 hr in the presence of 10 μM ZOL. To determine whether the ZOL-induced death in RCS cells was caused by apoptosis, Hoechst staining and caspase-1 and -3 activation were investigated. Both tests gave negative results: Hoechst staining showed no modification of nuclear morphology in the presence of ZOL as compared to control cells (data not shown), and no significant activation of caspase 1 (data not shown) and 3 (Fig. 3c) activity could be detected after ZOL treatment in these cells.
Moreover, flow cytometric analysis of DNA content was performed to identify cell cycle perturbations in RCS cells following treatment with ZOL over a 48-hr period. Results showed no modification of the cell cycle profiles in the presence of 10 μM ZOL for 48 hr of incubation (Fig. 3d).
The present study focuses on the potential effect of ZOL on tumor progression and residual disease after intralesional curettage in a rat model of chondrosarcoma, and demonstrates for the first time that bisphosphonate has a potent beneficial effect in vivo on chondrosarcoma tumor progression. Most of the literature on chondrosarcoma has confirmed that adequate surgery is the mainstay of treatment for local control which itself is a risk factor for survival.5, 6, 27, 28 Despite different definitions of adequate margins, this goal can be achieved in only 46–76% of patients.5, 6, 7, 28 Adequate surgery means wide margins of normal tissue, that leads to severe disability, mainly when tumor occurs on spine or pelvis, when possible. On the other hand, inadequate surgery is an independent risk factor for local recurrence (as well as tumor size greater than 10 cm). Adjuvant treatment is of great interest to improve local control of this tumor when wide margin is not possible to obtain metastatic spreading and even to consider intralesional treatment of low grade chondrosarcoma. Radiation therapy is not effective in local control of the tumor7, 27, 29 but can be used in exceptional situations delaying local recurrence probably only in patients who have minimum residual microscopic disease.30 Chemotherapy is recommended in high risk chondrosarcoma and dedifferentiated chondrosarcoma,6, 31, 32, 33 but it is not administered according to a specific protocol and no controlled study is available. Cryosurgery after intralesional curettage is much promising in grade 1 chondrosarcoma, with at least similar results of marginal excision in term of oncological control and mostly better than those of wide excisions in terms of functional results.34, 35, 36
A vicious cycle has been described in osteolytic metastasis; tumor cells release osteolytic mediators and bone resorption release growth factors which enhance tumor cell growth and further release of osteolytic mediators.9 One can speculate that this vicious cycle may also occur in the case of the primary bone tumor. Thus, previous study with RCS pointed out the role of bone microenvironment on tumor aggressiveness, interactions between bone and tumor-induced bone remodelling and modification of the grade (grade II with foci of grade III, according to O'Neal and Ackerman grading) when tumor tissue is transplanted in close contact to the scarified bone.26 Inhibitors of bone resorption such as BPs may interfere with primary tumor development at the skeletal site. To our knowledge, such hypothesis has never been tested in vivo and only one oral communication has been recently reported with chondrosarcoma.37 RCS simulates the conditions of human chondrosarcoma development. It has been well characterized histologically, biochemically and structurally.38, 39 Kenan and Steiner39 showed that RCS is a well-differentiated malignant tumor, histologically similar to well-differentiated human chondrosarcoma. In the present study, this RCS tumor tissue was transplanted in close contact to femur after mechanical scarifications to insure interaction between tumor progression and bone remodelling as previously described.26 Human low grade chondrosarcomas induce 3–12% of metastasis appearing along time after diagnosis (mean 47 months).5, 6 As the present corresponds to a low grade differentiated chondrosarcoma (mostly grade II),26 it is unsurprising that any metastasis was observed during this short experimental course (maximal 5 weeks postimplantation) with animals dying from local development of the tumor. Such condition is seldom observed in clinical situation.
In vivo results confirmed in all experiments of the present study, the efficacy of ZOL on RCS progression. On the other hand, ZOL treatment postponed local recurrence in treated group, but failed to prevent tumor recurrence after intralesional curettage. Because of extensive spreading of the tumor in the soft tissue, intralesional curettage in all cases simulated microscopic residual disease. Such situation is frequent in clinical conditions, and efficient medical adjuvant treatment such as bisphosphonate would be of greatest interest in potential microscopic residual disease, in order to reduce margins and improve functional results. Unfortunately, this condition is difficult to mimick with RCS. Indeed, intraosseous implantation, which could be a better model for an extensive curettage, leads to inconstant and small tumors in our experience. ZOL doses used in the present study are justified as the clinical dose (4 mg i.v. every 3–4 weeks) is equivalent to ∼100 μg/kg of the research grade disodium salt used in this study. However, even if dosing frequency of twice a week is greater than in human, it could be justified by the aggressive nature of the chondrosarcoma.
Initially, it was thought that the specific inhibition of osteoclastic bone resorption is the only mechanism of action of BPs, by which they are effective in the treatment of cancer patients bearing bone metastasis. However, evidence is emerging from both preclinical and clinical studies to suggest that BPs also have direct antitumor properties that may contribute to their therapeutic efficacy in malignant bone disease.40 Potential direct antitumor effect of pamidronate and clodronate have been suggested in 2 in vitro studies showing inhibition of osteosarcoma cell growth proliferation.20, 21 Morever, ZOL induced in a dose- and time-dependant decrease in cell proliferation in osteosarcoma cell lines in vitro, and reduced tumor progression and metastatic lung spreading in vivo.23, 24 The present study reports a significant decrease of chondrosarcoma cell proliferation and an increase of cell death at 1 and 10 μM in vitro with an IC50 comparable to those observed on osteosarcoma and breast carcinoma cells.1, 19, 24 ZOL appears to be a more potent inhibitor of cell proliferation compared to the other N-BPs.40 Indeed, while several BPs exert antitumor activities, their effects seem to vary among the different compounds, in relation to their basic structure. The nitrogen-containing BPs such as ZOL have been suggested to be clinically superior to their first generation counterparts.41 Moreover, the peak plasma levels of ZOL appear to be around 1 μM as shown by Skerjanec et al.42 which is in agreement with the IC50 of ZOL measured on cancer cells, thus then strengthening an in vivo potential effect of ZOL even at a low concentration. ZOL has been reported to inhibit cell cycle progression and increase the proportion of cells arrested in S-phase in rat and human osteosarcoma,21, 23 that was not confirmed in the present study with RCS cells. This observation can be explained by the slow proliferation rate of the RCS cells as shown in the control condition (Fig. 3d). Moreover, ZOL-induced RCS cell death is not mediated by activation of caspase 1 and 3 as in osteosarcoma cells.21, 22 Thus, our data support a cytotoxic effects of ZOL which could look like to an anoikis mechanism as already described in osteosarcoma cells.21 However, the precise molecular mechanisms implicated in the ZOL-induced cell death need further investigation.
The present study demonstrated for the first time, that in addition to surgery, the therapy of chondrosarcoma with BPs might be beneficial. Such results open the way to the development of effective adjuvant treatment associated with surgical approach for treatment of chondrosarcoma.
The authors wish to thank Dr. Jonathan Green for helpful discussions and C. Bailly, A. Hivonnait and C. Le Corre from the Experimental Therapy Unit of the IFR26 (Nantes, France) for their technical assistance.