Giant cell tumor (GCT) is a locally aggressive, benign bone lesion which shows extensive bone destruction that often leads to pain and pathologic fracture. It usually occurs in the epiphysis and metaphysis of bone near the joint. The destruction of subchondral bone impairs joint function.7,14,26 In rare instances, the tumor can spread to the lungs or can present as multifocal disease.5,27 On histologic examination, the tumor shows a mixture of cells consisting of osteoclastlike multinucleated giant cells, mononuclear cells, and stromal cells.7,26 This unique composition of different cell types has been the subject of the study of osteoclast biology.3,19,38
The preferred treatment options include surgical treatment such as curettage, in conjunction with chemical or physical adjuvant therapy, or extensive resection followed by major reconstructive surgery.7,13,26 The outcome after surgical resection has been affected negatively by tumor recurrence and additional bone destruction.6 Many topical adjuvant therapies such as phenol, hydrogen peroxide, or liquid nitrogen have been used in an attempt to decrease local recurrence by inducing physical or chemical damage to the remaining GCT cells after curettage, but a specific adjuvant therapy that directly targets osteoclasts and GCT cells has not been described.10,25 To decrease the local recurrence and additional destruction of bone by GCT, it is necessary to understand the pathophysiology of GCT. A medical or pharmacologic treatment that targets the osteolytic process may lower the recurrence rates of GCT and avoid major reconstructive surgery with its associated morbidity and mortality. Furthermore, this kind of therapy may be able to control other osteolytic bone lesions that share the common mechanism of osteoclastic activation.
Bisphosphonates are known to bind to bone in vivo and in vitro, to inhibit osteoclastic bone resorption, and to inhibit osteoclastogenesis.18,32 Multinucleated giant cells in GCT have the capacity to resorb bone and express markers similar to those in osteoclasts such as tartrate resistant acid phosphatase (TRAP) and cathepsin K.15,33,36 This functional and immunohistochemical similarity between osteoclasts and the multinucleated giant cells found in GCT suggests that bisphosphonates may have a similar action against GCT cells as they do against osteoclasts. The purpose of this study was to evaluate the efficacy of the bisphosphonates pamidronate and zoledronate (Novartis Pharmaceuticals, Basel, Switzerland) in inhibiting GCT cells and in inducing apoptosis in GCT cells in an in vitro model.
MATERIALS AND METHODS
The diagnosis of GCT was established by biopsy before surgical excision. Fresh GCT tumor tissues were obtained at the time of surgery from nine patients having tumor resection. Four specimens among nine samples were freshly minced with scissors in Dulbecco’s minimum essential medium (DMEM) producing a cell suspension with small fragments of tissue. The suspension was pelleted by centrifugation and the small fragments were digested enzymatically in phosphate buffered saline (PBS) containing 1 mg/mL collagenase, 0.15 mg/mL DNAse, and 0.15 mg/mL hyaluronidase for 1 hour at 37o C. The suspension then was passed through sterile gauze to remove any undigested fragments and the cells were either frozen in liquid nitrogen for later use or seeded in 75 cm2 flasks with DMEM supplemented with 10% fetal calf serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.1% amphotericin B. Cells were grown at 37o C in a humidified atmosphere of 5% CO2 and 95% air. Culture medium was changed every three to four days. Without supplemental receptor activator of NF-κB ligand (RANKL) and macrophage colony stimulating factor (MCSF), one of the four cell lines showed osteoclastlike giant cells with 3–4 nuclei consistently after more than six passages.
GCT were treated with 0 μm, 50 μm, 100 μm, 500 μm, and 1000 μm of pamidronate and zoledronate for 1 to 3 days. Cells were prepared for analyzing apoptosis and cycle cell using flow cytometry.
Immunohistochemical characterization of GCT cells was done on 5 μm thickness of tissue section. The sections were rehydrated in PBS and incubated with primary antibodies raised against markers for osteoclasts, hematopoietic mononuclear cells and mesenchymal stromal cells after blocking using VECTASTAIN ABC Systems (Vector Laboratories, CA). For osteoclasts, we used antibodies for TRAP. For hematopoietic mononuclear cells, we used CD14, CD34, and CD68.8,16,21 For mesenchymal stromal cells, we used SH2, Stro-1, and stromal–cell-derived factor-1α (SDF-1 α).34 Colorimetric detection was used to localize positive staining on the GCT tissue. Using the same antibodies, GCT cells also were stained in vitro.
For apoptosis analysis, control and treated cells were digested by trypsin and resuspended in 5 mL PBS. Cell concentration was measured and the cells then were pelleted again and resuspended in binding buffer (A.G. Scientific, San Diego, CA) to a final cell density of 5 × 105 cells/mL. Annexin V is an early apoptotic marker in the apoptotic cell membrane.30 Five microliters of Annexin V-FITC (A.G. Scientific) was added to 195 μL of cell suspension and incubated in the dark for 10 minutes. Ten microliters of 20 μg/mL propidium iodide (A.G. Scientific) was added to the cell suspension and flow cytometry was done within 30 minutes using a FACS Calibur (Becton Dickinson, Franklin Lakes, NJ). For cell cycle analysis, 5 × 105 cells/mL cells were fixed in 70% cold ethanol for an hour after harvest. And after RNase (50 μg/ml) digestion, 2 × 105 cells/mL cells in 190 μL as apoptosis analysis were stained with 10 μL of propidium iodide.23 Monoparametric cytograms of annexin V-FITC fluorescence (FL1) versus number of events were created using the CellQuest (Becton Dickinson) program gating for living cells and excluding dead cells on the basis of cell size and granularity. Using similar methods, cell cycle analysis was done.23
Western blotting for the cleaved forms of caspase-3 and poly-ADP ribose polymerase (PARP) then was done. Caspase-3 is a downstream effector of apoptosis and is present as an inactive, noncleaved form in the cytoplasm. The active, cleaved form can be detected using Western blotting. To confirm the functionality of caspase-3, we assessed the decreasing amount of PARP that is one of the substrates of activated caspase-3 using Western blotting.1
Cells were lysed using buffer IP (10 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1% Triton X-100, 0.25% Nonidet P-40, and 2 mM EDTA), supplemented with a protease inhibitor cocktail tablet (Roche Molecular Biochemicals, Indianapolis, IN). Protein quantification, SDS-PAGE, and Western blot analyses were done. Primary antibodies were used at the following dilutions: anticaspase-3 (Cell Signaling, MA) at 1:1000 and antiPARP (Cell Signaling, MA) at 1:500.
Data were expressed as means ± standard error of the mean. Differences between each treated group and controls were analyzed by Scheffe’s test using one-way ANOVA, and p < 0.05 was considered statistically significant.
Cell markers for TRAP, monocytes of hematopoietic stem cell origin, and stromal cells of mesenchymal cell origin were present universally in the GCT tissue. The first subset is a group of TRAP-positive multinucleated osteoclastlike giant cells or tumor osteoclasts (Fig 1A). Mononuclear tumor osteoclast precursor cells also were stained positively with TRAP. There was abundant RANKL localization in tumor tissue suggesting autocrine loop of osteoclastogenesis in GCT (Fig 1B).
One of the GCT cells grew and maintained consistent phenotypes without supplemental RANKL and M-CSF and there were subpopulations of cells fusing each other and beginning to form osteoclastlike giant cells with 3–4 nuclei (Fig 1C).
There was a dose-dependent increase in apoptosis with a higher dose of pamidronate increasing the fraction of cells undergoing apoptosis and in the number of dead cells (p = 0.00003 – 1.0) (Table 1). Treatment with pamidronate increased (p = 0.00003) the number of apoptotic cells (Table 1). After addition of pamidronate, GCT cells shrunk at a lower dose and showed more abundant round debris at a higher dose. These cells were stained positively with the Annexin V antibody that reflects early apoptotic change in the cell membrane (Fig 2). Quantitative analysis showed an increasing fraction of cells positively stained with Annexin V antibody according to the dose of treatment. Of note is a dose–response relationship between the dose of pamidronate and fraction of apoptotic cells by flow cytometric analysis (Fig 4). We also analyzed the fraction of apoptotic cells by cell cycle analysis, and the trend was comparable with the pattern seen with Annexin V binding. When we analyzed the fraction of live cells after treatment with pamidronate, the live cell fraction decreased in a dose-dependent manner.
Treating GCT cells with zoledronate increased the cell fraction undergoing apoptosis to a greater degree than after treatment with pamidronate (p = 0.001 – 1.0) (Fig 3). The cell cycle analysis showed increasing apoptotic population in a dose-dependent manner (Fig 4). Treatment with zoledronate increased (p = 0.001 for zoledronate concentration of 100 μmol/L) the number of apoptotic cells (Table 1).
Activation of caspase-3 (a downstream effector of apoptosis) and a corresponding decline in PARP (a substrate of activated caspase-3) by bisphosphonate was shown by Western blotting. Giant cell tumor cells treated with 100 μmol/L of pamidronate showed an increasing amount of the active form of caspase-3 from Day 1 to Day 3 and a decreasing amount of PARP from Day 1 to Day 3 (Fig 5).
Our data show a dose dependent increase in apoptosis by pamidronate and zoledronate on GCT cells as measured by the cell cycle analysis assay. The induction of apoptosis also was seen by the Western blot assay measuring the downstream effectors of apoptosis.
Bisphosphonates have a strong affinity for bone in vivo and in vitro, inhibit osteoclastic bone resorption, and inhibit osteoclastogenesis.32 Multinucleated giant cells in GCT resorb bone, and express markers like TRAP and cathepsin K that also are expressed by osteoclasts.15,33,36 This immunohistochemical and functional similarity between osteoclasts and the multinucleated giant cells of GCT can be used to explore new treatment options for GCT.
Giant cell tumor is a naturally occurring coculture of stromal cells and hematopoietic cells leading to osteoclastogenesis. This unique feature is an excellent model for understanding osteoclastogenesis and developing new therapeutic strategies targeting osteoclastogenesis. With recent advances in the biology of bone such as the discovery and functional analysis of osteoprotegerin (OPG), OPG ligand (OPGL, RANKL, TRANCE), receptor activator of NF-κB (RANK), TNF-α, and interleukins, the pathophysiology of osteolysis has become better understood.2,22,35,37 Briefly, RANKL is an osteoclastogenesis factor released by osteoblasts, stromal cells, and activated T cells. RANK is a receptor that is present on the cell membrane of osteoclasts, monocytes, and osteoblasts. Osteoprotegerin is a decoy receptor of RANKL and inactivates RANKL on binding. Studies have shown the role of RANKL in GCT as evidenced by the inhibitory effect of OPG on RANKL-mediated tumoral osteoclastogenesis.2,19 Although OPG-blocking or RANKL-blocking antibody can be used as a potential therapeutic agent for tumor-induced osteolysis, systemic use theoretically can induce altered immune function.4 For these reasons, we chose to investigate bisphosphonates as an alternative adjuvant therapy for GCT of bone.
Bisphosphonates are pyrophosphates that are used in the treatment of osteoporosis and metastatic cancers to bone.18 Pamidronate and zoledronate are available in an intravenously injectable form, but alendronate (Merck, Whitehouse Station, NJ), risedronate (Aventis, Bridgewater, NJ), and tiludronate (Sanofi-Synthelabo, Paris, France) are available in oral formulations. Etidronate (Procter and Gamble, Cincinnati, OH) is available in injectable and oral forms. Bisphosphonates can bind to bone or other calcium-containing substances in vivo and in vitro. After intravenous injection, as much as 50–80% of the absorbed dose is taken up by bone and the remainder is cleared rapidly in the urine. The bioavailability of an oral dose of a bisphosphonate is low, in the range of 1–10%. Molecular mechanisms of bisphosphonates involve inhibition of ATP by incorporating into intracellular analogues of ATP in nonnitrogen-containing bisphosphonates such as etidronate and tiludronate; and inhibition of prenylation and functioning of guanosine triphosphate-binding proteins required for osteoclast formation, function and survival in nitrogen-containing bisphosphonates such as risedronate, zoledronate, alendronate, and pamidronate.9,31 Bisphosphonates also are known to have a direct antitumor and antiangiogenesis effect.28 For these unique pharmacologic reasons, bisphosphonates can be considered novel alternative therapeutic agents for benign and possible malignant bone tumors. As long as pamidronate or zoledronate is given slowly in diluted form, side effects are minimal, limited to transient pyrexia, seen in 10% of the patients, which is popularly treated with acetaminophen and usually disappears within the first 3 days.12
Giant cell tumor involves remarkable tumor osteoclastogenesis, and once was referred to as an osteoclastoma.20 As shown in our results, GCT shows TRAP-positive multinucleated osteoclastlike giant cells, which are known therapeutic targets of bisphosphonates (Fig 1A). For this reason, the potential therapeutic effect of bisphosphonates is logical and appealing. As expected, based on the pathogenesis of GCT resulting from the molecular interaction between neoplastic stromal cells and hematopoietic mononuclear cells, pamidronate and zoledronate showed in vitro therapeutic effects by inducing apoptosis (Fig 4).
Apoptosis is a special form of cell death that is distinct from necrosis. In contrast to necrosis caused by heat, freezing, and chemical or physical damages, apoptosis is triggered by specific agents that activate the cell-death machinery leading to cell suicide. One of the major actions of bisphosphonates in the treatment of osteoporosis is induction of apoptosis in the osteoclasts in addition to the inhibition of osteoclastogenesis and functional impairment of osteoclasts. The treatment rationale in GCT is analogous to treatment of osteoporosis or metastatic cancers to bone. Zoledronate is known to be 100 times more potent than pamidronate in rodent models.17 In our experiments, zoledronate showed a 10-fold to 20-fold increase in apoptosis and decrease in live-cell fraction for a given dose compared with pamidronate (Fig 4). Zoledronate seemed to be a more potent therapeutic agent in GCT seemingly affecting all the cellular components of GCT and causing the entire cell to appear apoptotic on photomicrography (Fig 3C). In our study, the therapeutic effects of pamidronate and zoledronate seem obvious.
There are no data available regarding the mode of delivery of bisphosphonates, such as topical application, perioperative intravenous administration, or oral ingestion, in benign bone tumors. In osteogenesis imperfecta, fibrous dysplasia, or Paget’s disease, intravenous treatment seems to be logical because of the multifocal disease process. For unifocal bone tumors, topical delivery through direct application or use of carriers seems to be more reasonable because most surgeons have the advantage of exploring the region of interest in the bone. We did not explore the optimal delivery method for bisphosphonates because it was beyond the scope of the study.
We admit that the GCT culture is a coculture of different lineages, and we did not test the effect of pamidronate or zoledronate on an individual lineage such as the neoplastic stromal cells, mononuclear cells, or tumor osteoclasts. However, GCT by nature involves molecular interactions among different cell types and has an autocrine function of tumor osteoclastogenesis. An effect on any particular lineage would affect this autocrine interaction and disrupt the disease process.
The dose of zoledronate and pamidronate used in our study is similar to the dose range used in the in vitro study of osteoclasts, breast cancer cells, and myeloma, but we did not evaluate in vivo dosing.11 Our study provides insight into the potential therapeutic value of bisphosphonates. Traditionally, local adjuvant therapeutic agents, such as phenol, hydrogen peroxide, liquid nitrogen, and heat generated by polymethylmethacrylate cement have been applied directly to the remaining host bone boundary after resection of tumor.10,25,29 The application of bisphosphonates may have the additional theoretical advantage of an antiresorptive effect in the remaining host bone at the host–tumor interface. As far as bone healing and bone graft incorporation are concerned, bisphosphonates do not have detrimental effects on osteoblastic function or formation of reparative tissue.24
Our study shows that GCT cells consist of a heterogeneous subset of cells of hematopoietic and mesenchymal origin that have an autocrine control of tumor osteoclastogenesis (Fig 1). These cells undergo dose-related apoptosis when treated with pamidronate or zoledronate, with zoledronate showing more potency (Fig 4). Pamidronate and zoledronate show potential therapeutic effects on GCT in vitro. Bisphosphonates can be considered an alternative adjuvant therapy for GCT of bone or possibly other benign bone disorders.
1. Affar EB, Germain M, Winstall E, et al: Caspase-3 mediated processing of poly ADP-ribose) glycohydrolase during apoptosis. J Biol Chem 276:2935–2942, 2001.
2. Atkins GJ, Bouralexis S, Haynes DR, et al: Osteoprotegerin inhibits osteoclast formation and bone resorbing activity in giant cell tumors of bone. Bone 28:370–377, 2001.
3. Atkins GJ, Haynes DR, Graves SE, et al: Expression of osteoclast differentiation signals by stromal elements of giant cell tumors. J Bone Miner Res 15:640–649, 2000.
4. Bengtsson AK, Ryan EJ: Immune function of the decoy receptor osteoprotegerin. Crit Rev Immunol 22:201–215, 2002.
5. Bertoni F, Bacchini P, Staals EL: Malignancy in giant cell tumor of bone. Cancer 97:2520–2529, 2003.
6. Blackley HR, Wunder JS, Davis AM, et al: Treatment of giant-cell tumors of long bones with curettage and bone grafting. J Bone Joint Surg 81A:811–820, 1999.
7. Campanacci M, Baldini N, Boriani S, et al: Giant cell tumor of bone. J Bone Joint Surg 69A:106–114, 1987.
8. Civin CI, Strauss LC, Brovall C, et al: Antigenic analysis of hematopoiesis: III. A hematopoietic progenitor cell surface antigen defined by a monoclonal antibody raised against KG-1a cells. J Immunol 133:157–165, 1984.
9. David P, Nguyen H, BArbier A, et al: The bisphosphonate is a potent inhibitor of the osteoclast vacuolar H(+)-ATPase. J Bone Miner Res 11:1498–1507, 1996.
10. Durr HR, Maier M, Jansson V, et al: Phenol as an adjuvant for local control in the treatment of giant cell tumor of the bone. Eur J Surg Oncol 25:610–618, 1999.
11. Evans CE, Braidman IP: Effects of two novel bisphosphonates on bone cells in vitro. Bone Miner 26:95–107, 1994.
12. Fleisch HA: Bisphosphonates: Preclinical aspects and use in osteoporosis. Ann Med 29:55–62, 1997.
13. Gitelis S, Mallin BA, Piasecki P, et al: Intralesional excision compared with en bloc resection for giant-cell tumors of bone. J Bone Joint Surg 75A:1648–1655, 1993.
14. Goldenberg RR, Campbell CJ, Bonfiglio M: Giant-cell tumor of bone: An analysis of two hundred and eighteen cases. J Bone Joint Surg 52A:619–664, 1970.
15. Grano M, Colucci S, Portoghese A, et al: Functional and biochemical characterization of osteoclast-like cells derived from giant cell tumors of bone. Boll Soc Ital Biol Sper 68:249–253, 1992.
16. Greaves DR, Gordon S: Macrophage-specific gene expression: current paradigms and future challenges. Int J Hematol 76:6–15, 2002.
17. Green JR, Muller K, Jaeggi KA: Preclinical pharmacology of CGP 42’446, a new, potent, heterocyclic bisphosphonate compound. J Bone Miner Res 9:745–751, 1994.
18. Green JR: Antitumor effects of bisphosphonates. Cancer 97S:840–847, 2003.
19. Huang L, Xu J, Wood DJ, et al: Gene expression of osteoprotegerin ligand, osteoprotegerin, and receptor activator of NF-kappaB in giant cell tumor of bone: Possible involvement in tumor cell induced osteoclastlike cell formation. Am J Pathol 156:761–767, 2000.
20. Jaffe HL, Lichtenstein L, Portis RB: Giant cell tumor of bone: Its pathological appearance grading supposed variants and treatment. Arch Pathol 30:993–1031, 1940.
21. Keller R, Keist R, Joller P, et al: Mononuclear phagocytes from human bone marrow progenitor cells: Morphology, surface phenotype, and functional properties of resting and activated cells. Clin Exp Immunol 91:176–182, 1993.
22. Kim N, Odgren PR, Kim DK, et al: Diverse roles of the tumor necrosis factor family member TRANCE in skeletal physiology revealed by TRANCE deficiency and partial rescue by a lymphocyte expressed TRANCE transgene. Proc Natl Acad Sci USA 97:10905–10910, 2000.
23. Krishan A: Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J Cell Biol 66:188–193, 1975.
24. Madsen JE, Berg-Larsen T, Kirkeby OJ, et al: No adverse effects of clodronate on fracture healing in rats. Acta Orthop Scand 69:532–536, 1998.
25. Marcove RC, Lyden JP, Huvos AG, et al: Giant-cell tumors treated by cryosurgery: A report of twenty-five cases. J Bone Joint Surg 55A:1633–1644, 1973.
26. McDonald DJ, Sim FH, McLeod RA, Dahlin DC: Giant-cell tumor of bone. J Bone Joint Surg 68A:235–242, 1986.
27. Nascimento AG, Huvos AG, Marcove RC: Primary malignant giant cell tumor of bone: A study of eight cases and review of the literature. Cancer 44:1393–1402, 1979.
28. Neville-Webbe HL, Holen I, Coleman RE: The anti-tumor activity of bisphosphonates. Cancer Treat Rev 28:305–319, 2002.
29. O’Donnell RJ, Springfield DS, Motwani HK, et al: Recurrence of giant-cell tumors of the long bones after curettage and packing with cement. J Bone Joint Surg 76A:1827–1833, 1994.
30. Pepper C, Thomas A, Tucker H, Hoy T, Bentley P: Flow cytometric assessment of three different methods for the measurement of in vitro apoptosis. Leuk Res 22:439–444, 1998.
31. Reszka AA, Rodan GA: Bisphosphonate mechanism of action. Curr Rheumatol Rep 5:65–74, 2003.
32. Rogers MJ, Gordon S, Benford HL, et al: Cellular and molecular mechanisms of action of bisphosphonates. Cancer 88:2961–2978, 2000.
33. Sabokbar A, Kudo O, Athanasou NA: Two distinct cellular mechanisms of osteoclast formation and bone resorption in periprosthetic osteolysis. J Orthop Res 21:73–80, 2003.
34. Simmons PJ, Torok-Storb B: Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody, STRO-1. Blood 78:55–62, 1991.
35. Simonet WS, Lacey DL, Dunstan CR, et al: Osteoprotegerin: A novel secreted protein involved in the regulation of bone density. Cell 89:309–319, 1997.
36. Teitelbaum SL: Bone resorption by osteoclasts. Science 289:1504–1508, 2000.
37. Wong BR, Josien R, Choi Y: TRANCE is a TNF family member that regulates dendritic cell and osteoclast function. J Leukoc Biol 65:715–724, 1999.
38. Zheng MH, Fan Y, Smith A, et al: Gene expression of monocyte chemoattractant protein-1 in giant cell tumors of bone osteoclastoma: possible involvement in CD68+ macrophage-like cell migration. J Cell Biochem 70:121–129, 1998.