An In Vitro Osteoclast-Forming Assay to Measure Myeloma Cell-Derived Osteoclast-Activating Factors

  • Andrew C. W. Zannettino
  • Amanda N. Farrugia
  • L. Bik To
  • Gerald J. Atkins
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 113)


Much of the morbidity and mortality associated with the plasma cell (PC) malignancy, multiple myeloma (MM), is owing to the severe osteolytic bone disease seen in patients with this disease. Although the molecular mechanisms responsible for osteolysis remain to be fully elucidated, it is clear from numerous studies that it is owing, in part, to an increase in osteoclastic bone resorption. Several known osteoclast (OC)-activating factors (OAFs) are produced by myeloma PCs (MPCs), or by stromal cells in response to MPCs and include interleukin-1β (IL-1β); tumor necrosis factor-α (TNF-α); IL-6; parathyroid hormone-related protein; macrophage inflammatory protein-1α; and, most recently, the TNF-ligand family member receptor activator of nuclear factor-κB ligand (RANKL). The identification and significance of any one of these myeloma-derived OAFs is dependent on robust and reliable assays that measure the de novo formation and activation of OCs. A number of in vitro assay systems have been described that examine the requirements for normal OC formation and are easily adaptable for examining which MM-derived OAF and to what extent it is responsible for the bone loss observed in individuals with myeloma. This chapter describes one such in vitro model system.

Key Words

Osteoclast myeloma plasma cell bone resorption osteoclast activating factor in vitro assay 


  1. 1.
    Roodman G. D. (1997) Mechanisms of bone lesions in multiple myeloma and lymphoma. Cancer 80, 1557–1563.PubMedCrossRefGoogle Scholar
  2. 2.
    Mundy G. R. (1997) Mechanisms of bone metastasis. Cancer 80, 1546–1556.PubMedCrossRefGoogle Scholar
  3. 3.
    Bataille R., Chappard D., Marcelli C., et al. (1991) Recruitment of new osteo-blasts and osteoclasts is the earliest critical event in the pathogenesis of human multiple myeloma. J. Clin. Invest. 88, 62–66.PubMedCrossRefGoogle Scholar
  4. 4.
    Lichtenstein A., Berenson J., Norman D., Chang M. P., and Carlile A. (1989) Production of cytokines by bone marrow cells obtained from patients with multiple myeloma. Blood 74, 1266–1273.PubMedGoogle Scholar
  5. 5.
    Croucher P. I. and Apperley J. F. (1998) Bone disease in multiple myeloma. Br. J. Haematol. 103, 902–910.PubMedCrossRefGoogle Scholar
  6. 6.
    Garrett I. R., Durie B. G., Nedwin G. E., et al. (1987) Production of lympho-toxin, a bone-resorbing cytokine, by cultured human myeloma cells. N. Engl. J. Med. 317, 526–532.PubMedCrossRefGoogle Scholar
  7. 7.
    Bataille R., Klein B., Jourdan M., Rossi J. F., and Durie B. G. (1989) Spontaneous secretion of tumor necrosis factor-beta by human myeloma cell lines. Cancer 63, 877–880.PubMedCrossRefGoogle Scholar
  8. 8.
    Cozzolino F., Torcia M., Aldinucci D., et al. (1989) Production of interleukin-1 by bone marrow myeloma cells. Blood 74, 380–387.PubMedGoogle Scholar
  9. 9.
    Yamamoto I., Kawano M., Sone T., et al. (1989) Production of interleukin 1 beta, a potent bone resorbing cytokine, by cultured human myeloma cells. Cancer Res. 49, 4242–4246.PubMedGoogle Scholar
  10. 10.
    Sati H. L., Greaves M., Apperley J. F., Russell R. G., and Croucher P. I. (1999) Expression of interleukin-1 beta and tumour necrosis factor-alpha in plasma cells from patients with multiple myeloma. Br. J. Haematol. 104, 350–357.PubMedCrossRefGoogle Scholar
  11. 11.
    Barille S., Bataille R., and Amoit M. (2000) The role of interleukin-6 and interleukin-6/interleukin-6 receptor-alpha complex in the pathogenesis of multiple myeloma. Eur. Cytokine Netw. 11, 546–551.PubMedGoogle Scholar
  12. 12.
    Sati H. L., Apperley J. R., Greaves M., et al. (1998) Interleukin-6 is expressed by plasma cells from patients with multiple myeloma and monoclonal gammopathy of undetermined significance. Br. J. Haematol. 101, 287–295.PubMedCrossRefGoogle Scholar
  13. 13.
    Suzuki A., Takahashi T., Okuno Y., et al. (1994) Production of parathyroid hormone-related protein by cultured human myeloma cells. Am. J. Hematol. 45, 88–90.PubMedCrossRefGoogle Scholar
  14. 14.
    Otsuki T., Yamada O., Kurebayashi J., et al. (2001) Expression and in vitro modification of parathyroid hormone-related protein (PTHrP) and PTH/PTHrP-receptor in human myeloma cells. Leuk. Lymphoma 41, 397–409.PubMedCrossRefGoogle Scholar
  15. 15.
    Han J.-H., Choi S. J., Kurihara N., Koide M., Oba Y., and Roodman G. D. (2001) Macrophage inflammatory protein-1a is an osteoclastogenic factor in myeloma that is independent of receptor activator of nuclear factor kB ligand. Blood 97, 3349–3353.PubMedCrossRefGoogle Scholar
  16. 16.
    Farrugia A. N., Atkins G. J., To L. B., et al. (2003) Receptor activator of nuclear factor-kappaß ligand expression by human myeloma cells mediates osteoclast formation in vitro and correlates with bone destruction in vivo. Cancer Res. 63, 5438–5445.PubMedGoogle Scholar
  17. 17.
    Sezer O., Heider U., Jakob C., et al. (2002) Immunocytochemistry reveals RANKL expression of myeloma cells. Blood 99, 4646, 4647; discussion 4647.PubMedCrossRefGoogle Scholar
  18. 18.
    Heider U., Langelotz C., Jakob C., et al. (2003) Expression of receptor activator of nuclear factor kappaß ligand on bone marrow plasma cells correlates with osteolytic bone disease in patients with multiple myeloma. Clin. Cancer Res. 9, 1436–1440.PubMedGoogle Scholar
  19. 19.
    Haynes D. R., Atkins G. J., Loric M., Crotti T. N., Geary S. M., and Findlay D. M. (1999) Bidirectional signaling between stromal and hemopoietic cells regulates interleukin-1 expression during human osteoclast formation. Bone 25, 269–278.PubMedCrossRefGoogle Scholar
  20. 20.
    Shalhoub V., Elliott G., Chiu L., et al. (2000) Characterization of osteoclast precursors in human blood. Br. J. Haematol. 111, 501–512.PubMedCrossRefGoogle Scholar
  21. 21.
    Quinn J. M., Elliott J., Gillespie M. T., and Martin T. J. (1998) A combination of osteoclast differentiation factor and macrophage-colony stimulating factor is sufficient for both human and mouse osteoclast formation in vitro. Endocrinology 139, 4424–4427.PubMedCrossRefGoogle Scholar
  22. 22.
    Atkins G. J., Haynes D. R., Geary S. M., Loric M., Crotti T. N., and Findlay D. M. (2000) Coordinated cytokine expression by stromal and hematopoietic cells during human osteoclast formation. Bone 26, 653–661.PubMedCrossRefGoogle Scholar
  23. 23.
    James I. E., Lark M. W., Zembryki D., et al. (1999) Development and characterization of a human in vitro resorption assay: demonstration of utility using novel antiresorptive agents. J. Bone Miner. Res. 14, 1562–1569.PubMedCrossRefGoogle Scholar
  24. 24.
    Kudo O., Sabokbar A., Pocock A., Itonaga I., and Athanasou N. A. (2002) Isolation of human osteoclasts formed in vitro: hormonal effects on the bone-resorbing activity of human osteoclasts. Calcif. Tissue Int. 71, 539–546.PubMedCrossRefGoogle Scholar
  25. 25.
    Joyner C. J., Quinn J. M., Triffitt J. T., Owen M. E., and Athanasou N. A. (1992) Phenotypic characterisation of mononuclear and multinucleated cells of giant cell tumour of bone. Bone Miner. 16, 37–48.PubMedCrossRefGoogle Scholar
  26. 26.
    Fujikawa Y., Quinn J. M., Sabokbar A., McGee J. O., and Athanasou N. A. (1996) The human osteoclast precursor circulates in the monocyte fraction. Endocrinology 137, 4058–4060.PubMedCrossRefGoogle Scholar
  27. 27.
    Massey H. M. and Flanagan A. M. (1999) Human osteoclasts derive from CD14-positive monocytes. Br. J. Haematol. 106, 167–170.PubMedCrossRefGoogle Scholar
  28. 28.
    Pope B., Brown R. D., Gibson J., Yuen E., and Joshua D. (2000) B7-2-positive myeloma: incidence, clinical characteristics, prognostic significance, and implications for tumor immunotherapy. Blood 96, 1274–1279.PubMedGoogle Scholar
  29. 29.
    Nicholson G. C., Aitken C. J., Hodge J. M., et al. (2001) Limited RANKL exposure in vitro induces osteoclastogenesis in human PBMC. Bone 28(Suppl.), P271 (abstract).Google Scholar
  30. 30.
    Nicholson G. C., Moseley J. M., Sexton P. M., Mendelsohn F. A., and Martin T. J. (1986) Abundant calcitonin receptors in isolated rat osteoclasts: biochemical and autoradiographic characterization. J. Clin. Invest. 78, 355–360.PubMedCrossRefGoogle Scholar
  31. 31.
    Drake F. H., Dodds R. A., James I. E., et al. (1996) Cathepsin K, but not cathep-sins B, L, or S, is abundantly expressed in human osteoclasts. J. Biol. Chem. 271, 12,511–12,516.PubMedCrossRefGoogle Scholar
  32. 32.
    Vaananen H. K. (1984) Immunohistochemical localization of carbonic anhydrase isoenzymes I and II in human bone, cartilage and giant cell tumor. Histochemistry 81, 485–487.PubMedCrossRefGoogle Scholar
  33. 33.
    Davies J., Warwick J., Totty N., Philp R., Helfrich M., and Horton M. (1989) The osteoclast functional antigen, implicated in the regulation of bone resorption, is biochemically related to the vitronectin receptor. J. Cell Biol. 109, 1817–1826.PubMedCrossRefGoogle Scholar
  34. 34.
    Minkin C. (1982) Bone acid phosphatase: tartrate-resistant acid phosphatase as a marker of osteoclast function. Calcif. Tissue Int. 34, 285–290.PubMedCrossRefGoogle Scholar
  35. 35.
    Halleen J. M., Alatalo S. L., Suominen H., Cheng S., Janckila A. J., and Vaananen H. K. (2000) Tartrate-resistant acid phosphatase 5b: a novel serum marker of bone resorption. J. Bone Miner. Res. 15, 1337–1345.PubMedCrossRefGoogle Scholar
  36. 36.
    Hsu H., Lacey D. L., Dunstan C. R., et al. (1999) Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proc. Natl. Acad. Sci. USA 96, 3540–3545.PubMedCrossRefGoogle Scholar
  37. 37.
    Atkins G. J., Haynes D. R., Graves S. E., et al. (2000) Expression of osteoclast differentiation signals by stromal elements of giant cell tumors. J. Bone Miner. Res. 15, 640–649.PubMedCrossRefGoogle Scholar
  38. 38.
    Hattersley G. and Chambers T. J. (1989) Generation of osteoclastic function in mouse bone marrow cultures: multinuclearity and tartrate-resistant acid phosphatase are unreliable markers for osteoclastic differentiation. Endocrinology 124, 1689–1696.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Andrew C. W. Zannettino
    • 1
  • Amanda N. Farrugia
    • 2
  • L. Bik To
    • 3
  • Gerald J. Atkins
    • 4
  1. 1.Myeloma and Mesenchymal Research LaboratoryInstitute of Medical and Veterinary ScienceAdelaideAustralia
  2. 2.Myeloma and Mesenchymal Research LaboratoryInstitute of Medical and Veterinary ScienceAdelaideAustralia
  3. 3.Myeloma and Mesenchymal Research LaboratoryInstitute of Medical and Veterinary ScienceAdelaideAustralia
  4. 4.Department of Orthopaedics and TraumaUniversity of AdelaideAdelaideAustralia

Personalised recommendations