Mesenchymal mode of migration participates in pulmonary metastasis of mouse osteosarcoma LM8


The outcomes of osteosarcoma patients still remain poor because of intractable pulmonary metastasis. We previously established a highly metastatic osteosarcoma cell line, LM8 from Dunn mouse osteosarcoma by in vivo selection. We herein aimed to clarify the characteristic biological features related with high metastatic potential and new target molecules to suppress pulmonary metastasis of osteosarcoma, using this syngeneic spontaneous metastatic model. LM8 cells acquired fibroblastic morphology with striking filopodia on the cell surface. Immunostaining showed faint stress fiber formation and peripherally localized integrin β1, and biochemical analyses showed the activated Cdc42 and autophosphorylation of focal adhesion kinase (FAK) in LM8 cells when compared to Dunn cells. LM8 cells had activated motility in single cell migration mode. LM8 migration was increased by a Rho-associated kinase (ROCK) inhibitor, Y-27632, while decreased by Cdc42 silencing using RNA interference system. We found that a clinically approved camptothecin analog, irinotecan suppressed the migration, Cdc42 activity, and autophosphorylation of FAK, and attenuated integrin β1 distribution selectively in LM8 cells. Daily oral administration of irinotecan significantly reduced the rate and size of pulmonary metastasis in syngeneic C3H mice. The fibroblastic morphology and activated cell migration with the dependency on Cdc42 but not Rho-ROCK signaling pathway argued that LM8 moved in mesenchymal mode of cell migration. This activated mesenchymal migration was a key component of the pulmonary metastasis of LM8 cells. The inhibition of mesenchymal migration by irinotecan, in addition to its cytotoxic effects, might be effective in preventing pulmonary metastasis of osteosarcoma.

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Extracellular matrix


Three dimensions


Focal adhesion kinase


Matrix metalloproteinase


Rho-associated kinase


  1. 1.

    Lewis IJ, Nooij MA, Whelan J et al (2007) Improvement in histologic response but not survival in osteosarcoma patients treated with intensified chemotherapy: a randomized phase III trial of the European Osteosarcoma Intergroup. J Natl Cancer Inst 99(2):112–128

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Meyers PA, Schwartz CL, Krailo M et al (2005) Osteosarcoma: a randomized, prospective trial of the addition of ifosfamide and/or muramyl tripeptide to cisplatin, doxorubicin, and high-dose methotrexate. J Clin Oncol 23(9):2004–2011

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Goorin AM, Schwartzentruber DJ, Devidas M et al (2003) Presurgical chemotherapy compared with immediate surgery and adjuvant chemotherapy for nonmetastatic osteosarcoma: Pediatric Oncology Group Study POG-8651. J Clin Oncol 21(8):1574–1580

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    Kager L, Zoubek A, Potschger U et al (2003) Primary metastatic osteosarcoma: presentation and outcome of patients treated on neoadjuvant Cooperative Osteosarcoma Study Group protocols. J Clin Oncol 21(10):2011–2018

    Article  PubMed  Google Scholar 

  5. 5.

    Dunn TB, Andervont HB (1963) Histology of some neoplasms and non-neoplastic lesions found in wild mice maintained under laboratory conditions. J Natl Cancer Inst 31:873–901

    CAS  PubMed  Google Scholar 

  6. 6.

    Asai T, Ueda T, Itoh K et al (1998) Establishment and characterization of a murine osteosarcoma cell line (LM8) with high metastatic potential to the lung. Int J Cancer 76(3):418–422

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Sahai E (2007) Illuminating the metastatic process. Nat Rev Cancer 7(10):737–749

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Gupta GP, Massague J (2006) Cancer metastasis: building a framework. Cell 127(4):679–695

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Pantel K, Brakenhoff RH (2004) Dissecting the metastatic cascade. Nat Rev Cancer 4(6):448–456

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Yamaguchi H, Wyckoff J, Condeelis J (2005) Cell migration in tumors. Curr Opin Cell Biol 17(5):559–564

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Friedl P, Wolf K (2003) Tumour-cell invasion and migration: diversity and escape mechanisms. Nat Rev Cancer 3(5):362–374

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Friedl P (2004) Prespecification and plasticity: shifting mechanisms of cell migration. Curr Opin Cell Biol 16(1):14–23

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Thiery JP (2002) Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer 2(6):442–454

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Poste G, Fidler IJ (1980) The pathogenesis of cancer metastasis. Nature 283(5743):139–146

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Ren XD, Kiosses WB, Schwartz MA (1999) Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J 18(3):578–585

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Mettouchi A, Klein S, Guo W et al (2001) Integrin-specific activation of Rac controls progression through the G(1) phase of the cell cycle. Mol Cell 8(1):115–127

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Yoshioka K, Nakamori S, Itoh K (1999) Overexpression of small GTP-binding protein RhoA promotes invasion of tumor cells. Cancer Res 59(8):2004–2010

    CAS  PubMed  Google Scholar 

  18. 18.

    Sotobori T, Ueda T, Myoui A et al (2006) Bone morphogenetic protein-2 promotes the haptotactic migration of murine osteoblastic and osteosarcoma cells by enhancing incorporation of integrin beta1 into lipid rafts. Exp Cell Res 312(19):3927–3938

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Nitta N, Tsuchiya T, Yamauchi A et al (2007) Quantitative analysis of eosinophil chemotaxis tracked using a novel optical device—TAXIScan. J Immunol Methods 320(1–2):155–163

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Sano K, Yoshikawa M, Hayasaka S et al (2003) Simple non-ion-paired high-performance liquid chromatographic method for simultaneous quantitation of carboxylate and lactone forms of 14 new camptothecin derivatives. J Chromatogr B Analyt Technol Biomed Life Sci 795(1):25–34

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Itoh K, Yoshioka K, Akedo H et al (1999) An essential part for Rho-associated kinase in the transcellular invasion of tumor cells. Nat Med 5(2):221–225

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Furman WL, Crews KR, Billups C et al (2006) Cefixime allows greater dose escalation of oral irinotecan: a phase I study in pediatric patients with refractory solid tumors. J Clin Oncol 24(4):563–570

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Kuppens IE, Dansin E, Boot H et al (2006) Dose-finding phase I clinical and pharmacokinetic study of orally administered irinotecan in patients with advanced solid tumors. Clin Cancer Res 12(12):3774–3781

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Drengler RL, Kuhn JG, Schaaf LJ et al (1999) Phase I and pharmacokinetic trial of oral irinotecan administered daily for 5 days every 3 weeks in patients with solid tumors. J Clin Oncol 17(2):685–696

    CAS  PubMed  Google Scholar 

  25. 25.

    Wang W, Goswami S, Sahai E et al (2005) Tumor cells caught in the act of invading: their strategy for enhanced cell motility. Trends Cell Biol 15(3):138–145

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Wyckoff JB, Pinner SE, Gschmeissner S et al (2006) ROCK- and myosin-dependent matrix deformation enables protease-independent tumor-cell invasion in vivo. Curr Biol 16(15):1515–1523

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Philippar U, Roussos ET, Oser M et al (2008) A Mena invasion isoform potentiates EGF-induced carcinoma cell invasion and metastasis. Dev Cell 15(6):813–828

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Ellenbroek SI, Collard JG (2007) Rho GTPases: functions and association with cancer. Clin Exp Metastasis 24(8):657–672

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Condeelis J, Segall JE (2003) Intravital imaging of cell movement in tumours. Nat Rev Cancer 3(12):921–930

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Giampieri S, Manning C, Hooper S et al (2009) Localized and reversible TGFbeta signalling switches breast cancer cells from cohesive to single cell motility. Nat Cell Biol 11(11):1287–1296

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Vicente-Manzanares M, Cabrero JR, Rey M et al (2002) A role for the Rho-p160 Rho coiled-coil kinase axis in the chemokine stromal cell-derived factor-1alpha-induced lymphocyte actomyosin and microtubular organization and chemotaxis. J Immunol 168(1):400–410

    CAS  PubMed  Google Scholar 

  32. 32.

    Vega FM, Ridley AJ (2008) Rho GTPases in cancer cell biology. FEBS Lett 582(14):2093–2101

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Nobes CD, Hall A (1999) Rho GTPases control polarity, protrusion, and adhesion during cell movement. J Cell Biol 144(6):1235–1244

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Wilkinson S, Paterson HF, Marshall CJ (2005) Cdc42-MRCK and Rho-ROCK signalling cooperate in myosin phosphorylation and cell invasion. Nat Cell Biol 7(3):255–261

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Yoshikawa H, Yoshioka K, Nakase T et al (2009) Stimulation of ectopic bone formation in response to BMP-2 by Rho kinase inhibitor: a pilot study. Clin Orthop Relat Res 467(12):3087–3095

    Article  PubMed  Google Scholar 

  36. 36.

    Kashima T, Nakamura K, Kawaguchi J et al (2003) Overexpression of cadherins suppresses pulmonary metastasis of osteosarcoma in vivo. Int J Cancer 104(2):147–154

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Pommier Y, Redon C, Rao VA et al (2003) Repair of and checkpoint response to topoisomerase I-mediated DNA damage. Mutat Res 532(1–2):173–203

    CAS  PubMed  Google Scholar 

  38. 38.

    Pommier Y, Cherfils J (2005) Interfacial inhibition of macromolecular interactions: nature’s paradigm for drug discovery. Trends Pharmacol Sci 26(3):138–145

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    Nitiss J, Wang JC (1988) DNA topoisomerase-targeting antitumor drugs can be studied in yeast. Proc Natl Acad Sci USA 85(20):7501–7505

    CAS  Article  PubMed  Google Scholar 

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We thank Dr. Takahashi (The 21st Century COE Formation, Kyoto University Graduate School of Medicine, Japan) for the expressing vectors of GST-Rhotekin and GST-PAK, and Dr. Yukawa (GE Healthcare Bioscience, Tokyo, Japan) for the analysis of EZ-TAXIScan data.

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Correspondence to Yoshihiro Yui.

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Yui, Y., Itoh, K., Yoshioka, K. et al. Mesenchymal mode of migration participates in pulmonary metastasis of mouse osteosarcoma LM8. Clin Exp Metastasis 27, 619–630 (2010).

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  • Osteosarcoma
  • Metastasis
  • Mesenchymal migration
  • Cdc42
  • Irinotecan