Skip to main content

Advertisement

Log in

A highly bone marrow metastatic murine breast cancer model established through in vivo selection exhibits enhanced anchorage-independent growth and cell migration mediated by ICAM-1

  • Research Paper
  • Published:
Clinical & Experimental Metastasis Aims and scope Submit manuscript

Abstract

To understand the mechanisms underlying bone marrow metastasis precisely, we established the highly metastatic 4T1E/M3 murine breast cancer cell line. 4T1 murine breast cancer cells were transfected with the neomycin resistance gene, selected in G418, intravenously injected into mice, and harvested from bone marrow. By repeating this protocol three times, we established the 4T1E/M3 cells. The clonality of 4T1E/M3 cells was markedly high confirmed by genomic southern analysis using neo-gene probe. When tissues harvested from mice after intravenous injection of 4T1E/M3 cells were examined histologically, markedly enhanced bone marrow metastasis was observed; 77% of spines from 4T1E/M3-injected mouse showed metastasis as compared to 14% metastasis seen with the parent cells. In vitro, 4T1E/M3 cells attached more strongly to the plastic plate and to bone marrow-derived endothelial cells. DNA micro arrays, real time RT-PCR and FACS analyses revealed that the expression of ICAM-1 and β2 integrin was upregulated in 4T1E/M3 cells at both the mRNA and cell surface protein levels. 4T1E/M3 cells also showed greater anchorage-independent proliferation in soft agar, and migrated markedly faster than the parent cells in wound healing assays. Anti-ICAM-1 antibodies strongly inhibited both the colony formation and the migration activity of 4T1E/M3 suggesting the importance of the role of ICAM-1. Our newly established highly metastatic 4T1E/M3 cells may provide a potentially powerful tool to study the molecular mechanisms of bone marrow metastasis and to identify new molecular targets for therapeutic interventions.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Mundy GR (2002) Metastasis to bone: causes, consequences and therapeutic opportunities. Nat Rev Cancer 2:584–593

    Article  PubMed  CAS  Google Scholar 

  2. Parkin DM, Bray F, Ferlay J et al (2005) Global cancer statistics, 2002. CA Cancer J Clin 55:74–108

    Article  PubMed  Google Scholar 

  3. Bellahcene A, Bachelier R, Detry C et al (2007) Transcriptome analysis reveals an osteoblast-like phenotype for human osteotropic breast cancer cells. Breast Cancer Res Treat 101:135–148

    Article  PubMed  CAS  Google Scholar 

  4. Peyruchaud O, Winding B, Pecheur I et al (2001) Early detection of bone metastases in a murine model using fluorescent human breast cancer cells: application to the use of the bisphosphonate zoledronic acid in the treatment of osteolytic lesions. J Bone Miner Res 16:2027–2034

    Article  PubMed  CAS  Google Scholar 

  5. Yoneda T, Williams PJ, Hiraga T et al (2001) A bone-seeking clone exhibits different biological properties from the MDA-MB-231 parental human breast cancer cells and a brain-seeking clone in vivo and in vitro. J Bone Miner Res 16:1486–1495

    Article  PubMed  CAS  Google Scholar 

  6. Bandyopadhyay A, Elkahloun A, Baysa SJ et al (2005) Development and gene expression profiling of a metastatic variant of the human breast cancer MDA-MB-435 cells. Cancer Biol Ther 4:168–174

    PubMed  CAS  Google Scholar 

  7. Bandyopadhyay A, Agyin JK, Wang L et al (2006) Inhibition of pulmonary and skeletal metastasis by a transforming growth factor-beta type I receptor kinase inhibitor. Cancer Res 66:6714–6721

    Article  PubMed  CAS  Google Scholar 

  8. Sheridan C, Kishimoto H, Fuchs RK et al (2006) CD44+/CD24- breast cancer cells exhibit enhanced invasive properties: an early step necessary for metastasis. Breast Cancer Res 8:R59

    Article  PubMed  CAS  Google Scholar 

  9. Liotta LA, Kohn EC (2001) The microenvironment of the tumour-host interface. Nature 411:375–379

    Article  PubMed  CAS  Google Scholar 

  10. Luo Y, Zhou H, Krueger J et al (2006) Targeting tumor-associated macrophages as a novel strategy against breast cancer. J Clin Invest 116:2132–2141

    Article  PubMed  CAS  Google Scholar 

  11. Condeelis J, Pollard JW (2006) Macrophages: obligate partners for tumor cell migration, invasion, and metastasis. Cell 124:263–266

    Article  PubMed  CAS  Google Scholar 

  12. Aslakson CJ, Miller FR (1992) Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor. Cancer Res 52:1399–1405

    PubMed  CAS  Google Scholar 

  13. Pulaski BA, Ostrand-Rosenberg S (1998) Reduction of established spontaneous mammary carcinoma metastases following immunotherapy with major histocompatibility complex class II and B7.1 cell-based tumor vaccines. Cancer Res 58:1486–1493

    PubMed  CAS  Google Scholar 

  14. Pulaski BA, Terman DS, Khan S et al (2000) Cooperativity of Staphylococcal aureus enterotoxin B superantigen, major histocompatibility complex class II, and CD80 for immunotherapy of advanced spontaneous metastases in a clinically relevant postoperative mouse breast cancer model. Cancer Res 60:2710–2715

    PubMed  CAS  Google Scholar 

  15. Monzavi-Karbassi B, Artaud C, Jousheghany F et al (2005) Reduction of spontaneous metastases through induction of carbohydrate cross-reactive apoptotic antibodies. J Immunol 174:7057–7065

    PubMed  CAS  Google Scholar 

  16. Lewis JD, Shearer MH, Kennedy RC et al (2005) Surrogate tumor antigen vaccination induces tumor-specific immunity and the rejection of spontaneous metastases. Cancer Res 65:2938–2946

    Article  PubMed  CAS  Google Scholar 

  17. Demaria S, Kawashima N, Yang AM et al (2005) Immune-mediated inhibition of metastases after treatment with local radiation and CTLA-4 blockade in a mouse model of breast cancer. Clin Cancer Res 11:728–734

    PubMed  CAS  Google Scholar 

  18. Brilliant MH, Gondo Y, Eicher EM (1991) Direct molecular identification of the mouse pink-eyed unstable mutation by genome scanning. Science 252:566–569

    Article  PubMed  CAS  Google Scholar 

  19. Tominaga H, Ishiyama M, Ohseto F et al (1999) A Water-soluble tetrazolium salt useful for colorimetric cell viability assay. Anal Commun 36:47–50

    Article  CAS  Google Scholar 

  20. Carmichael J, DeGraff WG, Gazdar AF et al (1987) Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res 47:936–942

    PubMed  CAS  Google Scholar 

  21. Okada T, Li J, Kodaka M et al (1998) Enhancement of type IV collagenases by highly metastatic variants of HT1080 fibrosarcoma cells established by a transendothelial invasion system in vitro. Clin Exp Metastasis 16:267–274

    Article  PubMed  CAS  Google Scholar 

  22. Palmieri D, Halverson DO, Ouatas T et al (2005) Medroxyprogesterone acetate elevation of Nm23-H1 metastasis suppressor expression in hormone receptor-negative breast cancer. J Natl Cancer Inst 97:632–642

    Article  PubMed  CAS  Google Scholar 

  23. Okada T, Akikusa S, Okuno H et al (2003) Bone marrow metastatic myeloma cells promote osteoclastogenesis through RANKL on endothelial cells. Clin Exp Metastasis 20:639–646

    Article  PubMed  CAS  Google Scholar 

  24. Ohta H, Hamada J-I, Tada M et al (2006) HOXD3-overexpression increases integrin αvβ3 expression and deprives E-cadherin while it enhances cell motility in A549 cells. Clin Exp Metastasis 23:381–390

    Article  PubMed  CAS  Google Scholar 

  25. Coleman RE (2006) Clinical features of metastatic bone disease and risk of skeletal morbidity. Clin Cancer Res 12:6243s–6249s

    Article  PubMed  Google Scholar 

  26. Pulaski BA, Clements VK, Pipeling MR et al (2000) Immunotherapy with vaccines combining MHC class II/CD80+ tumor cells with interleukin-12 reduces established metastatic disease and stimulates immune effectors and monokine induced by interferon gamma. Cancer Immunol Immunother 49:34–45

    Article  PubMed  CAS  Google Scholar 

  27. Minn AJ, Gupta GP, Siegel PM et al (2005) Genes that mediate breast cancer metastasis to lung. Nature 436:518–524

    Article  PubMed  CAS  Google Scholar 

  28. Waghorne C, Thomas M, Lagarde A et al (1988) Genetic evidence for progressive selection and overgrowth of primary tumors by metastatic cell subpopulations. Cancer Res 48:6109–6114

    PubMed  CAS  Google Scholar 

  29. Frost P, Kerbel RS, Hunt B et al (1987) Selection of metastatic variants with identifiable karyotypic changes from a nonmetastatic murine tumor after treatment with 2′-deoxy-5-azacytidine or hydroxyurea: implications for the mechanisms of tumor progression. Cancer Res 47:2690–2695

    PubMed  CAS  Google Scholar 

  30. Eckhardt BL, Parker BS, van Laar RK et al (2005) Genomic analysis of a spontaneous model of breast cancer metastasis to bone reveals a role for the extracellular matrix. Mol Cancer Res 3:1–13

    PubMed  CAS  Google Scholar 

  31. Irshad S, Pedley RB, Anderson J et al (2004) The Brn-3β transcription factor regulates the growth, behavior, and invasiveness of human neuroblastoma cells in vitro and in vivo. J Biol Chem 279:21617–21627

    Article  PubMed  CAS  Google Scholar 

  32. Lee JY, Kim H, Ryu CH et al (2004) Merlin, a tumor suppressor, interacts with transactivation-responsive RNA-binding protein and inhibits its oncogenic activity. J Biol Chem 279:30265–30273

    Article  PubMed  CAS  Google Scholar 

  33. Muraoka-Cook RS, Kurokawa H, Koh Y et al (2004) Conditional overexpression of active transforming growth factor beta1 in vivo accelerates metastases of transgenic mammary tumors. Cancer Res 64:9002–9011

    Article  PubMed  CAS  Google Scholar 

  34. Glondu M, Liaudet-Coopman E, Derocq D et al (2002) Down-regulation of cathepsin-D expression by antisense gene transfer inhibits tumor growth and experimental lung metastasis of human breast cancer cells. Oncogene 21:5127–5134

    Article  PubMed  CAS  Google Scholar 

  35. Morimoto-Tomita M, Ohashi Y, Matsubara A et al (2005) Mouse colon carcinoma cells established for high incidence of experimental hepatic metastasis exhibit accelerated and anchorage-independent growth. Clin Exp Metastasis 22:513–521

    Article  PubMed  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  37. Hu J, Verkman AS (2006) Increased migration and metastatic potential of tumor cells expressing aquaporin water channels. FASEB J 20:1892–1894

    Article  PubMed  CAS  Google Scholar 

  38. Chen X, Lin J, Kanekura T et al (2006) A small interfering CD147-targeting RNA inhibited the proliferation, invasiveness, and metastatic activity of malignant melanoma. Cancer Res 66:11323–11330

    Article  PubMed  CAS  Google Scholar 

  39. Galaup A, Cazes A, Le Jan S et al (2006) Angiopoietin-like 4 prevents metastasis through inhibition of vascular permeability and tumor cell motility and invasiveness. Proc Natl Acad Sci USA 103:18721–18726

    Article  PubMed  CAS  Google Scholar 

  40. Rosette C, Roth RB, Oeth P et al (2005) Role of ICAM1 in invasion of human breast cancer cells. Carcinogenesis 26:943–950

    Article  PubMed  CAS  Google Scholar 

  41. Wang S, Coleman EJ, Pop LM et al (2006) Effect of an anti-CD54 (ICAM-1) monoclonal antibody (UV3) on the growth of human uveal melanoma cells transplanted heterotopically and orthotopically in SCID mice. Int J Cancer 118:932–941

    Article  PubMed  CAS  Google Scholar 

  42. Fanales-Belasio E, Zambruno G, Cavani A, Girolomoni G (1997) Antibodies against sialophorin (CD43) enhance the capacity of dendritic cells to cluster and activate T lymphocytes. J Immunol 159:2203–2211

    PubMed  CAS  Google Scholar 

  43. Rosenstein Y, Park JK, Hahn WC et al (1991) CD43, a molecule defective in Wiskott-Aldrich syndrome, binds ICAM-1. Nature 354:233–235

    Article  PubMed  CAS  Google Scholar 

  44. Ziprin P, Alkhamesi NA, Ridgway PF, Pech DH, Darzi AW (2004) Tumor-expressed CD43 (sialophorin) mediates tumour-mesothelial cell adhesion. Biol Chem 385:755–761

    Article  PubMed  CAS  Google Scholar 

  45. Kadaja-Saarepuu L, Laos S, Jããger K et al (2007) CD43 promotes cell growth and helps to evade FAS-mediated apoptosis in non-hematopoietic cancer cell lacking the tumor suppressors p53 or ARF. Oncogene (advance online publication). doi:10.1038/sj.onc.1210802

Download references

Acknowledgements

We thank Ms. Yoko Ezaki for her excellent technical assistance for cell culture, Dr. Toru Imamura at AIST for making his laboratory facilities available for this study. This work was financially supported by Talent in Nanobiotechnology Course, Promotion Budget for Science and Technology from Ministry of Education, Culture, Sports, Science and Technology (MEXT) and by Japan Science and Technology Agency (JST).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Tomoko Okada.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Takahashi, M., Furihata, M., Akimitsu, N. et al. A highly bone marrow metastatic murine breast cancer model established through in vivo selection exhibits enhanced anchorage-independent growth and cell migration mediated by ICAM-1. Clin Exp Metastasis 25, 517–529 (2008). https://doi.org/10.1007/s10585-008-9163-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10585-008-9163-5

Keywords

Navigation