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