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Assessing Cancer Cell Migration and Metastatic Growth In Vivo in the Chick Embryo Using Fluorescence Intravital Imaging

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In Vivo Cellular Imaging Using Fluorescent Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 872))

Abstract

Cell migration and metastasis are key features of aggressive tumors. These processes can be difficult to study, as they often occur deep within the body of a cancer patient or an experimental animal. In vitro assays are able to model some aspects of these processes, and a number of assays have been developed to assess cancer cell motility, migration, and invasion. However, in vitro assays have inherent limitations that may miss important aspects of these processes as they occur in vivo. The chick embryo provides a powerful model for studying these processes in vivo, facilitated by the external and accessible nature of the chorioallantoic membrane (CAM), a well-vascularized tissue that surrounds the embryo. When coupled with multiple fluorescent approaches to labeling both cancer cells and the embryonic vasculature, along with image analysis tools, the chick CAM model offers cost-effective, rapid assays for studying cancer cell migration and metastasis in a physiologically-relevant, in vivo setting. Here, we present recent developments of detailed procedures for using shell-less chick embryos, coupled with fluorescent labeling of cancer cells and/or chick vasculature, to study cancer cell migration and metastasis in vivo.

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References

  1. Hanahan, D., and Weinberg, R. A. (2000) The hallmarks of cancer, Cell 100, 57–70.

    Article  PubMed  CAS  Google Scholar 

  2. Fidler, I. J. (2001) Seed and soil revisited: contribution of the organ microenvironment to cancer metastasis, Surg Oncol Clin N Am 10, 257–269, vii–viiii.

    PubMed  CAS  Google Scholar 

  3. Chambers, A. F., Groom, A. C., and MacDonald, I. C. (2002) Dissemination and growth of cancer cells in metastatic sites, Nat Rev Cancer 2, 563–572.

    Article  PubMed  CAS  Google Scholar 

  4. Kauffman, E. C., Robinson, V. L., Stadler, W. M., Sokoloff, M. H., and Rinker-Schaeffer, C. W. (2003) Metastasis suppression: the evolving role of metastasis suppressor genes for regulating cancer cell growth at the secondary site, J Urol 169, 1122–1133.

    Article  PubMed  Google Scholar 

  5. Pantel, K., and Brakenhoff, R. H. (2004) Dissecting the metastatic cascade, Nat Rev Cancer 4, 448–456.

    Article  PubMed  CAS  Google Scholar 

  6. Minn, A. J., Gupta, G. P., Siegel, P. M., Bos, P. D., Shu, W., Giri, D. D., Viale, A., Olshen, A. B., Gerald, W. L., and Massague, J. (2005) Genes that mediate breast cancer metastasis to lung, Nature 436, 518–524.

    Article  PubMed  CAS  Google Scholar 

  7. Hoon, D. S., Kitago, M., Kim, J., Mori, T., Piris, A., Szyfelbein, K., Mihm, M. C., Jr., Nathanson, S. D., Padera, T. P., Chambers, A. F., Vantyghem, S. A., MacDonald, I. C., Shivers, S. C., Alsarraj, M., Reintgen, D. S., Passlick, B., Sienel, W., and Pantel, K. (2006) Molecular mechanisms of metastasis, Cancer Metastasis Rev 25, 203–220.

    Article  PubMed  CAS  Google Scholar 

  8. Suzuki, M., Mose, E. S., Montel, V., and Tarin, D. (2006) Dormant cancer cells retrieved from metastasis-free organs regain tumorigenic and metastatic potency, Am J Pathol 169, 673–681.

    Article  PubMed  CAS  Google Scholar 

  9. Eccles, S. A., and Welch, D. R. (2007) Metastasis: recent discoveries and novel treatment strategies, Lancet 369, 1742–1757.

    Article  PubMed  CAS  Google Scholar 

  10. Barkan, D., Kleinman, H., Simmons, J. L., Asmussen, H., Kamaraju, A. K., Hoenorhoff, M. J., Liu, Z. Y., Costes, S. V., Cho, E. H., Lockett, S., Khanna, C., Chambers, A. F., and Green, J. E. (2008) Inhibition of metastatic outgrowth from single dormant tumor cells by targeting the cytoskeleton, Cancer Res 68, 6241–6250.

    Article  PubMed  CAS  Google Scholar 

  11. Hunter, K. W., Crawford, N. P., and Alsarraj, J. (2008) Mechanisms of metastasis, Breast Cancer Res 10 Suppl 1, S2.

    Article  PubMed  Google Scholar 

  12. Taylor, J., Hickson, J., Lotan, T., Yamada, D. S., and Rinker-Schaeffer, C. (2008) Using metastasis suppressor proteins to dissect interactions among cancer cells and their microenvironment, Cancer Metastasis Rev 27, 67–73.

    Article  PubMed  Google Scholar 

  13. Weber, G. F. (2008) Molecular mechanisms of metastasis, Cancer Lett 270, 181–190.

    Article  PubMed  CAS  Google Scholar 

  14. Albini, A. (1998) Tumor and endothelial cell invasion of basement membranes. The matrigel chemoinvasion assay as a tool for dissecting molecular mechanisms, Pathol Oncol Res 4, 230–241.

    Article  PubMed  CAS  Google Scholar 

  15. Eccles, S. A., Box, C., and Court, W. (2005) Cell migration/invasion assays and their application in cancer drug discovery, Biotechnol Annu Rev 11, 391–421.

    Article  PubMed  CAS  Google Scholar 

  16. Leighton, J. (1964) Invasion and Metastasis of Heterologous Tumors in the Chick Embryo, Prog Exp Tumor Res 4, 98–125.

    PubMed  CAS  Google Scholar 

  17. Locker, J., Goldblatt, P. J., and Leighton, J. (1969) Hematogenous metastasis of Yoshida ascites Hepatoma in the chick embryo liver: ultrastructural changes in tumor cells, Cancer Res 29, 1245–1253.

    PubMed  CAS  Google Scholar 

  18. McAllister, R. M., Peer, M., Gilden, R. V., Klement, V., and Landing, B. H. (1974) Tumors formed by human rhabdomyosarcoma cells in chorioallantoic membrane of embryonated hens’ eggs, Int J Cancer 13, 886–890.

    Article  PubMed  CAS  Google Scholar 

  19. Chambers, A. F., Shafir, R., and Ling, V. (1982) A model system for studying metastasis using the embryonic chick, Cancer Res 42, 4018–4025.

    PubMed  CAS  Google Scholar 

  20. Chambers, A. F., and Wilson, S. (1985) Cells transformed with a ts viral src mutant are temperature sensitive for in vivo growth, Mol Cell Biol 5, 728–733.

    PubMed  CAS  Google Scholar 

  21. Gordon, J. R., and Quigley, J. P. (1986) Early spontaneous metastasis in the human epidermoid carcinoma HEp3/chick embryo model: contribution of incidental colonization, Int J Cancer 38, 437–444.

    Article  PubMed  CAS  Google Scholar 

  22. Chambers, A. F., Schmidt, E. E., MacDonald, I. C., Morris, V. L., and Groom, A. C. (1992) Early steps in hematogenous metastasis of B16F1 melanoma cells in chick embryos studied by high-resolution intravital videomicroscopy, J Natl Cancer Inst 84, 797–803.

    Article  PubMed  CAS  Google Scholar 

  23. MacDonald, I. C., Schmidt, E. E., Morris, V. L., Chambers, A. F., and Groom, A. C. (1992) Intravital videomicroscopy of the chorioallantoic microcirculation: a model system for studying metastasis, Microvasc Res 44, 185–199.

    Article  PubMed  CAS  Google Scholar 

  24. Koop, S., Khokha, R., Schmidt, E. E., MacDonald, I. C., Morris, V. L., Chambers, A. F., and Groom, A. C. (1994) Overexpression of metalloproteinase inhibitor in B16F10 cells does not affect extravasation but reduces tumor growth, Cancer Res 54, 4791–4797.

    PubMed  CAS  Google Scholar 

  25. Quigley, J. P., and Armstrong, P. B. (1998) Tumor cell intravasation alu-cidated: the chick embryo opens the window, Cell 94, 281–284.

    Article  PubMed  CAS  Google Scholar 

  26. Ossowski, L., Aguirre Ghiso, J., Liu, D., Yu, W., and Kovalski, K. (1999) The role of plasminogen activator receptor in cancer invasion and dormancy, Medicina (B Aires) 59, 547–552.

    CAS  Google Scholar 

  27. Aguirre-Ghiso, J. A., Ossowski, L., and Rosenbaum, S. K. (2004) Green fluorescent protein tagging of extracellular signal-regulated kinase and p38 pathways reveals novel dynamics of pathway activation during primary and metastatic growth, Cancer Res 64, 7336–7345.

    Article  PubMed  CAS  Google Scholar 

  28. Wilson, S. M., and Chambers, A. F. (2004) Experimental metastasis assays in the chick embryo, Curr Protoc Cell Biol Chapter 19, Unit 19.6.

    Google Scholar 

  29. Zijlstra, A., Lewis, J., Degryse, B., Stuhlmann, H., and Quigley, J. P. (2008) The inhibition of tumor cell intravasation and subsequent metastasis via regulation of in vivo tumor cell motility by the tetraspanin CD151, Cancer Cell 13, 221–234.

    Article  PubMed  CAS  Google Scholar 

  30. Deryugina, E. I., and Quigley, J. P. (2008) Chick embryo chorioallantoic membrane model systems to study and visualize human tumor cell metastasis, Histochem Cell Biol 130, 1119–1130.

    Article  PubMed  CAS  Google Scholar 

  31. Jilani, S. M., Murphy, T. J., Thai, S. N., Eichmann, A., Alva, J. A., and Iruela-Arispe, M. L. (2003) Selective binding of lectins to embryonic chicken vasculature, J Histochem Cytochem 51, 597–604.

    Article  PubMed  CAS  Google Scholar 

  32. Welch, D. R. (1997) Technical considerations for studying cancer metastasis in vivo, Clin Exp Metastasis 15, 272–306.

    Article  PubMed  CAS  Google Scholar 

  33. Chishima, T., Miyagi, Y., Wang, X., Yamaoka, H., Shimada, H., Moossa, A. R., and Hoffman, R. M. (1997) Cancer invasion and micrometastasis visualized in live tissue by green fluorescent protein expression, Cancer Res 57, 2042–2047.

    PubMed  CAS  Google Scholar 

  34. Naumov, G. N., Wilson, S. M., MacDonald, I. C., Schmidt, E. E., Morris, V. L., Groom, A. C., Hoffman, R. M., and Chambers, A. F. (1999) Cellular expression of green fluorescent protein, coupled with high-resolution in vivo videomicroscopy, to monitor steps in tumor metastasis, J Cell Sci 112 (Pt 12), 1835–1842.

    PubMed  CAS  Google Scholar 

  35. Lewis, J. D., Destito, G., Zijlstra, A., Gonzalez, M. J., Quigley, J. P., Manchester, M., and Stuhlmann, H. (2006) Viral nanoparticles as tools for intravital vascular imaging, Nat Med 12, 354–360.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  37. Hoffman, R. M. (2009) Imaging cancer dynamics in vivo at the tumor and cellular level with fluorescent proteins, Clin Exp Metastasis 26, 345–355.

    Article  PubMed  CAS  Google Scholar 

  38. Hoffman, R.M. (2005) The multiple uses of fluorescent proteins to visualize cancer in vivo. Nat Rev Cancer5, 796–806.

    Article  PubMed  CAS  Google Scholar 

  39. Hoffman, R. M., and Yang, M. (2006) Subcellular imaging in the live mouse. Nat Protoc1, 775–782.

    Article  PubMed  CAS  Google Scholar 

  40. Hoffman, R. M., and Yang, M. (2006) Color-coded fluorescence imaging of tumor-host interactions. Nat Protoc1, 928–935.

    Article  PubMed  CAS  Google Scholar 

  41. Hoffman, R. M., and Yang, M. (2006) Whole-body imaging with fluorescent proteins. Nat Protoc1, 1429–1438.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work is supported by the Canadian Cancer Society Research Institute (grant #18176) and the Canadian Institutes of Health Research (grants #845351 and #42511). H.S.L. is funded by a Post-Doctoral Fellowship from the Canadian Breast Cancer Foundation (Ontario Division). A.F.C. is Canada Research Chair in Oncology, supported by the Canada Research Chairs Program.

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Correspondence to John D. Lewis .

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© 2012 Springer Science+Business Media New York

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Leong, H.S., Chambers, A.F., Lewis, J.D. (2012). Assessing Cancer Cell Migration and Metastatic Growth In Vivo in the Chick Embryo Using Fluorescence Intravital Imaging. In: Hoffman, R. (eds) In Vivo Cellular Imaging Using Fluorescent Proteins. Methods in Molecular Biology, vol 872. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-797-2_1

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  • DOI: https://doi.org/10.1007/978-1-61779-797-2_1

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-796-5

  • Online ISBN: 978-1-61779-797-2

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