Abstract
Fluorescent proteins have revolutionized biology, allowing what was formerly invisible to be clearly seen. The Nobel Prize in Chemistry was awarded in 2008 for the discovery and early use of green fluorescent protein (GFP) as a genetic reporter. Our laboratory pioneered the use of GFP for in vivo imaging. In this chapter we review the developments within our research on subcellular imaging of metastatic trafficking of cancer cells carried out in real time in mice. Dual-color fluorescent cells, with one color fluorescent protein in the nucleus and another color fluorescent protein in the cytoplasm, enable real-time nuclear-cytoplasmic dynamics to be visualized in living cells in vivo as well as in vitro. In the dual-color cells, red fluorescent protein (RFP) is expressed in the cytoplasm of cancer cells, and GFP is linked to histone H2B and is expressed in the nucleus. Nuclear GFP expression enables visualization of nuclear dynamics, whereas simultaneous cytoplasmic RFP expression allows visualization of nuclear cytoplasmic ratios in addition to simultaneous cell and nuclear shape changes. With the use of dual-color fluorescent cells, it is possible to achieve subcellular real-time imaging of cancer cell trafficking in live mice. Extravasation can also be imaged in real time. Dual-color imaging has shown that cytoplasmic processes of cancer cells exit the vessels first, with nuclei following along the cytoplasmic projections [Yamauchi et al., Cancer Res 66:4208–4214, 2006]. Dual-color in vivo cellular imaging was used to visualize cancer cell trafficking blood vessels, as well as in the lymphatic systems of the mice. The real-time imaging of cancer cell seeding on the lung has now been achieved with dual-color cells. Subcellular in vivo imaging confers great promise for understanding metastasis at the cellular level in vivo.
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Acknowledgements
The author’s laboratory acknowledges the support of the National Cancer Institute grants CA099258, CA103563, and CA132971.
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Hoffman, R.M. (2014). Imaging Metastatic Cell Trafficking at the Cellular Level In Vivo with Fluorescent Proteins. In: Dwek, M., Schumacher, U., Brooks, S. (eds) Metastasis Research Protocols. Methods in Molecular Biology, vol 1070. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4614-8244-4_12
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DOI: https://doi.org/10.1007/978-1-4614-8244-4_12
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