The Use of Fluorescent Proteins for Intravital Imaging of Cancer Cell Invasion

  • James Hulit
  • Dmitriy Kedrin
  • Bojana Gligorijevic
  • David Entenberg
  • Jeffrey Wyckoff
  • John Condeelis
  • Jeffrey E. SegallEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 872)


The analysis of cancer cell behavior in the primary tumor in living animals provides an opportunity to explore the process of invasion and intravasation in the complex microenvironment that is present in vivo. In this chapter, we describe the methods that we have developed for performing intravital imaging of mammary tumors. We provide procedures for generating tumors through injection of tumor cell lines, and multiphoton imaging using a skin-flap tumor dissection and a mammary imaging window.

Key words

Intravital imaging Multiphoton imaging Live animal imaging 



The work described herein was supported by CA100324, CA113395, and CA126511 (B.G., D.E., J.W., and J.C.) and CA100324 and CA77522 (J.H., D.K., and J.E.S.).


  1. 1.
    Chambers, A.F., Groom, A.C., MacDonald, I.C. (2002) Dissemination and growth of cancer cells in metastatic sites.Nat Rev Cancer.2, 563–72.PubMedCrossRefGoogle Scholar
  2. 2.
    Tuettenberg, J., Grobholz, R., Seiz, M., et al. (2009) Recurrence pattern in glioblastoma multiforme patients treated with anti-angiogenic chemotherapy.J Cancer Res Clin Oncol. 135, 1239–44.PubMedCrossRefGoogle Scholar
  3. 3.
    Arons, M.S., Smith, R.R. (1961) Distant metastases and local recurrence in head and neck cancer.Annals of Surgery. 154, 235–40.PubMedCrossRefGoogle Scholar
  4. 4.
    Condeelis, J., Pollard, J.W. (2006) Macrophages: obligate partners for tumor cell migration, invasion, and metastasis.Cell. 124, 263–6.PubMedCrossRefGoogle Scholar
  5. 5.
    Queen, M.M., Ryan, R.E., Holzer, R.G., Keller-Peck, C.R., Jorcyk, C.L. (2005) Breast cancer cells stimulate neutrophils to produce oncostatin M: potential implications for tumor progression.Cancer Res. 65, 8896–904.PubMedCrossRefGoogle Scholar
  6. 6.
    Orimo, A., Gupta, P.B., Sgroi, D.C., et al. (2005) Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion.Cell. 121, 335–48.PubMedCrossRefGoogle Scholar
  7. 7.
    Gaggioli, C., Hooper, S., Hidalgo-Carcedo, C., et al. (2007) Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells.Nature Cell Biol. 9, 1392–400.PubMedCrossRefGoogle Scholar
  8. 8.
    Karnoub, A.E., Dash, A.B., Vo, A.P., et al. (2007) Mesenchymal stem cells within tumour stroma promote breast cancer metastasis.Nature. 449, 557–63.PubMedCrossRefGoogle Scholar
  9. 9.
    Wang, W., Wyckoff, J.B., Frohlich, V.C., et al. (2002) Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling.Cancer Res. 62, 6278–88.PubMedGoogle Scholar
  10. 10.
    Provenzano, P.P., Eliceiri, K.W., Campbell, J.M., Inman, D.R., White, J.G., Keely, P.J. (2006) Collagen reorganization at the tumor-stromal interface facilitates local invasion.BMC Medicine. 4, 38.PubMedCrossRefGoogle Scholar
  11. 11.
    Levental, K.R., Yu, H., Kass, L., et al. (2009) Matrix crosslinking forces tumor progression by enhancing integrin signaling.Cell. 139, 891–906.PubMedCrossRefGoogle Scholar
  12. 12.
    Pepper, M.S., Tille, J.C., Nisato, R., Skobe, M. (2003) Lymphangiogenesis and tumor metastasis.Cell Tissue Res. 314, 167–77.PubMedCrossRefGoogle Scholar
  13. 13.
    Alexander, S., Koehl, G.E., Hirschberg, M., Geissler, E.K., Friedl, P. (2008) Dynamic imaging of cancer growth and invasion: a modified skin-fold chamber model.Histochem Cell Biol. 130, 1147–54.PubMedCrossRefGoogle Scholar
  14. 14.
    Dvorak, H.F. (2003) Rous-Whipple Award Lecture. How tumors make bad blood vessels and stroma.Am J Pathol. 162, 1747–57.PubMedCrossRefGoogle Scholar
  15. 15.
    Hashizume, H., Baluk, P., Morikawa, S., et al. (2000) Openings between defective endothelial cells explain tumor vessel leakiness.Am J Pathol. 156, 1363–80.PubMedCrossRefGoogle Scholar
  16. 16.
    Hoffman, R.M. (2005) Orthotopic metastatic (MetaMouse) models for discovery and development of novel chemotherapy.Methods in Molecular Medicine. 111, 297–322.PubMedGoogle Scholar
  17. 17.
    Lohela, M., Werb, Z. (2009) Intravital imaging of stromal cell dynamics in tumors.Curr Opin Genet Dev. 20, 72–8.PubMedCrossRefGoogle Scholar
  18. 18.
    Frese, K.K., Tuveson, D.A. (2007) Maximizing mouse cancer models.Nat Rev Cancer 7, 645–58.PubMedCrossRefGoogle Scholar
  19. 19.
    Wyckoff, J.B., Wang, Y., Lin, E.Y., et al. (2007) Direct visualization of macrophage-assisted tumor cell intravasation in mammary tumors.Cancer Res. 67, 2649–56.PubMedCrossRefGoogle Scholar
  20. 20.
    Hillen, F., Kaijzel, E.L., Castermans, K., oude Egbrink, M.G., Lowik, C.W., Griffioen, A.W. (2008) A transgenic Tie2-GFP athymic mouse model; a tool for vascular biology in xenograft tumors.Biochem Biophys Res Commun.368, 364–7PubMedCrossRefGoogle Scholar
  21. 21.
    Yang, M., Reynoso, J., Jiang, P., Li, L., Moossa, A.R., and Hoffman, R.M. (2004) Transgenic nude mouse with ubiquitous green fluorescent protein expression as a host for human tumors.Cancer Res.64, 8651–6.PubMedCrossRefGoogle Scholar
  22. 22.
    Yang, M., Reynoso, J., Bouvet, M., and Hoffman, R.M. (2009) A transgenic red fluorescent protein-expressing nude mouse for color-coded imaging of the tumor microenvironment.J. Cell. Biochem.106, 279–84.PubMedCrossRefGoogle Scholar
  23. 23.
    Tran Cao, H.S., Reynoso, J., Yang M., Kimura, H., Kaushal, S., Snyder, C.S., Hoffman, R.M., and Bouvet M. (2009) Development of the transgenic cyan fluorescent protein (CFP)-expressing nude mouse for “Technicolor” cancer imaging.J. Cell. Biochem.107, 328–34.PubMedCrossRefGoogle Scholar
  24. 24.
    Centonze, V.E., White, J.G. (1998) Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal ­imaging.Biophys J.75, 2015–24.PubMedCrossRefGoogle Scholar
  25. 25.
    Brown, E., McKee, T., diTomaso, E., et al. (2003) Dynamic imaging of collagen and its modulation in tumors in vivo using ­second-harmonic generation.Nature Med. 9, 796–800.PubMedCrossRefGoogle Scholar
  26. 26.
    Wyckoff, J., Gligorijevic, B., Entenberg, J., Segall, J.E., Condeelis, J. (2010) “High-Resolution Multiphoton Imaging of Tumors in Vivo”,Live Cell Imaging: A Laboratory Manual.2nd Edition ed, CSHL Press.Google Scholar
  27. 27.
    Kedrin, D., Gligorijevic, B., Wyckoff, J., et al. (2008) Intravital imaging of metastatic behavior through a mammary imaging window.Nature Methods. 5, 1019–21.PubMedCrossRefGoogle Scholar
  28. 28.
    Gligorijevic, B., Kedrin, D., Segall, J.E., Condeelis, J., van Rheenen, J. (2009) Dendra2 photoswitching through the Mammary Imaging Window.J Vis Exp.(
  29. 29.
    Yang, M., Baranov, E., Wang, J-W., et al. (2002) Direct external imaging of nascent cancer, tumor progression, angiogenesis, and metastasis on internal organs in the fluorescent orthotopic model.Proc Natl Acad Sci USA99, 3824–3829.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • James Hulit
    • 1
    • 2
  • Dmitriy Kedrin
    • 1
  • Bojana Gligorijevic
    • 3
  • David Entenberg
    • 3
  • Jeffrey Wyckoff
    • 3
  • John Condeelis
    • 3
  • Jeffrey E. Segall
    • 4
    Email author
  1. 1.Department of Anatomy and Structural BiologyAlbert Einstein College of MedicineBronxUSA
  2. 2.Centre for Tumour Biology, Barts and the London Queen Mary’s Medical and Dental SchoolLondonUK
  3. 3.Department of Anatomy and Structural BiologyGruss-Lipper Biophotonics Center, Albert Einstein College of MedicineBronxUSA
  4. 4.Anatomy and Structural BiologyAlbert Einstein College of MedicineBronxUSA

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