Molecular Imaging of Hypoxia Using Genetic Biosensors

  • Pablo Iglesias
  • J. A. CostoyaEmail author
Part of the Computational Methods in Applied Sciences book series (COMPUTMETHODS, volume 19)


In the last years, the need for visualization of tumoral processes has become a high-top priority in molecular imaging. This is especially true for those methods dedicated to functional imaging that focus on revealing phenomena associated with biological processes such as hypoxia to cancer. Among them, optical imaging methods such as fluorescence are provided with a broad range of proteins and dyes used to visualize many types of these biological processes widely used in cell biology studies. Although the most popular of these proteins is the green fluorescent protein (GFP), autofluorescence due to the absorption of the exciting radiation by endogenous fluorophores and signal dispersion raises doubts about its suitability as an in vivotracer. In the last years a number of groups have developed several NIR fluorescent proteins that enables real-time imaging to take place without interference from autofluorescence events allowing at the same time to take a deep view into the tissues. With all of this in mind, we devised a novel fluorescence-bioluminescence genetically encoded biosensor activated by the neoangiogenesis-related transcription factor HIF-1α, which appears upregulated in growing tumors. At the same time, by fusing a NIR emitting flurochrome (mCherry) and the firefly luciferase together we obtained a bioluminescence resonance energy transfer (BRET) phenomenon turning this fusion protein into a new class of hypoxia-sensing genetically encoded biosensor.


BRET Biosensors 



We thank the members of Molecular Oncology Lab for helpful discussions. We also thank M.E. Vazquez for helpful discussions and assistance with the spectrophotometric analysis. This study was supported by Spanish Ministry of Science and Innovation, (SAF2005-00306; SAF2008-00543) and Xunta de Galicia grants (PGIDIT05PXIB20801PR); Grupos emerxentes 2007/064) (J.A.C.), and by Fundacion de Investigacion Medica Mutua Madrileña (J.A.C., P.I.).


  1. 1.
    Ai, H., Shaner, N.C., Cheng, Z., etal.: Exploration of new chromophore structures leads to the identification of improved blue fluorescent proteins. Biochemistry 46, 5904–5910(2007)CrossRefGoogle Scholar
  2. 2.
    Akagi, Y., et al.: Regulation of vascular endothelial growth factor expression in human colon cancer by insulin-like growth factor-I. Cancer Res. 58, 4008–4014 (1998)Google Scholar
  3. 3.
    Alavi, A., Lakhani, P., Mavi, A., etal.: PET: a revolution in medical Imaging. Radiol. Clin. North Am. 42, 983–1001(2004)CrossRefGoogle Scholar
  4. 4.
    Baird, G.S., Zacharias, D.A., Tsien, R.Y.: Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. USA 97, 11984–11989 (2000)CrossRefGoogle Scholar
  5. 5.
    Bárdos, J.I., Ashcroft, M.: Negative and positive regulation of HIF-1: a complex network. BioEssays 26, 262–269(2004)CrossRefGoogle Scholar
  6. 6.
    Dunn, A.K., Bolay, T., Moskowitz, M.A., Boas, D.A.: Dynamic imaging of cerebral blood flow using laser speckle. J. Cereb. Blood Flow Metab. 2, 195–201(2001)CrossRefGoogle Scholar
  7. 7.
    Evans, S.M., Judy, K.D., Dunphy, I., etal.: Hypoxia is important in the biology and aggression of human glial brain tumors. Clin. Cancer Res. 10, 8177–8184(2004)CrossRefGoogle Scholar
  8. 8.
    Furlan, D., Sahnane, N., Carnevali, I., etal.: Regulation and stabilization of HIF-1alpha in colorectal carcinomas. Surg. Oncol. 16, S25–S27(2007)CrossRefGoogle Scholar
  9. 9.
    Gordan, J.D., Simon, M.C.: Hypoxia-inducible factors: central regulators of the tumor phenotype. Curr. Opin. Genet. Dev. 17, 71–77(2007)CrossRefGoogle Scholar
  10. 10.
    Gould, S.J., Subramani, S.: Firefly luciferase as a tool in molecular and cell biology. Anal. Biochem. 175, 5–13(1988)CrossRefGoogle Scholar
  11. 11.
    Graham, F.L., van der Eb, E.J.: A new technique for the assay of infectivity of human adenovirus 5 DNA. J. Virol. 52, 456–467(1973)CrossRefGoogle Scholar
  12. 12.
    Hastings, J.W.: Biological diversity, chemical mechanisms, and the evolutionary origins of bioluminescent systems. J. Mol. Evol. 19, 309–321(1983)CrossRefGoogle Scholar
  13. 13.
    Hoffman, R.M.: Imaging tumor angiogenesis with fluorescent proteins. APMIS 112, 441–449 (2004)Google Scholar
  14. 14.
    Hoffman, R.M.: Recent advances on in vivo imaging with fluorescent proteins. Nat. Rev. Cancer 5, 796–806(2005)CrossRefGoogle Scholar
  15. 15.
    Iglesias, P., Costoya, J.A.: A novel BRET-based genetically encoded biosensor for functional imaging of hypoxia. Biosens. Bioelec. 10:3126–30(2009)CrossRefGoogle Scholar
  16. 16.
    Lee, J.Y., Lee, Y.S., Kim, K.L., etal.: A novel chimeric promoter that is highly responsive to hypoxia and metals. Gene Therapy 13, 857–868(2006)CrossRefGoogle Scholar
  17. 17.
    Maxwell, P.H., Wiesener, M.S., Chang, G.W., etal.: The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399, 271–275(1999)CrossRefGoogle Scholar
  18. 18.
    Michelini, E., Mirasoli, M., Karp, M., et al.: Development of a bioluminescence resonance energy-transfer assay for estrogen-like compound in vivo monitoring. Anal. Chem. 76, 7069–7076 (2004)CrossRefGoogle Scholar
  19. 19.
    Ntziachristos, V., Bremer, C., Weissleder, R.: Fluorescence imaging with near-infrared light: new technological advances that enable in vivo molecular imaging. Eur. Radiol. 13, 195–208 (2003)Google Scholar
  20. 20.
    Pfleger, K.D.G., Eidne, K.: Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET). Nat. Methods 3, 165–173(2006)CrossRefGoogle Scholar
  21. 21.
    Prasher, D.C., Eckenrodeb, V.K., Wardc, W.W., etal.: Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111, 229–233(1992)CrossRefGoogle Scholar
  22. 22.
    Prinz, A., Diskar, M., Herberg, F.W.: Application of bioluminescence resonance energy transfer (BRET) for biomolecular interaction studies. Chembiochemistry 7, 1007–1012(2006)CrossRefGoogle Scholar
  23. 23.
    Safran, M., Kim, W.Y., O’Connell, F., etal.: Mouse model for noninvasive imaging of HIF prolyl hydroxylase activity: assessment of an oral agent that stimulates erythropoietin production. Proc. Natl. Acad. Sci. USA 103, 105–110(2006)CrossRefGoogle Scholar
  24. 24.
    Sampath, L., Wang, W., Sevick-Muraca, E.M.: Near infrared fluorescent optical imaging for nodal staging. J. Biomed. Opt. 13, 041312(2008)CrossRefGoogle Scholar
  25. 25.
    Shaner, N.C., Campbell, R.E., Steinbach, P.A., etal.: Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572(2004)Google Scholar
  26. 26.
    Shaner, N.C., Steinbach, P.A., Tsien, R.Y.: A guide to choosing fluorescent proteins. Nature 2, 905–909(2005)Google Scholar
  27. 27.
    Shu, X., Shaner, N.C., Yarbrough, C.A., etal.: Novel chromophores and buried charges control color in mFruits. Biochemistry 45, 9639–9647(2006)CrossRefGoogle Scholar
  28. 28.
    So, M.K., Xu, C., Loening, A.M.: Self-illuminating quantum dot conjugates for in vivo imaging. Nat. Biotechnol. 24, 339–343(2006)CrossRefGoogle Scholar
  29. 29.
    Soltesz, E.G., Kim, S., Kim, S.W., etal.: Sentinel lymph node mapping of the gastrointestinal tract by using invisible light. Ann. Surg. Oncol. 13, 386–96(2006)CrossRefGoogle Scholar
  30. 30.
    Tanaka, E., Choi, H.S., Fujii, H., etal.: Image-guided oncologic surgery using invisible light: Completed pre-clinical development for sentinel lymph node mapping. Ann. Surg. Oncol. 13, 1671–81(2006)CrossRefGoogle Scholar
  31. 31.
    Torigian, D.A., Huang, S.S., Houseini, M.: Functional imaging of cancer with emphasis on molecular techniques. CA Cancer J. Clin. 57, 206–224(2007)CrossRefGoogle Scholar
  32. 32.
    Victor, N., Ivy, A., Jiang, B.H., Agani, F.H.: Involvement of HIF-1 in invasion of Mum2B uveal melanoma cells. Clin. Exp. Metastasis 23, 87–96(2006)CrossRefGoogle Scholar
  33. 33.
    Weissleder, R., Ntziachristos, V.: Shedding light onto live molecular targets. Nat. Medicine 9, 123–128(2003)CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  1. 1.Molecular Oncology Laboratory, Departamento de Fisioloxía, Facultade de MedicinaUniversidade de Santiago de CompostelaGaliciaSpain

Personalised recommendations