Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Molecular imaging of VEGF receptors in angiogenic vasculature with single-chain VEGF-based probes


We describe a new generation of protein-targeted contrast agents for multimodal imaging of the cell-surface receptors for vascular endothelial growth factor (VEGF). These receptors have a key role in angiogenesis and are important targets for drug development. Our probes are based on a single-chain recombinant VEGF expressed with a cysteine-containing tag that allows site-specific labeling with contrast agents for near-infrared fluorescence imaging, single-photon emission computed tomography or positron emission tomography. These probes retain VEGF activities in vitro and undergo selective and highly specific focal uptake into the vasculature of tumors and surrounding host tissue in vivo. The fluorescence contrast agent shows long-term persistence and co-localizes with endothelial cell markers, indicating that internalization is mediated by the receptors. We expect that multimodal imaging of VEGF receptors with these probes will be useful for clinical diagnosis and therapeutic monitoring, and will help to accelerate the development of new angiogenesis-directed drugs and treatments.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Site-specific modifications do not affect binding and internalization of scVEGF.
Figure 2: NIRF imaging with scVEGF/Cy and localization of Cy5.5 on histological sections.
Figure 3: Blood clearance, biodistribution and SPECT imaging of scVEGF/Tc.
Figure 4: Blood clearance, biodistribution and PET imaging of scVEGF/Cu.


  1. Miller, J.C. et al. Imaging angiogenesis: applications and potential for drug development. J. Natl. Cancer Inst. 97, 172–187 (2005).

    Article  CAS  PubMed  Google Scholar 

  2. Haubner, R. & Wester, H.J. Radiolabeled tracers for imaging of tumor angiogenesis and evaluation of anti-angiogenic therapies. Curr. Pharm. Des. 10, 1439–1455 (2004).

    Article  CAS  PubMed  Google Scholar 

  3. Wu, Y. et al. microPET imaging of glioma integrin αvβ3 expression using (64)Cu-labeled tetrameric RGD peptide. J. Nucl. Med. 46, 1707–1718 (2005).

    CAS  PubMed  Google Scholar 

  4. Ferrara, N., Gerber, H.P. & LeCouter, J. The biology of VEGF and its receptors. Nat. Med. 9, 669–676 (2003).

    Article  CAS  PubMed  Google Scholar 

  5. Manley, P.W. et al. Advances in the structural biology, design and clinical development of VEGF-R kinase inhibitors for the treatment of angiogenesis. Biochim. Biophys. Acta 1697, 17–27 (2004).

    Article  CAS  PubMed  Google Scholar 

  6. Couffinhal, T. et al. Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in normal and artherosclerotic human arteries. Am. J. Pathol. 150, 1673–1685 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Koukourakis, M.I. et al. Vascular endothelial growth factor/KDR activated microvessel density versus CD31 standard microvessel density in non-small cell lung cancer. Cancer Res. 60, 3088–3095 (2000).

    CAS  PubMed  Google Scholar 

  8. Witmer, A.N. et al. Expression of vascular endothelial growth factor receptors 1, 2, and 3 in quiescent endothelia. J. Histochem. Cytochem. 50, 767–777 (2002).

    Article  CAS  PubMed  Google Scholar 

  9. Vajkoczy, P. et al. Microtumor growth initiates angiogenic sprouting with simultaneous expression of VEGF, VEGF receptor-2, and angiopoietin-2. J. Clin. Invest. 109, 777–785 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Bikfalvi, A. et al. Interaction of vasculotropin/vascular endothelial cell growth factor with human umbilical vein endothelial cells: binding, internalization, degradation, and biological effect. J. Cell. Physiol. 149, 50–59 (1991).

    Article  CAS  PubMed  Google Scholar 

  11. Li, S. et al. Characterization of 123I-vascular endothelial growth factor-binding sites expressed on human tumour cells: possible implication for tumour scintigraphy. Int. J. Cancer 91, 789–796 (2001).

    Article  CAS  PubMed  Google Scholar 

  12. Li, S. et al. Imaging gastrointestinal tumours using vascular endothelial growth factor-165 (VEGF165) receptor scintigraphy. Annals Oncol. 14, 1274–1277 (2003).

    Article  CAS  Google Scholar 

  13. Li, S. et al. Iodine-123-vascular endothelial growth factor-165 (123I-VEGF165). Biodistribution, safety and radiation dosimetry in patients with pancreatic carcinoma. Q. J. Nucl. Med. Mol. Imag. 48, 198–206 (2004).

    CAS  Google Scholar 

  14. Lu, E. et al. Targeted in vivo labeling of receptors for vascular endothelial growth factor: approach to identification of ischemic tissue. Circulation 108, 97–103 (2003).

    Article  CAS  PubMed  Google Scholar 

  15. Chan, C. et al. A human transferrin(hnTf)-VEGF fusion protein containing an integrated binding site for 111In for imaging tumor angiogenesis. J. Nucl. Med. 46, 1745–1752 (2005).

    CAS  PubMed  Google Scholar 

  16. Blankenberg, F.G. et al. Tumor imaging using a standardized radiolabeled adapter protein docked to vascular endothelial growth factor (VEGF). J. Nucl. Med. 45, 1373–1380 (2004).

    CAS  PubMed  Google Scholar 

  17. Backer, M.V. et al. Vascular endothelial growth factor selectively targets boronated dendrimers to tumor vasculature. Mol. Cancer Ther. 4, 1423–1429 (2005).

    Article  CAS  PubMed  Google Scholar 

  18. Blankenberg, F.G. et al. In vivo tumor angiogenesis imaging with site-specific labeled 99mTc-HYNIC-VEGF. Eur. J. Nucl. Med. Mol. Imag. 33, 841–848 (2006).

    Article  Google Scholar 

  19. Backer, M.V., Patel, V., Jehning, B.T., Claffey, K. & Backer, J.M. Surface immobilization of active vascular endothelial growth factor via a cysteine-containing tag. Biomaterials 27, 5452–5458 (2006).

    Article  CAS  PubMed  Google Scholar 

  20. Backer, M.V. & Backer, J.M. Targeting endothelial cells overexpressing VEGFR-2: selective toxicity of shiga-like toxin-VEGF fusion proteins. Bioconjugate Chem. 12, 1066–1073 (2001).

    Article  CAS  Google Scholar 

  21. Backer, M.V. & Backer, J.M. Functionally active VEGF fusion proteins. Prot. Exp. Purif. 23, 1–7 (2001).

    Article  CAS  Google Scholar 

  22. Heppner, G.H. et al. Nontransgenic models of breast cancer. Breast Cancer Res. 2, 331–334 (2000).

    Article  CAS  PubMed  Google Scholar 

  23. Li, C.Y. et al. Initial stages of tumor cell-induced angiogenesis: evaluation via skin window chambers in rodent models. J. Natl. Cancer Inst. 92, 143–147 (2000).

    Article  CAS  PubMed  Google Scholar 

  24. Ono, M. et al. Intracellular metabolic fate of radioactivity after injection of technetium-99m-labeled hydrazino nicotinamide derivatized proteins. Bioconjugate Chem. 10, 386–394 (1999).

    Article  CAS  Google Scholar 

  25. Ono, M. et al. Control of radioactivity pharmacokinetics of 99mTc-HYNIC-labeled polypeptides derivatized with ternary ligand complexes. Bioconjugate Chem. 13, 491–501 (2002).

    Article  CAS  Google Scholar 

  26. Zhang, W. et al. A monoclonal antibody that blocks VEGF binding to VEGFR2 (KDR/Flk-1) inhibits vascular expression of Flk-1 and tumor growth in an orthotopic human breast cancer model. Angiogenesis 5, 35–44 (2002).

    Article  CAS  PubMed  Google Scholar 

  27. Lee, S. et al. Processing of VEGF-A by matrix metalloproteinases regulates bioavailability and vascular patterning in tumors. J. Cell Biol. 169, 681–691 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Jain, R.K. Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. Science 307, 58–62 (2005).

    Article  CAS  PubMed  Google Scholar 

  29. McCarty, M.F. et al. Promises and pitfalls of anti-angiogenic therapy in clinical trials. Trends Mol. Med. 9, 53–58 (2003).

    Article  CAS  PubMed  Google Scholar 

  30. Kerbel, R.S. & Kamen, B.A. The anti-angiogenic basis of metronomic chemotherapy. Nat. Rev. Cancer 4, 423–436 (2004).

    Article  CAS  PubMed  Google Scholar 

  31. Jain, R.K. et al. Lessons from phase III clinical trials on anti-VEGF therapy for cancer. Nature Clin. Practice Onc 3, 24–40 (2006).

    Article  CAS  Google Scholar 

Download references


We thank J. Pizzonia (KODAK Molecular Imaging Systems) for help with NIRF imaging; K. Tracht (Olympus America) for help with microscopy; P.T. Pienkos (Molecular Logix) for codon optimized human EGF; and R. Barth (The Ohio State University) for F98-EGFR(F) cells. This work was supported in part by NIH grants R43 CA113080 and R21 EB001946 to J.M.B., NIH 1 P50 CA114747 to F.G.B., and by support from NIH CA064436 and the Patrick and Catharine Weldon Donaghue Foundation to K.P.C.

Author information

Authors and Affiliations



M.V.B. and Z.L. contributed equally to this manuscript. M.V.B., F.G.B. and J.M.B. conceived of and initiated the project, coordinated discovery research and wrote the manuscript; M.V.B. designed imaging conjugates, validated all probes in tissue culture and performed colocalization studies; M.V.B. and J.M.B. designed and conducted optical imaging experiments; Z.L. and F.G.B. prepared radiolabeled SPECT and PET probes and performed all experiments with these probes; V.P. and B.T.G. made scVEGF-based conjugates; and K.C. performed confocal microscopy experiments. All authors discussed and commented on the manuscript.

Corresponding author

Correspondence to Joseph M Backer.

Ethics declarations

Competing interests

M.V.B. and J.M.B. own equity in privately held SibTech Inc.

Supplementary information

Supplementary Fig. 1

Scatchard analysis of scVEGF/Cu binding to VEGFR-2 on 293/KDR cells. (PDF 88 kb)

Supplementary Fig. 2

Long-term retention of Cy5.5 after imaging with scVEGF/Cy. (PDF 123 kb)

Supplementary Fig. 3

NIRF imaging with scVEGF/Cy in MDA-231luc orthotopic tumor model. (PDF 100 kb)

Supplementary Fig. 4

Functionally active Cys-EGF/Cy accumulates in tumor area, but does not co-localize with endothelial cells. (PDF 139 kb)

Supplementary Fig. 5

Stability of radionuclide-loaded scVEGF probes in vivo (PDF 130 kb)

Supplementary Fig. 6

SPECT imaging of 4T1luc tumor bearing mice. (PDF 170 kb)

Supplementary Fig. 7

scVEGF/Cu stability in murine plasma ex vivo. (PDF 110 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Backer, M., Levashova, Z., Patel, V. et al. Molecular imaging of VEGF receptors in angiogenic vasculature with single-chain VEGF-based probes. Nat Med 13, 504–509 (2007).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing