In vivo tumor angiogenesis imaging with site-specific labeled (99m)Tc-HYNIC-VEGF

Eur J Nucl Med Mol Imaging. 2006 Jul;33(7):841-8. doi: 10.1007/s00259-006-0099-1.


Purpose: We recently developed a cysteine-containing peptide tag (C-tag) that allows for site-specific modification of C-tag-containing fusion proteins with a bifunctional chelator, HYNIC (hydrazine nicotinamide)-maleimide. We then constructed and expressed C-tagged vascular endothelial growth factor (VEGF) and labeled it with HYNIC. We wished to test (99m)Tc-HYNIC-C-tagged VEGF ((99m)Tc-HYNIC-VEGF) for the imaging of tumor vasculature before and after antiangiogenic (low continuous dosing, metronomic) and tumoricidal (high-dose) cyclophosphamide treatment.

Methods: HYNIC-maleimide was reacted with the two thiol groups of C-tagged VEGF without any effect on biologic activity in vitro. (99m)Tc-HYNIC-VEGF was prepared using tin/tricine as an exchange reagent, and injected via the tail vein (200-300 microCi, 1-2 microg protein) followed by microSPECT imaging 1 h later.

Results: Sequencing analysis of HYNIC-containing peptides obtained after digestion confirmed the site-specific labeling of the two accessible thiol groups of C-tagged VEGF. Tumor vascularity was easily visualized with (99m)Tc/VEGF in Balb/c mice with 4T1 murine mammary carcinoma 10 days after implantation into the left axillary fat pad in controls (12.3+/-5.0 tumor/bkg, n=27) along with its decrease following treatment with high (150 mg/kg q.o.d. x 4; 1.14+/-0.48 tumor/bkg, n=9) or low (25 mg/kg q.d. x 7; 1.03+/-0.18 tumor/bkg, n=9) dose cyclophosphamide. Binding specificity was confirmed by observing a 75% decrease in tumor uptake of (99m)Tc/biotin-inactivated VEGF, as compared with (99m)Tc-HYNIC-VEGF.

Conclusion: (99m)Tc can be loaded onto C-tagged VEGF in a site-specific fashion without reducing its bioactivity. (99m)Tc-HYNIC-VEGF can be rapidly prepared for the imaging of tumor vasculature and its response to different types of chemotherapy.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Cell Line, Tumor
  • Female
  • Isotope Labeling
  • Mammary Neoplasms, Experimental / blood supply
  • Mammary Neoplasms, Experimental / diagnostic imaging*
  • Mammary Neoplasms, Experimental / metabolism*
  • Metabolic Clearance Rate
  • Mice
  • Mice, Inbred BALB C
  • Neovascularization, Pathologic / diagnostic imaging*
  • Neovascularization, Pathologic / metabolism*
  • Organ Specificity
  • Radionuclide Imaging
  • Radiopharmaceuticals / chemical synthesis
  • Radiopharmaceuticals / pharmacokinetics
  • Tissue Distribution
  • Vascular Endothelial Growth Factor A / chemistry
  • Vascular Endothelial Growth Factor A / pharmacokinetics*


  • Radiopharmaceuticals
  • Vascular Endothelial Growth Factor A
  • technetium 99m HYNIC-vascular endothelial growth factor