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


CONCLUSIONS
(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.
Blankenberg
Francis G
FG
Division of Nuclear Medicine/Department of Radiology and MIPS (Molecular Imaging Program at Stanford), Stanford University, Stanford, CA 94304, USA. blankenb@stanford.edu
Backer
Marina V
MV
Levashova
Zoia
Z
Patel
Vimalkumar
V
Backer
Joseph M
JM
D000818:Animals; D045744:Cell Line, Tumor; D005260:Female; D007553:Isotope Labeling; D008325:Mammary Neoplasms, Experimental / Q000098:blood supply / Q000000981:diagnostic imaging* / Q000378:metabolism*; D008657:Metabolic Clearance Rate; D051379:Mice; D008807:Mice, Inbred BALB C; D009389:Neovascularization, Pathologic / Q000000981:diagnostic imaging* / Q000378:metabolism*; D009928:Organ Specificity; D011877:Radionuclide Imaging; D019275:Radiopharmaceuticals / Q000138:chemical synthesis / Q000493:pharmacokinetics; D014018:Tissue Distribution; D042461:Vascular Endothelial Growth Factor A / Q000737:chemistry / Q000493:pharmacokinetics*
D019275:Radiopharmaceuticals; D042461:Vascular Endothelial Growth Factor A; C520135:technetium 99m HYNIC-vascular endothelial growth factor