In vivo tumor angiogenesis imaging with site-specific labeled 99mTc-HYNIC-VEGF

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 Tc-99m-HYNIC-C-tagged VEGF (Tc-99m-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. Tc-99m-HYNIC-VEGF was prepared using tin/tricine as an exchange reagent, and injected via the tail vein ( 200 - 300 mu Ci, 1 - 2 mu g 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 Tc-99m/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 Tc-99m/biotin-inactivated VEGF, as compared with Tc-99m-HYNIC-VEGF. Conclusion: (99m)Tccan be loaded onto C-tagged VEGF in a site-specific fashion without reducing its bioactivity. Tc-99m-HYNIC-VEGF can be rapidly prepared for the imaging of tumor vasculature and its response to different types of chemotherapy.