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Tumor growth, local invasion, and metastatic dissemination are dependent on the formation of new microvessels. Angiogenesis is therefore a crucial event in tumor progression. In recent years anti-angiogenic agents have been developed as a novel approach to cancer treatment. Successful intervention with tumor angiogenesis can induce tumor vasculature regression, leading to a complete cessation of tumor growth. Clinically, however, anti-angiogenesis inhibitors have been used with marginal success. For the development of novel effective anti-angiogenic therapies it is of crucial interest, therefore, to be able to screen new treatments for both the effects on the tumor vasculature as well as the tumor burden itself. We have engineered the murine breast cancer cell line 4T1 to stably express the luciferase gene of the North American firefly. This allowed us to visualize tumor burden by in vivo bioluminescence imaging. The 4T1 mouse mammary carcinoma is derived from Balb/c mice and very closely models advanced stage (stage IV) human breast cancer in immunogenicity, metastatic properties and growth characteristics. Additionally, we have developed the adapter/docking tag system based on interactions between an 18–127 amino acid fragment of human RNase I and a 1–15 amino acid fragment of RNase I fused to a targeting protein. To visualize angiogenesis we applied to the docking system labeling vascular endothelial growth factor with 99mTc in 4T1 breast cancer tumor-bearing mice. In preliminary studies we were able to detect neovascularization in mouse breast cancer tumor nodules as small as 2–3 mm in diameter. We found that the 99mTc-labeled vascular endothelial growth factor complexes selectively and specifically bound to tumor neovasculature. We expect that 99mTc-Adapter, a broadly applicable and general humanized radionuclide imaging 'payload' module, can be readily employed for a non-destructive labeling of many targeting proteins armed with the docking tag. Availability of multiple imaging proteins might have tremendous implications for the development and evaluation of novel anti-cancer and, specifically, anti-angiogenic therapies.

Imaging angiogenesis in early stages of breast cancer using a standardized radiolabeled adapter protein docked to vascular endothelial growth factor