Abstract 4934: Imaging and quantification of bombesin receptor expression in vivo using a NIR-labeled bombesin peptide

Gastrin-releasing peptide (GRP) is one of the mammalian analogs of bombesin-like peptides that function as growth factors in normal and neoplastic tissues upon binding to a family of bombesin-binding G protein-coupled receptors. Bombesin receptors are overexpressed in a variety of cancers, particularly prostate, breast, and gastrointestinal stromal tumors, and as such have been used to develop radiolabeled imaging tracers. As a pre-clinical alternative to using radiolabeled ligands, we developed a novel near infrared (NIR) fluorescent agent, BombesinRSense 680 (BRS680), designed to target and quantify bombesin receptors in vivo. BRS680 was produced from a modified 7 amino-acid GRP analog peptide labeled with a NIR fluorophore (ex/em 660/680 nm) and a pharmacokinetic modifier designed to improve its plasma availability (plasma t1/2 = 1.5 hours). In vitro labeling of human colonic adenocarcinoma HT-29 cells, which express GRP receptors, and blocking the signal by addition of unlabeled native bombesin, demonstrated the specificity of the agent by both fluorescence microscopy and flow cytometry. In vivo receptor expression was quantified by fluorescence tomography after BRS680 (2 nmol/mouse) was injected intravenously into nude mice bearing HT-29 tumor xenografts. HT-29 tumors showed a high level of receptor expression with approximately 30 pmol (1.5% injected dose) of BRS680 quantified in the tumors at 24 hours, and lower fluorescence in other tissues except for pancreas, a tissue known for high receptor expression, and kidneys, indicating renal clearance. In contrast to the fast clearance from circulation, the tumor tissue half-life of BRS680 was shown to be approximately 42 hours. In vivo targeting specificity was confirmed by collecting tumor tissue from injected mice and co-localizing BRS680 fluorescent signal with an anti-GRP receptor antibody on frozen sections. More importantly, treatment of HT-29 tumor-bearing mice with a tumor growth-arresting chemotherapy regime decreased in vivo BRS680 signal. Six days after beginning treatment with 5-fluorouracil and oxaliplatin in mice with established tumors, BRS680 fluorescent signal was significantly decreased in treated mice as compared to control mice (21.55 + 4.89 versus 34.10 + 2.90 pmoles, p=0.043) paralleling the inhibition of tumor growth (74.25 + 7.65 versus 141 + 19.39 mm3, p=0.003). Interestingly, chemotherapy did not consistently affect the fluorescent signal associated with ProSense 750 FAST, an ag