Studies On NGR-containing Molecular Probes For Tumor Targeted Multimodal Imaging And Radionuclide Therapy

Objective:Pathological angiogenesis is crucial in tumor growth, invasion and metastasis. The NGR(aspargine-glycine-arginine)-containing peptides can specifically bind to CD13(Aminopeptidase N) receptor which is overexpressed in angiogenic blood vessels and various tumor cells. In this study, monomeric and dimeric NGR peptides were synthesized and labeled with 64 Cu. Tumor-bearing mice were employed to investigate the difference of 64Cu-labeled monomeric and dimeric NGR peptides in biodistribution and tumor imaging. A novel NGR-VEGI fusion protein was produced and labeled with fluorescent dyes Cy5.5 and therapeutic radionuclide Re-188. The NGR-VEGI protein labeled with fluorescent dyes was used for near-infrared fluorescence(NIRF) optical imaging. The NGR-VEGI protein labeled with Re-188 was evaluated for tumor vasculature targeted imaging and therapy. Based on the NGR mediated tumor targeted specificity, these novel probes offer a noninvasive method for early detection of tumor angiogenesis and efficient monitoring of tumor response to anti-angiogenesis therapy. Methods:Western blot analysis and immunofluorescence staining were performed to identify CD13-positive and CD13-negative cell lines. 64Cu-labeled monomeric and dimeric NGR peptides were employed for microPET imaging of CD13 receptor expression in vivo. NGR-containing peptides were conjugated with 1, 4, 7, 10-tetraazadodecane-N, N′, N″, N?-tetraacetic acid(DOTA) and labeled with 64 Cu in ammonium acetate buffer. The resulting monomeric(64Cu-DOTA-NGR1) and dimeric(64Cu-DOTA-NGR2) peptides were then subjected to in vitro stability, cell uptake and efflux, small animal microPET, and biodistribution studies. The NGR–VEGI protein was labeled with a Cy5.5 fluorophore to afford Cy5.5-NGR-VEGI probe. Using the NIRF imaging technique, we visualized and quantified the specific delivery of Cy5.5-NGR-VEGI protein to subcutaneous HT-1080 fibrosarcoma tumors in mouse xenografts. Finally NGR-VEGI fusion protein was prepared and labeled with 188 Re for radioimaging and radiotherapy in mice bearing HT-1080 xenografts. Results:Western blot analysis and immunofluorescence staining results showed that CD13 receptor was highly expressed on HT-1080 colon and negative expression on HT-29 cells. The binding affinity of 64Cu-DOTA-NGR2 to HT-1080 cells was measured to be within nanomolar range and about 2-fold higher than that of 64Cu-DOTA-NGR1. For small animal microPET studies, 64Cu-DOTA-NGR2 displayed more favorable in vivo performance in terms of higher tumor uptake and slower tumor washout in CD13-positive HT-1080 tumor xenografts as compared to 64Cu-DOTA-NGR1. As expected, significantly lower tumor uptake and poorer tumor/normal organ contrast were observed for both 64Cu-DOTA-NGR1 and 64Cu-DOTA-NGR2 in CD13-negative HT-29 tumor xenografts in comparison with those in the HT-1080 tumor xenografts. The CD13-specific tumor activity accumulation of both 64Cu-DOTA-NGR1 and 64Cu-DOTA-NGR2 was further demonstrated by significant reduction of tumor uptake in HT-1080 tumor xenografts with a co-injected blocking dose of cyclic NGR peptide [c(CNGRC)]. The biodistribution results were consistent with the quantitative analysis of microPET imaging. The Cy5.5-NGR-VEGI probe exhibited rapid HT-1080 tumor targeting, and highest tumor-to-background contrast at 8 h post-injection(pi). Tumor specificity of Cy5.5-NGR-VEGI was confirmed by effective blocking of tumor uptake in the presence of unlabeled NGR-VEGI(20 mg/kg). Ex vivo NIRF imaging further confirmed in vivo imaging findings, demonstrating that Cy5.5-NGR-VEGI displayed an excellent tumor-to-muscle ratio(18.93 ± 2.88) at 8 h pi for the non-blocking group and significantly reduced ratio(4.92 ± 0.75) for the blocking group. SPECT imaging results revealed that 188Re-NGR?VEGI exhibits good tumor-to-background contrast in CD13-positive HT-1080 tumor xenografts. The CD13 specificity of 188Re-NGR-VEGI was further verified by significant reduction of tumor uptake in HT-1080 tumor xenografts with coinjection of the non-radiolabeled NGR-VEGI protein. The biodistribution results demonstrated good tumor-to-muscle ratio(4.98 ± 0.25) of 188Re-NGR-VEGI at 24 h, which is consistent with the results from SPECT imaging. For radiotherapy, 18.5 MBq of 188Re-NGR-VEGI showed excellent tumor inhibition effect in HT-1080 tumor xenografts with no observable biological toxicity. Conclusion:Both 64Cu-DOTA-NGR1 and 64Cu-DOTA-NGR2 have good and specific tumor uptake in CD13-positive HT-1080 tumor xenografts. 64Cu-DOTA-NGR2 showed higher tumor uptake and better tumor retention than 64Cu-DOTA-NGR1, presumably due to bivalency effect and increase in apparent molecular size. 64Cu-DOTA-NGR2 is a promising PET probe for noninvasive detection of CD13 receptor expressed tumor. Cy5.5-NGR-VEGI provided highly sensitive and target-specific imaging of CD13 positive expressed tumors. As a novel theranostic protein, Cy5.5-NGR-VEGI has the potential to improve cancer treatment by targeting tumor vasculature. 188Re-NGR-VEGI has the potential as a theranostic agent for CD13-targeted tumor imaging and therapy. These NGR-containning molecular probes could provide a new direction and method for tumor diagnosis and therapy.