A relatively small (approximately 100 kDa) Gd(3+)-based contrast agent, which gives positive contrast on MR images, can be used to detect tumor cell death in vivo, and future derivatives of it may be used to assess early tumor responses to treatment.

Cell death is an important target for imaging the early response of tumors to treatment. We describe here the validation of a phosphatidylserine-binding agent for detecting tumor cell death in vivo based on the C2A domain of synaptotagmin-I. Methods: The capability of nearinfrared fluorophore-labeled and 99m In-labeled derivatives of C2Am for imaging tumor cell death, using planar near-infrared fluorescence imaging and SPECT, respectively, was evaluated in implanted and genetically engineered mouse models of lymphoma and in a human colorectal xenograft. Results: The fluorophorelabeled C2Am derivative showed predominantly renal clearance and high specificity and sensitivity for detecting low levels of tumor cell death (2%-5%). There was a significant correlation (R . 0.9, P , 0.05) between fluorescently labeled C2Am binding and histologic markers of cell death, including cleaved caspase-3, whereas there was no such correlation with a site-directed mutant of C2Am (iC2Am) that does not bind phosphatidylserine. 99m In-C2Am also showed favorable biodistribution profiles, with predominantly renal clearance and low nonspecific retention in the liver and spleen at 24 h after probe administration. 99m In-C2Am generated tumor-to-muscle ratios in drug-treated tumors of 4.3· and 2.2·, respectively, at 2 h and 7.3· and 4.1·, respectively, at 24 h after administration. Conclusion: Given the favorable biodistribution profile of 99m In-labeled C2Am, and their ability to produce rapid and cell death-specific image contrast, these agents have potential for clinical translation.

A 110 kDa (ca. 5 nm in diameter) bivalent paramagnetic nanoprobe for detecting cell death using magnetic resonance imaging (MRI) is described, in which two biotinylated C2A domains of the protein synaptotagmin-I were complexed with a single avidin molecule, which had been labeled with gadolinium chelates. This nanoprobe bound with high affinity and specificity to the phosphatidylserine exposed by dying cells and was demonstrated to allow MRI detection of apoptotic tumor cells in vitro.