Radionuclides Optical Imaging And Molecular Imaging Of Melanoma Research

Molecular imaging is a relatively new yet fast growing research discipline. Practical molecular imaging enables researchers to study diseases non-invasively in living subjects at the molecular level. Numerous studies have demonstrated that molecular imaging techniques play a central role in the era of personalized medicine. A variety of imaging modalities have been developed that provide functional and anatomical information of diseases in living small animals and patients. Such modalities include positron emission tomography (PET), single photon emission computed tomography (SPECT), optical imaging (OI, bioluminescence and fluorescence), magnetic resonance imaging (MRI), ultrasound (US), and computed tomography (CT).OI has rapidly gained popularity in molecular imaging. It is an inexpensive imaging technique due to the low costs of detection devices. The technique is easy to learn and use and interpretation of the images is generally straightforward. Imaging in the near-infrared (NIR) region has advantages of improved tissue penetration and low tissue autofluorescence which enhances target to background ratios. While OI generally detects low energy light (visible or near-infrared light) emitted from bioluminescence or fluorescence probes, radioactive molecular probes are traditionally imaged with PET, SPECT or gamma (y) cameras that detect high energy y rays. We hypothesized that radionuclide radiation in the low energy window of light (1.2-3.1 eV, 400-1000 nm) could be imaged using OI techniques and be especially valuable for molecular OI. Thus we examined a variety of radionuclides with different types of emission properties (β+,β- orγ) using a commercially available OI instrument for in vitro and in vivo optical imaging.In this research, we have evaluated three radionuclides (18F,131I, and 90Y) for small animal radioactive OI because of their important roles in nuclear medicine.18F is the most often used PET radionuclide, and 131I and 90Y are two most widely used radionuclides for radiotherapy. In vivo optical images with reasonable sensitivity can be quickly obtained for all three radionuclides. These encouraging results suggest that the radioactive OI can be a powerful tool for fast preliminary evaluation of 18F,131I, and 90Y labeled compounds. This will be important for 90Y based agents development, since it has been relatively difficult to obtain in vivo information for an 90Y agent through non-invasive imaging method. Compared to conventional fluorescence and bioluminescence imaging, radioactive OI has some unique properties. It has wide emission spectrum as demonstrated here, so that a radioactive probe can be monitored at different wavelengths. More importantly, radioactive OI does not require excitation light, which is a significant advantage over traditional OI. The radioactive OI signal generated by a radioactive probe is constitutive, which is very different from fluorescence and bioluminescence probes. The radioactive OI can be performed by monitoring spectral windows that differ from typical FLuc spectrum. Therefore, it is possible to perform BLI and radioactive OI in the same animal with proper emission filters.The results presented here bridge the subfields of imaging by visualizing radioactive probes with OI. Our study demonstrates the feasibility of molecular imaging of living subjects using OI modalities in conjunction with a wide diversity of radioactive probes. It provides a new molecular imaging strategy and will likely have significant impact on both small animal and clinical imaging. Multivalent peptides have been explored as a useful strategy to construct molecular imaging probes and drug delivery carriers. It is generally accepted that multivalency has advantages over monovalency for improving binding affinity, activity and even in vivo performance of biomolecule. The alpha-melanocyte-stimulating hormone (a-MSH) receptor (melanocortin type 1 receptor, or MC1R) has been found to be over-expressed in most murine and human melanoma. a-MSH analogs tagged with radionuclides have been demonstrated to be a class of promising agents for melanoma imaging and/or peptide receptor-targeted radionuclide therapy.Herein by using multivalent a-melanocyte stimulating hormone (a-MSH) analogs, B16F10 melanoma-bearing mice and microPET imaging technology, we systematically investigated the influence of multivalent effect on a-MSH analogs'binding affinity and in vivo melanoma targeting profiles.Three a-MSH analogs named as MSH1, MSH2 and MSH4 were designed and synthesized, which contained one, two or four valency of an a-MSH core sequence, His-d, Phe-Arg-Trp, respectively (Fig. a). a-MSH analog tetramer was constructed using the multiple antigenic peptide (MAP) scaffold.1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the lysine residue of peptides for radiolabeling with a PET radioisotope,64Cu. In vitro binding affinity assays were performed with B16F10 melanoma cell line. After radiolabeling with 64Cu, the in vivo performances of the peptides were evaluated in subcutaneous B16F10 melanoma xenografted mice by micro-PET imaging followed by biodistribution studies.In the receptor competition binding assays, DOTA-MSH4 showed highest binding affinity (IC50= 1.00 nM) which is consistent with its highest ligand density. However, in vivo study demonstrated poor performance of MSH4 as an imaging agent due to its lowest tumor uptake and highest kidney accumulation, In comparison, DOTA-MSH2 displayed medium affinity (IC50=2.06 nM), yet highest tumor uptake and lowest kidney accumulation (Fig. b). Further blocking study of DOTA-MSH2 confirmed its tumor targeting specificity in vivo.Multivalency effects have complex impact to peptides' in vivo behaviors. Eventhough MSH tetramer shows the higher binding affinities in vitro, the better in vivo tumor targeting ability is achieved by MSH dimer. Cu-64 labeled dimeric DOTA-MSH2 has been identified as an ideal melanoma PET imaging probe.