Paired Agent Fluorescence Imaging of Cancer in a Living Mouse Using Preassembled Squaraine Molecular Probes with Emission Wavelengths of 690 and 830 nm

New methods are described for the construction of targeted fluorescence probes for imaging cancer and the assessment of tumor targeting performance in a living mouse model. A novel noncovalent assembly process was used to fabricate a set of structurally related targeted fluorescent probes with modular differences in three critical assembly components: the emission wavelength of the squaraine fluorochrome, the number of cRGDfK peptide units that target the cancer cells, and the length of the polyethylene glycol chains as pharmacokinetic controllers. Selective targeting of cancer cells was proven by a series of cell microscopy experiments followed by in vivo imaging of subcutaneous tumors in living mice. The mouse imaging studies included a mock surgery that completely removed a fluorescently labeled tumor. Enhanced tumor accumulation due to probe targeting was first evaluated by conducting Single Agent Imaging (SAI) experiments that compared tumor imaging performance of a targeted probe and untargeted probe in separate mouse cohorts. Although there was imaging evidence for enhanced tumor accumulation of the targeted probe, there was moderate scatter in the data due to tumor-to-tumor variability of the vasculature structure and interstitial pressure. A subsequent Paired Agent Imaging (PAI) study coinjected a binary mixture of targeted probe (with emission at 690 nm) and untargeted probe (with emission at 830 nm) into the same tumor-burdened animal. The conclusion of the PAI experiment also indicated enhanced tumor accumulation of the targeted probe, but the statistical significance was much higher, even though the experiment required a much smaller cohort of mice. The imaging data from the PAI experiment was analyzed to determine the targeted probe’s Binding Potential (BP) for available integrin receptors within the tumor tissue. In addition, pixelated maps of BP within each tumor indicated a heterogeneous spatial distribution of BP values. The results of this study show that the combination of fluorescent probe preassembly and PAI is a promising new way to rapidly develop targeted fluorescent probes for tumors with high BP and eventual use in clinical applications such as targeted therapy, image guided surgery, and personalized medicine.