Far-Red Spray-On Imaging Probes for FAP-Targeted Cancer Surgery

Fluorescence-guided surgery (FGS) using fluorescent imaging probes is an emerging technology that enables tumor margin detection at potentially higher accuracy than is possible with traditional means. Most FGS imaging probes are administered systemically, many hours before a planned surgery, and must have cleared the bloodstream once surgery is initiated, so as not to contaminate the operative field. Topical administration of specially designed conjugates in the form of spray-on probes (SOP) offers the advantage of much lower doses, on-demand use, and result in far higher achievable image contrast. Here we report the discovery and characterization of a far-red SOP imaging agent targeting fibroblast activation protein alpha. We show that only 1 out of 10 synthesized probes has appropriate imaging and biological characteristics, increasing tumor-to-background ratio by 5-15 fold. The optimal SOP (FTF-Cy5) was able to delineate tumor margins rapidly (<10 min) when sprayed on at a low dose (5 μM) followed by a short washing step. These results suggest that the lead far-red SOP has the potential to be utilized as a spray-on intraoperative tumor margin delineation tool using far-red fluorescence imaging systems in the operating room, thereby simplifying surgical workflows and enabling more complete cancer resections in the future.