Small-Molecule Far-Red Fluorescent Spray-on Probes Targeting Prostatic Acid Phosphatase for Fluorescence-Guided Surgery

Abstract Fluorescence-guided surgery (FGS) is an emerging approach for real-time tumor visualization and surgical margin assessment, yet its application to prostate cancer remains limited by the scarcity of clinically relevant molecular targets and on-demand targeted imaging agents. Most molecularly targeted imaging strategies for prostate cancer have focused on prostate-specific membrane antigen (PSMA), underscoring the need for complementary targets that could enable fluorescence imaging across molecularly distinct disease subtypes. Here, we developed and systematically evaluated three far-red fluorescent probes targeting prostatic acid phosphatase (ACP3) by conjugating distinct fluorophores to a small-molecule ACP3 affinity ligand. We characterized the probes across biochemical, physicochemical, cellular, and tissue-based assays. Although all three probes retained nanomolar ACP3 inhibitor potency, ACP3i-SulfoCy5 exhibited the most favorable overall imaging profile, combining high aqueous solubility, low nonspecific retention, and superior tumor-to-background contrast in cellular and ex vivo tissue assays. In a PC3-ACP3 prostate tumor model, topical application of ACP3i-SulfoCy5 generated high-contrast fluorescence that clearly delineated tumor margins. Collectively, these findings establish ACP3 as a promising complementary target for prostate cancer fluorescence imaging and support further investigation of ACP3i-SulfoCy5 for topical fluorescence-guided imaging during prostate cancer surgery.