PURPOSE: Complete and minimally invasive cancer surgery remains challenging. Targeting the fibroblast activation protein (FAP) offers valuable opportunities for surgical planning, intraoperative guidance and improved resection outcomes. Herein, we developed the first dimeric, dual-modality FAP-targeted imaging agents and investigated the influence of different near-infrared cyanine-7 dyes on their final properties. METHODS: Four dual-modality ligands based on the Fusarinine C scaffold were synthesized. Their FAP specificity and retention were evaluated in cellular and xenograft tumor models. The most promising candidates were labelled with 67/68Ga and assessed in vivo at early time points by PET/CT imaging and by comparative SPECT/CT and NIR fluorescence imaging (FI) up to two days post-injection. RESULTS: Distinct fluorophore influences on the properties of the final compounds were identified. The introduction of the s775z dye demonstrated a beneficial effect on the cellular uptake and on the in vivo biodistribution profile as revealed by the greatest improvement in blood clearance and the least off-target accumulation in liver and kidneys when compared to the control and to the other candidates respectively. Ex vivo experiments and in vivo PET/CT, SPECT/CT and FI studies in xenografted mice confirmed these findings and demonstrated sustained tumor uptake (> 7% ID/g and > 5% ID/g at 1 h and 1 day p.i. respectively) for 67Ga-s775z-FFAPi and 67Ga-IRDye-FFAPi. CONCLUSIONS: In this study we introduced and evaluated novel dimeric FAP-targeting agents for dual-modality applications. In the preclinical setting, within the group of compounds investigated, two candidates enabled tumor visualization through PET, SPECT and optical imaging, providing satisfactory background contrast after a single administration and supporting their potential for preoperative nuclear imaging and subsequent fluorescence-guided surgery.

Czech Advanced Technology and Research Institute Palacký University 77900 Olomouc Czech Republic

Department of Chemistry and Biochemistry University of Notre Dame 46556 Notre Dame Indiana USA

Department of Nuclear Medicine Medical University of Innsbruck 6020 Innsbruck Austria

Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacky University 77900 Olomouc Czech Republic

Institute of Pharmacy Department of Pharmaceutical Chemistry University of Innsbruck 6020 Innsbruck Austria

Institute of Physiology Medical University of Innsbruck 6020 Innsbruck Austria

University Hospital Olomouc 77900 Olomouc Czech Republic

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