Introduction: The increasing focus on developing tumor-targeted near-infrared fluorescent (NIRF) probes aims to assist surgeons in detecting and removing malignant lesions during surgeries, enhancing surgical outcomes, and improving patient prognosis. Fibroblast activation protein (FAP), highly expressed in cancer-associated fibroblasts, presents in most epithelial solid tumors while being minimally expressed in healthy tissue. FAP inhibitors based on small-molecules were reported as powerful imaging probes for many cancer types due to their rapid uptake and impressive tumor-to-background ratios (TBRs).[1] This highlights the potential of FAP in the application of fluorescent-guided surgery (FGS)[2], a technique that aids in the complete resection of tumors, including the tumor stroma. The stroma, an often overlooked component of tumors, its complete removal is vital for successful cancer treatment. To explore the possibility of using FAP-mediated FGS, we recently developed three fluorescent probes based on a quinoline core structure with enhanced pharmacokinetics, possessing the capability to image a broad range of cancer types. We aim to characterize their pharmacology through complementary in vitro and in vivo studies and to evaluate their suitability as NIRF probes for FGS.

Method: In this study, three novel FAP-targeted probes, eFAP-17, eFAP-24, and eFAP-27, were designed and synthesized. These probes consisted of a (4-quinolinoyl)-glycyl-2-cyanopyrrolidine (QCP) conjugated to the clinically approved NIRF dye IRDye800CW. Direct coupling of IRDye800CW onto the QCP scaffold resulted in eFAP-17, while eFAP-24 and eFAP-27 incorporated hydrophilic linkers between the QCP and the dye. Enzymatic assays against human/murine FAP, prolyl oligopeptidase (PREP) and dipeptidyl peptidase-4 (DPP4) were used to determine inhibition activity and specificity. Stability was monitored by high-performance liquid chromatography after incubating the eFAPs in PBS buffer, mouse serum (MS) and human serum (HS). Cell uptake experiments on cell lines displaying varying FAP expression levels (FAP+: U87; FAP-: U2OS) were performed to quantify uptake and specificity.[3] Subsequently, the binding efficacy of eFAP-17 and eFAP-24 was evaluated in a relevant pancreatic ductal adenocarcinoma (PDAC) model in tumor-bearing mice. This model involved co-culturing patient-derived organoid pancreatic stellate cells (hPS1) with a PDAC cell line (BxPC-3), mimicking the tumor-fibroblast interaction observed in PDAC [4]. After probe injection, NIRF imaging of tumors was performed at various time points using a clinical Quest Spectrum NIRF imaging system, followed by tracer biodistribution analysis of tumors and organs.

Results: All three IRDye800CW-equipped eFAP probes were successfully synthesized. They exhibited nanomolar inhibition of both human and murine FAP (IC50, human FAP < 6 nM, IC50, murine FAP < 5 nM), as well as high selectivity to FAP, as confirmed by their low affinity against PREP (IC50 0.32-2.00 µM) and DPP4 (IC50 in the low millimolar range). Additionally, all three eFAPs showed high stability in PBS, MS, and HS for up to 4 hours, with less than 5% degradation. Remarkably, eFAP-24 displayed the highest stability, retaining >97% in PBS, MS, and HS after 24 hours. All probes showed specific uptake in FAP-positive glioblastoma (U87) cells, with eFAP-24 exhibiting the highest uptake (27.57 ± 2.02% AD) compared to eFAP-17 (3.55 ± 0.42% AD) and eFAP-27 (6.54 ± 0.81% AD). In optical imaging studies, eFAP-24 exhibited a relatively high mean TBR of 3.1 ± 0.6 after 24h post-injection, allowing for clear tumor boundaries identification. Biodistribution analyses at 48h post-injection revealed high fluorescence signal in the tumors and minimal fluorescence signal in all other organs, with increased signal observed only in the liver and kidneys, reflecting the clearance pathway of eFAPs.

Conclusions: Our novel FAP-targeted NIRF probe, eFAP-24, showed a high binding affinity, selectivity, good stability in vitro, and provided clear tumor delineation at 24h post-injection in vivo. The promising results demonstrate the potential of eFAP-24 for fluorescence-guided solid tumor surgery.

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