Fluorescent Nanobodies for Enhanced Guidance in Digestive Tumors and Liver Metastasis Surgery
EJSO（2025）
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Abstract
Background: Fluorescence molecular imaging, a potent and non-invasive technique, has become indispensable in medicine for visualizing molecular processes. In surgical oncology, it aids treatment by allowing visualization of tumor cells during fluorescence-guided surgery (FGS). Targeting the urokinase plasminogen activator receptor (uPAR), overexpressed during tissue remodeling and inflammation, holds promise for advancing FGS by specifically highlighting tumors. This study explores the extended use of Nanobody-based (Nb) anti-uPAR tracers, evaluating their receptor binding, ability to visualize and demarcate colorectal (CRC) and gastric cancer (GC), and detect localized (PC) and metastatic (PC-M) pancreatic carcinoma. Methods: First, the receptor structure interactions of Nb15, which binds specifically to the human homologue of uPAR, were characterized in vitro to deepen our understanding of these interactions. Subsequently, Nbs 15 and 13-where Nb13 targets the murine uPAR homologue-were labeled with the s775z fluorescent dye and validated in a randomized study in mice (n = 4 per group) using orthotopic human CRC, GC, and PC models, as well as a mouse PC-M model. Results: Nb15, which binds to the D1 domain of uPAR and competes with urokinase's binding fragment, showed rapid and specific tumor accumulation. It exhibited higher tumor-to-background ratios in CRC (3.35 +/- 0.75) and PC (3.41 +/- 0.46), and effectively differentiated tumors in GC (mean fluorescence intensity: 0.084 +/- 0.017), as compared to control Nbs. Nb13 successfully identified primary tumors and liver metastases in PC-M models. Conclusion: The tested fluorescently-labeled anti-uPAR Nbs show significant preclinical and clinical potential for improving surgical precision and patient outcomes, with Nb15 demonstrating promise for real-time surgical guidance.
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Key words
Nanobodies,Urokinase plasminogen activator receptor,Fluorescence-guided surgery,Fluorescence molecular imaging,Liver metastasis
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