Validation of Fluorescent Nanobodies for Visualization of uPAR-expressing Colorectal Tumors and Tracking Liver Metastasis

Lukasz Mateusiak, Eveline de Jonge-Muller, Sarah Hakuno, Sam Floru, AL Vahrmeijer, C.F.M. Sier, Luuk JAC Hawinkels, Sophie Hernot

Research output: Unpublished contribution to conferenceUnpublished abstract

Abstract

Introduction: While traditional surgery remains crucial in cancer treatment, fluorescence-guided surgery (FGS) is gaining prominence. FGS enables the visualization of diseased tissue by targeting specific receptors with fluorescent agents. Targeting the urokinase receptor (uPAR) in FGS shows promise due to its specificity for tissue remodeling. Nanobodies (Nbs), characterized by high target affinity and rapid clearance, offer promise as tracers for FGS. This study assesses the potential of Nb-based anti-uPAR tracers, for visualizing colorectal cancer and detecting pancreatic cancer metastases.
Methods: The interaction of uPAR-targeting Nbs with the receptor structure was first assessed in vitro. Binding to specific receptor domains was investigated using flow cytometry. Subsequently, a surface plasmon resonance competition study was conducted to evaluate if the binding of Nbs was affected by the amino-terminal fragment (ATF) of the natural uPAR ligand, urokinase.
Finally, two high-affinity Nbs, labeled with the s775z fluorescent dye, were tested in vivo. Nb15, meant for clinical translation targeting the human uPAR homologue, was tested in a xenograft orthotopic colorectal cancer (CRC) model. Nb13, targeting the murine uPAR variant, was evaluated in an allograft metastatic pancreatic ductal adenocarcinoma (PDAC) model to compensate for signal loss from the stromal tumor component.
Results/Discussion: The interaction between the tested Nbs and uPAR appears to directly involve amino acids from the D1 domain, evidenced by the loss of binding upon D1 cleavage. Partial competition with ATF, which requires residues from all three receptor domains, indicates a shared binding region and strengthens the speculation that D1 is the sole binding site of the Nbs.
In vivo, both tested fluorescently-labeled Nbs exhibited rapid and specific uptake in primary tumors, providing high contrast one hour after intravenous injection. CRC lesions were clearly demarcated, with the Nb15 producing a significantly higher ex vivo TBR (3.35±0.75) compared to the control Nb (1.88±0.60) (Fig.1A). Additionally, the Nb13 used in the PDAC model, due to the lack of cross-reactivity of Nb15 with the murine uPAR homologue, specifically accumulated in both the primary tumor and distant liver metastases, outperforming a non-targeting control Nb (ex vivo TBR 3.11±1.36 vs. 1.13±0.15) (Fig.1B).
Conclusion: We conclude that receptor-level hindrances do not impede our Nbs' ability to specifically accumulate in cancer lesions and allow fast fluorescence imaging 1 hour post-injection reinforcing uPAR's potential for FGS. The favorable results observed with the leading fluorescently-labeled anti-uPAR Nbs suggest their potential for future preclinical and clinical applications. These Nbs could provide real-time guidance during surgical procedures for a range of cancer models, including more aggressive metastatic ones. Applying these recently developed Nbs bearing a novel fluorophore has the potential to increase the accuracy of surgical tumor resection, thereby potentially improving patient survival rates.
Original languageEnglish
Publication statusPublished - 15 Mar 2024
EventEMIM 2024 - Porto, Portugal
Duration: 12 Mar 202415 Mar 2024

Conference

ConferenceEMIM 2024
Country/TerritoryPortugal
CityPorto
Period12/03/2415/03/24

Keywords

  • nanobodies
  • uPAR
  • Fluorescence-guided surgery

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