Fluorescence molecular imaging (FMI) is becoming a powerful tool to improve surgical precision and diagnostics. Although significant advances have been made in imaging technology and contrast agents, debate continues regarding suitable standards for FMI. Standardizing all FMI aspects is crucial for adoption in clinical procedures and routine practice. Currently, the lack of clear-cut guidelines on FMI study reporting complicates comparison between studies and impedes standardization. This work presents community-driven REFLECT guidelines for FMI study reporting for fluorescence-guided surgery and interventions, covering preclinical, translational, and clinical studies. It aims to improve quality of reporting, enable comparison between studies, and enhance interpretation of results. The resulting structured checklist encompasses all essential details for reporting on the contrast agent, imaging device, imaging protocol, and image processing and analysis methods. Adoption of this framework is encouraged to enhance reproducibility and establish alignment with standardization efforts in the field - thus fostering advancements in FMI.

Fluorescence molecular imaging plays a vital role in image‐guided surgery. In this context, the urokinase plasminogen activator receptor (uPAR) is an interesting biomarker enabling the detection and delineation of various tumor types due to its elevated expression on both tumor cells and the tumor microenvironment. In this study, anti‐uPAR Nanobodies (Nbs) are generated through llama immunization with human and murine uPAR protein. Extensive in vitro characterization and in vivo testing with radiolabeled variants are conducted to assess their pharmacokinetics and select lead compounds. Subsequently, the selected Nbs are converted into fluorescent agents, and their application for fluorescence‐guided surgery is evaluated in various subcutaneous and orthotopic tumor models. The study yields a panel of high‐affinity anti‐uPAR Nbs, showing specific binding across multiple types of cancer cells in vitro and in vivo. Lead fluorescently‐labeled compounds exhibit high tumor uptake with high contrast at 1 h after intravenous injection across all assessed uPAR‐expressing tumor models, outperforming a non‐targeting control Nb. Additionally, rapid and accurate tumor localization and demarcation are demonstrated in an orthotopic human glioma model. Utilizing these Nbs can potentially enhance the precision of surgical tumor resection and, consequently, improve survival rates in the clinic.

Complexation of radiometals by chelators allows for convenient radiolabeling of molecules of interest for the preparation of radiopharmaceuticals. In the chelator family, triazacyclononane (TACN)-based macrocycles have been used ubiquitously over the last 40 years, and many bifunctional derivatives have been developed. Despite this diversity, researchers commonly make their chelator selection based on practical factors like (commercial) availability and compatibility with the desired radionuclide, acknowledging that these considerations often outweigh achieving ideal in vivo pharmacokinetics. In this study, we generated and preclinically evaluated four gallium-68-labeled anti-CEA Nanobody-based tracers carrying different TACN-derivatives: p-NCS-Bn-NOTA, p-NCS-Bn-NODAGA, NODAGA-Sq, and NODAGA-NHS. Since the macrocyclic chelator is highly similar in these derivativesthey all present a NOTA ((1,4,7-triazacyclononane-1,4,7-triacetic acid) scaffoldthe effect of the bioconjugation handle on the pharmacokinetic properties could be examined in a side-by-side comparison. The four PET tracers were prepared and could easily be labeled with gallium-68. Then, their stability, target affinity, and hydrophilic character were determined in vitro. Next, their in vivo biodistribution was evaluated using PET/CT imaging in a subcutaneous tumor mouse model. Despite their high structural similarity, notable differences in pharmacokinetics were observed in vivo, more specifically in the tumor and liver signal. Tracer [⁶⁸Ga]­Ga-NODAGA-Sq-NbCEA was found to be the best performer in our study, with a tumor signal 1.6-fold higher than [⁶⁸Ga]­Ga-NODAGA-NbCEA and a tumor-to-liver ratio 1.5-fold and 1.7-fold higher than that of [⁶⁸Ga]­Ga-NOTA-Bn-NCS-NbCEA and [⁶⁸Ga]­Ga-NODAGA-Bn-NCS-NbCEA, respectively. By comparing this tracer to the negative control [⁶⁸Ga]­Ga-NODAGA-Sq-R3b23, specific tumor targeting was demonstrated.

Despite the increasing clinical adoption of molecular fluorescence-guided surgery (FGS), the limited tumour specificity of currently available contrast agents remains a challenge for accurate tumour identification. This study aimed to validate a single-domain antibody (sdAb)-based tracer specifically targeting the human folate receptor alpha (hFRα) and evaluate its specificity and in vivo pharmacokinetics compared to an established folate receptor targeted tracer. The anti-hFRα sdAb 2BD42 was conjugated to the near-infrared dye s775z. Following quality control, in vitro studies assessed binding specificity towards hFRα and hFRβ. Subsequently, its longitudinal in vivo biodistribution profile in a subcutaneous xenograft model was compared to that of OTL-38. Finally, the applicability of sdAb 2BD42-s775z was evaluated as a proof-of-concept in ovarian carcinomatosis and orthotopic lung tumour models. Following successful fluorescence labelling, sdAb 2BD42-s775z retained sub-nanomolar affinity for hFRα and selectively discriminated hFRα from hFRβ in vitro, in contrast to OTL-38. Furthermore, sdAb 2BD42-s775z exhibited a rapid tumour accumulation and a relevant tumour-to-background ratio (TBR) of 4.1 ± 0.9 at 1 h post-injection which persisted for up to 1 week post-injection, while for OTL-38, a relevant TBR was developed more slowly from 24 h post-injection onwards. In addition, sdAb 2BD42-s775z was able to rapidly and specifically highlight tumour lesions in an ovarian carcinomatosis and orthotopic lung tumour model, whereas no tumour uptake was observed with an irrelevant sdAb. To conclude, 2BD42-s775z demonstrated high specificity for hFRα, favourable TBR contrast, and rapid tumour visualisation in preclinical ovarian and lung cancer models, highlighting its potential for FGS. Further studies are warranted to determine whether selective hFRα targeting improves the detection of lymph node metastases compared with currently available folate receptor-targeted tracers.