Curative oncologic surgery successfully removes all cancerous tissues from the body and is the most effective treatment modality for localized solid tumors. Outcomes from cancer surgery are primarily determined by the ability of the surgeon to identify the presence and extent of all lesions intraoperatively, and subsequently perform complete resections [1]. While multiple diagnostic imaging modalities are used preoperatively for surgical planning, methods for reliable tumor visualization in the operating room are limited [2]. Accordingly, technological advances in the fields of optics, chemistry, pharmacology, and molecular imaging have converged into the powerful intraoperative imaging technique known as fluorescence-guided surgery (FGS) [3]. Several recent reviews describe the principles of FGS and highlight emerging concepts in probe development, instrument design/requirements, and translational applications [2,4, 5, 6]. Contrast agents used for FGS can generate signal via passive accumulation or be actively targeted to tumors, and typically emit signal in the near-infrared (NIR) spectral range (750–900 nm) to maximize image contrast and detection sensitivity [3]. If they display sufficient tumor uptake and specificity, tumor-targeted FGS agents could reduce incidences of missed lesions, positive margins, and unnecessarily extended surgery that can result in organ insufficiency [2].

In 2021, the FDA approved the first receptor-targeted FGS agent (Cytalux, pafolacianine) for intraoperative detection of folate receptor-positive ovarian cancer after results from a randomized phase III study showed that 33% of patients that underwent NIRF imaging with pafolacianine had lesions that were not previously identified under white light inspection or by palpation (ClinicalTrials.gov identifier: NCT03180307) [7]. Encouraging results from that trial eventually led to the phase III ELUCIDATE study, which is currently investigating the efficacy and safety of pafolacianine in lung cancer to determine the potential for expanded utility of the agent (ClinicalTrials.gov identifier: NCT04241315). Additionally, there are numerous ongoing clinical trials with other tumor-targeted FGS agents that could build on the landmark approval of pafolacianine and bring new surgical imaging capabilities to other types of cancers [6,8].

Given the inherent risk associated with the clinical development of a novel FGS agent, a large fraction of clinical-stage studies use targeting moieties with known in vivo properties [9]. Most common are therapeutic antibodies, which are widely repurposed for FGS since their tumor targeting capabilities have been established in the treatment setting and dye conjugation has little effect on binding specificity and biodistribution. These pharmacological observations are primarily attributed to the minimal size and structural change in the immunoconjugate relative to the native antibody. The slow elimination of these macromolecules from the body is also well-characterized, as are potential off-target effects. Thus, intraoperative imaging with therapeutic antibodies comes with a level of understanding that reduces translational risk. Low molecular weight agents such as peptides and small molecules are another class of targeting moieties with high binding affinity that could offer important molecularly-driven FGS capabilities. Due to their smaller size, these agents are rapidly cleared from the body and non-target tissues surrounding the tumor (usually within hours), thus making them particularly well-suited for surgical workflows and clinical imaging at lower total doses (i.e., a few milligrams) compared to antibodies [6]. Indeed, the feasibility and benefits of FGS drug development derived from low molecular weight agents has been showcased by pafolacianine, which consists of a chemically modified analog of an essential vitamin (i.e., folate) for fluorescence imaging. However, the dye conjugation strategy (discussed elsewhere [10,11]) must be carefully considered in order to retain the desirable pharmacological properties of the ligand since the fluorophore is often comparable in size to the native ligand.

An alternative strategy for developing new FGS agents with potentially higher translational efficiency [12] is to use clinically approved radiopharmaceuticals as model systems. Over the last two decades, radiopharmaceutical development has increasingly focused on improving diagnostic accuracy by designing agents with high target selectivity [13]. Biomarkers expressed on cancer cells, blood vessels, and the associated microenvironment have been identified as ideal targets due to their overexpression relative to healthy tissues. Accordingly, targeting agents have been developed using well-established preclinical characterization methods that include quantitative measurements of binding affinity, target selectivity, and biodistribution [14]. Since the whole-body biodistribution of radiopharmaceuticals is already known via non-invasive PET imaging, the on-target (i.e., tumor) and off-target uptake of a fluorescent analog could also be reasonably approximated [13]. Furthermore, the clearance mechanisms of a radiopharmaceutical will strongly influence feasibility of use in certain sites (i.e., liver) and the time required after injection to achieve meaningful contrast. These findings, in combination with the application of clinically relevant animal models, have led to the growing use of radiopharmaceutical platforms as foundational pieces for developing FGS agents.