Design of a Pan-Tumor Fluorescence Imaging Cocktail for Fluorescence-Guided Surgery

Fluorescence-guided surgery (FGS) has emerged as a powerful tool for enhancing a surgeon's ability to locate and resect all malignant lesions, thereby improving a patient's probability of survival. While several tumor-targeted near-infrared (NIR) fluorophores have been developed to image selected cancer types, no single tumor-targeted dye has been found to image all cancers. To remedy this deficiency, we have examined the ability of a fibroblast activation protein (FAP)-targeted NIR fluorophore to broad-spectrum solid tumors when combined with pafolacianine (OTL-38, Cytalux). Pafolacianine was selected for this imaging cocktail because it highlights all cells that express either folate receptor α (a receptor that is expressed on ∼40% of human cancers) or folate receptor β (a related receptor expressed on tumor-associated macrophages and myeloid-derived suppressor cells). An FAP-targeted conjugate of the same near-infrared fluorescent dye (S0456) was also selected because it targets myofibroblasts that infiltrate virtually all solid tumors but are essentially absent from healthy tissues. In this paper, we describe the design, synthesis, and characterization of a novel FAP ligand (FAP9) and determine its affinity (<i>K</i>_d ∼ 2 nM) and specificity (>2000-fold over homologous enzymes) for FAP. We then quantify its ability to image many different cancer types in murine tumor models and combine it in different ratios with pafolacianine to determine the ratio that yields the highest total tumor fluorescence and tumor-to-background ratio in seven tumor models. Because the combination of the two fluorescent conjugates invariably images tumors better than either conjugate alone, and since the two-dye combination displays no obvious toxicities, we propose that a cocktail of FAP9-S0456 plus pafolacianine warrants evaluation as a candidate for intraoperative imaging of all human tumors.