Mucins (MUC1–MUC24) are a family of glycoproteins involved in cell signaling and barrier protection. They have been implicated in the progression of numerous malignancies including gastric, pancreatic, ovarian, breast, and lung cancer. Mucins have also been extensively studied with respect to colorectal cancer. They have been found to have diverse expression profiles amongst the normal colon, benign hyperplastic polyps, pre-malignant polyps, and colon cancers. Those expressed in the normal colon include MUC2, MUC3, MUC4, MUC11, MUC12, MUC13, MUC15 (at low levels), and MUC21. Whereas MUC5, MUC6, MUC16, and MUC20 are absent from the normal colon and are expressed in colorectal cancers. MUC1, MUC2, MUC4, MUC5AC, and MUC6 are currently the most widely covered in the literature regarding their role in the progression from normal colonic tissue to cancer.

Fluorescence-guided surgery (FGS) using tumor-specific antibodies conjugated with near-infrared fluorophores (NIR) have demonstrated the potential to visualize solid tumor lesions in preclinical and early stage clinical trials.^{1 – 3} Several commercial cancer therapeutic monoclonal antibodies (mAbs) have been repurposed as molecular optical imaging agents by their conjugation with NIR dyes and are in early-stage clinical trials.^{4 – 6} Compared to visible light fluorophores, NIR fluorophores have reduced light scattering, increased depth of penetration, and minimal autofluorescence. The use of NIR dyes in the 800-nm range enables the use of instrumentation available in most operating suites using the FDA-approved indocyanine green dye to monitor cardiac output and liver blood flow function. Our theranostics group has focused on the development of anti-carcinoembryonic antigen (CEA) mAbs for the radionuclide imaging and therapy of CEA-positive disease.^{7 – 12} CEA is surface GPI-linked glycoprotein highly expressed in gastrointestinal malignancies (GI), such as colorectal (95%), gastric (80%), pancreatic (60%), and breast (50%) cancers but with very limited expression in normal adults.^{13 – 17} We designed and produced a recombinant humanized anti-CEA hT84.66-M5A (M5A) mAb that is currently in early phase clinical trials.¹⁸ Radiolabeled with the positron emission tomography (PET) radionuclide Cu-64, the ${}^{64}\mathrm{Cu}$-DOTA-M5A mAb has provided promising PET images of CEA-positive disease in an ongoing pilot imaging trial (NCT02293954). Based on success with immunoPET imaging prior to surgery, a natural extension of the anti-CEA M5A mAb would be its development for fluorescent image-guided surgery. We have collaborated with the Bouvet group who have a long-standing expertise on the preclinical use of a chimeric anti-CEA antibody for FGS.^{19 – 23} Using the commercial NHS-IRDye800CW currently being used in antibody-targeted FGS clinical trials,^5,24 the M5A-IR800 exhibited strong tumor-specific labeling in a human colon cancer²⁵ as well as pancreatic cancer²⁶ orthotopic xenograft mouse models. However, these studies identified room for improvement, in that conjugation of the IRDye800CW fluorophore to M5A resulted in accelerated pharmacokinetics over the unmodified M5A mAb, requiring low antibody-to-dye ratios, limiting sensitivity of tumor detection over a shortened window for surgical planning.

Importance: Fluorescence-guided surgery (FGS) is a potentially powerful tool for hepatobiliary (HPB) surgery. The high sensitivity of fluorescence navigation is especially useful in settings where tactile feedback is limited. Objective: The present narrative review evaluates literature on the use of FDA-approved fluorophores such as methylene blue (MB), 5-aminolevulinic acid (5-ALA), and indocyanine green (ICG) for clinical intra-operative image-guidance during HPB surgery. Evidence Review: Approaches such as dosing, timing, imaging devices and comparative endpoints are summarized. The feasibility and safety of fluorophores in visualizing the biliary tree, identify biliary leaks, outline anatomic hepatic segments, identify tumors, and evaluate perfusion and graft function in liver transplants are discussed. Findings: Tumor-specific probes are a promising advancement in FGS with a greater degree of specificity. The current status of tumor-specific probes being evaluated in clinical trials are summarized. Conclusions and Relevance for Reviews: Relevant discussion of promising tumor-specific probes in pre-clinical development are discussed. Fluorescence-guidance in HPB surgery is relatively new, but current literature shows that the dyes are reliably able to outline desired structures with a variety of dosing, timing, and imaging devices to provide real-time intra-operative anatomic information to surgeons. Development of tumor-specific probes will further advance the field of HPB surgery especially during oncologic resections.

Tumor-specific targeting with fluorescent probes can enhance contrast for identification of cancer during surgical resection and visualize otherwise invisible tumor margins. Nanobodies are the smallest naturally-occurring antigen-binding molecules with rapid pharmacokinetics. The present work demonstrates the efficacy of a fluorescent anti-CEA nanobody conjugated to an IR800 dye to target and label patient derived pancreatic cancer xenografts. After intravenous administration, the probe rapidly localized to the pancreatic cancer tumors within an hour and had a tumor-to-background ratio of 2.0 by 3 h. The fluorescence signal was durable over a prolonged period of time. With the rapid kinetics afforded by fluorescent nanobodies, both targeting and imaging can be performed on the same day as surgery.