br Conclusions Claudin is a useful
Conclusions: Claudin-1 is a useful target for near-infrared antibody-based imaging for visualization of colorectal tumors for future use in fluorescence-guided surgery.
Published by Elsevier Inc.
Presented at the 13th Annual Academic Surgical Congress, Houston, TX, February 5-7, 2019.
Colorectal cancer remains one of the most prevalent cancers in the United States and is the second leading cause of cancer-related death.1 Although many technical advances have improved surgical options, surgical resection of colorectal cancer remains a challenge in part because of the difficulty in achieving negative margins in relatively small surgical fields. Positive margin rates of colorectal cancers have been previ-ously reported at 6.83%, which may contribute to higher rates of local recurrence and subsequent metastases.2
In current surgical practice, indocyanine green (ICG) is commonly used for visualization of biliary structures and to confirm adequate perfusion of tissues. Although ICG is generally not used in clinical practice techniques for colo-rectal cancer, the use of ICG creates a technological plat-form for novel fluorophores to be introduced to the operating room for various surgical fields. In comparison with ICG, tumor-specific fluorescent Prostaglandin E2 could pro-vide clearer margins between normal tissue and cells overexpressing specific antigens.3 This has been demon-strated in previous studies comparing ICG fluorescence with anti-carcinoembryonic antigen (CEA) antibodies in colon cancer.3,4 Anti-CEA antibodies have also been successful at labeling metastases in patient-derived pancreatic cancer mouse models.5 However, not all colon cancers express CEA and therefore other targets are needed.
The claudin family of proteins has been linked to tumorigenesis and development of colon cancer.6 Claudin proteins are tight-junction proteins, which help to maintain an epithelial barrier in normal colon cells.6,7 The deregula-tion of claudin expression leads to a loss of epithelial integrity and promotes tumorigenesis.8 Overexpression of Claudin-1 has been implicated in the development of colon cancer and cancer cells express a significantly higher amount of claudin when compared with normal colonic mucosa.8 Adenomatous popysosis coli (APC) mutations, which are implicated in development of colon cancer, cause an increase in Claudin-1 expression.9
The use of claudin as a target for tumor-specific imaging is beginning to gain interest. A recent publication utilized CpC-APC mice (mice in which APC allele has been deleted and polyps spontaneously form).10 A cyanine5.5 fluorophore that fluoresces around 700 nm was tagged to a peptide that competitively binds to Claudin-1. This was administered via enema and imaging was performed on the IVIS small animal system. This demonstrated enhancement of polyp visualiza-tion as well as flat lesions that were not detectable with bright-light imaging.10
In the present report, we aimed to develop clinically translatable claudin-targeted tumor-specific fluorescent an-tibodies. We used a near-infrared (NIR) 800 nm fluorophore that has compatibility with many of the fluorescent imaging systems currently used for ICG imaging.5 We show that Claudin-1 is a useful target for NIR imaging of colorectal cancer and metastases on patient-derived orthotopic xeno-grafts (PDOX) as well as orthotopic colon cancer cell line models in nude mice.
All studies were approved by the San Diego Veterans Administration Medical Center Institutional Animal Care and Use Committee (animal use protocol A17-020). Male nude (nu/ nu) mice, aged 4-6 wk, purchased from the Jackson Laboratory (Bar Harbor, ME), were used for this study. Only male mice were used to prevent colonization and breeding. The animals were fed an autoclaved laboratory diet. All surgical procedures were performed with anesthesia by intramuscular injection of ketamine, xylazine and acepromazine reconstituted in phos-phate buffered saline (PBS). Mice were treated with bupre-norphine for pain control after surgical procedures. At the conclusion of the study, or if tumor burden became too large, mice were euthanized with CO2 inhalation, which was confirmed with cervical dislocation.
Claudin-1 rabbit polyclonal antibody (lLS-C382732 CLDN1; LifeSpan BioSciences Inc, Seattle, WA) was conjugated with IR800CW NHS ester (LI-COR Biosciences Inc, Lincoln, NE) under basic conditions. Excess dye was removed using a Zeba desalting column (Thermo Fisher Scientific, Waltham, MA) and the product suspended in PBS. Protein concentration was measured using the Lowry assay on a microplate reader (Bio-Rad Laboratories Inc, Hercules, CA)