Near-Infrared Claudin-1 antibody enables accurate detection of primary and metastatic colorectal tumors in mice

Background: Claudin-1, a tight-junction protein frequently overexpressed in colorectal cancer (CRC), is an attractive target for antibody-mediated near-infrared (NIR) fluorescence imaging. This study evaluates an anti-Claudin-1 antibody conjugated to IRDye800CW for detecting and validating colorectal tumors using bioluminescence correlation in orthotopic and metastatic mouse models. Methods: Luciferase-expressing LS174T human colon cancer cells were implanted into athymic nude mice to establish orthotopic (n = 10) and hepatic (n = 9) tumor models. Mice received 50 µg of Claudin-1-IRDye800CW intravenously 48 hours before imaging. In vivo imaging was performed using both the Pearl Trilogy Small Animal Imaging System and the Arthrex Synergy Vision System equipped for 800 nm fluorescence. Mean fluorescence intensity (mFI) was used to calculate tumor-to-background (TBR) and tumor-to-liver (TLR) ratios. Co-registration with luciferase bioluminescence was used to confirm probe specificity, and ex vivo imaging validated biodistribution. Results: In the orthotopic model, Claudin-1-IRDye800CW achieved a mean TBR of 3.92, clearly delineating tumor margins, while sub-millimeter peritoneal metastases (< 1 mm) produced a mean TBR of 2.9. In the hepatic model, the mean TLR was 3.42, confirming selective tumor contrast over surrounding tissue. Ex vivo analysis further increased contrast (TBR = 16.4; TLR = 4.78). Fluorescent signals consistently co-localized with luciferase activity, confirming targeting specificity. Both imaging systems demonstrated high-resolution detection, and the Arthrex clinical laparoscope enabled real-time intraoperative visualization under clinically relevant conditions. Conclusion: The anti-Claudin-1-IRDye800CW conjugate selectively and accurately identifies both primary and metastatic colorectal tumors in vivo. Strong tumor contrast and spatial concordance with bioluminescence validate its specificity, while compatibility with a clinical laparoscopic platform underscores the translational promise of Claudin-1-targeted NIR fluorescence imaging for colorectal cancer detection and fluorescence-guided surgery.