J. C. DeLong1, T. Murakami1,2, P. J. Yazaki3, R. M. Hoffman1,2, M. Bouvet1 1University Of California – San Diego,Surgery,San Diego, CA, USA 2AntiCancer, Inc.,San Diego, CA, USA 3City Of Hope National Medical Center,Immunology,Duarte, CA, USA
Introduction: The success of a curative surgery for cancer is dependent on the complete removal of all cancer cells, an R0 resection. Intraoperative verification of clear tumor margins is not possible by the surgeon and requires a surgical pathologist to analyze frozen sections of the resected tumor. Tumor visualization by the surgeon can be enhanced through fluorescence-guided surgery (FGS) by delivering labeled tumor-specific antibodies. We selected humanized anti-carcinoembryonic antigen (CEA) conjugated to a near-infrared (NIR) dye to target orthotopically implanted human colon cancer in nude mice.
Methods: The HT-29 cell line for human colon cancer was grown in culture and subcutaneously injected subcutaneously in a nude mouse model. After 3 weeks of growth tumors were resected and cut into 2 mm3 fragments that were sutured to the cecum of 5 additional nude mice. The tumors were allowed to grow for 4 weeks at which point 3 had successful orthotopic tumor growth and were selected for injection of the humanized antibody for CEA that was convalently bound to the IR800 NIR dye (anti-CEA-IR800) through an ester reaction. Antibody-dye conjugate (75 μg ) was intravenously administered via tail vein injection. Images were taken with the Pearl Trilogy Small Animal Imaging System (Li-Cor, Lincoln, NE) pre-injection, 5 min post, 5 hours post, 24 hours post (with laparotomy views), and 48 hours post injection (with laparotomy views) with both 700 nm and 800 nm channels. Images were evaluated using Image Studio.
Results: Images taken at 5 min and 5 hours (through skin, no laparotomy) did not demonstrate appreciable accumulation in the tumor. At 24 hours laparotomy was performed and the tumors were strongly labeled with anti-CEA-IR800 when imaged through the 800 nm channel. At 48 hours laparotomy was repeated which again demonstrated strong labeling of the tumors through the 800 nm channel, but with a lower absolute intensity (in relative units), than at 24 hours for each of the 3 mice imaged. Normal bowel was fluorescent through the 700 nm channel due to the autofluorescence of plant chlorophylls in mouse chow.
Conclusion: Humanized anti-CEA-IR800 can rapidly and effectively label CEA-expressing human colon cancer in an orthotopic nude mouse model. Given the ability of this technology to target and label tumor with great specificity, anti-CEA-IR800 should be made available for clinical use for fluorescence guided surgery in the near future.