H. M. Hollandsworth1,2,5, T. M. Lwin1,2,5, S. Amirfakhri1,2,5, F. Filemoni1,2,5, D. Stupack2, S. K. Batra3, R. M. Hoffman1,2,4,5, P. Dhawan3, M. Bouvet1,2,5 1University Of California – San Diego,Department Of Surgery,San Diego, CA, USA 2University Of California – San Diego,Moores Cancer Center,San Diego, CA, USA 3University of Nebraska Medical Center,Department Of Biochemistry,Omaha, NE, USA 4AntiCancer, Inc.,San Diego, CA, USA 5VA San Diego Healthcare System,Surgical Services,San Diego, CA, USA
Introduction:
Colorectal cancer remains one of the most prevalent cancers in the United States and is the third leading cause of cancer related death. Claudins are tight junction proteins which maintain an epithelial barrier in normal colon cells. Overexpression of Claudin-1 has been implicated in the development of colon cancer. Tumor cells express a significantly higher amount of Claudin when compared with normal colonic mucosa. We postulated that Claudin-1 may be a useful target in near infrared imaging and fluorescence guided surgery for colorectal cancers.
Methods:
We conjugated Claudin-1 antibody to LI-COR IR800DyeCW (Claudin-1-IRDye800CW). Western blotting of 9 different human colon cancer lysates was performed with Claudin-1-IRDye800CW. Animal imaging was initially performed with LI-COR Pearl Trilogy imaging system on subcutaneous and orthotopic colon tumors with 75 mcg Claudin-1-IRDye800CW 72 hours after injection. A dose-response study was then completed on subcutaneous cell line models. Subcutaneous implantation of LS174T on the bilateral flanks of nude mice was performed. Three groups of mice were administered increasing doses of Claudin-1-IRDye800CW: 12.5, 25, and 50 micrograms reconstituted in 100 microliters PBS, via tail vein injection. 3 mice were used as controls (no treatment, antibody alone, and 1 dye alone). In vivo imaging was performed at 24, 48, and 72 hours after administration of the conjugated dye. The mice were then euthanized and laparotomy was performed to assess for enhancement of internal organs.
Results:
Western blotting revealed that 9 out of 10 colon cancer xenografts expressed varying amounts of Claudin-1. All tumors demonstrated strong and specific fluorescence labeling at 800 nm wavelength, even with the lowest dose of 12.5 micrograms. A representative image of mouse with an orthotopic LS174T colon tumor treated with 75 mcg of Claudin-1-IRDye800CW is shown in the Figure below. The control mice that did not receive treatment and received Claudin-1 antibody alone did not have any signal on the colon or tumor. The mouse that received IRDye800CW alone had diffuse signal that did not localize to tumors.
Conclusion:
Claudin-1 is a useful target for near infrared antibody-based imaging for visualization of colorectal tumors for future use in fluorescence-guided surgery.
