Abstract 3917: Breast cancer cells expose thymidine kinase 1 as a new immunotherapy target
Authors: Weagel Evita G. , Brog Rachel A. , Townsend Michelle H. , Velazquez Edwin J. , Becker Toshiko A. , Mejia Camilo A. , Downey Michael R. , Arroyo Juan A. , Robison Richard A. , O’Neill Kim L. 
Published: July 2016

Abstract
This project investigates the role of Thymidine Kinase 1 (TK1) as a possible target for cancer therapeutics in breast cancer. Currently, therapeutic cancer treatments frequently damage bystander cells and have a devastating impact on the immune system. Many attempts have been made to engineer therapeutic techniques that are specific to cancer cells, unfortunately, resulting in few breakthroughs. TK1 is a phosphotransferase enzyme that is consistently present at abnormally high levels in cancer patient serum. TK1 assists in DNA repair and is characteristically expressed in the cytosol. We have discovered that TK1 is overexpressed on the surface of breast cancer cells lines MCF-7, MDA-MB-231, and Sk-Br-3. Interestingly, TK1 is not found on the surface of normal cells. We developed a unique monoclonal anti-TK1 antibody specific to human TK1 (A72). A72 was used in conjunction with flow cytometry, confocal microscopy, electron microscopy, and immunohistochemistry to test for TK1 on the cell surface. We stained each breast cancer cell line with A72-FITC conjugate and prepared appropriate controls. The samples were then analyzed using an Attune Flow Cytometer showing a 6% binding of A72 on the normal lymphocytes, while A72 binding was elevated to 20% of the breast cancer cells. Further testing using confocal microscopy was performed. The cells were treated with A72-FITC conjugate and Cell Mask Deep Red (a red membrane dye), and consecutively imaged. Results showed that A72-FITC associate with the cells in the same region of the red membrane stain, demonstrating that A72 binds to the cell membrane. This was further confirmed using electron microscopy by probing the cells with anti-TK1 conjugated to biotin that complexed with gold-streptavidin. The normal cells expectedly showed no significant binding, while gold staining was clearly seen on the surface of the each breast cancer cell line. Immunohistochemistry was completed to confirm the presence of TK1 in actual breast tumors from patients. Normal breast tissues along with breast tumor tissues were obtained from the Utah Valley Regional Medical Center. After treating the slides with anti-TK1 antibodies in conjunction with MACH 4 HRP secondary, DAP peroxidase was used to develop the slides. The tissues were examined using a light microscope. Cancer tissues appeared brown, indicating an overexpression of TK1. As expected, healthy breast tissues showed no significant staining, suggesting little to no presence of TK1. These data strongly suggest that TK1 is located on the surface of breast cancer cells, but is not found on the surface of normal cells. Further testing may open the possibilities of using TK1 as a new breast cancer biomarker. Ultimately, this also indicates the possible use of TK1 monoclonal antibodies in a therapeutic approach.
Citation Format: Evita G. Weagel, Rachel A. Brog, Michelle H. Townsend, Edwin J. Velazquez, Toshiko A. Becker, Camilo A. Mejia, Michael R. Downey, Juan A. Arroyo, Richard A. Robison, Kim L. O’Neill. Breast cancer cells expose thymidine kinase 1 as a new immunotherapy target. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3917.