Laura L Bronsart1,2, Christian Stokes2, Christopher H Contag3,4. 1. Department of Biology, Stanford University, 318 Campus Drive, Stanford, CA, 94305, USA. 2. Department of Pediatrics, Stanford University, 318 Campus Drive, Stanford, CA, 94305, USA. 3. Department of Pediatrics, Stanford University, 318 Campus Drive, Stanford, CA, 94305, USA. ccontag@stanford.edu. 4. Departments of Radiology, Microbiology and Immunology, Stanford University, 318 Campus Drive, Stanford, CA, 94305, USA. ccontag@stanford.edu.
Abstract
PURPOSE: We evaluated the small molecule coelenterazine as a potential reporter of cancer-associated superoxide anion in cell culture and in mice. PROCEDURES: The superoxide anion concentrations of various cancer cell lines were quantified by coelenterazine chemiluminescence in vitro. Coelenteramide fluorescence was detected via flow cytometry and fluorescent microscopy. Coelenterazine was used for the in vivo detection of cancer-associated superoxide anion using the 4T1 breast adenocarcinoma mouse model. RESULTS: Various cell lines in culture demonstrated different superoxide anion concentrations, with a signal range of 3.15 ± 0.06 to 11.80 ± 0.24 times that of background. In addition to chemiluminescent detection of coelenterazine, we demonstrated fluorescent detection of coelenteramide within the cytoplasm of cells. 4T1 murine mammary adenocarcinoma tumors in mice demonstrated significantly higher 2.13 ± 0.19-fold coelenterazine-based chemiluminescence than that of surrounding normal tissues. CONCLUSIONS: Collectively, our results indicate that coelenterazine can be used to assay superoxide anion concentrations in cultured cancer cells and in tumors growing in mice.
PURPOSE: We evaluated the small molecule coelenterazine as a potential reporter of cancer-associated superoxide anion in cell culture and in mice. PROCEDURES: The superoxide anion concentrations of various cancer cell lines were quantified by coelenterazine chemiluminescence in vitro. Coelenteramide fluorescence was detected via flow cytometry and fluorescent microscopy. Coelenterazine was used for the in vivo detection of cancer-associated superoxide anion using the 4T1 breast adenocarcinomamouse model. RESULTS: Various cell lines in culture demonstrated different superoxide anion concentrations, with a signal range of 3.15 ± 0.06 to 11.80 ± 0.24 times that of background. In addition to chemiluminescent detection of coelenterazine, we demonstrated fluorescent detection of coelenteramide within the cytoplasm of cells. 4T1 murine mammary adenocarcinoma tumors in mice demonstrated significantly higher 2.13 ± 0.19-fold coelenterazine-based chemiluminescence than that of surrounding normal tissues. CONCLUSIONS: Collectively, our results indicate that coelenterazine can be used to assay superoxide anion concentrations in cultured cancer cells and in tumors growing in mice.
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