In vitro influence of hypoxia on bioluminescence imaging in brain tumor cells

Bioluminescence Imaging (BLI) has been employed as an imaging modality to identify and characterize fundamental processes related to cancer development and response at cellular and molecular levels. This technique is based on the reaction of luciferin with oxygen in the presence of luciferase and ATP. A major concern in this technique is that tumors are generally hypoxic, either constitutively and/or as a result of treatment, therefore the oxygen available for the bioluminescence reaction could possibly be reduced to limiting levels, and thus leading to underestimation of the actual number of luciferase-labeled cells during in vivo procedures. In this report, we present the initial in vitro results of the oxygen dependence of the bioluminescence signal in rat gliosarcoma 9L cells tagged with the luciferase gene (9L^luc cells). Bioluminescence photon emission from cells exposed to different oxygen tensions was detected by a sensitive CCD camera upon exposure to luciferin. The results showed that bioluminescence signal decreased at administered pO₂ levels below about 5%, falling by approximately 50% at 0.2% pO₂. Additional experiments showed that changes in BLI was due to the cell inability to maintain normal levels of ATP during the hypoxic period reducing the ATP concentration to limiting levels for BLI.