The importance of the pharmacology of luciferin and its effect on bioluminescent endpoints when conducting in vivo and in vitro experiments

Proc Amer Assoc Cancer Res, Volume 46, 2005 3834 Bioluminescent imaging of luciferase in genetically engineered cells is an emerging technology. Luciferase catalyzes conversion of the substrate luciferin + O2 into oxyluciferin (ATP is reduced) and a photon of light is emitted. The amount of light emitted is proportional to enzymatic activity and quantitative. Sensitive CCD cameras detect bioluminescence within the tissues of live animals and special software quantitates the signal. Thus, total tumor burden can be determined in mice inoculated with luciferase transfected cell lines and monitored for bioluminescence. By comparing bioluminescence at various time points during growth/treatment studies one can determine whether the tumor mass is increasing, decreasing or remaining stable. While this is an important tool in biological studies, the pharmacology of luciferin and its impact on the assay methodology has not been fully elucidated. A sensitive detection assay was developed and validated and the pharmacologic profile of luciferin following IP and IV administration was assessed. At the standard dose of 150 mg/kg given IP or IV, our studies demonstrate that luciferin is still detectable more than 8 hours post-dosing. In fact, plasma levels of 1 uM or greater can be detected 8 hr following a single IP administration of 150 mg/kg. This may explain, at least in part, the quenching of signal that is seen with repeated luciferin doses. The residual luciferin may be affecting tumor signaling for several reasons including continued consumption of ATP and O2 in the target cells, enzyme saturation/loss, altered pharmacokinetics due to repeated luciferin doses resulting in accumulation. Concurrent with the pharmacologic evaluations, we determined the minimum luciferin concentration needed to yield significant measurements. Initially, in vitro experiments were conducted to determine the minimum concentration required under optimal imaging conditions. Luciferase transfected cells (PC3M-Luc-C6) were seeded into 96 well plates 24 hours prior to the addition of luciferin. Cell density ranged from about 1000 cells to 0.5 cell/well. Varying concentrations of luciferin were added. The concentrations of luciferin ranged from 471 uM (standard used) to 0.094 uM. For this cell line, 125 cells can be detected with 0.094 uM luciferin and 1 cell can be measured at 0.188 uM luciferin. In vivo studies demonstrate a luciferin dose/luminescence sensitivity relationship as well. These experiments suggest that the optimal concentration of luciferin must be determined depending on the experimental design (imaging frequency, number and location of tumor cells, etc.) and goals (high sensitivity vs. repeated dosing). Because the substrate, luciferin, is costly it may be economically advantageous as well as scientifically justified to use lower concentrations where possible. This work was supported by NCI Contract NO1-CO-12400.