Bioluminescence imaging (BLI) is a relatively new noninvasive technology used for quantitative assessment of tumor growth and therapeutic effect in living animal models. BLI involves the generation of light by luciferase-expressing cells following administration of the substrate luciferin in the presence of oxygen and ATP. In the present study, the effects of hypoxia, hypoperfusion, and pH on BLI signal (BLS) intensity were evaluated in vitro using cultured cells and in vivo using a xenograft model in nude mice. The intensity of the BLS was significantly reduced in the presence of acute and chronic hypoxia. Changes in cell density, viability, and pH also affected BLS. Although BLI is a convenient non-invasive tool for tumor assessment, these factors should be considered when interpreting BLS intensity, especially in solid tumors that could be hypoxic due to rapid growth, inadequate blood supply, and/or treatment. 1. Introduction In vivo bioluminescence imaging (BLI) is a technology that is frequently used in the study of animal tumor models [1]. It has been successfully used to follow many different types of tumors, such as prostate, breast, colon, ovarian, and lung cancers [2–8]. The in vivo BLI method is based on the action of luciferase on luciferin which produces light emission within the xenograft [9, 10]. The light-producing reaction requires molecular oxygen and ATP for the oxidation of luciferin to oxyluciferin. The light produced is transmitted through tissue and detected by a sensitive charge-coupled device (CCD) camera; the acquired data can be presented as qualitative pseudocolor images or as quantitative photon counts. A significant advantage of in vivo BLI is the ability to noninvasively obtain several data points from the same group of animals by repeated monitoring. In addition, the sensitivity of in vivo BLI permits the detection of very small tumors or metastases [8, 11]. A major concern is that solid tumors frequently outgrow their oxygen supply and can develop central hypoxia [12]. Alternatively, tumor hypoxia can develop as a result of treatment [13]. In these settings, oxygen available for the BLI reaction could be reduced to limiting levels, which would result in a reduced BLI signal (BLS) and underestimation of the actual tumor size [14]. In the process of developing an in vivo BLI-based mouse model of U87 glioma cells for evaluation of radiotherapy, we noted that these solid tumors frequently become transiently or chronically hypoxic and that, in this situation, tumor growth determined by BLI may be an underestimate. We
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