Gene and Stem Cell Therapy Program, Centenary Institute, University of Sydney, Camperdown NSW 2050, Australia.
Mol Cancer. 2010 Nov 22;9:299. doi: 10.1186/1476-4598-9-299.
Live animal imaging is becoming an increasingly common technique for accurate and quantitative assessment of tumor burden over time. Bioluminescence imaging systems rely on a bioluminescent signal from tumor cells, typically generated from expression of the firefly luciferase gene. However, previous reports have suggested that either a high level of luciferase or the resultant light reaction produced upon addition of D-luciferin substrate can have a negative influence on tumor cell growth. To address this issue, we designed an expression vector that allows simultaneous fluorescence and luminescence imaging. Using fluorescence activated cell sorting (FACS), we generated clonal cell populations from a human breast cancer (MCF-7) and a mouse melanoma (B16-F10) cell line that stably expressed different levels of luciferase. We then compared the growth capabilities of these clones in vitro by MTT proliferation assay and in vivo by bioluminescence imaging of tumor growth in live mice. Surprisingly, we found that neither the amount of luciferase nor biophotonic activity was sufficient to inhibit tumor cell growth, in vitro or in vivo. These results suggest that luciferase toxicity is not a necessary consideration when designing bioluminescence experiments, and therefore our approach can be used to rapidly generate high levels of luciferase expression for sensitive imaging experiments.
活体动物成像是一种越来越常见的技术，可用于准确和定量评估肿瘤随时间的负担。生物发光成像系统依赖于肿瘤细胞的生物发光信号，通常是由萤火虫荧光素酶基因的表达产生的。然而，先前的报告表明，荧光素酶的高水平或加入 D-荧光素酶底物后产生的光反应可能对肿瘤细胞的生长产生负面影响。为了解决这个问题，我们设计了一个表达载体，允许同时进行荧光和发光成像。我们使用荧光激活细胞分选（FACS）从人乳腺癌（MCF-7）和小鼠黑色素瘤（B16-F10）细胞系中生成稳定表达不同水平荧光素酶的克隆细胞群。然后，我们通过 MTT 增殖测定法比较了这些克隆在体外的生长能力，并通过活体小鼠肿瘤生长的生物发光成像比较了它们在体内的生长能力。令人惊讶的是，我们发现无论是荧光素酶的数量还是生物光子活性都不足以抑制肿瘤细胞的生长，无论是在体外还是体内。这些结果表明，在设计生物发光实验时，荧光素酶毒性不是必需的考虑因素，因此我们的方法可用于快速生成高浓度的荧光素酶表达，以进行敏感的成像实验。
