Luciferase expression and bioluminescence does not affect tumor cell growth in vitro or in vivo
© Tiffen et al; licensee BioMed Central Ltd. 2010
Received: 12 May 2010
Accepted: 22 November 2010
Published: 22 November 2010
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.
List of abbreviations
Fluorescence activated cell sorting. MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. BLI: Bioluminescence imaging. P2A: Porcine teschovirus-1 2A sequence. SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis. PCR: Polymerase chain reaction.
Bioluminescence imaging (BLI) is an increasingly popular technique for quantitatively assessing tumor growth and the effects of therapy over time . The sensitivity and accuracy of in vivo BLI systems offers several advantages over traditional methods of measuring subcutaneous tumors using calipers [2–9]. Typically cancer cells are engineered to express the firefly luciferase gene and are engrafted into mice to form tumors . Following an intraperitoneal injection of D-luciferin, the luciferase enzyme will catalyze this substrate into oxyluciferin, requiring the presence of oxygen, and cofactors such as adenosine triphosphate (ATP) and Mg2+ ions . The resulting light photons generated by this reaction are captured non-invasively with a charge-coupled device (CCD) camera mounted within the BLI system . Successful BLI requires prior modification of the cancer cell line with the luciferase gene, however little is known about the effect this may have on normal cell function . To date, the only evidence of a detrimental effect of biophotonic emissions on cell function was in a luciferase-expressing ovarian cancer cell line that showed a high level of luciferase reduced tumor growth in vivo. It was suggested that build up of oxyluciferin during repeated BLI might cause oxidative damage to the cells. Limiting cofactors in the luciferase-luciferin reaction include oxygen and ATP ; therefore high levels of biophotonic activity may place extra demand for energy on the cells, possibly leading to growth inhibition. One report even suggests the use of luciferase in photodynamic therapy following a 90% reduction in the survival of NIH3T3 mouse fibroblasts, which were stably expressing luciferase and incubated with a photosensitizer . However, doubts remain as to whether luciferase can generate enough photons to significantly inhibit the growth of cancer cells.
Future work is needed to investigate the possibility of an immune response against tumor cells expressing firefly luciferase or P2A; however as most researchers perform xenograft experiments in immune-compromised animals, the immunogenicity of luciferase is unlikely to be a significant concern. The use of clonal cell populations is useful to ensure a homogenous expression of luciferase, but problems may arise due to interclonal variation (Figure 4A, B). Based on the stoichiometric expression of GFP and luciferase protein, our approach also allows selection of a polyclonal population of cells using a narrow band of GFP expression (Figure 1B, C), thus minimizing any interclonal variation. It has been suggested that a hypoxic tumor environment can lead to a reduction in intracellular ATP levels, that in turn may result in an underestimation of BLI . Our vector provides a solution to this problem in that imaging can be performed using luminescence or fluorescence, to ensure comparable measurements.
Our vector represents a versatile tool for BLI in that fluorescence from GFP-positive cells correlates directly with luciferase expression levels. Contrary to previous reports however [14, 16], we found that neither a high level of luciferase expression, nor biophotonic activity had a detrimental effect on cancer cell growth in vitro or in vivo. In light of these data, we conclude that oxyluciferin toxicity is not an important consideration when designing BLI experiments.
We thank the Centenary Institute animal house and flow cytometry staff for technical assistance and Trina Lum and Robyn Soper from the Royal Prince Alfred Hospital Pathology department for immunohistochemistry expertise. We also thank Don Anson for providing us with the vector and accessory plasmids. This work was generously supported by the Fred Barbagallo, Rotary Club of Dural, Australian Rotary Health PhD Scholarship in conjunction with the Cure the Future foundation. Funding was also provided by the National Health and Medical Research Council of Australia (512271), the Cancer Council NSW and the Cancer Institute of NSW.
- Burgos JS, Rosol M, Moats RA, Khankaldyyan V, Kohn DB, Nelson MD, Laug WE: Time course of bioluminescent signal in orthotopic and heterotopic brain tumors in nude mice. Biotechniques. 2003, 34: 1184-1188.PubMed
- Craft N, Bruhn KW, Nguyen BD, Prins R, Liau LM, Collisson EA, De A, Kolodney MS, Gambhir SS, Miller JF: Bioluminescent imaging of melanoma in live mice. J Invest Dermatol. 2005, 125: 159-165. 10.1111/j.0022-202X.2005.23759.xPubMed CentralView ArticlePubMed
- Deroose CM, De A, Loening AM, Chow PL, Ray P, Chatziioannou AF, Gambhir SS: Multimodality imaging of tumor xenografts and metastases in mice with combined small-animal PET, small-animal CT, and bioluminescence imaging. J Nucl Med. 2007, 48: 295-303.PubMed CentralPubMed
- Drake JM, Gabriel CL, Henry MD: Assessing tumor growth and distribution in a model of prostate cancer metastasis using bioluminescence imaging. Clin Exp Metastasis. 2005, 22: 674-684. 10.1007/s10585-006-9011-4View ArticlePubMed
- Gross S, Piwnica-Worms D: Spying on cancer: molecular imaging in vivo with genetically encoded reporters. Cancer Cell. 2005, 7: 5-15.PubMed
- Jenkins DE, Oei Y, Hornig YS, Yu SF, Dusich J, Purchio T, Contag PR: Bioluminescent imaging (BLI) to improve and refine traditional murine models of tumor growth and metastasis. Clin Exp Metastasis. 2003, 20: 733-744. 10.1023/B:CLIN.0000006815.49932.98View ArticlePubMed
- Lyons SK, Lim E, Clermont AO, Dusich J, Zhu L, Campbell KD, Coffee RJ, Grass DS, Hunter J, Purchio T, Jenkins D: Noninvasive bioluminescence imaging of normal and spontaneously transformed prostate tissue in mice. Cancer Res. 2006, 66: 4701-4707. 10.1158/0008-5472.CAN-05-3598View ArticlePubMed
- Nogawa M, Yuasa T, Kimura S, Kuroda J, Sato K, Segawa H, Yokota A, Maekawa T: Monitoring luciferase-labeled cancer cell growth and metastasis in different in vivo models. Cancer Lett. 2005, 217: 243-253. 10.1016/j.canlet.2004.07.010View ArticlePubMed
- Sadikot RT, Blackwell TS: Bioluminescence imaging. Proc Am Thorac Soc. 2005, 2: 537-540. 511-532, 10.1513/pats.200507-067DSPubMed CentralView ArticlePubMed
- Sato A, Klaunberg B, Tolwani R: In vivo bioluminescence imaging. Comp Med. 2004, 54: 631-634.PubMed
- Greer LF, Szalay AA: Imaging of light emission from the expression of luciferases in living cells and organisms: a review. Luminescence. 2002, 17: 43-74. 10.1002/bio.676View ArticlePubMed
- Massoud TF, Gambhir SS: Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Genes Dev. 2003, 17: 545-580. 10.1101/gad.1047403View ArticlePubMed
- Klerk CP, Overmeer RM, Niers TM, Versteeg HH, Richel DJ, Buckle T, Van Noorden CJ, van Tellingen O: Validity of bioluminescence measurements for noninvasive in vivo imaging of tumor load in small animals. Biotechniques. 2007, 43: 7-13. 30, 10.2144/000112515View ArticlePubMed
- Brutkiewicz S, Mendonca M, Stantz K, Comerford K, Bigsby R, Hutchins G, Goebl M, Harrington M: The expression level of luciferase within tumour cells can alter tumour growth upon in vivo bioluminescence imaging. Luminescence. 2007, 22: 221-228. 10.1002/bio.953View ArticlePubMed
- Moriyama EH, Niedre MJ, Jarvi MT, Mocanu JD, Moriyama Y, Subarsky P, Li B, Lilge LD, Wilson BC: The influence of hypoxia on bioluminescence in luciferase-transfected gliosarcoma tumor cells in vitro. Photochem Photobiol Sci. 2008, 7: 675-680. 10.1039/b719231bView ArticlePubMed
- Theodossiou T, Hothersall JS, Woods EA, Okkenhaug K, Jacobson J, MacRobert AJ: Firefly luciferin-activated rose bengal: in vitro photodynamic therapy by intracellular chemiluminescence in transgenic NIH 3T3 cells. Cancer Res. 2003, 63: 1818-1821.PubMed
- Koldej R, Cmielewski P, Stocker A, Parsons DW, Anson DS: Optimisation of a multipartite human immunodeficiency virus based vector system; control of virus infectivity and large-scale production. J Gene Med. 2005, 7: 1390-1399. 10.1002/jgm.803View ArticlePubMed
- Holst J, Vignali KM, Burton AR, Vignali DA: Rapid analysis of T-cell selection in vivo using T cell-receptor retrogenic mice. Nat Methods. 2006, 3: 191-197. 10.1038/nmeth858View ArticlePubMed
- Riley PA: Melanin. Int J Biochem Cell Biol. 1997, 29: 1235-1239. 10.1016/S1357-2725(97)00013-7View ArticlePubMed
- Schipper ML, Patel MR, Gambhir SS: Evaluation of firefly luciferase bioluminescence mediated photodynamic toxicity in cancer cells. Mol Imaging Biol. 2006, 8: 218-225. 10.1007/s11307-006-0048-1View ArticlePubMed
- Russell PJ, Raghavan D, Gregory P, Philips J, Wills EJ, Jelbart M, Wass J, Zbroja RA, Vincent PC: Bladder cancer xenografts: a model of tumor cell heterogeneity. Cancer Res. 1986, 46: 2035-2040.PubMed
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