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Validity of bioluminescence measurements for noninvasive in vivo imaging of tumor load in small animals
Clara P.W. Klerk1, Renée M. Overmeer1, Tatjana M.H. Niers1, Henri H. Versteeg1, Dick J. Richel1, Tessa Buckle2, Cornelis J.F. Van Noorden1, and Olaf van Tellingen2
1Academic Medical Center, University of Amsterdam, Amsterdam
2The Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands
BioTechniques, Vol. 43, No. S1, July 2007, pp. S7–S13, S30
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A relatively new strategy to longitudinally monitor tumor load in intact animals and the effects of therapy is noninvasive bioluminescence imaging (BLI). The validity of BLI for quantitative assessment of tumor load in small animals is critically evaluated in the present review. Cancer cells are grafted in mice or rats after transfection with a luciferase gene—usually that of a firefly. To determine tumor load, animals receive the substrate agent luciferin intraperitoneally, which luciferase converts into oxyluciferin in an ATP-dependent manner. Light emitted by oxyluciferin in viable cancer cells is captured noninvasively with a highly sensitive charge-coupled device (CCD) camera. Validation studies indicate that BLI is useful to determine tumor load in the course of time, with each animal serving as its own reference. BLI is rapid, easy to perform, and sensitive. It can detect tumor load shortly after inoculation, even when relatively few cancer cells (2500–10,000) are used. BLI is less suited for the determination of absolute tumor mass in an animal because of quenching of bioluminescence by tissue components and the exact location of tumors because its spatial resolution is limited. Nevertheless, BLI is a powerful tool for high-throughput longitudinal monitoring of tumor load in small animals and allows the implementation of more advanced orthotopic tumor models in therapy intervention studies with almost the same simplicity as when measuring traditional ectopic subcutaneous models in combination with calipers.


Animal models remain important experimental tools to investigate mechanisms of tumor development and effects of therapy. Since cancer progression consists of a wide variety of events, such as primary tumor growth, angiogenesis, invasion into surrounding tissues, extravasation of cancer cells, and growth of metastases (1,2), it is likely that (potential) therapies may be effective at certain stages of cancer progression only. Thus far, assessment of the effects of therapeutic intervention has usually been made using subcutaneously implanted tumors, which, however, do not recapitulate many of the essential features of tumor growth in patients (3). Whereas orthotopically implanted tumors mimic the clinical situation more closely and may be preferred (4,5), assessment of such tumor burden initially required the sacrifice of the animals at a certain time point after cancer cell inoculation and evaluation of tumor load at time of death. The course of tumor development in time was then assessed by comparing groups of animals that were sacrificed at different time points. Because of substantial inter-individual variation, large numbers of animals and laborious efforts were required, rendering the use of orthotopic models highly impractible (6). Noninvasive determination of tumor load over time in individual animals can reduce the numbers of animals in experiments considerably and provide information on the various stages of tumor development (7,8,9). Alternatively, tumor load in animals is determined by surgically opening the animals to inspect the organ affected by cancer (10), but this type of procedure causes considerable discomfort to the animal and may also affect cancer progression by operation-related effects such as activation of the coagulation cascade (11).

Tumors that are not visible or palpable noninvasively can also be visualized by special preparation of tissues. Skin-fold chambers and subcutaneous windows inserted with semitransparent material can be used to visualize tumor development at a cellular or even subcellular level for longer periods of time (12,13). These techniques can also be combined with externalization of organs of interest (14). However, the maintenance of windows for longer periods of time is rather difficult (14). Furthermore, investigations are limited to areas exposed by the chamber or window. The use of skin flap models can extend the areas of investigation considerably (15). Again, these approaches have the disadvantage that heterotopic tumors are used and operation-related effects may affect tumor development.

Noninvasive Imaging

Several noninvasive approaches to imaging tumor development in small animals have become available, such as magnetic resonance imaging (MRI), computerized tomography (CT), positron emission tomography (PET), single photon emission computed tomography (SPECT), and ultrasonography. Each approach has its own advantages and disadvantages. MRI, CT, and ultrasonography produce anatomical images of structures in the body, whereas PET and SPECT image physiological processes and thus produce functional images. Their use for the in vivo study of cancer progression in small animals has been reviewed recently (7,8,16). Alternatively, the use of two relatively new noninvasive approaches, fluorescence imaging (FLI) and bioluminescence imaging (BLI), is expanding rapidly.

Noninvasive FLI

For FLI, cancer cells are transfected with the gene for green fluorescent protein (GFP) or related fluorescent proteins that allow visualization of these cells in living animals using a sensitive charge-coupled device (CCD) camera (17,18,19). Large tumors can even be observed noninvasively with the naked eye (20,21). Other reporter genes have been applied as well that make cells fluorescent when a specific promoter is activated (22) or encode for an enzyme, such as β-galactosidase, that produces a fluorescent product (19). The use of skin flaps in combination with high-power microscopes even allows investigation of these tumors at a single-cell level (15). Additionally, nonfluorescent cells or tissue compartments can be studied against a fluorescent background of the cancer cells, as they contrast with this background. In this way, host (immune) cells and angio-genesis have been studied in tumors (15,17,23). A disadvantage is the fact that cancer cells have to be transfected with a reporter gene and that may alter their malignant behavior. Moreover, visualization of fluorescent cancer cells in the deeper parts of animals that cannot easily be exposed is often hard or even impossible to accomplish because of interference by autofluorescence and quenching of excitation and emission light by tissues (7). Excitation light and emission light are often of wavelengths below 600 nm, and at these wavelengths, tissue components quench strongly (21,24,25). Light at wavelengths between 600 and 900 nm is more optimal for use in live animals, as tissues are more translucent (8,19,26) and produce less autofluorescence (8,16,21,24). However, the food in the gastrointestinal tract may contain plant products that are fluorescent at longer wavelengths and thus interfere with the sensitivity of FLI (24).

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