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Bioluminescence imaging (BLI) has emerged during the past five years as the preeminent method for rapid, cheap, facile screening of tumor growth and spread in mice. Both subcutaneous and orthotopic tumor models are readily observed with high sensitivity and reproducibility. User friendly commercial instruments exist and increasingly luciferase expressing tumor cells are available in academic institutions or commercially. There is an increasing literature on routine use of BLI for assessing chemotherapeutic efficacy, drug combinations, dosing and timing. In addition, BLI may be applied to more sophisticated questions of molecular biology by including specific promoter sequences. This chapter will describe routine methods used to support multiple investigators in our small animal imaging resource.
The concept of bioluminescence to study biochemistry has been around for many years, for example, as the basis for quantifying ATP in snap frozen histological specimens or tissue extracts (1, 2). However, in vivo application has been spearheaded by Contag et al. (3) and promoted by Xenogen (now Caliper Lifesciences). In less than a decade, BLI has become a routine modality for use in cancer biology, particularly suited for assessing tumor burden and metastatic spread. In vivo BLI has been reviewed many times (3–6) and readers are directed to these papers and other chapters of this book for further insight.
In its most popular format, the bioluminescent reaction requires luciferase enzyme derived from the American firefly (Photinus pyralis) and D-luciferin substrate. Luciferase is generated by cells following transfection. It is important to select clones with high stable expression, usually based on lentiviral transfection, which tends to be more stable than plasmid transfection. It is important to recognize that clones isolated for high expression may not behave identically to parallel lines or the parental system (e.g., differential growth rates). Thus, tumor models can be highly effective in terms of assessing tumor development and response to therapy, but they may not perfectly replicate parental cell lines.
Pharmacokinetics of the luciferin substrate are important. Remarkably, luciferin appears to readily permeate every tissue including crossing the blood-brain and -placental barriers (4). However, the kinetics of light-emission can differ with tumor location, and thus, it is critical to establish reproducibility of light-emission curves prior to embarking on large scale studies. The most popular route of administration of luciferin is IP (intra peritoneal) (7), but while this is apparently facile, we find a significant failure rate (8), where no light-emission is observed following substrate administration, yet repeat one hour later gives expected bioluminescence. We attribute this to poor injection, possibly into the intestines. Intravenous (IV) administration can give much higher light-emission (9), but more transiently so that any variation in the timing of image capture and/or integration time can generate poorer reproducibility (8). Intravenous injection is also technically more challenging. Direct intra tumor (IT) injection generates the most intense bioluminescence, but is obviously invasive and only feasible for easily accessible tumors (7, 10). We favor subcutaneous (SC) administration of luciferin in the back neck region. The technique is facile with overwhelming success in observing expected signal and the kinetics provide intense light over several minutes (8, 11).
Light detection is strongest from subcutaneous tumor sites although in this case caliper measurements may be just as effective and cheaper for simple tumor volume assessment. However, BLI is particularly effective for low tumor burdens, and indeed, sub-palpable volumes can be detected and quantified. For large tumors, self absorption and scatter of light can bias apparent relative tumor volume. Planar BLI appears to accurately reflect the volume of small tumors, but becomes less linear for larger tumors, although continuing to increase monotonically (12, 13). Light is subject to significant absorption and scattering from deep tumors, and thus, equivalent tumors located at depth are expected to provide much less detectable light. Thus, for longitudinal studies it is crucial to view an animal from the same direction on successive occasions to ensure a reproducible solid viewing angle and consistent absorption by any intervening tissues. Nude mice are preferred, though light may also be detected from white or black mice with hair: some investigators prefer to shave the animals or apply depilating agents.
Bioluminescent imaging systems can be constructed quite easily and cheaply based on several recipes in the literature, primarily from the amateur astronomy field, where there is a similar need to detect weak signals against a low background based on long-term signal integration (14). To date, our BLI service uses a home built system, which has been described elsewhere (7, 8, 15). The primary protocol below describes the procedures with this system (Cyclops). However, the instrument is technically complex requiring a BLI technician and engineering support. Sophisticated commercial systems are available, which are user friendly (Caliper Xenogen1 and Berthtold2), and we have recently acquired both IVIS® Lumina and Spectrum systems for use by multiple research teams. These provide both bioluminescence and fluorescence imaging including depth resolved capabilities for the Spectrum. D-luciferin can cost $100 per 100 mg, but bulk purchases should allow better than $400 per gram, which is important for high-throughput screening.
Although BLI is simple, several properties require consideration. The light-emission can by characterized by parameters including area under the curve (AUC), maximum signal intensity, time to maximum intensity or light integration over a specified period. We routinely use a dose of 450 mg/kg administered subcutaneously into an anaesthetized nude mouse with imaging for a period of five minutes starting ten minutes after luciferin administration. Weak signals may require longer integration to achieve useful signal to noise, but many investigators prefer a constant acquisition method even though small tumors than provide essentially zero signal.
Supported in part by grants from the DOD Breast Cancer Initiative (IDEA award DAMD17-03-1-0343), the NIH Cancer Imaging Program (P20 CA86354 and U24 CA126608) and the Simmons Cancer Center. We are grateful to Drs. Li Liu, Robert Bollinger, Jerry Shay, and Peter Antich for bringing the vision of BLI to UT Southwestern.
3D-luciferin may be obtained from many sources as either synthetic or natural material. Sodium or potassium salts may be used. We have no evidence for differential quality. Other sources include D-Luciferin sodium salt (Catalog #10102; Biotium, Hayward, CA, USA); D-Luciferin Potassium Salt (P/N 122769) isolated from firefly (Caliper Life Sciences: http://www.caliperls.com/products/dluciferin-potassium-salt.htm)
4In earlier work we had used a TC245 Charge-coupled device camera-(Texas Instruments, Dallas TX) (7). and a system based on the French Audine astronomical camera with a high performance Kodak KAF-0402ME CCD (14).
5Other forms of anesthesia such as ketamine can also be used.
6Other doses of luciferin may be used. Caliper recommends a dose of 150 mg/kg for mice with its Lumina Imaging system. Our experience favors the higher dose. Caliper Lifesciences recommends 10 to 15 minutes between injection and imaging.
7The exposure time may be altered to avoid over-exposing intense signals or to detect weak signals. In practice, we may use anywhere from 1 to 30 mins. Many investigators like to maintain a constant imaging time, where 5 mins is typical.
8The macros are really outside the scope of this chapter and interested readers are referred to (8)
9Instructions derived from the instrument user manual.