MIR Preclinical Services: Bioluminescence technology

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TUMOR DIAGNOSIS

APOPTOSIS

P53 ACTIVATION

TRACKING METASTASIS

MIR has been licensed by the Xenogen Corporation to perform bioluminescence and fluorescence imaging for our clients, even if they do not have a license. MIR utilizes Xenogen's a state-of-the-art IVIS® imaging system to capture publication quality bioluminescence or fluorescence images of disease states and drug induced changes in physiology.

The Xenogen IVIS® system is ideally suited for biophotonic imaging, particularly bioluminescence (biolume) imaging. This system can image 3 animals at a time with an average scan time of one minute for all 3 animals. This high throughput imaging is ideal for tracking the spread of metastasis and tracking orthotopic syngeneic tumors and orthotopic human tumor xenografts in vivo. This technology is also an ideal instrument for the correlation of drug induced target modulation through the use of genetically altered syngeneic and xenograft cell lines. MIR Preclinical Services is a contract research organization (CRO) licensed by Xenogen to offer this imaging as a service to clients.

The IVIS® Imaging System is physically calibrated and measures absolute light emitted from an animal or sample. This allows MIR to make meaningful comparisons between different mice or experiments conducted at different times. This system also has Xenogens XFO-12 Fluorescence Option which provides the option of fluorescent imaging in addition to bioluminescent imaging for both in vivo and in vitro applications. MIR can readily image fluorescence from eGFP, FITC, Ds Red, Cell Tracker® orange, Cy5.5, Alexa fluor® 660 and 680 and indocanine Green as well as other similar dyes and fluorescent proteins.

Cells or animals can be genetically altered to express fluorescent proteins or enzymes capable of cleaving a luminescent substrate (luciferase). These genes can be constitutively expressed to indicate tumor burden or conditionally expressed and used as a reporter for a specific gene or function of interest. MIR has developed a number of assay systems for non-invasive assessment of drug function.

Applications

Tumor Diagnosis
Physiologic changes
Apoptosis
P53 Activation
Gene Expression
Tracking of Metasasis

Example images of xenograft cell lines that either constitutively express Luciferase or express Luciferase when a certain mechanism within the cell becomes active (reporter). Injecting the animals with luciferin cause the emission of light in luciferase expressing cell lines. Transgenic animals that utilize this technology are also available.

TUMOR DIAGNOSIS AND EFFICACY MEASUREMENT

This image is an example of a transgenic animal that utilizes biophotonic imaging to report proliferation. The Ef-luc transgenic animal (also called the Elux transgenic animal) will produce Luciferin in any proliferating tissue. This animal was crossed with a Nestin-TVa, a transgenic animal that develops gliomas when injected with an avian leukosis virus that causes the over expression of PDGF. The resulting transgenic animal has gliomas that emit a bioluminescence signal when proliferating. This image is an animal that shows disease progression over time.

Therapeutic effect can be measured when tumor regression correlates with a decrease in fluorescent intensity levels. Similar technology can also be used to measure changes in gene expression for a functional gene of interest.

The figure to the left shows an example using the Ef-luc transgenic mouse. This mouse has a luciferase reporter gene downstream of the E2F1 promoter. Cells with a pathologically active Rb pathway (tumor cells) are detected by their E2F1-driven expression of luciferase. Luciferase drives light production in vivo in the presence of luciferin.

Pharmacodynamics (primarily level II biomarkers)

Drug induced effects at the molecular level
Protein expression/degradation
Enzyme activity (proteases)
Protein-protein interactions (secondary to phosphorylation, etc – under development)

APOPTOSIS

Mitochondrial (left) and Death Receptor (right) pathways

MIR proprietary assay:

Bioluminescence imaging of apoptosis via caspase 3 activation

Attachment of a regulatory domain to a reporter gene results in silencing of reporter activity

Incorporation of a proteolytic cleavage site specific to a given protease enables release from the regulatory domain thus activating the reporter

Central role for caspase 3

Igney and Kramer Nature Reviews 2002

Strategy for Non-invasive Imaging of Apoptosis

Utilizing a proprietary caspase 3-sensitive bioluminescence reporter that is transfected into, and expressed in tumor cells, MIR has undertaken extensive validation experiments, using multiple standard treatments that span the spectrum of cytotoxic mechanistic classes. By correlating the image-based apoptotic response with later determined growth inhibition, we are able to correlate apoptosis with growth delay for temozolomide, paclitaxel, BCNU, doxorubicin and avastin treatments. MIR is a contract research organization with exclusive rights to this technology.

Fusion with ER silences luciferase activity due to sequestration

Activation of caspase 3 during apoptosis results in cleavage at DEVD site releasing luciferase

Free luciferase in the presence of luciferin generates bioluminescence which can be imaged in vitro and in vivo

Laxman, Hall, Bhoiana, Chenevert, Ross and Rehemtulla. Proc. Natl. Acad. Sci. USA99: 16551-16555 (2002)

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This image is an example of using biophotonic imaging to correlate disease progression. This is an example of a D54 human glioma tumor xenograft that constitutively expresses luciferase. The tissues emit light after the animal is given an injection of Luciferin.

In vivo bioluminescence image superimposed on a conventional photograph of a mouse with a subcutaneous Luciferase expressing (D54-Luc-ER) tumor (glioma) obtained before (A) and 60–75 min after (B) TRAIL treatment. Activation of caspase-3 was imaged non-invasively by tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL) treatment during tumor growth evaluation in mice.

P53 ACTIVATION

P53 is expressed ubiquitously but is sequestered and degraded under normal conditions.

DNA damage causes P53 accumulation (approx. 100-fold).

P53 transcription factor activity is induced, activating the MDM2 promoter and luciferase expression.

TRACKING METASTASIS

The recent technological advancements in biophotonic/bioluminescence imaging have allowed a concurrent increase in the use of preclinical models of metastasis, models that had traditionally been associated with tedious, expensive and highly variable serial sacrifice methods for efficacy determination. MIR has taken advantage of new imaging innovations by validating reproducibility and treatment response in mouse models of metastasis. Luciferase-expressing cell lines are utilized to determine appearance and site of metastases in bone and lung. These models rely on intracardiac, intravenous, or intrasplenic injection of tumor cells. MIR is currently validating breast, glioma, lung, melanoma, pancreas, prostate and ovarian orthotopic models of metastasis, enabling testing of early stage, metastasis prevention therapies.

Tracking in vivo metastatic tumor models can be of great importance in anti-metastatic drug studies. Bioluminescent and fluorescent (biophotonic) imaging is a rapid way of determining where cells migrate and and relative tumor burden at those sites*. Since Biophotonic imaging is non-invasive, the same cohort of animals can be studied over time. This reduces problems associated with variability in metastatic spread and growth rates. Biophotonic imaging can also be performed concurently with other imaging modalities such as CT and MRI which have greater resolution, are clinically relevant and can measure other pharmacodynamic parameters.

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