Edmond Richer

Validating Bioluminescence Imaging as a High-Throughput, Quantitative Modality for Assessing Tumor Burden

Bioluminescence imaging (BLI) is a highly sensitive tool for visualizing tumors, neoplastic development, metastatic spread, and response to therapy. Although BLI has engendered much excitement due to its apparent simplicity and ease of implementation, few rigorous studies have been presented to validate the measurements. Here, we characterize the nature of bioluminescence output from mice bearing subcutaneous luciferase-expressing tumors over a 4-week period. Following intraperitoneal or direct intratumoral administration of luciferin substrate, there was a highly dynamic kinetic profile of light emission. Although bioluminescence was subject to variability, strong correlations (r >.8, p <.001) between caliper measured tumor volumes and peak light signal, area under light signal curve and light emission at specific time points were determined. Moreover, the profile of tumor growth, as monitored with bioluminescence, closely resembled that for caliper measurements. The study shows that despite the dynamic and variable nature of bioluminescence, where appropriate experimental precautions are taken, single time point BLI may be useful for noninvasive, high-throughput, quantitative assessment of tumor burden.

The Role of Cdk5 in Neuroendocrine Thyroid Cancer

Medullary thyroid carcinoma (MTC) is a neuroendocrine cancer that originates from calcitonin-secreting parafollicular cells, or C cells. We found that Cdk5 and its cofactors p35 and p25 are highly expressed in human MTC and that Cdk5 activity promotes MTC proliferation. A conditional MTC mouse model was generated and corroborated the role of aberrant Cdk5 activation in MTC. C cell-specific overexpression of p25 caused rapid C cell hyperplasia leading to lethal MTC, which was arrested by repressing p25 overexpression. A comparative phosphoproteomic screen between proliferating and arrested MTC identified the retinoblastoma protein (Rb) as a crucial Cdk5 downstream target. Prevention of Rb phosphorylation at Ser807/Ser811 attenuated MTC proliferation. These findings implicate Cdk5 signaling via Rb as critical to MTC tumorigenesis and progression.

Antivascular effects of combretastatin A4 phosphate in breast cancer xenograft assessed using dynamic bioluminescence imaging and confirmed by MRI

Bioluminescence imaging (BLI) has found significant use in evaluating long‐term cancer therapy in small animals. We have now tested the feasibility of using BLI to assess acute effects of the vascular disrupting agent combretastatin A4 phosphate (CA4P) on luciferase‐expressing MDA‐MB‐231 human breast tumor cells growing as xenografts in mice. Following administration of luciferin substrate, there is a rapid increase in light emission reaching a maximum after about 6 min, which gradually decreases over the following 20 min. The kinetics of light emission are highly reproducible;however, following i.p. administration of CA4P (120 mg/kg), the detected light emission was decreased between 50 and 90%, and time to maximum was significantly delayed. Twenty‐four hours later, there was some recovery of light emission following further administration of luciferin substrate. Comparison with dynamic contrast‐enhanced MRI based on the paramagnetic contrast agent Omniscan showed comparable changes in the tumors consistent with the previous literature. Histology also confirmed shutdown of tumor vascular perfusion. We believe this finding provides an important novel application for BLI that could have widespread application in screening novel therapeutics expected to cause acute vascular changes in tumors.—Zhao, D., Richer, E., Antich, P. P., Mason, R. P. Antivascular effects of combretastatin A4 phosphate in breast cancer xenograft assessed using dynamic bioluminescence imaging and confirmed by MRI. FASEB J. 22, 2445–2451 (2008)

Iterative reconstruction method for light emitting sources based on the diffusion equation

Bioluminescent imaging (BLI) of luciferase-expressing cells in live small animals is a powerful technique for investigating tumor growth, metastasis, and specific biological molecular events. Three-dimensional imaging would greatly enhance applications in biomedicine since light emitting cell populations could be unambiguously associated with specific organs or tissues. Any imaging approach must account for the main optical properties of biological tissue because light emission from a distribution of sources at depth is strongly attenuated due to optical absorption and scattering in tissue. Our image reconstruction method for interior sources is based on the deblurring expectation maximization method and takes into account both of these effects. To determine the boundary of the object we use the standard iterative algorithm-maximum likelihood reconstruction method with an external source of diffuse light. Depth-dependent corrections were included in the reconstruction procedure to obtain a quantitative measure of light intensity by using the diffusion equation for light transport in semi-infinite turbid media with extrapolated boundary conditions.

Striatal dysregulation of Cdk5 alters locomotor responses to cocaine, motor learning, and dendritic morphology

Motor learning and neuro-adaptations to drugs of abuse rely upon neuronal signaling in the striatum. Cyclin-dependent kinase 5 (Cdk5) regulates striatal dopamine neurotransmission and behavioral responses to cocaine. Although the role for Cdk5 in neurodegeneration in the cortex and hippocampus and in hippocampal-dependent learning has been demonstrated, its dysregulation in the striatum has not been examined. Here we show that strong activation of striatal NMDA receptors produced p25, the truncated form of the Cdk5 co-activator p35. Furthermore, inducible overexpression of p25 in the striatum prevented locomotor sensitization to cocaine and attenuated motor coordination and learning. This corresponded with reduced dendritic spine density, increased neuro-inflammation, altered dopamine signaling, and shifted Cdk5 specificity with regard to physiological and aberrant substrates, but no apparent loss of striatal neurons. Thus, dysregulation of Cdk5 dramatically affects striatal-dependent brain function and may be relevant to non-neurodegenerative disorders involving dopamine neurotransmission.

BONE MATERIAL ULTRASOUND VELOCITY IS PREDICTIVE OF WHOLE BONE STRENGTH

In humans, bone strength is assessed indirectly by the noninvasive measurement of structure or mass. Recent clinical application of an ultrasonic critical-angle reflectometry technique (UCR) has demonstrated the measurement of the regional and directional distribution of mechanical stiffness. This study investigates the specific question: are these measurements of a local material level property predictive of the strength of whole bone? Maximum values of pressure wave velocity and breaking strength were recorded at two locations (midshaft and base of neck) on rat femurs from growing rats. The results demonstrate a strong empirical relationship between material-level ultrasound (US) velocity and whole bone mechanical strength. However, the US velocity at a specific bone site can be used to assess bone strength at that site only, explaining discrepancies in other published studies that negate a relationship between strength and US velocity. The results indicate an important role for US velocity measurement in clinical evaluation of bone health.

High-throughput quantitative bioluminescence imaging for assessing tumor burden.

Bioluminescence imaging (BLI) has emerged during the past 5 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.

Construction, calibration and evaluation of a tissue phantom with reproducible optical properties for investigations in light emission tomography

We describe in details the materials and methods used to construct, calibrate and evaluate tissue phantoms and light sources; we describe the method for acquiring the data and present quantitative results for light intensity reconstruction using the reproducible phantoms optical properties as a priori information. We consider a tissue phantom imaging with optical properties which match those for typical mouse tissues, as a useful part of light diffusion studies in heterogeneous biological media in order to obtain important parameters for any reconstruction technique. Solid tissue phantoms are particularly useful for the study of light-tissue interaction and are still an active area of investigation.

Development and Evaluation of an EMCCD Based Gamma Camera for Preclinical SPECT Imaging

The electron-multiplying charge-coupled device (EMCCD) offers improved quantum efficiencies (40 to 95%) over a broader range of wavelengths (400 to 900 nm) and a higher intrinsic resolution (<100 mum using photon counting) when compared to photomultiplier tubes. The electron gain achieved in the multiplication register of an EMCCD effectively reduces the readout noise to less than 1 electron/pixel, making them sensitive to single photoelectrons. Our prototype camera uses the Texas Instruments Impactrontrade EMCCD model TC253SPD-B0 (7.4 mum square pixels) which is cooled under vacuum to -50degC using a four stage Peltier and liquid heat exchanger. Shuttered lens-coupling is used to image the optical light from a 3 mm thick monolithic CsI(Tl) crystal. Precise clocking for the EMCCD is provided by a National Instruments FPGA controller (PCI-7811R) and LabVIEW FPGA module (version 8.0). A custom built electronics box contains the clock driver circuitry and 16-bit video board for digital conversion of the video signal. Our optical coupling method differs from other EMCCD SPECT systems by using lenses rather than fiber optic bundles for transfer. TC253 characterization tests measured a maximum gain just over 1000times, dark current rate of 0.14 e/p/s, read noise of 18.2 e/p, and spurious charge generation of 4 e/p. A light integration intrinsic resolution of 110 mum FWHM was measured. Light integration images of a line phantom using a single pinhole collimator were used for SPECT reconstruction. We found the relative high spurious charge generation and low quantum efficiency of the TC253 made it incapable of photon counting for low energy sources using lens coupling.

UTSW Small Animal Positron Emission Imager

A Small Animal Imager (SAI) for PET has been designed, built, tested in phantoms, and applied to investigations in mice and rats. The device uses principles based on gamma-ray induced scintillation in crossed fiber optic detectors connected to Position Sensitive Photomultiplier Tubes (PSPMT). Each detector consists of an epoxied stack of 28 layers of 135 round 1 mm BCF-10 scintillating plastic fibers. The overlap region forms a 13.5times13.5times2.8 cm3 detector volume. Scintillating light from the fibers is detected by two (X and Y directions) Hamamatsu R-2486 PSPMTs with 16 anode wires in each of two orthogonal directions. A centroid-finding algorithm gives the position of a light cluster on the face (photocathode) of a PSPMT. The accuracy of the reconstruction of an interaction position is essentially independent of light cluster position. This translates to a nearly isotropic photon response for the entire detector. The system has been used to test several 3D image reconstruction algorithms, software modifications, and improvements. The sensitivity (~12.6 cps/kBq at 9 cm inner diameter) and sub-millimeter spatial resolution (better than 1 mm in phantoms) obtained with an iterative algorithm incorporating system modeling make the SAI a relatively inexpensive high performance animal imager. The SAI is currently being used for imaging experiments in mice and rats