Functional Imaging in Preclinical Models

The ability to apply imaging capabilities to cell and small animal models has enabled great strides in the biological sciences and expands the scope of biomarker assays of utility in drug discovery and development. Advances in imaging technologies, many of which were first developed for use in human healthcare rather than for laboratory science, can allow for visualization and quantification of molecular and cellular events noninvasively and in real time. Assessments of physiological endpoints such as vascular blood flow in angiogenesis models or tumor size and anatomy in cancer models can now be performed over time in the same animal (to monitor drug effect, for example), and thus the need for sacrifice prior to histological analysis is reduced or eliminated. Applications in small animal preclinical models have been enabled by miniaturization of imaging equipment and by expansion of the number of reporter probes and contrast agents.53 It is not within the scope of this chapter to provide an in-depth discussion of imaging technologies (see 3.31 Imaging); rather, examples of how imaging applications can be used in biomarker studies will be discussed in brief. Positron emission tomography and magnetic resonance imaging

Positron emission tomography (PET) and magnetic resonance imaging (MRI) are powerful technologies that have revolutionized clinical medicine and are now making inroads into the preclinical arena, expanding biomarker methodologies in the process. Cancer treatment and research have perhaps benefited the most from PET and MRI, as some aspects of tumor physiology and anatomy can now be monitored noninvasively and serially, in the same animal.54,55 PET imaging scanners are capable of measuring the presence and concentration of positron emitting isotopes in living tissues, and thus tracer probes can be synthetically generated for monitoring of molecular transport or enzymatic processes dynamically. The most mature tracer that utilizes PET imaging is a glucose derivative, 2-[18F]fluoro-2-deoxy -D-glucose (FDG), which is transported into metabolically active cells and tissues where it becomes trapped after phosphorylation by the hexokinase enzyme. Since tumors are usually highly metabolically active, FDG-PET imaging can be used to detect and subsequently monitor changes in tumor tissue metabolic activity. Likewise, another PET probe relevant to monitoring changes in tumor physiology is 3'-deoxy-3'[18F]fluorothymidine (FLT), which is a thymidine analog that is preferentially processed by cells that are actively replicating genomic DNA; thus antiproliferative effects induced by cancer targeted agents can be monitored. The application of PET in monitoring specific inducible gene expression events in vivo is rapidly growing via the generation of a number of radiolabeled molecular probes that are captured by specific enzymes or receptors, e.g., herpes tymidine kinase, dopamine D2 receptor, or somatostatin receptor,56'57 that are placed under the transcriptional control of regulatory sequences in transgenic models. Degradation of a specific protein in cells can also be monitored, as illustrated by the application of a 68Ga-labeled F(ab')2 fragment of the anti-HER2 therapeutic antibody trastuzumab (Herceptin). This was used to pharmacodynamically monitor heat shock protein inhibitor-induced HER2 protein degradation and subsequent recovery.58 With the advent of additional PET tracers and small animal scanners (micro-PET systems) with higher resolution (coupled with lowered costs), the range of biomarker studies that can be pursued preclinically will continue to expand.

Magnetic resonance imaging does not rely on reporter probes and is a versatile technology for measuring biophysical magnetic properties in tissues. The primary impact thus far in animal studies has been in imaging tumor anatomy and changes in tumor mass. However, MRI has also enabled great strides in the noninvasive imaging of tissue vasculature (especially tumor vascular changes). A variant of MRI called dynamic contrast enhanced (DCE) MRI relies on tracking time-dependent intensity changes of an injected contrast agent and thus provides information on several physiological parameters in tumor vasculature such as blood flow and vessel permeability. The resolution of MRI allows for imaging even in tissues deep within the body and thus DCE-MRI can be applied to various animal models as well as humans. The DCE-MRI method has been used to assess the activity of antiangiogenesis agents in animal models59 as well as in the clinic60 and thus can be added to the repertoire of biomarker approaches that are relevant in characterizing targets with known or predicted roles in angiogenesis. Optical imaging with fluorescent and luminescent probes

Measurement of specific molecular targets and processes in living cells and tissues, both microscopically and macroscopically, has been greatly enabled in the last decade by advances in molecular probes that can be optically tracked in real time and engineered to serve as reporters. The engineering of the Aequorea victoria green fluorescent protein (GFP) as a protein-based fluorescent probe in vivo was a key event in optical molecular imaging; subsequent genetic engineering has led to the GFP variants that emit at different wavelengths, and additional fluorescent proteins (such as more readily detectable, higher-wavelength red fluorescent proteins) have been isolated from other organisms.61,62 A key application of relevance to target biomarkers is the ability to generate specific fusion proteins containing a fluorescent protein tag; this can be used to track the subcellular localization of the fusion target of interest, and to follow spatial patterns dynamically. As an approach to target discovery, fluorescent proteins can be used in a variant of shotgun cloning, in which random fusions generated with expression libraries are expressed in a cell population of interest, allowing for visualization and subsequent cloning of the fusion partner motif that is driving a specific localization pattern of interest.63 Another application for fluorescent probes is in fluorescence resonance energy transfer (FRET), which provides a readout of close molecular interaction between two distinct fluorophores, often linked together in the same engineered polypeptide chain. FRET probes can provide real-time monitoring of enzymatic processes such as proteolysis64,65 and protein kinase activity.66,67

A limitation of utilizing fluorescent proteins in whole-body in vivo imaging, at least in mammalian species, is the relatively shallow depth of penetration (2 mm or less) afforded by the excitation/emission wavelengths of these proteins. Imaging based on bioluminescence has emerged as a complementary approach, particularly for whole-body imaging with aims of tracking the movement of cells throughout the body or monitoring tumor response after treatment with experimental agents. Bioluminescent imaging typically relies on monitoring enzymatic activity of the bioluminescent enzyme luciferase, from the firefly. Transfected cells that express the luciferase transgene can be visualized based on light transmitted; the method has been used, for example, to monitor clearance of engrafted tumor cells68 or to visualize the effects of antibacterial agents against labeled bacteria in vivo.69 As is the case with PET and fluorescent protein imaging, gene expression regulation can be visualized through the use of luciferase reporter genes.70 The bioluminescent method is fairly straightforward although somewhat limited in that absolute signal quantification is not feasible and enzyme substrate must be administered exogenously. Both fluorescent and bioluminescent in vivo imaging approaches utilize reporter gene transfection and thus as biomarker assays they are somewhat artificial. However the unique ability to monitor cell signaling and physiology noninvasively in real time ensures that these and other functional imaging modalities will continue to be powerful tools in cell-based assays in target or lead identification as well as in pharmacology studies in animals.

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