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A systematic approach to the development of fluorescent contrast agents for optical imaging of mouse cancer models

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Principles of fluorescence imaging

The use of fluorochrome-labeled optical agents such as labeled antibodies, receptor-binding ligands, small molecules, peptides, and activatable probes offers a flexible and direct imaging methodology. The fluorescent labels can be visualized by excitation with an appropriate light source and capture of the emitted photons with a charge-coupled device camera or other optical detector. Several commercially available imaging systems enable visualization of these probes in mice. These include

Near infrared fluorochromes

A key to enabling optical imaging has been the development of suitable NIR fluorochromes. Important criteria for effective optical imaging fluorochromes include excitation and emission maxima in the NIR between 700 and 900 nm, high quantum yield [3], [4], [5], [6], [7], chemical and optical stability, and suitable pharmacological properties including aqueous solubility, low nonspecific binding, rapid clearance of the free dye, and low toxicity. The dyes most commonly used for fluorescent optical

Targets and ligands

Many targeted optical probes have been described in the literature. Targets include cell surface receptors, metabolic pathways, hormone receptors, apoptotic markers, and enzymatic activities [11]. High-affinity probes may be developed by rational investigation, combinatorial chemical synthesis, or phage display. An effective agent reaches the target at a sufficient concentration and/or is retained there for a sufficient length of time to be visible at the time of imaging. Obstacles such as

Antibody conjugates

Many of the first fluorochrome-conjugated targeted imaging agents were antibodies. For example, indocyanine-conjugated monoclonal antibodies against cells derived from a squamous cell carcinoma have been used to image A431 cell xenografts in nude mice [13]. Detection of the xenografts was sensitive and specific. Cy3-, Cy5-, and Cy5.5-conjugated monoclonal antibodies have been used to direct SSEA-1 for detection of MH-15 teratocarcinoma xenografts [14]. In this study, fluorescence did

Tumor surface proteins

Tumor surface proteins offer diverse possibilities for targeting of optical probes. An excellent example is a receptor-binding ligand. Growth factors are a popular choice for optical imaging agents because, in addition to high-affinity targeting, the ligand and its fluorescent label are often internalized by the normal endocytic pathway, amplifying the signal in the tumor cells. Fluorochrome-labeled epidermal growth factor (EGF) has been a versatile tumor imaging agent because the epidermal

Peptides and small molecules

Because of their small size, convenience of handling, and attractive pharmacokinetic properties, peptides are useful agents for in vivo imaging. Peptides targeting integrins, a family of cell surface receptors that broadly regulate tumor growth, metastasis, and angiogenesis, have been successful agents for imaging neovascular density [34], [35], [36], [37], [38], [39], [40], [41]. Chen et al. [37] and Achilefu et al. [41] used an NIR peptide conjugate, arginine-glycine-aspartic acid (RGD),

Activatable probes

The last category of fluorescent agents, activatable probes or “molecular beacons”[43], [44], specifically yield a fluorescent signal only when activated by an enzyme target. Most activatable probes are protease substrates. Protease levels are elevated in the extracellular space of many tumors, where they play roles in invasion and metastasis [45], [46], [47], [48], [49] and present a physically accessible target. Typically, these probes contain multiple NIR fluorochromes coupled to peptide

Developing an optical imaging agent

Below, we will discuss some of the critical parameters involved in developing, validating, and using an optical imaging agent. We present an overview of the process as it applies to tumor imaging, using as an example the IRDye 800CW EGF imaging agent that we recently developed [27], [28]. Again, although we use this agent as an example, the principles described will be applicable to any dye-conjugate optical agent.

Conjugating probes with NIR dyes

Development of an optical targeting agent begins with covalent attachment of an NIR dye to the targeting compound. Many dyes are available with an N-hydroxysuccinimidyl (NHS) ester, which is activated for simple one-step coupling to free amines. NHS esterified NIR dyes may be purchased in bulk or in prepackaged labeling kits. Methods for removal of unreacted dye may include HPLC, FPLC, or dialysis. Purification will be dictated by the molecular weight and chemical properties of the conjugates.

Testing the performance and specificity of an optical imaging agent in vitro

Validation of targeting agent efficacy and specificity in vitro is an important prelude to animal studies. Specificity can be demonstrated on cells in culture or in suspension by blocking the target with an antibody or by competition with the unlabeled agent. In targeting somatostatin receptors, Becker et al. [53] used a flow cytometric assay to quantify binding of the agent. As mentioned above, Ntziachristos et al. [31] used whole-cell competition assays to demonstrate both probe specificity

Animal care and use

All research animals must be handled according to protocols that comply with the animal care and use regulations of the country and institution where the research will be performed. In the United States, these regulations are described in a document compiled by the National Institutes of Health Organization for Lab Animal Welfare, available at http://grants.nih.gov/grants/olaw/GuideBook.pdf.

Considerations for tumor model selection

An ideal tumor model system exhibits minimal background interference. Although much of the autofluorescence in the animal is ameliorated by imaging at NIR wavelengths, some anatomic regions inherently maintain higher fluorescent signals. For example, natural fluorescence from compounds in the animal’s diet accumulates and can be visualized in the abdominal cavity. Organs involved in clearance of the dye, such as the liver and kidney, may also accumulate signal. Tumors arising in areas remote

Reducing optical interference for tumor imaging

Chlorophyll, which is often present in animal chow, absorbs at 655 and 411 nm and fluoresces at 673 nm, producing strong signal in the abdominal cavity. For optimal fluorescent imaging performance, purified food formulations that do not contain plant products may be used. Fasting of the animal prior to imaging has been used in some studies and requires prior approval from the institutional committee governing animal care.

The hairless phenotype of the nude mouse makes it an ideal choice for NIR

Establishing tumors in the animal

For assays of tumorigenic and metastatic potential using cultured human cells, cells or tissue may be implanted in animals subcutaneously, intravenously, intraperitonally, or orthotopically (i.e., prostate cells implanted in mouse prostate). These assays are called xenografts, since they involve transplantation of cells, tissue, or organs from one species to another.

Depending on the aggressiveness of the cell line, we have established subcutaneous xenografts in mice by injection of 0.5–1 × 106 

Probe dosage and administration

Tumor type may dictate the optical imaging agent selected and its optimal parameters for use. For example, A431 epidermoid carcinoma cells express EGFR at a much higher level than normal cells. However, HeLa ovarian carcinoma cells have low expression of EGFR. If IRDye 800CW EGF is used as an optical probe for both of these cell types, binding of the labeled ligand would be expected to vary dramatically.

An optimal dosage of the imaging agent will afford the best signal-to-background, clearance,

Evaluation of dye and optical agent clearance

Performing initial imaging time courses following injection of the chosen targeting agent will establish the optimal time point for sensitive tumor analysis. First, the time course analysis begins with the unconjugated fluorochrome chosen for optical imaging, which should not be appreciably retained in the animal. An example of measurement of the clearance of IRDye 800CW is shown in Fig. 6. Other dyes may have different clearance characteristics. Second, a time course of agent clearance from

Confirming probe specificity in vivo

Clearance studies and optimization of timing will minimize nonspecific fluorescence, but imaging artifacts may be misinterpreted nonetheless. For example, an apparent tumor detected in the liver upon targeting with an antibody probe that pooled in the liver should be confirmed by a competition test. One method is to preinject tumor-bearing animals with an excess (for example, 100-fold) of the unlabeled form of the optical agent. The labeled agent is injected shortly thereafter. Probe

Conclusions

Fluorochrome-labeled molecular probes are valuable tools for noninvasive longitudinal study of tumorigenesis and metastasis, preclinical studies of the effects of therapeutic agents, and pharmacokinetic and pharmacodynamic studies of drug–target interactions. Because of this, these probes have significant potential for translation to human clinical use.

Several applications may expand the clinical utility of fluorochrome-labeled probes. For example, accurate definition of tumor margins is

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