In Vivo Cellular Imaging Using Fluorescent Proteins

Abstract

Target-specifi c imaging probes represent a promising tool in the molecular imaging of human cancer. Fluorescently-labeled target-specifi c probes are useful in imaging cancers because of their ability to bind a target receptor with high sensitivity and specifi city. The development of probes relies upon preclinical testing to validate the sensitivity and specifi city of these agents in animal models. However, this process involves both conventional histology and immunohistochemistry, which require large numbers of animals and samples with costly handling. In this chapter, we describe a novel validation tool that takes advantage of genetic engineering technology, whereby cell lines are transfected with genes that induce the target cell to produce fl uorescent proteins with characteristic emission spectra, thus enabling their easy identifi cation as cancer cells in vivo. Combined with multicolor fl uorescence imaging, this can provide rapid validation of newly-developed exogenous probes that fl uoresce at different wavelengths. For example, the plasmid containing the gene encoding red fl uorescent protein (RFP) was transfected into cell lines previously developed to either express or not express specifi c cell surface receptors. Various antibody-based or ligandbased optical-contrast agents, with green fl uorophores were developed to concurrently target cancer cells and validate their positive and negative controls, such as the β - D -galactose receptor, HER1, and HER2 in a single animal/organ. Spectrally-resolved multicolor fl uorescence imaging was used to detect separate fl uorescence emission spectra from the exogenous green fl uorophore and RFP. Here, we describe the use of “co-staining” (matching the exogenous fl uorophore and the endogenous fl uorescent protein to the positive control cell line) and “counter-staining” (matching the exogenous fl uorophore to the positive control and the endogenous fl uorescent protein to the negative control cell line) to validate the sensitivity and specifi city of target-specifi c probes. Using these in vivo imaging techniques, we are able to determine the sensitivity and specifi city of target-specifi c optical contrast agents in several distinct animal models of cancer in vivo, thus exemplifying the versatility of our technique, while reducing the number of animals needed to conduct these experiments.