William M. Volcheck

Characterization and performance of a near-infrared 2-deoxyglucose optical imaging agent for mouse cancer models

Malignant neoplasms exhibit an elevated rate of glycolysis over normal cells. This characteristic can be exploited for optical imaging of tumors in mice. A near-infrared fluorophore, IRDye 800CW, emission maximum 794 nm, was conjugated to 2-deoxyglucose (2-DG). An immunofluorescent cell-based assay was used to evaluate specificity and sensitivity of the conjugate in cultured cell monolayers. Dose-dependent uptake was established with increasing concentrations of IRDye 800CW 2-DG for epithelial and prostate carcinomas. IRDye 800CW 2-DG was specifically blocked by an antibody against GLUT1 glucose transporter, and by excess unlabeled 2-DG or d-glucose. Signal was increased by a phorbol ester activator of glucose transport. Fluorescence microscopy data confirmed localization of the conjugate in the cytoplasm. Subsequent in vivo studies optimized dose, clearance, and timing for signal capture in nude mouse xenografts. In all cases, tumors were clearly imaged with good signal-to-noise characteristics. These data indicate that IRDye 800CW 2-DG is a broadly applicable optical imaging agent for in vivo imaging of neoplasms in mice.

A nonfluorescent, broad-range quencher dye for Förster resonance energy transfer assays

We report here a novel, water-soluble, nonfluorescent dye that efficiently quenches fluorescence from a broad range of visible and near-infrared (NIR) fluorophores in Förster resonance energy transfer (FRET) systems. A model FRET-based caspase-3 assay system was used to test the performance of the quencher dye. Fluorogenic caspase-3 substrates were prepared by conjugating the quencher, IRDye QC-1, to a GDEVDGAK peptide in combination with fluorescein (emission maximum approximately 540 nm), Cy3 (approximately 570 nm), Cy5 (approximately 670 nm), IRDye 680 (approximately 700 nm), IRDye 700DX (approximately 690 nm), or IRDye 800CW (approximately 790 nm). The Förster distance R(0) values are calculated as 41 to 65A for these dye/quencher pairs. The fluorescence quenching efficiencies of these peptides were determined by measuring the fluorescence change on complete cleavage by recombinant caspase-3 and ranged from 97.5% to 98.8%. The fold increase in fluorescence on caspase cleavage of the fluorogenic substrates ranged from 40 to 83 depending on the dye/quencher pair. Because IRDye QC-1 effectively quenches both the NIR fluorophores (e.g., IRDye 700DX, IRDye 680, IRDye 800CW) and the visible fluorophores (e.g., fluorescein, Cy3, Cy5), it should find broad applicability in FRET assays using a wide variety of fluorescent dyes.

Development of fluorescent contrast agents for optical imaging of mouse disease models

Optical imaging is a rapidly developing field of research aimed at non-invasively interrogating animals for disease progression, determining the effects of a drug on a particular pathology, assessing the pharmacokinetic behavior of a drug, or identifying molecular biomarkers of disease. One of the key components of molecular imaging is the development of specific, targeted imaging contrast agents to assess these biological processes. The development of robust fluorochrome-labeled optical agents is a process that is often underestimated in terms of its complexity. We describe here the development process and performance issues for three different optical agents: IRDye 800CW EGF (epidermal growth factor), IRDye(R) 800CW 2-DG (2-deoxy D-glucose), and an IRDye 680 BoneTagTM. In vitro competitive assays were developed for two of the markers to demonstrate specificity. Specificity was confirmed in animal studies. Uptake of IRDye 800CW 2-DG was also examined by near-infrared confocal microscopy. Histological examinations were performed on target and non-target tissues following the completion of the imaging studies. The issues unique to the development of each labeled marker are discussed.