Fluorescence Labeling of Circulating Tumor Cells with Folate Receptor Targeted Molecular Probes for DiffuseIn VivoFlow Cytometry

Abstract

AbstractPurposeWe recently developed a new instrument called ‘diffusein vivoflow cytometry’ (DiFC) for enumeration of rare fluorescently-labeled circulating tumor cells (CTCs) in small animals without drawing blood samples. Until now, we have used cell lines that express fluorescent proteins, or were pre-labeled with a fluorescent dyeex-vivo. In this work, we investigated the use of two folate receptor (FR)-targeted fluorescence molecular probes forin vivolabeling of FR+ CTCs for DiFC.MethodsWe used EC-17 and Cy5-PEG-FR fluorescent probes. We studied the affinity of these probes for L1210A and KB cancer cells, both of which over-express FR. We tested the labeling specificity in cells in culturein vitro, in whole blood, and in micein vivo. We also studied detectability of labeled cells with DiFC.ResultsBoth EC-17 and Cy5-PEG-FR probes had high affinity for FR+ CTCs in cell culturein vitro. However, only EC-17 had sufficient specificity for CTCs in whole blood. EC-17 labeled CTCs were also readily detectable in circulation in mice with DiFC.ConclusionsThis work demonstrates the feasibility of labeling CTCs for DiFC with a cell surface receptor targeted probe, greatly expanding the utility of the method for pre-clinical animal models. Because DiFC uses diffuse light, this method could be also used to enumerate CTCs in larger animal models and potentially even in humans.