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

Purpose We recently developed a new instrument called ‘diffuse in vivo flow 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 dye ex-vivo . In this work, we investigated the use of two folate receptor (FR)-targeted fluorescence molecular probes for in vivo labeling of FR+ CTCs for DiFC. Methods We 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 culture in vitro , in whole blood, and in mice in vivo . We also studied detectability of labeled cells with DiFC. Results Both EC-17 and Cy5-PEG-FR probes had high affinity for FR+ CTCs in cell culture in 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. Conclusions This 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.