Fluorescence Labeling of Circulating Tumor Cells with Folate Receptor Targeted Molecular Probes for Diffuse In Vivo Flow 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 in humans.