Fluorescence Labeling of Circulating Tumor Cells with a Folate Receptor-Targeted Molecular Probe for Diffuse In Vivo Flo
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RESEARCH ARTICLE
Fluorescence Labeling of Circulating Tumor Cells with a Folate Receptor-Targeted Molecular Probe for Diffuse In Vivo Flow Cytometry Roshani A. Patil,1 Madduri Srinivasarao,2 Mansoor M. Amiji,3 Philip S. Low,2 Mark Niedre
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Department of Bioengineering, Northeastern University, Boston, MA, 02115, USA Department of Chemistry, Purdue University, West Lafayette, IN, 47906, USA 3 Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University, Boston, MA, 02115, USA 2
Abstract 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 a folate receptor (FR)-targeted fluorescence molecular probe for in vivo labeling of FR+ CTCs for DiFC. Procedures: We used EC-17, a FITC-folic acid conjugate that has been used in clinical trials for fluorescence-guided surgery. We studied the affinity of EC-17 for FR+ L1210A and KB cancer cells. We also tested FR− MM.1S cells. We tested the labeling specificity in cells in culture in vitro and in whole blood. We also studied the detectability of labeled cells in mice in vivo with DiFC. Results: EC-17 showed a high affinity for FR+ L1210A and KB cells in vitro. In whole blood, 85.4 % of L1210A and 80.9 % of KB cells were labeled above non-specific background with EC17, and negligible binding to FR− MM.1S cells was observed. In addition, EC-17-labeled CTCs were readily detectable in circulation in mice with DiFC. Conclusions: This work demonstrates the feasibility of labeling CTCs with a cell-surface receptor-targeted probe for DiFC, greatly expanding the potential utility of the method for preclinical 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. Key words: Circulating tumors cells (CTCs), Folate receptor, Diffuse imaging, Fluorescence, In vivo flow cytometry
Introduction Cancer metastasis is a multi-step process, by which tumor cells colonize distant organs and tissues. The circulatory system is one of the most common pathways, wherein tumor cells intravasate into the peripheral blood, circulate, and form metastases at secondary sites [1–3]. Circulating tumor cells (CTCs) in the bloodstream are
Correspondence to: Mark Niedre; e-mail: [email protected]
therefore of great interest in cancer research. Numerous studies have demonstrated that CTC numbers correlate with overall survival, disease progression, and response to treatment for many cancers [4–9]. CTCs are extremely rare: fewer than 1 CTC per milliliter of peripheral blood is considered prognostically negative [10]. Normally, CTCs are studied using “liquid biopsy,” where relatively small blood samples are drawn from the patient or small animal (in the case of
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