Real-time Detection of Circulating Apoptotic Cells by in Vivo Flow Cytometry

Abstract

Survival of cancer cells in the circulation is an important step in metastasis. However, the fate of circulating tumor cells is difficult to assess with conventional methods that require blood sampling. We report the first in situ measurement of circulating apoptotic cells in live animals using in vivo flow cytometry, a novel method [1–3] that enables real-time detection and quantification of circulating cells without blood extraction. Injected cancer cells undergo cell death within 1–2 hr after entering the mouse circulation. Apoptotic cells are rapidly cleared from the circulation with a half-life of ~10 min. Real-time monitoring of circulating apoptotic cells can be useful for detecting early changes in disease processes, as well as for monitoring response to therapeutic intervention. To detect circulating apoptotic cells in vivo, annexin-V conjugated to Alexa Fluor 647 (AF647) was used to label exposed phosphatidylserine on the cell surface. The long wavelength of AF647 fluorescence (Molecular Probes, OR) allows its detection through blood with minimum attenuation by red blood cells. In initial studies conducted to demonstrate that our instrument had sufficient sensitivity to detect individual annexin-Vlabeled apoptotic cells in vivo, we used MatLyLu prostate cancer cells pretreated with camptothecin [4] as a positive control. We verified that >80% of the camptothecin-treated cells undergo apoptosis by conventional flow cytometry (i.e., >80% of the treated cells were FITC–annexin V positive and propidium iodide negative). The camptothecin-treated cells were then labeled with the AF647-conjugated annexin-V and injected into the mouse intravenously. The circulating annexin-V + cells were measured by focusing a He–Ne laser beam onto an ear vessel and detecting the fluorescent bursts as individual cells flowed