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

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-V-labeled 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 through the probe beam (Figure 1, inset). The cell count dropped from >200/min to <50/min within 20 min of injection, indicating rapid clearance (Figure 1, solid circles).

Figure 1.

Detection of circulating apoptotic cells with in vivo flow cytometry. (Solid circle) Camptothecin-treated MatLyLu cells (~1 × 10⁶) in 100 μL PBS were labeled with annexin-V probe conjugated to AF647 (Molecular Probes; 2.95 μg), washed, and injected into the mouse tail vein. Mice were anesthetized and placed on a heated stage (37°C). An appropriate vessel (usually a small artery) for obtaining measurements was chosen in the ear. Fluorescence signal was recorded as individual labeled cells passed through a slit of He – Ne laser (633 nm) focused across the blood vessel. Confocal detection of the excited fluorescence enabled continuous monitoring of labeled cells flowing through the vessel over time. Signal was detected by a photomultiplier tube and digitized for analysis with Matlab software developed in-house [1 – 3]. The number of annexin-V⁺ cells in the circulation was measured by taking sequential 60-sec data traces and plotted as a function of time after injection. Inset shows a representative data trace where two annexin-V⁺ cells could be detected during the 1-sec time period (A.U. denotes arbitrary unit). (Open square) Number of annexin-V⁺ cells detected in the mouse circulation when unlabeled, camptothecin-treated MatLyLu cells (~1 × 10⁶) in 100 μL PBS were injected, followed immediately by injection of the annexin-V probe (11.8 μg). (Cross) Control experiments with an injection of the annexin-V probe alone.

Next, unlabeled, camptothecin-treated apoptotic MatLyLu cells were introduced into the circulation, followed immediately by injection of the annexin-V probe. The cell count increased during the first 5 min (Figure 1, open squares) due to the in vivo labeling process. Subsequently, the cells were cleared with the same rate as with in vitro labeled cells. Control experiments with injection of annexin-V alone yielded negligible cell count (Figure 1, crosses).

When untreated, viable MatLyLu tumor cells were injected into the circulation, no apoptotic cells were detected initially. Circulating annexin-V⁺ cells were detected starting at 1 hr and reached a plateau at 3 hr (Figure 2, solid circles). Because the total number of circulating MatLyLu cells decreased precipitously during the first 3 hours [2], we conclude that the fraction of MatLyLu cells undergoing cell death steadily increases during this time. Analysis of extracted blood with standard flow cytometry indicated that about 9% of the injected cells were annexin-V⁺ at the 3-hr time point.

Figure 2.

(Solid circles) Time course of annexin-V⁺ cell count following injection of ~1 million untreated MatLyLu cells. Injection of the annexin-V probe at each time point (10 min, 1 hr, 2 hr, 3 hr, 5.5 hr, and 8.5 hr) was necessary because of its short circulation time [8]. Measurement was taken 5 min after each probe injection to allow binding of the probe to the apoptotic cells. (Standard errors were shown; n = 3). The experiments using ~1 million LNCaP prostate cancer cells and ~3 million leukemic Nalm-6 cells gave similar results (open squares and solid triangles, respectively; standard errors were not shown for the clarity of the figure).

Metastasis is an inefficient process [5]. Only a small fraction of the circulating tumor cells form metastasis in distant organs. Indeed, it has been reported that <0.1% tumor cells survive after being injected into the animal [6]. It remains unclear, however, whether tumor cell death initiates while in the circulation. Our results suggest that MatLyLu cells undergo cell death in circulation within 1–2 hr after injection, possibly due to lack of survival signal from cell adhesion, and the harsh environment imparted by the sheer stress [7]. The studies using LNCaP prostate cancer cells and Nalm-6 leukemic cells gave similar results (Figure 2, open squares and solid triangles, respectively). Because annexin-V labels both apoptotic cells and necrotic cells, further experiments with two-color in vivo flow cytometry are required to verify the mechanisms for the cell death. Nonetheless, a therapeutic strategy to prolong the tumor cell circulation time may force the tumor cells to undergo cell death, ultimately reducing metastasis.