A simple biological imaging system for detecting viable human circulating tumor cells
Toru Kojima, … , Noriaki Tanaka, Toshiyoshi Fujiwara
Toru Kojima, … , Noriaki Tanaka, Toshiyoshi Fujiwara
Published September 1, 2009
Citation Information: J Clin Invest. 2009;119(10):3172-3181. https://doi.org/10.1172/JCI38609.
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Technical Advance Oncology

A simple biological imaging system for detecting viable human circulating tumor cells

Abstract

The presence of circulating tumor cells (CTCs) in the peripheral blood is associated with short survival, making the detection of CTCs clinically useful as a prognostic factor of disease outcome and/or a surrogate marker of treatment response. Recent technical advances in immunocytometric analysis and quantitative real-time PCR have made it possible to detect a few CTCs in the blood; however, there is no sensitive assay to specifically detect viable CTCs. Here, we report what we believe to be a new approach to visually detect live human CTCs among millions of peripheral blood leukocytes, using a telomerase-specific replication-selective adenovirus expressing GFP. First, we constructed a GFP-expressing attenuated adenovirus, in which the telomerase promoter regulates viral replication (OBP-401; TelomeScan). We then used OBP-401 to establish a simple ex vivo method that was able to detect viable human CTCs in the peripheral blood. The detection method involved a 3-step procedure, including the lysis of rbc, the subsequent addition of OBP-401 to the cell pellets, and an automated scan using fluorescence microscopy. OBP-401 infection increased the signal-to-background ratio as a tumor-specific probe, because the fluorescent signal was amplified only in viable, infected human tumor cells, by viral replication. This GFP-expressing virus-based method is remarkably simple and allows precise enumeration of CTCs.

Authors

Toru Kojima, Yuuri Hashimoto, Yuichi Watanabe, Shunsuke Kagawa, Futoshi Uno, Shinji Kuroda, Hiroshi Tazawa, Satoru Kyo, Hiroyuki Mizuguchi, Yasuo Urata, Noriaki Tanaka, Toshiyoshi Fujiwara

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Figure 2

A simple method to detect telomerase-positive cells in the blood.

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A simple method to detect telomerase-positive cells in the blood. (A) St...
(A) Steps in the sample preparation for GFP fluorescence detection. Blood samples (5-ml samples) are collected in heparinized tubes and incubated with rbc lysis buffer containing ammonium chloride (NH4Cl) for 15 minutes. After centrifugation, cell pellets are mixed with 104 PFUs of OBP-401 and incubated at room temperature for another 24 hours. Cells are resuspended in 15 μl of PBS following centrifugation and then placed onto the slide under a coverslip. (B) System for automated fluorescence molecular imaging. The automated optical scan system serially captures segmented tile images in the area of the coverslip under fluorescence microscopy. (C) A high-resolution large view of the reconstructed tile images. The zoomed image allows easy visualization of GFP-expressing cells among millions of blood leukocytes. Original magnification, ×40 (left panels); ×100 (middle panels); ×400 (right panels).