A simple biological imaging system for detecting viable human circulating tumor cells
Toru Kojima, Yuuri Hashimoto, Yuichi Watanabe, Shunsuke Kagawa, Futoshi Uno, Shinji Kuroda, Hiroshi Tazawa, Satoru Kyo, Hiroyuki Mizuguchi, Yasuo Urata, Noriaki Tanaka, Toshiyoshi Fujiwara
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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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 1

Schematic DNA structure of OBP-401 and selective replication of OBP-401 in human cancer cells.

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Schematic DNA structure of OBP-401 and selective replication of OBP-401 ...
(A) OBP-401 is a telomerase-specific replication-competent adenovirus variant, in which the hTERT promoter element drives expression of the E1A and E1B genes linked with an IRES, and the gfp gene is inserted under the CMV promoter into the E3 region for monitoring viral replication. hTERT-p, hTERT promoter; Ad-E1A, adenoviral E1A; Ad-E1B, adenoviral E1B; CMV-p, CMV promoter; poly-A, polyadenylation signal; ITR, inverted terminal repeat. (B) Relationship between hTERT expression and GFP fluorescence. Relative hTERT mRNA expression and GFP fluorescence in human tumor cell lines were determined by real-time RT-PCR analysis and fluorescence microplate reader, respectively. The relative hTERT mRNA expression ratios normalized by dividing the value of H1299 cells are presented for each sample. Relative GFP fluorescence was measured 48 hours after OBP-401 infection at an MOI of 10. (C) Time-lapse images of H1299 human lung cancer cells were recorded for 48 hours after OBP-401 infection at an MOI of 10. Selected images taken at the indicated time points show cell morphology by phase-contrast microscopy (top panels) and GFP expression under fluorescence microscopy (bottom panels). Original magnification, ×200.