Abstract 1605: Dielectrophoretic microwell array system for detection circulating tumor cells in patients with solid tumors and single cell analysis of detected circulating tumor cells
Authors: Sawada Takeshi , Morimoto Atsushi , Shimoyama Tatsu , Yamashita Toshinari , Mogami Toshifumi , Iijima Kazuki , Yunokawa Mayu , Kanda Shintaro , Akiyama Yasuyuki , Katayama Koji , Watanabe Masaru , Koh Yasuhiro , Tamura Kenji , Tamura Tomohide , Futami Toru , Koizumi Fumiaki 
Published: August 2015

Abstract
Background
Detection and analysis of circulating tumor cells (CTCs) is expected to be a non-invasive real-time monitoring of response to therapy and tumor cell status, however it remains technically challenging. Besides, biological feature of CTCs is still unclear. We have developed a capture system for detection and molecular characterization of single CTCs based on a high-density dielectrophoretic microwell array technology.
Material and methods
CTC enrichment was performed by density gradient centrifugation with RosetteSep®. After CTC enrichment, samples were loaded into the cell entrapment chamber, followed by application of 20 Vp-p AC voltage (1 MHz for 3 minutes), so as to entrap cells in the microwells using the dielectrophoretic force. Then samples were fixed and labeled with DAPI, FITC-anti-cytokeratin (CK), PE-anti-CD45 antibodies, and detected by image-based analysis using a fluorescence microscope. The results of the CTC enumeration were compared with those by CellSearch® system. In this study, we investigated the DAPI+, CK-, CD45- cells with tumor cell morphology in addition to cells with classical definition of CTCs (DAPI+, CK+, CD45-). Those cells were picked up by micropipette, and isolated single cells were subjected to whole genome amplification followed by sequencing in 50 cancer-related genes on the Ion Torrent PGM platform. Fifteen patients with metastatic breast cancer and 6 patients with metastatic non-small lung cancer were examined in this study.
Results
The number of CK-positive(+) and CK-negative(-) cells (DAPI+, CD45-) detected by our system ranged from 1 - 38 CTCs (median: 11 / 3 mL), and 100% (21 / 21) of the samples were above the threshold level (≧ 1 / 3 mL). On the other hand, with CellSearch® system, 57.9% of the samples had a ≧ 2 / 7.5 mL threshold level (median: 2 / 3 mL). In 2 blood samples from the healthy donors, both systems detected 0 CTC. Single cell sequencing was performed on 9 of the 21 samples. All analyzed samples contained cells with at least one mutation in 50 cancer-related mutations, indicating that those isolated single cells were possible CTCs including CK-negative(-) cells.
Discussion
We established a platform enable us to capture and characterize CTCs at the single-cell level. The results of the present study show that single cells isolated by our system are useful for further genomic analysis. In addition, the results of mutation analysis of isolated single CTCs suggested that CTCs have mutational heterogeneity in cancer-related genes. Further investigation is going to be conducted to evaluate the capability of capture and characterize CTCs in other types of cancer.
Citation Format: Takeshi Sawada, Atsushi Morimoto, Tatsu Shimoyama, Toshinari Yamashita, Toshifumi Mogami, Kazuki Iijima, Mayu Yunokawa, Shintaro Kanda, Yasuyuki Akiyama, Koji Katayama, Masaru Watanabe, Yasuhiro Koh, Kenji Tamura, Tomohide Tamura, Toru Futami, Fumiaki Koizumi. Dielectrophoretic microwell array system for detection circulating tumor cells in patients with solid tumors and single cell analysis of detected circulating tumor cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1605. doi:10.1158/1538-7445.AM2015-1605