Development of on-chip multi-imaging flow cytometry for identification of imaging biomarkers of clustered circulating tumor cells

Hyonchol Kim; Hideyuki Terazono; Yoshiyasu Nakamura; Kazuko Sakai; Akihiro Hattori; Masao Odaka; Mathias Girault; Tokuzo Arao; Kazuto Nishio; Yohei Miyagi; Kenji Yasuda

doi:10.1371/journal.pone.0104372

Development of on-chip multi-imaging flow cytometry for identification of imaging biomarkers of clustered circulating tumor cells

PLoS One. 2014 Aug 20;9(8):e104372. doi: 10.1371/journal.pone.0104372. eCollection 2014.

Authors

Affiliations

¹ Kanagawa Academy of Science and Technology, Takatsu, Kawasaki, Japan.
² Kanagawa Academy of Science and Technology, Takatsu, Kawasaki, Japan; Department of Biomedical Information, Division of Biosystems, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Chiyoda, Tokyo, Japan.
³ Molecular Pathology and Genetics Division, Kanagawa Cancer Center Research Institute, Asahi-ku, Yokohama, Japan.
⁴ Department of Genome Biology, School of Medicine, Kinki University, Osaka-Sayama, Osaka, Japan.

Abstract

An on-chip multi-imaging flow cytometry system has been developed to obtain morphometric parameters of cell clusters such as cell number, perimeter, total cross-sectional area, number of nuclei and size of clusters as "imaging biomarkers", with simultaneous acquisition and analysis of both bright-field (BF) and fluorescent (FL) images at 200 frames per second (fps); by using this system, we examined the effectiveness of using imaging biomarkers for the identification of clustered circulating tumor cells (CTCs). Sample blood of rats in which a prostate cancer cell line (MAT-LyLu) had been pre-implanted was applied to a microchannel on a disposable microchip after staining the nuclei using fluorescent dye for their visualization, and the acquired images were measured and compared with those of healthy rats. In terms of the results, clustered cells having (1) cell area larger than 200 µm2 and (2) nucleus area larger than 90 µm2 were specifically observed in cancer cell-implanted blood, but were not observed in healthy rats. In addition, (3) clusters having more than 3 nuclei were specific for cancer-implanted blood and (4) a ratio between the actual perimeter and the perimeter calculated from the obtained area, which reflects a shape distorted from ideal roundness, of less than 0.90 was specific for all clusters having more than 3 nuclei and was also specific for cancer-implanted blood. The collected clusters larger than 300 µm2 were examined by quantitative gene copy number assay, and were identified as being CTCs. These results indicate the usefulness of the imaging biomarkers for characterizing clusters, and all of the four examined imaging biomarkers-cluster area, nuclei area, nuclei number, and ratio of perimeter-can identify clustered CTCs in blood with the same level of preciseness using multi-imaging cytometry.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Biomarkers, Tumor / analysis*
Cell Line, Tumor
Diagnostic Imaging
Flow Cytometry / methods*
Male
Neoplastic Cells, Circulating / pathology*
Prostatic Neoplasms / diagnosis*
Prostatic Neoplasms / pathology
Rats

Substances

Biomarkers, Tumor

Grants and funding

This work was supported by Kanagawa Prefector's local basic science funding for the On-chip Cellomics Project at the Kanagawa Academy of Science and Technology. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.