Abstract 3415: Development of a new circulating tumor cell detection and isolation assay based on automated on-chip imaging flow cytometry technology
Authors: Miyagi Yohei , Kim Hyonchol , Arao Tokuzo , Terazono Hideyuki , Hayashi Masato , Otsu Takashi , Nishio Kazuto , Yasuda Kenji 
Published: August 2012

Abstract
Backgrounds: Counting of circulating tumor cells (CTCs) is now a promising tumor biomarker. Although CTCs could also serve as a source of tumor cells, this application is limited under the now established methods due to shortage and uncertain viability of CTCs identified and isolated. The aim of the present study is to develop a non-destructive approach to efficiently detect and isolate viable CTCs using the indices of their detailed shapes acquired from the imaging flow cytometry. Method: The device was designed based on the automated on-chip imaging flow cytometry system, consisted of a disposable microfludic cell sorting chip, a phase-contrast/fluorescent microscopy module, and a real-time image-processing unit with a 10,000 images/s high-speed camera. Following two kinds of samples were prepared to evaluate the method: in vitro cultured human leukemia HL60 cells labeled with GFP, and a peripheral venous blood obtained from a rat model of CTC consisted of tumor autografts of highly metastatic syngenic cancer cells labeled with GFP. After 3 weeks of implantation, 2 ml of heparinized blood were obtained from the subclavicular vein, and used for the analysis. We confirmed the existence of CTCs by culturing a part of blood sample followed by detection of GFP doped cancer cells grew up in vitro. Results and Discussion: HL60 cells in PBS flew smoothly through the microfludic chip at fluidic velocity up to 100 mm/sec, even at a high concentration of 5×107 cells/ml, and the high-speed camera clearly detected each single image of GFP doped cell. For blood samples, as direct injection of undiluted samples caused clog of microfluidic channels, we used the samples 3 times diluted with PBS, and we have successfully detected the fluorescent signals and the images of doped cancer cells at fluid velocity of 75 mm/sec with 50 µl of the diluted sample. Because the number of CTC is believed to be limited, the preceding devices for CTC analysis are based on enrichment of CTC with immunomagnetic technology or with size filtration, those may loss and also give some damage to CTCs. However, our imaging flowcytometry assay has succeeded in detection of CTCs to the single cell level with simultaneous detection and real-time analysis of single cells’ fluorescence intensities and their detailed shapes with 10,000 events/s intervals in the continuous flow of directly applied blood samples without enrichment steps. Although confirmation by cell culture from the isolated CTC is now undergoing, the results indicated that this technology could give to avoid cell damage and to obtain viable cells based on their new indices, i.e., shape and cluster formation. Conclusion: The system applied for this study based on the automated on-chip imaging flow cytometry with the cell image-based sorting with high-speed camera could be a promising tool for CTC analysis and widen the applications of CTCs in research field.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3415. doi:1538-7445.AM2012-3415