Background: Liquid biopsy using noninvasive sampling of blood is rapidly advancing in cancer diagnostics. Circulating tumor cells (CTCs) are intact tumor-derived cells and are potentially utilized for further molecular analyses. Capturing rare CTCs with high purity, however, remains technically challenging. Here we report the results of preclinical evaluation of the multistep sorting method using On-chip Sort system (On-Chip Biotechnologies) for high-purity capture and isolation of rare cells in the blood for further molecular analyses.

Materials and Methods: Non-small-cell lung cancer cell lines PC-9, NCI-H1975 and A549 were used for this preclinical study. Cells were spiked into 4 mL of peripheral blood form healthy donors. After hemolysis and fixation, the samples were labeled with PE/Cy7-CD45 and FITC-cytokeratin (CK) antibodies and Hoechst for nuclei staining followed by three multiple sorting steps based on positive staining for CK and negative staining for CD45. For evaluating the potential of recovered tumor cells for genome sequencing, recovered NCI-H1975 cells, known to harbor EGFR L858R and T790M mutations, from spike-in samples were subjected to next-generation sequencing after whole genome amplification. To further establish the rare cells detection using the preserved blood samples, PC-9 cells were spiked the peripheral blood from healthy donors and transferred to the preservative tubes (Streck, Omaha, NE) and processed and sorted between 24 and 48 hours after the sample preparation.

Results: Up to 50 PC-9 and A549 cells were spiked into 4 mL of peripheral blood and recovery rate of both cells were as high as 70% and recovered white blood cells were as low as 20% after three cycles of sorting steps, suggesting the sufficient recovery and purity of tumor cells for further analyses. One NCI-H1975 cells were also spiked into 4 mL of peripheral blood and recovered cells were subjected to next-generation sequencing after whole genome amplification. Both EGFR L858R and T790M mutations were successfully called and mutant allele frequencies of these gene mutations were as high as those called from the control DNA which were extracted from 1000 NCI-H1975 cells. PC-9 cells (0, 10, 100, 500 cells) were spiked into 20 mL of peripheral blood and split into two preservative tubes and the recovery rate ranged from 55.0 to 61.3%, suggesting the robustness of the multistep sorting method when preservative tubes were used. Good correlation was also observed between the observed number and the expected number of cancer cells demonstrating the linearity with a correlation coefficient (R2) of 0.99.

Conclusions: We confirmed that rare cancer cells in the blood can be captured and isolated by the multistep sorting method using On-chip Sort system and can be utilized for further molecular analyses Results also suggest that this method allows us to deal with the preserved

Citation Format: Yasuhiro Koh, Mio Ikeda, Shunsuke Teraoka, Masayuki Ishige, Yuu Fujimura, Kazuo Takeda, Nahomi Tokudome, Yuichi Ozawa, Hiroki Ueda, Nobuyuki Yamamoto. High-purity isolation method of rare cells for molecular analyses using microfluidic chip type cell sorter [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2223.