Copy Number Variation Analysis of Circulating Tumor Cells at a Single Cell Level Based on Hydrogel Encapsulation
Authors: Iwata Reito , Negishi Ryo , Tanaka Tsuyoshi , Matsunaga Tadashi , Yoshino Tomoko 
Published: February 2020

Circulating tumor cells (CTCs) are defined as tumor cells circulating in peripheral blood of patients with metastatic cancer. CTCs have potential as a biomarker for cancer diagnosis or drug discovery. Recently, much attention has been paid to genomic analyses such as copy number variation (CNV) analysis of CTCs, because it will provide important information related to treatment for personalized therapy. These analyses should be performed at a single cell level because of the heterogeneity of cancer cells. However, CTCs are extremely rare cell in blood (1 in 109blood cells), so highly efficient technique for CTC recovery and single-cell isolation is required. Our group has developed “microcavity array system” on which rare CTCs can be efficiently captured from whole blood on the basis of differences in the size and deformability between tumor and blood cells. Furthermore, a novel technique for a single cell manipulation using a photo-polymerized hydrogel, “hydrogel-based cell encapsulation” has been demonstrated. In this study, to evaluate the performance of “microcavity array system” and “hydrogel-based cell encapsulation”, whole genome amplification (WGA) was performed using single-CTCs isolated by our proposed method. Furthermore, the utility of the proposed method in CNV analysis were evaluated.
The microcavity array (φ= 8 μm, 4×103 cavities) was created by electroforming technique as microfabricated filter. The microcavity array was fabricated with PDMS structure equipped with a vacuum microchannel to apply negative pressure for cell entrapment. A549 cells (lung cancer cell line), SKBR-3 cells (breast cancer cell line) or MCF-7 cells (breast cancer cell line) were used as model CTCs. These cells were stained with fluorescent dyes, and suspended in PBS or healthy whole blood. These cell suspensions were introduced into the microcavity array at a flow rate of 150 µl/min. After cell recovery process, the number of cancer cells captured on the microcavity array was counted under a fluorescence microscope. As a results, more than 95% of cancer cells was successfully recovered on the microcavity array from PBS and whole blood. Then, poly (ethylene glycol) diacrylate (PEGDA) hydrogel was introduced onto the MCA. An excitation light (λ=365 nm) was irradiated to the targeted cancer cells for 30 seconds, leading to cross-linking reaction of PEGDA hydrogel and encapsulation of a single-cell. Solidified PEGDA hydrogel can be easily recovered from microcavity array by tweezers. Fluorescent microscopic analysis revealed that targeted single-cells were successfully encapsulated with PEGDA hydrogel. The hydrogel-based cell encapsulation process for 20 single-cells was completed within 10 min. To evaluate the effect of hydrogel on genome amplification, hydrogels with single-cells were subjected to the WGA, and the yields of WGA products were measured by PicoGreen assay. Genome amplification was also successfully carried out in all single-cells encapsulated on the hydrogel, and the yields of WGA products were the same with those obtained by using the micromanipulation as a conventional method. The amplification bias of WGA was evaluated based on the success rate of PCR amplification using 9 different primer sets (multiple loci). The PCR amplification of 9 fragments revealed that no significant bias was observed between WGA samples from single-cells isolated by hydrogel encapsulation and micromanipulation. Based on these results, we concluded that our single cell isolation method could be utilized for WGA at single cell level. Furthermore, quantitative PCR was performed to evaluate CNV analysis using SKBR-3 and MCF-7 cells, which have different HER2 status. As a result, the level of HER2 copy number in WGA products obtained from single-cells was similar with that from bulk analysis (103cells).Determination and monitoring of HER2 status in genome is well known to serve as a useful biomarker for diagnosis of breast cancer. Therefore, our proposed method for CNV analysis of CTCs is expected to be widely utilized towards real-time cancer diagnosis without repeating surgical biopsy.