Multicolor detection of rare tumor cells in blood using a novel flow cytometry‐based system

Watanabe, Masaru; Uehara, Y.; Yamashita, Naoto; Fujimura, Yuu; Nishio, Kaori; Sawada, Takeshi; Takeda, Kazuo; Koizumi, Fumiaki; Koh, Yasuhiro

doi:10.1002/cyto.a.22422

Cited by 33 publications

(39 citation statements)

References 39 publications

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“…Discrimination of CTCs from the bulk of the blood cells was achieved by negative enrichment using anti-human CD45 microbeads [30]. A typical example for the full gating strategy is shown in Figure 1C.…”

Section: Resultsmentioning

confidence: 99%

A novel flow cytometry-based cell capture platform for the detection, capture and molecular characterization of rare tumor cells in blood

Watanabe

Serizawa

Sawada³

et al. 2014

J Transl Med

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BackgroundPersonalized cancer treatment relies on the accurate detection of actionable genomic aberrations in tumor cells. Circulating tumor cells (CTCs) could provide an alternative genetic resource for diagnosis; however, the technical difficulties in isolating and analyzing rare CTCs have limited progress to date. In this preclinical study, we aimed to develop an improved capture system for molecular characterization of CTCs based on a novel cell sorting technology.MethodsWe developed a cell capture platform using On-chip Sort (On-Chip Biotechnologies), a novel bench-top cell sorter equipped with a disposable microfluidic chip. Spike-in experiments comprising a series of lung cancer cell lines with varying epithelial cell adhesion molecule (EpCAM) expression levels were conducted to assess the capture and purification efficiency of the platform. Samples were negatively enriched using anti-CD45-coated magnetic beads to remove white blood cells, followed by sample fixation and labeling. The enriched and labeled samples were then sorted by On-chip Sort based on cytokeratin, vimentin, and CD45 expression. Captured cells were immediately subjected to whole genome amplification followed by mutation analysis using deep targeted sequencing, and copy number analysis using quantitative polymerase chain reaction (qPCR).ResultsSpike-in experiments revealed an excellent overall mean capture rate of 70.9%. A 100% success rate in the detection of EGFR, KRAS and BRAF mutations from captured cells was achieved using pyrosequencing and deep sequencing. The mutant variant detection rates were markedly higher than those obtained with the CellSearch profile kit. qPCR analysis of amplified DNA demonstrated reproducible detection of copy number changes of the EGFR in captured tumor cells.ConclusionsUsing a novel cell sorter, we established an efficient and convenient platform for the capture of CTCs. Results of a proof-of-principle preclinical study indicated that this platform has potential for the molecular characterization of captured CTCs from patients.

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Section: Resultsmentioning

confidence: 99%

A novel flow cytometry-based cell capture platform for the detection, capture and molecular characterization of rare tumor cells in blood

Watanabe

Serizawa

Sawada³

et al. 2014

J Transl Med

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“…After a CD45-depletion cells were fixed and labelled with fluorescently labelled antibodies against CD45, EpCAM, and CK, helping to distinguish different levels of EpCAM expression. Additional antibodies against EMT-markers could also be used in this system, allowing a characterization of the tumor cells [72]. A high throughput system for EMT-CTC detection could also be a microchip filter device, which sorts cells for Caveolin-1, a marker, which was shown to be upregulated during EMT-process [73].…”

Section: New Ctc-detection Strategiesmentioning

confidence: 99%

The Significance of Epithelial-to-Mesenchymal Transition for Circulating Tumor Cells

Kölbl

Jeschke

Andergassen

2016

IJMS

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Epithelial to mesenchymal transition (EMT) is a process involved in embryonic development, but it also plays a role in remote metastasis formation in tumor diseases. During this process cells lose their epithelial features and adopt characteristics of mesenchymal cells. Thereby single tumor cells, which dissolve from the primary tumor, are enabled to invade the blood vessels and travel throughout the body as so called “circulating tumor cells” (CTCs). After leaving the blood stream the reverse process of EMT, the mesenchymal to epithelial transition (MET) helps the cells to seed in different tissues, thereby generating the bud of metastasis formation. As metastasis is the main reason for tumor-associated death, CTCs and the EMT process are in the focus of research in recent years. This review summarizes what was already found out about the molecular mechanisms driving EMT, the consequences of EMT for tumor cell detection, and suitable markers for the detection of CTCs which underwent EMT. The research work done in this field could open new roads towards combating cancer.

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“…This method is unable to capture CTCs of all cancer types, however, because some CTCs are known to be EpCAM negative [7,8]. Thus, many methods that do not apply anti-EpCAM antibodies have been under development worldwide to address this issue [9–13]. …”

Section: Introductionmentioning

confidence: 99%

Development of an automated size-based filtration system for isolation of circulating tumor cells in lung cancer patients

et al. 2017

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Circulating tumor cells (CTCs), defined as tumor cells circulating in the peripheral blood of patients with solid tumors, are relatively rare. Diagnosis using CTCs is expected to help in the decision-making for precision cancer medicine. We have developed an automated microcavity array (MCA) system to detect CTCs based on the differences in size and deformability between tumor cells and normal blood cells. Herein, we evaluated the system using blood samples from non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) patients. To evaluate the recovery of CTCs, preclinical experiments were performed by spiking NSCLC cell lines (NCI-H820, A549, NCI-H23 and NCI-H441) into peripheral whole blood samples from healthy volunteers. The recovery rates were 70% or more in all cell lines. For clinical evaluation, 6 mL of peripheral blood was collected from 50 patients with advanced lung cancer and from 10 healthy donors. Cells recovered on the filter were stained. We defined CTCs as DAPI-positive, cytokeratin-positive, and CD45-negative cells under the fluorescence microscope. The 50 lung cancer patients had a median age of 72 years (range, 48–85 years); 76% had NSCLC and 20% had SCLC, and 14% were at stage III disease whereas 86% were at stage IV. One or more CTCs were detected in 80% of the lung cancer patients (median 2.5). A comparison of the CellSearch system with our MCA system, using the samples from NSCLC patients, confirmed the superiority of our system (median CTC count, 0 versus 11 for CellSearch versus MCA; p = 0.0001, n = 17). The study results suggest that our MCA system has good clinical potential for diagnosing CTCs in lung cancer.

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Multicolor detection of rare tumor cells in blood using a novel flow cytometry‐based system

Cited by 33 publications

References 39 publications

A novel flow cytometry-based cell capture platform for the detection, capture and molecular characterization of rare tumor cells in blood

A novel flow cytometry-based cell capture platform for the detection, capture and molecular characterization of rare tumor cells in blood

The Significance of Epithelial-to-Mesenchymal Transition for Circulating Tumor Cells

Development of an automated size-based filtration system for isolation of circulating tumor cells in lung cancer patients

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