X-ray enabled detection and eradication of circulating tumor cells with nanoparticles

The early detection and eradication of circulating tumor cells (CTCs) play an important role in cancer metastasis management. This paper describes a new nanoparticle-enabled technique for integrated enrichment, detection and killing of CTCs by using magnetic nanoparticles and bismuth nanoparticles, X-ray fluorescence spectrometry, and X-ray radiation. The nanoparticles are modified with tumor targeting agents and conjugated with tumor cells through folate receptors over-expressed on cancer cells. A permanent micro-magnet is used to collect CTCs suspended inside a flowing medium that contains phosphate buffered saline (PBS) or whole blood. The characteristic X-ray emissions from collected bismuth nanoparticles, upon excitation with collimated X-rays, are used to detect CTCs. Results show that the method is capable of selectively detecting CTCs at concentrations ranging from 100-100,000cells/mL in the buffer solution, with a detection limit of ～100CTCs/mL. Moreover, the dose of primary X-rays can be enhanced to kill the localized CTCs by radiation induced DNA damage, with minimal invasiveness, thus making in vivo personalized CTC management possible.     

Sensitive and Specific Biomimetic Lipid Coated Microfluidics to Isolate Viable Circulating Tumor Cells and Microemboli for Cancer Detection

Here we presented a simple and effective membrane mimetic microfluidic device with antibody conjugated supported lipid bilayer (SLB) “smart coating” to capture viable circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) directly from whole blood of all stage clinical cancer patients. The non-covalently bound SLB was able to promote dynamic clustering of lipid-tethered antibodies to CTC antigens and minimized non-specific blood cells retention through its non-fouling nature. A gentle flow further flushed away loosely-bound blood cells to achieve high purity of CTCs, and a stream of air foam injected disintegrate the SLB assemblies to release intact and viable CTCs from the chip. Human blood spiked cancer cell line test showed the ~95% overall efficiency to recover both CTCs and CTMs. Live/dead assay showed that at least 86% of recovered cells maintain viability. By using 2 mL of peripheral blood, the CTCs and CTMs counts of 63 healthy and colorectal cancer donors were positively correlated with the cancer progression. In summary, a simple and effective strategy utilizing biomimetic principle was developed to retrieve viable CTCs for enumeration, molecular analysis, as well as ex vivo culture over weeks. Due to the high sensitivity and specificity, it is the first time to show the high detection rates and quantity of CTCs in non-metastatic cancer patients. This work offers the values in both early cancer detection and prognosis of CTC and provides an accurate non-invasive strategy for routine clinical investigation on CTCs.     

Quantification of Rare Cancer Cells in Patients With Gastrointestinal Cancer by Nanostructured Substrate

Detecting the cancer cells in the peripheral blood, i.e. circulating tumor cell (CTC), have been considered as the “liquid biopsy” and become a particular area of focus. A deep insight into CTC provides a potential alternative method for early diagnosis of solid tumor. Previous studies showed that CTC counts could be regarded as an indicator in tumor diagnosis, predicting clinical outcomes and monitoring treatment responses. In this report, we utilize our facile and efficient CTC detection device made of hydroxyapatite/chitosan (HA/CTS) for rare cancer cells isolation and enumeration in clinical use. A biocompatible and surface roughness controllable nanofilm was deposited onto a glass slide to achieve enhanced topographic interactions with nanoscale cellular surface components, anti-EpCAM (epithelial cell adhesion molecule, EpCAM) were then coated onto the surface of nanosubstrate for specific capture of CTCs. This device performed a considerable and stable capture yields. We evaluated the relationship performance between serial CTC changes and the changes of tumor volume/serum tumor marker in gastrointestinal cancer patients undergoing anti-cancer treatments. The present study results showed that changes in the number of CTC were associated with tumor burden and progression. Enumeration of CTCs in cancer patients may predict clinical response. Longitudinal monitoring of individual patients during the therapeutic process showed a close correlation between CTC quantity and clinical response to anti-cancer therapy. Effectively capture of this device is capable of CTCs isolation and quantification for monitoring of cancer and predicting treatment response.     

A label-free microfluidic chip for the highly selective isolation of single and cluster CTCs from breast cancer patients

BackgroundCirculating tumor cells (CTCs) existing in peripheral blood can be used to predict the prognosis and survival of cancer patients. The study was designed to detect circulating tumor cells and circulating tumor single cell genes by applying microfluidic chip technology. It was used to explore the clinical application value in breast cancer.MethodsWe have developed a size-based CTCs sorting microfluidic chip, which contains a hexagonal array and a micro-pipe channel array to isolate and confirm both single CTCs and CTCs clusters. The sorting performance of the as-fabricated chip was tested by analyzing the clinical samples collected from 129 breast cancer patients and 50 healthy persons.ResultsIn this study, the chip can detect different immunophenotypes of CTCs in breast cancer patients. It was found that the new microfluidic device had high sensitivity (73.6%) and specificity (82.0%) in detecting CTCs. By detecting the blood samples of 129 breast cancer patients and 50 healthy blood donors, it was found that the number of CTCs was not associated with clinical factors such as age, gender, pathological type, and tumor size of breast cancer patients (P > 0.05), but was associated with TNM staging of breast cancer, with or without metastasis (P < 0.005). There was a statistically significant difference in the number of CTCs between luminal A (ER+/PR+/HER2-) and HER-2+ (ER-/PR-/HER2+) (P < 0.05). The best cut-off level distinguished by CTC between the breast cancer patients and the healthy persons was 3.5 cells/mL, with 0.845 for AUC-ROC, 0.790–0.901 for 95% CI, 73.6% for sensitivity, and 82% for specificity (P = 0.000). The combination of CTC, CEA, CA125 and CA153 can provide more effective breast cancer screening.ConclusionsThe CTCs analysis method presented here doesn''t rely on the specific antibody, such as anti-EpCAM, which would avoid the missed inspection caused by antibody-relied methods and offer more comprehensive biological information for clinical breast cancer diagnosis and treatment.     

SELEX Aptamer Used as a Probe to Detect Circulating Tumor Cells in Peripheral Blood of Pancreatic Cancer Patients

Many studies have shown that the quantity and dynamics of circulating tumor cells (CTCs) in peripheral blood of patients afflicted with solid tumours have great relevance in therapeutic efficacy and prognosis. Different methods based on various strategies have been developed to isolate and identify CTCs, but their efficacy needs to be improved because of the rarity and complexity of CTCs. This study was designed to examine the possibility of using a SELEX aptamer (BC-15) as a probe to identify rare CTCs out of background nucleated cells. Aptamer BC-15 was selected from a random oligonucleotide library screened against human breast cancer tissue. Fluorescence staining showed that BC-15 had a high affinity for nuclei of human cancer cell lines of various origins as well as CTCs isolated from pancreatic cancer patients, whereas its binding capacity for non-tumor breast epithelial cells and leukocytes was almost undetectable. BC-15⁺/CD45^- cells in cancer patient blood were also found to be cytokeratins 18-positive and aneuploid by immunofluorescence staining and fluorescent in situ hybridization, respectively. Finally, the aptamer method was compared with the well-established anti-cytokeratin method using 15 pancreatic cancer patient blood samples, and enumeration indicated no difference between these two methods. Our study establishes a novel way to identify CTCs by using a synthetic aptamer probe. This new approach is comparable with the anti-cytokeratin-based CTC identification method.     

Predicting therapy response in live tumor cells isolated with the flexible micro spring array device

Cells disseminated from primary epithelial tumors into peripheral blood, called circulating tumor cells (CTCs), can be monitored to assess metastases and to provide a surrogate marker of treatment response. Here, we demonstrate how the flexible micro spring array (FMSA) device—a novel microfluidic device that enriches CTCs by two physical parameters: size and deformability—could be used in the rational development of treatment intervention and as a method to study the fundamental biology of CTCs. Cancer cells of different origins were spiked into healthy samples of donor blood to mimic blood samples of metastatic cancer patients. This spiked human blood was filtered using the FMSA device, and the recovered cells were successfully expanded in vitro and in a novel in vivo system. A series of experiments were performed to characterize these cells and to investigate the effect of chemotherapy on the resulting cultures. As few as 20 colon cancer cells in 7.5 mL blood could be isolated with the FMSA device, expanded both in vitro and in vivo and used at 25 cells per well to obtain significant and reliable chemosensitivity data. We also show that isolating a low number of viable patient CTCs and maintaining them in culture for a few weeks is possible. The isolation of viable cancer cells from human blood using the FMSA device provides a novel and realistic means for studying the biology of viable CTCs and for testing drug efficacy on these rare cells—a hypothesis that can be tested in future clinical trials.     

基于微纳技术的循环肿瘤细胞免疫检测新方法北大核心CSCD

本研究旨在探索一种高灵敏度、高特异性检测循环肿瘤细胞(circulating tumor cells, CTCs)的免疫检测新方法,以尽早地检出结直肠癌,提高该疾病的检出率。首先制备含有线性微柱结构的微芯片,通过在其表面孵育氧化石墨烯-链霉亲和素(graphite oxide-streptavidin, GO-SA)及偶联广谱一抗(antibody1, Ab₁),即上皮特异性黏附分子(epithelial cell adhesion molecule, EpCAM)单克隆抗体以捕获CTCs。运用羧基化多壁碳纳米管(carboxylated multi-walled carbon nanotubes, MWCNTs-COOH)与结直肠癌相关抗体,即特异性二抗(antibody 2, Ab₂)偶联制备抗体复合物。在捕获CTCs的微芯片上孵育该抗体复合物,构建以Ab₁-CTCs-Ab₂为主体的超级三明治结构,通过电化学工作站检测并验证其高灵敏度和高特异性。结果发现,在免疫传感器的构建中结合应用微纳技术,极大地提高了CTCs的检测灵敏度和特异性。本研究验证了该免疫传感器应用于临床血样检测的可行性,并通过该免疫传感器对结直肠癌患者外周血中CTCs进行检测和计数。结果表明,基于微纳技术的超级三明治式免疫传感器为CTCs的检测提供了新的途径,对临床工作中的疾病诊断及病情实时监控方面均具有潜在的应用价值。     

Clinical Validation of an Ultra High-Throughput Spiral Microfluidics for the Detection and Enrichment of Viable Circulating Tumor Cells

Background

Circulating tumor cells (CTCs) are cancer cells that can be isolated via liquid biopsy from blood and can be phenotypically and genetically characterized to provide critical information for guiding cancer treatment. Current analysis of CTCs is hindered by the throughput, selectivity and specificity of devices or assays used in CTC detection and isolation.

Methodology/Principal Findings

Here, we enriched and characterized putative CTCs from blood samples of patients with both advanced stage metastatic breast and lung cancers using a novel multiplexed spiral microfluidic chip. This system detected putative CTCs under high sensitivity (100%, n = 56) (Breast cancer samples: 12–1275 CTCs/ml; Lung cancer samples: 10–1535 CTCs/ml) rapidly from clinically relevant blood volumes (7.5 ml under 5 min). Blood samples were completely separated into plasma, CTCs and PBMCs components and each fraction were characterized with immunophenotyping (Pan-cytokeratin/CD45, CD44/CD24, EpCAM), fluorescence in-situ hybridization (FISH) (EML4-ALK) or targeted somatic mutation analysis. We used an ultra-sensitive mass spectrometry based system to highlight the presence of an EGFR-activating mutation in both isolated CTCs and plasma cell-free DNA (cf-DNA), and demonstrate concordance with the original tumor-biopsy samples.

Conclusions/Significance

We have clinically validated our multiplexed microfluidic chip for the ultra high-throughput, low-cost and label-free enrichment of CTCs. Retrieved cells were unlabeled and viable, enabling potential propagation and real-time downstream analysis using next generation sequencing (NGS) or proteomic analysis.     

CA19-9及CA72-4联合检测在胰腺癌诊断中的应用价值

目的:探究联合检测血清糖类抗原(CA)19-9和CA72-4水平在胰腺癌诊断中的应用价值。方法:回顾性选取我院2016年1月～2017年12月收治的72例胰腺癌患者作为胰腺癌组,以同期住院的68例良性胰腺病患者作为良性胰腺疾病组,同时纳入67例健康体检者作为对照组。检测三组人群血清CA19-9和CA72-4水平,采用受试者工作特征曲线(ROC曲线)及曲线下面积(AUC)分析评估各单项检测指标及联合检测指标对胰腺癌特异性诊断的价值。结果:胰腺癌组患者血清CA19-9和CA72-4水平分别为(137.69±25.32)U/mL和(6.96±1.25)U/mL,显著高于良性胰腺疾病组和对照组(P0.05)。血清CA19-9和CA72-4联合检测诊断胰腺癌的ROC曲线AUC高于其单独检测(P0.05),CA19-9和CA72-4的最佳临界值分别为86.94 U/m L和4.23 U/m L,此时联合检测诊断胰腺癌的敏感性为94.7%,特异性为95.2%。结论:联合检测血清CA19-9和CA72-4诊断胰腺癌的临床价值明显优于其单独检测。     

Development of an Automated and Sensitive Microfluidic Device for Capturing and Characterizing Circulating Tumor Cells (CTCs) from Clinical Blood Samples

Current analysis of circulating tumor cells (CTCs) is hindered by sub-optimal sensitivity and specificity of devices or assays as well as lack of capability of characterization of CTCs with clinical biomarkers. Here, we validate a novel technology to enrich and characterize CTCs from blood samples of patients with metastatic breast, prostate and colorectal cancers using a microfluidic chip which is processed by using an automated staining and scanning system from sample preparation to image processing. The Celsee system allowed for the detection of CTCs with apparent high sensitivity and specificity (94% sensitivity and 100% specificity). Moreover, the system facilitated rapid capture of CTCs from blood samples and also allowed for downstream characterization of the captured cells by immunohistochemistry, DNA and mRNA fluorescence in-situ hybridization (FISH). In a subset of patients with prostate cancer we compared the technology with a FDA-approved CTC device, CellSearch and found a higher degree of sensitivity with the Celsee instrument. In conclusion, the integrated Celsee system represents a promising CTC technology for enumeration and molecular characterization.     

Size-Based Isolation of Circulating Tumor Cells in Lung Cancer Patients Using a Microcavity Array System

Background

Epithelial cell adhesion molecule (EpCAM)-based enumeration of circulating tumor cells (CTC) has prognostic value in patients with solid tumors, such as advanced breast, colon, and prostate cancer. However, poor sensitivity has been reported for non-small cell lung cancer (NSCLC). To address this problem, we developed a microcavity array (MCA) system integrated with a miniaturized device for CTC isolation without relying on EpCAM expression. Here, we report the results of a clinical study on CTCs of advanced lung cancer patients in which we compared the MCA system with the CellSearch system, which employs the conventional EpCAM-based method.

Methods

Paired peripheral blood samples were collected from 43 metastatic lung cancer patients to enumerate CTCs using the CellSearch system according to the manufacturer’s protocol and the MCA system by immunolabeling and cytomorphological analysis. The presence of CTCs was assessed blindly and independently by both systems.

Results

CTCs were detected in 17 of 22 NSCLC patients using the MCA system versus 7 of 22 patients using the CellSearch system. On the other hand, CTCs were detected in 20 of 21 small cell lung cancer (SCLC) patients using the MCA system versus 12 of 21 patients using the CellSearch system. Significantly more CTCs in NSCLC patients were detected by the MCA system (median 13, range 0–291 cells/7.5 mL) than by the CellSearch system (median 0, range 0–37 cells/7.5 ml) demonstrating statistical superiority (p = 0.0015). Statistical significance was not reached in SCLC though the trend favoring the MCA system over the CellSearch system was observed (p = 0.2888). The MCA system also isolated CTC clusters from patients who had been identified as CTC negative using the CellSearch system.

Conclusions

The MCA system has a potential to isolate significantly more CTCs and CTC clusters in advanced lung cancer patients compared to the CellSearch system.     

A Novel Approach for the Detection and Genetic Analysis of Live Melanoma Circulating Tumor Cells

Background

Circulating tumor cell (CTC) detection and genetic analysis may complement currently available disease assessments in patients with melanoma to improve risk stratification and monitoring. We therefore sought to establish the feasibility of a telomerase-based assay for detecting and isolating live melanoma CTCs.

Methods

The telomerase-based CTC assay utilizes an adenoviral vector that, in the presence of elevated human telomerase activity, drives the amplification of green fluorescent protein. Tumor cells are then identified via an image processing system. The protocol was tested on melanoma cells in culture or spiked into control blood, and on samples from patients with metastatic melanoma. Genetic analysis of the isolated melanoma CTCs was then performed for BRAF mutation status.

Results

The adenoviral vector was effective for all melanoma cell lines tested with sensitivity of 88.7% (95%CI 85.6-90.4%) and specificity of 99.9% (95%CI 99.8-99.9%). In a pilot trial of patients with metastatic disease, CTCs were identified in 9 of 10 patients, with a mean of 6.0 CTCs/mL. At a cutoff of 1.1 CTCs/mL, the telomerase-based assay exhibits test performance of 90.0% sensitivity and 91.7% specificity. BRAF mutation analysis of melanoma cells isolated from culture or spiked control blood, or from pilot patient samples was found to match the known BRAF mutation status of the cell lines and primary tumors.

Conclusions

To our knowledge, this is the first report of a telomerase-based assay effective for detecting and isolating live melanoma CTCs. These promising findings support further studies, including towards integrating into the management of patients with melanoma receiving multimodality therapy.     

肺癌循环肿瘤细胞的单细胞EGFR基因突变检测

循环肿瘤细胞（Circulating tumor cells,CTCs）是从肿瘤原发病灶脱落并侵入外周血循环的肿瘤细胞。由于CTCs存在较大的异质性,其与癌症发展转移密切相关,但目前尚缺乏有效的CTCs单细胞异质性检测方法。鉴于此,本文发展了在单细胞层面对CTCs进行基因突变的检测方法并用于单个肺癌CTC的EGFR（Epidermal growth factor receptor）基因突变检测。首先用集成式微流控系统完成血液中稀有CTCs的捕获,接着将CTCs释放入含有多个微孔的微阵列芯片中,得到含有单个CTC的微孔,通过显微操作将单个CTC转入PCR管内完成单细胞基因组的放大,并进行单细胞的EGFR基因突变检测。以非小细胞肺癌细胞系A549、NCI-H1650和NCI-H1975为样本,通过芯片与毛细管修饰、引物扩增条件（复性温度、循环次数）的优化,结果显示在复性温度59℃、30个循环次数的条件下,引物扩增效果最优。利用该方法成功地对非小细胞肺癌（Non-small cell lung cancer, NSCLC）患者的血液样本进行了测试。从患者2 mL血液中获取5个CTCs,分别对其EGFR基因的第18、19、20、21外显子进行测序,发现该患者CTCs均为EGFR野生型。研究结果证明此检测方法可以灵敏地用于单个CTC基因突变的检测,在临床研究上具有重要的指导意义。     

Clinical Implications and Future Perspectives of Circulating Tumor Cells and Biomarkers in Clinical Outcomes of Colorectal Cancer

Colorectal cancer (CRC) is a major public health problem. Early CRC detection, pretherapeutic responsiveness prediction, and postoperative micrometastasis monitoring are the hallmarks for successful CRC treatment. Here, the methodologies used for detecting circulating tumor cells (CTCs) from CRC are reviewed. In addition to the traditional CRC biomarkers, the persistent presence of posttherapeutic CTCs indicates resistance to adjuvant chemotherapy and/or radiotherapy; hence, CTCs also play a decisive role in the subsequent relapse of CRC. Moreover, the genetic and phenotypic profiling of CTCs often differs from that of the primary tumor; this difference can be used to select the most effective targeted therapy. Consequently, studying CTCs can potentially individualize treatment strategies for patients with CRC. Therefore, CTC detection and characterization may be valuable tools for refining prognosis, and CTCs can be used in a real-time tumor biopsy for designing individually tailored therapy against CRC.     

Circulating Tumor Cells: Clinically Relevant Molecular Access Based on a Novel CTC Flow Cell

Background

Contemporary cancer diagnostics are becoming increasing reliant upon sophisticated new molecular methods for analyzing genetic information. Limiting the scope of these new technologies is the lack of adequate solid tumor tissue samples. Patients may present with tumors that are not accessible to biopsy or adequate for longitudinal monitoring. One attractive alternate source is cancer cells in the peripheral blood. These rare circulating tumor cells (CTC) require enrichment and isolation before molecular analysis can be performed. Current CTC platforms lack either the throughput or reliability to use in a clinical setting or they provide CTC samples at purities that restrict molecular access by limiting the molecular tools available.

Methodology/Principal Findings

Recent advances in magetophoresis and microfluidics have been employed to produce an automated platform called LiquidBiopsy®. This platform uses high throughput sheath flow microfluidics for the positive selection of CTC populations. Furthermore the platform quantitatively isolates cells useful for molecular methods such as detection of mutations. CTC recovery was characterized and validated with an accuracy (<20% error) and a precision (CV<25%) down to at least 9 CTC/ml. Using anti-EpCAM antibodies as the capture agent, the platform recovers 78% of MCF7 cells within the linear range. Non specific recovery of background cells is independent of target cell density and averages 55 cells/mL. 10% purity can be achieved with as low as 6 CTCs/mL and better than 1% purity can be achieved with 1 CTC/mL.

Conclusions/Significance

The LiquidBiopsy platform is an automated validated platform that provides high throughput molecular access to the CTC population. It can be validated and integrated into the lab flow enabling CTC enumeration as well as recovery of consistently high purity samples for molecular analysis such as quantitative PCR and Next Generation Sequencing. This tool opens the way for clinically relevant genetic profiling of CTCs.     

HER2-positive circulating tumor cells in breast cancer

Purpose

Circulating Tumor Cells (CTCs) detection and phenotyping are currently evaluated in Breast Cancer (BC). Tumor cell dissemination has been suggested to occur early in BC progression. To interrogate dissemination in BC, we studied CTCs and HER2 expression on CTCs across the spectrum of BC staging.

Methods

Spiking experiments with 6 BC cell lines were performed and blood samples from healthy women and women with BC were analyzed for HER2-positive CTCs using the CellSearch®.

Results

Based on BC cell lines experiments, HER2-positive CTCs were defined as CTCs with HER2 immunofluoresence intensity that was at least 2.5 times higher than the background. No HER2-positive CTC was detected in 42 women without BC (95% confidence interval (CI) 0–8.4%) whereas 4.1% (95%CI 1.4–11.4%) of 73 patients with ductal/lobular carcinoma in situ (DCIS/LCIS) had 1 HER2-positive CTC/22.5 mL, 7.9%, (95%CI 4.1–14.9%) of 101 women with non metastatic (M0) BC had ≥1 HER2-positive CTC/22.5 mL (median 1 cell, range 1–3 cells) and 35.9% (95%CI 22.7–51.9%) of 39 patients with metastatic BC had ≥1 HER2-positive CTC/7.5 mL (median 1.5 cells, range 1–42 cells). In CTC-positive women with DCIS/LCIS or M0 BC, HER2-positive CTCs were more commonly detected in HER2-positive (5 of 5 women) than HER2-negative BC (5 of 12 women) (p = 0.03).

Conclusion

HER2-positive CTCs were detected in DCIS/LCIS or M0 BC irrespective of the primary tumor HER2 status. Nevertheless, their presence was more common in women with HER2-positive disease. Monitoring of HER2 expression on CTCs might be useful in trials with anti-HER2 therapies.     

Circulating tumor cells in the diagnosis and management of pancreatic cancer

Pancreatic cancers are typically resistant to chemo and radiation therapy and are predisposed to distant metastases. Circulating tumor cells (CTCs) are tumor cells disseminated from primary and metastatic sites and can be isolated from peripheral blood. CTC may overcome the limitation of the current available tumor markers, CA19-9. As a surrogate for 'real-time biopsy', CTCs allow recurrent assessment of a tumor's biological activity. We review the current methodologies for CTC extraction and characterization including antibody-based immunological assays, PCR-based assays, and novel technologies based on the physical or biological characteristics of CTCs. CTCs also provide an accessible link to the existence of epithelial to mesenchymal transition, tumor stem cell markers, and ongoing clonal mutations and epigenetic changes in the tumor. We also explore the potential of using CTC profiling in diagnosis, selection of neoadjuvant and adjuvant therapy, detection of recurrent disease, examination of pharmacodynamic biomarkers, as well as in gene therapy and immunotherapy for pancreatic cancer. Ongoing CTC characterization not only has the potential to represent all cells shed from primary pancreatic tumor and each metastatic site, but also allows dynamic sampling at multiple time points during the clinical course to identify the subpopulations of CTCs and the specific molecules driving metastasis and chemo resistance. We predict that CTC genotyping and phenotyping will play an increasing role in personalized therapy and in identification of novel therapeutic targets as well as monitoring the course and status of the disease.     

Cancer Cell Analyses at the Single Cell-Level Using Electroactive Microwell Array Device

Circulating tumor cells (CTCs), shed from primary tumors and disseminated into peripheral blood, are playing a major role in metastasis. Even after isolation of CTCs from blood, the target cells are mixed with a population of other cell types. Here, we propose a new method for analyses of cell mixture at the single-cell level using a microfluidic device that contains arrayed electroactive microwells. Dielectrophoretic (DEP) force, induced by the electrodes patterned on the bottom surface of the microwells, allows efficient trapping and stable positioning of single cells for high-throughput biochemical analyses. We demonstrated that various on-chip analyses including immunostaining, viability/apoptosis assay and fluorescent in situ hybridization (FISH) at the single-cell level could be conducted just by applying specific reagents for each assay. Our simple method should greatly help discrimination and analysis of rare cancer cells among a population of blood cells.     

High-Density Dielectrophoretic Microwell Array for Detection,Capture, and Single-Cell Analysis of Rare Tumor Cells in Peripheral Blood

Development of a reliable platform and workflow to detect and capture a small number of mutation-bearing circulating tumor cells (CTCs) from a blood sample is necessary for the development of noninvasive cancer diagnosis. In this preclinical study, we aimed to develop a capture system for molecular characterization of single CTCs based on high-density dielectrophoretic microwell array technology. Spike-in experiments using lung cancer cell lines were conducted. The microwell array was used to capture spiked cancer cells, and captured single cells were subjected to whole genome amplification followed by sequencing. A high detection rate (70.2%–90.0%) and excellent linear performance (R² = 0.8189–0.9999) were noted between the observed and expected numbers of tumor cells. The detection rate was markedly higher than that obtained using the CellSearch system in a blinded manner, suggesting the superior sensitivity of our system in detecting EpCAM− tumor cells. Isolation of single captured tumor cells, followed by detection of EGFR mutations, was achieved using Sanger sequencing. Using a microwell array, we established an efficient and convenient platform for the capture and characterization of single CTCs. The results of a proof-of-principle preclinical study indicated that this platform has potential for the molecular characterization of captured CTCs from patients.