Nanoparticle‐Mediated Binning and Profiling of Heterogeneous Circulating Tumor Cell Subpopulations

The analysis of circulating tumor cells (CTCs) is an important capability that may lead to new approaches for cancer management. CTC capture devices developed to date isolate a bulk population of CTCs and do not differentiate subpopulations that may have varying phenotypes with different levels of clinical relevance. Here, we present a new device for CTC spatial sorting and profiling that sequesters blood‐borne tumor cells with different phenotypes into discrete spatial bins. Validation data are presented showing that cancer cell lines with varying surface expression generate different binning profiles within the device. Working with patient blood samples, we obtain profiles that elucidate the heterogeneity of CTC populations present in cancer patients and also report on the status of CTCs within the epithelial‐to‐mesenchymal transition (EMT).     

Highly efficient circulating tumor cell isolation from whole blood and label-free enumeration using polymer-based microfluidics with an integrated conductivity sensor

A novel microfluidic device that can selectively and specifically isolate exceedingly small numbers of circulating tumor cells (CTCs) through a monoclonal antibody (mAB) mediated process by sampling large input volumes (>/=1 mL) of whole blood directly in short time periods (<37 min) was demonstrated. The CTCs were concentrated into small volumes (190 nL), and the number of cells captured was read without labeling using an integrated conductivity sensor following release from the capture surface. The microfluidic device contained a series (51) of high-aspect ratio microchannels (35 mum width x 150 mum depth) that were replicated in poly(methyl methacrylate), PMMA, from a metal mold master. The microchannel walls were covalently decorated with mABs directed against breast cancer cells overexpressing the epithelial cell adhesion molecule (EpCAM). This microfluidic device could accept inputs of whole blood, and its CTC capture efficiency was made highly quantitative (>97%) by designing capture channels with the appropriate widths and heights. The isolated CTCs were readily released from the mAB capturing surface using trypsin. The released CTCs were then enumerated on-device using a novel, label-free solution conductivity route capable of detecting single tumor cells traveling through the detection electrodes. The conductivity readout provided near 100% detection efficiency and exquisite specificity for CTCs due to scaling factors and the nonoptimal electrical properties of potential interferences (erythrocytes or leukocytes). The simplicity in manufacturing the device and its ease of operation make it attractive for clinical applications requiring one-time use operation.     

Integration of Lateral Filter Arrays with Immunoaffinity for Circulating‐Tumor‐Cell Isolation

Circulating tumor cells (CTCs) are an important biomarker for cancer prognosis and treatment monitoring. However, the heterogeneity of the physical and biological properties of CTCs limits the efficiency of various approaches used to isolate small numbers of CTCs from billions of normal blood cells. To address this challenge, we developed a lateral filter array microfluidic (LFAM) device to integrate size‐based separation with immunoaffinity‐based CTC isolation. The LFAM device consists of a serpentine main channel, through which most of a sample passes, and an array of lateral filters for CTC isolation. The unique device design produces a two‐dimensional flow, which reduces nonspecific, geometric capture of normal cells as typically observed in vertical filters. The LFAM device was further functionalized by immobilizing antibodies that are specific to the target cells. The resulting devices captured pancreatic cancer cells spiked in blood samples with (98.7±1.2) % efficiency and were used to isolate CTCs from patients with metastatic colorectal cancer.     

Label-free enrichment of MCF7 breast cancer cells from leukocytes using continuous flow dielectrophoresis

Circulating tumor cells (CTCs) present in the bloodstream are strongly linked to the invasive behavior of cancer; therefore, their detection holds great significance for monitoring disease progression. Currently available CTC isolation tools are often based on tumor-specific antigen or cell size approaches. However, these techniques are limited due to the lack of a unique and universal marker for CTCs, and the overlapping size between CTCs and regular blood cells. Dielectrophoresis (DEP), governed by the intrinsic dielectric properties of the particles, is a promising marker-free, accurate, fast, and low-cost technique that enables the isolation of CTCs from blood cells. This study presents a continuous flow, antibody-free DEP-based microfluidic device to concentrate MCF7 breast cancer cells, a well-established CTC model, in the presence of leukocytes extracted from human blood samples. The enrichment strategy was determined according to the DEP responses of the corresponding cells, obtained in our previously reported DEP spectrum study. It was based on the positive-DEP integrated with hydrodynamic focusing under continuous flow. In the proposed device, the parylene microchannel with two inlets and outlets was built on top of rectangular and equally spaced isolated planar electrodes rotated certain degree relative to the main flow (13°). The recovery of MCF7 cells mixed with leukocytes was 74%–98% at a frequency of 1 MHz and a magnitude of 10–12 V_pp. Overall, the results revealed that the presented system successfully concentrates MCF7 cancer cells from leukocytes, ultimately verifying our DEP spectrum study, in which the enrichment frequency and separation strategy of the microfluidic system were determined.     

Diagnostic microchip to assay 3D colony-growth potential of captured circulating tumor cells

Microfluidic technology has been successfully applied to isolate very rare tumor-derived epithelial cells (circulating tumor cells, CTCs) from blood with relatively high yield and purity, opening up exciting prospects for early detection of cancer. However, a major limitation of state-of-the-art CTC-chips is their inability to characterize the behavior and function of captured CTCs, for example to obtain information on proliferative and invasive properties or, ultimately, tumor re-initiating potential. Although CTCs can be efficiently immunostained with markers reporting phenotype or fate (e.g. apoptosis, proliferation), it has not yet been possible to reliably grow captured CTCs over long periods of time and at single cell level. It is challenging to remove CTCs from a microchip after capture, therefore such analyses should ideally be performed directly on-chip. To address this challenge, we merged CTC capture with three-dimensional (3D) tumor cell culture on the same microfluidic platform. PC3 prostate cancer cells were isolated from spiked blood on a transparent PDMS CTC-chip, encapsulated on-chip in a biomimetic hydrogel matrix (QGel?) that was formed in situ, and their clonal 3D spheroid growth potential was assessed by microscopy over one week in culture. The possibility to clonally expand a subset of captured CTCs in a near-physiological in vitro model adds an important element to the expanding CTC-chip toolbox that ultimately should improve prediction of treatment responses and disease progression.     

A combined micromagnetic-microfluidic device for rapid capture and culture of rare circulating tumor cells

Here we describe a combined microfluidic-micromagnetic cell separation device that has been developed to isolate, detect and culture circulating tumor cells (CTCs) from whole blood, and demonstrate its utility using blood from mammary cancer-bearing mice. The device was fabricated from polydimethylsiloxane and contains a microfluidic architecture with a main channel and redundant 'double collection' channel lined by two rows of dead-end side chambers for tumor cell collection. The microdevice design was optimized using computational simulation to determine dimensions, magnetic forces and flow rates for cell isolation using epithelial cell adhesion molecule (EpCAM) antibody-coated magnetic microbeads (2.8 μm diameter). Using this device, isolation efficiencies increased in a linear manner and reached efficiencies close to 90% when only 2 to 80 breast cancer cells were spiked into a small volume (1.0 mL) of blood taken from wild type mice. The high sensitivity visualization capabilities of the device also allowed detection of a single cell within one of its dead-end side chambers. When blood was removed from FVB C3(1)-SV40 T-antigen mammary tumor-bearing transgenic mice at different stages of tumor progression, cells isolated in the device using anti-EpCAM-beads and magnetically collected within the dead-end side chambers, also stained positive for pan-cytokeratin-FITC and DAPI, negative for CD45-PerCP, and expressed SV40 large T antigen, thus confirming their identity as CTCs. Using this isolation approach, we detected a time-dependent rise in the number of CTCs in blood of female transgenic mice, with a dramatic increase in the numbers of metastatic tumor cells appearing in the blood after 20 weeks when tumors transition to invasive carcinoma and exhibit increased growth of metastases in this model. Importantly, in contrast to previously described CTC isolation methods, breast tumor cells collected from a small volume of blood removed from a breast tumor-bearing animal remain viable and they can be easily removed from these devices and expanded in culture for additional analytical studies or potential drug sensitivity testing.     

Microchip-based immunomagnetic detection of circulating tumor cells

Screening for circulating tumor cells (CTCs) in blood has been an object of interest for evidence of progressive disease, status of disease activity, recognition of clonal evolution of molecular changes and for possible early diagnosis of cancer. We describe a new method of microchip-based immunomagnetic CTC detection, in which the benefits of both immunomagnetic assay and the microfluidic device are combined. As the blood sample flows through the microchannel closely above arrayed magnets, cancer cells labeled with magnetic nanoparticles are separated from blood flow and deposited at the bottom wall of the glass coverslip, which allows direct observation of captured cells with a fluorescence microscope. A polydimethylsiloxane (PDMS)-based microchannel fixed on a glass coverslip was used to screen blood samples. The thin, flat dimensions of the microchannel, combined with the sharp magnetic field gradient in the vicinity of arrayed magnets with alternate polarities, lead to an effective capture of labeled cells. Compared to the commercially available CellSearch? system, fewer (25%) magnetic particles are required to achieve a comparable capture rate, while the screening speed (at an optimal blood flow rate of 10 mL h(-1)) is more than five times faster than those reported previously with a microchannel-based assay. For the screening experiment, blood drawn from healthy subjects into CellSave? tubes was spiked with cultured cancer cell lines of COLO205 and SKBR3. The blood was then kept at room temperature for 48 hours before the screening, emulating the actual clinical cases of blood screening. Customized Fe(3)O(4) magnetic nanoparticles (Veridex Ferrofluid?) conjugated to anti-epithelial cell adhesion molecule (EpCAM) antibodies were introduced into the blood samples to label cancer cells, and the blood was then run through the microchip device to capture the labelled cells. After capture, the cells were stained with fluorescent labelled anti-cytokeratin, DAPI and anti-CD45. Subsequent immunofluorescence images were taken for the captured cells, followed by comprehensive computer aided analysis based on fluorescence intensities and cell morphology. Rare cancer cells (from ～1000 cells down to ～5 cells per mL) with very low tumor cell to blood cell ratios (about 1?:?10(7) to 10(9), including red blood cells) were successfully detected. Cancer cell capture rates of 90% and 86% were demonstrated for COLO205 and SKBR3 cells, respectively.     

Characterization of a hybrid dielectrophoresis and immunocapture microfluidic system for cancer cell capture

The capture of circulating tumor cells (CTCs) from cancer patient blood enables early clinical assessment as well as genetic and pharmacological evaluation of cancer and metastasis. Although there have been many microfluidic immunocapture and electrokinetic techniques developed for isolating rare cancer cells, these techniques are often limited by a capture performance tradeoff between high efficiency and high purity. We present the characterization of shear‐dependent cancer cell capture in a novel hybrid DEP–immunocapture system consisting of interdigitated electrodes fabricated in a Hele‐Shaw flow cell that was functionalized with a monoclonal antibody, J591, which is highly specific to prostate‐specific membrane antigen expressing prostate cancer cells. We measured the positive and negative DEP response of a prostate cancer cell line, LNCaP, as a function of applied electric field frequency, and showed that DEP can control capture performance by promoting or preventing cell interactions with immunocapture surfaces, depending on the sign and magnitude of the applied DEP force, as well as on the local shear stress experienced by cells flowing in the device. This work demonstrates that DEP and immunocapture techniques can work synergistically to improve cell capture performance, and it will aid in the design of future hybrid DEP–immunocapture systems for high‐efficiency CTC capture with enhanced purity.     

核酸适配体功能化纳米界面用于循环肿瘤细胞捕获与释放

循环肿瘤细胞(CTCs)是肿瘤研究和临床癌症诊断中的重要对象,也是"液体活检"的重要标志物.CTCs携带着肿瘤组织的遗传和表型信息,有助于肿瘤的早期诊断、个体化治疗和预后监测.然而,CTCs是一种极其罕见的细胞群体,在癌症患者外周血中十分稀少,这对从患者血液中分离CTCs并无损释放进行下游分析提出了挑战.目前,基于CT...     

Ex vivo identification of circulating tumor cells in peripheral blood by fluorometric “turn on” aptamer nanoparticles

The detection of the circulating tumor cells (CTCs) detached from solid tumors has emerged as a burgeoning topic for cancer diagnosis and treatment. The conventional CTC enrichment and identification mainly rely on the specific binding of the antibodies on the capture interface of the magnetic nanoparticles with the corresponding biomarkers on the cell membranes. However, these methods could easily generate false-negative results due to the extremely low concentration of CTCs and the internal heterogeneity of the tumor cells. Herein, with the aim of selectively identifying CTCs and improving the detection accuracy in peripheral blood, we designed the fluorometric “turn on” Au nanoparticles (DHANs) with the modification of a tumor-targeted moiety, dehydroascorbic acid (DHA) and a fluorometric aptamer, which could be “switched-on” by an over-expressed intracellular protein, namely hypoxia-inducible factor-1α (HIF 1α). This novel nanoformulated detection platform demonstrated the great capacity for visualizing various CTCs in peripheral blood with significantly improved detection efficiency and sensitivity. As a result, the nanoplatform has a great potential to be further applied for CTC detection in vitro or in vivo, which holds promise for extensive CTC studies.

The detection of the circulating tumor cells (CTCs) detached from solid tumors has emerged as a burgeoning topic for cancer diagnosis and treatment.     

Determination of EGFR mutations in single cells microdissected from enriched lung tumor cells in peripheral blood

A minimally invasive and repeatable approach for real-time epidermal growth factor receptor (EGFR) mutation surveillance would be highly beneficial for individualized therapy of late stage lung cancer patients whose surgical specimens are often not available. We aim to develop a viable method to detect EGFR mutations in single circulating tumor cells (CTCs). Using a model CTC system of spiked tumor cells in whole blood, we evaluated EGFR mutation determination in single tumor cells enriched from blood. We used magnetic beads labeled with antibody against leukocyte surface antigens to deplete leukocytes and enrich native CTCs independent of epithelial marker expression level. We then used laser cell microdissection (LCM) to isolate individual CTCs, followed by whole-genome amplification of the DNA for exon 19 microdeletion, L858R and T790M mutation detection by PCR sequencing. EGFR mutations were successfully measured in individual spiked tumor cells enriched from 7.5 ml whole blood. Whole-genome amplification provided sufficient DNA for mutation determination at multiple sites. Ninety-five percent of the single CTCs microdissected by LCM (19/20) yielded PCR amplicons for at least one of the three mutation sites. The amplification success rates were 55 % (11/20) for exon 19 deletion, 45 % (9/20) for T790M, and 85 % (17/20) for L858R. Sequencing of the amplicons showed allele dropout in the amplification reactions, but mutations were correctly identified in 80 % of the amplicons. EGFR mutation determination from single captured tumor cells from blood is feasible with the approach described here. However, to overcome allele dropout and to obtain reliable information about the tumor's EGFR status, multiple individual tumor cells should be assayed.

Pinched flow coupled shear-modulated inertial microfluidics for high-throughput rare blood cell separation

Blood is a highly complex bio-fluid with cellular components making up >40% of the total volume, thus making its analysis challenging and time-consuming. In this work, we introduce a high-throughput size-based separation method for processing diluted blood using inertial microfluidics. The technique takes advantage of the preferential cell focusing in high aspect-ratio microchannels coupled with pinched flow dynamics for isolating low abundance cells from blood. As an application of the developed technique, we demonstrate the isolation of cancer cells (circulating tumor cells (CTCs)) spiked in blood by exploiting the difference in size between CTCs and hematologic cells. The microchannel dimensions and processing parameters were optimized to enable high throughput and high resolution separation, comparable to existing CTC isolation technologies. Results from experiments conducted with MCF-7 cells spiked into whole blood indicate >80% cell recovery with an impressive 3.25 × 10(5) fold enrichment over red blood cells (RBCs) and 1.2 × 10(4) fold enrichment over peripheral blood leukocytes (PBL). In spite of a 20× sample dilution, the fast operating flow rate allows the processing of ～10(8) cells min(-1) through a single microfluidic device. The device design can be easily customized for isolating other rare cells from blood including peripheral blood leukocytes and fetal nucleated red blood cells by simply varying the 'pinching' width. The advantage of simple label-free separation, combined with the ability to retrieve viable cells post enrichment and minimal sample pre-processing presents numerous applications for use in clinical diagnosis and conducting fundamental studies.     

SSA-MOA: a novel CTC isolation platform using selective size amplification (SSA) and a multi-obstacle architecture (MOA) filter

Circulating tumor cells (CTCs) have gained increasing attention as physicians and scientists learn more about the role these extraordinarily rare cells play in metastatic cancer. In developing CTC technology, the critical criteria are high recovery rates and high purity. Current isolation methods suffer from an inherent trade-off between these two goals. Moreover, ensuring minimal cell stress and robust reproducibility is also important for the clinical application of CTCs. In this paper, we introduce a novel CTC isolation technology using selective size amplification (SSA) for target cells and a multi-obstacle architecture (MOA) filter to overcome this trade-off, improving both recovery rate and purity. We also demonstrate SSA-MOA's advantages in minimizing cell deformation during filter transit, resulting in more stable and robust CTC isolation. In this technique, polymer microbeads conjugated with anti-epithelial cell adhesion molecules (anti-EpCAM) were used to selectively size-amplify MCF-7 breast cancer cells, definitively differentiating from the white blood cells (WBCs) by avoiding the size overlap that compromises other size selection methods. 3 μm was determined to be the optimal microbead diameter, not only for size discrimination but also in maximizing CTC surface coverage. A multi-obstacle architecture filter was fabricated using silicon-on-glass (SOG) technology-a first such application of this fabrication technique-to create a precise microfilter structure with a high aspect ratio. The filter was designed to minimize cell deformation as simulation results predicted that cells captured via this MOA filter would experience 22% less moving force than with a single-obstacle architecture. This was verified by experiments, as we observed reliable cell capture and reduced cell deformation, with a 92% average recovery rate and 351 peripheral blood leukocytes (PBL) per millilitre (average). We expect the SSA-MOA platform to optimize CTC recovery rates, purity, and stability, increasing the sensitivity and reliability of such tests, thereby potentially expanding the utilization of CTC technologies in the clinic.     

Continuous separation of breast cancer cells from blood samples using multi-orifice flow fractionation (MOFF) and dielectrophoresis (DEP)

Circulating tumor cells (CTCs) are highly correlated with the invasive behavior of cancer, so their isolations and quantifications are important for biomedical applications such as cancer prognosis and measuring the responses to drug treatments. In this paper, we present the development of a microfluidic device for the separation of CTCs from blood cells based on the physical properties of cells. For use as a CTC model, we successfully separated human breast cancer cells (MCF-7) from a spiked blood cell sample by combining multi-orifice flow fractionation (MOFF) and dielectrophoretic (DEP) cell separation technique. Hydrodynamic separation takes advantage of the massive and high-throughput filtration of blood cells as it can accommodate a very high flow rate. DEP separation plays a role in precise post-processing to enhance the efficiency of the separation. The serial combination of these two different sorting techniques enabled high-speed continuous flow-through separation without labeling. We observed up to a 162-fold increase in MCF-7 cells at a 126 μL min(-1) flow rate. Red and white blood cells were efficiently removed with separation efficiencies of 99.24% and 94.23% respectively. Therefore, we suggest that our system could be used for separation and detection of CTCs from blood cells for biomedical applications.     

微流控芯片系统在循环肿瘤细胞分离检测中的应用进展

循环肿瘤细胞(CTCs)的分离分析一直是肿瘤相关研究中的热点方向,作为液体活检的重要标志物之一,其在外周血中的含量与癌症病发状况密切相关.然而人体血液中CTCs的含量非常低,通常来说仅有0～10个/mL,因此在开展临床血液样本中CTCs的检测前,往往需要对样本进行前处理,以实现CTCs的分离和富集.微流控芯片技术凭借样...     

循环肿瘤细胞体内检测技术及其应用研究

从实体瘤脱落进入血液循环系统的肿瘤细胞即循环肿瘤细胞(CTCs)与肿瘤转移密切相关,因此CTCs检测对癌症患者的诊断、治疗监测、病情评估以及肿瘤转移机制研究具有重要意义.由于CTCs在体内含量极少、异质性、分布不均一,通过体外采血发展的CTCs检测技术虽然已取得很大进展,但仍然面临肿瘤细胞损失、失活、失真以及灵敏度低等...     

Beyond the Capture of Circulating Tumor Cells: Next‐Generation Devices and Materials

Over the last decade, significant progress has been made towards the development of approaches that enable the capture of rare circulating tumor cells (CTCs) from the blood of cancer patients, a critical capability for noninvasive tumor profiling. These advances have leveraged new insights in materials chemistry and microfluidics and allowed the capture and enumeration of CTCs with unprecedented sensitivity. However, it has become increasingly clear that simply capturing and counting tumor cells launched into the bloodstream may not provide the information needed to advance our understanding of the biology of these rare cells, or to allow us to better exploit them in medicine. A variety of advances have now emerged demonstrating that more information can be extracted from CTCs with next‐generation devices and materials featuring tailored physical and chemical properties. In this Minireview, the last ten years of work in this area will be discussed, with an emphasis on the groundbreaking work of the last five years, during which the focus has moved beyond the simple capture of CTCs and gravitated towards approaches that enable in‐depth analysis.     

Bioinspired Engineering of a Multivalent Aptamer‐Functionalized Nanointerface to Enhance the Capture and Release of Circulating Tumor Cells

Circulating tumor cell (CTC)‐enrichment by using aptamers has a number of advantages, but the issue of compromised binding affinities and stabilities in real samples hinders its wide applications. Inspired by the high efficiency of the prey mechanism of the octopus, we engineered a deterministic lateral displacement (DLD)‐patterned microfluidic chip modified with multivalent aptamer‐functionalized nanospheres (AP‐Octopus‐Chip) to enhance capture efficiency. The multivalent aptamer–antigen binding efficiency improves 100‐fold and the capture efficiency is enhanced more than 300 % compared with a monovalent aptamer‐modified chip. Moreover, the captured cancer cells can be released through a thiol exchange reaction with up to 80 % efficiency and 96 % viability, which is fully compatible with downstream mutation detection and CTC culture. Using the chip, we were able to find CTCs in all cancer samples analyzed.     

Isolation,Detection, and Antigen‐Based Profiling of Circulating Tumor Cells Using a Size‐Dictated Immunocapture Chip

Even though the diagnostic and prognostic value of circulating tumor cells (CTCs) has been demonstrated, their clinical utility and widespread adoption have been limited. Herein, we describe a new device, size‐dictated immunocapture chip (SDI‐Chip), for efficient, sensitive, and spatially resolved capture and detection of CTCs. SDI‐Chip enables selective, frequent, and extended interaction of CTCs with hydrodynamically optimized immunocoated micropillar surfaces. CTCs with different antigen expression levels can be efficiently captured and spatially resolved around the micropillars. Capture efficiency greater than 92 % with a purity of 82 % was achieved with blood samples. CTCs were detected in non‐metastasis colorectal (CRC) patients, while none was detected from healthy volunteers. We believe that SDI‐Chip will facilitate the transition of tumor diagnosis from anatomical pathology to molecular pathology in localized CRC patients.     

Isolation,Detection, and Antigen‐Based Profiling of Circulating Tumor Cells Using a Size‐Dictated Immunocapture Chip

Even though the diagnostic and prognostic value of circulating tumor cells (CTCs) has been demonstrated, their clinical utility and widespread adoption have been limited. Herein, we describe a new device, size‐dictated immunocapture chip (SDI‐Chip), for efficient, sensitive, and spatially resolved capture and detection of CTCs. SDI‐Chip enables selective, frequent, and extended interaction of CTCs with hydrodynamically optimized immunocoated micropillar surfaces. CTCs with different antigen expression levels can be efficiently captured and spatially resolved around the micropillars. Capture efficiency greater than 92 % with a purity of 82 % was achieved with blood samples. CTCs were detected in non‐metastasis colorectal (CRC) patients, while none was detected from healthy volunteers. We believe that SDI‐Chip will facilitate the transition of tumor diagnosis from anatomical pathology to molecular pathology in localized CRC patients.