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).     

Spectrally Combined Encoding for Profiling Heterogeneous Circulating Tumor Cells Using a Multifunctional Nanosphere-Mediated Microfluidic Platform

Comprehensive phenotypic profiling of heterogeneous circulating tumor cells (CTCs) at single-cell resolution has great importance for cancer management. Herein, a novel spectrally combined encoding (SCE) strategy was proposed for multiplex biomarker profiling of single CTCs using a multifunctional nanosphere-mediated microfluidic platform. Different cellular biomarkers uniquely labeled by multifunctional nanosphere barcodes, possessing identical magnetic tags and distinct optical signatures, enabled isolation of heterogeneous CTCs with over 91.6 % efficiency and in situ SCE of phenotypes. By further trapping individual CTCs in ordered microstructures on chip, composite single-cell spectral signatures were conveniently and efficiently obtained, allowing reliable spectral-readout for multiplex biomarker profiling. This SCE strategy exhibited great potential in multiplex profiling of heterogeneous CTC phenotypes, offering new avenues for cancer study and precise medicine.     

Spectrally Combined Encoding for Profiling Heterogeneous Circulating Tumor Cells Using a Multifunctional Nanosphere‐Mediated Microfluidic Platform

Comprehensive phenotypic profiling of heterogeneous circulating tumor cells (CTCs) at single‐cell resolution has great importance for cancer management. Herein, a novel spectrally combined encoding (SCE) strategy was proposed for multiplex biomarker profiling of single CTCs using a multifunctional nanosphere‐mediated microfluidic platform. Different cellular biomarkers uniquely labeled by multifunctional nanosphere barcodes, possessing identical magnetic tags and distinct optical signatures, enabled isolation of heterogeneous CTCs with over 91.6 % efficiency and in situ SCE of phenotypes. By further trapping individual CTCs in ordered microstructures on chip, composite single‐cell spectral signatures were conveniently and efficiently obtained, allowing reliable spectral‐readout for multiplex biomarker profiling. This SCE strategy exhibited great potential in multiplex profiling of heterogeneous CTC phenotypes, offering new avenues for cancer study and precise medicine.     

Microfluidic applications on circulating tumor cell isolation and biomimicking of cancer metastasis

The prognosis of malignant tumors is challenged by insufficient means to effectively detect tumors at early stage. Liquid biopsy using circulating tumor cells (CTCs) as biomarkers demonstrates a promising solution to tackle the challenge, because CTCs play a critical role in cancer metastatic process via intravasation, circulation, extravasation, and formation of secondary tumor. However, the effectiveness of the solution is compromised by rarity, heterogeneity, and vulnerability associated with CTCs. Among a plethora of novel approaches for CTC isolation and enrichment, microfluidics leads to isolation and detection of CTCs in a cost-effective and operation-friendly way. Development of microfluidics also makes it feasible to model the cancer metastasis in vitro using a microfluidic system to mimick the in vivo microenvironment, thereby enabling analysis and monitor of tumor metastasis. This paper aims to review the latest advances for exploring the dual-roles microfluidics has played in early cancer diagnosis via CTC isolation and investigating the role of CTCs in cancer metastasis; the merits and drawbacks for dominating microfluidics-based CTC isolation methods are discussed; biomimicking cancer metastasis using microfluidics are presented with example applications on modelling of tumor microenvironment, tumor cell dissemination, tumor migration, and tumor angiogenesis. The future perspectives and challenges are discussed.     

循环肿瘤细胞捕获方法的研究进展

血液中的循环肿瘤细胞（CTCs）携带着肿瘤组织的遗传和表型信息，是液体活检的重要标志物。监测和分析血液中CTCs的数量和性质对癌症的早期诊断、治疗方案的确定和疗效评估具有重要意义。然而CTCs在血液中的含量极低，实现对CTCs的捕获与检测极具挑战。该文综述了基于生物物理原理、生物亲和原理以及人工抗体的CTCs捕获方法，并从捕获效率、捕获纯度和释放活性保持等方面进行了评述。此外，该文还对CTCs捕获方法的发展趋势进行了展望。     

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...     

A Method for Detecting Circulating Tumor Cells Based on the Measurement of Single‐Cell Metabolism in Droplet‐Based Microfluidics

The number of circulating tumor cells (CTCs) in blood is strongly correlated with the progress of metastatic cancer. Current methods to detect CTCs are based on immunostaining or discrimination of physical properties. Herein, a label‐free method is presented exploiting the abnormal metabolic behavior of cancer cells. A single‐cell analysis technique is used to measure the secretion of acid from individual living tumor cells compartmentalized in microfluidically prepared, monodisperse, picoliter (pL) droplets. As few as 10 tumor cells can be detected in a background of 200 000 white blood cells and proof‐of‐concept data is shown on the detection of CTCs in the blood of metastatic patients.     

Emerging landscapes of nanosystems based on pre-metastatic microenvironment for cancer theranostics

The emergence of disseminated metastasis is the leading cause of mortality in patients with malignant tumor. The pre-metastatic microenvironment, including the primary tumor-derived components, pre-metastatic niche (PMN), circulating tumor cells (CTCs), micro-metastases, and tumor immune microenvironment (TIM), are the crucial factors to initiate metastasis and form macro-metastases. It may be a more promising strategy for directly targeting pre-metastatic microenvironment-interrelated factors and cells before they have the chance to form secondary tumors to prevent metastasis. During recent years, a variety of nanosystems, with specific microstructures and functional properties, have been devised to selectively target pre-metastatic cells components and interrelated molecular, and exhibited strong potential on anti-metastatic therapy by absorbing and neutralizing primary tumor-derived components, preventing establishment of the PMN, eliminating the CTCs, eradicating the micro-metastases and modulating the TIM. In this review, we comprehensively review the emerging nanosystems based on the pre-metastatic microenvironments. Hopefully, this review can cast new lights for early preventing and attenuating metastatic progression.     

Phenotype-related drug sensitivity analysis of single CTCs for medicine evaluation

Due to the heterogeneous and variable drug sensitivity of tumor cells, real-time monitoring of a patient''s drug response is desirable for implementing personalized and dynamic therapy. Although considerable efforts have been directed at drug screening in living cells, performing repeated drug sensitivity analysis using patient-derived primary tumor cells at the single-cell level remains challenging. Here, we present an efficient approach to assess phenotype-related drug sensitivity at the single-cell level using patient-derived circulating tumor cells (CTCs) based on a drug sensitivity microfluidic chip (DS-Chip). The DS-Chip consists of a drug gradient generator and parallel cell traps, achieving continuous single CTC capture, drug gradient distributions, drug stimulation, fluorescent probe labeling and three-color fluorescence imaging. Based on the established DS-Chip, we investigated the drug sensitivity of single cells by simultaneously monitoring epithelial–mesenchymal transition (EMT) biomarkers and apoptosis in living cells, and verified the correlation between EMT gradients and drug sensitivity. Using the new approach, we further tested the optimal drug response dose in individual CTCs isolated from 5 cancer patients through fluorescence analysis of EMT and apoptosis. The DS-Chip allows noninvasive and real-time measurements of the drug sensitivity of a patient''s tumor cells during therapy. This developed approach has practical significance and can effectively guide drug selection and therapeutic evaluation for personalized medicine.

Due to the heterogeneous and variable drug sensitivity of tumor cells, real-time monitoring of a patient''s drug response is desirable for implementing personalized and dynamic therapy.     

On the design of deterministic dielectrophoresis for continuous separation of circulating tumor cells from peripheral blood cells

Detection and analysis of circulating tumor cells (CTCs) have emerged as a promising way to diagnose cancer, study its cellular mechanism, and test or develop potential treatments. However, the rarity of CTCs among peripheral blood cells is a big challenge toward CTC detection. In addition, in cases where there is similar size range between certain types of CTCs (e.g. breast cancer cells) and white blood cells (WBCs), high‐resolution techniques are needed. In the present work, we propose a deterministic dielectrophoresis (DEP) method that combines the concept of deterministic lateral displacement (DLD) and insulator‐based dielectrophoresis (iDEP) techniques that rely on physical markers such as size and dielectric properties to differentiate different type of cells. The proposed deterministic DEP technology takes advantage of frequency‐controlled AC electric field for continuous separation of CTCs from peripheral blood cells. Utilizing numerical modeling, different aspects of coupled DLD‐DEP design such as the required applied voltages, velocities, and geometrical parameters of DLD arrays of microposts are investigated. Regarding the inevitable difference and uncertainty ranges for the reported crossover frequencies of cells, a comprehensive analysis is conducted on applied electric field frequency as design's determinant factor. Deterministic DEP design provides continuous sorting of CTCs from WBCs even with similar size and has the future potential for high throughput and efficiency.     

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.     

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.     

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.     

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...     

Highlighting the uniqueness in dielectrophoretic enrichment of circulating tumor cells

Circulating tumor cells (CTCs) play an essential role in the metastasis of tumors, and thus can serve as a valuable prognostic factor for malignant diseases. As a result, the ability to isolate and characterize CTCs is essential. This review underlines the potential of dielectrophoresis for CTCs enrichment. It begins by summarizing the key performance parameters and challenges of CTCs isolation using microfluidics. The two main categories of CTCs enrichment—affinity‐based and label‐free methods—are analysed, emphasising the advantages and disadvantages of each as well as their clinical potential. While the main argument in favour of affinity‐based methods is the strong specificity of CTCs isolation, the major advantage of the label‐free technologies is in preserving the integrity of the cellular membrane, an essential requirement for downstream characterization. Moving forward, we try to answer the main question: “What makes dielectrophoresis a method of choice in CTCs isolation?” The uniqueness of dielectrophoretic CTCs enrichment resides in coupling the specificity of the isolation process with the conservation of the membrane surface. The specificity of the dielectrophoretic method stems from the differences in the dielectric properties between CTCs and other cells in the blood: the capacitances of the malignantly transformed cellular membranes of CTCs differ from those of other cells. Examples of dielectrophoretic devices are described and their performance evaluated. Critical requirements for using dielectrophoresis to isolate CTCs are highlighted. Finally, we consider that DEP has the potential of becoming a cytometric method for large‐scale sorting and characterization of cells.     

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.     

Examination of the dielectrophoretic spectra of MCF7 breast cancer cells and leukocytes

The detection of circulating tumor cells (CTCs) in blood is crucial to assess metastatic progression and to guide therapy. Dielectrophoresis (DEP) is a powerful cell surface marker-free method that allows intrinsic dielectric properties of suspended cells to be exploited for CTC enrichment/isolation from blood. Design of a successful DEP-based CTC enrichment/isolation system requires that the DEP response of the targeted particles should accurately be known. This paper presents a DEP spectrum method to investigate the DEP spectra of cells without directly analyzing their membrane and cytoplasmic properties in contrast to the methods in literature, which employ theoretical assumptions and complex modeling. Integrating electric field simulations based on DEP theory with the experimental data enables determination of the DEP spectra of leukocyte subpopulations, polymorphonuclear and mononuclear leukocytes, and MCF7 breast cancer cells as a model of CTC due to their metastatic origin over the frequency range 100 kHz–50 MHz at 10 V_pp. In agreement with earlier findings, differential DEP responses were detected for mononuclear and polymorphonuclear leukocytes due to the richness of the cell surface features and morphologies of the different leukocyte types. The data reveal that the strength of the DEP force exerted on MCF7 cells was particularly high between 850 kHz and 20 MHz. These results illustrate that the proposed technique has the potential to provide a generic platform to identify DEP responses of different biological particles.     

纳米生物无机界面的构筑与循环肿瘤细胞分离

纳米生物无机界面的研究是无机化学学科新兴的前沿领域之一。纳米结构的无机材料在仿生界面、细胞界面、生物检测界面等领域扮演着越来越重要的角色。近几年来, 无机纳米结构被尝试用于痕量循环肿瘤细胞(Circulating Tumor Cells, CTCs)分离的基础探索研究中, 并展现出非常吸引人的应用前景。痕量CTCs的高效分离对于癌症早期检测、术后监测及生物学研究等具有重要的意义。本文主要综述纳米生物无机界面在CTCs分离中的应用, 详细介绍其发展现状, 并对未来做一展望。     

Three‐dimensional microfluidic chip with twin‐layer herringbone structure for high efficient tumor cell capture and release via antibody‐conjugated magnetic microbeads

Harvesting rare circulating tumor cells (CTCs) from human blood is distinctly substantial to monitor tumor stage and evaluate therapeutic efficacy. As a proof‐of‐concept study, a microfluidic chip with twin‐layer herringbone grooves was developed to isolate and recover tumor cells with high efficiency based on the immunoreaction between cells and antibody‐conjugated microbeads (MBs) under local magnetic field. Functional MBs were initially localized onto the internal channel wall through the magnetic guidance. Then, infused tumor cells were deviated into the herringbone groove via passive microvortex and were further trapped through an irreversible interaction with MBs. Upon the removal of magnet, the captured cells and residual MBs were released from the channel and collected for further analysis in cell adhesion and proliferation in vitro. Capture efficiency of tumor cells reached up to ∼90% and limit of detection was down to 50 cells per mL based on this approach. Furthermore, recovery rate of tumor cells was as high as ∼94%, and potencies of cell attachment and proliferation was well maintained in retrieved cells. Hence, the present technique has a great potential for the isolation, quantitation and recovery of CTCs for cancer theranostic guidance and biomolecular analysis.