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.     

Direct Detection of Circulating Tumor Cells in Whole Blood Using Time‐Resolved Luminescent Lanthanide Nanoprobes

The detection of circulating tumor cells (CTCs) is crucial to early cancer diagnosis and the evaluation of cancer metastasis. However, it remains challenging due to the scarcity of CTCs in the blood. Herein, we report an ultrasensitive platform for the direct detection of CTCs using luminescent lanthanide nanoprobes. These were designed to recognize the epithelial cell adhesion molecules on cancer cells, allowing signal amplification through dissolution‐enhanced time‐resolved photoluminescence (TRPL) and the elimination of short‐lived autofluorescence interference. This enabled the direct detection of blood breast‐cancer cells with a limit of detection down to 1 cell/well of a 96‐well plate. Moreover, blood CTCs (≥10 cells mL^?1) can be detected in cancer patients with a detection rate of 93.9 % (14/15 patients). We envision that this ultrasensitive detection platform with excellent practicality may provide an effective strategy for early cancer diagnosis and prognosis evaluation.     

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.     

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.     

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.     

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.     

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.     

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.     

Chemical maps of patterned samples by microline‐imaging laser‐induced plasma spectrometry

The potential of a microline‐imaging laser‐induced plasma spectrometry (LIPS) system for surface and depth analysis of heterogeneous solid samples in air at atmospheric pressure has been demonstrated. A pulsed Nd : YAG laser beam operating at 532 nm, with a homogeneous energy distribution (flat top laser), was used to generate a microline plasma on the sample surface. Subsequent light from the microline plasma was resolved spectrally and spatially and detected with an imaging spectrograph and an intensified charged‐coupled device detector. A patterned metal sample was chosen as the most appropriate for this study. Three‐dimensional chemical maps of Ni and Cu from the edge connectors of a printed circuit board have been obtained. With this experimental configuration, the lateral resolution (limited by crater width) was 42 µm and the spatial resolution along the spectrometer slit was 17.4 µm. The results illustrate the capability of microline imaging for fast mapping of large‐area samples and for depth profiling purposes. Copyright © 2003 John Wiley & Sons, Ltd.     

Profiling Functional and Biochemical Phenotypes of Circulating Tumor Cells Using a Two‐Dimensional Sorting Device

During cancer progression, tumors shed circulating tumor cells (CTCs) into the bloodstream. CTCs that originate from the same primary tumor can have heterogeneous phenotypes and, while some CTCs possess benign properties, others have high metastatic potential. Deconstructing the heterogeneity of CTCs is challenging and new methods are needed that can sort small numbers of cancer cells according to their phenotypic properties. Here we describe a new microfluidic approach that profiles, along two independent phenotypic axes, the behavior of heterogeneous cell subpopulations. Cancer cells are first profiled according to expression of a surface marker using a nanoparticle‐enabled approach. Along the second dimension, these subsets are further separated into subpopulations corresponding to migration profiles generated in response to a chemotactic agent. We deploy this new technique and find a strong correlation between the surface expression and migration potential of CTCs present in blood from mice with xenografted tumors. This system provides an important new means to characterize functional diversity in circulating tumor cells.     

Novel Aryl‐Modified Benzoylamino‐N‐(5,6‐dimethoxy‐1H‐benzoimidazol‐2‐yl)‐heteroamides as Potent Inhibitors of Vascular Endothelial Growth Factor Receptors 1 and 2

Tumor angiogenesis has become an important target for antitumor therapy, with most current therapies aimed at blocking the vascular endothelial growth factor (VEGF) pathway. The VEGF and its receptors have been implicated as key factors in tumor angiogenesis and are major targets in cancer therapy. A series of aryl‐modified benzoylamino‐N‐(5,6‐dimethoxy‐1H‐benzoimidazol‐2‐yl)‐heteroamides were synthesized from 2‐amino‐5,6‐dimethoxy benzimidazole and aryl‐substituted benzoylamino hetero acids. The new compounds were tested for inhibition of VEGF receptors I and II (VEGFR‐1 and VEGFR‐2). Compound 6e displayed VEGFR‐2 inhibitory activity with a 50% inhibition concentration value as low as 0.020 μM in a homogeneous time‐resolved fluorescence enzymatic assay. VEGFR‐2 active compounds display good activity against VEGFR‐1 as well.     

Click‐Assembled,Oxygen‐Sensing Nanoconjugates for Depth‐Resolved,Near‐Infrared Imaging in a 3 D Cancer Model

Hypoxia is an important contributing factor to the development of drug‐resistant cancer, yet few nonperturbative tools exist for studying oxygenation in tissues. While progress has been made in the development of chemical probes for optical oxygen mapping, penetration of such molecules into poorly perfused or avascular tumor regions remains problematic. A click‐assembled oxygen‐sensing (CAOS) nanoconjugate is reported and its properties demonstrated in an in vitro 3D spheroid cancer model. The synthesis relies on the sequential click‐based ligation of poly(amidoamine)‐like subunits for rapid assembly. Near‐infrared confocal phosphorescence microscopy was used to demonstrate the ability of the CAOS nanoconjugates to penetrate hundreds of micrometers into spheroids within hours and to show their sensitivity to oxygen changes throughout the nodule. This proof‐of‐concept study demonstrates a modular approach that is readily extensible to a wide variety of oxygen and cellular sensors for depth‐resolved imaging in tissue and tissue models.     

6,7‐Dimethoxy‐quinazolin‐4‐yl‐amino‐thiophene‐2‐carboxamides as Potent Inhibitors of VEGF Receptors 1 and 2

The identification of agents with antiproliferative activity against endothelial cells has significant value for the treatment of many angiogenesis‐dependent pathologies. The vascular endothelial growth factor (VEGF) and its receptors have been implicated as key factors in tumor angiogenesis and are major targets in cancer therapy. A series of novel 6,7‐dimethoxy‐quinazolin‐4‐yl‐amino‐thiophene‐2‐carboxamides were synthesized and evaluated as antagonists of VEGFR‐1 and VEGFR‐2. More specifically, several analogues exhibited low micromolar to nanomolar potency in the inhibition of VEGFR‐1 and VEGFR‐2. The most potent compound in this series, compound 7b , was found to be a potent inhibitor of VEGFR‐2 in a homogeneous time‐resolved fluorescence enzymatic assay with an IC₅₀ as low as 87 nm.     

Click‐Assembled,Oxygen‐Sensing Nanoconjugates for Depth‐Resolved,Near‐Infrared Imaging in a 3 D Cancer Model

Hypoxia is an important contributing factor to the development of drug‐resistant cancer, yet few nonperturbative tools exist for studying oxygenation in tissues. While progress has been made in the development of chemical probes for optical oxygen mapping, penetration of such molecules into poorly perfused or avascular tumor regions remains problematic. A click‐assembled oxygen‐sensing (CAOS) nanoconjugate is reported and its properties demonstrated in an in vitro 3D spheroid cancer model. The synthesis relies on the sequential click‐based ligation of poly(amidoamine)‐like subunits for rapid assembly. Near‐infrared confocal phosphorescence microscopy was used to demonstrate the ability of the CAOS nanoconjugates to penetrate hundreds of micrometers into spheroids within hours and to show their sensitivity to oxygen changes throughout the nodule. This proof‐of‐concept study demonstrates a modular approach that is readily extensible to a wide variety of oxygen and cellular sensors for depth‐resolved imaging in tissue and tissue models.     

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...     

Near‐Infrared Light‐Initiated Hybridization Chain Reaction for Spatially and Temporally Resolved Signal Amplification

Precise control over signal amplification provides unparalleled opportunities for diverse applications. However, spatiotemporally controlled amplification has not been realized because of the lack of a design methodology. The aim of this study was thus to develop a conceptual approach for remote control over signal amplification at a chosen time and site in living cells. This system was constructed by re‐engineering the functional units of the hybridization chain reaction (HCR) and combination with upconversion photochemistry, thus resulting in an activatable HCR with the high spatial and temporal precision of near‐infrared (NIR) light. As a proof of concept, we demonstrate the spatially and temporally resolved amplified imaging of messenger RNA (mRNA) with ultrahigh sensitivity in vitro and in vivo. Furthermore, by using a system targeting subcellular sites we have developed a new technique for NIR‐initiated amplified imaging of mRNA exclusively within a specific organelle.     

Ferrocene‐modified thiopyrimidines: synthesis,enantiomeric resolution,antitumor activity

Ferrocenylalkyl thiopyrimidines ( 6a–d to 9a–d ) were prepared via the reaction of the α‐(hydroxy)alkyl ferrocenes, FcCHR(OH) ( 1a–d ; Fc = ferrocenyl; R = H, Me, Et, Ph), with 2‐thiopyrimidines ( 2 – 5 ) in acetone at room temperature in the presence of TFA, yielding 50–95%. The resulting enantiomers were resolved using HPLC on modified cellulose as chiral selector. The antitumor activities of S‐ferrocenylethyl 2‐thiopyrimidine ( 6b ) against two murine solid tumor models, carcinoma 755 (Ca755) and Lewis lung carcinoma (LLC) were evaluated in vivo. The strong antitumor effect of compound 6b on Ca755 and LLC was demonstrated. The index of tumor growth inhibition on Ca755 equaled 95% in comparison with control. Copyright © 2010 John Wiley & Sons, Ltd.     

Fluorescence Lifetime Spectroscopy of Glioblastoma Multiforme¶

Fluorescence spectroscopy of the endogenous emission of brain tumors has been researched as a potentially important method for the intraoperative localization of brain tumor margins. We investigated the use of time‐resolved, laser‐induced fluorescence spectroscopy for demarcation of primary brain tumors by studying the time‐resolved spectra of gliomas. The fluorescence of human brain samples (glioblastoma multiforme, cortex and white matter: six patients, 23 sites) was induced ex vivo with a pulsed nitrogen laser (337 nm, 3 ns). The time‐resolved spectra were detected in a 360–550 nm wavelength range using a fast digitizer and gated detection. Parameters derived from both the spectral‐ (intensities from narrow spectral bands) and the time domain (average lifetime) measured at 390 and 460 nm were used for tissue characterization. We determined that high‐grade gliomas are characterized by fluorescence lifetimes that varied with the emission wavelength (>3 ns at 390 nm, <1 ns at 460 nm) and their emission is overall longer than that of normal brain tissue. Our study demonstrates that the use of fluorescence lifetime not only improves the specificity of fluorescence measurements but also allows a more robust evaluation of data collected from brain tissue. Combined information from both the spectraland the time domain can enhance the ability of fluorescencebased techniques to diagnose and detect brain tumor margins intraoperatively.     

6,7‐Dimethoxy‐Quinazolin‐4‐yl‐Amino‐Nicotinamide Derivatives as Potent Inhibitors of VEGF Receptor II

The sprouting of new blood vessels, or angiogenesis, is necessary for any solid tumor to grow large enough to cause life‐threatening disease. Vascular endothelial growth factor (VEGF) is one of the key promoters of tumor‐induced angiogenesis. Inhibition of the VEGF signaling pathway has emerged as one of the most promising new approaches for cancer therapy. A series of 6,7‐dimethoxy‐quinazolin‐4‐yl‐amino‐nicotinamides were synthesized and evaluated as antagonists of VEGF receptor II (VEGFR‐2). Many compounds display VEGFR‐2 inhibitory activity, and compound 7a was found to be a potent inhibitor of VEGFR‐2 in an homogeneous time‐resolved fluorescence enzymatic assay with an IC₅₀ as low as 48 nM (comparable activity to ZD‐6474).