Circulating Tumor Cells In Whole Blood Research Articles

Dual-source powered nanomotors coupled with dual-targeting ligands for efficient capture and detection of CTCs in whole blood and in vivo tumor imaging

Circulating tumor cells (CTCs) are important biomarkers in cancer diagnosis. However, the specific labeling of CTCs with high capture efficiency in whole blood remains a problem. Herein, a dual-source-driven nanomotor coupled with dual-targeting ligands (CD@NM) was designed for efficient capture, specific imaging and quantitative detection of CTCs. In both water and biological fluid, CD@NMs moved autonomously under the propulsion of a magnetic field and H2O2 solution, which improved the capture efficiency of CTCs to 97.50 ± 2.38%. More importantly, specific labeling of CTCs was achieved by fluorescence quenching and recovery of fluorescent carbon dots modified on the CD@NMs. As a result, the CD@NMs exhibited efficient CTC capture, specific CTC imaging and recognition in whole blood. CD@NMs were also successfully deployed in the specific imaging of tumor tissues in vivo. On this basis, CD@NMs are expected to provide a new platform for tumor diagnosis both in vitro and in vivo.

Chemically-Modified Sepharose 6B Beads for Collection of Circulating Tumor Cells.

The isolation and quantitative characterization of circulating tumor cells (CTCs) are of great importance in cancer diagnosis and prognosis. However, isolating and detecting CTCs in whole blood presents a significant challenge due to the low numbers of CTCs (often ranging from one to five) in samples containing billions of erythrocytes. Recently, point-of-care devices that use antibody trapping coupled with remote immunofluorescence analyses have been described to identify the number and type of CTCs in blood. In this study, we propose a novel method for trapping and quantifying CTCs using Sepharose 6B beads of 45-160 μm size that are engineered with capture antibodies. Specifically, we employed CD44 antibody conjugates (bearing a maleimide group) that are specific to the CTCs of breast cancer to thiol-Sepharose beads 6B. These beads, when mixed with MDAMB231 and Jurkat cells and filtered through a 40 μm filter, can capture ~80% of MDAMB231 cells. Furthermore, the antibody-modified Sepharose 6B can be stored at four degrees Celsius for a period exceeding six months.

Detection of mRNAs of Ribosomal Protein L15 and E-Cadherin in Living Circulating Tumor Cells at Single Cell Resolution To Study Tumor Heterogeneity.

To study the heterogeneity of circulating tumor cells (CTCs) is of crucial importance to analyze cancer progression and metastasis. However, in situ detection of highly heterogeneous CTCs in peripheral blood still faces an elusive challenge. Here, we show direct detection of two metastasis-related mRNAs of diverse CTCs in whole blood by a triple-targeting nanoprobe. In the nanoprobe, two kinds of molecular beacons, MB1 to detect RPL15 mRNA and MB2 to detect E-cadherin (E-cad) mRNA, are loaded in a highly efficient delivery vector decorated with EpCAM-targeted SYL3C, EGFR-targeted CL4, and CD44-targeted hyaluronic acid chains to specifically deliver MB1/MB2 into epithelial, mesenchymal, and stem CTCs in unprocessed peripheral blood. The numbers of RPL15+ and E-cad+ CTCs are positively correlated with the metastasis stages of cancer patients. This study provides an effective strategy to realize direct observation on diverse metastasis-related genes in living CTCs with different phenotypes to provide accurate information on cancer heterogeneity and metastasis.

Direct detection of intracellular miRNA in living circulating tumor cells by tumor targeting nanoprobe in peripheral blood

Molecular analysis of circulating tumor cells (CTCs) is of critical significance for the non-invasive early detection of tumors. However, in situ detection of intracellular nucleic acids of CTCs in whole blood still remains challenge. By using a highly efficient tumor targeting nanoprobe, we realize in situ detection of microRNA-21 (miR-21) of living CTCs in unprocessed whole blood. In the nanoprobe, a catalytic hairpin assembly (CHA) system is complexed with protamine sulfate (PS), and then decorated by SYL3C conjugated hyaluronic acid (SHA) and hyaluronic acid (HA). The CHA system can be specifically delivered into living CTCs in whole blood, followed by hybridization between the CHA system and intracellular miR-21 in CTCs to induce strong fluorescence emission. After isolation of CTCs by membrane filtration, CTCs of cancer patients can be directly visualized by a fluorescence microscope for miR-21 detection at a single-cell level. Our study provides an efficient strategy to realize in situ genomic analysis of living CTCs in whole blood.

In Situ-Generated Multivalent Aptamer Network for Efficient Capture and Sensitive Electrochemical Detection of Circulating Tumor Cells in Whole Blood.

Monitoring circulating tumor cells (CTCs) in human blood can offer useful information for convenient metastasis diagnosis, prognosis, and treatment of cancers. However, it remains a substantial challenge to detect CTCs because of their particular scarcity in complex peripheral blood. Herein, we describe an in situ-generated multivalent aptamer network-modified electrode interface for efficiently capturing and sensitively detecting CTCs in whole blood by electrochemistry. Such an interface was fabricated via rolling circle amplification extension of the electrode-immobilized primer/circular DNA complexes for the yield of long ssDNA strands with many repeated aptamer segments, which could achieve efficient capture of rare CTCs in a multivalent cooperative manner. The antibody and horseradish peroxidase-functionalized gold nanoparticles further specifically associated with the surface-bound CTCs and generated electrocatalytically amplified current outputs for highly sensitive detection of CTCs with an attractive detection limit of five cells. Also, the multivalent aptamer network interface could successfully distinguish the target cells from other control cells and achieve CTC detection in whole blood, demonstrating its promising potential for monitoring different rare CTCs in human blood.

Mechanical Low-Pass Filtering of Cells for Detection of Circulating Tumor Cells in Whole Blood.

The detection of circulating tumor cells (CTCs) from liquid biopsies using microfluidic devices is attracting a considerable amount of attention as a new, less-invasive cancer diagnostic and prognostic method. One of the drawbacks of the existing antibody-based detection systems is the false negatives for epithelial cell adhesion molecule detection of CTCs. Here we report a mechanical low-pass filtering technique based on a microfluidic constriction and electrical current sensing system for the novel CTC detection in whole blood without any specific antigen-antibody interaction or biochemical modification of the cell surface. The mechanical response of model cells of CTCs, such as HeLa, A549, and MDA-MB-231 cells, clearly demonstrated different behaviors from that of Jurkat cells, a human T-lymphocyte cell line, when they passed through the 6-μm wide constriction channel. A 6-μm wide constriction channel was determined as the optimum size to identify CTCs in whole blood with an accuracy greater than 95% in tens of milliseconds. The mechanical filtering of cells at a single cell level was achieved from whole blood without any pretreatment (e.g., dilution of lysing) and prelabeling (e.g., fluorophores or antibodies).

Highly specific and sensitive point-of-care detection of rare circulating tumor cells in whole blood via a dual recognition strategy

Despite the fact that the identification and detection of circulating tumor cells (CTCs) plays a critical role in cancer monitoring and diagnosis, it remains a major challenge to isolate and detect these cells, due to their extreme scarcity in peripheral blood. In this work, by coupling a dual recognition strategy and the commercial personal glucose meter, we established a point-of-care approach for detecting rare CTCs in whole blood with high sensitivity and selectivity. The antibody-conjugated magnetic beads lead to the capture and isolation of the CTCs while the enzyme- and second antibody-modified microspheres yield the signal for detection. Because of the dual recognition format, the developed method is highly selective, and a low detection limit of 7 cells can be realized as well, owing to the great signal amplification through the enzyme-loaded microbead labels. More importantly, the detection of CTCs in whole blood can be achieved in a point-of-care fashion with the using of the glucose meter transducer, offering our method a convenient and attractive alternative to traditional biopsy for the diagnosis of various cancers.

Single-Cell Phenotypic Profiling of CTCs in Whole Blood Using an Integrated Microfluidic Device.

Single-cell phenotypic profiling of circulating tumor cells (CTCs) in the blood of cancer patients can reveal vital tumor biology information. Even though various approaches have been provided to enrich and detect CTCs, it remains challenging for consecutive CTC sorting, enumeration, and single-cell characterizations. Here, we report an integrated microfluidic device (IMD) for single-cell phenotypic profiling of CTCs that enables automated CTCs sorting from whole blood following continuous single-cell phenotypic analysis while satisfying the requirements of both high purity (92 ± 3%) of cell sorting and high-throughput processing capacity (5 mL whole blood/3 h). Using this new technique we test the phenotypes of individual CTCs collected from xenograft tumor-bearing mice and colorectal (CRC) patients at different tumor stages. We obtained a correlation between CTC characterization and clinical tumor stage and treatment response. The developed IMD offers a high-throughput, convenient, and rapid strategy to study individual CTCs toward minimally invasive cancer therapy prediction and disease monitoring and has the potential to be translated to clinic for liquid biopsy.

Unraveling the Vascular Fate of Deformable Circulating Tumor Cells Via a Hierarchical Computational Model

Distant spreading of primary lesions is modulated by the vascular dynamics of circulating tumor cells (CTCs) and their ability to establish metastatic niches. While the mechanisms regulating CTC homing in specific tissues are yet to be elucidated, it is well documented that CTCs possess different size, biological properties and deformability. A computational model is presented to predict the vascular transport and adhesion of CTCs in whole blood. A Lattice–Boltzmann method, which is employed to solve the Navier-Stokes equation for the plasma flow, is coupled with an Immersed Boundary Method. The vascular dynamics of a CTC is assessed in large and small microcapillaries. The CTC shear modulus $${k}_{\text{ctc}}$$ is varied returning CTCs that are stiffer, softer and equally deformable as compared to RBCs. In large microcapillaries, soft CTCs behave similarly to RBCs and move away from the vessel walls; whereas rigid CTCs are pushed laterally by the fast moving RBCs and interact with the vessel walls. Three adhesion behaviors are observed—firm adhesion, rolling and crawling over the vessel walls—depending on the CTC stiffness. On the contrary, in small microcapillaries, rigid CTCs are pushed downstream by a compact train of RBCs and cannot establish any firm interaction with the vessel walls; whereas soft CTCs are squeezed between the vessel wall and the RBC train and rapidly establish firm adhesion. These findings document the relevance of cell deformability in CTC vascular adhesion and provide insights on the mechanisms regulating metastasis formation in different vascular districts.

Design of a Biocompatible and Ratiometric Fluorescent probe for the Capture, Detection, Release, and Reculture of Rare Number CTCs.

Circulating tumor cells (CTCs) served as an important biomarker for tumor recurrence and prediction of prognosis. However, selective capture and quantification of CTCs from whole blood was still full of challenge due to the extremely scare number of CTCs. Moreover, how to keep a high cell viability after capture remained to be solved. Here, we described a ratiometric fluorescent probe for the efficient capture and accurate determination of CTCs by conjugating graphitic carbon nitride quantum dots (g-CNQDs) with gold nanoclusters (AuNCs) and further linking with anti-EpCAM antibody to acquire the CTC-specific immune probe. In this probe, AuNCs protected by albumin V bovine played the role as the fluorophore reference and anti-EpCAM-attached g-CNQDs acted as both the response signal and specific recognition element for sensing CTCs. In the presence of CTCs, the quenched fluorescence of the immune probe at 500 nm was recovered due to the detachment of anti-EpCAM from the probe, whereas the intensity at 650 nm was essentially unchanged. This strategy realized the highly sensitive detection of CTCs in whole blood down to one CTC. Furthermore, it was demonstrated that the designed probe allowed capturing living CTCs with minimal cell damage. The subsequent reculture of captured cells for proliferation revealed that after a 7 day proliferation, almost 28 MCF-7 cells were obtained from one target cell. The immune probe was successfully applied into capture and detection of CTCs from clinical cancer patients. Our data suggested the good potential of fluorescent probe for the clinical diagnosis of cancers.

Immunomagnetic antibody plus aptamer pseudo-DNA nanocatenane followed by rolling circle amplication for highly-sensitive CTC detection

Biosensing and detecting the rare circulating tumor cells (CTCs) in complex blood samples are a great challenge but necessary for cancer metastasis prevention. Here we show a novel highly-sensitive biosensing system for detecting CTCs in whole blood. The system is composed of Her2-coated immunomagnetic beads and an anti-EpCAM aptamer assembled pseudo-DNA nanocatenane (PDN) for dual targeting and separating CTCs, in conjunction with the rolling circle amplification (RCA) and molecular beacon (MB) system for CTCs signal amplification. The Her-2-coated beads separated CTCs from blood after their elution from a magnetic column. The unique PDN, which is a tailor-designed self-assembly of three circular DNAs that are inter-locked with independent and non-interfered templates for periodic RCA process, binds EpCAM-rich CTCs. In the presence of the RCA primer, phi29 DNA polymerase and MB, the system collaboratively generated the amplified fluorescent signals for highly-sensitive detection of CTCs. Through this system, we achieved the limit of detection less than 10 CTCs/mL blood, and quantified the number of CTCs in patient blood, which is proportional to the patient cancer status. Our technique is highly-sensitive, practicable and convenient enough for clinical detection of breast CTCs.

DNA Hydrogel with Aptamer-Toehold-Based Recognition, Cloaking, and Decloaking of Circulating Tumor Cells for Live Cell Analysis.

Circulating tumor cells (CTCs) contain molecular information on the primary tumor and can be used for predictive cancer diagnostics. Capturing rare live CTCs and their quantification in whole blood remain technically challenging. Here we report an aptamer-trigger clamped hybridization chain reaction (atcHCR) method for in situ identification and subsequent cloaking/decloaking of CTCs by porous DNA hydrogels. These decloaked CTCs were then used for live cell analysis. In our design, a DNA staple strand with aptamer-toehold biblocks specifically recognizes epithelial cell adhesion molecule (EpCAM) on the CTC surface that triggers subsequent atcHCR via toehold-initiated branch migration. Porous DNA hydrogel based-cloaking of single/cluster of CTCs allows capturing of living CTCs directly with minimal cell damage. The ability to identify a low number of CTCs in whole blood by DNA hydrogel cloaking would allow high sensitivity and specificity for diagnosis in clinically relevant settings. More significantly, decloaking of CTCs using controlled and defined chemical stimuli can release living CTCs without damages for subsequent culture and live cell analysis. We expect this liquid biopsy tool to open new powerful and effective routes for cancer diagnostics and therapeutics.

A cancer cell-activatable aptamer-reporter system for one-step assay of circulating tumor cells.

The current antibody-mediated numeration assays of circulating tumor cells (CTCs) require multiple steps and are time-consuming. To overcome these technical limitations, a cancer cell-activatable aptamer-reporter was formulated by conjugating a biomarker-specific aptamer sequence with paired fluorochrome-quencher molecules. In contrast to the antibody probes, the intact aptamer-reporter was optically silent in the absence of cells of interest. However, when used in an assay, the aptamer selectively targeted cancer cells through interaction with a specific surface biomarker, which triggered internalization of the aptamer-reporter and, subsequently, into cell lysosomes. Rapid lysosomal degradation of the aptamer-reporter resulted in separation of the paired fluorochrome-quencher molecules. The released fluorochrome emitted bright fluorescent signals exclusively within the targeted cancer cells, with no background noise in the assay. Thus, the assays could be completed in a single step within minutes. By using this one-step assay, CTCs in whole blood and marrow aspirate samples of patients with lymphoma tumors were selectively highlighted and rapidly detected with no off-target signals from background blood cells. The development of the cancer cell-activatable aptamer-reporter system allows for the possibility of a simple and robust point-of-care test for CTC detection, which is currently unavailable.

Sensitive and Direct Detection of Circulating Tumor Cells by Multimarker µ-Nuclear Magnetic Resonance

Identifying circulating tumor cells (CTCs) with greater sensitivity could facilitate early detection of cancer and rapid assessment of treatment response. Most current technologies use EpCAM expression as a CTC identifier. However, given that a significant fraction of cancer patients have low or even absent EpCAM levels, there is a need for better detection methods. Here, we hypothesize that a multimarker strategy combined with direct sensing of CTC in whole blood would increase the detection of CTC in patients. Accordingly, molecular profiling of biopsies from a patient cohort revealed a four-marker set (EpCAM, HER-2, EGFR, and MUC-1) capable of effectively differentiating cancer cells from normal host cells. Using a point-of-care micro-nuclear magnetic resonance (µNMR) system, we consequently show that this multimarker combination readily detects individual CTC directly in whole blood without the need for primary purification. We also confirm these results in a comparative trial of patients with ovarian cancer. This platform could potentially benefit a broad range of applications in clinical oncology.

Perioperative search for circulating tumor cells in patients undergoing radical cystectomy for bladder cancer

ObjectiveDespite having an organ confined tumor stage at the time of radical cystectomy, a certain number of bladder cancer patients will develop local or distant metastases over time. Currently there are no reliable serum markers for monitoring and evaluating risk profiles of urothelial cancers. Several studies suggest that detection of Circulating Tumor Cells (CTC) may correlate with disease status and prognosis at baseline and early in the treatment of cancers. The presence of CTCs in whole blood before and during radical cystectomy could provide further information on disease status, and could be used as an indicator to determine the need for adjuvant or even perioperative chemotherapy.MethodsFrom 03/2009 to 05/2009, five patients with histologically proven transitional cell carcinoma of the urinary bladder participated in this study. All patients were admitted to the hospital for radical cystectomy (rCx). A standard or extended lymph node dissection was performed in all cases. Preoperative CT or MRI scans revealed no distant or local metastases. Median age was 66.8 years (55-81 yrs). After obtaining informed consent from each patient, approximately 30 mL of peripheral blood was taken immediately before rCx and again during surgical removal of the urinary bladder from the patients' body. As additional parameters, operation time (OR) for surgical removal of the bladder and the amount of blood volume that was used for the detection of CTCs were recorded. Obtained blood samples were processed using the Cell-Search System (Veridex©) within 48 hours of collection. CTCs were identified and quantitated using the Cell-Search System, followed by re-evaluation of the provided results by specially trained and experienced personal. (CS, SH)ResultsCTCs were detected before and during surgical removal of the urinary bladder in one of five patients (20%). In the one patient positive for CTC, two CTCs were detected in the blood sample that was obtained before surgery (analyzed blood volume was 25 mL). There was one CTC detected in the blood sample that was obtained during surgical removal of the urinary bladder (analyzed blood volume was 27 mL). There was no rise in the amount of CTCs during surgical procedure. The final pathological report of this patient showed an advanced tumor stage (T3b, N0, R1). In the other patients, no CTCs were detected at all, neither before rCX nor right after surgical removal of the bladder. Pathological stage for these patients ranged from pT1m G3 -pT2b G3. None of these patients showed lymph node involvement. An average of 14.6 lymph nodes (5-40 LNs) were obtained. OR time to surgical removal of the urinary bladder ranged from 60 minutes to 150 minutes (mean 82 min.).ConclusionsAlthough only a very small group of patients was analyzed in this study, the presence of CTCs seems to be correlated with an advanced tumor stage. Therefore the detection of CTCs could be used for an optimized assessment of a patient's disease status in urothelial cancer. A further aim of this study was to assess whether surgical manipulation during radical cystectomy is associated with a release of CTCs into the vascular system. None of the patients who were negative for CTCs before surgery showed CTCs during surgical removal of the bladder, suggesting that there was no release of CTCs during surgery. However, further study is needed to prove these findings and evaluate the significance of CTCs as an indicator for therapeutic decisions.