Surface Of Circulating Tumor Cells Research Articles

Novel Isolating Approaches to Circulating Tumor Cell Enrichment Based on Microfluidics: A Review.

Circulating tumor cells (CTCs), derived from the primary tumor and carrying genetic information, contribute significantly to the process of tumor metastasis. The analysis and detection of CTCs can be used to assess the prognosis and treatment response in patients with tumors, as well as to help study the metastatic mechanisms of tumors and the development of new drugs. Since CTCs are very rare in the blood, it is a challenging problem to enrich CTCs efficiently. In this paper, we provide a comprehensive overview of microfluidics-based enrichment devices for CTCs in recent years. We explore in detail the methods of enrichment based on the physical or biological properties of CTCs; among them, physical properties cover factors such as size, density, and dielectric properties, while biological properties are mainly related to tumor-specific markers on the surface of CTCs. In addition, we provide an in-depth description of the methods for enrichment of single CTCs and illustrate the importance of single CTCs for performing tumor analyses. Future research will focus on aspects such as improving the separation efficiency, reducing costs, and increasing the detection sensitivity and accuracy.

Surfaceome: a new era in the discovery of immune evasion mechanisms of circulating tumor cells.

Circulating tumor cells (CTCs) are cancer cells that detach from the original site and reach the bloodstream. The most aggressive CTCs survive various immune system attacks and initiate metastasis formation. Importantly, CTCs are not specifically targeted by the current immunotherapies due to the limited knowledge on specific targets. Proteomic profiling can be a powerful tool for understanding some of the immune evasion mechanisms used by cancer cells and particularly CTCs. These mechanisms are generally linked to the expression of specific surface proteins/peptides (i.e. the surfaceome). The study of the peptides that bind to class I molecules of the major histocompatibility complex (MHC-I) and of the various glycoproteins expressed on CTC surface may open a completely new avenue for the discovery of novel mechanisms of immune evasion. In this review, we discuss how immunopeptidomic and glycoproteomic studies of CTCs that interact with immune cells could help to better understand how metastasis-initiator CTCs escape the host immune response. We also describe how immunopeptidomic and glycoproteomic studies are carried out.

Precise Capture and Dynamic Release of Circulating Liver Cancer Cells with Dual-Histidine-Based Cell Imprinted Hydrogels.

Circulating tumor cells (CTCs) detection presents significant advantages in diagnosing liver cancer due to its noninvasiveness, real-time monitoring, and dynamic tracking. However, the clinical application of CTCs-based diagnosis is largely limited by the challenges of capturing low-abundance CTCs within a complex blood environment while ensuring them alive. Here, an ultrastrong ligand, l-histidine-l-histidine (HH), specifically targeting sialylated glycans on the surface of CTCs, is designed. Furthermore, HH is integrated into a cell-imprinted polymer, constructing a hydrogel with precise CTCs imprinting, high elasticity, satisfactory blood compatibility, and robust anti-interference capacities. These features endow the hydrogel with excellent capture efficiency (>95%) for CTCs in peripheral blood, as well as the ability to release CTCs controllably and alive. Clinical tests substantiate the accurate differentiation between liver cancer, cirrhosis, and healthy groups using this method. The remarkable diagnostic accuracy (94%), lossless release of CTCs, material reversibility, and cost-effectiveness ($6.68 per sample) make the HH-based hydrogel a potentially revolutionary technology for liver cancer diagnosis and single-cell analysis.

Functionalized tetrahedral DNA frameworks for the capture of circulating tumor cells.

Identification and characterization of circulating tumor cells (CTCs) from blood samples of patients with cancer can help monitor parameters such as disease stage, disease progression and therapeutic efficiency. However, the sensitivity and specificity of current multivalent approaches used for CTC capture is limited by the lack of control over the ligands' position. In this Protocol Update, we describe DNA-tetrahedral frameworks anchored with aptamers that can be configured with user-defined spatial arrangements and stoichiometries. The modified tetrahedral DNA frameworks, termed 'n-simplexes', can be used as probes to specifically target receptor-ligand interactions on the cell membrane. Here, we describe the synthesis and use of n-simplexes that target the epithelial cell adhesion molecule expressed on the surface of CTCs. The characterization of the n-simplexes includes measuring the binding affinity to the membrane receptors as a result of the spatial arrangement and stoichiometry of the aptamers. We further detail the capture of CTCs from patient blood samples. The procedure for the preparation and characterization of n-simplexes requires 11.5 h, CTC capture from clinical samples and data processing requires ~5 h per six samples and the downstream analysis of captured cells typically requires 5.5 h. The protocol is suitable for users with basic expertise in molecular biology and handling of clinical samples.

Self-assembly circular multi-DNA mediated rolling circle amplification for sensitive detection of PD-L1+ circulating tumor cells in the peripheral blood of cancer patients

It has been found that evaluating the status of programmed cell death ligand (PD-L1) in the circulating tumor cells (CTCs) was associated with improved efficacy to anti-PD-L1 inhibitors. However, high-quality detection of PD-L1 positive (PD-L1+) CTCs remains a huge challenge. In this study, we propose a short time, high efficiency and applicability method that fabricated by self-assembly circular multi-DNA (CMD) mediating rolling circle amplification (RCA) for highly sensitive and rapid analysis of PD-L1+ cells. Firstly, a short single-stranded circular DNA which could bind specifically with PD-L1 protein was designed. In addition, CMD were designed as self-assembly which could serve as a template in RCA process. Next, the exponential amplification of DNA was achieved by the rolling circle amplification and the fluorescence signal was detected by using the molecular beacons. The unique CMD mediated rolling circle amplification facilitated quantitative detection of PD-L1+ CTCs in the blood of different cancer patients. To visualize the results of CMD on the target cells, we used confocal microscope to assess PD-L1 expression in MCF-7 (breast cancer), OE33 (esophaguscancer) and HCC827 (lung cancer) cells. The results indicated that the CMD mediating RCA can perfectly combine with different kinds of cells, amplify the signal and the recovery rate was over 99 %. What’s more, we performed clinical tests in the blood of the corresponding patient (14 cases of breast cancer, 15 esophageal cancer and 11 lung cancer). We further quantified the number of CTCs in patient blood through establishing the standard curve and the limit of detection (LOD) was calculated to be 1 cell/mL. Moreover, the ring structure was designed to have replaceable sequence and to realize combining different CTCs surface markers (such as Her-2). This method has the advantages of high sensitivity, good expansibility, low cost and can be completed in a short time as well. Therefore, it is expected to develop a new CTCs typing detection kit and might thus provide reliable diagnostic results to provide individualized treatment strategies.

Sensitive detection of folate receptor-positive circulating tumor cells based on intracellular uptake of the PbS nanoparticle cluster-loaded phospholipid micelles decorated with folic acid in combination with E-DNA sensor

Developing methods for the circulating tumor cells (CTCs) detection is important for noninvasive cancer diagnosis. A sensitive method for homogeneous electrochemical detection of CTCs was developed based on the affinity of folic acid (FA) for folate receptor (FR), which is overexpressed on the CTC surface. The PbS nanoparticle cluster-loaded phospholipid micelles decorated with FA were prepared and used as a nanoprobe. The specific binding of FA to FR-positive CTCs leads to the uptake of the nanoprobes into the CTCs. Cellular imaging assay showed the excellent biocompatibility and selectivity of nanoprobes. After separation of intracellular nanoprobes and the residual free nanoprobes in the solution by centrifugation, nanoprobes in the cell were dissolved using acid. The amount of the target cells was quantified by the electrochemical detection of Pd2+ using the electrochemical DNA (E-DNA) sensor, which was fabricated based on the self-assembly of probe DNA on the flowerlike gold nanostructures modified the screen-printed carbon electrode (SPCE). Because of the large quantities of detectable Pd2+ contained in the nanoprobes, the signal amplification effect was achieved. The developed electrochemical assay allows the detection of ∼5 cells from 1 mL of sample. Studies with both healthy individuals and cancer patient blood samples demonstrate that the assay is sensitive and FR-positive CTCs specific.

An Application of Tumor-Associated Macrophages as Immunotherapy Targets: Sialic Acid-Modified EPI-Loaded Liposomes Inhibit Breast Cancer Metastasis.

Breast cancer metastasis is an important cause of death in patients with breast cancer and is closely related to circulating tumor cells (CTCs) and the metastatic microenvironment. As the most infiltrating immune cells in the tumor microenvironment (TME), tumor-associated macrophages (TAMs), which highly express sialic acid (SA) receptor (Siglec-1), are closely linked to tumor progression and metastasis. Furthermore, the surface of CTCs also highly expressed receptor (Selectin) for SA. A targeting ligand (SA-CH), composed of SA and cholesterol, was synthesized and modified on the surface of epirubicin (EPI)-loaded liposomes (EPI-SL) as an effective targeting delivery system. Liposomes were evaluated for characteristics, stability, in vitro release, cytotoxicity, cellular uptake, pharmacokinetics, tumor targeting, and pharmacodynamics. In vivo and in vitro experiments showed that EPI-SL enhanced EPI uptake by TAMs. In addition, cellular experiments showed that EPI-SL could also enhance the uptake of EPI by 4T1 cells, resulting in cytotoxicity second only to that of EPI solution. Pharmacodynamic experiments have shown that EPI-SL has optimal tumor inhibition with minimal toxicity, which can be ascribed to the fact that EPI-SL can deliver drugs to tumor based on TAMs and regulate TME through the depletion of TAMs. Our study demonstrated the significant potential of SA-modified liposomes in antitumor metastasis. Schematic diagram of the role of SA-CH modified EPI-loaded liposomes in the model of breast cancer metastasis.

SERS Multiplex Profiling of Melanoma Circulating Tumor Cells for Predicting the Response to Immune Checkpoint Blockade Therapy.

Immune checkpoint blockade (ICB) therapy has achieved remarkable success in many cancers including melanoma. However, ICB therapy benefits only a small proportion of patients and produces severe side effects for some patients. Thus, there is an urgent need to identify patients who are more likely to respond to ICB therapy to improve outcomes and minimize side effects. To predict ICB therapy responses, we design a surface-enhanced Raman scattering (SERS) assay for multiplex profiling of circulating tumor cells (CTCs) under basal and interferon-γ (IFN-γ) stimulation. Through simultaneous ensemble and single-cell measurements of CTCs, the SERS assay can reveal tumor heterogeneity and offer a comprehensive CTC phenotype for decision-making. Anisotropic gold-silver alloy nanoboxes are utilized as SERS plasmonic substrates for improved signal readouts of CTC surface biomarkers. By generating a unique CTC signature with four surface biomarkers, the developed assay enables the differentiation of CTCs from three different patient-derived melanoma cell lines. Significantly, in a cohort of 14 melanoma patients who received programmed cell death-1 blockade therapy, the changes of CTC signature induced by IFN-γ stimulation to CTCs show the potential to predict responders. We expect that the SERS assay can help select patients for receiving ICB therapy in other cancers.

In situ signal amplification improves the capture efficiency of circulating tumor cells with low expression of EpCAM

Circulating tumor cells (CTCs) as non-invasive biomarkers have great potential in evaluating tumor progression and prognosis. However, effective enrichment of CTCs and minimizing phenotypic bias remain a serious challenge. Herein, a DNA tetrahedron-aptamer complex-mediated rolling circle amplification (TDN-RCA) strategy is developed for cell surface protein signal amplification and CTC enrichment, employing DNA tetrahedron-EpCAM aptamer complex as a scaffold and initiating rolling circle amplification (RCA) reaction on the surface of CTCs in situ. The DNA tetrahedron-aptamer complex enables the cell-specific recognition and enhances cell membrane anchoring ability, generating a large number of magnetic beads binding sites through the RCA reaction in situ. Thus, the signals of cell surface markers with low expression levels are amplified in situ and then efficient CTC enrichment is achieved. This method improves the capture efficiency of CTCs with low expression of EpCAM, which has great potential in clinical application.

Simultaneous in situ detection of protein expression of multiple tumor markers of circulating tumor cells and heteroploid of chromosome 8 in primary lung cancer

BackgroundPreviously published studies have shown that circulating tumor cells (CTC) play an important role in clinical staging, efficacy monitoring and prognostic evaluation of lung cancer. The aim of the present study was to investigate the significance of simultaneous in situ detection of multiple tumor marker protein expression and chromosome 8 aneuploid of CTC in the diagnosis and treatment of primary lung cancer.MethodsWe investigated the expression of cytokeratin 18 (CK18) and Vimentin on the surface of CTC and the aneuploidy of chromosome 8 in the peripheral blood of 24 patients with metastatic primary lung cancer, which were detected by subtraction enrichment (SE) and immunofluorescence in situ hybridization (iFISH). Their correlation with clinicopathological features, curative effect, and prognosis was analyzed.ResultsThe positive rate of CTC was 95.83% (23/24). There was a certain correlation between the positive rate of CTC and smoking, and a correlation between the number of CTC and histopathological type. The number of monoploid, diploid, triploid, and polyploid CTC had no statistical correlation with progression-free survival (PFS) or overall survival (OS). However, a tetraploid CTC count of ≥2 was an unfavorable predictor of response; a tetraploid CTC count ≥1 was an unfavorable prognostic predictor of poor OS. The rate of CK18+ CTC in all 24 patients was 4.35% (1/23). Seven out of these 24 patients were also tested for Vimentin, and the rate of Vimentin+ CTC was 85.71% (6/7). Small-cell (≤5 µm) CTC was found in 6 of these 7 patients and accounted for 11.83% (11/93) of the total CTC. The tumor marker phenotype of small-cell CTC was CK18– and Vimentin+. In addition, circulating tumor microthrombi (CTM) were found in 2 of these 7 patients (28.57%).ConclusionsSE-iFISH has a high detection rate of CTC in the peripheral blood of patients with metastatic primary lung cancer, and it can identify small cell CTC. Tetraploid CTC count ≥2 can predict poor PFS in patients with advanced lung cancer. Tetraploid CTC count ≥1 could predict poor OS in patients with advanced lung cancer. CTM can predict poor prognosis in patients with lung cancer.

CD44v6 Defines a New Population of Circulating Tumor Cells Not Expressing EpCAM.

Simple SummaryIn the present work, we describe (for the first time) the use of the transmembrane protein, CD44v6, to detect CTCs from blood samples of several patients with colorectal or breast cancer. We used CD44v6 antibodies to demonstrate that live CTCs can be specifically purified from CRC patient blood samples via magnetic bead- or FACS-based isolation techniques. Finally, we demonstrated that CD44v6-positive CTCs rarely expressed EpCam, which is currently the gold standard to enumerate CTCs, suggesting the need to use a combination of markers for a more comprehensive view of CTC heterogeneity.Circulating tumor cells (CTCs) are promising diagnostic and prognostic tools for clinical use. In several cancers, including colorectal and breast, the CTC load has been associated with a therapeutic response as well as progression-free and overall survival. However, counting and isolating CTCs remains sub-optimal because they are currently largely identified by epithelial markers such as EpCAM. New, complementary CTC surface markers are therefore urgently needed. We previously demonstrated that a splice variant of CD44, CD44 variable alternative exon 6 (CD44v6), is highly and specifically expressed by CTC cell lines derived from blood samples in colorectal cancer (CRC) patients. Two different approaches—immune detection coupled with magnetic beads and fluorescence-activated cell sorting—were optimized to purify CTCs from patient blood samples based on high expressions of CD44v6. We revealed the potential of the CD44v6 as a complementary marker to EpCAM to detect and purify CTCs in colorectal cancer blood samples. Furthermore, this marker is not restricted to colorectal cancer since CD44v6 is also expressed on CTCs from breast cancer patients. Overall, these results strongly suggest that CD44v6 could be useful to enumerate and purify CTCs from cancers of different origins, paving the way to more efficacious combined markers that encompass CTC heterogeneity.

Dual-Multivalent-Aptamer-Conjugated Nanoprobes for Superefficient Discerning of Single Circulating Tumor Cells in a Microfluidic Chip with Inductively Coupled Plasma Mass Spectrometry Detection.

The efficient recognition of circulating tumor cells (CTCs) with an aptamer probe confers numerous benefits; however, the stability and binding affinity of aptamers are significantly hampered in real biological sample matrices. Inspired by the efficient preying mechanism by multiplex tubing feet and endoskeletons of sea urchins, we engineered a superefficient biomimetic single-CTC recognition platform by conjugating dual-multivalent-aptamers (DMAs) Sgc8 and SYL3C onto AuNPs to form a sea urchin-like nanoprobe (sea urchin-DMA-AuNPs). Aptamers Sgc8 and SYL3C selectively bind with the biomarker proteins PTK7 and EpCAM expressed on the surface of CTCs. CTCs were captured with 100% efficiency, followed by sorting on a specially designed multifunctional microfluidic configuration, integrating a single-CTC separation unit and a hydrodynamic filtrating purification unit. After sorting, background-free analysis of biomarker proteins in single CTCs was undertaken with inductively coupled plasma mass spectrometry by measuring the amount of 197Au isotope in sea urchin-DMA-AuNPs. With respect to a single-aptamer nanoprobe/-interface, the dual-aptamer nanoprobe improves the binding efficiency by more than 200% (Kd < 0.35 nM). The microchip facilitates the recognition of single CTCs with a sorting separation rate of 93.6% at a flow rate of 60 μL min-1, and it exhibits 73.8 ± 5.0% measurement efficiency for single CTCs. The present strategy ensures the manipulation and detection of a single CTC in 100 μL of whole blood within 1 h.

In Situ Single Cell Proteomics Reveals Circulating Tumor Cell Heterogeneity during Treatment.

Cancer is a dynamic disease with heterogenic molecular signatures and constantly evolves during the course of the disease. Single cell proteomic analysis could offer a suitable pathway to monitor cancer cell heterogeneity and deliver critical information for the diagnosis, recurrence, and drug-resistant mechanisms in cancer. Current standard techniques for proteomic analysis such as ELISA, mass spectrometry, and Western blots are time-consuming, expensive, and often require fluorescence labeling that fails to provide accurate information about the multiple protein expression changes at the single cell level. Herein, we report a surface-enhanced Raman spectroscopy-based simple microfluidic device that enables the screening of single circulating tumor cells (CTC) in a dynamic state to precisely understand the heterogeneous expression of multiple protein biomarkers in response to therapy. It further enables identifying intercellular heterogeneous expression of CTC surface proteins which would be highly informative to identify the cancer cells surviving treatment and potentially responsible for drug resistance. Using a bead and cell line-based model system, we successfully detect single bead and single cell spectra when flowed through the device. Using SK-MEL-28 melanoma cells, we demonstrate that our system is capable of monitoring heterogeneous expressions of multiple surface protein markers (MCSP, MCAM, and LNGFR) before and during drug treatment. Integrating a label-free electrochemical system with the device, we also monitor the expression of an intracellular protein (here, BRAFV600E) under drug treatment. Finally, we perform a longitudinal study with 15 samples from five different melanoma patients who underwent therapy. We find that the average expression of receptor proteins in a patient fails to determine the therapy response particularly when the disease progresses. However, single CTC analysis with our device shows a high level of intercellular heterogeneity in the receptor expression profiles of patient-derived CTCs and identifies heterogeneity within CTCs. More importantly, we find that a fraction of CTCs still shows a high expression of these receptor proteins during and after therapy, indicating the presence of resistant CTCs which may evolve after a certain time and progress the disease. We believe this automated assay will have high clinical importance in disease diagnosis and monitoring treatment and will significantly advance the understanding of cancer heterogeneity on the single cell level.

Abstract 2448: Modeling the mechanisms behind the biphasic expression of the PTK7 tyrosine kinase receptor in colorectal cancer

Abstract Colorectal cancer (CRC) is the second deadliest cancer worldwide. Discovery of new biomarkers and therapeutic targets is thus essential (PMID: 27922044). CRC cells frequently reactivate developmental pathways such as WNT, Hedgehog and Hippo pathways, which participate to tumorigenesis and metastatic dissemination. The tyrosine kinase receptor PTK7 is a cell surface component from the WNT pathway involved in embryonic development in many species (PMID: 15229603). In retrospective studies, PTK7 was previously found overexpressed in CRC, an event associated with metastatic development, reduced metastases-free survival of non-metastatic patients, and resistance to chemotherapy (PMID: 25962058 and 21103379). PTK7 has pro-migratory and pro-metastatic functions in vitro and in vitro, but the mechanisms behind are not well understood yet. We recently conducted a prospective study collecting primary tumors, circulating tumor cells (CTCs) and metastases from CRC patients and evaluated expression of PTK7 in all types of samples at the mRNA and protein levels. PTK7 was confirmed as an independent poor prognosis marker in CRC with strong expression in primary tumors and metastases. Surprisingly, a large majority of CTCs were tested negative for the presence of PTK7 at their cell surface. We recapitulated this observation in a xenografted mouse model, as well as in an in vitro microfluidic setup preventing cell-matrix and cell-cell interactions of circulating CRC cells. Both results demonstrated the cell autonomous origin of PTK7 disappearance from CRC cells once in circulation. Interestingly, knockdown of PTK7 with small interfering RNA impaired the adhesion pattern of now PTK7-negative CRC cells on several matrix and promoted resistance to anoikis. A screen combining proximity labeling and mass spectrometry from CRC cells identified partners of PTK7 involved in cell adhesion, including integrins. In the absence of PTK7, two of them (ITGβ1 and ITGαV) had disabled function and expression, and their deregulation was associated with an increased phosphorylation of Focal Adhesion Kinase (FAK), a major actor of cancer cell survival and resistance to anoikis. We are currently investigating the mechanism of disappearance of PTK7 from the cell surface of CTCs and its consequence on drug resistance and metastatic dissemination of CRC cells. Citation Format: Laetitia Ganier, Anais Aulas, Olivier Cabaud, Alexia Lopresti, Kostyantyn Semenchenko, Pascal Finetti, Marine Gilabert, Anthony Goncalves, Claire Acquaviva, Daniel Birnbaum, Emilie Mamessier, Jean-Paul Borg. Modeling the mechanisms behind the biphasic expression of the PTK7 tyrosine kinase receptor in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2448.

Magnetic bead-based electrochemical and colorimetric assays of circulating tumor cells with boronic acid derivatives as the recognition elements and signal probes

Simple, sensitive and rapid detection of circulating tumor cells (CTCs) is of great importance for early diagnosis and therapy of cancers. Overexpression of sugar units on cell surface is related to the phenotypes of many cancers. Based on the boronate ester interaction, we reported the electrochemical and colorimetric detection of CTCs with high simplicity and sensitivity. Specifically, ferroceneboronic acid (FcBA) can be measured by differential pulse voltammetry and 4-mercaptophenylboronic acid (MPBA) can induce the aggregation and color change of gold nanoparticles (AuNPs). CTCs captured by the aptamer-modified magnetic beads (Apt-MBs) can sequestrate FcBA or MPBA molecules by the formation of boronate ester bonds, thus leading to the decrease in the electrochemical signal of FcBA or preventing the MPBA-triggered aggregation of AuNPs. Due to the overexpression of sugar groups on the surface of CTCs, the amplification-free methods exhibited high sensitivity and obviated the use of additional antibody or aptamer for the recognition of captured cells. With MCF-7 cancer cell as the model, 50 cells can be readily determined by the electrochemical and colorimetric methods. The proposed strategy is valuable for probing of cell glycosylation and designing of novel sensing devices for detection of sugar-containing biological macromolecules and cells.

Encapsulation and release of living tumor cells using hydrogels with the hybridization chain reaction.

Circulating tumor cells (CTCs) enable noninvasive liquid biopsy and identification of cancer. Various approaches exist for the capture and release of CTCs, including microfluidic methods and those involving magnetic beads or nanostructured solid interfaces. However, the concomitant cell damage and fragmentation that often occur during capture make it difficult to extensively characterize and analyze living CTCs. Here, we describe an aptamer-trigger-clamped hybridization chain reaction (atcHCR) method for the capture of CTCs by porous 3D DNA hydrogels. The 3D environment of the DNA networks minimizes cell damage, and the CTCs can subsequently be released for live-cell analysis. In this protocol, initiator DNAs with aptamer-toehold biblocks specifically bind to the epithelial cell adhesion molecule (EpCAM) on the surface of CTCs, which triggers the atcHCR and the formation of a DNA hydrogel. The DNA hydrogel cloaks the CTCs, facilitating quantification with minimal cell damage. This method can be used to quantitively identify as few as 10 MCF-7 cells in a 2-µL blood sample. Decloaking of tumor cells via gentle chemical stimulus (ATP) is used to release living tumor cells for subsequent cell culture and live-cell analysis. We also describe how to use the protocol to encapsulate and release cells of cancer cell lines, which can be used in preliminary experiments to model CTCs. The whole protocol takes ~2.5 d to complete, including downstream cell culture and analysis.

Aminophenylboronic Acid-Functionalized Thorny-Trap-Shaped Monolayer Microarray for Efficient Capture and Release of Circulating Tumor Cells.

An aminophenylboronic acid (APBA)-functionalized thorny-trap-shaped monolayer microarray as a 3D fractal structures' substrate was fabricated to capture and release circulating tumor cells (CTCs) efficiently. The microarray can not only trap cells inside by microbowls but also enhance the interaction between cell and substrate by providing more binding sites and facilitating the spread of cell filopodias via the growth of nanorods. Modification of APBA enhanced the interaction further by binding with sialic acid of CTCs surface. The special topological structure achieved a high capture efficiency of 79.5%. The captured cancer cells were released without introducing any affinity molecules by a ligand exchange reaction with up to 70% efficiency and good proliferation. This substrate can isolate 33 tumor cells from a mimic blood sample even at a low spiked number of 50 cancer cells. This study provides valuable guidance for isolation and release of CTCs and is significant for the further study of tumors.

Photoelectrochemical detection of circulating tumor cells based on aptamer conjugated Cu2O as signal probe

In this work, a sensitive and reliable photoelectrochemical (PEC) biosensor was proposed based on hexagonal carbon nitride tubes (HCNT) as photoactive material for detection of circulating tumor cells (CTCs). Magnetic Fe3O4 nanospheres (MNs) and Cu2O nanoparticles (Cu2O NPs) were utilized for highly efficient magnetic capture of CTCs and for signal amplification, respectively. First, anti-epithelial cell adhesion molecule (EpCAM) antibody was linked onto MNs for capture and enrichment of CTCs. With the captured MCF-7 coated onto the electrode, the photocurrent intensity of HCNT was decreased due to the steric hindrance derived from MCF-7. Then, when the Cu2O-aptamer probe was bound onto the CTC surface, the photocurrent intensity was further decreased because Cu2O NPs competed with HCNT for absorption of exciting light and the aptamer molecules increased the steric hindrance, which leads to significantly decreased photocurrent response, thus realizing dual signal amplification. Using the breast cancer cell MCF-7 as a model, the proposed PEC biosensor displays good performances with a linear range from 3 to 3000 cell mL−1 and limit of detection down to 1 cell mL−1. The HCNT-based PEC biosensor shows good performance for detection of CTCs, which may have potential applications in cancer diagnostics and therapeutics.

Electrochemical Detection of Circulating Tumor Cells Based on DNA Generated Electrochemical Current and Rolling Circle Amplification

Circulating tumor cells (CTCs) are important indicators for tumor diagnosis and tumor metastasis. However, the extremely low levels of CTCs in peripheral blood challenges the precise detection of CTCs. Herein, we report DNA generated electrochemical current combined with rolling circle amplification (RCA) as well as magnetic nanospheres for highly efficient magnetic capture and ultrasensitive detection of CTCs. The antiepithelial cell adhesion molecule (EpCAM) antibody-modified magnetic nanospheres were used to capture and enrich CTCs. The following binding of an aptamer onto the CTC surface and the subsequent RCA assembled a significant amount of DNA molecules onto the electrode. The reaction of the DNA molecules with molybdate can then form redox molybdophosphate and produce an electrochemical current. Using the breast cancer cell MCF-7 as a model, the sensor displays good performances toward detection of MCF-7 that was spiked into peripheral blood. The signal amplification strategy integrated with a magnetic nanosphere platform exhibits good performance in the efficient capture and detection of CTCs, which may find wide potential in cancer diagnostics and therapeutics.

Folic Acid Targeting for Efficient Isolation and Detection of Ovarian Cancer CTCs from Human Whole Blood Based on Two-Step Binding Strategy.

Studies regarding circulating tumor cells (CTCs) have great significance for cancer prognosis, treatment monitoring, and metastasis diagnosis. However, due to their extremely low concentration in peripheral blood, isolation and enrichment of CTCs are the key steps for early detection. To this end, targeting the folic acid receptors (FRs)on the CTC surface for capture with folic acid (FA) using bovine serum albumin (BSA)-tether for multibiotin enhancement in combination with streptavidin-coated magnetic nanoparticles (MNPs-SA) was developed for ovarian cancer CTC isolation. The streptavidin-biotin-system-mediated two-step binding strategy was shown to capture CTCs from whole blood efficiently without the need for a pretreatment process. The optimized parameters for this system exhibited an average capture efficiency of 80%, which was 25% higher than that of FA-decorated magnetic nanoparticles based on the one-step CTC separation method. Moreover, the isolated cells remained highly viable and were cultured directly without detachment from the MNPs-SA-biotin-CTC complex. Furthermore, when the system was applied for the isolation and detection of CTCs in ovarian cancer patients' peripheral blood samples, it exhibited an 80% correlation with clinical diagnostic criteria. The results indicated that FA targeting, in combination with BSA-based multibiotin enhancement magnetic nanoparticle separation, is a promising tool for CTC enrichment and detection of early-stage ovarian cancer.