Cancer-leukocyte hybrid membrane-cloaked magnetic beads for the ultrasensitive isolation, purification, and non-destructive release of circulating tumor cells.
Chang Zhi-Min,Zhang Rui,Yang Chao,Shao Dan,Tang Yuguo,Dong Wen-Fei,Wang Zheng
Most of the current circulating tumor cell (CTC) isolation techniques are based on immunomagnetic beads with antibodies or aptamers that specifically target epithelial cell adhesion molecules (EpCAMs). However, these techniques are unsuitable for the isolation and purification of circulating tumor cells because they fail to recognize EpCAM-negative CTCs and thus lead to the non-specific adsorption of background leucocytes and EpCAM-positive circulating epithelial cells. Moreover, releasing the CTCs from the capture platform without disruption is a big challenge. To address these issues, herein, we developed biomimetic magnetic beads (MBs) by cloaking a cancer cell-leukocyte hybrid membrane on the MBs. These biomimetic MBs inherited homologous CTC binding capability from the cancer cell membrane and less affinity for the background cells from the leukocyte membrane, exhibitng a higher CTC capture efficiency and separation purity than EpCAM-based MBs. Importantly, the captured CTCs could be rapidly released by a facile method i.e. co-incubation with a trypsin-EDTA solution. We demonstrated the excellent performance of these MBs for the highly pure separation and non-destructive release of CTCs in metastatic mammary carcinoma models. Our results indicate that the proposed homologous cancer-leukocyte membrane coating strategy may provide a promising method for the ultrahigh-specific and sensitive detection of CTCs.
Exploring Circulating Tumor Cells in Cholangiocarcinoma Using a Novel Glycosaminoglycan Probe on a Microfluidic Platform.
Gopinathan Priya,Chiang Nai-Jung,Bandaru Anandaraju,Sinha Anirban,Huang Wen-Yen,Hung Shang-Cheng,Shan Yan-Shen,Lee Gwo-Bin
Advanced healthcare materials
The search of alternative approaches to epithelial cell adhesion molecule (EpCAM), for the isolation of circulating tumor cells (CTC), is on the rise. This work attempts at evaluating the feasibility of using a new glycosaminoglycan, SCH45, as a probe to isolate CTCs from the peripheral blood of 65 advanced/metastatic cholangiocarcinoma (CCA) patients. The positive enrichment of CTCs from 1 mL of blood using SCH45-bound magnetic beads and subsequent staining on an integrated microfluidic platform is demonstrated. Results detailing CTC concentrations averaging ≥1 CTCs mL of blood are shown, and a conventional protein biomarker, EpCAM, has been used to corroborate the finding that 100% of the patients possess CTCs in their blood. Studies detailing the use of CTCs in the prognostic monitoring and treatment effectiveness of advanced/metastatic CCA are scarce, and the isolation of CTCs from all CCA patients tested has not been reported yet. A strong correlation between CTC counts and disease progression at the time of and/or in advance of radiographic imaging in patients receiving chemotherapy is also reported. This study is one of its kind with the new probe and reduced sample volume and has potential for use in CCA diagnosis and prognosis in the near future.
Vertical Magnetic Separation of Circulating Tumor Cells for Somatic Genomic-Alteration Analysis in Lung Cancer Patients.
Yoo Chang Eun,Park Jong-Myeon,Moon Hui-Sung,Joung Je-Gun,Son Dae-Soon,Jeon Hyo-Jeong,Kim Yeon Jeong,Han Kyung-Yeon,Sun Jong-Mu,Park Keunchil,Park Donghyun,Park Woong-Yang
Efficient isolation and genetic analysis of circulating tumor cells (CTCs) from cancer patients' blood is a critical step for clinical applications using CTCs. Here, we report a novel CTC-isolation method and subsequent genetic analysis. CTCs from the blood were complexed with magnetic beads coated with antibodies against the epithelial cell adhesion molecule (EpCAM) and separated vertically on a density-gradient medium in a modified well-plate. The recovery rate of model CTCs was reasonable and the cell purity was enhanced dramatically when compared to those parameters obtained using a conventional magnetic isolation method. CTCs were recovered from an increased number of patient samples using our magnetic system vs. the FDA-approved CellSearch system (100% vs. 33%, respectively). In 8 of 13 cases, targeted deep sequencing analysis of CTCs revealed private point mutations present in CTCs but not in matched tumor samples and white blood cells (WBCs), which was also validated by droplet digital PCR. Copy-number alterations in CTCs were also observed in the corresponding tumor tissues for some patients. In this report, we showed that CTCs isolated by the EpCAM-based method had complex and diverse genetic features that were similar to those of tumor samples in some, but not all, cases.
Efficient capture and simple quantification of circulating tumor cells using quantum dots and magnetic beads.
Min Hyegeun,Jo Seong-Min,Kim Hak-Sung
Small (Weinheim an der Bergstrasse, Germany)
Circulating tumor cells (CTCs) are valuable biomarkers for monitoring the status of cancer patients and drug efficacy. However, the number of CTCs in the blood is extremely low, and the isolation and detection of CTCs with high efficiency and sensitivity remain a challenge. Here, we present an approach to the efficient capturing and simple quantification of CTCs using quantum dots and magnetic beads. Anti-EpCAM antibody-conjugated quantum dots are used for the targeting and quantification of CTCs, and quantum-dot-attached CTCs are isolated using anti-IgG-modified magnetic beads. Our approach is shown to result in a capture efficiency of about 70%-80%, enabling the simple quantification of captured CTCs based on the fluorescence intensity of the quantum dots. The present method can be used effectively in the capturing and simple quantification of CTCs with high efficiency for cancer diagnosis and monitoring.
Enrichment and detection of circulating tumor cells by immunomagnetic beads and flow cytometry.
Hu Lei,Chen Xueran,Chen Meng,Fang Jinman,Nie Jinfu,Dai Haiming
OBJECTIVE:The purpose of the article is to establish a quick enrichment and detection method using immunomagnetic beads and flow cytometry to analyze circulating tumor cells (CTCs) in the peripheral blood. RESULTS:After incubation with CD326-PE and CD45-APC antibodies, more than 60% MCF7 cells in M-Buffer could be detected while less than 10% of the same cells could be detected by flow cytometry (FCM) if spiked into blood. However, in combination with CD326 and CD45 immunomagnetic beads, detection rate of MCF7 cells in blood reached 57%. For circulating tumor cells, enrichment by CD326 and CD45 immunomagnetic beads improve the detection rate from nearly undetectable to more than 24.14%. CONCLUSIONS:Live CTCs in peripheral blood can be effectively and sensitively detected by using a combination of immunomagnetic beads (CD45 and CD326) and flow cytometry.
Heterogeneous detection of circulating tumor cells in patients with colorectal cancer by immunomagnetic enrichment using different EpCAM-specific antibodies.
Antolovic Dalibor,Galindo Luis,Carstens Anina,Rahbari Nuh,Büchler Markus W,Weitz Jürgen,Koch Moritz
BACKGROUND:Circulating tumor cells (CTC) and disseminated tumor cells (DTC) are thought to be responsible for metastasis, so the detection of CTC may serve as individual prognostic factor in patients suffering from colorectal cancer. Therefore, a series of immunomagnetic enrichment methods for CTC have been developed using a variety of monoclonal antibodies against the Epithelial Cell Adhesion Molecule (EpCAM). However, it remains unclear whether all commercially available EpCAM antibodies show the same sensitivity and specificity. Furthermore, it remains unclear which method of sample preparation and cell extraction is most suitable for immunomagnetic enrichment and detection of CTC. In this study, we aimed to investigate whether the detection of CTC by a cytokeratin 20 reverse transcriptase-polymerase chain reaction (CK20 RT-PCR) may be influenced by the use of various Epithelial Cell Adhesion Molecule (EpCAM) antibodies for immunomagnetic isolation of CTC. RESULTS:Using both EpCAM antibodies (mAb BerEP4 and mAb KS1/4) for immunomagnetic enrichment in blood samples of 39 patients with colorectal cancer we found heterogenous results in each patient with regard to tumor cell detection. In the tumor cell spiking experiments with whole blood samples the sensitivity of the CK 20 RT-PCR assay was higher using immunomagnetic beads coated with mAb KS1/4 compared to precoated mAb BerEP4 Dynabeads. Extraction of MNC fraction with Ficoll gradient centrifugation prior to immunomagnetic enrichment resulted in a higher sensitivity of the CK 20 RT-PCR assay. CONCLUSIONS:We concluded that isolation and detection of CTC with immunomagnetic enrichment methods is critically dependent on the used EpCAM clone. Further studies with a larger number of patients should clarify if the enrichment protocol influences the prognostic value of the tumor cell detection protocol.
EpCAM-Independent Enrichment of Circulating Tumor Cells in Metastatic Breast Cancer.
Schneck Helen,Gierke Berthold,Uppenkamp Frauke,Behrens Bianca,Niederacher Dieter,Stoecklein Nikolas H,Templin Markus F,Pawlak Michael,Fehm Tanja,Neubauer Hans,
Circulating tumor cells (CTCs) are the potential precursors of metastatic disease. Most assays established for the enumeration of CTCs so far-including the gold standard CellSearch-rely on the expression of the cell surface marker epithelial cell adhesion molecule (EpCAM). But, these approaches may not detect CTCs that express no/low levels of EpCAM, e.g. by undergoing epithelial-to-mesenchymal transition (EMT). Here we present an enrichment strategy combining different antibodies specific for surface proteins and extracellular matrix (ECM) components to capture an EpCAMlow/neg cell line and EpCAMneg CTCs from blood samples of breast cancer patients depleted for EpCAM-positive cells. The expression of respective proteins (Trop2, CD49f, c-Met, CK8, CD44, ADAM8, CD146, TEM8, CD47) was verified by immunofluorescence on EpCAMpos (e.g. MCF7, SKBR3) and EpCAMlow/neg (MDA-MB-231) breast cancer cell lines. To test antibodies and ECM proteins (e.g. hyaluronic acid (HA), collagen I, laminin) for capturing EpCAMneg cells, the capture molecules were first spotted in a single- and multi-array format onto aldehyde-coated glass slides. Tumor cell adhesion of EpCAMpos/neg cell lines was then determined and visualized by Coomassie/MitoTracker staining. In consequence, marginal binding of EpCAMlow/neg MDA-MB-231 cells to EpCAM-antibodies could be observed. However, efficient adhesion/capturing of EpCAMlow/neg cells could be achieved via HA and immobilized antibodies against CD49f and Trop2. Optimal capture conditions were then applied to immunomagnetic beads to detect EpCAMneg CTCs from clinical samples. Captured CTCs were verified/quantified by immunofluorescence staining for anti-pan-Cytokeratin (CK)-FITC/anti-CD45 AF647/DAPI. In total, in 20 out of 29 EpCAM-depleted fractions (69%) from 25 metastatic breast cancer patients additional EpCAMneg CTCs could be identified [range of 1-24 CTCs per sample] applying Trop2, CD49f, c-Met, CK8 and/or HA magnetic enrichment. EpCAMneg dual-positive (CKpos/CD45pos) cells could be traced in 28 out of 29 samples [range 1-480]. By single-cell array-based comparative genomic hybridization we were able to demonstrate the malignant nature of one EpCAMneg subpopulation. In conclusion, we established a novel enhanced CTC enrichment strategy to capture EpCAMneg CTCs from clinical blood samples by targeting various cell surface antigens with antibody mixtures and ECM components.
Isolation, Detection, and Antigen-Based Profiling of Circulating Tumor Cells Using a Size-Dictated Immunocapture Chip.
Ahmed Metages Gashaw,Abate Mahlet Fasil,Song Yanling,Zhu Zhi,Yan Feng,Xu Yao,Wang Xiaomin,Li Qingbiao,Yang Chaoyong
Angewandte Chemie (International ed. in English)
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.
Circulating tumor cells: biology and clinical significance.
Signal transduction and targeted therapy
Circulating tumor cells (CTCs) are tumor cells that have sloughed off the primary tumor and extravasate into and circulate in the blood. Understanding of the metastatic cascade of CTCs has tremendous potential for the identification of targets against cancer metastasis. Detecting these very rare CTCs among the massive blood cells is challenging. However, emerging technologies for CTCs detection have profoundly contributed to deepening investigation into the biology of CTCs and have facilitated their clinical application. Current technologies for the detection of CTCs are summarized herein, together with their advantages and disadvantages. The detection of CTCs is usually dependent on molecular markers, with the epithelial cell adhesion molecule being the most widely used, although molecular markers vary between different types of cancer. Properties associated with epithelial-to-mesenchymal transition and stemness have been identified in CTCs, indicating their increased metastatic capacity. Only a small proportion of CTCs can survive and eventually initiate metastases, suggesting that an interaction and modulation between CTCs and the hostile blood microenvironment is essential for CTC metastasis. Single-cell sequencing of CTCs has been extensively investigated, and has enabled researchers to reveal the genome and transcriptome of CTCs. Herein, we also review the clinical applications of CTCs, especially for monitoring response to cancer treatment and in evaluating prognosis. Hence, CTCs have and will continue to contribute to providing significant insights into metastatic processes and will open new avenues for useful clinical applications.