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Skovborg G, Svejsø FH, Müller C, Jensen BN, Jensen JG, Majidi SE, Matthiesen CL, Chen M. Replication of patient specific circulating tumor cells on a microfibrous filter for drug screening. NANOSCALE 2025; 17:11592-11604. [PMID: 40242908 DOI: 10.1039/d4nr05294c] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/18/2025]
Abstract
Personalized medicine in cancer treatment has the potential to enhance therapeutic efficacy while simultaneously reducing adverse effects. Molecular characterization of circulating tumor cells (CTCs) offers invaluable insight into metastatic tumor heterogeneity, making them a perfect candidate for metastatic cancer drug screening. However, they are extremely rare. This study presents the development of melt-electrowritten membrane filters designed for the capture, culture, and drug testing of CTCs. By varying the collector speeds, filters with optimized pore sizes and polymer densities were produced, enabling selective capture of CTCs while minimizing co-capture of white blood cells. Biocompatibility tests showed that the filter supported the proliferation of multiple cancer cell lines. The filter successfully captured and cultured colorectal cancer patient-derived CTC44 and CTC45 cells, which formed 3D clusters observable over several weeks. Drug testing with chemotherapeutic agents 5-fluorouracil/oxaliplatin (FOX) and 5-fluorouracil/irinotecan (FIRI) revealed that CTCs in 3D clusters on the filters exhibited significantly higher drug resistance compared to 2D monolayers. These findings demonstrate the potential of the filter as a versatile platform for studying CTC biology and for screening anticancer drugs, providing a more physiologically relevant environment than traditional 2D cultures.
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Affiliation(s)
- Grith Skovborg
- Department of Biological and Chemical Engineering, Aarhus University, Aarhus, DK-8000, Denmark.
| | - Frederik Høbjerg Svejsø
- Department of Biological and Chemical Engineering, Aarhus University, Aarhus, DK-8000, Denmark.
| | - Christoph Müller
- Department of Biological and Chemical Engineering, Aarhus University, Aarhus, DK-8000, Denmark.
| | | | - Jesper Godrim Jensen
- Department of Biological and Chemical Engineering, Aarhus University, Aarhus, DK-8000, Denmark.
| | - Sara Egsgaard Majidi
- Department of Biological and Chemical Engineering, Aarhus University, Aarhus, DK-8000, Denmark.
| | | | - Menglin Chen
- Department of Biological and Chemical Engineering, Aarhus University, Aarhus, DK-8000, Denmark.
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2
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Shen C, Fan S, Li X, Guo F, Li J, Yang M. A novel electrochemiluminescent cytosensor using dual-target magnetic probe recognition and nanozymes-catalyzed cascade signal amplification for precise phenotypic enumeration of CTCs. Mikrochim Acta 2024; 191:736. [PMID: 39531095 DOI: 10.1007/s00604-024-06825-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2024] [Accepted: 11/03/2024] [Indexed: 11/16/2024]
Abstract
The inability of surgical biopsy to monitor the dynamic evolution of cancer cells hampers its capacity to reflect real-time tumor heterogeneity. Circulating tumor cells (CTCs), as a crucial target in liquid biopsy, offer a novel approach for accurate monitoring of tumors. However, the rarity and complex phenotype resulting from epithelial-mesenchymal transition pose challenges for conventional methods such as CellSearch and immunohistochemistry, which have insufficient ability for simultaneous phenotyping and enumeration of CTCs. The enumeration of a single phenotype CTCs is insufficient for accurately assessing disease progression. Herein, we propose a strategy to address this issue by fabricating an electrochemiluminescence cytosensor via the integration of dual-target enrichment and nanozymes-catalyzed cascade signal amplification. The graphene oxide@hollow mesoporous Prussian blue/Pt (GO@HMPB/Pt) complex, possessing a large specific surface area and exceptional catalytic activity, is employed for loading a substantial amount of luminol as the signal probe. Dual-target magnetic PPy@Fe3O4/Au-antibody/aptamer is utilized for the magnetic capture of both epithelial and interstitial CTCs. Glutathione (GSH) can disrupt the Au-S bond on aptamer by a thiol exchange reaction and selectively releases a specific subset of phenotypic CTCs, thereby facilitating the efficient capture, accurate classification, and ultrasensitive detection of CTCs in peripheral blood. Using the epithelial MCF-7 and mesenchymal Hela cells as models, the ECL cytosensor demonstrates excellent performance in identifying cells spiked into whole blood. This study presents a novel approach for early detection of metastasis, tracking tumor recurrence, and monitoring therapeutic efficacy.
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Affiliation(s)
- Congcong Shen
- Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, School of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang, 453007, Henan, China.
| | - Simin Fan
- Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, School of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang, 453007, Henan, China
| | - Xiaoqing Li
- College of Chemistry and Chemical Engineering, Central South University, Changsha, 410083, China
| | - Fanshu Guo
- Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, School of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang, 453007, Henan, China
| | - Junru Li
- Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, School of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang, 453007, Henan, China
| | - Minghui Yang
- College of Chemistry and Chemical Engineering, Central South University, Changsha, 410083, China.
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3
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Smithers JP, Sheu J, Richardson B, Hayes MA. NanoRidge filters: Fabrication strategies and performance optimization for nano-scale microfluidic particle filtration. BIOMICROFLUIDICS 2024; 18:054102. [PMID: 39247800 PMCID: PMC11379496 DOI: 10.1063/5.0210149] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/25/2024] [Accepted: 07/13/2024] [Indexed: 09/10/2024]
Abstract
Filters with high throughput, minimal dead volume, and greater sensitivity to particle size are needed, which traditional benchtop filtration cannot provide. Leveraging microfabrication techniques developed by the electronics and optics industries, the filters presented here feature a unique serpentine "NanoRidge" structure, offering a continuous filtration gap spanning over three meters on a compact 4 × 14.5 mm2 footprint. This design provides more precise size filtration cut-offs and consistent flow paths compared to traditional membrane filtration systems. Despite challenges associated with glass substrate deformation impacting uniform filter gap sizes, the study provides valuable insights into the development of NanoRidge filters (NRFs) for enhancing filtration efficiency in preparatory techniques and sample analysis. This study describes the fabrication and testing of these new filter types and directly compares the performance to traditional membrane filters using the metrics of particle size cut-off (the smallest difference in particle size which can be filtered vs passed) and particle loss. The NanoRidge filters were characterized using imaging (during fabrication, post-fabrication and use, fluorescent particles captured and small molecule dye), pressure and flow measurements, and a series of particle sizes "filter or pass" studies. Particle capacity (100-250 nm) ranged from 5 × 108 to 7 × 109 in 1 ml samples at a flow rate of 100 μl/min with backpressure in the range of 1-3 Bar. The optimized fabrication procedure for the 150 nm NRF yielded a small particle recovery of 95% while also achieving a large particle filtration of 73%. High filtration efficiency was also proven in the final 60 and 80 nm NRF fabrication procedures at 96% and 91%, respectively.
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Affiliation(s)
- Jared P. Smithers
- School of Molecular Sciences, Arizona State University, Tempe, Arizona 85282, USA
| | - Jerry Sheu
- School of Molecular Sciences, Arizona State University, Tempe, Arizona 85282, USA
| | - Brian Richardson
- Imagine TF, LLC, 1350 Dell Ave. #102, Campbell, California 95008, USA
| | - Mark A. Hayes
- School of Molecular Sciences, Arizona State University, Tempe, Arizona 85282, USA
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4
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AK N, Kumar S. Integration of 2D Nanoporous Membranes in Microfluidic Devices. ACS OMEGA 2024; 9:22305-22312. [PMID: 38799317 PMCID: PMC11112725 DOI: 10.1021/acsomega.4c01688] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/21/2024] [Revised: 04/22/2024] [Accepted: 04/29/2024] [Indexed: 05/29/2024]
Abstract
2D material-based membranes have emerged as promising candidates for next-generation separation technology due to their exceptional permeability and selectivity. Integration of these membranes into microfluidic devices has offered significant potential for improving the efficiency, throughput, and precision. However, designing compact and reliable microfluidic devices with membranes has many challenges, including complexities in membrane integration, analyte measurement, and contamination issues. Addressing these challenges is critical for unlocking the full potential of membrane-integrated devices. This paper proposes a systematic procedure for integrating membranes into a microfluidic device by creating a pore in the middle layer. Furthermore, an ion transport experiment is carried out across various stacked graphene and poly carbonate track etch membranes in an Ostemer-based device. The resulting device is capable of facilitating the concurrent measurement, a task that is cumbersome in standard macroscopic diffusion cells. The transparency and compactness of the microfluidic device allowed for the in situ and real-time optical characterization of analytes. The integration of microfluidic devices with 2D nanoporous membranes has enabled the incorporation of several analytical modalities, resulting in a highly versatile platform with numerous applications.
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Shen F, Gao J, Zhang J, Ai M, Gao H, Liu Z. Vortex sorting of rare particles/cells in microcavities: A review. BIOMICROFLUIDICS 2024; 18:021504. [PMID: 38571909 PMCID: PMC10987199 DOI: 10.1063/5.0174938] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/04/2023] [Accepted: 03/18/2024] [Indexed: 04/05/2024]
Abstract
Microfluidics or lab-on-a-chip technology has shown great potential for the separation of target particles/cells from heterogeneous solutions. Among current separation methods, vortex sorting of particles/cells in microcavities is a highly effective method for trapping and isolating rare target cells, such as circulating tumor cells, from flowing samples. By utilizing fluid forces and inertial particle effects, this passive method offers advantages such as label-free operation, high throughput, and high concentration. This paper reviews the fundamental research on the mechanisms of focusing, trapping, and holding of particles in this method, designs of novel microcavities, as well as its applications. We also summarize the challenges and prospects of this technique with the hope to promote its applications in medical and biological research.
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Affiliation(s)
- Feng Shen
- Authors to whom correspondence should be addressed: and
| | - Jie Gao
- School of Mathematics, Statistics and Mechanics, Beijing University of Technology, Beijing 100124, People’s Republic of China
| | - Jie Zhang
- School of Mathematics, Statistics and Mechanics, Beijing University of Technology, Beijing 100124, People’s Republic of China
| | - Mingzhu Ai
- School of Mathematics, Statistics and Mechanics, Beijing University of Technology, Beijing 100124, People’s Republic of China
| | - Hongkai Gao
- Department of General Surgery, First Medical Center of Chinese PLA General Hospital, Beijing 100853, People’s Republic of China
| | - Zhaomiao Liu
- Authors to whom correspondence should be addressed: and
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Lv W, Fu B, Liu W, Huang W, Li M, Liu Y, Kang Y, Wang J, Bai S, Lu C, Dai X. Efficient detection of single circulating tumor cell in blood using Raman mapping based on Aptamer-SERS bio-probe coupled with micropore membrane filtration. Talanta 2024; 267:125220. [PMID: 37783108 DOI: 10.1016/j.talanta.2023.125220] [Citation(s) in RCA: 10] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2023] [Revised: 08/15/2023] [Accepted: 09/16/2023] [Indexed: 10/04/2023]
Abstract
Rapid and accurate detection of rare circulating tumor cells (CTCs) in human blood still remains a challenge. We present a surface enhanced Raman spectroscopy (SERS) method based on aptamer-SERS bio-probe recognition coupled with micropore membrane filtration capture for the detection of CTCs at single cell level. The parylene micropore membrane with optimized micropore size installed on a filtration holder could capture bio-probe labeled CTCs by gravity in less than 10 s, and only with very less white blood cells (WBCs) residual. In order to facilitate the synthesis of the aptamer-SERS bio-probe, ethyl acetate dehydration method was established. The bio-probe can be rapidly synthesized within 2 h by binding SH-aptamer to 4- mercaptobenzoic acid (4-MBA) modified AuNPs with the help of ethyl acetate. The SERS bio-probe with selected specific aptamer could distinguish single human non-small cell lung cancer A549 cells from residual WBCs on membrane efficiently and reliably based on their Raman signal intensity difference at 1075 cm-1. Through the filter membrane coupled with aptamer-SERS bio-probe system, even 20 A549 cells in blood solution simulating CTCs sample can be detected, which the recovery rate and recognition rate are more than 90%. This method is rapid, reliable and cost-effective, which indicates a good prospect in clinical application for CTCs detection.
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Affiliation(s)
- Wanxue Lv
- State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing, 100029, China; National Institute of Metrology China, Beijing, 100029, China
| | - Boqiang Fu
- National Institute of Metrology China, Beijing, 100029, China.
| | - Wencheng Liu
- National Institute of Metrology China, Beijing, 100029, China
| | - Wenfeng Huang
- National Institute of Metrology China, Beijing, 100029, China
| | - Manli Li
- National Institute of Metrology China, Beijing, 100029, China
| | - Yingying Liu
- National Institute of Metrology China, Beijing, 100029, China
| | - Yu Kang
- State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing, 100029, China
| | - Jing Wang
- National Institute of Metrology China, Beijing, 100029, China
| | - Shouli Bai
- State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing, 100029, China
| | - Chao Lu
- State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing, 100029, China.
| | - Xinhua Dai
- National Institute of Metrology China, Beijing, 100029, China.
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7
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Hazra RS, Kale N, Boyle C, Molina KB, D'Souza A, Aland G, Jiang L, Chaturvedi P, Ghosh S, Mallik S, Khandare J, Quadir M. Magnetically-activated, nanostructured cellulose for efficient capture of circulating tumor cells from the blood sample of head and neck cancer patients. Carbohydr Polym 2024; 323:121418. [PMID: 37940250 DOI: 10.1016/j.carbpol.2023.121418] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2022] [Revised: 09/14/2023] [Accepted: 09/18/2023] [Indexed: 11/10/2023]
Abstract
In this report, the relative efficiency of cellulose nanocrystals (CNCs) and nanofibers (CNFs) to capture circulating tumor cells (CTCs) from the blood sample of head and neck cancer (HNC) patients was evaluated. Detection and enumeration of CTCs are critical for monitoring cancer progression. Both types of nanostructured cellulose were chemically modified with Epithelial Cell Adhesion Molecule (EpCAM) antibody and iron oxide nanoparticles. The EpCAM antibody facilitated the engagement of CTCs, promoting entrapment within the cellulose cage structure. Iron oxide nanoparticles, on the other hand, rendered the cages activatable via the use of a magnet for the capture and separation of entrapped CTCs. The efficiency of the network structures is shown in head and neck cancer (HNC) patients' blood samples. It was observed that the degree of chemical functionalization of hydroxyl groups located within the CNCs or CNFs with anti-EpCAM determined the efficiency of the system's interaction with CTCs. Further, our result indicated that inflexible scaffolds of nanocrystals interacted more efficiently with CTCs than that of the fibrous CNF scaffolds. Network structures derived from CNCs demonstrated comparable CTC capturing efficiency to commercial standard, OncoDiscover®. The output of the work will provide the chemical design principles of cellulosic materials intended for constructing affordable platforms for monitoring cancer progression in 'real time'.
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Affiliation(s)
- Raj Shankar Hazra
- Department of Mechanical Engineering, North Dakota State University, Fargo, ND 58108, USA; Department of Coatings and Polymeric Materials, North Dakota State University, Fargo 58108, ND, USA
| | - Narendra Kale
- Department of Coatings and Polymeric Materials, North Dakota State University, Fargo 58108, ND, USA; Department of Pharmaceutical Sciences, North Dakota State University, Fargo 58108, ND, USA
| | - Camden Boyle
- Department of Engineering and Technology, Southeast Missouri State University, One University Plaza, MS6825, Cape Girardeau, MO 63701, USA
| | - Kayla B Molina
- Department of Biomedical Engineering, The University of Minnesota Twin Cities, Minneapolis, MN 55455, USA
| | - Alain D'Souza
- Actorius Innovations and Research, Pune, India; Actorius Innovations and Research, Simi Valley, CA 93063, USA
| | - Gourishankar Aland
- Actorius Innovations and Research, Pune, India; Actorius Innovations and Research, Simi Valley, CA 93063, USA
| | - Long Jiang
- Department of Mechanical Engineering, North Dakota State University, Fargo, ND 58108, USA
| | - Pankaj Chaturvedi
- Department of Head and Neck Surgical Oncology, Tata Memorial Hospital, Mumbai, India
| | - Santaneel Ghosh
- Department of Engineering and Technology, Southeast Missouri State University, One University Plaza, MS6825, Cape Girardeau, MO 63701, USA
| | - Sanku Mallik
- Department of Pharmaceutical Sciences, North Dakota State University, Fargo 58108, ND, USA
| | - Jayant Khandare
- Actorius Innovations and Research, Pune, India; School of Pharmacy, Dr. Vishwananth Karad MIT World Peace University, Pune 411038, India; School of Consciousness, Dr. Vishwananth Karad MIT World Peace University, Pune 411038, India; Actorius Innovations and Research, Simi Valley, CA 93063, USA.
| | - Mohiuddin Quadir
- Department of Coatings and Polymeric Materials, North Dakota State University, Fargo 58108, ND, USA.
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8
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Caballero D, Reis RL, Kundu SC. Trapping metastatic cancer cells with mechanical ratchet arrays. Acta Biomater 2023; 170:202-214. [PMID: 37619895 DOI: 10.1016/j.actbio.2023.08.034] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2023] [Revised: 07/26/2023] [Accepted: 08/17/2023] [Indexed: 08/26/2023]
Abstract
Current treatments for cancer, such as chemotherapy, radiotherapy, immunotherapy, and surgery, have positive results but are generally ineffective against metastatic tumors. Treatment effectiveness can be improved by employing bioengineered cancer traps, typically utilizing chemoattractant-loaded materials, to attract infiltrating cancer cells preventing their uncontrolled spread and potentially enabling eradication. However, the encapsulated chemical compounds can have adverse effects on other cells causing unwanted responses, and the generated gradients can evolve unpredictably. Here, we report the development of a cancer trap based on mechanical ratchet structures to capture metastatic cells. The traps use an array of asymmetric local features to mechanically attract cancer cells and direct their migration for prolonged periods. The trapping efficiency was found to be greater than isotropic or inverse anisotropic ratchet structures on either disseminating cancer cells and tumor spheroids. Importantly, the traps exhibited a reduced effectiveness when targeting non-metastatic and non-tumorigenic cells, underscoring their particular suitability for capturing highly invasive cancer cells. Overall, this original approach may have therapeutic implications for fighting cancer, and may also be used to control cell motility for other biological processes. STATEMENT OF SIGNIFICANCE: Current cancer treatments have limitations in treating metastatic tumors, where cancer cells can invade distant organs. Biomaterials loaded with chemoattractants can be implanted to attract and capture metastatic cells preventing uncontrolled spread. However, encapsulated chemical compounds can have adverse effects on other cells, and gradients can evolve unpredictably. This paper presents an original concept of "cancer traps" based on using mechanical ratchet-based structures to capture metastatic cancer cells, with greater trapping efficiency and stability than previously studied methods. This innovative approach has significant potential clinical implications for fighting cancer, particularly in treating metastatic tumors. Additionally, it could be applied to control cell motility for other biological processes, opening new possibilities for biomedicine and tissue engineering.
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Affiliation(s)
- David Caballero
- 3B's Research Group, I3Bs - Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark - Parque da Ciência e Tecnologia, 4805-017 Barco, Guimarães, Portugal; ICVS/3B's-PT Government Associate Laboratory, Braga, Guimarães, Portugal.
| | - Rui L Reis
- 3B's Research Group, I3Bs - Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark - Parque da Ciência e Tecnologia, 4805-017 Barco, Guimarães, Portugal; ICVS/3B's-PT Government Associate Laboratory, Braga, Guimarães, Portugal
| | - Subhas C Kundu
- 3B's Research Group, I3Bs - Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark - Parque da Ciência e Tecnologia, 4805-017 Barco, Guimarães, Portugal; ICVS/3B's-PT Government Associate Laboratory, Braga, Guimarães, Portugal
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Akashi T, Okumura T, Terabayashi K, Yoshino Y, Tanaka H, Yamazaki T, Numata Y, Fukuda T, Manabe T, Baba H, Miwa T, Watanabe T, Hirano K, Igarashi T, Sekine S, Hashimoto I, Shibuya K, Hojo S, Yoshioka I, Matsui K, Yamada A, Sasaki T, Fujii T. The use of an artificial intelligence algorithm for circulating tumor cell detection in patients with esophageal cancer. Oncol Lett 2023; 26:320. [PMID: 37332339 PMCID: PMC10272959 DOI: 10.3892/ol.2023.13906] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2023] [Accepted: 05/25/2023] [Indexed: 06/20/2023] Open
Abstract
Despite recent advances in multidisciplinary treatments of esophageal squamous cell carcinoma (ESCC), patients frequently suffer from distant metastasis after surgery. For numerous types of cancer, circulating tumor cells (CTCs) are considered predictors of distant metastasis, therapeutic response and prognosis. However, as more markers of cytopathological heterogeneity are discovered, the overall detection process for the expression of these markers in CTCs becomes increasingly complex and time consuming. In the present study, the use of a convolutional neural network (CNN)-based artificial intelligence (AI) for CTC detection was assessed using KYSE ESCC cell lines and blood samples from patients with ESCC. The AI algorithm distinguished KYSE cells from peripheral blood-derived mononuclear cells (PBMCs) from healthy volunteers, accompanied with epithelial cell adhesion molecule (EpCAM) and nuclear DAPI staining, with an accuracy of >99.8% when the AI was trained on the same KYSE cell line. In addition, AI trained on KYSE520 distinguished KYSE30 from PBMCs with an accuracy of 99.8%, despite the marked differences in EpCAM expression between the two KYSE cell lines. The average accuracy of distinguishing KYSE cells from PBMCs for the AI and four researchers was 100 and 91.8%, respectively (P=0.011). The average time to complete cell classification for 100 images by the AI and researchers was 0.74 and 630.4 sec, respectively (P=0.012). The average number of EpCAM-positive/DAPI-positive cells detected in blood samples by the AI was 44.5 over 10 patients with ESCC and 2.4 over 5 healthy volunteers (P=0.019). These results indicated that the CNN-based image processing algorithm for CTC detection provides a higher accuracy and shorter analysis time compared to humans, suggesting its applicability for clinical use in patients with ESCC. Moreover, the finding that AI accurately identified even EpCAM-negative KYSEs suggested that the AI algorithm may distinguish CTCs based on as yet unknown features, independent of known marker expression.
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Affiliation(s)
- Takahisa Akashi
- Department of Surgery and Science, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama 930-0194, Japan
| | - Tomoyuki Okumura
- Department of Surgery and Science, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama 930-0194, Japan
| | - Kenji Terabayashi
- Department of Mechanical and Intellectual Systems Engineering, Faculty of Engineering, University of Toyama, Toyama 930-8555, Japan
| | - Yuki Yoshino
- Department of Mechanical and Intellectual Systems Engineering, Faculty of Engineering, University of Toyama, Toyama 930-8555, Japan
| | - Haruyoshi Tanaka
- Department of Surgery and Science, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama 930-0194, Japan
| | - Takeyoshi Yamazaki
- Department of Surgery and Science, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama 930-0194, Japan
| | - Yoshihisa Numata
- Department of Surgery and Science, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama 930-0194, Japan
| | - Takuma Fukuda
- Department of Surgery and Science, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama 930-0194, Japan
| | - Takahiro Manabe
- Department of Surgery and Science, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama 930-0194, Japan
| | - Hayato Baba
- Department of Surgery and Science, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama 930-0194, Japan
| | - Takeshi Miwa
- Department of Surgery and Science, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama 930-0194, Japan
| | - Toru Watanabe
- Department of Surgery and Science, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama 930-0194, Japan
| | - Katsuhisa Hirano
- Department of Surgery and Science, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama 930-0194, Japan
| | - Takamichi Igarashi
- Department of Surgery and Science, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama 930-0194, Japan
| | - Shinichi Sekine
- Department of Surgery and Science, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama 930-0194, Japan
| | - Isaya Hashimoto
- Department of Surgery and Science, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama 930-0194, Japan
| | - Kazuto Shibuya
- Department of Surgery and Science, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama 930-0194, Japan
| | - Shozo Hojo
- Department of Surgery and Science, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama 930-0194, Japan
| | - Isaku Yoshioka
- Department of Surgery and Science, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama 930-0194, Japan
| | - Koshi Matsui
- Department of Surgery and Science, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama 930-0194, Japan
| | - Akane Yamada
- Department of Mechanical and Intellectual Systems Engineering, Faculty of Engineering, University of Toyama, Toyama 930-8555, Japan
| | - Tohru Sasaki
- Department of Mechanical and Intellectual Systems Engineering, Faculty of Engineering, University of Toyama, Toyama 930-8555, Japan
| | - Tsutomu Fujii
- Department of Surgery and Science, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama 930-0194, Japan
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Surappa S, Multani P, Parlatan U, Sinawang PD, Kaifi J, Akin D, Demirci U. Integrated "lab-on-a-chip" microfluidic systems for isolation, enrichment, and analysis of cancer biomarkers. LAB ON A CHIP 2023; 23:2942-2958. [PMID: 37314731 PMCID: PMC10834032 DOI: 10.1039/d2lc01076c] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/15/2023]
Abstract
The liquid biopsy has garnered considerable attention as a complementary clinical tool for the early detection, molecular characterization and monitoring of cancer over the past decade. In contrast to traditional solid biopsy techniques, liquid biopsy offers a less invasive and safer alternative for routine cancer screening. Recent advances in microfluidic technologies have enabled handling of liquid biopsy-derived biomarkers with high sensitivity, throughput, and convenience. The integration of these multi-functional microfluidic technologies into a 'lab-on-a-chip' offers a powerful solution for processing and analyzing samples on a single platform, thereby reducing the complexity, bio-analyte loss and cross-contamination associated with multiple handling and transfer steps in more conventional benchtop workflows. This review critically addresses recent developments in integrated microfluidic technologies for cancer detection, highlighting isolation, enrichment, and analysis strategies for three important sub-types of cancer biomarkers: circulating tumor cells, circulating tumor DNA and exosomes. We first discuss the unique characteristics and advantages of the various lab-on-a-chip technologies developed to operate on each biomarker subtype. This is then followed by a discussion on the challenges and opportunities in the field of integrated systems for cancer detection. Ultimately, integrated microfluidic platforms form the core of a new class of point-of-care diagnostic tools by virtue of their ease-of-operation, portability and high sensitivity. Widespread availability of such tools could potentially result in more frequent and convenient screening for early signs of cancer at clinical labs or primary care offices.
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Affiliation(s)
- Sushruta Surappa
- Canary Center at Stanford for Cancer Early Detection, Bio-Acoustic MEMS in Medicine (BAMM) Lab, Department of Radiology, School of Medicine, Stanford University, Palo Alto, CA 94304, USA.
| | - Priyanka Multani
- Canary Center at Stanford for Cancer Early Detection, Bio-Acoustic MEMS in Medicine (BAMM) Lab, Department of Radiology, School of Medicine, Stanford University, Palo Alto, CA 94304, USA.
| | - Ugur Parlatan
- Canary Center at Stanford for Cancer Early Detection, Bio-Acoustic MEMS in Medicine (BAMM) Lab, Department of Radiology, School of Medicine, Stanford University, Palo Alto, CA 94304, USA.
| | - Prima Dewi Sinawang
- Canary Center at Stanford for Cancer Early Detection, Bio-Acoustic MEMS in Medicine (BAMM) Lab, Department of Radiology, School of Medicine, Stanford University, Palo Alto, CA 94304, USA.
- Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA
| | - Jussuf Kaifi
- Department of Surgery, School of Medicine, University of Missouri, Columbia, MO 65212, USA
- Harry S. Truman Memorial Veterans' Hospital, Columbia, MO 65201, USA
| | - Demir Akin
- Canary Center at Stanford for Cancer Early Detection, Bio-Acoustic MEMS in Medicine (BAMM) Lab, Department of Radiology, School of Medicine, Stanford University, Palo Alto, CA 94304, USA.
- Center for Cancer Nanotechnology Excellence for Translational Diagnostics (CCNE-TD), School of Medicine, Stanford University, Stanford, CA 94305, USA
| | - Utkan Demirci
- Canary Center at Stanford for Cancer Early Detection, Bio-Acoustic MEMS in Medicine (BAMM) Lab, Department of Radiology, School of Medicine, Stanford University, Palo Alto, CA 94304, USA.
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11
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Parvin D, Hashemi ZS, Shokati F, Mohammadpour Z, Bazargan V. Immunomagnetic Isolation of HER2-Positive Breast Cancer Cells Using a Microfluidic Device. ACS OMEGA 2023; 8:21745-21754. [PMID: 37360498 PMCID: PMC10286087 DOI: 10.1021/acsomega.3c01287] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/26/2023] [Accepted: 05/25/2023] [Indexed: 06/28/2023]
Abstract
Analysis of circulating tumor cells (CTCs) as a tool for monitoring metastatic cancers, early diagnosis, and evaluation of disease prognosis paves the way toward personalized cancer treatment. Developing an effective, feasible, and low-cost method to facilitate CTC isolation is, therefore, vital. In the present study, we integrated magnetic nanoparticles (MNPs) with microfluidics and used them for the isolation of HER2-positive breast cancer cells. Iron oxide MNPs were synthesized and functionalized with the anti-HER2 antibody. The chemical conjugation was verified by Fourier transform infrared spectroscopy, energy-dispersive X-ray spectroscopy, and dynamic light scattering/zeta potential analysis. The specificity of the functionalized NPs for the separation of HER2-positive from HER2-negative cells was demonstrated in an off-chip test setting. The off-chip isolation efficiency was 59.38%. The efficiency of SK-BR-3 cell isolation using a microfluidic chip with a S-shaped microchannel was considerably enhanced to 96% (a flow rate of 0.5 mL/h) without chip clogging. Besides, the analysis time for the on-chip cell separation was 50% faster. The clear advantages of the present microfluidic system offer a competitive solution in clinical applications.
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Affiliation(s)
- Delaram Parvin
- School
of Mechanical Engineering, College of Engineering, University of Tehran, North Amirabad, 1439957131 Tehran, Iran
| | - Zahra Sadat Hashemi
- ATMP
Department, Breast Cancer Research Center, Motamed Cancer Institute, ACECR, No. 146, South Gandhi Street, Vanak Square, 1517964311 Tehran, Iran
| | - Farhad Shokati
- Biomaterials
and Tissue Engineering Department, Breast Cancer Research Center, Motamed Cancer Institute, No. 146, South Gandhi Street, Vanak Square, 1517964311 Tehran, Iran
| | - Zahra Mohammadpour
- Biomaterials
and Tissue Engineering Department, Breast Cancer Research Center, Motamed Cancer Institute, No. 146, South Gandhi Street, Vanak Square, 1517964311 Tehran, Iran
| | - Vahid Bazargan
- School
of Mechanical Engineering, College of Engineering, University of Tehran, North Amirabad, 1439957131 Tehran, Iran
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12
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Wang J, Dallmann R, Lu R, Yan J, Charmet J. Flow Rate-Independent Multiscale Liquid Biopsy for Precision Oncology. ACS Sens 2023; 8:1200-1210. [PMID: 36802518 PMCID: PMC10043932 DOI: 10.1021/acssensors.2c02577] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2022] [Accepted: 02/06/2023] [Indexed: 02/22/2023]
Abstract
Immunoaffinity-based liquid biopsies of circulating tumor cells (CTCs) hold great promise for cancer management but typically suffer from low throughput, relative complexity, and postprocessing limitations. Here, we address these issues simultaneously by decoupling and independently optimizing the nano-, micro-, and macro-scales of an enrichment device that is simple to fabricate and operate. Unlike other affinity-based devices, our scalable mesh approach enables optimum capture conditions at any flow rate, as demonstrated with constant capture efficiencies, above 75% between 50 and 200 μL min-1. The device achieved 96% sensitivity and 100% specificity when used to detect CTCs in the blood of 79 cancer patients and 20 healthy controls. We demonstrate its postprocessing capacity with the identification of potential responders to immune checkpoint inhibition (ICI) therapy and the detection of HER2 positive breast cancer. The results compare well with other assays, including clinical standards. This suggests that our approach, which overcomes major limitations associated with affinity-based liquid biopsies, could help improve cancer management.
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Affiliation(s)
- Jie Wang
- Institute
for Advanced Materials, School of Material Science and Engineering, Jiangsu University, Zhenjiang 212013, China
| | - Robert Dallmann
- Division
of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry CV4 7AL, U. K.
| | - Renquan Lu
- Department
of Clinical Laboratory, Fudan University
Shanghai Cancer Center, Shanghai 200032, China
| | - Jing Yan
- Holosensor
Medical Technology Ltd., Suzhou 215000, China
| | - Jérôme Charmet
- Division
of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry CV4 7AL, U. K.
- WMG
University of Warwick, Coventry CV4 7AL, U.K.
- School of
Engineering − HE-Arc Ingénierie, HES-SO University of Applied Sciences Western Switzerland, 2000 Neuchâtel, Switzerland
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13
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Zhang X, Jiang X, Wang W, Luo S, Guan S, Li W, Situ B, Li B, Zhang Y, Zheng L. A simple and sensitive electrochemical biosensor for circulating tumor cell determination based on dual-toehold accelerated catalytic hairpin assembly. Mikrochim Acta 2023; 190:65. [PMID: 36692585 DOI: 10.1007/s00604-023-05649-7] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2022] [Accepted: 01/04/2023] [Indexed: 01/25/2023]
Abstract
Tumor cells in blood circulation (CTCs) are vital biomarkers for noninvasive cancer diagnosis. We developed a simple and sensitive electrochemical biosensor based on dual-toehold accelerated catalytic hairpin assembly (DCHA) to distinguish CTCs from blood cells. In the presence of CTCs, the aptamer probe initiates the DCHA process, which produces amplified electrochemical signals. Compared with conventional catalytic hairpin assembly (CHA), the proposed DCHA showed high sensitivity, which led to a broader working range of 10-1000 cells mL-1 with a limit of detection of 4 cells mL-1. Furthermore, our method exhibited an excellent capability of distinguishing malignant breast cancers from healthy people, with a sensitivity of 97.4%. In summary, we have established an enzyme-free, easy-to-operate, and nondisruptive method for detecting circulating tumor cells in blood circulation based on the DCHA strategy. Its versatility and simplicity will make it more widely used in clinical diagnosis and biomedical research.
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Affiliation(s)
- Xiaohe Zhang
- Laboratory Medicine Center, Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.,Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensors, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Xiujuan Jiang
- Laboratory Medicine Center, Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.,Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensors, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Wen Wang
- Laboratory Medicine Center, Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.,Medical Laboratory of Shenzhen Luohu People's Hospital, Shenzhen, 518003, Guangdong Province, China
| | - Shihua Luo
- Laboratory Medicine Center, Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.,Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensors, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Shujuan Guan
- Laboratory Medicine Center, Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.,Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensors, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Wenbin Li
- Laboratory Medicine Center, Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.,Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensors, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Bo Situ
- Laboratory Medicine Center, Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.,Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensors, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Bo Li
- Laboratory Medicine Center, Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.,Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensors, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Ye Zhang
- Laboratory Medicine Center, Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China. .,Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensors, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.
| | - Lei Zheng
- Department of Clinical Laboratory, Shunde Hospital, Southern Medical University, (The First People's Hospital of Shunde), Foshan, 528300, Guangdong Province, China. .,Laboratory Medicine Center, Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China. .,Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensors, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.
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14
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Tretyakova MS, Menyailo ME, Schegoleva AA, Bokova UA, Larionova IV, Denisov EV. Technologies for Viable Circulating Tumor Cell Isolation. Int J Mol Sci 2022; 23:ijms232415979. [PMID: 36555625 PMCID: PMC9788311 DOI: 10.3390/ijms232415979] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2022] [Revised: 12/07/2022] [Accepted: 12/13/2022] [Indexed: 12/23/2022] Open
Abstract
The spread of tumor cells throughout the body by traveling through the bloodstream is a critical step in metastasis, which continues to be the main cause of cancer-related death. The detection and analysis of circulating tumor cells (CTCs) is important for understanding the biology of metastasis and the development of antimetastatic therapy. However, the isolation of CTCs is challenging due to their high heterogeneity and low representation in the bloodstream. Different isolation methods have been suggested, but most of them lead to CTC damage. However, viable CTCs are an effective source for developing preclinical models to perform drug screening and model the metastatic cascade. In this review, we summarize the available literature on methods for isolating viable CTCs based on different properties of cells. Particular attention is paid to the importance of in vitro and in vivo models obtained from CTCs. Finally, we emphasize the current limitations in CTC isolation and suggest potential solutions to overcome them.
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Affiliation(s)
- Maria S. Tretyakova
- Laboratory of Cancer Progression Biology, Cancer Research Institute, Tomsk National Research Medical Center, Russian Academy of Sciences, 634009 Tomsk, Russia
| | - Maxim E. Menyailo
- Laboratory of Cancer Progression Biology, Cancer Research Institute, Tomsk National Research Medical Center, Russian Academy of Sciences, 634009 Tomsk, Russia
- Single Cell Biology Laboratory, Research Institute of Molecular and Cellular Medicine, Peoples’ Friendship University of Russia (RUDN University), 117198 Moscow, Russia
| | - Anastasia A. Schegoleva
- Laboratory of Cancer Progression Biology, Cancer Research Institute, Tomsk National Research Medical Center, Russian Academy of Sciences, 634009 Tomsk, Russia
- Single Cell Biology Laboratory, Research Institute of Molecular and Cellular Medicine, Peoples’ Friendship University of Russia (RUDN University), 117198 Moscow, Russia
| | - Ustinia A. Bokova
- Laboratory of Cancer Progression Biology, Cancer Research Institute, Tomsk National Research Medical Center, Russian Academy of Sciences, 634009 Tomsk, Russia
| | - Irina V. Larionova
- Laboratory of Cancer Progression Biology, Cancer Research Institute, Tomsk National Research Medical Center, Russian Academy of Sciences, 634009 Tomsk, Russia
| | - Evgeny V. Denisov
- Laboratory of Cancer Progression Biology, Cancer Research Institute, Tomsk National Research Medical Center, Russian Academy of Sciences, 634009 Tomsk, Russia
- Single Cell Biology Laboratory, Research Institute of Molecular and Cellular Medicine, Peoples’ Friendship University of Russia (RUDN University), 117198 Moscow, Russia
- Correspondence: ; Tel./Fax: +7-3822-282676 (ext. 3375)
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15
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Lai ZX, Wu CC, Huang NT. A Microfluidic Platform with an Embedded Miniaturized Electrochemical Sensor for On-Chip Plasma Extraction Followed by In Situ High-Sensitivity C-Reactive Protein (hs-CRP) Detection. BIOSENSORS 2022; 12:1163. [PMID: 36551130 PMCID: PMC9775575 DOI: 10.3390/bios12121163] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 10/28/2022] [Revised: 12/02/2022] [Accepted: 12/05/2022] [Indexed: 06/17/2023]
Abstract
Blood testing is a clinical diagnostic tool to evaluate physiological conditions, the immune system response, or the presence of infection from whole blood samples. Although conventional blood testing can provide rich biological information, it usually requires complicated and tedious whole blood processing steps operated by benchtop instruments and well-experienced technicians, limiting its usage in point-of-care (POC) settings. To address the above problems, we propose a microfluidic platform for on-chip plasma extraction directly from whole blood and in situ biomarker detection. Herein, we chose C-reactive protein (CRP) as the target biomarker, which can be used to predict fatal cardiovascular disease (CVD) events such as heart attacks and strokes. To achieve a rapid, undiluted, and high-purity on-chip plasma extraction, we combined two whole blood processing methods: (1) anti-D immunoglobulin-assisted sedimentation, and (2) membrane filtration. To perform in situ CRP detection, we fabricated a three-dimensional (3D) microchannel with an embedded electrochemical (EC) sensor, which has a modular design to attach the blood collector and buffer reservoir with standard Luer connectors. As a proof of concept, we first confirmed that the dual plasma extraction design achieved the same purity level as the standard centrifugation method with smaller sample (100 µL of plasma extracted from 400 µL of whole blood) and time (7 min) requirements. Next, we validated the functionalization protocol of the EC sensor, followed by evaluating the detection of CRP spiked in plasma and whole blood. Our microfluidic platform performed on-chip plasma extraction directly from whole blood and in situ CRP detection at a 0.1-10 μg/mL concentration range, covering the CVD risk evaluation level of the high-sensitivity CRP (hs-CRP) test. Based on the above features, we believe that this platform constitutes a flexible way to integrate the processing of complex samples with accurate biomarker detection in a sample-to-answer POC platform, which can be applied in CVD risk monitoring under critical clinical situations.
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Affiliation(s)
- Zhi-Xuan Lai
- Graduation Institute of Biomedical Electronics and Bioinformatics, National Taiwan University, Taipei 10617, Taiwan
| | - Chia-Chien Wu
- Graduation Institute of Biomedical Electronics and Bioinformatics, National Taiwan University, Taipei 10617, Taiwan
| | - Nien-Tsu Huang
- Graduation Institute of Biomedical Electronics and Bioinformatics, National Taiwan University, Taipei 10617, Taiwan
- Department of Electrical Engineering, National Taiwan University, Taipei 10617, Taiwan
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16
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Li A, He X, Wu J, Zhang J, Xu G, Xu B, Zhao G, Shen Z. Ultrathin silicon nitride membrane with slit-shaped pores for high-performance separation of circulating tumor cells. LAB ON A CHIP 2022; 22:3676-3686. [PMID: 35997043 DOI: 10.1039/d2lc00703g] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/15/2023]
Abstract
In this study, we developed an ultrathin filtering membrane with slit-shaped pores which can achieve circulating tumor cell (CTC) separation from whole blood with high performance (high capture efficiency, high white blood cell (WBC) depletion, and high viability). The silicon nitride (Si3N4) filtering membrane was fabricated via the standard microfabrication technology, which can be easily scaled up to mass-production. 6 μm was determined as the optimum width of the filtering pores to better separate CTCs in whole blood, which can reach a high capture efficiency of ∼96%. Meanwhile, the filtering membrane with a high porosity of 34% demonstrated high WBC depletion (∼99.99%). Furthermore, the ultrathin (thickness: 200 nm) Si3N4 membrane facilitated the capture of CTCs with high viability (∼90%). Finally, the microfluidic chip was successfully applied to separate CTCs in whole blood samples from cancer patients and used for molecular examination. These results indicate that this microfluidic chip facilitates the clinical application of CTC-based liquid biopsy technology.
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Affiliation(s)
- Ang Li
- Department of Clinical Laboratory, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, 230001, China.
| | - Xiaodong He
- Department of Clinical Laboratory, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, 230001, China.
| | - Jing Wu
- Department of Clinical Laboratory, Anhui Provincial Hospital Affiliated to Anhui Medical University, Hefei, Anhui, 230001, China
| | - Juan Zhang
- Department of Clinical Laboratory, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, 230001, China.
| | - Guoyong Xu
- School of Engineering Science, University of Science and Technology of China, Hefei, Anhui, 230026, China.
| | - Bing Xu
- School of Mechanical Engineering, Suzhou University of Science and Technology, Suzhou, 215009, China.
| | - Gang Zhao
- School of Engineering Science, University of Science and Technology of China, Hefei, Anhui, 230026, China.
| | - Zuojun Shen
- Department of Clinical Laboratory, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, 230001, China.
- Department of Clinical Laboratory, Anhui Provincial Hospital Affiliated to Anhui Medical University, Hefei, Anhui, 230001, China
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17
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Yao X, Liu Y, Chu Z, Jin W. Membranes for the life sciences and their future roles in medicine. Chin J Chem Eng 2022; 49:1-20. [PMID: 35755178 PMCID: PMC9212902 DOI: 10.1016/j.cjche.2022.04.027] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2022] [Revised: 04/15/2022] [Accepted: 04/15/2022] [Indexed: 01/12/2023]
Abstract
Since the global outbreak of COVID-19, membrane technology for clinical treatments, including extracorporeal membrane oxygenation (ECMO) and protective masks and clothing, has attracted intense research attention for its irreplaceable abilities. Membrane research and applications are now playing an increasingly important role in various fields of life science. In addition to intrinsic properties such as size sieving, dissolution and diffusion, membranes are often endowed with additional functions as cell scaffolds, catalysts or sensors to satisfy the specific requirements of different clinical applications. In this review, we will introduce and discuss state-of-the-art membranes and their respective functions in four typical areas of life science: artificial organs, tissue engineering, in vitro blood diagnosis and medical support. Emphasis will be given to the description of certain specific functions required of membranes in each field to provide guidance for the selection and fabrication of the membrane material. The advantages and disadvantages of these membranes have been compared to indicate further development directions for different clinical applications. Finally, we propose challenges and outlooks for future development.
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Affiliation(s)
- Xiaoyue Yao
- State Key Laboratory of Materials-Oriented Chemical Engineering, College of Chemical Engineering, Nanjing Tech University, Nanjing 211816, China
| | - Yu Liu
- State Key Laboratory of Materials-Oriented Chemical Engineering, College of Chemical Engineering, Nanjing Tech University, Nanjing 211816, China
| | - Zhenyu Chu
- State Key Laboratory of Materials-Oriented Chemical Engineering, College of Chemical Engineering, Nanjing Tech University, Nanjing 211816, China
| | - Wanqin Jin
- State Key Laboratory of Materials-Oriented Chemical Engineering, College of Chemical Engineering, Nanjing Tech University, Nanjing 211816, China
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18
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Rahmanian M, Sartipzadeh Hematabad O, Askari E, Shokati F, Bakhshi A, Moghadam S, Olfatbakhsh A, Al Sadat Hashemi E, Khorsand Ahmadi M, Morteza Naghib S, Sinha N, Tel J, Eslami Amirabadi H, den Toonder JMJ, Majidzadeh-A K. A micropillar array-based microfluidic chip for label-free separation of circulating tumor cells: The best micropillar geometry? J Adv Res 2022; 47:105-121. [PMID: 35964874 PMCID: PMC10173300 DOI: 10.1016/j.jare.2022.08.005] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2022] [Revised: 07/22/2022] [Accepted: 08/07/2022] [Indexed: 10/15/2022] Open
Abstract
INTRODUCTION The information derived from the number and characteristics of circulating tumor cells (CTCs), is crucial to ensure appropriate cancer treatment monitoring. Currently, diverse microfluidic platforms have been developed for isolating CTCs from blood, but it remains a challenge to develop a low-cost, practical, and efficient strategy. OBJECTIVES This study aimed to isolate CTCs from the blood of cancer patients via introducing a new and efficient micropillar array-based microfluidic chip (MPA-Chip), as well as providing prognostic information and monitoring the treatment efficacy in cancer patients. METHODS We fabricated a microfluidic chip (MPA-Chip) containing arrays of micropillars with different geometries (lozenge, rectangle, circle, and triangle). We conducted numerical simulations to compare velocity and pressure profiles inside the micropillar arrays. Also, we experimentally evaluated the capture efficiency and purity of the geometries using breast and prostate cancer cell lines as well as a blood sample. Moreover, the device's performance was validated on 12 patients with breast cancer (BC) in different states. RESULTS The lozenge geometry was selected as the most effective and optimized micropillar design for CTCs isolation, providing high capture efficiency (>85 %), purity (>90 %), and viability (97 %). Furthermore, the lozenge MPA-chip was successfully validated by the detection of CTCs from 12 breast cancer (BC) patients, with non-metastatic (median number of 6 CTCs) and metastatic (median number of 25 CTCs) diseases, showing different prognoses. Also, increasing the chemotherapy period resulted in a decrease in the number of captured CTCs from 23 to 7 for the metastatic patient. The MPA-Chip size was only 0.25 cm2 and the throughput of a single chip was 0.5 ml/h, which can be increased by multiple MPA-Chips in parallel. CONCLUSION The lozenge MPA-Chip presented a novel micropillar geometry for on-chip CTC isolation, detection, and staining, and in the future, the possibilities can be extended to the culture of the CTCs.
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Affiliation(s)
- Mehdi Rahmanian
- Biomaterials and Tissue Engineering Research Group, Interdisciplinary Technologies Department, Breast Cancer Research Center, Motamed Cancer Institute, ACECR, Tehran, Iran; Microsystems Research Section, Department of Mechanical Engineering, Eindhoven University of Technology, Eindhoven, the Netherlands
| | - Omid Sartipzadeh Hematabad
- Biomaterials and Tissue Engineering Research Group, Interdisciplinary Technologies Department, Breast Cancer Research Center, Motamed Cancer Institute, ACECR, Tehran, Iran
| | - Esfandyar Askari
- Biomaterials and Tissue Engineering Research Group, Interdisciplinary Technologies Department, Breast Cancer Research Center, Motamed Cancer Institute, ACECR, Tehran, Iran
| | - Farhad Shokati
- Biomaterials and Tissue Engineering Research Group, Interdisciplinary Technologies Department, Breast Cancer Research Center, Motamed Cancer Institute, ACECR, Tehran, Iran
| | - Atin Bakhshi
- Biomaterials and Tissue Engineering Research Group, Interdisciplinary Technologies Department, Breast Cancer Research Center, Motamed Cancer Institute, ACECR, Tehran, Iran
| | - Shiva Moghadam
- Breast Diseases Group, Breast Cancer Research Center, Motamed Cancer Institute, ACECR, Tehran, Iran
| | - Asiie Olfatbakhsh
- Breast Diseases Group, Breast Cancer Research Center, Motamed Cancer Institute, ACECR, Tehran, Iran
| | - Esmat Al Sadat Hashemi
- Breast Diseases Group, Breast Cancer Research Center, Motamed Cancer Institute, ACECR, Tehran, Iran
| | - Mohammad Khorsand Ahmadi
- Microsystems Research Section, Department of Mechanical Engineering, Eindhoven University of Technology, Eindhoven, the Netherlands
| | - Seyed Morteza Naghib
- Nanotechnology Department, School of Advanced Technologies, Iran University of Science and Technology, Tehran, Iran
| | - Nidhi Sinha
- Laboratory of Immunoengineering, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, the Netherlands; Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, the Netherlands
| | - Jurjen Tel
- Laboratory of Immunoengineering, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, the Netherlands; Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, the Netherlands
| | - Hossein Eslami Amirabadi
- Microsystems Research Section, Department of Mechanical Engineering, Eindhoven University of Technology, Eindhoven, the Netherlands; Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, the Netherlands; AZAR Innovations, Utrecht, the Netherlands
| | - Jaap M J den Toonder
- Microsystems Research Section, Department of Mechanical Engineering, Eindhoven University of Technology, Eindhoven, the Netherlands; Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, the Netherlands.
| | - Keivan Majidzadeh-A
- Genetics Department, Breast Cancer Research Center, Motamed Cancer Institute, ACECR, Tehran, Iran.
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19
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Nasiłowska B, Bogdanowicz Z, Kasprzycka W, Bombalska A, Mierczyk Z. Studies on the Effect of Graphene Oxide Deposited on Gold and Nickel Microsieves on Prostate Cancer Cells DU 145. Int J Mol Sci 2022; 23:ijms23126567. [PMID: 35743008 PMCID: PMC9224325 DOI: 10.3390/ijms23126567] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2022] [Revised: 05/30/2022] [Accepted: 06/10/2022] [Indexed: 02/05/2023] Open
Abstract
This work shows the effect of graphene oxide deposition on microsieves’ surfaces of gold and nickel foils, on DU 145 tumor cells of the prostate gland. The sieves were made by a laser ablation process. The graphene oxide (GO) deposition process was characterized by the complete covering of the inner edges of the microholes and the flat surface between the holes with GO. Electron microscanning studies have shown that due to the deposition method applied, graphene oxide flakes line the interior of the microholes, reducing the unevenness of the downstream surfaces during the laser ablation process. The presence of graphene oxide was confirmed by Fourier infrared spectroscopy. During the screening (sieving) process, the microsieves were placed in a sieve column. Gold foil is proven to be a very good material for the screening of cancer cells, but even more so after screening as a substrate for re-culture of the DU 145. This allows a potential recovery of the cells and the development of a targeted therapy. The sieved cells were successfully grown on the microsieves used in the experiment. Graphene oxide remaining on the surface of the nickel sieve has been observed to increase the sieving effect. Although graphene oxide improved separation efficiency by 9.7%, the nickel substrate is not suitable for re-culturing of the Du 145 cells and the development of a targeted therapy compared to the gold one.
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Affiliation(s)
- Barbara Nasiłowska
- Institute of Optoelectronics, Military University of Technology, gen. S. Kaliskiego 2, 00-908 Warsaw, Poland; (W.K.); (A.B.); (Z.M.)
- Correspondence:
| | - Zdzisław Bogdanowicz
- Faculty of Mechanical Engineering, Military University of Technology, gen. S. Kaliskiego 2, 00-908 Warsaw, Poland;
| | - Wiktoria Kasprzycka
- Institute of Optoelectronics, Military University of Technology, gen. S. Kaliskiego 2, 00-908 Warsaw, Poland; (W.K.); (A.B.); (Z.M.)
| | - Aneta Bombalska
- Institute of Optoelectronics, Military University of Technology, gen. S. Kaliskiego 2, 00-908 Warsaw, Poland; (W.K.); (A.B.); (Z.M.)
| | - Zygmunt Mierczyk
- Institute of Optoelectronics, Military University of Technology, gen. S. Kaliskiego 2, 00-908 Warsaw, Poland; (W.K.); (A.B.); (Z.M.)
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20
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Cai S, Ma Z, Ge Z, Yang W. Recent advances in optically induced di-electrophoresis and its biomedical applications. Biomed Microdevices 2022; 24:22. [PMID: 35689721 DOI: 10.1007/s10544-022-00620-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/27/2022] [Indexed: 11/02/2022]
Abstract
The development of the micro/nano science and technology has promoted the evolvement of human civilization tremendously. The advancement of the micro/nano science and technology highly depends on the progress of the micro/nano manipulation techniques, and the micro/nano-scaled manipulation level is the critical sign of the micro/nano science and technology. This review, aimed at the demand and the challenge of the micro/nano material and biomedical fields and related to the scientific issues and implementation techniques of the optically induced di-electrophoresis (ODEP). We explained its working principle, manipulating method, and influencing factors of ODEP force to a certain extent. A number of application fields based-ODEP technology and specific applications so far are summarized and reviewed. Finally, some perspectives are provided on current development trends, future research directions, and challenges of ODEP.
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Affiliation(s)
- Shuxiang Cai
- School of Electromechanical and Automotive Engineering, Yantai University, Yantai, 264005, China
| | - Zheng Ma
- School of Electromechanical and Automotive Engineering, Yantai University, Yantai, 264005, China
| | - Zhixing Ge
- State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang, 110016, China
| | - Wenguang Yang
- School of Electromechanical and Automotive Engineering, Yantai University, Yantai, 264005, China.
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21
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Lucas K, Oh J, Hoelzl J, Weissleder R. Cellular point-of-care diagnostics using an inexpensive layer-stack microfluidic device. LAB ON A CHIP 2022; 22:2145-2154. [PMID: 35514273 PMCID: PMC9214713 DOI: 10.1039/d2lc00162d] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/14/2023]
Abstract
Cellular analyses are increasingly used to diagnose diseases at point-of-care and global healthcare settings. Some analyses are simple as they rely on chromogenic stains (blood counts, malaria) but others often require higher multiplexing to define and quantitate cell populations (cancer diagnosis, immunoprofiling). Simplifying the latter with inexpensive solutions represents a current bottleneck in designing start-end pipelines. Based on the hypothesis that novel film adhesives could be used to create inexpensive disposable devices, we tested a number of different designs and materials, to rapidly perform 12-15 channel single-cell imaging. Using an optimized passive pumping layer-stack microfluidic (PLASMIC) device (<1 $ in supplies) we show that rapid, inexpensive cellular analysis is feasible.
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Affiliation(s)
- Kilean Lucas
- Center for Systems Biology, Massachusetts General Hospital, 185 Cambridge St, CPZN 5206, Boston, MA 02114, USA.
| | - Juhyun Oh
- Center for Systems Biology, Massachusetts General Hospital, 185 Cambridge St, CPZN 5206, Boston, MA 02114, USA.
| | - Jan Hoelzl
- Center for Systems Biology, Massachusetts General Hospital, 185 Cambridge St, CPZN 5206, Boston, MA 02114, USA.
| | - Ralph Weissleder
- Center for Systems Biology, Massachusetts General Hospital, 185 Cambridge St, CPZN 5206, Boston, MA 02114, USA.
- Department of Systems Biology, Harvard Medical School, 200 Longwood Ave, Boston, MA 02115, USA
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22
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Li C, He W, Wang N, Xi Z, Deng R, Liu X, Kang R, Xie L, Liu X. Application of Microfluidics in Detection of Circulating Tumor Cells. Front Bioeng Biotechnol 2022; 10:907232. [PMID: 35646880 PMCID: PMC9133555 DOI: 10.3389/fbioe.2022.907232] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2022] [Accepted: 04/11/2022] [Indexed: 12/22/2022] Open
Abstract
Tumor metastasis is one of the main causes of cancer incidence and death worldwide. In the process of tumor metastasis, the isolation and analysis of circulating tumor cells (CTCs) plays a crucial role in the early diagnosis and prognosis of cancer patients. Due to the rarity and inherent heterogeneity of CTCs, there is an urgent need for reliable CTCs separation and detection methods in order to obtain valuable information on tumor metastasis and progression from CTCs. Microfluidic technology is increasingly used in various studies of CTCs separation, identification and characterization because of its unique advantages, such as low cost, simple operation, less reagent consumption, miniaturization of the system, rapid detection and accurate control. This paper reviews the research progress of microfluidic technology in CTCs separation and detection in recent years, as well as the potential clinical application of CTCs, looks forward to the application prospect of microfluidic technology in the treatment of tumor metastasis, and briefly discusses the development prospect of microfluidic biosensor.
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Affiliation(s)
- Can Li
- Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, School of Artificial Intelligence and Information Technology, Nanjing University of Chinese Medicine, Nanjing, China
| | - Wei He
- Department of Clinical Medical Engineering, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China
| | - Nan Wang
- Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, School of Artificial Intelligence and Information Technology, Nanjing University of Chinese Medicine, Nanjing, China
| | - Zhipeng Xi
- Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, School of Artificial Intelligence and Information Technology, Nanjing University of Chinese Medicine, Nanjing, China
| | - Rongrong Deng
- Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, School of Artificial Intelligence and Information Technology, Nanjing University of Chinese Medicine, Nanjing, China
| | - Xiyu Liu
- Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, School of Artificial Intelligence and Information Technology, Nanjing University of Chinese Medicine, Nanjing, China
| | - Ran Kang
- Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, School of Artificial Intelligence and Information Technology, Nanjing University of Chinese Medicine, Nanjing, China
- Department of Orthopedics, Nanjing Lishui Hospital of Traditional Chinese Medicine, Nanjing, China
| | - Lin Xie
- Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, School of Artificial Intelligence and Information Technology, Nanjing University of Chinese Medicine, Nanjing, China
| | - Xin Liu
- Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, School of Artificial Intelligence and Information Technology, Nanjing University of Chinese Medicine, Nanjing, China
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23
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Zhang S, Wang Y, Yang C, Zhu J, Ye X, Wang W. On-chip circulating tumor cells isolation based on membrane filtration and immuno-magnetic bead clump capture. NANOTECHNOLOGY AND PRECISION ENGINEERING 2022. [DOI: 10.1063/10.0009560] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Affiliation(s)
- Shuai Zhang
- State Key Laboratory of Precision Measurement Technology and Instruments, Department of Precision Instruments, Tsinghua University, Beijing 100084, China
| | - Yue Wang
- State Key Laboratory of Precision Measurement Technology and Instruments, Department of Precision Instruments, Tsinghua University, Beijing 100084, China
| | - Chaoqiang Yang
- State Key Laboratory of Precision Measurement Technology and Instruments, Department of Precision Instruments, Tsinghua University, Beijing 100084, China
| | - Junwen Zhu
- State Key Laboratory of Precision Measurement Technology and Instruments, Department of Precision Instruments, Tsinghua University, Beijing 100084, China
| | - Xiongying Ye
- State Key Laboratory of Precision Measurement Technology and Instruments, Department of Precision Instruments, Tsinghua University, Beijing 100084, China
| | - Wenhui Wang
- State Key Laboratory of Precision Measurement Technology and Instruments, Department of Precision Instruments, Tsinghua University, Beijing 100084, China
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24
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Xu K, Jiao X, Wang P, Chen C, Chen C. Isolation of circulating tumor cells based on magnetophoresis. CHINESE JOURNAL OF ANALYTICAL CHEMISTRY 2022. [DOI: 10.1016/j.cjac.2022.100058] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
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25
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Topa J, Grešner P, Żaczek AJ, Markiewicz A. Breast cancer circulating tumor cells with mesenchymal features-an unreachable target? Cell Mol Life Sci 2022; 79:81. [PMID: 35048186 PMCID: PMC8770434 DOI: 10.1007/s00018-021-04064-6] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2021] [Revised: 11/26/2021] [Accepted: 11/27/2021] [Indexed: 12/13/2022]
Abstract
Circulating tumor cells (CTCs) mediate dissemination of solid tumors and can be an early sign of disease progression. Moreover, they show a great potential in terms of non-invasive, longitudinal monitoring of cancer patients. CTCs have been extensively studied in breast cancer (BC) and were shown to present a significant phenotypic plasticity connected with initiation of epithelial-mesenchymal transition (EMT). Apart from conferring malignant properties, EMT affects CTCs recovery rate, making a significant portion of CTCs from patients’ samples undetected. Wider application of methods and markers designed to isolate and identify mesenchymal CTCs is required to expand our knowledge about the clinical impact of mesenchymal CTCs. Therefore, here we provide a comprehensive review of clinical significance of mesenchymal CTCs in BC together with statistical analysis of previously published data, in which we assessed the suitability of a number of methods/markers used for isolation of CTCs with different EMT phenotypes, both in in vitro spike-in tests with BC cell lines, as well as clinical samples. Results of spiked-in cell lines indicate that, in general, methods not based on epithelial enrichment only, capture mesenchymal CTCs much more efficiently that CellSearch® (golden standard in CTCs detection), but at the same time are not much inferior to Cell Search®, though large variation in recovery rates of added cells among the methods is observed. In clinical samples, where additional CTCs detection markers are needed, positive epithelial-based CTCs enrichment was the most efficient in isolating CTCs with mesenchymal features from non-metastatic BC patients. From the marker side, PI3K and VIM were contributing the most to detection of CTCs with mesenchymal features (in comparison to SNAIL) in non-metastatic and metastatic BC patients, respectively. However, additional data are needed for more robust identification of markers for efficient detection of CTCs with mesenchymal features.
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Affiliation(s)
- Justyna Topa
- Laboratory of Translational Oncology, Intercollegiate Faculty of Biotechnology, University of Gdańsk and Medical University of Gdańsk, Debinki 1, 80-211, Gdansk, Poland
| | - Peter Grešner
- Laboratory of Translational Oncology, Intercollegiate Faculty of Biotechnology, University of Gdańsk and Medical University of Gdańsk, Debinki 1, 80-211, Gdansk, Poland
| | - Anna J Żaczek
- Laboratory of Translational Oncology, Intercollegiate Faculty of Biotechnology, University of Gdańsk and Medical University of Gdańsk, Debinki 1, 80-211, Gdansk, Poland
| | - Aleksandra Markiewicz
- Laboratory of Translational Oncology, Intercollegiate Faculty of Biotechnology, University of Gdańsk and Medical University of Gdańsk, Debinki 1, 80-211, Gdansk, Poland.
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26
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Cheng J, Zhang L, Zhang Y, Ye Y, Zhao W, Zhang L, Li Y, Liu Y, Zhang W, Guo H, Li M, Zhao Y, Huang C. 3D spiral channels combined with flexible micro-sieve for high-throughput rare tumor cell enrichment and assay from clinical pleural effusion samples. Biodes Manuf 2022. [DOI: 10.1007/s42242-021-00167-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/02/2022]
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27
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Chelakkot C, Yang H, Shin YK. Relevance of Circulating Tumor Cells as Predictive Markers for Cancer Incidence and Relapse. Pharmaceuticals (Basel) 2022; 15:75. [PMID: 35056131 PMCID: PMC8781286 DOI: 10.3390/ph15010075] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2021] [Revised: 12/31/2021] [Accepted: 01/03/2022] [Indexed: 02/04/2023] Open
Abstract
Shedding of cancer cells from the primary site or undetectable bone marrow region into the circulatory system, resulting in clinically overt metastasis or dissemination, is the hallmark of unfavorable invasive cancers. The shed cells remain in circulation until they extravasate to form a secondary metastatic lesion or undergo anoikis. The circulating tumor cells (CTCs) found as single cells or clusters carry a plethora of information, are acknowledged as potential biomarkers for predicting cancer prognosis and cancer progression, and are supposed to play key roles in determining tailored therapies for advanced diseases. With the advent of novel technologies that allow the precise isolation of CTCs, more and more clinical trials are focusing on the prognostic and predictive potential of CTCs. In this review, we summarize the role of CTCs as a predictive marker for cancer incidence, relapse, and response to therapy.
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Affiliation(s)
- Chaithanya Chelakkot
- Bio-MAX/N-Bio, Bio-MAX Institute, Seoul National University, Seoul 08226, Korea
- Genobio Corp., Seoul 08394, Korea
| | - Hobin Yang
- Research Institute of Pharmaceutical Science, Department of Pharmacy, College of Pharmacy, Seoul National University, Seoul 08226, Korea
| | - Young Kee Shin
- Bio-MAX/N-Bio, Bio-MAX Institute, Seoul National University, Seoul 08226, Korea
- Research Institute of Pharmaceutical Science, Department of Pharmacy, College of Pharmacy, Seoul National University, Seoul 08226, Korea
- Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University, Seoul 08226, Korea
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28
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Li D, Wang C, Ni Y, Liu Y, Wang W, Zhang S, Chang HC, Senapati S. Development of a Multi-target Protein Biomarker Assay for Circulating Tumor Cells. Methods Mol Biol 2022; 2394:3-18. [PMID: 35094318 DOI: 10.1007/978-1-0716-1811-0_1] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/14/2023]
Abstract
We report a highly sensitive and selective CNT-switch liquid biopsy platform that detects and quantifies protein biomarker expressions from circulating tumor cells in blood for early detection of metastatic breast cancer and its relapse. This platform first isolates and enriches more than 99% of tumor cells with an off-chip micro-size membrane filtration technique and then conducts on-chip detection of the membrane and internal protein biomarkers of the tumor cells with high sensitivity and selectivity. High sensitivity is achieved with complete association of the antibody-antigen-antibody (Ab-Ag-Ab) complex by precisely and rapidly assembling carbon nanotubes (CNTs) across two parallel electrodes via sequential DC electrophoresis and dielectrophoresis (DEP) deposition. Each bridged CNT acts as a switch that connects the electrodes and closes the circuit to generate an electrical signal. The high selectivity is achieved with a critical hydrodynamic shear rate that irreversibly removes non-target linkers of the aligned CNTs. At present, we are able to detect the protein biomarkers from 5 spiked breast cancer tumor cells of different types within 7.5 ml of human blood samples. This demonstrates the potential of this platform as an inexpensive and noninvasive alternative to MRI scans and tissue biopsies currently used to detect early metastatic breast cancer and its relapse.
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Affiliation(s)
- Diya Li
- Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, IN, USA
| | - Ceming Wang
- Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, IN, USA
| | - Yingjia Ni
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, USA
| | - Yaoping Liu
- Institute of Microelectronics, Peking University, Beijing, China
| | - Wei Wang
- Institute of Microelectronics, Peking University, Beijing, China
- National Key Laboratory of Science and Technology on Micro/Nano Fabrication, Beijing, China
| | - Siyuan Zhang
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, USA
- Harper Cancer Research Institute, University of Notre Dame, Notre Dame, IN, USA
| | - Hsueh-Chia Chang
- Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, IN, USA
- Harper Cancer Research Institute, University of Notre Dame, Notre Dame, IN, USA
| | - Satyajyoti Senapati
- Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, IN, USA.
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29
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Danila DC. Liquid biopsy as a cancer biomarker-potential, and challenges. Cancer Biomark 2022. [DOI: 10.1016/b978-0-12-824302-2.00013-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
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30
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Pathak N, Chitikela S, Malik PS. Recent advances in lung cancer genomics: Application in targeted therapy. ADVANCES IN GENETICS 2021; 108:201-275. [PMID: 34844713 DOI: 10.1016/bs.adgen.2021.08.004] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Genomic characterization of lung cancer has not only improved our understanding of disease biology and carcinogenesis but also revealed several therapeutic opportunities. Targeting tumor dependencies on specific genomic alterations (oncogene addiction) has accelerated the therapeutic developments and significantly improved the outcomes even in advanced stage of disease. Identification of genomic alterations predicting response to specific targeted treatment is the key to success for this "personalized treatment" approach. Availability of multiple choices of therapeutic options for specific genomic alterations highlight the importance of optimum sequencing of drugs. Multiplex gene testing has become mandatory in view of constantly increasing number of therapeutic targets and effective treatment options. Influence of genomic characteristics on response to immunotherapy further makes comprehensive genomic profiling necessary before therapeutic decision making. A comprehensive elucidation of resistance mechanisms and directed treatments have made the continuum of care possible and transformed this deadly disease into a chronic condition. Liquid biopsy-based approach has made the dynamic monitoring of disease possible and enabled treatment optimizations accordingly. Current lung cancer management is the perfect example of "precision-medicine" in clinical oncology.
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Affiliation(s)
- Neha Pathak
- Department of Medical Oncology, Dr. B.R.A.I.R.C.H., All India Institute of Medical Sciences, New Delhi, India
| | - Sindhura Chitikela
- Department of Medical Oncology, Dr. B.R.A.I.R.C.H., All India Institute of Medical Sciences, New Delhi, India
| | - Prabhat Singh Malik
- Department of Medical Oncology, Dr. B.R.A.I.R.C.H., All India Institute of Medical Sciences, New Delhi, India.
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31
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Hakim M, Khorasheh F, Alemzadeh I, Vossoughi M. A new insight to deformability correlation of circulating tumor cells with metastatic behavior by application of a new deformability-based microfluidic chip. Anal Chim Acta 2021; 1186:339115. [PMID: 34756251 DOI: 10.1016/j.aca.2021.339115] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2021] [Revised: 09/22/2021] [Accepted: 09/23/2021] [Indexed: 11/18/2022]
Abstract
Isolation and characterization of circulating tumor cells (CTCs) found in blood samples of cancer patients have been considered as a reliable source for cancer prognosis and diagnosis. A new continuous microfluidic platform has been designed in this investigation for simultaneous capture and characterization of CTCs based on their deformability. The deformability-based chip (D-Chip) consists of two sections of separation and characterization where slanted weirs with a gap of 7 μm were considered. Although sometimes CTCs and leukocytes have the same size, the deformability differs in such a way that can be exploited for enrichment purposes. MCF7 and MDA-MB-231 cell lines were used for the initial evaluation of the D-Chip performance. In the separation section, cancer cells were isolated based on deformability differences with an efficiency of higher than 93% (∼average capturing capacity of 2085 out of 2200 cancer cells ml-1) and with significantly high purity (15-40 WBCs ml-1; ∼5 log depletion of WBCs). Cancer cells were categorized based on the deformability difference in the characterization section. Subsequently, 15 clinical blood samples from breast cancer patients were analyzed by the D-Chip. Suggest 'The chip detected CTCs in all patient samples, processed the blood sample at a high throughput of 5.3 ml/h, and properly categorized CTCs based on deformability differences. Further characterization showed that the highly deformable breast cancer CTCs in our patient samples also showed higher potential of metastasis in support of a broader correlation between deformability of CTCs and metastatic behavior.
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Affiliation(s)
- Maziar Hakim
- Department of Chemical and Petroleum Engineering, Sharif University of Technology, Tehran, Iran
| | - Farhad Khorasheh
- Department of Chemical and Petroleum Engineering, Sharif University of Technology, Tehran, Iran
| | - Iran Alemzadeh
- Department of Chemical and Petroleum Engineering, Sharif University of Technology, Tehran, Iran
| | - Manouchehr Vossoughi
- Department of Chemical and Petroleum Engineering, Sharif University of Technology, Tehran, Iran.
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32
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Shi L, Esfandiari L. Emerging on-chip electrokinetic based technologies for purification of circulating cancer biomarkers towards liquid biopsy: A review. Electrophoresis 2021; 43:288-308. [PMID: 34791687 DOI: 10.1002/elps.202100234] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2021] [Revised: 11/12/2021] [Accepted: 11/12/2021] [Indexed: 12/11/2022]
Abstract
Early detection of cancer can significantly reduce mortality and save lives. However, the current cancer diagnosis is highly dependent on costly, complex, and invasive procedures. Thus, a great deal of effort has been devoted to exploring new technologies based on liquid biopsy. Since liquid biopsy relies on detection of circulating biomarkers from biofluids, it is critical to isolate highly purified cancer-related biomarkers, including circulating tumor cells (CTCs), cell-free nucleic acids (cell-free DNA and cell-free RNA), small extracellular vesicles (exosomes), and proteins. The current clinical purification techniques are facing a number of drawbacks including low purity, long processing time, high cost, and difficulties in standardization. Here, we review a promising solution, on-chip electrokinetic-based methods, that have the advantage of small sample volume requirement, minimal damage to the biomarkers, rapid, and label-free criteria. We have also discussed the existing challenges of current on-chip electrokinetic technologies and suggested potential solutions that may be worthy of future studies.
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Affiliation(s)
- Leilei Shi
- Department of Electrical Engineering and Computer Science, College of Engineering and Applied Science, University of Cincinnati, Cincinnati, Ohio, USA
| | - Leyla Esfandiari
- Department of Electrical Engineering and Computer Science, College of Engineering and Applied Science, University of Cincinnati, Cincinnati, Ohio, USA.,Department of Biomedical Engineering, College of Engineering and Applied Science, University of Cincinnati, Cincinnati, Ohio, USA
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33
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Gou Y, Chen Z, Sun C, Wang P, You Z, Yalikun Y, Tanaka Y, Ren D. Specific capture and intact release of breast cancer cells using a twin-layer vein-shaped microchip with a self-assembled surface. NANOSCALE 2021; 13:17765-17774. [PMID: 34558589 DOI: 10.1039/d1nr04018a] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/13/2023]
Abstract
Breast cancer is the most fatal disease among female cancers yet its detection still relies on needle biopsy. The unique physical and immune characteristics of breast cancer cells different from blood cells make them suitable to be employed as excellent biomarkers in liquid biopsy, through which breast cancer cells are collected from peripheral blood for further cancer diagnosis, medical treatment monitoring, and drug screening. Although the separation and enrichment of breast cancer cells from peripheral blood have been studied for years, there are still two problems to be solved in these methods: the low efficiency of on-chip immunologic capture in the flow state and the influence of the conjugated antibodies for the following analyses during cell release. In this paper, a vein-shaped microchip with self-assembled surface was developed for the specific and robust capture (91.2%) of breast cancer cells in the flow state. A protein-recovery process was proposed, in which trypsin served as a mild release reagent, releasing 92% of cells with high viability (96%), normal adherent proliferation, and complete proteins on the cell membrane, avoiding disturbance of the conjugated chemical molecules in the following clinical study. The excellent performance demonstrated in isolating free breast cancer cells from real peripheral blood sample, originating from the orthotopic 4T1 breast cancer metastatic models, suggest the microchip could be utilized as a multiple circulating tumor cell capture and release platform that could allow providing more reliable information in liquid biopsies.
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Affiliation(s)
- Yixing Gou
- State Key Laboratory of Precision Measurement Technology and Instruments, Tianjin University, Tianjin, 300072, China
- State Key Laboratory of Precision Measurement Technology and Instruments, Department of Precision Instrument, Tsinghua University, Beijing, 100084, China.
| | - Zhuyuan Chen
- Department of Basic Sciences, School of Medicine, Tsinghua University, Beijing 100084, China
| | - Changku Sun
- State Key Laboratory of Precision Measurement Technology and Instruments, Tianjin University, Tianjin, 300072, China
| | - Peng Wang
- State Key Laboratory of Precision Measurement Technology and Instruments, Tianjin University, Tianjin, 300072, China
| | - Zheng You
- State Key Laboratory of Precision Measurement Technology and Instruments, Department of Precision Instrument, Tsinghua University, Beijing, 100084, China.
| | - Yaxiaer Yalikun
- Center for Biosystems Dynamics Research (BDR), RIKEN, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan
- Division of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayamacho, Ikoma, Nara 630-0192, Japan
| | - Yo Tanaka
- Center for Biosystems Dynamics Research (BDR), RIKEN, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan
| | - Dahai Ren
- State Key Laboratory of Precision Measurement Technology and Instruments, Department of Precision Instrument, Tsinghua University, Beijing, 100084, China.
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34
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Li YH, Zhou S, Jian X, Zhang X, Song YY. Asymmetrically coating Pt nanoparticles on magnetic silica nanospheres for target cell capture and therapy. Mikrochim Acta 2021; 188:361. [PMID: 34601637 DOI: 10.1007/s00604-021-05009-3] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2021] [Accepted: 08/25/2021] [Indexed: 12/15/2022]
Abstract
A Janus cargo has been developed via the combination of magnetic mesoporous silica (MMS) with asymmetric decoration of Pt nanoparticles (PtNPs). Mesoporous morphology of MMS provides plenty of space for loading photosensitizers and targeting agents; the magnetic feature endows the as-formed nanospheres with satisfactory isolation function in removal of low abundant target cells. The excellent catalytic ability of PtNPs can effectively alleviate the hypoxia condition of tumor microenvironment via the decomposition of hydrogen peroxide (H2O2), as well as an O2-drived nanomotor for highly efficient drug release. Using CCRF-CEM as the model target cell, the Janus cargo is demonstrated to possess significantly improved performance in cell capture and photodynamic therapy. Specially, owing to the patchy Pt decoration, the loaded photosensitizers exhibit a more efficient release behavior. More importantly, asymmetric O2-emission from one side of the nanocargo acts as a driving force, which could effectively accelerate the motion ability of cargo in cell media, thus leading to an enhanced therapeutic effect compared with the traditionally symmetric nanocargo. This Janus cargo would offer a new paradigm to design highly efficient drug carrier for gaining an improved photodynamic therapy in hypoxic cancer cells.
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Affiliation(s)
- Ya-Hang Li
- College of Sciences, Northeastern University, Shenyang, 110004, China
- College of Chemistry and Materials Engineering, Bohai University, Jinzhou, 121013, China
| | - Shanshan Zhou
- College of Sciences, Northeastern University, Shenyang, 110004, China
| | - Xiaoxia Jian
- College of Sciences, Northeastern University, Shenyang, 110004, China
| | - Xi Zhang
- College of Sciences, Northeastern University, Shenyang, 110004, China.
| | - Yan-Yan Song
- College of Sciences, Northeastern University, Shenyang, 110004, China.
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35
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Fabrication of Formalin-Fixed, Paraffin-Embedded (FFPE) Circulating Tumor Cell (CTC) Block Using a Hydrogel Core-Mediated Method. MICROMACHINES 2021; 12:mi12091128. [PMID: 34577771 PMCID: PMC8466852 DOI: 10.3390/mi12091128] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/16/2021] [Revised: 09/17/2021] [Accepted: 09/18/2021] [Indexed: 12/24/2022]
Abstract
Circulating tumor cells (CTCs) are extremely low-frequency cells in the bloodstream. As those cells have detached from the primary tumor tissues and it circulates throughout the whole body, they are considered as promising diagnostic biomarkers for clinical application. However, the analysis of CTC is often restricted due to their rarity and heterogeneity, as well as their short-term presence. Here we proposed formalin-fixed, paraffin-embedded (FFPE) CTC block method, in combination manner with the hydrogel core-mediated CTC accumulation and conventional paraffin tissue block preparation. The hydrogel core specifically captures and releases cancer cells with high efficiency with an immunoaffinity manner. An additional shell structure protects the isolated cancer cells during the FFPE CTC block preparation process. The fabricated FFPE CTC block was sectioned and cytopathologically investigated just the same way as the conventional tissue block. Our results demonstrate that rare cells such as CTCs can also be prepared for FFPE cell blocks and shows great promise for cytopathological CTC studies.
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Lemma S, Perrone AM, De Iaco P, Gasparre G, Kurelac I. Current methodologies to detect circulating tumor cells: a focus on ovarian cancer. Am J Cancer Res 2021; 11:4111-4126. [PMID: 34659879 PMCID: PMC8493391] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2021] [Accepted: 07/19/2021] [Indexed: 06/13/2023] Open
Abstract
Identification of circulating tumor cells (CTC) in liquid biopsies opens a window of opportunities for the optimization of clinical management of oncologic patients. In ovarian cancer (OC), which involves atypical routes of metastatic spread, CTC analyses may also offer novel insights about the mechanisms behind malignant progression of the disease. However, current methodologies struggle to precisely define CTC number in the peripheral blood of OC patients, and the isolation of viable cells for further characterization is still challenging. The biggest limitation is the lack of methodological standardization for OC CTC detection, preventing comprehensive definition of their clinical potential required for the transfer to practice. Here we describe and compare methods for CTC analysis that have been implemented for OC thus far, discussing pros, cons and improvements needed. We identify biophysical separation approaches as optimal for CTC enrichment. On the other hand, the identification of specific tumor antigens or gene transcripts, despite displaying drawbacks related to tumor heterogeneity, still remains the best approach for OC CTC detection.
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Affiliation(s)
- Silvia Lemma
- Unit of Medical Genetics, Department of Medical and Surgical Sciences (DIMEC), University of BolognaVia Massarenti 9, 40138 Bologna, Italy
- Study and Research Center on Gynecological Neoplasias, Department of Medical and Surgical Sciences (DIMEC), University of BolognaVia Massarenti 9, 40138 Bologna, Italy
- Center for Applied Biomedical Research (CRBA), University of Bologna40138 Bologna, Italy
| | - Anna M Perrone
- Study and Research Center on Gynecological Neoplasias, Department of Medical and Surgical Sciences (DIMEC), University of BolognaVia Massarenti 9, 40138 Bologna, Italy
- Division of Oncologic Gynecology, IRCCS-Azienda Ospedaliero-Universitaria di Bologna40138 Bologna, Italy
| | - Pierandrea De Iaco
- Study and Research Center on Gynecological Neoplasias, Department of Medical and Surgical Sciences (DIMEC), University of BolognaVia Massarenti 9, 40138 Bologna, Italy
- Division of Oncologic Gynecology, IRCCS-Azienda Ospedaliero-Universitaria di Bologna40138 Bologna, Italy
| | - Giuseppe Gasparre
- Unit of Medical Genetics, Department of Medical and Surgical Sciences (DIMEC), University of BolognaVia Massarenti 9, 40138 Bologna, Italy
- Study and Research Center on Gynecological Neoplasias, Department of Medical and Surgical Sciences (DIMEC), University of BolognaVia Massarenti 9, 40138 Bologna, Italy
- Center for Applied Biomedical Research (CRBA), University of Bologna40138 Bologna, Italy
| | - Ivana Kurelac
- Unit of Medical Genetics, Department of Medical and Surgical Sciences (DIMEC), University of BolognaVia Massarenti 9, 40138 Bologna, Italy
- Study and Research Center on Gynecological Neoplasias, Department of Medical and Surgical Sciences (DIMEC), University of BolognaVia Massarenti 9, 40138 Bologna, Italy
- Center for Applied Biomedical Research (CRBA), University of Bologna40138 Bologna, Italy
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Li R, Gong Z, Liu Y, Zhao X, Guo S. Detection of circulating tumor cells and single cell extraction technology: principle, effect and application prospect. NANO FUTURES 2021; 5:032002. [DOI: 10.1088/2399-1984/ac1325] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2025]
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Lucas K, Dehghani M, Khire T, Gaborski T, Flax JD, Waugh RE, McGrath JL. A predictive model of nanoparticle capture on ultrathin nanoporous membranes. J Memb Sci 2021. [DOI: 10.1016/j.memsci.2021.119357] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
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Schmidt M, Franken A, Wilms D, Fehm T, Neubauer HJ, Schmidt S. Selective Adhesion and Switchable Release of Breast Cancer Cells via Hyaluronic Acid Functionalized Dual Stimuli-Responsive Microgel Films. ACS APPLIED BIO MATERIALS 2021; 4:6371-6380. [PMID: 35006876 DOI: 10.1021/acsabm.1c00586] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
The detection of tumor cells from liquid biopsy samples is of critical importance for early cancer diagnosis, malignancy assessment, and treatment. In this work, coatings of hyaluronic acid (HA)-functionalized dual-stimuli responsive poly(N-isopropylacrylamide) (PNIPAM) microgels are used to study the specificity of breast cancer cell binding and to assess cell friendly release mechanisms for further diagnostic procedures. The microgels are established by straightforward precipitation polymerization with amine bearing comonomers and postfunctionalization with a UV-labile linker that covalently binds HA to the microgel network. Well-defined microgel coatings for cell binding are established via simple physisorption and annealing. The HA-presenting PNIPAM microgel films are shown to specifically adhere CD44 expressing breast cancer cell lines (MDA-MB-231 and MCF-7), where an increase in adhesion correlates with higher CD44 expression and HA functionalization. Upon cooling below the lower critical solution temperature of PNIPAM microgels, the cells could be released; however, 10-30% of the cells still remained on the surface even after prolonged cooling and mild mechanical agitation. A complete cell release is achieved after applying the light stimulus by short UV treatment cleaving HA units from the microgels. Owing to the comparatively straightforward preparation procedures, such dual-responsive microgel films could be considered for the effective capture, release, and diagnostics of tumor cells.
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Affiliation(s)
- Melanie Schmidt
- Institute for Organic Chemistry and Macromolecular Chemistry, Heinrich-Heine-University, Universitätsstr. 1, 40225, Düsseldorf, Germany
| | - André Franken
- Department of Obstetrics and Gynecology, Life Science Center, University Hospital and Medical Faculty, Heinrich-Heine University Duesseldorf, Merowingerplatz 1A, 40225 Düsseldorf, Germany
| | - Dimitri Wilms
- Institute for Organic Chemistry and Macromolecular Chemistry, Heinrich-Heine-University, Universitätsstr. 1, 40225, Düsseldorf, Germany
| | - Tanja Fehm
- Department of Obstetrics and Gynecology, Life Science Center, University Hospital and Medical Faculty, Heinrich-Heine University Duesseldorf, Merowingerplatz 1A, 40225 Düsseldorf, Germany
| | - Hans J Neubauer
- Department of Obstetrics and Gynecology, Life Science Center, University Hospital and Medical Faculty, Heinrich-Heine University Duesseldorf, Merowingerplatz 1A, 40225 Düsseldorf, Germany
| | - Stephan Schmidt
- Institute for Organic Chemistry and Macromolecular Chemistry, Heinrich-Heine-University, Universitätsstr. 1, 40225, Düsseldorf, Germany
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Fukuyama S, Kumamoto S, Nagano S, Hitotsuya S, Yasuda K, Kitamura Y, Iwatsuki M, Baba H, Ihara T, Nakanishi Y, Nakashima Y. Detection of cancer cells in whole blood using a dynamic deformable microfilter and a nucleic acid aptamer. Talanta 2021; 228:122239. [DOI: 10.1016/j.talanta.2021.122239] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2020] [Revised: 02/15/2021] [Accepted: 02/16/2021] [Indexed: 01/22/2023]
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Schneider S, Gruner D, Richter A, Loskill P. Membrane integration into PDMS-free microfluidic platforms for organ-on-chip and analytical chemistry applications. LAB ON A CHIP 2021; 21:1866-1885. [PMID: 33949565 DOI: 10.1039/d1lc00188d] [Citation(s) in RCA: 32] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/12/2023]
Abstract
Membranes play a crucial role in many microfluidic systems, enabling versatile applications in highly diverse research fields. However, the tight and robust integration of membranes into microfluidic systems requires complex fabrication processes. Most integration approaches, so far, rely on polydimethylsiloxane (PDMS) as base material for the microfluidic chips. Several limitations of PDMS have resulted in the transition of many microfluidic approaches to PDMS-free systems using alternative materials such as thermoplastics. To integrate membranes in those PDMS-free systems, novel alternative approaches are required. This review provides an introduction into microfluidic systems applying membrane technology for analytical systems and organ-on-chip as well as a comprehensive overview of methods for the integration of membranes into PDMS-free systems. The overview and examples will provide a valuable resource and starting point for any researcher that is aiming at implementing membranes in microfluidic systems without using PDMS.
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Affiliation(s)
- Stefan Schneider
- Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, 70569 Stuttgart, Germany
| | - Denise Gruner
- Institut für Halbleiter- und Mikrosystemtechnik, Technische Universität Dresden, 01062 Dresden, Germany and Universitätsklinikum Carl Gustav Carus Dresden, Institut für Klinische Chemie und Laboratoriumsmedizin, 01307 Dresden, Germany
| | - Andreas Richter
- Institut für Halbleiter- und Mikrosystemtechnik, Technische Universität Dresden, 01062 Dresden, Germany
| | - Peter Loskill
- Department of Biomedical Science, Faculty of Medicine, Eberhard Karls University Tübingen, 72076 Tübingen, Germany. and NMI Natural and Medical Sciences Institute at the University of Tübingen, 72770 Reutlingen, Germany
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Smolle E, Taucher V, Lindenmann J, Pichler M, Smolle-Juettner FM. Liquid biopsy in non-small cell lung cancer-current status and future outlook-a narrative review. Transl Lung Cancer Res 2021; 10:2237-2251. [PMID: 34164273 PMCID: PMC8182706 DOI: 10.21037/tlcr-21-3] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2021] [Accepted: 03/11/2021] [Indexed: 12/24/2022]
Abstract
Lung cancer ranks first as the cause of cancer-associated deaths gobally. The American Cancer Society estimates for 228,820 new cases and 135,720 deaths from lung cancer in the United States for the year 2020. Targeted treatment options have rapidly emerged for non-small cell lung cancer (NSCLC) within the past decade. Screening for molecular aberrations is mainly done by tissue biopsy. However, in some cases a biopsy is not possible, or patients do not consent to it. Hence, liquid biopsy remains the only option. Relevant data about the topic of liquid biopsy, with a special focus on NSCLC, was obtained via a PubMed search. We included mainly literature published from 2010 onwards, omitting older studies whenever possible. With this review of the literature, we give an overview of different liquid biopsy approaches, as well as their respective advantages and disadvantages. We have reviewed the assessment of epidermal growth factor receptor (EGFR) mutation status in particular, and go into detail with current use of liquid biopsy in everyday clinical practice. Today, liquid biopsy is still infrequently used, depending on the treatment center, but popularity is steadily increasing. Various different approaches are already available, but costs and level of sensitivity significantly differ between techniques. By using liquid biopsy more widely in selected patients, complication rates can be reduced, and constant disease monitoring is made considerably easier.
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Affiliation(s)
- Elisabeth Smolle
- Division of Pulmonology, Department of Internal Medicine, Medical University of Graz, Graz, Austria
| | - Valentin Taucher
- Division of Cardiology, Department of Internal Medicine, Hospital Barmherzige Schwestern Ried, Ried, Austria
| | - Jörg Lindenmann
- Department of Thoracic Surgery, Medical University of Graz, Graz, Austria
| | - Martin Pichler
- Division of Oncology, Department of Internal Medicine, Medical University of Graz, Graz, Austria
- Department of Experimental Therapeutics, The UT MD Anderson Cancer Center, Houston, TX, USA
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Zhang Y, Zhou Y, Yang Y, Pappas D. Microfluidics for sepsis early diagnosis and prognosis: a review of recent methods. Analyst 2021; 146:2110-2125. [PMID: 33751011 DOI: 10.1039/d0an02374d] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Sepsis is a complex disorder of immune system response to infections that can be caused by a wide range of clinical contexts. Traditional methods for sepsis detection include molecular diagnosis, biomarkers either based on protein concentration or cell surface expression, and microbiological cultures. Development of point-of-care (POC) instruments, which can provide high accuracy and consume less time, is in unprecedented demand. Within the past few years, applications of microfluidic systems for sepsis detection have achieved excellent performance. In this review, we discuss the most recent microfluidic applications specifically in sepsis detection, and propose their advantages and disadvantages. We also present a comprehensive review of other traditional and current sepsis diagnosis methods to obtain a general understanding of the present conditions, which can hopefully direct the development of a new sepsis roadmap.
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Affiliation(s)
- Ye Zhang
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, USA.
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SHEN CC, WU CK, CHEN YH, WANG JX, YANG MH, ZHANG H. Advance in Novel Methods for Enrichment and Precise Analysis of Circulating Tumor Cells. CHINESE JOURNAL OF ANALYTICAL CHEMISTRY 2021. [DOI: 10.1016/s1872-2040(21)60089-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
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Liu Y, Xu H, Li T, Wang W. Microtechnology-enabled filtration-based liquid biopsy: challenges and practical considerations. LAB ON A CHIP 2021; 21:994-1015. [PMID: 33710188 DOI: 10.1039/d0lc01101k] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/12/2023]
Abstract
Liquid biopsy, an important enabling technology for early diagnosis and dynamic monitoring of cancer, has drawn extensive attention in the past decade. With the rapid developments of microtechnology, it has been possible to manipulate cells at the single-cell level, which dramatically improves the liquid biopsy capability. As the microtechnology-enabled liquid biopsy matures from proof-of-concept demonstrations towards practical applications, a main challenge it is facing now is to process clinical samples which are usually of a large volume while containing very rare targeted cells in complex backgrounds. Therefore, a high-throughput liquid biopsy which is capable of processing liquid samples with a large volume in a reasonable time along with a high recovery rate of rare targeted cells from complex clinical liquids is in high demand. Moreover, the purity, viability and release feasibility of recovered targeted cells are the other three key impact factors requiring careful considerations. To date, among the developed techniques, micropore-type filtration has been acknowledged as the most promising solution to address the aforementioned challenges in practical applications. However, the presently reported studies about micropore-type filtration are mostly based on trial and error for device designs aiming at different cancer types, which requires lots of efforts. Therefore, there is an urgent need to investigate and elaborate the fundamental theories of micropore-type filtration and key features that influence the working performances in the liquid biopsy of real clinical samples to promote the application efficacy in practical applications. In this review, the state of the art of microtechnology-enabled filtration is systematically and comprehensively summarized. Four key features of the filtration, including throughput, purity, viability and release feasibility of the captured targeted cells, are elaborated to provide the guidelines for filter designs. The recent progress in the filtration mode modulation and sample standardization to improve the filtration performance of real clinical samples is also discussed. Finally, this review concludes with prospective views for future developments of filtration-based liquid biopsy to promote its application efficacy in clinical practice.
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Affiliation(s)
- Yaoping Liu
- Institute of Microelectronics, Peking University, Beijing, 100871, China.
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The Mechanical Fingerprint of Circulating Tumor Cells (CTCs) in Breast Cancer Patients. Cancers (Basel) 2021; 13:cancers13051119. [PMID: 33807790 PMCID: PMC7961579 DOI: 10.3390/cancers13051119] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2021] [Revised: 02/17/2021] [Accepted: 02/26/2021] [Indexed: 12/11/2022] Open
Abstract
Simple Summary Detection of circulating tumor cells (CTCs) in the blood of cancer patients is a challenging issue, since they adapt to the biochemical and physical landscape of the bloodstream. We approached the issue of CTC identification on a biophysical level. For the first time, we recorded the mechanical deformation profiles of potential CTCs, which were isolated from the blood of breast cancer patients, at the force regime of the deforming blood flow. Mechanical fingerprints of CTCs were significantly different from healthy white blood cells. We used machine learning to further evaluate the differences and identify discrimination criteria. Our results suggest that mechanical characterization of CTCs at low forces is a promising path towards CTC detection. Abstract Circulating tumor cells (CTCs) are a potential predictive surrogate marker for disease monitoring. Due to the sparse knowledge about their phenotype and its changes during cancer progression and treatment response, CTC isolation remains challenging. Here we focused on the mechanical characterization of circulating non-hematopoietic cells from breast cancer patients to evaluate its utility for CTC detection. For proof of premise, we used healthy peripheral blood mononuclear cells (PBMCs), human MDA-MB 231 breast cancer cells and human HL-60 leukemia cells to create a CTC model system. For translational experiments CD45 negative cells—possible CTCs—were isolated from blood samples of patients with mamma carcinoma. Cells were mechanically characterized in the optical stretcher (OS). Active and passive cell mechanical data were related with physiological descriptors by a random forest (RF) classifier to identify cell type specific properties. Cancer cells were well distinguishable from PBMC in cell line tests. Analysis of clinical samples revealed that in PBMC the elliptic deformation was significantly increased compared to non-hematopoietic cells. Interestingly, non-hematopoietic cells showed significantly higher shape restoration. Based on Kelvin–Voigt modeling, the RF algorithm revealed that elliptic deformation and shape restoration were crucial parameters and that the OS discriminated non-hematopoietic cells from PBMC with an accuracy of 0.69, a sensitivity of 0.74, and specificity of 0.63. The CD45 negative cell population in the blood of breast cancer patients is mechanically distinguishable from healthy PBMC. Together with cell morphology, the mechanical fingerprint might be an appropriate tool for marker-free CTC detection.
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Wei X, Chen K, Guo S, Liu W, Zhao XZ. Emerging Microfluidic Technologies for the Detection of Circulating Tumor Cells and Fetal Nucleated Red Blood Cells. ACS APPLIED BIO MATERIALS 2021; 4:1140-1155. [DOI: 10.1021/acsabm.0c01325] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Affiliation(s)
- Xiaoyun Wei
- Key Laboratory of Artificial Micro- and Nano-Structures of Ministry of Education, School of Physics and Technology, Wuhan University, Wuhan 430072, China
- Key Laboratory of Medical Information and 3D Bioprinting of Zhejiang Province, Hangzhou Dianzi University, Hangzhou 310018, China
| | - Keke Chen
- Key Laboratory of Artificial Micro- and Nano-Structures of Ministry of Education, School of Physics and Technology, Wuhan University, Wuhan 430072, China
| | - Shishang Guo
- Key Laboratory of Artificial Micro- and Nano-Structures of Ministry of Education, School of Physics and Technology, Wuhan University, Wuhan 430072, China
| | - Wei Liu
- Key Laboratory of Artificial Micro- and Nano-Structures of Ministry of Education, School of Physics and Technology, Wuhan University, Wuhan 430072, China
| | - Xing-Zhong Zhao
- Key Laboratory of Artificial Micro- and Nano-Structures of Ministry of Education, School of Physics and Technology, Wuhan University, Wuhan 430072, China
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Farshchi F, Hasanzadeh M. Microfluidic biosensing of circulating tumor cells (CTCs): Recent progress and challenges in efficient diagnosis of cancer. Biomed Pharmacother 2020; 134:111153. [PMID: 33360045 DOI: 10.1016/j.biopha.2020.111153] [Citation(s) in RCA: 30] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2020] [Revised: 12/11/2020] [Accepted: 12/14/2020] [Indexed: 10/22/2022] Open
Abstract
Cancer metastasis is one of the foremost causes of cancer incidence and fatality in the whole of the world. Circulating tumor cells (CTC) have been confirmed to be among the most significant stimuli of metastasis in recent years and presently are the subject of extensive research aiming to be accurately identified by using biological and physical properties. Among the various studies conducted for isolation, identification, and characterization of CTCs, microfluidic systems have aroused great attention owing to their unique advantages such as low-cost, simplicity, reduction in reagent consumption, miniaturization, fast and precise control. The purpose of this review is to provide an overview of current state of the microfluidic biosensors for the screening of CTCs. Additionally, given the recent progress in this field, future outlook for the development of the microfluidics biosensing is briefly discussed.
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Affiliation(s)
- Fatemeh Farshchi
- Pharmaceutical Analysis Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; Food and Drug Safety Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Mohammad Hasanzadeh
- Pharmaceutical Analysis Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
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Hu D, Liu H, Tian Y, Li Z, Cui X. Sorting Technology for Circulating Tumor Cells Based on Microfluidics. ACS COMBINATORIAL SCIENCE 2020; 22:701-711. [PMID: 33052651 DOI: 10.1021/acscombsci.0c00157] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Circulating tumor cells (CTCs) carry reliable clinical information for the diagnosis and treatment of cancer that is a malignant disease with a high mortality rate. However, the amount of CTCs in the blood is quite low. To obtain credible clinical information, an efficient method of extracting CTCs is necessary. Microfluidic technology has proven its effectiveness on CTCs separation in recent years. Here, we present a comprehensive review of CTC sorting methods based on microfluidics. Specifically, we introduce four different microfluidic sorting methods of CTCs and compare their advantages and disadvantages. Finally, we summarize the analysis of CTCs based on microfluidics and present a prospective view of future research.
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Affiliation(s)
- Dayu Hu
- College of Medicine and Biological Information Engineering, Northeastern University, Shenyang 110169, China
| | - He Liu
- College of Medicine and Biological Information Engineering, Northeastern University, Shenyang 110169, China
| | - Ye Tian
- College of Medicine and Biological Information Engineering, Northeastern University, Shenyang 110169, China
| | - Zhi Li
- Department of Medical Oncology, The First Hospital of China Medical University, Shenyang 110001, China
| | - Xiaoyu Cui
- College of Medicine and Biological Information Engineering, Northeastern University, Shenyang 110169, China
- Minist Educ, Key Lab Intelligent Comp Med Image MIIC, Shenyang 110169, Liaoning, China
- Key Laboratory of Data Analytics and Optimization for Smart Industry, Northeastern University, Shenyang 110169, China
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Takagi H, Dong L, Kuczler MD, Lombardo K, Hirai M, Amend SR, Pienta KJ. Analysis of the Circulating Tumor Cell Capture Ability of a Slit Filter-Based Method in Comparison to a Selection-Free Method in Multiple Cancer Types. Int J Mol Sci 2020; 21:ijms21239031. [PMID: 33261132 PMCID: PMC7730626 DOI: 10.3390/ijms21239031] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2020] [Revised: 11/10/2020] [Accepted: 11/11/2020] [Indexed: 11/16/2022] Open
Abstract
Circulating tumor cells (CTCs) are a promising biomarker for cancer liquid biopsy. To evaluate the CTC capture bias and detection capability of the slit filter-based CTC isolation platform (CTC-FIND), we prospectively compared it head to head to a selection-free platform (AccuCyte®-CyteFinder® system). We used the two methods to determine the CTC counts, CTC positive rates, CTC size distributions, and CTC phenotypes in 36 patients with metastatic cancer. Between the two methods, the median CTC counts were not significantly different and the total counts were correlated (r = 0.63, p < 0.0001). The CTC positive rate by CTC-FIND was significantly higher than that by AccuCyte®-CyteFinder® system (91.7% vs. 66.7%, p < 0.05). The median diameter of CTCs collected by CTC-FIND was significantly larger (13.0 μm, range 5.2–52.0 vs. 10.4 μm, range 5.2–44.2, p < 0.0001). The distributions of CTC phenotypes (CK+EpCAM+, CK+EpCAM− or CK−EpCAM+) detected by both methods were similar. These results suggested that CTC-FIND can detect more CTC-positive cases but with a bias toward large size of CTCs.
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Affiliation(s)
- Hidenori Takagi
- Research and Development Division, ARKRAY, Inc. Yousuien-nai, 59 Gansuin-cho, Kamigyo-ku, Kyoto 602-0008, Japan;
- The James Buchanan Brady Urological Institute, Johns Hopkins University School of Medicine, 600 N. Wolfe Street, Baltimore, MD 21287, USA; (L.D.); (M.D.K.); (K.L.); (S.R.A.); (K.J.P.)
- Correspondence: ; Tel.: +81-75-662-8979; Fax: +81-75-431-1202
| | - Liang Dong
- The James Buchanan Brady Urological Institute, Johns Hopkins University School of Medicine, 600 N. Wolfe Street, Baltimore, MD 21287, USA; (L.D.); (M.D.K.); (K.L.); (S.R.A.); (K.J.P.)
- Department of Urology and Renji Hospital, Shanghai Jiao Tong University School of Medicine, 1630 Dongfang Road, Shanghai 200025, China
| | - Morgan D. Kuczler
- The James Buchanan Brady Urological Institute, Johns Hopkins University School of Medicine, 600 N. Wolfe Street, Baltimore, MD 21287, USA; (L.D.); (M.D.K.); (K.L.); (S.R.A.); (K.J.P.)
| | - Kara Lombardo
- The James Buchanan Brady Urological Institute, Johns Hopkins University School of Medicine, 600 N. Wolfe Street, Baltimore, MD 21287, USA; (L.D.); (M.D.K.); (K.L.); (S.R.A.); (K.J.P.)
| | - Mitsuharu Hirai
- Research and Development Division, ARKRAY, Inc. Yousuien-nai, 59 Gansuin-cho, Kamigyo-ku, Kyoto 602-0008, Japan;
| | - Sarah R. Amend
- The James Buchanan Brady Urological Institute, Johns Hopkins University School of Medicine, 600 N. Wolfe Street, Baltimore, MD 21287, USA; (L.D.); (M.D.K.); (K.L.); (S.R.A.); (K.J.P.)
| | - Kenneth J. Pienta
- The James Buchanan Brady Urological Institute, Johns Hopkins University School of Medicine, 600 N. Wolfe Street, Baltimore, MD 21287, USA; (L.D.); (M.D.K.); (K.L.); (S.R.A.); (K.J.P.)
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