scholarly journals Immuno-Acoustic Sorting of Disease-Specific Extracellular Vesicles by Acoustophoretic Force

Micromachines ◽  
2021 ◽  
Vol 12 (12) ◽  
pp. 1534
Author(s):  
Junyuan Liu ◽  
Yuxin Qu ◽  
Han Wang
Keyword(s):  
Extracellular Vesicles ◽  
Processing Time ◽  
Life Science ◽  
Biological Fluids ◽  
Label Free ◽  
Her2 Positive ◽  
Diagnosis And Prognosis ◽  
Efficient Recovery ◽  
Downstream Analysis ◽  
Disease Specific

Methods for the isolation and analysis of extracellular vesicles (EVs) have been extensively explored in the field of life science and in clinical diagnosis in recent years. The separation and efficient recovery of high-purity target EVs from biological samples are important prerequisites in the study of EVs. So far, commonly used methods of EV separation include ultracentrifugation, filtration, solvent precipitation and immunoaffinity capturing. However, these methods suffer from long processing time, EV damage and low enrichment efficiency. The use of acoustophoretic force facilitates the non-contact label-free manipulation of cells based on their size and compressibility but lacks specificity. Additionally, the acoustophoretic force exerted on sub-micron substances is normally weak and insufficient for separation. Here we present a novel immuno-acoustic sorting technology, where biological substances such as EVs, viruses, and biomolecules, can be specifically captured by antibody/receptor coated microparticles through immunoaffinity, and manipulated by an acoustophoretic force exerted on the microparticles. Using immuno-acoustic sorting technology, we successfully separated and purified HER2-positive EVs for further downstream analysis. This method holds great potential in isolating and purifying specific targets such as disease-related EVs from biological fluids and opens new possibilities for the EV-based early diagnosis and prognosis of diseases.

Separation of Extracellular Vesicles by DNA-Directed Immunocapturing Followed by Enzymatic Release

2020 ◽  
Author(s):  
Dario Brambilla ◽  
Laura Sola ◽  
Elisa Chiodi ◽  
Natasa Zarovni ◽  
Diogo Fortunato ◽  
...  
Keyword(s):  
Cell Culture ◽  
Extracellular Vesicles ◽  
Culture Media ◽  
Biological Fluids ◽  
Imaging Techniques ◽  
Cell Communication ◽  
Disease Diagnosis ◽  
Cell Culture Supernatant ◽  
Transmission Electron ◽  
Downstream Analysis

Extracellular vesicles (EVs) have attracted great interest among researchers due to their role in cell-cell communication, disease diagnosis, and drug delivery. In spite of their potential in the medical field, there is no consensus on the best method for separating microvesicles from cell culture supernatant and complex biological fluids. Obtaining a good recovery yield and preserving physical characteristics is critical for the diagnostic and therapeutic use of EVs. The separation is made complex by the fact that blood and cell culture media, contain a large number of nanoparticles in the same size range. Methods that exploit immunoaffinity capture provide high purity samples and overcome the issues of currently used separation methods. However, the release of captured nanovesicles requires harsh conditions that hinder their use in certain types of downstream analysis. Herein, a novel capture and release approach for small extracellular vesicles (sEVs), based on DNAdirected immobilization of antiCD63 antibody is presented. The flexible DNAlinker increases the capture efficiency and allows releasing of EVs by exploiting the endonucleasic activity of DNAse I. This separation protocol works under mild conditions, enabling the release of intact vesicles that can be successfully analyzed by imaging techniques. In this article sEVs recovered from plasma were characterized by established techniques for EVs analysis including nanoparticle tracking and transmission electron microscopy.<br>

scholarly journals Separation of Extracellular Vesicles by DNA-Directed Immunocapturing Followed by Enzymatic Release

2020 ◽  
Author(s):  
Dario Brambilla ◽  
Laura Sola ◽  
Elisa Chiodi ◽  
Natasa Zarovni ◽  
Diogo Fortunato ◽  
...  
Keyword(s):  
Cell Culture ◽  
Extracellular Vesicles ◽  
Culture Media ◽  
Biological Fluids ◽  
Imaging Techniques ◽  
Cell Communication ◽  
Disease Diagnosis ◽  
Cell Culture Supernatant ◽  
Transmission Electron ◽  
Downstream Analysis

Extracellular vesicles (EVs) have attracted great interest among researchers due to their role in cell-cell communication, disease diagnosis, and drug delivery. In spite of their potential in the medical field, there is no consensus on the best method for separating microvesicles from cell culture supernatant and complex biological fluids. Obtaining a good recovery yield and preserving physical characteristics is critical for the diagnostic and therapeutic use of EVs. The separation is made complex by the fact that blood and cell culture media, contain a large number of nanoparticles in the same size range. Methods that exploit immunoaffinity capture provide high purity samples and overcome the issues of currently used separation methods. However, the release of captured nanovesicles requires harsh conditions that hinder their use in certain types of downstream analysis. Herein, a novel capture and release approach for small extracellular vesicles (sEVs), based on DNAdirected immobilization of antiCD63 antibody is presented. The flexible DNAlinker increases the capture efficiency and allows releasing of EVs by exploiting the endonucleasic activity of DNAse I. This separation protocol works under mild conditions, enabling the release of intact vesicles that can be successfully analyzed by imaging techniques. In this article sEVs recovered from plasma were characterized by established techniques for EVs analysis including nanoparticle tracking and transmission electron microscopy.<br>

scholarly journals Extracellular vesicles in lung health, disease, and therapy

2019 ◽  
Vol 316 (6) ◽  
pp. L977-L989 ◽  
Author(s):  
Mark J. McVey ◽  
Mazharul Maishan ◽  
Kaj E. C. Blokland ◽  
Nathan Bartlett ◽  
Wolfgang M. Kuebler
Keyword(s):  
Lung Disease ◽  
Extracellular Vesicles ◽  
Biological Fluids ◽  
Biologically Active ◽  
Target Cells ◽  
Molecular Physiology ◽  
Diagnosis And Prognosis ◽  
Lung Physiology ◽  
Subcellular Membrane ◽  
Lung Health

Both physiological homeostasis and pathological disease processes in the lung typically result from complex, yet coordinated multicellular responses that are synchronized via paracrine and endocrine intercellular communication pathways. Of late, extracellular vesicles have emerged as important information shuttles that can coordinate and disseminate homeostatic and disease signals. In parallel, extracellular vesicles in biological fluids such as sputum, mucus, epithelial lining fluid, edema fluid, the pulmonary circulation, pleural fluid, and lymphatics have emerged as promising candidate biomarkers for diagnosis and prognosis in lung disease. Extracellular vesicles are small, subcellular, membrane-bound vesicles containing cargos from parent cells such as lipids, proteins, genetic information, or entire organelles. These cargos endow extracellular vesicles with biologically active information or functions by which they can reprogram their respective target cells. Recent studies show that extracellular vesicles found in lung-associated biological fluids play key roles as biomarkers and effectors of disease. Conversely, administration of naïve or engineered extracellular vesicles with homeostatic or reparative effects may provide a promising novel protective and regenerative strategy to treat lung disease. To highlight this rapidly developing field, the American Journal of Physiology-Lung Cellular and Molecular Physiology is now launching a special Call for Papers on extracellular vesicles in lung health, disease, and therapy. This review aims to set the stage for this call by introducing extracellular vesicles and their emerging roles in lung physiology and pathobiology.

scholarly journals Proteomic Approach for Searching for Universal, Tissue-Specific, and Line-Specific Markers of Extracellular Vesicles in Lung and Colorectal Adenocarcinoma Cell Lines

2020 ◽  
Vol 21 (18) ◽  
pp. 6601
Author(s):  
Svetlana Novikova ◽  
Natalia Shushkova ◽  
Tatiana Farafonova ◽  
Olga Tikhonova ◽  
Roman Kamyshinsky ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Extracellular Vesicles ◽  
Cancer Progression ◽  
Biological Fluids ◽  
Cancer Cell Lines ◽  
Label Free ◽  
Human Colon Cancer ◽  
Tissue Specific ◽  
Proteomic Approach

Tumor-derived extracellular vesicles (EVs), including exosomes, contain proteins that mirror the molecular landscape of producer cells. Being potentially detectible in biological fluids, EVs are of great interest for the screening of cancer biomarkers. To reveal universal, tissue-specific, and line-specific markers, we performed label-free mass spectrometric profiling of EVs originating from the human colon cancer cell lines Caco-2, HT29, and HCT-116, as well as from the lung cancer cell lines NCI-H23 and A549. A total of 651 proteins was identified in the EV samples using at least two peptides. These proteins were highly enriched in exosome markers. We found 11 universal, eight tissue-specific, and 29 line-specific markers, the levels of which were increased in EVs compared to the whole lysates. The EV proteins were involved in the EGFR, Rap1, integrin, and microRNA signaling associated with metastasis and cancer progression. An EV protein-based assay could be developed as a liquid biopsy tool.

scholarly journals Intraoperative visualization of the tumor microenvironment and quantification of extracellular vesicles by label-free nonlinear imaging

2018 ◽  
Vol 4 (12) ◽  
pp. eaau5603 ◽  
Author(s):  
Yi Sun ◽  
Sixian You ◽  
Haohua Tu ◽  
Darold R. Spillman ◽  
Eric J. Chaney ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Extracellular Vesicles ◽  
Cancer Progression ◽  
Spatial Information ◽  
Imaging System ◽  
Label Free ◽  
Breast Reduction Surgery ◽  
Diagnosis And Prognosis ◽  
Nonlinear Imaging

Characterization of the tumor microenvironment, including extracellular vesicles (EVs), is important for understanding cancer progression. EV studies have traditionally been performed on dissociated cells, lacking spatial information. Since the distribution of EVs in the tumor microenvironment is associated with cellular function, there is a strong need for visualizing EVs in freshly resected tissues. We intraoperatively imaged untreated human breast tissues using a custom nonlinear imaging system. Label-free optical contrasts of the tissue, correlated with histological findings, enabled point-of-procedure characterization of the tumor microenvironment. EV densities from 29 patients with breast cancer were found to increase with higher histologic grade and shorter tumor-to-margin distance and were significantly higher than those from 7 cancer-free patients undergoing breast reduction surgery. Acquisition and interpretation of these intraoperative images not only provide real-time visualization of the tumor microenvironment but also offer the potential to use EVs as a label-free biomarker for cancer diagnosis and prognosis.

scholarly journals Circulating Tumor Cells from Enumeration to Analysis: Current Challenges and Future Opportunities

Cancers ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2723
Author(s):  
Yu-Ping Yang ◽  
Teresa M. Giret ◽  
Richard J. Cote
Keyword(s):  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Ex Vivo ◽  
Detection Sensitivity ◽  
Clinical Utilization ◽  
Diagnosis And Prognosis ◽  
Early Cancer Diagnosis ◽  
Potential Marker ◽  
Potential Applications ◽  
Downstream Analysis

Circulating tumor cells (CTCs) have been recognized as a major contributor to distant metastasis. Their unique role as metastatic seeds renders them a potential marker in the circulation for early cancer diagnosis and prognosis as well as monitoring of therapeutic response. In the past decade, researchers mainly focused on the development of isolation techniques for improving the recovery rate and purity of CTCs. These developed techniques have significantly increased the detection sensitivity and enumeration accuracy of CTCs. Currently, significant efforts have been made toward comprehensive molecular characterization, ex vivo expansion of CTCs, and understanding the interactions between CTCs and their associated cells (e.g., immune cells and stromal cells) in the circulation. In this review, we briefly summarize existing CTC isolation technologies and specifically focus on advances in downstream analysis of CTCs and their potential applications in precision medicine. We also discuss the current challenges and future opportunities in their clinical utilization.

scholarly journals Temporal Quantitative Proteomics Analysis of Neuroblastoma Cells Treated with Bovine Milk-Derived Extracellular Vesicles Highlights the Anti-Proliferative Properties of Milk-Derived Extracellular Vesicles

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 750
Author(s):  
Pamali Fonseka ◽  
Taeyoung Kang ◽  
Sing Chee ◽  
Sai V. Chitti ◽  
Rahul Sanwlani ◽  
...  
Keyword(s):  
High Risk ◽  
Extracellular Vesicles ◽  
Quantitative Proteomics ◽  
Cell Metabolism ◽  
Bovine Milk ◽  
Neuroblastoma Cells ◽  
Treatment Strategies ◽  
Treatment Resistance ◽  
Label Free ◽  
The Impact

Neuroblastoma (NBL) is a pediatric cancer that accounts for 15% of childhood cancer mortality. Amplification of the oncogene N-Myc occurs in 20% of NBL patients and is considered high risk as it correlates with aggressiveness, treatment resistance and poor prognosis. Even though the treatment strategies have improved in the recent years, the survival rate of high-risk NBL patients remain poor. Hence, it is crucial to explore new therapeutic avenues to sensitise NBL. Recently, bovine milk-derived extracellular vesicles (MEVs) have been proposed to contain anti-cancer properties. However, the impact of MEVs on NBL cells is not understood. In this study, we characterised MEVs using Western blotting, NTA and TEM. Importantly, treatment of NBL cells with MEVs decreased the proliferation and increased the sensitivity of NBL cells to doxorubicin. Temporal label-free quantitative proteomics of NBL cells highlighted the depletion of proteins involved in cell metabolism, cell growth and Wnt signalling upon treatment with MEVs. Furthermore, proteins implicated in cellular senescence and apoptosis were enriched in NBL cells treated with MEVs. For the first time, this study highlights the temporal proteomic profile that occurs in cancer cells upon MEVs treatment.

Protein biomarkers in breast cancer-derived extracellular vesicles for use in liquid biopsies

2021 ◽  
Author(s):  
Dan Li ◽  
Wenjia Lai ◽  
Di Fan ◽  
Qiaojun Fang
Keyword(s):  
Breast Cancer ◽  
Early Diagnosis ◽  
Extracellular Vesicles ◽  
Liquid Biopsy ◽  
Breast Cancer Diagnosis ◽  
Extracellular Vesicle ◽  
Detection Methods ◽  
Protein Markers ◽  
Liquid Biopsies ◽  
Diagnosis And Prognosis

Breast cancer is the most common malignant disease in women worldwide. Early diagnosis and treatment can greatly improve the management of breast cancer. Liquid biopsies are becoming convenient detection methods for diagnosing and monitoring breast cancer due to their non-invasiveness and ability to provide real-time feedback. A range of liquid biopsy markers, including circulating tumor proteins, circulating tumor cells, and circulating tumor nucleic acids, have been implemented for breast cancer diagnosis and prognosis, with each having its own advantages and limitations. Circulating extracellular vesicles are messengers of intercellular communication that are packed with information from mother cells and are found in a wide variety of bodily fluids; thus, they are emerging as ideal candidates for liquid biopsy biomarkers. In this review, we summarize extracellular vesicle protein markers that can be potentially used for the early diagnosis and prognosis of breast cancer or determining its specific subtypes.

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