Protein Profiling and Sizing of Extracellular Vesicles from Colorectal Cancer Patients <i>via</i> Flow Cytometry

Tian, Ye; Ma, Ling; Gong, Manfei; Su, Guoqiang; Zhu, Shaobin; Zhang, Wenqiang; Wang, Shuo; Li, Zhibin; Chen, Chaoxiang; Li, Lihong; Wu, Lina; Yan, Xiaomei

doi:10.1021/acsnano.7b07782

Cited by 375 publications

(396 citation statements)

References 45 publications

(93 reference statements)

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“…The Yan laboratory employed high‐sensitivity flow cytometry (HSFCM), which allows detection of single EVs (down to 40 nm size), to compare and characterize protein profiles of EVs isolated from normal colorectal cells and colorectal cancer cells, as well as plasma EVs from colorectal cancer patients . HSFCM identified 85–90% of particles isolated from cultured cells as EVs, while only 70% of particles isolated from platelet‐free patient plasma corresponded to vesicles (probably due to large number of contaminating lipoprotein particles in plasma).…”

Section: Emerging Approaches To Study Ev Heterogeneitymentioning

confidence: 99%

Protein Composition Reflects Extracellular Vesicle Heterogeneity

et al. 2019

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Extracellular vesicles (EVs) are membrane‐enclosed particles that are released by virtually all cells from all living organisms. EVs shuttle biologically active cargo including protein, RNA, and DNA between cells. When shed by cancer cells, they function as potent intercellular messangers with important functional consequences. Cells produce a diverse spectrum of EVs, spanning from small vesicles of 40–150 nm in diameter, to large vesicles up to 10 μm in diameter. While this diversity was initially considered to be purely based on size, it is becoming evident that different classes of EVs, and different populations within one EV class may harbor distinct molecular cargo and play specific functions. Furthermore, there are considerable cell type‐dependent differences in the cargo and function of shed EVs. This review focuses on the most recent proteomic studies that have attempted to capture the EV heterogeneity by directly comparing the protein composition of different EV classes and EV populations derived from the same cell source. Recent studies comparing protein composition of the same EV class(es) derived from different cell types are also summarized. Emerging approaches to study EV heterogeneity and their important implications for future studies are also discussed.

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Section: Emerging Approaches To Study Ev Heterogeneitymentioning

confidence: 99%

Protein Composition Reflects Extracellular Vesicle Heterogeneity

et al. 2019

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“…To determine if this human EVmarking reagent could also identify intact EVs we incubated our EV fraction with commercial Alexa Fluorconjugated anti-CD63 (BD Biosciences San Jose, CA; Cat# 561983). This method has been used in the past in FACS analysis of human EVs 43,44,45,46 . About a third of the total events between 100 and 300 nm were anti-CD63 positive and these positive events disappeared with Triton-X 100 treatment, indicating that this anti-CD63 reagent can also be used to bind C. elegans EVs (FIG 4D).…”

Section: Canonical Human Ev Transmembrane Proteins Can Be Used To Idementioning

confidence: 99%

Isolation and characterization of extracellular vesicles from Caenorhabditis elegans for multi-omic analysis

et al. 2018

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Cells from bacteria to human release vesicles into their extracellular environment. These extracellular vesicles (EVs) contain multiple classes of molecules, including nucleic acids, proteins, and lipids. The isolation and analysis of EV cargos from mammalian cell culture and liquid biopsy samples has become a powerful approach for uncovering the messages that are packaged into these organelles. However, this approach has not been tenable in invertebrate model systems due to lack of sufficient amounts of pure EVs. Here we report a robust and reproducible procedure to isolate EVs from Caenorhabditis elegans with yields similar to those obtained from human cell culture. Through nanoparticle tracking, transmission electron microscopy, flow cytometry, mass spectrometry, RNAseq, and immunoaffinity analysis we provide the first ever detailed characterization of C.elegans EV composition and demonstrate that C. elegans EVs share fundamentally similar properties with their mammalian counterparts. These include vesicle size, enrichment for lipid rafts, and similar types of RNA and protein cargos. This ability of isolate pure EVs on a scale amenable to multiple types of downstream analyses permits, multi-omics characterization of EV cargos in an invertebrate model system. BACKGROUNDThe cellular secretion of small membrane-bound extracellular vesicles (EVs) into the external environment is an ancient capacity conserved throughout evolution 1,2,3 . EVs range in size from 30-1000 nm in diameter and can be internalized into recipient cells via endocytosis or membrane fusion. There is growing recognition that EVs may play important roles in facilitating intercellular communication through transferring protein, lipid, and genetic cargos 4,5 . The content of EVs are influenced by the physiological state of the cells and are thought to play critical roles in diverse cellular processes as well as multiple types of pathological conditions including cancer, immunity, and neurodegenerative diseases.Many studies have characterized the composition of mammalian EVs. Such EVs are highly enriched in the lipid raft species cholesterol, and sphingomyelin, and in proteins that associate with lipid rafts, including glycosylphosphatidylinositol-anchored (GPI) proteins 6,7,8 . The two main types of EVs studied so far are exosomes, which release from the endosomal network and microvesicles which bud directly from the plasma membrane 9,10 . Mammalian exosomes commonly contain membrane proteins such as CD9, CD63, and CD81, as well as lysosomal and endosomal-marking proteins, and various amounts of extracellular matrix proteins while are largely free of nuclear proteins 11 . Microvesicles have less defined protein markers but may contain proteins of mitochondria and endoplasmic reticulum origin 11 . However, the methods utilized to purify EVs do not separate these types of vesicles, so it is currently unclear how to definitively distinguish the cargos from different types of EVs 12 . EVs from diverse species, including humans, are known to carry RNA ...

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“…[9] Similarly,i tc ould be used to study the heterogeneous EVs.H owever,t he sizes of EVs fall well below the detection limit of conventional flow cytometers,m aking it impossible to do single-EV analysis without significant instrumentation development. [10] Herein, we report the first single-EV flow cytometry analysis (FCA) in conventional flow cytometers enabled by target-initiated engineering (TIE) of DNAnanostructures on individual EVs. This technique employs ac onformation-switchable DNA probe to bind to the EV surface marker,w hich triggers the engineering of aD NA nanostructure by hybridization chain reaction (HCR).…”

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confidence: 99%

A Single Extracellular Vesicle (EV) Flow Cytometry Approach to Reveal EV Heterogeneity

et al. 2018

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Extracellular vesicles (EVs) actively participate in intercellular communication and pathological processes. Studying the molecular signatures of EVs is the key to reveal their biological functions and clinical values, which, however, is greatly hindered by their sub-100-nm dimensions, the low quantities of biomolecules each EV carries, and the large population heterogeneity. Here, we report the single-EV Flow Cytometry Analysis technique that realizes single EV counting and phenotyping in a conventional flow cytometer for the first time, enabled by Target-Initiated Engineering (TIE) of DNA nanostructures on each EV. By illuminating multiple markers on single EVs, we reveal statistically significant differences among the molecular signatures of EVs originated from several breast cancer cell lines, and successfully recognize the cancer cell-derived EVs among the heterogeneous EV populations. Thus, our approach holds great potential for various biological and biomedical applications.

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Protein Profiling and Sizing of Extracellular Vesicles from Colorectal Cancer Patients via Flow Cytometry

Cited by 375 publications

References 45 publications

Protein Composition Reflects Extracellular Vesicle Heterogeneity

Protein Composition Reflects Extracellular Vesicle Heterogeneity

Isolation and characterization of extracellular vesicles from Caenorhabditis elegans for multi-omic analysis

A Single Extracellular Vesicle (EV) Flow Cytometry Approach to Reveal EV Heterogeneity

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