Multiplexed Profiling of Single Extracellular Vesicles

Lee, Kyungheon; Fraser, Kyle; Ghaddar, Bassel; Yang, Katy; Kim, Eunha; Balaj, Leonora; Chiocca, E. Antonio; Breakefield, Xandra O.; Lee, Hakho; Weissleder, Ralph

doi:10.1021/acsnano.7b07060

Cited by 300 publications

(353 citation statements)

References 56 publications

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“…82 In this approach, EVs are immobilized inside a microfluidic chamber, immuno-stained, and imaged. With the vesicles immobilized on the chip surface, the achievable signal-to-noise ratio from each vesicle is generally much higher than that when the vesicles are free-floating in solution or under flow condition.…”

Section: Physical Characterizationmentioning

confidence: 99%

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New Technologies for Analysis of Extracellular Vesicles

et al. 2018

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Extracellular vesicles (EVs) are diverse, nanoscale membrane vesicles actively released by cells. Similar sized vesicles can be further classified (e.g., exosomes, microvesicles) based on their biogenesis, size and biophysical properties. Although initially thought to be cellular debris, and thus under-appreciated, EVs are now increasingly recognized as important vehicles of intercellular communication and circulating biomarkers for disease diagnoses and prognosis. Despite their clinical potential, the lack of sensitive preparatory and analytical technologies for EVs poses a barrier to clinical translation. New analytical platforms including molecular ones are thus actively being developed to address these challenges. Recent advances in the field are expected to have far-reaching impact in both basic and translational studies. This article aims to present a comprehensive and critical overview of emerging analytical technologies for EV detection, and their clinical applications.

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Section: Physical Characterizationmentioning

confidence: 99%

“…EVs from Gli36-WT and Gli36-IDH1R132 cells lines clustered similarly, whereas EVs from Gli36-EGFRvIII cells showed distinct clusters. Reprinted with permission from Ref 82. Copyright 2017 American Chemical Society.…”

Section: Figurementioning

confidence: 99%

New Technologies for Analysis of Extracellular Vesicles

et al. 2018

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“…[5] However, subtle molecular differences at the single EV level may yield significant variation in EV biological functions, [6] and the highly heterogeneous nature of EV population demands the development of techniques capable of profiling individual EVs. [4] Most recently, single EV counting with a nanochip [7] and imaging single vesicles with advanced fluorescence microscopy [8] have been reported, and showed that EVs from different cell of origin can carry distinct surface markers mimicking their parent cells. Still, they require EV immobilization steps, limiting the down-stream investigations on EV functions and biogenesis.…”

mentioning

confidence: 99%

“…If the signals from both markers can be acquired simultaneously, the flow plots should directly show the presence of EV sub-populations in a heterogeneous EV mixture without the help of statistical tools, which is necessary for single vesicle analysis using microscopic methods. [8] Herein, we designed a “dual hybridization cascade system” to simultaneously amplify signals from HER2 and CD63 on a single EV by two separate hybridization cascade reactions (Table S1). In this system, CD63 signal was still derived from the streptavidin-conjugated QDs, but HER2 signal was from the Alexa660 labelled DNA tag hybridized with the overhang in H3 on the HCR product (Fig.…”

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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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“…As an example, antibodies against the tetraspanin CD63 have been widely applied to exosome isolation from the plasma of patients with ovarian cancer, breast cancer, and glioblastoma . However, anti‐CD63 is not a specific biomarker for any one cancer, and its expression is known to vary depending on the type of cancer . Recent studies using clinical samples showed that only 69.56% of lung cancer patients have CD63 positive exosomes with comparably low absolute expression level compared to other exosomal markers .…”

Section: Introductionmentioning

confidence: 99%

Isolation and Profiling of Circulating Tumor‐Associated Exosomes Using Extracellular Vesicular Lipid–Protein Binding Affinity Based Microfluidic Device

Kang

Purcell

Palacios-Rolston

et al. 2019

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Extracellular vesicles (EVs) are emerging as a potential diagnostic test for cancer. Owing to the recent advances in microfluidics, on‐chip EV isolation is showing promise with respect to improved recovery rates, smaller necessary sample volumes, and shorter processing times than ultracentrifugation. Immunoaffinity‐based microfluidic EV isolation using anti‐CD63 is widely used; however, anti‐CD63 is not specific to cancer‐EVs, and some cancers secrete EVs with low expression of CD63. Alternatively, phosphatidylserine (PS), usually expressed in the inner leaflet of the lipid bilayer of the cells, is shown to be expressed on the outer surface of cancer‐associated EVs. A new exosome isolation microfluidic device (newExoChip), conjugated with a PS‐specific protein, to isolate cancer‐associated exosomes from plasma, is presented. The device achieves 90% capture efficiency for cancer cell exosomes compared to 38% for healthy exosomes and isolates 35% more A549‐derived exosomes than an anti‐CD63‐conjugated device. Immobilized exosomes are then easily released using Ca2+ chelation. The recovered exosomes from clinical samples are characterized by electron microscopy and western‐blot analysis, revealing exosomal shapes and exosomal protein expressions. The newExoChip facilitates the isolation of a specific subset of exosomes, allowing the exploration of the undiscovered roles of exosomes in cancer progression and metastasis.

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Multiplexed Profiling of Single Extracellular Vesicles

Cited by 300 publications

References 56 publications

New Technologies for Analysis of Extracellular Vesicles

New Technologies for Analysis of Extracellular Vesicles

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

Isolation and Profiling of Circulating Tumor‐Associated Exosomes Using Extracellular Vesicular Lipid–Protein Binding Affinity Based Microfluidic Device

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