Imaging of surface microdomains on individual extracellular vesicles in 3‐D

McNamara, Ryan P.; Zhou, Yijun; Eason, Anthony; Landis, Justin T.; Chambers, Meredith G.; Willcox, Smaranda; Peterson, Tiffany A.; Schouest, Blake; Maness, Nicholas J.; MacLean, Andrew G.; Costantini, Lindsey M.; Griffith, Jack D.; Dittmer, Dirk P.

doi:10.1002/jev2.12191

Cited by 42 publications

(37 citation statements)

References 61 publications

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“…More recently, there have been efforts to develop single-vesicle analysis tools. Some single-vesicle techniques have been used for characterizing EVs, that is, nanoparticle tracking analysis, [16] high-resolution flow cytometry, [15a,17] super-resolution microscopy, [6,18] immunosequencing, [5b] and droplet PCR. [7] Some of these techniques can directly visualize EVs and provide information concerning the biomolecular composition of the surface, size, and shape of single EVs, while others rely on known markers for the quantification of specific targets in individual EVs or small identical subpopulations.…”

Section: Discussionmentioning

confidence: 99%

Transcriptomic Features in a Single Extracellular Vesicle via Single‐Cell RNA Sequencing

Luo

Chen

Qiu³

et al. 2022

Small Methods

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body fluids. EVs are highly heterogenous in size, ranging from 50 to 1000 nm in diameter. [1] Furthermore, they play key roles in the cell microenvironment and intercellular communication by transferring biological molecules including proteins, lipids, and nucleic acids. [2] Because of the relatively stable state in body fluids, EVs are recognized as potential noninvasive biomarkers in disease diagnostics and mediators in drug delivery. [3] EVs carry the characteristics of their parental cells; therefore, they are highly heterogeneous in molecular cargos. [4] Thus, EVs demonstrate a marked diversity, even in homogeneous or monoclonal cell populations. [5] The different molecular cargos in EVs are related to their function and effectiveness as disease biomarkers. Therefore, it is important to investigate the molecular cargos and their heterogeneity at the single-EV level.Traditional analytical methods to examine the molecular cargos of EVs, such as western blotting, polymerase chain reaction (PCR), and RNA-seq, usually rely on bulk assays to determine the overall molecular content from an entire EV population. The molecular cargo of an individual EV and the heterogeneity within an EV population is lost in bulk-level analyses. Recently, several new analytical methods, such as total internal reflection fluorescence microscopy (TIRFM), [6] digital PCR, [7] microarraybased chip, [8] and high-sensitivity flow cytometry combined with qPCR, [9] have been used to quantify a specific miRNA or mRNA in an single-EV or small identical subpopulations. However, a high-throughput analysis for the RNA profile of a single-EV is still lacking. Therefore, even though many studies have investigated the RNA cargos in bulk EVs, we still do not know how many genes or RNAs there are in a single-EV. This is a basic issue that needs to be addressed. Investigations of transcriptome for a single-EV are of great value to better define EV features, EV subpopulations, and clinical applications.The development of single-cell RNA sequencing (scRNA-seq) technologies, such as 10x Genomics Chromium, enable the detection of gene expression at the single-cell level, as well as the detection of cell heterogeneity and cell types. [10] It has been reported that EV populations are more heterogeneous than their parental cells. [11] Therefore, it is very important to build an experimental and analytical protocol to perform single-EV sequencing to reveal the transcriptome features of individual EVs.Although many studies have investigated functional molecules in extracellular vesicles (EVs), the exact number of ribonucleic acid molecules in a single-EV is unknown. Therefore, it is critical to explore the transcriptomic features and heterogeneity at the level of a single-EV. Here, using the 10x Genomics platform, the RNA cargos are profiled in single EVs derived from human K562 and mesenchymal stem cells. The key steps are labeling intact EVs using calcein-AM, detecting the EV concentration via flow cytometry, and using the CB2 algorithm with adaptive thres...

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Section: Discussionmentioning

confidence: 99%

Transcriptomic Features in a Single Extracellular Vesicle via Single‐Cell RNA Sequencing

Luo

Chen

Qiu³

et al. 2022

Small Methods

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“…A new class of photoswitching polymer dots has been used to map of the surface proteins on sEVs [18]. SMLM, including PALM [19] and STORM [20,21], offers high spatial resolution in the range of 20-50 nm, but are low throughput typically requiring several minutes for collection and reconstruction of multiple single molecule images.…”

Section: Introductionmentioning

confidence: 99%

Upconversion nanoparticles for super-resolution quantification of single small extracellular vesicles

Huang

Liu

Wang

et al. 2022

eLight

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Although small EVs (sEVs) have been used widely as biomarkers in disease diagnosis, their heterogeneity at single EV level has rarely been revealed. This is because high-resolution characterization of sEV presents a major challenge, as their sizes are below the optical diffraction limit. Here, we report that upconversion nanoparticles (UCNPs) can be used for super-resolution profiling the molecular heterogeneity of sEVs. We show that Er3+-doped UCNPs has better brightness and Tm3+-doped UCNPs resulting in better resolution beyond diffraction limit. Through an orthogonal experimental design, the specific targeting of UCNPs to the tumour epitope on single EV has been cross validated, resulting in the Pearson’s R-value of 0.83 for large EVs and ~ 65% co-localization double-positive spots for sEVs. Furthermore, super-resolution nanoscopy can distinguish adjacent UCNPs on single sEV with a resolution of as high as 41.9 nm. When decreasing the size of UCNPs from 40 to 27 nm and 18 nm, we observed that the maximum UCNPs number on single sEV increased from 3 to 9 and 21, respectively. This work suggests the great potentials of UCNPs approach “digitally” quantify the surface antigens on single EVs, therefore providing a solution to monitor the EV heterogeneity changes along with the tumour progression progress.

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“…An application note (https://oni.bio/oni-articles) suggested the possibility of visualizing generic EV DNA cargo (not a specific gene) by direct stochastic optical reconstruction microscopy (dSTORM, ONI). This elegant technique, available only a few centres, requires tedious set up and troubleshooting that hinders ease of applicability, which may explain the small number of publications to date that use this technology (Brunner et al, 2021;Mcnamara et al, 2022). Recently Yu et al summarized conventional and novel technologies for EVs isolation, characterization, and content detection, particularly in the context of EVs in liquid biopsy.…”

Section:  Discussionmentioning

confidence: 99%

In situ hybridization to detect DNA amplification in extracellular vesicles

Casadei

Sarchet

Faria

et al. 2022

J of Extracellular Vesicle

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EVs have emerged as an important component in tumour initiation, progression and metastasis. Although notable progresses have been made, the detection of EV cargoes remain significantly challenging for researchers to practically use; faster and more convenient methods are required to validate the EV cargoes, especially as biomarkers. Here we show, the possibility of examining embedded EVs as substrates to be used for detecting DNA amplification through ultrasensitive in situ hybridization (ISH). This methodology allows the visualization of DNA targets in a more direct manner, without time consuming optimization steps or particular expertise. Additionally, formalin‐fixed paraffin‐embedded (FFPE) blocks of EVs allows long‐term preservation of samples, permitting future studies. We report here: (i) the successful isolation of EVs from liposarcoma tissues; (ii) the EV embedding in FFPE blocks (iii) the successful selective, specific ultrasensitive ISH examination of EVs derived from tissues, cell line, and sera; (iv) and the detection of MDM2 DNA amplification in EVs from liposarcoma tissues, cell lines and sera. Ultrasensitive ISH on EVs would enable cargo study while the application of ISH to serum EVs, could represent a possible novel methodology for diagnostic confirmation. Modification of probes may enable researchers to detect targets and specific DNA alterations directly in tumour EVs, thereby facilitating detection, diagnosis, and improved understanding of tumour biology relevant to many cancer types.

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Imaging of surface microdomains on individual extracellular vesicles in 3‐D

Cited by 42 publications

References 61 publications

Transcriptomic Features in a Single Extracellular Vesicle via Single‐Cell RNA Sequencing

Transcriptomic Features in a Single Extracellular Vesicle via Single‐Cell RNA Sequencing

Upconversion nanoparticles for super-resolution quantification of single small extracellular vesicles

In situ hybridization to detect DNA amplification in extracellular vesicles

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