Biogenesis of extracellular vesicles (EV): exosomes, microvesicles, retrovirus-like vesicles, and apoptotic bodies

Akers, Johnny; Gonda, David; Kim, Ryan; Carter, Bob S.; Chen, Clark C.

doi:10.1007/s11060-013-1084-8

Cited by 1,126 publications

(1,084 citation statements)

References 124 publications

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“…EVs from urine can reveal urological diseases or tumours and their progression [2,4,5]. Despite the potential applications of EVs, their detection, isolation and characterisation from different bodily fluids are currently not optimal or standardised due to their small size and low refractive index (RI) [6, 7, 8, 9, 10]. …”

Section: Introductionmentioning

confidence: 99%

Ultrafiltration and size exclusion chromatography combined with asymmetrical‐flow field‐flow fractionation for the isolation and characterisation of extracellular vesicles from urine

Oeyen

Mol²,

Baggerman

et al. 2018

J of Extracellular Vesicle

113

107

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Extracellular vesicles (EVs) have a great potential in clinical applications. However, their isolation from different bodily fluids and their characterisation are currently not optimal or standardised. Here, we report the results of examining the performance of ultrafiltration combined with size exclusion chromatography (UF-SEC) to isolate EVs from urine. The results reveal that UF-SEC is an efficient method and provides high purity. Furthermore, we introduce asymmetrical-flow field-flow fractionation coupled with a UV detector and multi-angle light-scattering detector (AF4/UV-MALS) as a characterisation method and compare it with current methods. We demonstrate that AF4/UV-MALS is a straightforward and reproducible method for determining size, amount and purity of isolated urinary EVs.

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Section: Introductionmentioning

confidence: 99%

Ultrafiltration and size exclusion chromatography combined with asymmetrical‐flow field‐flow fractionation for the isolation and characterisation of extracellular vesicles from urine

Oeyen

Mol²,

Baggerman

et al. 2018

J of Extracellular Vesicle

113

107

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“…Microvesicles are typically larger than exosomes, having diameters of 100 nm to 2 μm, and are formed by outward budding of the plasma membrane [2]. Finally, apoptotic bodies are generated during apoptotic cell death and have diameters ranging from 1 μm to 5 μm [3]. …”

Section: Introductionmentioning

confidence: 99%

Proteome profiling of extracellular vesicles captured with the affinity peptide Vn96: comparison of Laemmli and TRIzol© protein‐extraction methods

Joy

Ayre

Chute

et al. 2018

J of Extracellular Vesicle

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Sample amount is often a limiting factor for multi-parametric analyses that encompass at least three areas of ‘-omics’ research: genomics, transcriptomics and proteomics. Limited sample amounts are also an important consideration when these multi-parametric analyses are performed on extracellular vesicles (EVs), as the amount of EVs (and EV cargo) that can be isolated is often very low. It is well understood that a monophasic solution of phenol and guanidine isothiocyanate (i.e. TRIzol©) can simultaneously isolate DNA, RNA and proteins from biological samples; however, it is most commonly used for the extraction of RNA. Validation of this reagent for the isolation of multiple classes of biological molecules from EVs would provide a widely applicable method for performing multi-parametric analyses of EV material. In this report, we describe a comparison of proteins identified from EVs processed with either TRIzol© or the conventional Laemmli buffer protein-extraction reagents. EVs were isolated from 3 mL of cell-culture supernatant derived from MCF-10A, MCF-7 and MDA-MB-231 cells using the Vn96 EV capture technology. For the TRIzol© extraction protocol, proteins were precipitated with acetone from the organic phase and then re-solubilized in a mixture of 8M urea, 0.2% SDS and 1 M Tris-HCl pH 6.8, followed by dilution in 5× loading buffer prior to fractionation with 1D SDS-PAGE. NanoLC-MS/MS of the trypsin-digested proteins was used to generate proteomic profiles from EV protein samples extracted with each method. Of the identified proteins, 57.7%, 69.2% and 57.0% were common to both extraction methods for EVs from MCF-10A, MCF-7 and MDA-MB-231, respectively. Our results suggest that TRIzol© extraction of proteins from EVs has significant equivalence to the traditional Laemmli method. The advantage of using TRIzol© reagent is the ability to accumulate multi-parametric data (e.g., RNA and protein profiles) on the same limited EV sample while minimizing sample preparation and processing time.

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“…Exosomes have long been implicated in immunity due to their importance in the communication between immune cells and cancer cells. 19 Additionally, exosomes have previously been shown to alter the tumor microenvironment by transferring important cellular cargo, such as cytokines and growth factors. 19 We therefore hypothesized that PC-Exo downregulate HLA-DR expression on CD14 C monocytes.…”

Section: Exosomes Are Abundantly Secreted In Pc and Readily Enter Monmentioning

confidence: 99%

Immunosuppressive CD14⁺HLA-DR^lo/negmonocytes are elevated in pancreatic cancer and “primed” by tumor-derived exosomes

et al. 2016

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Immunological strategies to treat pancreatic cancer offer new therapeutic approaches to improve patient outcomes. Understanding alterations in the immune systems of pancreatic cancer patients will likely lead to advances in immunotherapy for the disease. We profiled peripheral blood leukocytes from pancreatic cancer patients (n D 22) and age-matched controls (n D 20) using flow cytometry. Immune profiling of pancreatic cancer patients identified phenotypic changes in various immune cell populations, including a population of immunosuppressive monocytes (CD14 C HLA-DR lo/neg ), which were shown to be increased in these patients. There was a correlation between the levels of CD14 C monocytes and the levels of CD14 C HLA-DR lo/neg monocytes in peripheral blood from pancreatic cancer patients. HLA-DR downregulation of monocytes was shown to occur through pancreatic cancer-derived exosome interactions with monocytes. In an in vitro model, exosomes from patient-derived xenograft cell lines and patient plasma decreased HLA-DR expression on CD14 C monocytes. Additionally, tumor-derived exosomes caused immune suppression in monocytes through altered STAT3 signaling, induction of arginase expression, and reactive oxygen species. These findings provide novel insights into the mechanisms that govern immunosuppression in pancreatic cancer. Understanding monocyte-exosome interactions could lead to novel immunotherapies for this disease.

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Biogenesis of extracellular vesicles (EV): exosomes, microvesicles, retrovirus-like vesicles, and apoptotic bodies

Cited by 1,126 publications

References 124 publications

Ultrafiltration and size exclusion chromatography combined with asymmetrical‐flow field‐flow fractionation for the isolation and characterisation of extracellular vesicles from urine

Ultrafiltration and size exclusion chromatography combined with asymmetrical‐flow field‐flow fractionation for the isolation and characterisation of extracellular vesicles from urine

Proteome profiling of extracellular vesicles captured with the affinity peptide Vn96: comparison of Laemmli and TRIzol© protein‐extraction methods

Immunosuppressive CD14⁺HLA-DR^lo/negmonocytes are elevated in pancreatic cancer and “primed” by tumor-derived exosomes

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Biogenesis of extracellular vesicles (EV): exosomes, microvesicles, retrovirus-like vesicles, and apoptotic bodies

Cited by 1,126 publications

References 124 publications

Ultrafiltration and size exclusion chromatography combined with asymmetrical‐flow field‐flow fractionation for the isolation and characterisation of extracellular vesicles from urine

Ultrafiltration and size exclusion chromatography combined with asymmetrical‐flow field‐flow fractionation for the isolation and characterisation of extracellular vesicles from urine

Proteome profiling of extracellular vesicles captured with the affinity peptide Vn96: comparison of Laemmli and TRIzol© protein‐extraction methods

Immunosuppressive CD14+HLA-DRlo/negmonocytes are elevated in pancreatic cancer and “primed” by tumor-derived exosomes

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Immunosuppressive CD14⁺HLA-DR^lo/negmonocytes are elevated in pancreatic cancer and “primed” by tumor-derived exosomes