Optimization of small extracellular vesicle isolation from expressed prostatic secretions in urine for in‐depth proteomic analysis

Correll, Vanessa L; Otto, Joseph J.; Risi, Cristina; Main, Brian; Boutros, Paul C.; Kislinger, Thomas; Galkin, Vitold E.; Nyalwidhe, Julius O.; Semmes, O. John; Yang, Lifang

doi:10.1002/jev2.12184

Cited by 29 publications

(29 citation statements)

References 87 publications

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“…For clinical translation, EV assays will require high sensitivity and reliability while remaining cost-effective with highthroughput capabilities. 28,29,32,[37][38][39][40][41] We developed a sensitive and reproducible method for identifying exosomes and microvesicles and their molecular features to address these limitations. The immuno-magnetic beads allow for higher sample purity by increasing the wash capacity by substantially decreasing the wash time compared to ultracentrifugation.…”

Section: Discussionmentioning

confidence: 99%

“…Annexin A1 and ARF6 are specific markers for microvesicles shed from the plasma membrane and CD63, CD81, and CD9 are exosome markers confirmed in several studies. [27][28][29][30][31][32] Our first aim in this study is to optimize the immuno-magnetic affinity assay for the detection of HCC-specific exosomes using a small volume of serum. This EV-based immune-magnetic affinity assay described here is reliable, and reproducible with a rapid turnaround time appropriate for biomarker testing in a clinical laboratory.…”

Section: Introductionmentioning

confidence: 99%

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Impaired Autophagy Response in Hepatocellular Carcinomas Enriches Glypican-3 in Exosomes, Not in the Microvesicles

Köksal¹,

Thevenot²,

Aydın³

et al. 2022

JHC

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Background and Aim HCC development in liver cirrhosis is associated with impaired autophagy leading to increased production of extracellular vesicles (EVs) including exosomes and microvesicles. The goal of the study is to determine which of these particles is primarily involved in releasing of HCC-specific biomarker glypican-3 (GPC3) when autophagy is impaired. Methods Streptavidin-coated magnetic beads were coupled with either biotinylated CD63 or Annexin A1 antibodies. Coupled beads were incubated with EVs isolated from either HCC culture or serum. EVs captured by immuno-magnetic beads were then stained with FITC or PE fluorescent-conjugated antibodies targeting exosomes (CD81), and microvesicles (ARF6). The percentage of GPC3 enrichment in the microvesicles and exosomes was quantified by flow cytometry. The impact of autophagy modulation on GPC3 enrichment in exosomes and microvesicles was assessed by treating cells with Torin 1 and Bafilomycin A1. For clinical validation, GPC3 content was quantified in microvesicles, and exosomes were isolated from the serum of patients with a recent HCC diagnosis. Results The immune-magnetic bead assay distinguishes membrane-derived microvesicles from endosome-derived exosomes. The GPC3 expression was only seen in the CD63 beads group but not in the Annexin A1 beads group, confirming that in HCC, GPC3 is preferentially released through exosomes. Furthermore, we found that autophagy induction by Torin1 decreased GPC3-positive exosome secretion and decreased microvesicle release. Conversely, autophagy inhibition by Bafilomycin A1 increased the secretion of GPC3-positive exosomes. Serum analysis showed CD81+ve EVs were detected in exosomes and ARF6+ve vesicles were detected in microvesicles, suggesting that immunoaffinity assay is specific. The exosomal GPC3 enrichment was confirmed in isolated EVs from the serum of patients with HCC. The frequency of GPC3-positive exosomes was higher in patients with HCC (12.4%) compared to exosomes isolated from non-cirrhotic and healthy controls (3.7% and 1.3% respectively, p<0.001). Conclusion Our results show that GPC3 is enriched in the endolysosomal compartment and released in exosome fractions when autophagy is impaired.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Impaired Autophagy Response in Hepatocellular Carcinomas Enriches Glypican-3 in Exosomes, Not in the Microvesicles

Köksal¹,

Thevenot²,

Aydın³

et al. 2022

JHC

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“…20) in which Conventional differential UC is a heavy workload due to requiring consecutive centrifugation steps (Ref. 106). Both small and large size EVs can be isolated using differential UC (Ref.…”

Section: Protocols For Isolation and Detection Of Extracellular Vesiclesmentioning

confidence: 99%

“…A suggested approach for effective recovery of THP-entrapped urinary EVs and reducing contamination with proteins is the incorporation of alkaline wash and THP polymer reduction (Ref. 106).…”

Section: Protocols For Isolation and Detection Of Extracellular Vesiclesmentioning

confidence: 99%

Extracellular vesicle isolation, purification and evaluation in cancer diagnosis

Mortezaee

Majidpoor

Fathi

2022

Expert Rev. Mol. Med.

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Strategies for non-invasive biomarker discovery in early detection of cancer are an urgent need. Extracellular vesicles (EVs) have generated increasing attention from the scientific community and are under intensive investigations due to their unique biological profiles and their non-invasive nature. EVs are membrane-enclosed vesicles with variable sizes and function. Such vesicles are actively secreted from multiple cell types and are considered as key vehicles for inter-cellular communications and signalling. The stability and potential to easily cross biological barriers enable EVs for exerting durable effects on target cells. These along with easy access to such vesicles, the consistent secretion from tumour during all stages of tumorigenesis and their content providing a reservoir of molecules as well as mirroring the identity of the cell of origin are virtues that have made EVs appealing to be assessed in liquid biopsy approaches and for using as a promising resource of biomarkers in cancer diagnosis and therapy and monitoring targeted cancer therapy. Early detection of EVs will guide time-scheduled personalised therapy. Surveying reliable and sensitive methods for rapid isolation of EVs from biofluids, the purity of isolated vesicles and their molecular profiling and marker specification for clinical translation in patients with cancer are issues in the area and the hot topics of many recent studies. Here, the focus is over methods for EV isolation and stratification for digging more information about liquid biopsy-based diagnosis. Extending knowledge regarding EV-based strategies is a key to validate independent patient follow-up for cancer diagnosis at early stages and inspecting the efficacy of therapeutics.

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“…Apoptotic-derived EVs or apoptotic bodies (>1000 nm in diameter) are generated from plasma membrane blebbing during cell apoptosis [ 18 , 19 , 20 ]. Exo and MVs are the main focus of the EV-based method development for several cancers, i.e., head and neck [ 16 ], brain [ 21 ], lung [ 22 ], colon [ 23 ], bladder [ 24 ], endometrial [ 25 ], ovarian [ 26 ] and prostate [ 27 ]. To the best of our knowledge, the EV-based method has never been developed for detecting the MYCN status of pediatric NB.…”

Section: Introductionmentioning

confidence: 99%

Extracellular Vesicle-Based Method for Detecting MYCN Amplification Status of Pediatric Neuroblastoma

Panachan

Rojsirikulchai

Pongsakul

et al. 2022

Cancers

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MYCN amplification is the strongest predictor of high-risk neuroblastoma (NB). The standard procedure to detect MYCN status requires invasive procedures. Extracellular vesicles (EVs) contain molecular signatures of originated cells, present in biofluids, and serve as an invaluable source for cancer liquid biopsies. This study aimed to establish an EV-based method to detect the MYCN status of NB. Two EV subtypes, i.e., microvesicles (MVs) and exosomes, were sequentially isolated from the culture supernatant by step-wise centrifugation, ultrafiltration, and size-exclusion chromatography. Quantitative RT-PCR was performed to detect MYCN mRNA. As a result, MYCN mRNA was detectable in the MVs, but not exosomes, of MYCN-amplified NB cells. MYCN mRNA-containing MVs (MYCN-MV) were successfully detected in three distinct MYCN-amplified NB cell lines but absent in three MYCN non-amplification cells. The simulated samples were prepared by pulsing MVs into human serum. MYCN–MV detection in the simulated samples showed a less interfering effect from the human blood matrix. Validation using clinical specimens (2 mL bone marrow plasma) obtained from patients at various disease stages showed a promising result. Five out of six specimens of MYCN-amplified patients showed positive results, while there were no false positives in four plasma samples of the MYCN non-amplification group. This study communicated a novel EV-based method for detecting the MYCN status of pediatric NB based on MYCN mRNA contents in MVs. Future studies should be pursued in a prospective cohort to determine its true diagnostic performance.

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Optimization of small extracellular vesicle isolation from expressed prostatic secretions in urine for in‐depth proteomic analysis

Cited by 29 publications

References 87 publications

Impaired Autophagy Response in Hepatocellular Carcinomas Enriches Glypican-3 in Exosomes, Not in the Microvesicles

Impaired Autophagy Response in Hepatocellular Carcinomas Enriches Glypican-3 in Exosomes, Not in the Microvesicles

Extracellular vesicle isolation, purification and evaluation in cancer diagnosis

Extracellular Vesicle-Based Method for Detecting MYCN Amplification Status of Pediatric Neuroblastoma

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