Urinary extracellular vesicle biomarkers in urological cancers: From discovery towards clinical implementation

Dhondt, Bert; Deun, Jan Van; Vermaerke, Silke; Marco, Ario de; Lumen, Nicolaas; Wever, Olivier De; Hendrix, An

doi:10.1016/j.biocel.2018.04.009

Cited by 51 publications

(48 citation statements)

References 237 publications

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“…Urine contains a large number of EVs, which originate from different cell types and have proven to be remarkably stable [ 23 , 52 ]. Particularly small uEVs carry a majority of urinary miRNAs and assure an even greater protection of urinary miRNAs compared to cells [ 20 , 22 , 69 , 70 ].…”

Section: Discussionmentioning

confidence: 99%

“…By now, those small EVs were already investigated by numerous research groups from various biological fluids, with the major focus on blood derived compartments [ [9] , [10] , [11] , [12] ], saliva [ 13 ], milk [ 14 , 15 ], cerebrospinal fluid [ 16 ], semen [ 17 ], ascites [ 18 ], and urine [ 19 , 20 ]. Previous studies focusing on urine demonstrated intense uEV secretion from all parts of the nephron and collecting duct, whose content is characteristic for the urinary tract, including kidney, ureter and bladder [ [21] , [22] , [23] ]. Since Valadi et al discovered in 2007 that particularly small EVs contain functional RNAs including microRNA (miRNA) [ 24 ], small EV-miRNA has come to the fore.…”

Section: Introductionmentioning

confidence: 99%

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Comparing small urinary extracellular vesicle purification methods with a view to RNA sequencing—Enabling robust and non-invasive biomarker research

Mussack

Wittmann²,

Pfaffl

2019

Biomolecular Detection and Quantification

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Small extracellular vesicles (EVs) are 50–200 nm sized mediators in intercellular communication that reflect both physiological and pathophysiological changes of their parental cells. Thus, EVs hold great potential for biomarker detection. However, reliable purification methods for the downstream screening of the microRNA (miRNA) cargo carried within urinary EVs by small RNA sequencing have yet to be established. To address this knowledge gap, RNA extracted from human urinary EVs obtained by five different urinary EV purification methods (spin column chromatography, immunoaffinity, membrane affinity, precipitation and ultracentrifugation combined with density gradient) was analyzed by small RNA sequencing. Urinary EVs were further characterized by nanoparticle tracking analysis, Western blot analysis and transmission electron microscopy. Comprehensive EV characterization established significant method-dependent differences in size and concentration as well as variances in protein composition of isolated vesicles. Even though all purification methods captured enough total RNA to allow small RNA sequencing, method-dependent differences were also observed with respect to library sizes, mapping distributions, number of miRNA reads and diversity of transcripts. Whereas EVs obtained by immunoaffinity yielded the purest subset of small EVs, highly comparable with results attained by ultracentrifugation combined with density gradient, precipitation and membrane affinity, sample purification by spin column chromatography indicated a tendency to isolate different subtypes of small EVs, which might also carry a distinct subset of miRNAs. Based on our results, different EV purification methods seem to preferentially isolate different subtypes of EVs with varying efficiencies. As a consequence, sequencing experiments and resulting miRNA profiles were also affected. Hence, the selection of a specific EV isolation method has to satisfy the respective research question and should be well considered. In strict adherence with the MISEV (minimal information for studies of extracellular vesicles) guidelines, the importance of a combined evaluation of biophysical and proteomic EV characteristics alongside transcriptomic results was clearly demonstrated in this present study.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Comparing small urinary extracellular vesicle purification methods with a view to RNA sequencing—Enabling robust and non-invasive biomarker research

Mussack

Wittmann²,

Pfaffl

2019

Biomolecular Detection and Quantification

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“…Exosomes are small, 50 and 150 nm in diameter, cell-derived vesicles, that are released in several fluids, including blood, urine, and the medium of cell cultures [7,8]. Exosomes are released either by the cell, when multivesicular bodies fuse with the plasma membrane, or directly from the plasma membrane, under several stimuli [9]. Exosomes behave as paracrine effectors that influence the recipient cell, because they contain genetic material, proteins and factors essential for cell-cell communication [2], which can be transferred from one cell to another [10].…”

Section: Introductionmentioning

confidence: 99%

ARPE-19-derived VEGF-containing exosomes promote neovascularization in HUVEC: the role of the melanocortin receptor 5

Maisto¹,

Oltra

Vidal-Gil

et al. 2019

Cell Cycle

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ARPE-19 retinal pigment epithelial cells cultured in a medium containing 35 mM D-glucose led to an augmented ROS formation and release of vascular endothelial factor (VEGF)-containing exosomes compared to ARPE-19 cells cultured in a medium containing 5 mM D-glucose (standard medium). Exposing these cells to the melanocortin 5 receptor agonist (MCR 5) PG-901 (10 −10 M), for 9 d reduced ROS generation, the number of exosomes released and their VEGF content. In contrast, incubating the cells with the melanocortin receptor MCR 1 agonist BMS-470539 (10 −5 M) or with the mixed MCR 3/4 agonist MTII (0.30 nmol) did not produce any significant decrease in ROS levels. ARPE-19-derived VEGFcontaining exosomes promoted neovascularization in human umbilical vein endothelial cells (HUVEC), an effect that was markedly reduced by PG-901 (10 −10 M) but not by the MCR 3/4 agonist MTII (0.30 nmol) or the MCR 1 agonist BMS-470539 (10 −5 M). The MCR 5-related action in the ARPE-19 cells was accompanied by the increased expression of two coupled factors, cytochrome p4502E1 (CYP2E1) and nuclear factor kappa b (Nf-κB). These are both involved in high glucose signalling, in ROS generation and, interestingly, were reduced by the MCR 5 agonist in the ARPE-19 cells. Altogether, these data suggest that MCR 5 is a modulator of the responses stimulated by glucose in ARPE-19 cells, which might possibly be translated into a modulation of the retinal pigment epithelium response to diabetes in vivo.

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“…The gold-labeled antibody is bound to an antigen to form an immune complex, and similarly, another is used as a detection antibody to capture the immune complex by binding to different sites of the antigen. ELISA experiments are performed here to achieve screening of antibody pairs [43,44]. When the immune complex reaches a certain concentration, it will cause a chromogenic reaction, and more accurate results can be obtained by gray value analysis.…”

Section: Discussionmentioning

confidence: 99%

Bladder cancer hunting: A microfluidic paper‐based analytical device

Jiang

Han

Ren³

et al. 2020

Electrophoresis

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Bladder cancer is the fourth most common cancer in men, and it is becoming a prevalent malignancy. Most of the regular clinical examinations are prompt evaluations with cystoscopy, renal function testing, which require high‐precision instrument, well‐trained operators, and high cost. In this study, a microfluidic paper‐based analytical device (μPAD) was fabricated to detect nuclear matrix protein 22 (NMP22) and bladder cancer antigen (BTA) from the urine samples. Urine samples were collected from 11 bladder cancer patients and 10 well‐beings as experiment and control groups, respectively, to verify the working efficiency of μPAD. A remarkable checkout efficiency of up to 90.91% was found from the results. Meanwhile, this method is feasible for home‐based self‐detection from urine samples within 10 min for the total process, which provides a new way for quick, economical, and convenient tumor diagnosis, prognosis evaluation, and drug response.

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Urinary extracellular vesicle biomarkers in urological cancers: From discovery towards clinical implementation

Cited by 51 publications

References 237 publications

Comparing small urinary extracellular vesicle purification methods with a view to RNA sequencing—Enabling robust and non-invasive biomarker research

Comparing small urinary extracellular vesicle purification methods with a view to RNA sequencing—Enabling robust and non-invasive biomarker research

ARPE-19-derived VEGF-containing exosomes promote neovascularization in HUVEC: the role of the melanocortin receptor 5

Bladder cancer hunting: A microfluidic paper‐based analytical device

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