Nanoscale flow cytometry for immunophenotyping and quantitating extracellular vesicles in blood plasma

Salmond, Nikki; Khanna, Karan; Owen, Gethin; Williams, Karla C.

doi:10.1039/d0nr05525e

Cited by 28 publications

(31 citation statements)

References 31 publications

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“…Nanoscale flow cytometry analysis of EVs was performed as we have previously described [19]. Briefly, ten microliters of whole plasma or isolated SEC fraction was incubated with fluorescently labeled antibodies for 30 min at room temperature in the dark.…”

Section: Nanoscale Flow Cytometry Of Evsmentioning

confidence: 99%

“…In order to quantitate the number of circulating STEAP1 positive EVs in plasma, nanoscale flow cytometry was employed. Nanoscale flow cytometry can be used to detect and quantify nanosized particles between~80 nm and 1 µm at single event resolution [15,19,24]. EVs derived from cell culture, plasma, and serum have been successfully analysed using various nanoscale flow cytometry platforms [15,[25][26][27][28].…”

Section: Circulating Steap1 Evs Are Identified In Plasmamentioning

confidence: 99%

“…3A). It has been reported that platelets and platelet-derived EVs are abundant in plasma and are detected by nanoscale flow cytometry [19,24,30,31]. Importantly, as previous work has demonstrated that particles in a plasma can non-specifically associate or aggregate with platelet and EV membranes, to ensure that our identified STEAP1-positive EVs were not associated with platelets and platelet-derived EVs we analyzed 10 healthy and 10 PCa plasma samples for platelet specific markers CD9, CD41, and CD42a (Supp.…”

Section: Elevated Steap1 Ev Levels Associate With Pcamentioning

confidence: 99%

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Clinical significance of STEAP1 extracellular vesicles in prostate cancer

Khanna

Salmond

Lynn

et al. 2021

Prostate Cancer Prostatic Dis

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Background Extracellular vesicles (EVs) are cell-derived lipid bilayer enclosed structures shed from the plasma membrane by all cell types. Evidence of EV presence in biological fluids has led to considerable efforts focused on identifying their cargo and determining their utility as a non-invasive diagnostic platform for cancer. In this study, we identify circulating STEAP1 (six-transmembrane epithelial antigen of the prostate 1)-positive EVs in the plasma of healthy males and prostate cancer patients and evaluate its diagnostic and prognostic significance. Methods STEAP1 was identified on EVs in prostate cancer patient plasma. EVs were validated using electron microscopy, Western blot, nanoparticle tracking analysis, and nanoscale flow cytometry. STEAP1-positive EVs were quantified for 121 males with prostate cancer and 55 healthy age-matched control males. An evaluation of STEAP1 in prostate cancer tissue was also performed using established prostate cancer cohort data (TCGA, MSKCC, and SU2C/PCF Dream Team). Results Evaluation of STEAP1-positive EVs by nanoscale flow cytometry identified a significant increase in prostate cancer patient plasma compared to healthy males. However, no association was found between total STEAP1 EV levels and disease recurrence or overall survival. Cohort data from prostate cancer tissue also found STEAP1 to be elevated in prostate cancer while no significant association with recurrence or overall survival was identified. Conclusions STEAP1 is known to be enriched on the cells of the prostate with potential clinical significance in prostate cancer. Our results identify and quantitate STEAP1-positive EVs in plasma and provide rationale for a STEAP1 EV-based liquid biopsy as a diagnostic strategy in prostate cancer.

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Section: Nanoscale Flow Cytometry Of Evsmentioning

confidence: 99%

Section: Circulating Steap1 Evs Are Identified In Plasmamentioning

confidence: 99%

Section: Elevated Steap1 Ev Levels Associate With Pcamentioning

confidence: 99%

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Clinical significance of STEAP1 extracellular vesicles in prostate cancer

Khanna

Salmond

Lynn

et al. 2021

Prostate Cancer Prostatic Dis

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“…Proper instrumentation, antibodies and controls must be used to ensure accurate EV detection. [104][105][106][107][108][109] In characterizing EV content, it is important to show that the RNA/DNA/protein of interest is truly EV associated and not simply co-puried with EVs. For example, albumin and lipoproteins are abundantly present in plasma and serum and coisolate with EVs by most currently used isolation techniques.…”

Section: Ev Characterizationmentioning

confidence: 99%

“…However, membrane associated particles may be more difficult to replicate in cross validation studies. 107,[110][111][112] To validate nucleic acids/proteins of interest are encapsulated by EV membranes, EVs can be treated with RNase and/or DNase or protease with and without tritonX-100/detergent lysis. If the cargo is detected when EVs are treated with RNase/DNase/ protease, but are not detected if detergent lysis of EVs is followed by RNase/DNase/protease treatment, this indicates that the cargo resides within EVs and is protected by the EV membrane.…”

Section: Ev Characterizationmentioning

confidence: 99%

Isolation and characterization of extracellular vesicles for clinical applications in cancer – time for standardization?

Salmond

Williams

2021

Nanoscale Adv.

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Engineering a tunable micropattern‐array assay to sort single extracellular vesicles and particles to detect RNA and protein in situ

Zhang,

Rima,

Wang

et al. 2023

J of Extracellular Vesicle

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The molecular heterogeneity of extracellular vesicles (EVs) and the co‐isolation of physically similar particles, such as lipoproteins (LPs), confounds and limits the sensitivity of EV bulk biomarker characterization. Herein, we present a single‐EV and particle (siEVP) protein and RNA assay (siEVPPRA) to simultaneously detect mRNAs, miRNAs, and proteins in subpopulations of EVs and LPs. The siEVPPRA immobilizes and sorts particles via positive immunoselection onto micropatterns and focuses biomolecular signals in situ. By detecting EVPs at a single‐particle resolution, the siEVPPRA outperformed the sensitivities of bulk‐analysis benchmark assays for RNA and protein. To assess the specificity of RNA detection in complex biofluids, EVs from various glioma cell lines were processed with small RNA sequencing, whereby two mRNAs and two miRNAs associated with glioblastoma multiforme (GBM) were chosen for cross‐validation. Despite the presence of single‐EV‐LP co‐isolates in serum, the siEVPPRA detected GBM‐associated vesicular RNA profiles in GBM patient siEVPs. The siEVPPRA effectively examines intravesicular, intervesicular, and interparticle heterogeneity with diagnostic promise.

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Nanoscale flow cytometry for immunophenotyping and quantitating extracellular vesicles in blood plasma

Abstract: Nanoscale flow cytometry permits analysis of small particles in unpurified plasma and supports identification and quantitation of different particle populations.

Cited by 28 publications

References 31 publications

Clinical significance of STEAP1 extracellular vesicles in prostate cancer

Clinical significance of STEAP1 extracellular vesicles in prostate cancer

Isolation and characterization of extracellular vesicles for clinical applications in cancer – time for standardization?

Engineering a tunable micropattern‐array assay to sort single extracellular vesicles and particles to detect RNA and protein in situ

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