Employing Flow Cytometry to Extracellular Vesicles Sample Microvolume Analysis and Quality Control

Maia, Joana; Batista, Sílvia; Couto, Nuno; Gregório, Ana C.; Bodo, Cristian; Elzanowska, Julia; Moraes, Maria Carolina Strano; Costa-Silva, Bruno

doi:10.3389/fcell.2020.593750

Cited by 40 publications

(41 citation statements)

References 70 publications

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“…Brain endothelial EV content in plasma with BCBM formation was assessed by flow cytometry. We employed the flow cytometry strategy for EV population analysis, which does not require EV isolation or concentration prior to staining but relies on the staining of vesicular particles with carboxyfluorescein diacetate succinimidyl ester (CFSE) previously described by us [ 33 ]. In each sample, the CFSE labelling allowed the quantification of the proportion of vesicular particles (CFSE + ), distinguishing them from non-vesicular particles (CFSE − ), and further evaluation of the events within the vesicular (CFSE + ) population ( Figure 1 D).…”

Section: Resultsmentioning

confidence: 99%

“…Ideally, a new biomarker should fulfill an unmet need in cancer detection; alternatively, it should provide advantages over preexisting biomarker(s), such as being more accurate and simpler to measure, faster, or cheaper [ 35 ]. Here, we employed a flow cytometry methodology recently established by our team, which enables population analysis of EVs in samples characterized by challenging small volumes while reducing overall sample processing time [ 33 ]. Such methodology has the advantage of not requiring EV isolation or concentration prior to staining, enabling the analysis of EVs in both purified and non-purified biological samples.…”

Section: Discussionmentioning

confidence: 99%

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MicroRNAs and Extracellular Vesicles as Distinctive Biomarkers of Precocious and Advanced Stages of Breast Cancer Brain Metastases Development

Figueira

Godinho-Pereira

Galego

et al. 2021

IJMS

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Triple negative breast cancer presents higher mortality and poorer survival rates than other breast cancer (BC) types, due to the proneness to brain metastases formation, which are usually diagnosed at advanced stages. Therefore, the discovery of BC brain metastases (BCBM) biomarkers appears pivotal for a timely intervention. With this work, we aimed to disclose microRNAs (miRNAs) and extracellular vesicles (EVs) in the circulation as biomarkers of BCBM formation. Using a BCBM animal model, we analyzed EVs in plasma by nanoparticle tracking analysis and ascertained their blood-brain barrier (BBB) origin by flow cytometry. We further evaluated circulating miRNAs by RT-qPCR and their brain expression by in situ hybridization. In parallel, a cellular model of BCBM formation, combining triple negative BC cells and BBB endothelial cells, was used to differentiate the origin of biomarkers. Established metastases were associated with an increased content of circulating EVs, particularly of BBB origin. Interestingly, deregulated miRNAs in the circulation were observed prior to BCBM detection, and their brain origin was suggested by matching alterations in brain parenchyma. In vitro studies indicated that miR-194-5p and miR-205-5p are expressed and released by BC cells, endothelial cells and during their interaction. These results highlight miRNAs and EVs as biomarkers of BCBM in early and advanced stages, respectively.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

MicroRNAs and Extracellular Vesicles as Distinctive Biomarkers of Precocious and Advanced Stages of Breast Cancer Brain Metastases Development

Figueira

Godinho-Pereira

Galego

et al. 2021

IJMS

Self Cite

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“…The protocol we used as a prototype [ 17 ] included the staining of plasma with CFSE membrane-permeable dye. In our study, we used the lipophilic compound CM-Dil, which is expected to interact with various lipid-containing plasma components, as well as vesicles.…”

Section: Resultsmentioning

confidence: 99%

“…However, the common disadvantages, such as micelle formation [ 14 , 15 ] and the labeling of non-vesicular plasma components [ 16 ], require attention. An elegant strategy to overcome the inherent limitations of lipophilic compounds was demonstrated recently by J. Maia et al [ 17 ]. The authors of that study stained plasma with CFSE, fractionated it via size exclusion chromatography (SEC) to separate stained vesicles from other plasma components and micelles, and analyzed the stained vesicles using hrFCM.…”

Section: Introductionmentioning

confidence: 99%

CM-Dil Staining and SEC of Plasma as an Approach to Increase Sensitivity of Extracellular Nanovesicles Quantification by Bead-Assisted Flow Cytometry

Nikiforova¹,

Chumachenko

Nazarova³

et al. 2021

Membranes

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The quantification of the specific disease-associated populations of circulating extracellular membrane nanovesicles (ENVs) has opened up new opportunities for liquid biopsy in cancer and other chronic diseases. However, the sensitivity of such methods is mediated by an optimal combination of the isolation and labeling approaches, and is not yet sufficient for routine clinical application. The presented study aimed to develop, characterize, and explore a new approach to non-specific ENV staining, followed by size-exclusive chromatography (SEC), which allows us to increase the sensitivity of bead-assisted flow cytometry. Plasma from healthy donors was purified from large components, stained with lipophilic CM-Dil dye, and fractionated by means of SEC. The obtained fractions were analyzed in terms of particle size and concentration using NTA, as well as vesicular markers and plasma protein content via dot-blotting. We characterized the process of CM-Dil-stained plasma fractionation in detail and indicated the fractions with optimal characteristics. Finally, we explored the sensitivity of on-bead flow cytometry for the analysis of specific populations of plasma ENVs and demonstrated the advantages and limitations of the proposed technique.

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“…Perhaps not surprisingly, this indicates the advantage of using multiple antibodies for ENV capture. Other, perhaps more efficient methods of ENV labeling, e.g., using lipophilic dyes [55], may help to further improve sensitivity.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Colon-Specific Plasma Nanovesicles as New Markers of Colorectal Cancer

Nazarova¹,

Slyusarenko²,

Sidina³

et al. 2021

Cancers

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Purpose: Developing new and efficient approaches for the early diagnosis of colorectal cancer (CRC) is an important issue. Circulating extracellular nanovesicles (ENVs) present a promising class of cancer markers. Cells of well-differentiated adenocarcinomas retain the molecular characteristics of colon epithelial cells, and the ENVs secreted by these cells may have colon-specific surface markers. We hypothesize that an increase in the number of ENVs carrying colon-specific markers could serve as a diagnostic criterion for colorectal cancer. Experimental design: Potential colon-specific markers were selected based on tissue-specific expression profile and cell surface membrane localization data. Plasma was collected from CRC patients (n = 48) and healthy donors (n = 50). The total population of ENVs was isolated with a two-phase polymer system. ENVs derived from colon epithelium cells were isolated using immune-beads with antibodies to colon-specific markers prior to labelling with antibodies against exosomal tetraspanins (CD63 and CD9) and quantification by flow cytometry. Results: The number of ENVs positive for single colon cancer markers was found to be significantly higher in the plasma of CRC patients compared with healthy donors. The efficacy of detection depends on the method of ENV labelling. The diagnostic efficacy was estimated by ROC analysis (the AUC varied between 0.71 and 0.79). The multiplexed isolation of colon-derived ENVs using immune-beads decorated with antibodies against five markers allowed for a further increase in the diagnostic potency of the method (AUC = 0.82). Conclusions: ENVs derived from colon epithelium may serve as markers of differentiated CRC (adenocarcinomas). The composition of ligands used for capturing colon-derived ENVs and their method of labelling are critical for the efficacy of this proposed diagnostic approach.

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Employing Flow Cytometry to Extracellular Vesicles Sample Microvolume Analysis and Quality Control

Cited by 40 publications

References 70 publications

MicroRNAs and Extracellular Vesicles as Distinctive Biomarkers of Precocious and Advanced Stages of Breast Cancer Brain Metastases Development

MicroRNAs and Extracellular Vesicles as Distinctive Biomarkers of Precocious and Advanced Stages of Breast Cancer Brain Metastases Development

CM-Dil Staining and SEC of Plasma as an Approach to Increase Sensitivity of Extracellular Nanovesicles Quantification by Bead-Assisted Flow Cytometry

Evaluation of Colon-Specific Plasma Nanovesicles as New Markers of Colorectal Cancer

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