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

Nikiforova, Nadezhda; Chumachenko, Maria S; Nazarova, Inga; Zabegina, Lidia; Slyusarenko, Maria; Sidina, Elena; Malek, Anastasia

doi:10.3390/membranes11070526

Cited by 5 publications

(9 citation statements)

References 26 publications

(42 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…First, the sensitivity of bead-assisted flow cytometry can be greatly improved by staining the plasma with lipophilic dye followed by size-exclusive chromatography. As we demonstrated recently [57], such an approach allowed us to detect a very small vesicular population. Second, alternative methods of nanozyme-based sensing [58] or plasmon resonance [59] can provide a higher sensitivity than flow cytometry and can by optimized for clinical application.…”

Section: Discussionmentioning

confidence: 86%

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

Nazarova¹,

Slyusarenko²,

Sidina³

et al. 2021

Cancers

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 86%

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

Nazarova¹,

Slyusarenko²,

Sidina³

et al. 2021

Cancers

Self Cite

View full text Add to dashboard Cite

show abstract

“…Prostate-specific membrane antigen, PSMA (or folate hydrolase 1 (FOLH1)), a type 2 membrane glycoprotein, is expressed in various tissues [ 14 ], and is enhanced in the brain, intestine, and prostate [ 16 ]. PSMA overexpression is associated with PC development [ 17 ], with a high Gleason score of PC [ 18 ], whereas PSMA can be explored as a marker for the detection of recurrent disease and therapy with PSMA-targeted radioligands [ 19 ].…”

Section: Resultsmentioning

confidence: 99%

“…An equal amount of SPMB (1 µg) with a diameter of 1 µm modified either by an –N 3 or by a streptavidin group was functionalized either by PSMA–Apt or by PSMA-Ab as described in Section 2.9 and Section 2.11 . Both complexes were incubated with equal amounts of SEVs stained with lipophilic dye (Vibrant Dil) and isolated from plasma of healthy male donors (n = 8) by SEC ( Section 2.3 and [ 14 ]). The efficacy of PSMA(+)SEVs capture was estimated by flow cytometry; a representative example of one sample’s analysis is presented in Figure 5 .…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

A New Approach for Prostate Cancer Diagnosis by miRNA Profiling of Prostate-Derived Plasma Small Extracellular Vesicles

Zabegina¹,

Nazarova²,

Nikiforova³

et al. 2021

Cells

Self Cite

View full text Add to dashboard Cite

Vesicular miRNA has emerged as a promising marker for various types of cancer, including prostate cancer (PC). In the advanced stage of PC, the cancer-cell-derived small extracellular vesicles (SEVs) may constitute a significant portion of circulating vesicles and may mediate a detectable change in the plasma vesicular miRNA profile. However, SEVs secreted by small tumor in the prostate gland constitute a tiny fraction of circulating vesicles and cause undetectable miRNA pattern changes. Thus, the isolation and miRNA profiling of a specific prostate-derived fraction of SEVs can improve the diagnostic potency of the methods based on vesicular miRNA analysis. Prostate-specific membrane antigen (PSMA) was selected as a marker of prostate-derived SEVs. Super-paramagnetic beads (SPMBs) were functionalized by PSMA-binding DNA aptamer (PSMA–Apt) via a click reaction. The efficacy of SPMB–PSMA–Apt complex formation and PSMA(+)SEVs capture were assayed by flow cytometry. miRNA was isolated from the total population of SEVs and PSMA(+)SEVs of PC patients (n = 55) and healthy donors (n = 30). Four PC-related miRNAs (miR-145, miR-451a, miR-143, and miR-221) were assayed by RT-PCR. The click chemistry allowed fixing DNA aptamers onto the surface of SPMB with an efficacy of up to 89.9%. The developed method more effectively isolates PSMA(+)SEVs than relevant antibody-based technology. The analysis of PC-related miRNA in the fraction of PSMA(+)SEVs was more sensitive and revealed distinct diagnostic potency (AUC: miR-145, 0.76; miR-221, 0.7; miR-451a, 0.65; and miR-141, 0.64) than analysis of the total SEV population. Thus, isolation of prostate-specific SEVs followed by analysis of vesicular miRNA might be a promising PC diagnosis method.

show abstract

“…18,19,20,21 In the EV-field, different approaches have described the use of membrane-permeant enzyme substrates to label EVs, including carboxyfluorescein diacetate-acetoxymethyl ester (CFDA), carboxyfluorescein diacetate succinimidyl ester (CFSE), and calcein acetoxymethyl ester (calcein-AM). 18,22,23,24 Similarly to FDA, these molecules are cleaved by enzymes which are present inside cells as well as in secreted EVs. These experimental approaches have been developed to make EV fluorescent and detectable for flow cytometry applications or for fluorescent microscopy; so these methods are not quantitative and do not directly provide information on EV-functional activity and quality.…”

Section: Beyond Fluorescein Diacetate Based-methods For Evsmentioning

confidence: 99%

DetectEV: a functional enzymatic assays to predict the potency of extracellular vesicles

Adamo,

Picciotto,

Gargano

et al. 2023

Preprint

View full text Add to dashboard Cite

The establishment of an extracellular vesicle (EV) functional assay is essential for defining the bioactivity, quality, batch-to- batch reproducibility and stability of EV preparations to facilitate subsequent applications as therapeutics or carriers for cell- free therapies. The high heterogeneity of EV preparations requires a sensitive test that supports gold-standard-methods reported in the Minimal Information for Studies of Extracellular Vesicles (MISEV, 2018) guidelines for the functional characterization of EVs. Here, we developed a ubiquitous enzymatic-based assay for EVs, a single-step functional analysis to predict their potency that simultaneously allows a quantitative measure of EV-bioactivity and vesicle integrity, using small- size EV samples. In particular, we set up and validated this robust and simple test using nanoalgosomes as EV-model system, selecting a specific substrate for enzymes present the vesicular cargo of several type of EVs, including mammalian's one. We also explored the proposed assay's potential for EV-quality check after different EV-isolation, storage, and loading methods.

show abstract

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

Cited by 5 publications

References 26 publications

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

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

A New Approach for Prostate Cancer Diagnosis by miRNA Profiling of Prostate-Derived Plasma Small Extracellular Vesicles

DetectEV: a functional enzymatic assays to predict the potency of extracellular vesicles

Contact Info

Product

Resources

About