Orally Administered 5-aminolevulinic Acid for Isolation and Characterization of Circulating Tumor-Derived Extracellular Vesicles in Glioblastoma Patients

Background: In glioblastoma (GB), tissue is required for accurate diagnosis and subtyping. Tissue can be obtained through resection or (stereotactic) biopsy, but these invasive procedures provide risks for patients. Extracellular vesicles (EVs) are small, cell-derived vesicles that contain miRNAs, proteins, and lipids, and possible candidates for liquid biopsies. GB-derived EVs can be found in the blood of patients, but it is difficult to distinguish them from circulating non-tumor EVs. 5-aminolevulinic acid (5-ALA) is orally administered to GB patients to facilitate tumor visualization and maximal resection, as it is metabolized to fluorescent protoporphyrin IX (PpIX) that accumulates in glioma cells. In this study, we assessed whether PpIX accumulates in GB-derived EVs and whether these EVs could be isolated and characterized to enable a liquid biopsy in GB. Methods: EVs were isolated from the conditioned media of U87 cells treated with 5-ALA by differential ultracentrifugation. Blood samples were collected and processed from healthy controls and patients undergoing 5-ALA guided surgery for GB. High-resolution flow cytometry (hFC) enabled detection and sorting of PpIX-positive EVs, which were subsequently analyzed by digital droplet PCR (ddPCR). Results: PpIX-positive EVs could be detected in conditioned cell culture media as well as in patient samples after administration of 5-ALA. By using hFC, we could sort the PpIX-positive EVs for further analysis with ddPCR, which indicated the presence of EVs and GB-associated miRNAs. Conclusion: GB-derived EVs can be isolated from the plasma of GB patients by using 5-ALA induced fluorescence. Although many challenges remain, our findings show new possibilities for the development of blood-based liquid biopsies in GB patients.

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Isolation and characterization of extracellular vesicles

Trillium Exctracellular Vesicles ◽

10.47184/tev.2019.01.02 ◽

2019 ◽

Vol 1 (1) ◽

pp. 18-26

Author(s):

Fabia Fricke ◽

Dominik Buschmann ◽

Michael W. Pfaffl

Keyword(s):

Cell Culture ◽

Extracellular Vesicles ◽

Culture Media ◽

Body Fluids ◽

Cell Culture Media ◽

Advantages And Disadvantages ◽

The Past ◽

Isolation And Characterization ◽

Basic Biology

Research into extracellular vesicles (EVs) gained significant traction in the past decade and EVs have been investigated in a wide variety of studies ranging from basic biology to diagnostic and therapeutic applications. Since EVs are secreted by most, if not all, eukaryotic and prokaryotic cells, they have been detected in body fluids as diverse as blood, urine and saliva as well as in cell culture media. In this chapter, we will provide an overview of EV isolation and characterization strategies and highlight their advantages and disadvantages.

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Orally administered 5-aminolevulinic acid for isolation and characterization of circulating tumor-derived extracellular vesicles in glioblastoma patients

Brain and Spine ◽

10.1016/j.bas.2021.100475 ◽

2021 ◽

Vol 1 ◽

pp. 100475

Author(s):

S. Maas ◽

T.S. van Solinge ◽

R. Schnoor ◽

A. Yekula ◽

J. Senders ◽

...

Keyword(s):

Extracellular Vesicles ◽

Aminolevulinic Acid ◽

Isolation And Characterization

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The size of extracellular vesicles secreted by different types of stem cells

Nauchno-prakticheskii zhurnal «Patogenez» ◽

10.25557/gm.2018.4.9747 ◽

2018 ◽

pp. 38-42

Author(s):

И.Б. Алчинова ◽

М.В. Полякова ◽

И.Н. Сабурина ◽

М.Ю. Карганов

Keyword(s):

Stem Cells ◽

Extracellular Vesicles ◽

Culture Media ◽

Living Organism ◽

Isolation And Characterization ◽

Transmission Electron ◽

Study Results ◽

Multipotent Mesenchymal Stem Cells ◽

Rat Adipose Tissue ◽

Almost All

Механизм терапевтического действия мультипотентных мезенхимных стволовых клеток (ММСК) на облученный организм в последнее время вызывает повышенный интерес исследователей. В качестве активного участника паракринного механизма реализации этого эффекта предлагают рассматривать внеклеточные везикулы, секретируемые практически всеми клетками живого организма. Цель работы: выделить и охарактеризовать внеклеточные везикулы, продуцируемые стволовыми клетками различной природы. Материалы и методы. Суспензии внеклеточных везикул, выделенных по модифицированному протоколу дифференциального центрифугирования из культуральных жидкостей от культур ММСК костного мозга человека 2-го пассажа и ММСК жировой ткани крысы 4-го пассажа, были проанализированы методом просвечивающей электронной микроскопии и методом анализа траекторий наночастиц. Результаты. Исследование показало наличие в обоих образцах микрочастиц размерами до и около 100 нм, однако процентное содержание частиц разных размеров в суспензии различалось для двух анализируемых типов клеток. Заключение. Полученные результаты могут свидетельствовать о специфике секреции, обусловленной клеточным типом. A mechanism of the therapeutic effect of multipotent mesenchymal stem cells (MMSC) on irradiated body has recently arisen much interest of researchers. Extracellular vesicles (EVs) secreted by almost all cells of a living organism were suggested to actively contribute to the paracrine mechanism of this effect. The aim of the study was isolation and characterization of extracellular vesicles produced by various types of stem cells. Materials and methods. Suspensions of EVs were isolated from culture media of passage 2 human bone marrow-derived MMSC and passage 4 rat adipose tissue-derived MMSC using a modified protocol of differential centrifugation and then studied using transmission electron microscopy and nanoparticle tracking analysis. Results. The study showed the presence of microparticles with a size of >100 nm in the examined samples. However, the percent content of particles with different sizes in the suspension was different in two analyzed types of cell culture. Conclusion. The study results might reflect a specificity of secretion determined by the cell type.

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Immunomagnetic sequential ultrafiltration (iSUF) platform for enrichment and purification of extracellular vesicles from biofluids

Scientific Reports ◽

10.1038/s41598-021-86910-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Jingjing Zhang ◽

Luong T. H. Nguyen ◽

Richard Hickey ◽

Nicole Walters ◽

Xinyu Wang ◽

...

Keyword(s):

Extracellular Vesicles ◽

Culture Media ◽

Metastatic Breast ◽

Clinical Samples ◽

Clinical Use ◽

Immunomagnetic Beads ◽

Liquid Biopsies ◽

Cell Culture Media ◽

Non Invasive ◽

Healthy Donors

AbstractExtracellular vesicles (EVs) derived from tumor cells have the potential to provide a much-needed source of non-invasive molecular biomarkers for liquid biopsies. However, current methods for EV isolation have limited specificity towards tumor-derived EVs that limit their clinical use. Here, we present an approach called immunomagnetic sequential ultrafiltration (iSUF) that consists of sequential stages of purification and enrichment of EVs in approximately 2 h. In iSUF, EVs present in different volumes of biofluids (0.5–100 mL) can be significantly enriched (up to 1000 times), with up to 99% removal of contaminating proteins (e.g., albumin). The EV recovery rate by iSUF for cell culture media (CCM), serum, and urine corresponded to 98.0% ± 3.6%, 96.0% ± 2.0% and 94.0% ± 1.9%, respectively (p > 0.05). The final step of iSUF enables the separation of tumor-specific EVs by incorporating immunomagnetic beads to target EV subpopulations. Serum from a cohort of clinical samples from metastatic breast cancer (BC) patients and healthy donors were processed by the iSUF platform and the isolated EVs from patients showed significantly higher expression levels of BC biomarkers (i.e., HER2, CD24, and miR21).

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109 Isolation and characterization of myometrium and decidual cell-derived extracellular vesicles

American Journal of Obstetrics and Gynecology ◽

10.1016/j.ajog.2020.12.131 ◽

2021 ◽

Vol 224 (2) ◽

pp. S75-S76

Author(s):

Megan Shepherd ◽

Enkhtuya Radnaa ◽

Rheanna Urrabaz-Garza ◽

Talar Kechichian ◽

Ourlad Alzeus G. Tantengco ◽

...

Keyword(s):

Extracellular Vesicles ◽

Decidual Cell ◽

Isolation And Characterization

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Emerging techniques in the isolation and characterization of extracellular vesicles and their roles in cancer diagnostics and prognostics

The Analyst ◽

10.1039/c5an01775k ◽

2016 ◽

Vol 141 (2) ◽

pp. 371-381 ◽

Cited By ~ 46

Author(s):

Vijaya Sunkara ◽

Hyun-Kyung Woo ◽

Yoon-Kyoung Cho

Keyword(s):

Extracellular Vesicles ◽

Cancer Diagnostics ◽

Clinical Implementation ◽

Characterization Methods ◽

Cancer Management ◽

Isolation And Characterization

We present an overview of current isolation, detection, and characterization methods of extracellular vesicles and their applications and limitations as a potential emerging biomarker in cancer management and their clinical implementation.

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