Dataset Information

Isolation of biologically active and morphologically intact exosomes from plasma of patients with cancer.

ABSTRACT:

Objective

Isolation from human plasma of exosomes that retain functional and morphological integrity for probing their protein, lipid and nucleic acid content is a priority for the future use of exosomes as biomarkers. A method that meets these criteria and can be scaled up for patient monitoring is thus desirable.

Methods

Plasma specimens (1 mL) of patients with acute myeloid leukaemia (AML) or a head and neck squamous cell carcinoma (HNSCC) were differentially centrifuged, ultrafiltered and fractionated by size exclusion chromatography in small disposable columns (mini-SEC). Exosomes were eluted in phosphate-buffered saline and were evaluated by qNano for particle size and counts, morphology by transmission electron microscopy, protein content, molecular profiles by western blots, and for ability to modify functions of immune cells.

Results

Exosomes eluting in fractions #3-5 had a diameter ranging from 50 to 200 nm by qNano, with the fraction #4 containing the bulk of clean, unaggregated exosomes. The exosome elution profiles remained constant for repeated runs of the same plasma. Larger plasma volumes could be fractionated running multiple mini-SEC columns in parallel. Particle concentrations per millilitre of plasma in #4 fractions of AML and HNSCC were comparable and were higher (p<0.003) than those in normal controls. Isolated AML exosomes co-incubated with normal human NK cells inhibited NKG2D expression levels (p<0.004), and HNSCC exosomes suppressed activation (p<0.01) and proliferation of activated T lymphocytes (p<0.03).

Conclusions

Mini-SEC allows for simple and reproducible isolation from human plasma of exosomes retaining structural integrity and functional activity. It enables molecular/functional analysis of the exosome content in serial specimens of human plasma for clinical applications.

SUBMITTER: Hong CS

PROVIDER: S-EPMC4808740 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Publications

Isolation of biologically active and morphologically intact exosomes from plasma of patients with cancer.

Hong Chang-Sook CS Funk Sonja S Muller Laurent L Boyiadzis Michael M Whiteside Theresa L TL

Journal of extracellular vesicles 20160324

<h4>Objective</h4>Isolation from human plasma of exosomes that retain functional and morphological integrity for probing their protein, lipid and nucleic acid content is a priority for the future use of exosomes as biomarkers. A method that meets these criteria and can be scaled up for patient monitoring is thus desirable.<h4>Methods</h4>Plasma specimens (1 mL) of patients with acute myeloid leukaemia (AML) or a head and neck squamous cell carcinoma (HNSCC) were differentially centrifuged, ultra ...[more]

PMID: 27018366

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Project description:Gliomas including glioblastoma (GBM) are the most common primary malignant brain tumors. Glioma extracellular vesicles (EVs) including exosomes have biological effects (e.g., immunosuppression) and contain tumor-specific cargo that could facilitate liquid biopsies. We aimed to develop a simple, reproducible technique to isolate plasma exosomes in glioma patients. Glioma patients' and normal donors' plasma exosomes underwent brief centrifugation to remove cells/debris followed by serial density gradient ultracentrifugation (DGU). EV size/concentration was determined by nanoparticle tracking. Protein cargo was screened by array, western blot, and ELISA. Nanoscale flow cytometry analysis quantified exosome and microvesicle populations pre- and post-DGU. One-step DGU efficiently isolates exosomes for nanoparticle tracking. Wild type isocitrate dehydrogenase glioma patients' (i.e., more aggressive tumors) plasma exosomes are smaller but higher concentration than normal donors. A second DGU efficiently concentrates exosomes for subsequent cargo analysis but results in vesicle aggregation that skews nanoparticle tracking. Cytokines and co-stimulatory molecules are readily detected but appeared globally reduced in GBM patients' exosomes. Surprisingly, immunosuppressive programmed death-ligand 1 (PD-L1) is present in both patients' and normal donors' exosomes. Nanoscale flow cytometry confirms efficient exosome (<100 nm) isolation post-DGU but also demonstrates increase in microvesicles (>100 nm) in GBM patients' plasma pre-DGU. Serial DGU efficiently isolates plasma exosomes with distinct differences between GBM patients and normal donors, suggesting utility for non-invasive biomarker assessment. Initial results suggest global immunosuppression rather than increased circulating tumor-derived immunosuppressive exosomes, though further assessment is needed. Increased glioma patients' plasma microvesicles suggest these may also be a key source for biomarkers.

Dataset Information

Isolation of biologically active and morphologically intact exosomes from plasma of patients with cancer.

Objective

Methods

Results

Conclusions

Publications

Isolation of biologically active and morphologically intact exosomes from plasma of patients with cancer.

Similar Datasets

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