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. 2016 Mar 24;5:29289.
doi: 10.3402/jev.v5.29289. eCollection 2016.

Isolation of Biologically Active and Morphologically Intact Exosomes From Plasma of Patients With Cancer

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Free PMC article

Isolation of Biologically Active and Morphologically Intact Exosomes From Plasma of Patients With Cancer

Chang-Sook Hong et al. J Extracell Vesicles. .
Free PMC article

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.

Keywords: exosome functions; exosome isolation; exosome morphology; human cancer; human plasma; mini size-exclusion column chromatography.

Figures

Fig. 1
Fig. 1
The protein content of vesicle-containing fractions after mini-SEC of cancer patients’ or normal donors’ plasma. 50 µg aliquots of each fraction (unconcentrated) were separated by PAGE and gels were stained with a Coomassie blue dye. Exosomes were visualized in western blots using anti-CD9- and anti-TSG101-specific Abs. The #3 fraction contains little protein but vesicles are present as shown by TEM in Fig. 2. Fractions #4 and #5 are enriched in exosomes but contain protein bands. Note that the heavy chain of IgG and albumin are the major proteins in fractions # 5, while fractions #3 and #4 are relatively free of IgG and albumin. Shown are representative data from 1 of more than 15 PAGE performed with different column eluates.
Fig. 2
Fig. 2
Transmission electron microscopy (TEM) of vesicles eluted in mini-SEC fractions #3, #4 and #5. Following mini-SEC of (a) AML plasma, (b) HNSCC plasma and (c) NC plasma, TEM was performed. Unconcentrated aliquots of vesicle-containing fractions were placed on grids and negatively stained with uranyl acetate. Note the presence of single exosomes in the size range of 30–100 nm. (d) Exosome aggregates present in #5 fractions of HNSCC and AML plasma are shown. Representative images from 1 of 6 experiments performed are presented. AML=acute myeloid leukaemia; HNSCC=head and neck squamous cell carcinoma; NC=normal control.
Fig. 3
Fig. 3
Sizing and counting of particles by qNano. Fractions #4 were isolated from plasma of 2 AML patients, 2 HNSCC patients and 2 NCs by mini-SEC and evaluated by qNano as described in Materials and Methods. AML=acute myeloid leukaemia; HNSCC=head and neck squamous cell carcinoma; NC=normal control.
Fig. 4
Fig. 4
A. Comparisons of the protein content in mini-SEC fractions # 4 isolated from plasma of cancer patients or NCs. (a) Exosome protein levels (in µg/mL plasma) in fractions #4 obtained from plasma of AML or HNSCC patients and NCs. Note higher protein levels in in exosome fractions of cancer patients relative to those of NCs. (b) Reproducibility of mini-SEC: plasma specimens (1 mL) obtained from an NC) and a HNSCC patient were repeatedly chromatographed (×3) using different mini-SEC columns. Protein levels measured in #4 fractions of repeated samples are not significantly different. AML=acute myeloid leukaemia; HNSCC=head and neck squamous cell carcinoma; NC =normal control.
Fig. 5
Fig. 5
Western blot profiles of exosome proteins in fraction #4. Following mini-SEC, western blots were performed as described in Materials and Methods using concentrated aliquots of fractions #4 isolated from plasma specimens of (a) 2 patients with AML and 2 different NCs and (b) 2 patients with HNSCC and 2 different NCs. All lanes were loaded with 10 µg exosomal proteins. TSG101 was used as an exosome marker and a “loading control.” Immunoblotted proteins and their molecular weights are listed. AML=acute myeloid leukaemia; HNSCC=head and neck squamous cell carcinoma; NC=normal control; SEC=size exclusion chromatography.
Fig. 6
Fig. 6
Biological activity of exosomes in #4 fractions tested in vitro using normal human lymphocytes. Exosomes were co-incubated with PBMC (a) or with isolated and in vitro activated T cells (b) under conditions described in Materials and Methods. (a) AML exosomes in fractions #3 and #4, but not in fraction #5 down-regulated NKG2D expression levels (MFI) in activated NK cells and reduced the frequency of NKG2D+ in these cells (see table) as determined by flow cytometry with the gate set on CD3-CD56+ NK cells. The MFI data are representative from 1 of 3 experiments performed with exosomes isolated from different AML patients. (b) HNSCC exosomes in fractions #4 down-regulated expression of CD69 (percentages of expression are shown) in activated human CD4+ T cells (upper panels) and suppressed proliferation of T cells activated via the TcR (lower panels). Data shown are representative for different HNSCC patients as shown in the tables. AML=acute myeloid leukaemia; HNSCC=head and neck squamous cell carcinoma; NC=normal control; PBMC=peripheral blood mononuclear cell.

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