Cell culture

The human breast cancer cell line MCF7 was obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, Hanover Park, IL, USA), supplemented with 10% heat-inactivated fetal bovine serum (HyClone FBS; GE Healthcare Life Sciences, Logan, UT, USA), 100 units/mL penicillin, and 0.1 mg/mL streptomycin in a humidified incubator with 95% air and 5% CO2 at 37 °C. After 48 h (cells reached a confluency of about 80%), the media was changed to a serum-free synthetic medium (advanced DMEM, Thermo Fisher Scientific) followed by a 48-h incubation.

Isolation of EVs by differential ultracentrifugation

The conditioned medium described above was collected and centrifuged at 2000 × g for 10 min and then at 10,000 × g for 30 min. The resulting supernatant was filtered through a 0.2-µm membrane to remove cells and cellular debris, as only EVs < 0.2 µm, representing exosomes and smaller MVs, were the focus of this study, and then stored as a culture supernatant at − 80 °C. To prepare EVs, the MCF7 culture supernatant was thawed and centrifuged at 100,000 × g for 70 min at 4 °C using an Optima TLX equipped with a TLA100.3 fixed-angle rotor (Beckman Coulter, Miami, FL, USA). The supernatant was discarded, and the pellet was resuspended in phosphate-buffered saline (PBS, pH 7.4). The mixture was re-centrifuged at 100,000 × g for 70 min at 4 °C and the pellet was resuspended in PBS.

Preparation of affinity peptide-immobilized magnetic beads

The N-terminal biotinylated peptide comprising three GGGS linkers and 8 lysine residues (K8-peptide: GGGSGGGSGGGSKKKKKKKK) was synthesized by Eurofin Genomics (Tokyo, Japan). Similarly, peptides comprising 4 lysine residues (K4-peptide: GGGSGGGSGGGSKKKK) and 16 lysine residues (K16-peptide: GGGSGGGSKKKKKKKKKKKKKKKK) were synthesized by Eurofin Genomics. Twenty microliters of streptavidin magnetic beads (GE Healthcare Life Sciences) were recovered using a magnetic stand and washed with 200 µL of BW buffer (20 mM Bis–Tris, pH 6.0; 150 mM NaCl; 0.005% Tween 20) and resuspended in 180 µL of BW buffer. To immobilize the biotinylated lysine-rich affinity peptides onto the streptavidin magnetic beads, 20 µL of biotinylated lysine-rich affinity peptides (100 µM) were added to the suspended beads and mixed using a microtube mixer (MT-400; Tomy, Tokyo, Japan) for 20 min. The beads were then washed three times with 200 µL of BW buffer, and finally resuspended in 50 µL of BW buffer and stored at 4 °C until use.

Detection of the EV-CD9 marker bound to affinity peptide-immobilized magnetic beads

Two hundred microliters of the MCF7 culture supernatant was mixed with 20 µL of BT buffer (220 mM Bis–Tris, pH 6.0; 0.055% Tween 20) and 20 µL of the affinity peptide-immobilized magnetic beads solution followed by mixing using a microtube mixer MT-400 for 30 min. The beads were washed three times with 300 µL of BW buffer. Non-specific binding sites were blocked by treating the beads with 100 µL of TBS-T (Tris-buffered saline purchased from Nacalai Tesque, Kyoto, Japan and 0.05% Tween 20) containing 20 mg/mL fatty-acid-free bovine serum albumin (BSA; Wako, Osaka, Japan) for 20 min. Then, 100 µL of anti-CD9 antibody (Cosmo Bio Co., Ltd, Tokyo, Japan; diluted 1/1000 with TBS-T buffer containing BSA) was added and incubated for 30 min. After the beads were washed two times with TBS-T, 100 µL of horse-radish peroxidase (HRP)-conjugated anti-mouse IgG antibody (Abcam, Cambridge, UK; diluted 1/50,000 with TBS-T buffer containing BSA) was added followed by incubation for 30 min. After the beads were washed three times with TBS-T, the HRP activity was detected using a luminescent substrate with a microplate reader (Wallac 1420 ARVO MX; Perkin-Elmer, Boston, MA, USA).

Manual purification of EVs using affinity peptide-immobilized magnetic beads

To isolate EVs, 500 µL of the MCF7 culture supernatant was mixed with 50 µL of BT buffer and 50 µL of affinity peptide-immobilized magnetic beads, followed by rotary mixing for 1 h. The beads were separated using a magnet stand, and then washed three times with 1 mL of BW buffer. After washing, the beads were mixed with 50 µL of elution buffer (20 mM Tris–HCl, pH 7.5; 500 mM NaCl) and incubated for 10 min. The beads were separated using the magnet stand and the supernatant (EV fraction) was transferred to a new 1.5-mL tube.

Automated purification of EVs using Magtration Technology

The magLEAD 6gC (Precision System Science Co., Ltd., Matsudo, Japan) that can parallelly process a maximum of six samples was originally developed for DNA purification using the automated Magtration Technology. Before starting purification of EVs, 500 µL of the MCF7 culture supernatant was mixed with 50 µL of BT-2 buffer (220 mM Bis–Tris, pH 6.0; 0.275% Tween 20). The culture supernatant samples were then transferred to the magLEAD 6gC in which 20 µL of biotinylated K8- or K16-peptide, 50 µL of magnetic beads, 800 µL of BW-2 buffer (20 mM Bis–Tris, pH 6.0; 150 mM NaCl; 0.025% Tween 20), disposable tips, 50 µL of elution buffer, and tubes had been preset. The automated EV purification method involved the following steps: (1) mixing of the affinity peptide and streptavidin magnetic beads for 20 min; (2) mixing of the supernatant sample and the affinity peptide-immobilized magnetic beads for 60 min; (3) washing of the magnetic beads with BW-2 buffer; (4) elution of the EVs from the magnetic beads with elution buffer; and (5) the transfer of EVs into new tubes.

Western blotting

The purified EV fractions were mixed with 4 × SDS sample buffer (0.2 M Tris–HCl, pH 6.8; 8% SDS; 40% glycerol; 0.4% bromophenol blue) and incubated at 37 °C for 30 min prior to electrophoresis (for CD9 and CD63 detection). The purified EV fractions were mixed with 4 × SDS sample buffer containing 2-mercaptoethanol and incubated at 65 °C for 5 min prior to electrophoresis (for TSG101 and Calnexin detection). EVs bound to the K8-peptide magnetic beads were eluted and lysed with 1 × SDS sample buffer (50 mM Tris–HCl, pH 6.8; 2% SDS; 10% glycerol; 0.1% bromophenol blue). MCF7 cells were disrupted by ultrasonication (Branson, Danbury, CT, USA) and used as a cell lysate. These samples were subjected to SDS–polyacrylamide gel electrophoresis (PAGE) and immunoblotting was performed using an anti-human CD9 antibody (1:2000, clone 12A12, Cosmo Bio Co., Ltd), an anti-human CD63 antibody (1:2000, clone 3–13, Wako), an anti-human TSG101 antibody (1:1000, ab30871, Abcam), or an anti-human Calnexin antibody (1:2000, ab22595, Abcam). HRP-conjugated anti-mouse IgG antibody (1:10,000, Abcam) and HRP-conjugated anti-rabbit IgG antibody (1:10,000, Abcam) were used as the secondary antibodies as appropriate. Immunoreactive proteins were detected and analyzed using a EzWestBlue substrate (ATTO, Tokyo, Japan).

Phospholipid binding experiments

Phospholipid binding experiments were performed as previously described37. Briefly, phospholipids (100 µL, 0.75 µg/mL) suspended in methanol were added to 96-well F16 Black Polysorp Fluoronunc Plates (Thermo Fisher Scientific) and air-dried. Non-specific binding sites were blocked by treating the wells with a PBS-blocking solution containing 10 mg/mL fatty-acid-free BSA (Wako). The biotinylated K8-peptide was diluted to 100 nM with the PBS-blocking solution, and then added to the wells, followed by incubation for 30 min. Unbound K8-peptides were removed by washing the wells with 100 µL of PBS containing 0.05% Tween 20. Then, HRP-conjugated streptavidin (Thermo Fischer Scientific), diluted 10,000-fold with the PBS-blocking solution containing 0.05% Tween 20, was added to the wells, followed by incubation for 30 min. After the wells were washed with PBS containing 0.05% Tween 20, HRP activity was assessed by adding a luminescent peroxidase substrate and measuring the resulting luminescence on a microplate reader (Wallac 1420 ARVO MX). The luminescence data were fitted to the Langmuir isotherm to determine the equilibrium dissociation constant (Kd). The Kd was obtained by fitting the luminescence intensity obtained from specific binding at each concentration of peptide using the equation:\({\text{Y}} = {{{\text{B}}_{{\max}} \cdot {\text{X}}} \mathord{\left/ {\vphantom {{{\text{B}}_{{\max}} \cdot {\text{X}}} {\left( {{\text{Kd}} + {\text{X}}} \right)}}} \right. \kern-\nulldelimiterspace} {\left( {{\text{Kd}} + {\text{X}}} \right)}},\) where Y represents the luminescence intensity, X represents the peptide concentration, and Bmax represents the maximum luminescence intensity (maximum binding capacity of the peptide to PS).

For the competitive assay, the wells were blocked with Tris–HCl buffer (10 mM Tris, pH 7.4; 150 mM NaCl) containing 10 mg/mL BSA. The biotinylated K8-peptide (100 nM) was added to the wells in the presence of 30 mM sodium phosphate, 10 mM sodium triphosphate, or 2 mM sodium polyphosphate (15 phosphate residues) solution, followed by incubation for 30 min. After washing the wells with Tris–HCl buffer containing 0.05% Tween 20, HRP-conjugated streptavidin, diluted 10,000-fold with Tris–HCl buffer containing 10 mg/mL BSA and 0.05% Tween 20, was added to the wells, and incubated for 30 min. After the wells were washed with Tris–HCl buffer containing 0.05% Tween 20, HRP activity was determined as described above.

Quantification of EVs using CD9/CD63 ELISA

The EV samples were diluted to the appropriate concentrations and assayed following the instructions provided along with the CD9/CD63 ELISA kit (Cosmo Bio Co., Ltd). A calibration curve was developed using different amounts of the CD9-CD63 fusion protein included in this kit. EV concentrations in the samples were calculated as equivalent to the CD9-CD63 fusion protein. Total protein concentration was measured using the Micro-BCA protein assay (Thermo Fisher Scientific).

Scanning electron microscopy

Purified EVs, which had been dialyzed against ultrapure water, were placed onto the carbon tape (Nisshin EM Co., Ltd, Tokyo, Japan) and fixed with 2.5% glutaraldehyde. After washing with ultrapure water, the samples were dried overnight at 37 °C. Carbon-shadow coating was performed using CADE-E (Meiwafosis, Osaka, Japan). The samples were then observed using a field-emission scanning electron microscope (FE-SEM, Sigma VP; Carl Zeiss Microscopy GmbH, Jena, Germany).

Transmission electron microscopy

Purified EVs were added onto glow-discharged carbon-film grids. The grid was dried with filter paper and stained with 2% uranyl acetate in double distilled water for 10 s. After the stain was completed, the grid was washed with distilled water and blotted dry with filter paper. The grid was air dried and visualized under the Hitachi H-7600 TEM (Hitachi, Japan) operating at 100 kV.

Chemicals

l-Phosphatidylcholine and l-phosphatidylinositol were purchased from Nacalai Tesque (Kyoto, Japan). l-Phosphatidylethanolamine and l-phosphatidyl-l-serine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Other chemicals were purchased from Wako.

Statistical analysis

The data are presented as means of three independent experiments. The standard deviations of each set of experiments are represented in figures (as bars) and in the table. Statistical analysis was carried out using Student’s t-test. A value of p < 0.05 was considered significant.

Source