Paclitaxel was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-CD9, anti-Alix and anti-vinculin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-β-actin, anti-integrin β4 and anti-GAPDH antibodies were from Cell Signaling Technology (Danvers, MA, USA). Anti-TSG101 antibody was obtained from Abcam (Cambridge, UK).

Human prostate cancer PC-3 cells were purchased from the American Type Cell Collection (ATCC; Manassas, VA, USA) and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere containing 5% CO₂. Taxane-resistant PC-3 cells (PC-3R) that were resistant to both paclitaxel and docetaxel were generated from parental PC-3 cells as previously described (12). In the present study, we used PC-3R cells viable in the presence of 20 nM paclitaxel.

The conditioned medium was harvested from cells cultured in advanced RPMI-1640 medium (Life Technologies, Carlsbad, CA, USA). Exosomes were isolated from the medium according to the previously described method with minor modifications (13). Briefly, 1×10⁶ cells were seeded on 150-mm dish and the conditioned medium was collected 72 h later. The medium was centrifuged at 2,000 × g for 10 min to get rid of cells and the supernatant was centrifuged at 12,000 × g for 30 min to remove debris. Lastly, exosomes were pelleted by ultracentrifugation at 110,000 × g for 70 min, resuspended in PBS and stored at −80°C until use.

Dynabeads M-270 Epoxy magnetic beads (Life Technologies) were conjugated with mouse monoclonal anti-CD9 antibody (BioLegend, San Diego, CA, USA) according to the manufacturer’s protocol. The cell culture medium was centrifuged at 2,000 × g for 10 min and the supernatant was centrifuged at 12,000 × g for 30 min. After filtration through 0.20 μm polyvinylidene difluoride (PVDF) filter, 30 ml of the filtrate were incubated with 1 mg of the anti-CD9 antibody-conjugated beads for 90 min with rotation at 4°C. Beads were then washed 3 times with ice-cold PBS, resuspended in RIPA buffer containing protease inhibitor cocktail and stored at −20°C until use.

Whole cell lysates were prepared by resuspending cells in ice-cold lysis buffer [1% Igepal CA-630, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 25 mM Tris-HCl (pH 7.6)] containing protease inhibitor cocktail. Cell lysates and exosomes were subjected to electrophoresis on 7.5% SDS-polyacrylamide gels and transferred to PVDF membranes (GE Healthcare, Little Chalfont, UK). After blocking in 5% skim milk, membranes were probed with a primary antibody and subsequently with a horseradish peroxidase-linked secondary antibody. After washing, bound proteins were visualized using the ECL Prime Western blotting detection system (GE Healthcare).

After fixation with 2.5% glutaraldehyde and post-fixation with 2% osmium tetroxide, exosomes were embedded in polymeric resin Quetol 812 (Nisshin EM, Tokyo, Japan) and sectioned by ultramicrotomy into 80-nm thick slices, which were deposited onto Cu grids. Exosomes were then stained with uranyl acetate and lead citrate and examined with the transmission electron microscope (Hitachi H-7500; Hitachi, Tokyo, Japan).

Size distribution analysis of exosomes was performed with the qNano system (Izon Science, Christchurch, New Zealand). After dilution in PBS, the size of exosomes was measured using the NP100 nanopores (particle size analysis range; 70–200 nm) and CPC100 calibration particles and analyzed by the Izon Control Suite 2.2 software according to the manufacturer’s instructions.

Exosomes were labeled with iTRAQ reagents using the iTRAQ multiplex kit (AB Sciex, Redwood City, CA, USA) according to the manufacturer’s protocol. In brief, exosomes were isolated from two independent cell cultures for PC-3 and PC-3R cells. Exosomes (5 μg) diluted in PBS was denatured by SDS, reduced by Tris (2-carboxyethyl) phosphine (TCEP) at 60°C for 1 h and alkylated by cysteine blocking reagent methyl methanethiosulfonate (MMTS) at room temperature for 10 min. Samples were then trypsinized at 37°C for 5 h and labeled at room temperature for 1 h with iTRAQ reagent 114, 115, 116 or 117. Samples from PC-3 cells were labeled with 114 or 115 and those from PC-3R cells were labeled with 116 or 117. Labeled samples were cleaned up by using an ICAT cation exchange column (AB Sciex) and desalted using a MonoSpin C18 column (GL Sciences, Tokyo, Japan). After evaporation and reconstitution in 0.1% trifluoroacetic acid (TFA), samples were subjected to LC-MALDI-TOF/TOF analysis.

Peptides were separated and automatically spotted onto a MALDI plate using direct nanoLC and MALDI fraction system DiNa-MaP (KYA Technologies, Tokyo Japan). Solvent A was 2% MeCN/0.1% TFA and solvent B was 70% MeCN/0.1% TFA. The matrix solution was prepared at a concentration of 4 mg/ml α-cyano-4-hydroxycinnamic acid (CHCA) and 0.08 mg/ml ammonium citrate in 70% MeCN/0.1% TFA. After sample was loaded onto a trap column (HiQ sil C18W-3, 0.5×1 mm; KYA Technologies), valve was switched and peptides were separated by an analytical column (HiQ sil C18W-3, 0.1×50 mm; KYA Technologies) at a flow rate of 300 nl/min. The LC gradient was as follows: 0–2 min, 0–5% solvent B; 2–60 min, 5–45% solvent B; 60–75 min, 45–100% solvent B; 75–85 min, 100% solvent B; 85–100 min, 0% solvent B. The matrix solution was pumped at a flow rate of 1400 nl/min. The column end was connected directly to the spotter needle. LC eluent was mixed with matrix solution and spotted onto a MALDI plate at 30-sec intervals between 5–90 min for a total of 171 spots. Mass spectra were acquired using the AB Sciex TOF/TOF 5800 system operated on TOF/TOF Series Explorer software version 4.1 (AB Sciex). For each spot, MS spectra were acquired in positive ion mode between m/z 800 and 4000 and accumulated from 800 laser shots in a randomized raster. MS/MS spectra were acquired using parameters and methods as follows: collision energy, 1 kV; CID control, OFF; laser shots, 4000; minimum S/N filter, 50; mininum mass, 800 Da; maximum mass, 4000 Da; acquisition order/fraction, strongest precursors first; maximum precursors/fraction, 100; precursor mass window, 200 resolution (FWHM); metastable suppression, ON.

All MS/MS data were submitted to ProteinPilot software version 4.5 (AB Sciex). Search parameters were as follows: sample type, iTRAQ 4plex (peptide labeled); Cys. alkylation, MMTS; digestion, trypsin; instrument, 5800; species, Homo sapiens; ID focus, biological modifications; database, uniprot_sprot_can+iso_20100622+Contams+.fasta (AB Sciex). Protein identification was considered to be correct based on the following selection criteria: protein having at least 2 peptides with an ion score >95% confidence; and protein with protein score (ProtScore) >1.3 (unused, P<0.05, 95% confidence). The average fold-change (PC-3R/PC-3) was calculated as an average of 4 pairwise comparisons (116/114, 116/115, 117/114 and 117/115).

Two small interfering RNAs (siRNAs) for human ITGB4 and VCL as well as siGENOME non-targeting siRNA control gene were purchased from Thermo Fisher Scientific (Waltham, MA, USA). siRNAs were transfected into cells at 2 nM using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) as previously described (14).

The number of viable cells in 12-well plate was determined by trypan blue dye exclusion assay.

To determine the cell viability after paclitaxel treatment, WST assay was performed in 96-well plate using a kit obtained from Roche (Basel, Switzerland).

Transfected cells were cultured in 6-well plate until confluent. Cell layers were wounded by using a 1-ml tip and cultured for 24 h. The wound area was determined by analyzing the images taken at 0 and 24 h using Photoshop software (Adobe Systems, San Jose, CA, USA). The migration area was determined by subtracting the area free of cells at 24 h from that at 0 h and expressed as a percentage of that at 0 h.

Twenty-four hours after seeding 1×10⁵ transfected cells onto the Matrigel invasion chamber (Corning Incorp., Corning, NY, USA), cells were fixed with 100% methanol and stained with crystal violet and the number of invaded cells were counted. The invasion rate was expressed as a percentage of the number of invaded cells in siRNA-transfected cells to that in control siRNA-transfected cells.

Statistical differences were determined by the Student’s t-test. P<0.05 was considered statistically significant.

We isolated exosomes from the cell culture medium by differential centrifugation. In both PC-3 and PC-3R cells, exosomal marker proteins such as CD9, Alix and TSG101 were enriched in exosomes compared to cell lysates and there was no difference in the abundance of the marker proteins between two cell lines (Fig. 1A). The transmission electron microscopic analysis revealed morphology that is typical of exosomes in both PC-3 and PC-3R cells and showed no differences in size and shape of exosomes isolated from both cell lines (Fig. 1B). Consistent with these findings, the particle size distribution of exosomes isolated from PC-3 cells was similar to that from PC-3R cells as measured by the qNano particle size analyzer (Fig. 1C). These results indicate that exosomes were successfully isolated by differential centrifugation.

It has been previously reported that expression of a variety of genes is altered in taxane-resistant cancer cells including prostate cancer cells, which are involved in taxane-resistance, progression and aggressiveness of cancer (15–17). In order to identify differentially expressed proteins in exosomes between taxane-sensitive PC-3 and -resistant PC-3R cells, we conducted iTRAQ-based quantitative proteomic analysis of exosomes isolated by differential centrifugation. Exosomes were isolated from two independent cell cultures for PC-3 and PC-3R cells. Samples from PC-3 cells were labeled with 114 or 115 reporter and those from PC-3R cells were labeled with 116 or 117 reporter. We detected a total of 110 proteins (data not shown). The fold-changes were 1.00±0.07 (mean ± SD) for PC-3 cells (115/114) and 1.01±0.11 for PC-3R cells (117/116). The average fold-change (PC-3R/PC-3) was calculated as an average of 4 pairwise comparisons (116/114, 116/115, 117/114 and 117/115). As a result, we identified 4 and 4 proteins upregulated and downregulated in exosomes from PC3-R cells compared with those from PC-3 cells (>1.5-fold and <0.67-fold), respectively (Tables I and II). Among upregulated proteins, we selected ITGB4 and VCL for further studies, because integrin and VCL are involved in focal adhesion signaling pathways and have been implicated in cancer progression (18,19).

In order to confirm expression changes, cell lysates as well as exosomes were subjected to western blot analysis. As shown in Fig. 2A, expression of both ITGB4 and VCL was higher in exosomes isolated from PC-3R cells than in those from PC-3 cells. Similarly, ITGB4 and VCL expression was increased in cell lysates of PC-3R cells compared to PC-3 cells. The expression changes were more pronounced in cell lysates than in exosomes. These results suggest that expression of ITGB4 and VCL in exosomes reflected that within cells and that both were elevated in exosomes isolated from PC-3R cells by differential centrifugation.

In the present study, we isolated exosomes from the cell culture media by using anti-CD9 antibody-conjugated magnetic beads and performed western blot analysis. The results showed that the levels of ITGB4 and VCL in exosomes captured by anti-CD9 antibody from PC-3R cells were higher than those from PC-3 cells, while the level of CD9 in exosomes were similar between two cell lines. It is noteworthy that the difference in the ITGB4 and VCL levels between PC-3 and PC-3R cells was more evident in exosomes isolated by the immunoaffinity method than in those isolated by differential centrifugation. These results suggest that the levels of ITGB4 and VCL in CD9-positive exosomes from PC-3R cells were elevated compared to those from PC-3 cells.

Although previous studies demonstrated that ITGB4 and VCL play critical roles in various types of cancer including prostate cancer (19–26), their roles in taxane-resistant prostate cancer have not been clarified. We therefore knocked down ITGB4 and VCL expression by siRNA transfection and examined the effect of silencing on proliferation and taxane-resistance. As shown in Fig. 3A, ITGB4 and VCL expression was effectively silenced by siRNAs. There was no difference in proliferation among control siRNA-, ITGB4 siRNA- and VCL siRNA-transfected cells (Fig. 3B). The taxane-resistance was also not affected by ITGB4 and VCL knockdown (Fig. 3C). These result suggest that ITGB4 and VCL that are upregulated in PC-3R cells may not be involved in proliferation and taxane-resistance.

We next examined if silencing of ITGB4 and VCL expression could influence on migration and invasion of PC-3R cells. As shown in Fig. 4A, the migration activity was decreased in ITGB4 siRNA-transfected cells but not in VCL siRNA-transfected cells compared with control siRNA-transfected cells. In contrast, both ITGB4 and VCL knockdown diminished invasion of PC-3R cells (Fig. 4B). These results suggest that ITGB4 and VCL play an important role in migration and/or invasion of PC-3R cells.