Cells culture and reagents
The MIAPaCa-2 human pancreatic cancer cell line was obtained from Japanese Collection of Research Bioresources Cell Bank (JCRB) and maintained in minimal essential medium (MEM) medium containing 10% exosome-free fetal bovine serum (FBS) and nonessential amino acids at 37°C in 5% CO2. The following antibodies were purchased: anti‑cytochrome C and anti-Phosphorylated histone H2AX (γ-H2AX) from Cell Signaling Technology Inc. (Danvers, MA, USA), anti-CD9, anti-catalase (Cat), and anti-glutathione peroxidase 1 (GPx1) from Abcam (UK); anti-CD63 from Medical and Biological Laboratories (MBL, Nagoya, Japan); and anti-actin from Santa Cruz Biotechnology, Inc., (Dallas, TX, USA); anti-SOD1 and anti-SOD2 from Merck (Darmstadt, Germany); and tetramethyl rhodamine isothiocyanate (TRITC)-conjugated anti-rabbit secondary antibody from Agilent Technology (Tokyo, Japan). The following reagents were purchased: PKH-26, a lipophilic dye, and N-acetyl-L-cysteine (NAC) from Sigma-Aldrich Co. (St. Louis, MO, USA); Hoechst 33342 (Hoechst) and 2ʹ,7ʹ-dichlorodihydrofluorescein diacetate (C-H2DCF) from Thermo Fisher Diagnostics K.K. (Tokyo, Japan); Methylene Blue from Wako (Osaka, Japan); and 4ʹ,6-diaidino-2-phenylindole (DAPI) from Agilent Technology.
Isolation and morphological evaluation of exosomes
Exosomes were isolated from media-conditioned cells by ultracentrifugation [21]. Briefly, MIAPaCa-2 cells were seeded at 2.0 × 106 cells per T75 cm2 flask and irradiated after substituting the media with exosome-depleted 10% FBS cell culture media. The cell culture media was centrifuged at 2,000 ×g for 10 min at 4°C, and the supernatants were filtered through a 0.22 µm minisart syringe filter (Sartorius, Goettingen, Germany). The supernatants were ultracentrifuged at 150,000 ×g for 90 min at 4°C. The pellet was washed with phosphate-buffered saline (PBS) and ultracentrifuged at 150,000 ×g for 90 min at 4°C and resuspended in 50 μL PBS.
The exosomes isolated from nonirradiated cells (0 Gy-Exo) and those isolated from 5 Gy irradiated cells (5 Gy-Exo) were evaluated by transmission electron microscopy (TEM). Briefly, 4 mL of the PBS suspension of isolated exosomes was loaded onto carbon-coated 200 mesh copper grids for 1 min at room temperature. Excessive fluid was slightly drained using filter papers. The adsorbed exosomes were negatively stained with 2% uranyl acetate for 30 seconds. Finally, the air-dried exosome-containing grids were observed under TEM (JEM-1400plus, Japan) operating at 120 kV. Exosome size, concentration, and distribution were analyzed by nanoparticle tracking analysis (NTA) software using NanoSight NS300 (Malvern, Kobe, Japan). The software was optimized to first identify and then track each particle on a frame-by-frame basis, and its Brownian movement was tracked and measured from frame to frame (See Additional file 1).
Labelling of exosomes
Exosomes were labeled with the fluorescent dye PKH-26 using the PKH-26 labeling kit (Sigma-Aldrich Co.) [22]. Briefly, 0 Gy-Exo and 5 Gy-Exo were labeled with 2 μM PKH-26 for 15 min at room temperature. Thereafter, free PKH-26 was removed by ultrafiltration using the VIVACON 500 ultracentrifugation device (100,000 MWCO; Sartorius Stedim Biotech GmbH, Goettingen, Germany). The labeled exosomes were added to MIAPaCa-2 cell samples and the nuclei of cells were stained with Hoechst 33342 for 5 min.
Irradiation
Cells were exposed to a 5 Gy or 8 Gy dose of X-rays delivered at 0.57 Gy/min from an MBR-1505R2 generator (Hitachi Medical Corporation, Tokyo, Japan). The beam was filtered through a 1-mm aluminum board [23].
Colony-forming assay
Cell survival after irradiation was evaluated by performing a colony-forming assay with or without 5 Gy-Exo. Cells were reseeded into 6-well cell culture plates (Corning. Co., Tokyo, Japan) at a density of 200–4000 cells/well and incubated for 7 days. At the end of each experiment with 0 Gy-Exo, or with 5 Gy-Exo, the cells were fixed with a solution of 10% methanol and 20% acetic acid for 30 min, and stained with Methylene Blue for 30 min [24]. Colonies with ≥ 50 cells were counted. The experiment was performed in duplicates and repeated at least twice.
Determining intracellular ROS levels
Intracellular ROS levels were determined using the oxidation-sensitive fluorescent probe dye C-H2DCF. Cells were seeded in 6-well plates (1.5 × 105 cells/well) overnight and treated with 5 Gy of radiation with or without 10 mg/mL 5 Gy-Exo and 1 mM NAC for 24 h [25]. After washing twice with FBS-free media, the cells were stained with 50 mM C-H2DCF for 1 h at 37°C. Then, the nuclei of cells were stained with Hoechst 33342 for 5 min. The fluorescence of C-H2DCF was visualized by using a fluorescence microscope (BZ-9000, Keyence, Osaka, Japan).
Detection of DNA damage after exosome uptake
Induction of DNA damage was investigated by detecting phosphorylated histone 2AX (γ-H2AX) foci using immunocytochemistry as described previously [26]. Cells were subcultured on 35-mm dishes, and treated with 10 mg/mL 5 Gy-Exo and 1 mM NAC for 24 h and/or 5 Gy of irradiation. Thereafter, the cells were fixed in 4% paraformaldehyde in PBS for 20 min, permeabilized with 0.1% Triton X-100 in PBS for 5 min, and blocked in 5% bovine serum albumin in PBS for 60 min. The cells were incubated with 1:200 rabbit anti γ-H2AX antibody overnight at 4°C. Then, the cells were incubated with 1:20 TRITC-conjugated secondary antibody for 90 min at room temperature. The nuclei were stained with DAPI. The stained cells were observed using a fluorescence microscope. The cells expressing nuclear γ-H2AX foci were then counted manually from 100 cells for each treatment [27].
Immunoblotting
The expression levels of CD9, CD67, and cytochrome C were analyzed by western blotting. Briefly, 3 g of exosome or 30 g whole cell lysates were loaded onto 12% gels. After electrophoresis (30 mA), the proteins were transferred onto polyvinylidene difluoride membranes that were blocked with 5% nonfat milk and then incubated with primary antibodies. After washing, the membrane was incubated with horseradish peroxidase conjugated anti-CD9 or anti-CD63, and the expression levels were detected under nonreducing conditions. The expression levels of SOD1, SOD2, catalase, and glutathione peroxidase 1 were analyzed by western blotting. Briefly, the cells were seeded at 1.0 × 105 cells per T25 cm2 flask and subject to 8 Gy radiation or 8 Gy-Exo was added (30 mg). Each protein was collected 24 h after these treatments. The intensity of each signal was analyzed by using Image J software and then, the ratios of SOD1, SOD2 catalase, glutathione peroxidase, and actin levels were calculated.
Total RNA extraction from exosomes and miRNA microarray analysis
Toray’s 3D-Gene RNA-extraction reagent from a liquid sample kit was used (Toray Industries, Inc.). Comprehensive miRNA expression analysis was performed using a 3D-Gene miRNA Labeling Kit and a 3D-Gene Human miRNA Oligo Chip Ver. 21 (Toray Industries, Inc.) according to the manufacturer’s protocol that was designed to detect 2565 human miRNA sequences. The expression level of each miRNA was expressed as the background-subtracted signal intensity of all the miRNAs in each microarray. Any signal intensity in both the duplicate spots at >1.5 standard deviation (SD) of the background signal intensity was considered a valid measurement.
Database processing analyses and miRNA identification
MiRNAs from exosomes isolated from cells either after 5 Gy or 8 Gy of radiation were visualized in the form of a heat map generated using the R software (version 3.5.3) and heatmap.2 from the gplots package (version 3.0.1.1). The analysis was performed by using the miRNAs whose ratios of expression levels between control and 5 Gy-Exo or 8 Gy-Exo were <0.5 or >1.5. The expression of miRNAs was normalized and then converted loge values. In addition, we searched the targets of these miRNAs that caused ROS production from the databases using TargetScan and miRTarBase. MiRNAs were then clustered using hierarchical clustering with Euclidean distance and complete linkage.