Reagents and cells
Reagents were purchased from sigma, unless otherwise stated.
The human metastatic melanoma cell lines Ma-Mel-55 and Ma-Mel-86c were previously described , and genotyped for MICA. Both cell lines only express MICA A5.1, which is recruited to exosomes. Cells were grown in RPMI-1640 supplemented with 10% FCS, 1 mM glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 10 mM Hepes, 100 U/mL penicillin and 100 U/mL streptomycin (Biowest). The bladder cancer cell line RT-112 is available from the ATCC [kindly provided by Dr. FX Real (CNIO, Madrid) and genotyped using the StemElite ID System (Promega) at the Genomics Service (IIB-CSIC)]. These cells were grown in EMEM medium (Lonza) supplemented with 10% FCS, 1 mM glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 100 U/mL penicillin, and 100 U/mL streptomycin (Biowest).
Peripheral blood mononuclear cells (PBMC) were purified from healthy volunteer buffy coats (Regional Transfusion Centre, Madrid). Approval from local ethical committees (Transfusion Centre and CSIC) and informed consent from all participants were obtained. After Ficoll isolation, PBMCs were stimulated with 10 U/mL IL-12 (Peprotech) and 25 mg/mL IL-18 (MBL) to allow enrichment of the Natural Killer (NK) cell population. Buffy coats (Regional Transfusion Centre, Madrid) were used under approval from local ethical committees (Transfusion Centre and CSIC) and informed consent from all participants.
Exosome isolation, quantitation and EM visualization
For exosome enrichment, cells were cultured for 3–5 days in their appropriate medium, but with 1% exosome-free FCS (prepared by centrifugation at 100,000×g for 18 h and sterile-filtered). Cell culture supernatants were centrifuged for 10 min at 200×g to discard cells and exosomes were purified by sequential centrifugation as previously described [20, 21]. Briefly, supernatants were centrifuged for 10 min at 500×g, 30 min at 10,000×g and finally ultracentrifuged at 100,000×g for 2 h at 4 °C (Beckman Instruments). The pellet, containing exosomes, was resuspended in HEPES-buffered saline buffer (HBS: 10 mM HEPES pH 7.2, 150 mM NaCl). For liophylization the buffer was supplemented with 8% sucrose, samples were frozen immediately at − 80 °C and lyophilized using a Flexi-Dry Lyophilizer (FTS Systems).
Exosome concentration was determined by nanoparticle tracking analysis (NTA) in a NanoSight NS500 (Malvern Instruments Ltd, Malvern, UK). A 405-nm laser beam was used to highlight the particles, which act as point scatters. Analysis was performed using the NTA 3.1 software (Malvern). These experiments were carried out at the laboratory of Dr. H. Peinado, Spanish National Centre for Oncological Research (CNIO). Electron microscope examination of exosomes was carried out as previously described .
The same number of purified exosomes (7 × 109) resuspended in Laemmli buffer (non-reducing condition for tetraspanins) was run on 12% SDS-PAGE gels. Samples were transferred to Immobilon-P (Millipore) membrane. The membrane was blocked using PBS containing 0.1% Tween-20 (PBS-T) and 5% non-fat dry milk. The tetraspanins, CD63, CD9 and CD81, were detected by incubation with MEM-63, MEM-9 and MEM-81, respectively [kind gift from Vaclav Horejsi (Czech Republic)]. For MICA detection, the membrane was incubated with biotinylated goat polyclonal anti-MICA antibody (R&D) (BAF1300). Mouse anti- β-actin (AC-15) was from Sigma. The secondary antibody goat anti-mouse Ig-HRP (Dako) or streptavidin-HRP (Biolegend) were used before developing using the ECL system (GE Pharmaceuticals).
The day before the experiments, PBMC were stimulated with 20 ng/mL IL-15 (Peprotech) for 18 h. The percentage of NK cells in PBMC cultures was assessed by flow cytometry using CD3 and CD56-specific mAbs. PBMC (105 NK cells) were incubated in 96-well flat-bottomed plates with 1–5 × 1010 melanoma-derived exosomes (or with exosome-free RPMI medium, as negative control). After 24 h, NKG2D surface expression on NK cells was monitored by staining with PE-conjugated anti-NKG2D antibody, FITC-anti-CD3 (BioLegend) and APC-anti-CD56 (e-Bioscience), and flow cytometry analysis using the cytometer Cytomics FC 500 (Beckman Coulter) and Kaluza (Beckman Coulter) software.
MICA and tetraspanins were detected in melanoma-derived exosomes using sandwich ELISA. Plates were coated with purified mAb R&D against MICA (MAB13002) or anti-tetraspanin antibodies (anti-CD9 VJ1/20  or anti-CD63 Tea3/18  in BBS (Borate Buffered saline) overnight at 4 °C. After blocking the plates with 2% BSA-HBS for 2 h at 37 °C, melanoma-derived exosomes were added (or HBS as negative control) and incubated overnight at 4 °C. Biotinylated secondary antibodies [CD9 VJ1/20  and MICA (BAF1300, R&D)] were added and followed by streptavidin-HRP (Amersham). The reaction was developed using the peroxidase substrate system (ABTS; Roche). Absorbance was measured at 405 nm with a reference wavelength of 490 nm with Thermo Scientific™ Multiskan™ FC Filter-based Microplate Photometer (Thermo Fisher Scientific).
Size distribution was measured with a Zetasizer Nano ZS ZEN3600 (Malvern Instruments, Malvern, UK). The samples were diluted (50- to 100-fold) fractions and 3 readings were performed. Each reading was composed of 15 measurements of the backscattered (173°) intensity. Zetasizer software version 7.03 was used for data processing and analysis.
Lateral flow immunoassay
The LFIA was carried out in a dipstick format as described in . Capture antibodies [anti-MICA (1H10), anti-CD63 (Tea3/18), anti-CD81 (5A6, provided by Dr. S. Levy, Department of Oncology, Stanford University School of Medicine, Stanford, CA), anti-CD9 (VJ1/20, Immunostep)] and control anti-IgG antibody were loaded onto a nitrocellulose membrane to make a test and a control line in the strip, using a dispenser IsoFlow (Imagene Technology, USA) at a flow rate of 0.100 µL/mm. For trial experiments, the lines were prepared manually, dispensing 3 × 0.3 µL of the corresponding antibody (1 mg/mL). The membrane was dried for 20 min at 37 °C.
Detection antibodies [anti-CD9 (VJ1/20), anti-CD63 (Tea3/18), anti-MICA (1H10,  or MAB13002 from R&D)] were conjugated to 40 nm gold nanoparticles (AuNP) from BB International (UK). A gold colloid titration was done to estimate the optimal concentration of the antibody to stabilize the gold nanoparticles . For anti-CD63 and anti-MICA labelling, a non-covalent bioconjugation protocol was followed, while a covalent procedure was necessary for anti-CD9, protocols previously described in .
Purified exosomes were prepared in running buffer (10 mM HEPES, pH 7.4, with 150 mM NaCl, 0.05% Tween-20 and 1% BSA) and transferred to a microtube containing 20 µL of antibody-coupled AuNP (in the experiments shown in Additional file 1: Figure 1A and B, 10 µL) in a final volume of 100 µL. For negative control, buffer without exosomes was added to the detection antibody. For most of the experiments, a pre-incubation of 1 h was used prior to introduction of the dipstick in the microtube for a run of the LFIA of 15 min. 100 µL of running buffer were added after the sample. When other pre-incubation times were used, it is indicated in figure legend.
After the run, the signal obtained in the test line was quantitated by scanning the strips using ChemiDoc™ MP Imaging System and analyzing with Image Lab Software (Bio-Rad).
Student’s t-test was done for paired samples using GraphPad Prism 5. P-values are shown in each figure; only significant differences are indicated.