Sample collection and crude extract preparationBlood plasma

Venous blood from breast cancer patients was collected using citrate blood collection tubes (455,322, Greiner Bio-one). Platelet-depleted plasma was prepared by two serial centrifugations at 2500 g for 15 min at room temperature. All blood samples were first characterized (complete blood count) using the hematology analyzer (XP-300, Sysmex). All blood samples were processed within 120 min after blood collection and platelet-depleted plasma was stored as 1 mL aliquots at −80 °C. Patient characteristics are summarized in Additional file 1: Fig. S1A. Crude extracts from total blood plasma were prepared using SEC columns with Sepharose CL-2B as previously described [10] (Fig. 1A). A SEC column was prepared by placing a nylon net with 20 μm pore size (NY2002500, Merck Millipore) on the bottom of a 10 mL syringe (3SYR-10ML, Romed), followed by stacking of 10 mL pre-washed Sepharose CL-2B (17,014,001, GE Healthcare). On top of one SEC column, 2 mL blood plasma was loaded followed by elution and collection of 6 sequential 1 mL eluate fractions. Following SEC, eluted fractions 4–5–6 were pooled and concentrated to 1 mL using a 10 kDa centrifugal filter (Amicon Ultra-2 mL, UFC201024, Merck Millipore) (referred to as the crude extract). The crude extracts were pooled and aliquots of 1 mL were stored at −80 °C [20].

Urine

Urine from healthy volunteers was collected and crude extracts were prepared as previously described [8]. Urine samples were centrifuged for 10 min at 1000 g and 4 °C. Cell-free urine supernatants were collected (leaving approximately 0.5 cm urine above the cell pellet). Cell-free urine samples (50 mL) were concentrated to 800 µL using a 10 kDa centrifugal filter device (Centricon Plus-70, UFC701008, Merck Millipore) (Fig. 1A) and stored at −80 °C.

All samples were collected in compliance with the Ethical Committee from Ghent University Hospital (approval EC/2014/0655) and relevant guidelines.

EV separation by density gradient centrifugationOperators and automated workstation

Manual density gradient preparation and fraction collection was performed by operators using a P1000 single channel pipette. The operators were divided in two groups based on their experience. Experienced operators were defined as having prepared more than 15 density gradients. Inexperienced operators had prepared one to five gradients maximum. All inexperienced operators were educated by the same instructor in the procedure by demonstration of the technique, received pipetting technique training, and were guided during the preparation and collection procedure.

Robot-assisted density gradient preparation and fraction collection was performed using the Biomek 4000 laboratory automation workstation (Beckman Coulter, A99749) with a custom-made script as previously described [10] (details are provided in the Additional file 1). The Biomek 4000 automation workstation has 12 deck positions and can pipet 1 µL up to 1000 µL by liquid-level sensing. To ensure sterility during the procedures, the automatic liquid handler was equipped with a positive-pressure HEPA enclosure. The workstation was used for the preparation of density gradients, sample loading, and collection of density gradient fractions.

Preparation of top-down and bottom-up OptiPrep density gradients

OptiPrep (60% (w/v) aqueous iodixanol solution, AXS-1,114,542, Axis-Shield) density gradients were prepared as previously described [9, 10]. Solutions of 5, 10, 20, and 40% iodixanol were made by mixing appropriate volumes of homogenization buffer (0.25 M sucrose (S0389, Sigma-Aldrich), 1 mM EDTA (1,084,180,100, Merck Millipore), 10 mM Tris (103,154 M, VWR) - HCL (44,921.K2, Alfa Aesar) (pH 7.4)) and iodixanol working solution. This working solution was prepared by combining a working solution buffer (0.25 M sucrose, 6 mM EDTA, 60 mM Tris-HCl (pH 7.4)) and a stock solution of OptiPrep. rEV were generated by transfection of HEK293T cells (CRL-11,268, ATCC) with gag-EGFP DNA followed by rEV separation from conditioned medium using density gradient centrifugation as described previously [29, 39].

A discontinuous top-down (TD) OptiPrep density gradient was made by layering 4 mL of 40%, 4 mL of 20%, 4 mL of 10% and 3.5 mL of 5% iodixanol solutions on top of each other in a 16.8 mL open top polyallomer tube (337,986, Beckman Coulter). 1 mL crude extract from blood plasma or phosphate-buffered saline (PBS, TMS-012-A, Merck Millipore) spiked with 1.85 × 1010 rEV (as quantified by fluorescent nanoparticles tracking analysis (fNTA) was overlaid on top of the gradient (Fig. 1B). (r)EV suspension was made by resuspending 800 µL crude extract from urine or PBS spiked with 1.85 × 1010 rEV (as quantified by fNTA) in 3.2 mL working solution, obtaining a 40% iodixanol suspension. A discontinuous bottom-up (BU) density gradient was prepared by overlaying 4 mL (r)EV suspension with 4 mL 20%, 4 mL 10% and 3.5 mL 5% iodixanol solutions, and 1 mL PBS (Fig. 1B).

TD and BU density gradients were centrifuged for 18 h at 100,000 g and 4 °C (SW 32.1 Ti rotor, Beckman Coulter).

The technique of manual preparing density gradients is described in Fig. 1C, Additional file 1: Box S1, and Additional file 2: Video S1.

For the automated preparation of density gradients, the single- and eight-channel P1000 pipetting tools, tip boxes (B01122, Beckman Coulter), pre-cooled iodixanol solutions reservoir, pre-cooled tube rack with the centrifuge tube(s), and sample rack were placed in one of the deck positions of the workstation (Fig. 1D). For density gradient preparation the eight-channel MP1000 tool was used (Additional file 3: Video S2), and for sample loading the single-channel P1000SL tool (Additional file 4: Video S3). The custom-made script allows the preparation of up to eight density gradients within one run.

Collection of density gradient fractions

After ultracentrifugation, density gradient fractions of 1 mL were collected from top to bottom manually or by the liquid handler.

The technique of manual fraction collection is described in Fig. 1C, Additional file 1: Box S1, and Additional file 5: Video S4.

The automated fraction collection requires the single-channel P1000 pipetting tool, tip box(es), pre-cooled tube rack with density gradient(s), and fraction rack(s) (Fig. 1D, Additional file 6: Video S5).

EV recovery by ultracentrifugation or size-exclusion chromatography

To perform LC-MS/MS, ultracentrifugation was preferred as the most practical method to remove OptiPrep from individual EV-enriched density fractions from blood plasma and urine in a reproducible way, as previously described [10]. Density fractions 9 and 10 were separately transferred to centrifuge tubes. 14 mL of pre-cooled PBS was added to each sample and the solution was mixed in the tube by pipetting up and down. The tubes were centrifuged for 3 h at 100,000 g and 4 °C (SW 32.1 Ti rotor, Beckman Coulter). After ultracentrifugation, the supernatant was discarded leaving 50 µL at the bottom of the tube. The pellet was diluted to 100 µL with pre-cooled PBS. To prevent loss of EV sticking to the bottom of the tube, the EV pellet was directly lysed in the tube. Lysates were prepared by mixing samples with SDT-lysis buffer (2% SDS (436143-25G, Sigma-Aldrich), 500 mM Tris (103,154 M, VWR) - HCL (44,921.K2, Alfa Aesar) (pH 7.6), 0.5 M dithiothreitol (39759.02, Serva)) at a 4:1 sample to buffer ratio. The pellet was pipetted up and down and the bottom of the tube was rinsed with SDT-lysis buffer. The lysates were collected and incubated at 95 °C for 5 min. Lysates were stored at − 80 °C until processing for LC-MS/MS.

To perform transmission electron microscopy (TEM), EV were separated from pooled density fractions 9–10 obtained from blood plasma and urine samples by including a second SEC, following previously mentioned protocol unless stated otherwise. From this second SEC, eluted size-based fractions 4-5-6-7 were pooled and concentrated to 100 µL using a 10 kDa centrifugal filter (Amicon Ultra-2 mL, UFC201024, Merck Millipore) and stored at −80 °C.

Density measurement

The density of the density-gradient fractions was calculated using a standard curve of the absorbance values at 340 nm (SpectraMax Paradigm, Molecular Devices) of 1:1 aqueous dilution of 5, 10, 20 and 40% iodixanol solutions. This standard curve was used to determine the density of fractions collected from a control gradient overlaid with 1 mL of PBS.

Interface mixing

To determine interface mixing, the image of colored test density gradients prepared by an inexperienced, experienced, and automated operator was analyzed using ImageJ software version 1.53. Each 10% iodixanol layer was individually circumscribed with the rectangular region of interest (ROI) selection. Colors were converted to binary. Profile plots of the ROIs were generated. Area measurement of the peaks (corresponding with the spilling of the 5% and 20% iodixanol solution in the 10% layer) was performed with the wand tool.

Fluorescent nanoparticle tracking analysis

Fractions of rEV spiked density gradients were analyzed by fluorescent nanoparticle tracking analysis (fNTA) using a NanoSight LM10-HS microscope (Malvern Instruments Ltd) equipped with a 488 nm laser, an additional 500 nm longpass filter and an automatic syringe pump system (infusion speed: 20) (Fig. 1E). For each analysis, three videos of 60 s were recorded and analyzed with camera level 16 and detection threshold 3. Temperature was monitored during recording. Recorded videos were analyzed with the NTA Software version 3.3. For optimal measurements, samples were diluted with PBS until particle concentration was within the concentration range for the NTA Software (3 × 108-109 particles/mL). For recovery calculations the number of fluorescent particles was measured before spiking.

Anti-p24 ELISA

Gag-EGFP protein concentrations in fractions of rEV spiked density gradients were determined with the anti-p24 ELISA kit Innotest HIV antigen mAb (80,563, Fujirebio) (Fig. 1E) according to the manufacturer’s instructions. For recovery calculations a rEV standard curve, from the same batch as used for spiking, was included ranging from 1 × 106-107 fluorescent particles as previously measured with fNTA.

Western blot

Protein concentrations of rEV were measured, after lysis with 0.2% SDS (436143-25G, Sigma-Aldrich), with the Qubit Protein Assay (ThermoFisher) and Qubit fluorometer 3.0 following manufacturer’s instructions. For protein analysis, samples were dissolved in reducing sample buffer (0.5 M Tris-HCl [pH 6.8], 40% glycerol, 9.2% SDS, 3% 2-mercaptoethanol, 0.005% bromophenol blue) and boiled at 95 °C for 5 min. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked for 30 min in blocking buffer (5% non-fat milk in PBS with 0.5% Tween-20) and incubated overnight at 4 °C with primary antibodies (mouse monoclonal anti-ALIX (1:1000, #2171); rabbit monoclonal anti-CD9 clone D3H4P (1:1000, #13403S) (Cell Signaling Technology); and mouse monoclonal anti-flotillin-1 (1:1000, #610,820) (BD Biosciences)). Secondary antibodies (sheep anti-mouse horseradish peroxidase-linked antibody (1:3000, #NA931V) and donkey anti-rabbit horseradish peroxidase-linked antibody (1:4000, #NA934V) (GE Healthcare Life Sciences)) were added for 60 min at room temperature after extensive washing with blocking buffer. After final washing, chemiluminescence substrate (WesternBright Sirius, Advansta) was added and imaging was performed using Proxima 2850 Imager (IsoGen Life Sciences).

Protein measurements

Protein concentrations of the lysed EV preparations obtained from blood plasma and urine samples were measured using the fluorometric Qubit Protein Assay (ThermoFisher) and the Qubit Fluorometer 3.0 (ThermoFisher) according to the manufacturer’s instructions.

LC-MS/MS

EV preparations obtained from blood plasma and urine samples were processed for LC-MS/MS by filter-aided sample preparation (FASP) [40] (Fig. 1E). After thawing and clarification by centrifugation (16,000 g for 5 min), lysates were mixed with 300 µL UA (8 M urea (U5128, Sigma-Aldrich), 0.1 M Tris-HCl (pH 8.5)) in a Microcon-10 kDa centrifugal filter device (MRCPRT010, Merck Millipore). Filters were centrifuged twice (14,000 g for 40 min at 20 °C) with the addition of 200 µL UA in between. Proteins were alkylated by addition of 100 µL IAA solution (0.05 M iodoacetamide (I1149, Sigma-Aldrich) in UA buffer) and incubated for 30 min at room temperature, followed by centrifugation. Samples were treated twice by addition of 100 µL UA and centrifugation. Subsequently, samples were twice treated by addition of 100 µL DB buffer (1 M urea, 0.1 M Tris-HCl (pH 8.5) and centrifugation. Filter units were transferred to new collection tubes and proteins were resuspended in 40 µL DB with Trypsin/Lys-C mix (V5073, Promega) for overnight proteolytic digestion at 37 °C. Digests were collected by addition of 100 µL DB and centrifugation for 15 min at 14,000 g. This step was repeated once. Collected peptides were acidified with 1% trifluoroacetic acid to a pH of 2–3, followed by desalting with Peptide Cleanup C18 Spin Tubes (5188 − 2750, Aligent). Desalted peptides were vacuum dried, dissolved in 0.1% formic acid and analyzed by LC-MS/MS. Equal amounts of peptides of each sample were loaded on a nanoflow HPLC system (Easy- nLC1000, Thermo Fisher Scientific) coupled to a Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific) equipped with a nano-electrospray ionization source. The mobile phase consisted of 0.1% formic acid (solvent A) and acetonitrile/water (95:5 (v/v)) with 0.1% formic acid (solvent B). The peptides were separated with a 40 min gradient from 8 to 35% of solvent B. Before the end of the run, the percentage of solvent B was raised to 100% in 2 min and kept there for 8 min. Full MS scan over the mass-to-charge (m/z) range of 300–1750 with a resolution of 120,000, followed by data dependent acquisition with an isolation window of 2.0 m/z and a dynamic exclusion time of 30 s was performed. The top 12 ions were fragmented by higher energy collisional dissociation (HCD) with a normalized collision energy of 27% and scanned over the m/z range of 200–2000 with a resolution of 15,000. After the MS2 scan for each of the top 12 ions had been obtained, a new full mass spectrum scan was acquired, and the process repeated until the end of the 50 min run. Three repeated runs per sample were performed. Tandem mass spectra were searched using the MaxQuant software (version 1.6.10.43) against a database containing reviewed sequences of homo sapiens of UniProtKB release 2019_11. Peptide-spectrum-match- and protein-level false discovery rates were set at 0.01. Carbamidomethyl (C), as a fixed modification, and oxidation (M) and acetylation of the protein N-terminus as dynamic modifications were included. A maximum of two missed cleavages was allowed. The LC-MS profiles were aligned, and the identifications were transferred to non-sequenced or non-identified MS features in other LC-MS runs (matching between runs). The protein was determined as detected in the sample if its identification had been derived from at least two unique peptide identifications. Filtering for contaminating proteins, reverse identification and identification by site was used. Label-free quantification (LFQ) was performed using the MaxLFQ algorithm integrated in the MaxQuant software.

Proteomic data analysis

Identified proteins were analyzed and visualized using Perseus software version 1.6.15.0 [41]. Proteins showing valid values in at least 70% of at least one group were selected. Reverse database hits and potential contaminant proteins were removed. Missing values were imputed from the observed normal distribution of intensities. LFQ intensities were normalized using the Width adjustment method in Perseus. For selected analyses, the normalized LFQ intensities were log2 transformed. Coefficient of variation (CV) analysis was based on the 100 highest quantified proteins within each sample type. Principal component analysis (PCA) was performed using the Perseus software. Unsupervised hierarchical clustering heat maps, using 1-Pearson correlation, were generated using the Morpheus tool. Analysis of similarities (anosim) was performed using Past3 software [42]. The Vesiclepedia database was explored to identify the 100 most common EV-associated proteins [43]. Quantitative expression profile based functional enrichment analysis was performed FunRich software version 3.1.3 [44].

Transmission electron microscopy

EV preparations obtained from blood plasma and urine were qualitatively analyzed with transmission electron microscopy (TEM) (Fig. 1E). Samples were deposited on a formvar coated grids stabilized with evaporated carbon film and glow discharged before sample application (AGS162-3 H, Agar Scientific). Neutral uranyl acetate (2% in AD) (21447-25, Polysciences) was used for staining after which grids were coated with 2% methyl cellulose (M7027, Sigma-Aldrich) / uranyl acetate (0,4%) solution. These grids were examined using a Tecnai G2 Spirit transmission electron microscope (Thermo Fisher Scientific FEI) operated at 100 kV and images were captured with a Quemesa charge-coupled device camera (Olympus Soft Imaging Solutions).

Information on density measurement, interface mixing, and characterization methods (Fig. 1E) of rEV (fNTA and anti-p24 ELISA) and EV (mass-spectrometry based proteomics and TEM) is provided in the Additional file 1.

Statistical analysis

Data analysis and visualization was performed using GraphPad Prism version 8 (GraphPad Software). Data are expressed as median with interquartile range (IQR). Correlations were calculated using the Pearson product-moment (r). Differences of mean ranks were evaluated by Mann Whitney U test and differences of variance by F-test of equality of variances. P-values smaller than 0.05 were considered statistically significant.