Cell lines and culturing
MDA-MB-231 (malignant) and MCF 10A (non-malignant) breast cell lines were purchased from ATCC and maintained in DMEM (Dulbecco’s Modified Eagle Medium) and DMEM-F12 respectively. DMEM was supplemented with 10% fetal bovine serum (FBS) and DMEM-F12 was supplemented with 5% horse serum (HS). In experiments using SEVs, culture media were supplemented with SEV depleted serum (SEV-). To prepare FBS SEV- and HS EV-, the sera were ultracentrifuged at 100,000 x g overnight, and the supernatant was collected. Cells were cultured at 37 ºC in 5 % of CO2 atmosphere. Cell number was counted using a TC20 automated cell counter (Bio-Rad, Hercules, CA, USA) with 0.4% trypan blue (Thermo Scientific, Waltham, MA, USA) prior experiments in order to check cell viability.
pLenti-GFP-CD63 plasmid, previously described by Hoshino et. al., was used to make MDA-MB-231 cells stably expressing GFP-CD63. Human MDA-MB-231 cells (ATCC) and 293 FT packaging cells were grown in DMEM + 10% FBS. 293 FT cells transfection, viral harvest, and transduction of MDA-MB-231 cells were performed as previously described . Transduced cells were selected with 4 µg/ml puromycin for 7 days.
Recombinant DisBa-01 was isolated from inclusion bodies of E. coli BL21(DE3)-pET28a-DisBa-01 culture and purified to homogeneity as previously described.  Purified disintegrin was labeled with Alexa Fluor 546 (Invitrogen) according to the manufacturer’s instructions.
Isolation and purification of EVs from conditioned media
For EV isolation, 2.0 x 106 MDA-MB-231 cells were plated in T-150 flasks containing 15 mL culture media (total 10 flasks) and cultured for 48 h until reach 80% confluence. The culture media was replaced with 15 mL of Opti-MEM without serum and cells were further cultured for 48 h to obtain conditioned media. Conditioned media was submitted to serial centrifugation to respectively sediment live cells (300 x g for 10 min), dead cells (2,000 x g for 25 min), and large EVs (10,000 x g, Ti40 rotor for 30 min). The supernatant was concentrated to 30 ml volume in a concentrator (Sartorius, VS6041Cat# and 100 k MWCO), layered over 2 ml of 60% iodixanol in an ultracentrifuge tube (25 x 89 mm for SW 32 Ti rotor), and further centrifuged at 100,000 x g for 4 h. 3 ml was collected from the bottom of the tube and layered in a new centrifuge tube (40% iodixanol). Iodixanol solutions (20% wt/vol, 10% wt/vol, and 5% wt/vol) were layered over from the bottom to the top. Iodixanol solutions were prepared by diluting OptiPrep (60% wt/vol aqueous iodixanol; Axis-Shield PoC) with 0.25 M sucrose/10 mMTris, pH 7.5. The gradient was centrifuged at 100,000 x g for 18 h using a SW45 Ti rotor and 12 fractions of 1 ml each were collected. 2 ml of PBS was added to 1 ml fractions and ultracentrifuged in a tabletop centrifuge at 100,000 x g for 3 h using a TLA 100.3 rotor. Vesicles were resuspended in 50 mL PBS for subsequent studies.
Purified LEVs and SEVs were quantitated by Particle Metrix ZetaView PMX 110 and the protein amount was measured using microBCA.
We also uploaded all relevant data of our experiments to the EV-TRACK knowledgebase (EV-TRACK ID: EV190006) (Van Deun J, et al. EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research. Nature methods. 2017;14(3):228-32).
Characterization of purified EVs
Transmission electron microscopy: For negative staining of purified SEVs, 5 mL of EV samples was added to Formvar carbon film-coated grids (FCF-200-Cu; Electron Microscopy Sciences; Hatfield, PA) for 60 sec. Grids were immediately fixed with 4% paraformaldehyde in water for 20 min, stained with 2% unranyl acetate for 2 min, and allowed to air-dry. For each step, the excess of solution was removed by wicking with a filter paper. The grids were imaged using a TEM Tecnai F20 G2, 200Kv in 40000 x magnification.
Western blotting: Purified EVs were lysed with 1% SDS 50mM Tris pH 7.6-lysis solution, mixed with SDS sample buffer, and loaded onto 8% acrylamide gels (10 ml). Gels were transferred to nitrocellulose membranes and blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.05% Tween 20 (TBS-T) for 1-2 h. Membranes were probed with antibodies for EV markers, anti-CD63 (Abcam, ab59479), anti-Flotillin (BD, 610821), and anti-Alix (Sigma, SAB 4200476). As a negative control, anti-calnexin (Cell Signaling, mAb 2679) was used. Appropriate secondary antibodies were added and detected by ECL (Thermo Scientific, 32106 and 34095). The same procedure was applied to detect integrins and ECM proteins, fibronectin (Abcam, ab2413) and collagen (Abcam, ab34710).
Adhesion of isolated SEVs to different ECM proteins
A ninety-six well plates were coated with collagen (10 mg/ml) or fibronectin (2 mg/ml) overnight at 4 ºC. For the experiment, isolated SEVs were labeled with ExoGlow (System Bioscience Uniscience) according to the manufacturer’s instructions. Prior to incubation, vesicles were incubated with DisBa-01 in different concentrations (250, 500 and 1000 nM) in ice for 30 min and then plated (1.0 x 108 vesicles/well) over the coating for 4 h. After incubation, non-adherent SEVs were washed out and photomicrographs were acquired using a Nikon Plan Apo 60x/1.40 NA oil immersion lens in a Nikon Eclipse TE2000E microscope. For fluorescence intensity analysis, adhered green fluorescent vesicles were segmented from the background by thresholding and measured for integrated intensity using Image J Fiji (Analyze tab/Measure).
Cell adhesion on EV coating
Isolated small or large EVs were resuspended in PBS and added to a 96 well plate overnight (50 mg/ml). Prior to adhesion experiments, the wells were blocked with 1% BSA for 1 h. Fibronectin-coated wells (10 mg/ml) were used as a positive control of cell adhesion. 100 nM DisBa-01 was incubated on EV coating for 30 min. After washing unbound DisBa-01 using PBS, calcein-labeled MDA-MB-231 cells (5 x 106) were added and allowed to attach for 1 h. To measure adhesion, green fluorescence intensity was read in a SpectraMax I3 (Molecular Devices) plate reader.
SEV uptake by healthy breast cells
Uptake of purified SEVs: One day before the experiment, MCF 10A cells were plated in a 96 well plate in a density of 2.5 x 103 cells/well. Small EVs were labeled with ExoGlow kit (System Biosciences) and subsequently treated with DisBa-01 (100 or 1,000 nM). After treatment with the integrin inhibitor, 1.4 x 107 vesicles/well were added over the MCF 10A cells and allowed to internalize for 4 h. After incubation, the supernatant was removed and cells were washed. The uptake of ExoGlow-labeled SEVs was analyzed by epifluorescence microscopy in automated system ImageXpress Micro XLS (Molecular Devices) using a Nikon S Plan Fluor ELWD 40X /0.60 NA magnification lens.
Co-culture in transwell system: MCF10-A cells (1 x 103) were plated on glass coverslips inside a 24-well plate. After MCF 10A cells adhesion, transwell inserts with pore size of 0.4 mm were added to the wells. MDA-MB-231cells expressing GFP-CD63 (1 x 104, treated and non- treated with DisBa-01 at 1000 nM for 30 min prior incubation) were added to the transwell inserts and incubated for 6h. After incubation, MCF 10A cells were stained with DAPI and imaged in a Zeiss LSM 780 confocal microscope using a 63 X/1.3 NA objective lens.
To measure the vesicles in MCF 10A cells, the green fluorescence was quantitated by integrated intensity analysis. Percentage of inhibition was calculated by comparing the integrated intensity of green fluorescence in MCF 10A cells incubated with DisBa-01-treated MDA-MB-231 cells expressing GFP-CD63 to that incubated with DisBa-01-non-treated MDA-MB-231 cells expressing GFP-CD63.
Imaging for co-culture
Fluorescence microscopy: MCF 10A cells (1.5 x 105) were cultured on glass coverslips for 48 h. After adhesion, cells were labeled with Cell Tracker Red CMTPX according to manufacturer instructions (Invitrogen, C34552). MDA-MB-231 cells (1.0 x 106) labeled with Cell Trace CFSE (Life Technologies, C34554) according to manufacturer instructions or stably expressing GFP-CD63 were treated with 100 nM or 1,000 nM DisBa-01 for 30 min (untreated cells were used as a control), plated over an MCF 10A monolayer, and incubated for 24 h. Cells were fixed with 4% paraformaldehyde and stained with DAPI. Z-stack images were acquired in a Zeiss LSM 880 with Airyscan confocal microscope, using a 63x/1.40 Plan-APOCHROMAT oil lens and quantitated by integrated intensity using Fiji (Analyze tab/Measure). For extracellular SEV quantification, cell bodies were carefully selected and deleted from each image. GFP-CD63 deposits surrounding cells were segmented from the background by thresholding and measured for area and integrated intensity using Fiji (Analyze tab/Measure).
Scanning electronic microscopy (SEM): MCF 10A cells (2.0 x 104) were plated in a Lab-TeK® chamber slideTM (LOBOV, Catalog Number: 177402) and incubated at 37 ºC overnight. At the next day, MDA-MB-231 cells (DisBa-01-treated or non-treated) were plated at the same density over the MCF 10A cells and allowed to adhere for additional 24 h. Cells were then washed with PBS, fixed with 4% glutaraldehyde (Sigma-Aldrich®) for 1 h at 37 ºC, and dehydrated by increasing ethanol concentration (50, 60, 70, 80, 90 and 100%, 10 min for each steps) before drying with hexamethyldisilazane (Sigma-Aldrich®). Cell morphology was characterized by scanning electronic microscopy (Inspect F50 - FEI®).
DisBa-01 uptake assay
DisBa-01 was labeled using Alexa Fluor® 546 dye (Invitrogen, Thermo Scientific) according to manufacturer’s instructions. MDA-MB-231 expressing GFP-CD63 cells (1 x 104) were plated in 8-well Nunc™ Lab-Tek™ Chamber Slide (Thermo Scientific) and incubated overnight. At the next day, cells were incubated with DisBa-01 (1,000 nM) for 1 h and 4 h, fixed with 4% paraformaldehyde, and stained with DAPI. Slides were mounted with mounting media ProlongTM (Thermo Fisher Scientific). Confocal images were acquired in a in a Zeiss LSM 780 confocal microscope using a 63 X/1.3 NA objective lens. Zen software was used to acquire images and laser power was same for all conditions in order to compare the fluorescence intensity between different conditions.
Data analysis and statistics
At least three independent experiments were performed to acquire data for quantitation. All data sets were tested for normality using Shapiro-Wilk normality test in GraphPad Prism software. Mean ± standard error of the mean (SEM) were calculated and intergroup comparisons were made using One-way ANOVA or t Test (two-tailed paired or unpaired with Welch’s correction) analysis. Values of P < 0.05 were considered statistically significant.