Cell lines and cell culture

Transitional carcinoma cell lines T24 was from the Cell Bank of the Chinese Academy of Sciences (Shanghai, Cat: SCSP-536). Human normal bladder mucosal epithelial HCV29, benigh non-muscle-invasive bladder cancer KK47, and highly malignant invasive bladder cancer YTS-1 cell lines [19,20,21], were kindly gifted by Dr. Sen-itiroh Hakomori (The Biomembrane Institute; Seattle, WA, USA). All cell lines were cultured in RPMI 1640 medium (HyClone; Provo, UT, USA) with 10% fetal bovine serum (FBS) (Biological Industries; Beit Haemek, Israel), 100 IU/mL penicillin, and 100 μg/mL streptomycin at 37 °C in 5% CO2 atmosphere.

Generation of stable transfectants

Human full-length and site-directed mutant integrin β1 were constructed in our laboratory [22]. Full-length and mutant integrin β1 with flag tag were cloned into the pLVX-AcGFP-N1 plasmid (Takara; Shiga, Japan), and transfected into YTS-1 cells as described previously [18]. Stable transfectants were selected using puromycin and confirmed by western blotting.

Chemically synthesized oligonucleotides encoding integrin β1 short hairpin RNA (shRNA) were inserted in lentiviral plasmid Tet-pLKO-puro (Addgene plasmid #21915), and transfected into YTS-1 (termed Y-shβ1).

Western blotting analysis

Total proteins were isolated from cells with RIPA buffer (1% Triton X-100, 5% glycerol, 0.5% sodium deoxycholate, 50 mM Tris, pH 7.2, 0.1% SDS, 150 mM NaCl, 10 mM MgCl2) containing 1% protease inhibitor and phosphatase inhibitor. The lysate was centrifuged at 14,000×g for 15 min at 4 °C. The supernatant was collected, and protein concentration was quantified by bicinchoninic acid (BCA) assay (Beyotime Biotechnology; Jiangsu, China). Proteins were separated by SDS-PAGE and transferred onto PVDF membranes (Bio-Rad; Hercules, CA, USA). Membranes were blocked with 5% (w/v) bovine serum albumin (BSA) in Tris-buffered saline with Tween-20 (TBST) for 1 h at 37 °C, probed with primary antibodies overnight at 4 °C, and incubated with appropriate HRP-conjugated secondary antibody. Bands were visualized by enhanced chemiluminescence (ECL) (Vazyme Biotech; Nanjing, China).

Primary antibodies involved in this study: TSG101 (ab83), Neu1 (ab197020), Integrin β1 (ab183666), CD63 (ab134045; Abcam; Cambridge, UK); Calnexin (2679), β-Tubulin (2146S), Alix (2171S; Cell Signaling Technology; Danvers, Massachusetts, USA); GAPDH (60004-1-lg; Proteintech Group; Inc Rosemont, USA); Fibronectin (sc271098; Santa Cruz Biotechnology; USA); Flag (M20008L; PharmaTech; Shanghai, China). All Primary antibodies were used at a ratio of 1:1000 (v/v).

Lectin blotting analysis

Proteins were separated by SDS-PAGE as described above. PVDF membranes were blocked with phosphate-buffered saline with Tween-20 (PBST) containing 3% (w/v) BSA, incubated with SNA (B-1305) or MAL-II (B-B-1265, Vector Laboratories; Newark, CA, USA) for 12 h at 4 °C, washed with PBST, and incubated with HRP labeled streptavidin (ABC reagent, VECTASTAIN ABC Kit; Vector Laboratories). Bands were visualized by ECL. All lectins are used at a ratio of 1:1000 (v/v).

Immunoprecipitation (IP)

Total proteins (1 mg) were incubated with 1 μg primary antibody for 2 h at 4 °C, then added with 20 μL Protein A/G Plus-Agarose. The mixture was incubated overnight at 4 °C with rotation, washed with PBS, denatured with loading buffer for 10 min at 100 °C, and analyzed by western blotting. Antibodies used in IP assay: Integrin β1 (ab183666); Flag (M20008L).

Preparation of conditioned medium (CM)

Cells were incubated in the FBS-free medium for 48 h. The supernatant was collected and centrifuged at 500×g for 10 min, and then filtered with 0.22 μm filter as CM.

sEV purification by differential ultracentrifugation

To prepare sEV, CM was sequentially centrifuged at 2000×g for 20 min, 10,000×g for 30 min at 4 °C, and ultracentrifuged twice at 100,000×g (model Optima XE-100; Beckman Coulter Life Sciences; Indianapolis, IN, USA) for 70 min. Pellets were resuspended in PBS and stored at − 80 °C.

sEV purification by density gradient centrifugation

sEV were separated by density gradient centrifugation as described previously [23]. Briefly, 40%, 20%, 10%, and 5% (w/v) iodixanol solutions (OptiPrep; Axis-Shield PoC; Oslo, Norway) were prepared by diluting with 0.25 M sucrose/10 mM Tris, pH 7.5, placed in 14 × 89 mm Ultra-Clear tubes, and ultracentrifuged at 100,000×g for 18 h. Twelve fractions were collected, pelleted by ultracentrifugation (100,000×g for 3 h), resuspended in PBS, loaded on SDS-PAGE, and analyzed by western blotting.

Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA)

Purified sEV were morphologically characterized by TEM (model H-7650; Hitachi; Tokyo) at 80 kV as described previously [18]. sEV size distribution was evaluated by NTA (model NanoSight LM10; Malvern Instruments; Malvern, UK).

Removal of sialic acids

Cells in 12-well plates or sEV were treated with 0.05 μg/μL sialidase (S10170, Yuanye Bio-Technology Co.; Shanghai, China) for 1.5 h at 37 °C, which could catalyze the release of the α2,3- and α2,6-sialic acids from glycoprotein. The desialylated sEV were confirmed by lectin blotting and used for further uptake assay.

Identification of sEV proteins

sEV proteins (50 μg) were added in a size-exclusion spin ultrafiltration unit (10 kD; Millipore, Marlborough, MA, USA), denatured with urea, reduced with dithiothreitol (DTT), alkylated with iodoacetamide (IAM), digested with trypsin, desalted with C18 reversed-phase column, lyophilized, and analyzed by LTQ Orbitrap mass spectrometry (MS) (Thermo Fisher Scientific; San Jose, CA, USA) as described previously [24].

The proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE, partner repository with the PXD036973 and https://doi.org/10.6019/PXD036973.

sEV uptake

50 μg sEV were labeled with ExoTracker as described previously [25]. In brief, sEV were incubated with ExoTracker for 1 h at room temperature, and excess ExoTracker were removed using 10 kD ultrafiltration unit. Cells in 24 wells plates were incubated with labeled sEV for 1 h, detached with trypsin, rinsed with PBS, and analyzed by flow cytometry (ACEA Biosciences; San Diego, CA, USA).

ExoTracker labeling efficiency of sEV

50 μg sEV or sialidase-treated sEV were labeled with ExoTracker as described above, and incubated with 50 μL CD63 exosome capture beads (ab239686; Abcam; Cambridge, UK) in the dark overnight at room temperature. Fluorescence signal of sEV were analyzed by flow cytometry.

sEV biotin labeling and uptake detection

sEV were extracted and resuspended to 0.5 μg/μL with PBS buffer, and incubated with 10 μmol/L NHS-LC-biotin (Sigma-Aldrich) with shaking for 15 min at room temperature. Excess NHS-LC-biotin were removed using 10 kD ultrafiltration unit, and the protein concentration was determined. Cells were incubated with 50 μg of labeled sEV for 2 h or 24 h. The amount of sEV entering the cells was detected by western blotting. Recipient cells treated with non-labeled sEV and sialidase were used as control group.

sEV integrin β1 blocking

sEV were incubated with anti-integrin β1 neutralizing antibodies (102201, BioLegend Inc., San Diego, CA, USA) or mouse IgG (A7028, Beyotime Biotechnology; Jiangsu, China) at 1:50 (w/w) for 1 h on the ice.

Patient samples

Plasma samples from bladder cancer patients and healthy volunteers were obtained from Shaanxi Provincial People’s Hospital. Written informed consent was obtained from all patients, in accordance with the Declaration of Helsinki guidelines. Experiments using human plasma were approved by the Research Ethics Committee of Northwest University. Characteristics of bladder cancer patients and healthy volunteers were shown in Additional file 1: Tables S1 and S2.

Enzyme-linked immunosorbent assay (ELISA) of sialic acids on sEV

To determine levels of sialic acid on sEV, sialic acid on CD63 or sialic acid on integrin β1, plasma samples or plasma lysates (plasma lysed with RIPA buffer) were respectively added onto multi-well ELISA plates pre-coated with anti-CD63 antibody or anti-integrin β1 antibody, and incubated for 12 h at 4 °C. The plates were washed with PBST, blocked with BSA, and incubated with biotinylated SNA/ MAL-II for 30 min at 37 °C. The plates were washed with PBST, and incubated with HRP labeled streptavidin for 30 min at 37 °C. The mixture was incubated with 3,3′,5,5′-tetramethylbenzidine (TMB) reagent (Promega; Madison, USA), and quenched with 2 M sulfuric acid. The absorbance at 450 nm was measured.

To determine sialic acid levels, total CD63 levels or total integrin β1 levels in plasma, plasma samples or plasma lysates (plasma lysed with RIPA buffer) were respectively added onto ELISA plates, and incubated for 12 h at 4 °C. The plates were blocked, probed with lectins, antibody against CD63 or antibody against integrin β1, and visualized with TMB reagent as described above.

To determine levels of sialylated integrin β1 on sEV from plasma samples, sEV separated from plasma by ultra-centrifugation was added onto multi-well ELISA plates pre-coated anti-integrin β1 antibody, blocked with BSA, probed with biotinylated SNA/MAL-II, and visualized with TMB reagent as described above.

Cell apoptosis assay

Cells were incubated with sEV (final concentration of 100 μg/mL) for 48 h at 37 °C, detached with trypsin, centrifuged at 1000×g for 5 min, washed with PBS, resuspended in 100 μL 1× binding buffer containing 2.5 μL APC-Annexin V and 2.5 μL 7-AAD (BioLegend; San Diego, CA, USA), incubated for 20 min in the dark, and analyzed by flow cytometry.

Cell proliferation assay

Cells with a confluence of 40%-50% were incubated with CM or sEV (final concentration of 100 μg/mL) for 24 h, processed for EdU incorporation using baseclick EdU Kit (Promega; Madison, USA), and analyzed by flow cytometry.

Transwell assay

Cell culture inserts (pore size 8 μm; Corning; Corning, NY, USA) were used as per the manufacturer’s instructions. Cells (1 × 104) were starved in serum-free medium added sEV (final concentration of 100 μg/mL) for 24 h, seeded in upper chambers, added with RPMI 1640 complete medium to the bottom chamber, and incubated for 36 h. Cells on the upper surface of each filter were removed with cotton swabs, which migrated across the membrane were stained with 0.1% crystal violet, and photographed under microscopy.

sEV pre-conditioning of mice

All mouse experiments were approved by the Animal Care and Use Committee of Northwest University. sEV (20 μg in 100 μL PBS) were centrifuged at 4600×g for 1 min at 4 °C to remove sedimentable aggregates, then i.v. injected into 6- to 8-week-old Balb/c nu/nu mice twice per week.

Liver/lung colonization studies

Six- to 8-week-old female Balb/c nu/nu mice pre-conditioned with sEV were injected with 2 × 106 YTS-1 cells via tail vein. Mice were euthanized 8 weeks after injection. Lungs were fixed, sectioned, and stained with haematoxylin and eosin (H&E).

Data analysis

Each experiment was performed with three or more replicates. Statistical analyses were performed using the software program GraphPad Prism V. 8.0. Data from two groups were compared by two-tailed Student’s t-test, and results were presented as mean ± SD. Differences with p < 0.05 were considered statistically significant. Notations in figures: *, p < 0.05; **, p < 0.01; ***, p < 0.001.