Cell lines and cell culture
MDA-MB-231 and MCF-7 human breast cancer cells as well as HUVECs were purchased from the Cell Resource Center, Shanghai Academy of Life Sciences, Chinese Academy of Sciences. The purchased HUVECs were primary cells, and during the experiment, the cells passed through about 5-6 generations. MDA-MB-231 and MCF-7 cells were cultured in High Glucose-Dulbecco's Modified Eagle Medium (H-DMEM; Gibco, USA) with 10% fetal bovine serum (FBS; Bioind, ISR), 100 U/ml penicillin, and streptomycin (Beyotime BioteCNology, #C0222, CN) under mycoplasma‑free conditions at 37°C in 5% CO2 for the collection of culture supernatants and EVs. HUVECs were grown in DMEM-F12 (Gibco, USA) with 10% FBS, 100 U/ml penicillin, and streptomycin under mycoplasma‑free conditions at 37°C in 5% CO2. Mycoplasma detection was performed before culture using a mycoplasma detection kit (ThermoFisher, #4460626, USA). Cells in the log-growth phase were used for subsequent experiments.
EV isolation and characterization
MDA-MB-231 and MCF-7 cells were grown in H-DMEM containing 10% FBS until they reached 70%–80% confluency. The supernatant was then discarded and the cells were washed twice with phosphate-buffered saline (PBS). Then, H-DMEM containing 10% EV/exosome-depleted FBS (Thermo Fisher Scientific, #A2720803, USA) was used for subsequent 8-h culture. Next, the culture supernatant was collected and centrifuged at 2,000 × g for 30 min at 4°C to remove the cells and debris. Cell-free supernatants were transferred to a special ultracentrifuge tube and centrifuged at 10,0000 × g for 60 min. About 2 ml of the supernatant was retained and then centrifuged at 10,0000 × g for 60 min. During this step, the centrifuge tube was at least three-quarters full. PBS was used to make up the necessary volume when necessary. Finally, the supernatant was discarded, leaving a sample of about 100 μl. EVs were isolated using the above steps and stored at ‑80°C until further use. The morphological characteristics of EVs were observed using a transmission electron microscope after isolation. The EV size was determined via nanotracking analysis using a zetasizer (Malvern, UK). The expression of EV surface biosignature protein CD9 and CD63 in breast cancer cell lines was detected using western blotting. The quantity of EVs in subsequent experiments was determined by the protein content measured using a BCA Protein Assay Kit (Beyotime BioteCNology, #P0012, CN).
EV internalization
MDA-MB-231 and MCF-7 cell-derived EVs were labeled with CM-Dil and PBS was used as the control. After incubation with HUVECs for 6 h, HUVEC membranes were labeled with DiO (Beyotime BioteCNology, #C1993S, CN), while HUVEC nuclei were labeled with Hoechst 33342 (Beyotime BioteCNology, #C1026, CN). The internalization of EVs into HUVECs was observed using laser scanning confocal microscopy.
Cell proliferation assay
The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to examine HUVEC proliferation. HUVECs were harvested and seeded into 96-well plates at a density of 2 × 103 cells/well and incubated at 37°C in 5% CO2. The medium was discarded on the following day and 10, 20, or 40 μg/ml of MDA-MB-231 and MCF-7 cell-derived EVs were added and incubated for 0 h, 24 h, 48 h, and 72 h. PBS was used as the control. The 96-well plate medium was discarded at each time point and 20 μl of 5 mg/ml MTT (Amresco, C4825, USA) were added to each well and incubated for 4 h. When the reaction was terminated, the reagent was discarded and 150 μl of dimethyl sulfoxide were added to each well. The samples were then placed on an oscillator and agitated for 10 min in the dark to ensure adequate crystal dissolution. Optical density of each sample was determined at 490 nm using a microplate reader (BioTek, Winooski, VT, USA) [29]. Repeat the experiment three times.
Colony formation assay
HUVECs were treated with 10, 20, and 40 μg/ml of MDA-MB-231 and MCF-7 cell-derived EVs. PBS was used as the control. In some experiments, HUVECs were treated with 10 µM AG490 (CST, #14704, USA), a specific tyrosine phosphorylation inhibitor that inhibits the JAK2/STAT3 pathway, for 30 min prior to the addition of MDA-MB-231 and MCF-7 cell-derived EVs. The collected cells were plated at a density of 500 cells/well in a six-well plate and cultured at 37°C with 5% CO2 for 10 days. The cells were then fixed with 4% formaldehyde for 30 min and stained using crystal violet for 15 min at room temperature, followed by manual counting of the cell colony numbers. Repeat the experiment three times.
Transwell migration assay
HUVECs were treated with 10, 20, and 40 μg/ml of MDA-MB-231 and MCF-7 cell-derived EVs for 24 h in a transwell chamber (Corning, USA). PBS was used as the control. Cells were then counted, resuspended at a concentration of 500 cells/well in 100 μl of serum-free medium, and added into the upper transwell chamber. The lower chamber was filled with 800 μl of DMEM-F12. In some experiments, HUVECs were treated with 10 µM AG490 for 30 min prior to the addition of MDA-MB-231 and MCF-7 cell-derived EVs. Following incubation at 37°C and 5% CO2 for 8 h, the medium in the upper chamber was removed and the non-migrated cells were removed with cotton swabs. Cells in the lower chamber were fixed with 4% formaldehyde for 30 min and stained using crystal violet for 15 min at room temperature, followed by observation of three random visual fields under a microscope. Repeat the experiment three times.
Scratch migration assay
HUVECs were treated with 10, 20, and 40 μg/ml of MDA-MB-231 and MCF-7 cell-derived EVs for 24 h. PBS was used as the control. In some experiments, HUVECs were treated with 10 µM AG490 for 30 min prior to the addition of MDA-MB-231 and MCF-7 cell-derived EVs. The cells were then counted, seeded at a density of 1 × 105 cells/well into six-well plates, and cultured at 37°C and 5% CO2 until reaching 80% confluency. The six-well plates were scraped in a straight line with a pipette tip, washed with serum-free culture medium to remove unattached cells, and examined under a microscope. Representative images were obtained at 0 h, 12 h, and 24 h. Cell migration was determined by the final scratch healing rate from 0 to 24 h and was calculated using the following equation: (scratch length at 0 h - distance of cell migration at 24 h/scratch length at 0 h) × 100%. Repeat the experiment three times.
Tube formation assay
Formation of HUVEC tubes was carried out in matrigel-coated 96-well plates (Corning, USA). The 96-well plates were pre-chilled overnight, coated with 50 μl of pre-chilled matrigel, and kept at 37°C for 30 min before the assay. HUVECs were treated with 10, 20, and 40 μg/ml of MDA-MB-231 and MCF-7 cell-derived EVs for 24 h. PBS was used as the control. In some experiments, HUVECs were treated with 10 µM AG490 for 30 min prior to the addition of MDA-MB-231 and MCF-7 cell-derived EVs. The collected cells were trypsinized and seeded onto the coated plates at a density of 3 × 104 cells/well in 10% FBS DMEM-F12 medium followed by an 8-h incubation. Tube formation was observed under a microscope and photographed. Five visual fields were randomly selected, where the number of junctions was measured and analyzed using Image J (NIH). Repeat the experiment three times.
Western blot analysis
HUVECs were treated with 10, 20, and 40 μg/ml of MDA-MB-231 and MCF-7 cell-derived EVs for 24 h. Cell lysate was used as the control. In some experiments, HUVECs were treated with 10 µM AG490 for 30 min prior to the addition of MDA-MB-231 and MCF-7 cell-derived EVs. The culture medium was then discarded, the cells were lysed with RIPA buffer (Beyotime BioteCNology, #P0013, CN), and total protein samples were extracted on ice. The protein concentration was quantified using a BCA kit. Briefly, the extracted protein samples were loaded and separated in 10% sodium dodecyl sulphate-polyacrylamide gels (SDS-PAGE) and then transferred onto polyvinylidene difluoride membranes using the wet spinning method. The membranes were sealed with 5% skimmed milk at room temperature for 2 h and then incubated at 4°C overnight with the following primary antibodies: 1:1,000 rabbit anti-human CD63 (ABclonal, #A19023, USA), 1:1,000 rabbit anti-human CD9 (ABclonal, #A1703, USA), 1:1,000 rabbit anti-human VEGFA (Cohesion Biosciences, #CPA7272, UK), 1:1,000 rabbit anti-human JAK2 (CST, #3230, USA), 1:1,000 rabbit anti-human p-JAK2 (Tyr1007; CST, #4406, USA), 1:1,000 rabbit anti-human STAT3 (Absin, #143297, CN), 1:1,000 rabbit anti-human p-STAT3 (Ser727; WanleiBio, #WLP2412, CN), and 1:1,000 mouse anti-human β-actin as the loading control (Santa Cruz, #sc-47778, USA). The membranes were then washed for 15 min using tris-buffered saline-Tween (TBST) three times and incubated with 1:5,000 horseradish peroxidase-labeled goat anti-rabbit/goat anti-mouse IgG (Absin, CN) at room temperature for 2 h. Then, wash three times with TBST for 15 minutes each time, the membranes were exposed and developed to visualize the target protein bands. This was followed by the quantitative analysis of the ratio of protein gray value to β-actin [30]. Repeat the experiment three times.
Statistical analysis
Statistical analysis was performed using GraphPad Prism 7.0 software (GraphPad, San Diego, CA, USA). Measurement data were expressed as mean ± standard deviation (± s). One-way ANOVA was used for mean array comparison of multiple samples. further Dunnett-t test. P < 0.05 was considered to indicate statistical significance.