Patient and specimen collection
Serum samples from 60 patients with breast cancer (female) and 20 healthy volunteers (female) were collected at Tianjin Medical University Cancer Institute and Hospital between September 2019 and November 2019. Thirty cases of serum were collected from patients with early-stage operable invasive breast cancer, which did not receive any systemic treatment before specimen collection. Another 30 cases of serum were collected from patients with advanced breast cancer; these patients have received at least one prior line of systemic therapy. The serum samples were also collected from 20 healthy volunteers with matching ages and genders to the patients. Table 1 provides the clinicopathological characteristics of these patients enrolled in this study. The serum samples were centrifuged at 1,000 g for 10 min. Serum specimens were stored at −80 °C. This study was approved by the Ethics Committee of Tianjin Medical University Cancer Institute and Hospital and was conducted in accordance with the Declaration of Helsinki.
Cell lines and cell culture
Human embryonic kidney 293T (HEK-293T) and human breast cancer cell lines MDA-MB-468 and T47D were obtained from American Type Culture Collection. The drug-resistant cell line MDA-MB-468/EPR was established by our group in previous study. Human breast cancer cell line MCF-7 and its drug-resistant cell line MCF-7/ADR were provided by Henry Ford Hospital in Detroit, Mississippi, USA. T47D, MCF-7, and MCF-7/ADR cells were cultured in RPMI-1640 medium (Hyclone, Logan, UT, USA). MDA-MB-468 and MDA-MB-468/EPR cells were cultured in DMEM/F12 medium (Hyclone, Logan, UT, USA). HEK-293T cells were cultured in DMEM/high-glucose medium (Hyclone, Logan, UT, USA). All media were supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA). FBS exosomes were depleted by ultracentrifugation at 100,000 g for 16 h, followed by sterile filtering with 0.22 µm filters, to exclude the influence of serum exosomes on the cell functional activities. The conditioned medium (CM) was prepared by incubating cells for 12 h in serum-free medium and filtered through a 0.22 µm filter to remove cells and cellular debris.
Exosomes isolation from cells
Exosomes were obtained from cell culture medium as previously described [39]. In brief, the cell culture medium was collected and centrifuged at 300 g for 10 min to remove cells, and then the supernatant was centrifuged at 3,000 g for 10 min to remove cell debris, followed by centrifugation at 10,000 g for 30 min at 4 °C to remove large vesicles. The supernatant was further centrifuged at 100,000 g for 90 min at 4 °C. The exosomal pellets were resuspended in PBS and then centrifuged again at the same speed. The purified exosomes were further characterized and analyzed.
Exosomes isolation from human serum samples
In the method established by Kahlert C. et al. [40, 41], 500 μL of serum samples was thawed on ice. The serum was diluted in 12.5 mL PBS and then ultracentrifuged at 160,000 g overnight at 4 °C. Next, the exosomal pellets were washed in PBS, followed by a second step of ultracentrifugation at 160,000 g at 4 °C for 2 h. The supernatant was discarded, and the exosomal pellets were resuspended in 100 μL of PBS.
Characterization of purified exosomes
For transmission electron microscopy (TEM) analysis, exosomes suspended in PBS were dropped on formvar carbon-coated grid, incubated for 5 min, and then stained with 2% phosphotungstic acid for 2 min. The grid was dried in air for 5–10 min. Images were obtained using a TEM device (HT7700, HITACHI Company) at 80 kV. In addition, the size and concentration of exosomes were tracked using the NanoSight NS300 device (Malvern Instruments).
PKH26 staining
Exosomes were stained with the PKH26 Red Fluorescent Cell Linker Kit (Sigma-Aldrich), according to the manufacturer’s instructions with minor modifications. First, exosomes were diluted in 250 μL of diluent C. Second, 1 μL of PKH26 dye was added to another 250 μL of diluent C, and then the exosomes and dye were mixed together by gently pipetting, followed by incubating at room temperature for 3 min. Then, 500 μL of FBS was added to the mixture to quench the excess dye. Finally, the sample was diluted in 12.5 mL PBS and ultracentrifuged at 100,000 g at 4 °C for 90 min, followed by resuspending in fresh medium.
Enzyme-Linked Immunosorbent Assays (ELISA)
96-well ELISA plates (Biolegend, CA, USA) were coated with 50 µL/well of a 1:100 dilution of anti-human CD81 antibodies (0.2 mg/mL, R&D Systems) and incubated overnight at 4 °C. After washing three times with PBS, the plates were blocked with 5% BSA in PBS with 0.05% Tween-20 (PBST) at room temperature for 2 h (50 µL/well). Then, serum exosome samples (100 µL/well) were added into the plate and incubated overnight at 4 °C. After three washes with PBST, 50 µL of anti-human EphA2 antibodies (0.2 μg/mL, Novus) was added and incubated at 37 °C for 1 h. The plates were then washed three times with PBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (BIORAD) at room temperature for 1 h (100 μL/well). After three times final washes with PBST, plates were incubated with 50 μL/well TMB reagent (CST) at room temperature for 10–15 min, followed by the addition of 50 μL/well of stop solution (2M H2SO4). The absorbance was read at 450 nm using a micro-ELISA reader.
Western blotting
Western blotting was performed as described previously [17]. In brief, whole-cell lysates or exosomal proteins were separated by SDS–PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% milk for 1 h at room temperature and then incubated with the corresponding primary antibodies overnight at 4 °C. The following antibodies were used: TSG101 (1:200, Santa Cruz, CA), CD81 (1:200, Santa Cruz, CA), Alix (1:1000, CST, MA, USA), ERK (1:1000, CST, MA, USA), p-ERK (1:1000, CST, MA,USA), Akt (1:1000, CST, MA, USA), p-Akt (1:1000, CST, MA, USA), STAT3 (1:1000, CST, MA, USA), p-STAT3 (1:1000, CST, MA, USA), EphA2 (1:1000, CST, MA, USA), EphA2 (1:200, Santa Cruz, CA, USA), Rab27a (1:1000, Abcam), and β-actin (1:10000, Sigma-Aldrich). After washing three times with TBST, the membrane was incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. The signals were visualized with the ECL kit. CD81, Alix, and TSG101 were used as exosomal markers. β-actin was used as a loading control.
Wound healing and transwell assay
Wound healing assay was performed as described previously [17]. Cells were cultured to confluence in 6-well plates and then treated with CM for 12 h. Then, a 10 μL pipette tip was used to scrape a wound on the cell monolayer. After washing two times with PBS to remove the detached cells, the medium was replaced with fresh CM containing 2% exosome-depleted FBS. The plates were then incubated at 37 °C for 48 h in 5% CO2. The width of the wound gap was captured under an inverted microscope. Transwell assay was performed by using a Boyden chamber with a pore size of 8 μm as described previously [17]. The cells were pre-treated with exosomes for 24 h to study the effect of exosomes on migration and invasion of cancer cells. Then, transwell assays were performed with or without Matrigel. For cell migration assay, 5 × 104 cells suspended in 200 μL of serum-free medium were loaded onto the upper chambers. 600 μL of medium with 10% FBS was added into the lower chamber. For cell invasion assay, 1 × 105 cells suspended in 200 μL of serum-free medium were loaded onto the upper chambers coated with Matrigel. After incubation at 37 °C for 24 h, the migrated or invaded cells were fixed, stained, and captured by a microscope at 200 ×.
Immunofluorescence assay
Immunofluorescence assay was carried out as described previously [15]. In brief, cells were seeded in 12-well plates containing glass coverslips and incubated at 37 °C for 12 h in 5% CO2. Afterward, the cells were fixed with 4% PFA/PBS and permeabilized with 0.1% Triton X-100 in PBS for 10 min, followed by blocking with 3% BSA/PBS for 1 h. Then, the cells were incubated with primary antibodies overnight at 4 °C. After washing three times with PBS, the cells were then stained with Alexa Fluor 488-conjugated secondary antibodies at room temperature for 1 h in the dark, followed by nuclear staining by using 1 ng/mL of DAPI. The coverslips were mounted and observed by using a laser scanning confocal microscope (Zeiss Axio Imager).
Vector construction and stable transfection
EphA2, Ephrin A1, and Rab27a-specific shRNA sequences were subcloned into a lentiviral vector, pLko.1-hygromycin, in the BamH Ӏ and Age Ӏ cloning sites. The sequences of the shRNAs are listed in Supplementary Table 1. The EphA2-coding sequences were cloned from cDNA plasmid purchased from ORIGENE (Beijing, China) using polymerase chain reaction (PCR). The truncation mutants tagged with mCherry (EphA2-ΔS and EphA2-ΔL) were created by overlapping PCR and cloned into a linearized pCDNA3.1 vector using a ClonExpress II one-step cloning kit (Vazyme Biotech, Nanjing, China). The point mutation of mCherry-tagged EphA2 (S987A) was introduced by PCR-based site-directed mutagenesis and cloned into the pCDNA3.1 vector. The Flag-tagged Ephrin-A1 was amplified from human cDNA using PCR and cloned into a linearized pCDNA3.1 vector using a ClonExpress II one-step cloning kit (Vazyme Biotech). All the plasmids were confirmed by restriction digestion and DNA sequencing. The primers used for amplification of Ephrin A1, EphA2, and its mutants are listed in Supplementary Table 2. Plasmid transfections were performed using Lipofectamine 3000 (Thermo Fisher Scientific, CA, USA), according to the manufacturer’s instructions.
Co-immunoprecipitation assay
Co-immunoprecipitation assay was performed as described previously [42]. In brief, cells were washed three times with ice-cold PBS, solubilized with lysis buffer (40 mM Tris, 150 mM NaCl, 1% Triton X-100, 50 mM NaF, 5 mM Na3VO4, 2 mM EDTA, and protease inhibitor cocktail), and incubated on ice for 1 h. Lysates were then centrifuged at 12,000 g for 15 min at 4 °C. The supernatants were pre-cleared for 1 h with protein A-conjugated agarose beads, followed by incubation with 1 μg of anti-Flag antibody overnight at 4 °C. The immunocomplex was incubated with protein A agarose beads for 1 h at room temperature. The beads were then washed three times with cell lysis buffer. The final pellets were resuspended with 2× SDS sample buffer. The samples were then analyzed by Western blotting.
Mass spectrometric analysis and bioinformatics analysis
The exosome samples were prepared in three biological replicates from the CM of MDA-MB-468 and MDA-MB-468/EPR cells. Then, the exosome samples were processed for tandem mass tag (TMT) quantitative proteomic analysis by PTM BioLab (Hangzhou, China). The detailed procedure was described in supplemental methods.
For gene set enrichment analysis (GSEA), Pearson’s correlation value was calculated between EPHA2 and all protein-coding genes in TCGA-BRCA RNAseq data and subjected to WebGsetalt database (http://www.webgestalt.org/). The GSEA was performed using the KEGG gene sets.
In vivo metastasis assay
Four-week-old female SCID mice were purchased from Beijing Charles River (Beijing, China). All animal work procedures were approved by the Animal Ethical and Welfare Committee of Tianjin Medical University Cancer Institute and Hospital. The mice were randomly allocated to five groups (six mice/group). 5×106 cells (MDA-MD-468, MDA-MD-468/EPR, control, and EphA2 stable knockdown MDA-MD-468/EPR cells) were subcutaneously injected into the mammary fat pad of SCID mice. After injection, mouse weight and tumor size were measured once a week, and the subcutaneous tumor volume was calculated via the standard modified formula volume (mm3) = 1/2 (height2 × length). 1×106 GFP-labeled T47D cells were injected into SCID mice via tail veins when the tumor size reached 1 mm3. Two months after injection, the mice were anesthetized, and their peripheral blood was collected. Then, the mice were sacrificed, and the lung tissues were dissected and fixed in 4% neutral-buffered formalin. Afterward, the tissues were paraffin-embedded for H&E staining and immunohistochemical staining. The metastatic nodules were counted by H&E-stained tissues. Immunohistochemical staining was performed with anti-GFP antibodies to confirm the origin of the metastatic cancer cells.
Statistical analysis
All data were presented as mean ± SD of at least three independent experiments. GraphPad Prism 7.0 software was used to conduct statistical analysis. One-way or two-way ANOVA tests were performed for statistical analysis of the differences among groups. P< 0.05 was considered statistically significant.