Tissue specimens(n=134) were collected from female patients with TNBC, defined as ER negative, PR negative, HER2 negative or 1+ or 2+ but fluorescence in situ hybridization (FISH) negative, who underwent operation of tumor in Fudan University Shanghai Cancer Center (Shanghai, China) between August of 2015 and May of 2016. After selection of formalin-fixed paraffin embedded (FFPE) tissue blocks of tumor sections, 5 tissue microarrays (TMA) blocks with 50 cores per TMA were constructed. Sections were cut at 4µM thick for immunohistochemistry. Clinical characteristics were obtained from the Electronic Medical Record System and telephone follow-up. The research protocol was approved by the Ethics Committee of the Fudan University Shanghai Cancer Center, and written informed consent was obtained from all patients prior to enrollment.
Standard immunohistochemical procedures were carried out using anti-MMP-1(26585-1-AP, Proteintech), anti-PAR1(26366-1-AP, Proteintech) in human breast cancer TMAs and lung tumor sections of nude mice. The TMAs were digitized via whole-slide scanning with the Aperio T2 scanner (Aperio Technologies) and IOD values were assessed independently by two pathologists in from the Department of Pathology (Fudan University Shanghai Cancer Center); discrepancies were resolved by discussion until a consensus was reached. Lung tumor sections of nude mice were observed and imaged by microscope (OLYMPUS, BX43).
Cell lines and culture
MDA‑MB‑231, MDA-MB-468 and BT549 (human breast cancer cell line) were acquired from the Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). MDA-MB-231-HM cell line and was established from MDA-MB-231 using an in vivo stepwise selection scheme in Fudan University Shanghai Cancer Center (Shanghai, China), has a high potential for lung metastasis14. MDA‑MB‑231, MDA-MB-468 and MDA-MB-231-HM were grown in Leibovitz’s L-15 medium, BT549 was cultured in RPMI 1640, containing 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 mg/ml) in incubator at 37˚C (both Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA).
Tumor cell migration and invasion assays
24‑well plates inserted with 8‑mm pore size filters (Corning Life Sciences, Corning, NY, USA), for tumor cell invasion ability, the filter membranes were pre‑coated with 20µl Matrigel (dilution, 1:5; BD Biosciences, Franklin Lakes, NJ, USA). Cells were seeded into the upper chambers, with L-15 containing 20% FBS in the lower chambers. Cells were incubated for 24-36h at 37˚C, and cells that had invaded or migrated to the reverse side of the membrane were detected by staining with crystal violet, viewed and counted in at least five random fields under a light microscope (OLYMPUS, IX51).
A Cell Counting Kit-8 (PN812; Dojindo, Japan) was used for cell proliferation assays. Cells were seeded into a 96-well plate at a density of 2×103 cells per well in quintuplicate wells. At day 1-6 after culture, 10μl of CCK-8 solution was added to each well and incubated for 2h at 37°C, the absorbance of cells was measured at a wavelength of 450nm for calculation of the optical density (OD) values (Synergy H1, Biotek).
Wound healing assay
Cells were seeded in 6-well plates and grew to almost complete confluence, then a 10μl pipette tip was used to scratch a gap on the cell layer. Cells were incubated in serum-free medium. The gap widths were measured and imaged under an inverted microscope (OLYMPUS, IX51) at 0h, 24h and 36h after scratching.
Purification of exosomes from cell and human plasma
When cells grew at about 40–50% confluence, they were moved to medium containing exosome-depleted FBS, which was obtained by ultracentrifugation of standard FBS at 100,000g for 10h at 4°C followed by filtration through a 0.22µm vacuum filtration bottle. Supernatants were collected from 24-36h cell cultures. Exosomes were purified by differential centrifugation at 3,000g for 20 min to remove cell debris and dead cells. Vesicles were pelleted after centrifugation at 16,500g for 45 min and resuspended in PBS, then centrifuged at 100,000g for 2h at 4°C (Beckman Coulter, Optima XPN-100). After resuspending in an appropriate amount of PBS, the protein concentration was measured, recorded and stored for subsequent experiments.
For purification of circulating exosomes by differential centrifugation, blood from patients was centrifuged at 1,500g for 30min to obtain cell-free plasma. Plasma was centrifuged at 15,000g for 30min. The pelleted vesicles were suspended in PBS and then centrifuged at 100,000g for 2h at 4 °C.
Transmission electron microscopy (TEM) and Nanosight analysis (NTA) of exosomes
Purified exosomes were prepared on copper TEM grids (3.05mm; 200 mesh) by negative staining. 10μl sample was dropped on copper grids and incubated for 3mins. 10μl 2% uranyl acetate was pipetted on the grid and incubated for 10min. Excess solution was removed by with filter paper. Grid was stored in the dark grid box at RT until imaging. Imaging was conducted using a 120kV Biology Transmission Electron Microscope (Tecnai G2 SpiritBiotwin).
Nanoparticle tracking analysis was conducted by NanoSight NS300. Exosomes in 20μl PBS were diluted to 1000μl with DPBS, then loaded with a 1ml syringe to be tested. Three recordings of 30s at 37°C in camera were obtained and processed using NTA software.
Exosomes were resuspended in PBS and stained with DIO dye for 20 min at room temperature. The labeled exosomes were incubated with MDA-MB-231 cells pre-seeded on the small discs with medium containing exosome-depleted FBS for 24h at 37°C. Then the cells were washed three times in PBS-T, fixed with 4% paraformaldehyde for 15 min at room temperature, then washed 3 times with PBS-T. The cell nuclei were counterstained with 5ug/ml DAPI (Cat.0100-20, SouthernBiotech) for 5 min. The uptake of the labeled exosomes by MDA-MB-231 cells was assessed using an inverted confocal microscope (Leica TCS SP8, Germany).
Enzyme-linked immunosorbent assay (ELISA)
100ul patient serum, exosomes purified from 250μl patient serum and 10ml cell culture media suspended in 100μl PBS were transferred to a clean tube. The concentrations of MMP-1 were determined using ELISA kits (Cat. EHC134, Quanticyto, Neobiscience, Inc, Shenzhen, China) according to the manufacturer's protocols.
Construction and transfection of MMP-1 short hairpin RNA (shRNA) lentivirus into tumor cells
An MMP‑1 shRNA lentivirus was purchased (HanyinBiotech, Ltd., Shanghai, China) to knock down the expression of MMP‑1 in MDA‑MB‑231-HM cells. The target sequences against MMP‑1 were 5'-GCCTTCCAACTCTGGAGTAAT-3'、5'-GCTAGCTCAGGATGACATTGA-3' and 5'-GATTCTACATGCGCACAAATC-3'. MDA‑MB‑231-HM cells were plated into a 6‑well plate, grown to reach 60-80% confluence and then transfected with viral supernatant diluted 1:1000 (lentivirus) in the presence of 10μg/ml polybrene for 24h, according to the manufacturer's protocol. Cells were selected with 3 g/ml puromycin for 7 days since 48h after transduction.
Cell and exosome samples were lysed using RIPA buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors (Epizyme, Shanghai, China). The protein concentration was quantified using a BCA protein assay kit (Beyotime). Proteins were separated by 10% (gradient) SDS–PAGE (Epizyme) and transferred onto PVDF membranes (Amersham Hybond 0.45µm; GE Healthcare). The membranes were blocked with 5% milk in trisbuffered saline-Tween (TBS-T) for 2h, then incubated with primary antibodies (anti-CD9(ab92726,Abcam),anti-CD63(ab134045,Abcam), anti-MMP-1(ab134184,Abcam), anti-PAR1(26366-1-AP,Proteintech),anti-Zeb-1(21544-1-AP,Proteintech), anti-Slug(12129-1-AP,Proteintech), anti-SNAI1(13099-1-AP,Proteintech), anti-E-cadherin(20874-1-AP,Proteintech), anti-Vimentin(10366-1-AP,Proteintech), anti-GAPDH(60004-1-Ig,Proteintech)) for 12h at 4°C. the membranes were washed with TBS-T for 3times,15min per time, then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at RT. The proteins were visualized using the ECL western blotting substrate (cat. WBKLS0100, Millipore) and a Tanon-5200Multi device.
Lung colonization study
Six- to eight-week-old nude mice were injected in tail vein with 1×106 MDA-MB-231 cells. Cell-derived exosomes (15μg in 100µl PBS) were intravenous injected every other day for 2 weeks. The mice were euthanized six weeks after the cancer cell injection and their lungs were fixed, sectioned and analyzed for H&E quantify the metastatic tumor burden. Paraformaldehyde-fixed lungs were sectioned into 8-μm-thick sections, respectively, at 100μm intervals. Three large lung sections were stained with H&E and tumor nodules were counted and their area measured using the OlyVIA (Olympus) and ImageJ software. The number of the metastatic nodules were calculated by averaging data from individual sections.
Co-immunoprecipitation of MMP-1 or PAR1 from MDA-MB-231 cell lysates was carried out as follow. MMP-1 and PAR1 were immunoprecipitated using a rabbit anti-MMP-1 or anti-PAR1 antibody. Rabbit IgG were used as negative controls. Immune complexes were captured using the Protein A/G-agarose beads (LOT. 70474621, Roche), and immunoprecipitates were analyzed by SDS-PAGE followed by immunoblotting with rabbit monoclonal anti-MMP-1 antibody (26585-1-AP, Proteintech) or anti-PAR1 antibody (26366-1-AP, Proteintech). Immunoreactive bands were visualized using enhanced chemiluminescence.
Chemicals and inhibitors
Vorapaxar (HY‑10119) was obtained from MedChemExpress.
All experiments in the study were performed in triplicate. Statistical analyses were performed with the SPSS v20.0 software (SPSS Inc., Chicago, IL, USA). Data are presented as the mean±standard deviation (SD). Quantitative data were compared with a two‑tailed Student's t‑test between groups and a one‑way analysis of variance among multiple groups followed by Lease Significant Difference post hoc test. Kaplan-Meier curves of disease-free survival were plotted and survival in the groups was compared by log-rank test. The significance levels of the data are denoted by * symbols as follows: *p<0.05; **p<0.01; ***p<0.001; and ****p<0.0001. p<0.05 was considered to indicate a statistically significant difference.
The association between MMP1 expression and prognosis in triple-negative breast cancer patients was analyzed by using the Kaplan Meier plotter, which is an online database that provides assessment of the effect of 54,675 genes on survival using 10,293 cancer samples, including 22,277 genes in 5,143 breast cancer samples (http://kmplot.com/analysis/).