Exosomal MMP-1 transfers metastasis potential in triple-negative breast cancer through PAR1-mediated EMT

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with high risk of distant metastasis, in which the intercellular communication between tumor cells also plays a role. Exosomes can be released by tumor cells and promote distant metastasis through intercellular communication or changes in tumor microenvironment, it is an optimized transportation facility for biologically active payloads. This was a hypothesis-generating research on role of exosomal payload in TNBC distant metastasis. Exosomes isolated from supernatant of MDA-MB-231 and MDA-MB-231-HM (a highly pulmonary metastatic variant of parental MDA-MB-231 cells) were characterized. MMP-1 level was detected using mass spectrometry and western blot. Transwell assay, wound healing and CCK-8 assay were employed to explore the effect of exosomal MMP-1 on the metastatic capability of TNBC cells in vitro. Human breast cancer lung metastasis model in nude mice was established to observe the effect of exosomal MMP-1 in vivo. Tissue microarray and blood samples of TNBC patients were applied to analyze the relevance between MMP-1 with metastasis. MDA-MB-231-HM cells secrete exosomes enriched MMP-1, which can be taken up and enhance invasion and migration activities of TNBC cells, including MDA-MB-231, MDA-MB-468 and BT549. After ingesting exosomes enriched with MMP-1, cells secret more MMP-1, which may interact with membrane G protein receptor protease activated receptor 1 (PAR1), thereby initiating epithelial-mesenchymal transition (EMT) to enhance capability of migration and invasion. The lung colonization model shows that the expressions of MMP-1 and PAR1 in the metastases of the 231-HM-exo treated mice were both upregulated. Clinically, the enrichment of MMP-1 can be detected in exosomes extracted from serum of patients with metastasis at higher concentration than that in pre-operative patients. Moreover, in patients with multiple distant metastases, the level of MMP-1 in exosomes is also higher than that in patients with single lesion. MMP-1 from TNBC cells of high metastasis potential can promote the distant metastasis of transform those with low metastasis potential through PAR1-mediated EMT and is likely to be a potential molecular marker.


Background
Breast cancer is the most commonly diagnosed malignant tumor and the sixth cause of cancer-related deaths in women [1]. In China, breast cancer is usually diagnosed at 45 to 55 years old, 15-20% of which is triple-negative breast cancer (TNBC) [2]. TNBC is a subtype of breast cancer with high malignancy, specifically referring to negativity of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2). TNBC is characterized by a high risk of recurrence and metastasis, a short progression-free survival (PFS) and overall survival (OS) [3]. TNBC also has a tendency to metastasize to different organs including the lung, liver, brain and bones, leading to different prognosis and response to treatment [4]. Regarding the mechanism of breast cancer metastasis, most prior studies have focused on the process of metastasis. The interaction between cancer cell clones was less to be explored in terms of teamwork in the formation of distant metastasis. Exosome is a subtype of extracellular vesicles (EV) with a diameter of 30 to 150 nm, which can be secreted by almost all cells and detected in body fluids such as blood, saliva, cerebrospinal fluid, urine, semen and tumor microenvironment [5]. Exosomes carry proteins, lipids, mRNA and circRNA, which are considered to play an important role in tumor development such as tumor growth, invasion, metastasis, and regulation of tumor microenvironment [6]. There is increasing evidence showing that exosomes play an important role in tumor spread [7] (for example, establishing a pre-metastasis niche [8], promoting angiogenesis [9], destroying the peritoneum [10] or the blood-brain barrier [11]), inducing drug resistance [12] and producing cancerrelated fibroblast heterogeneity [13]. We speculate that cells with high metastasis ability can deliver metastasis-promoting substances to cells with normal metastasis ability through exosomes, initiate a series of downstream cascade reactions to enhance their metastasis ability. Our experiments in vitro showed that after co-cultivation of exosomes derived from classic TNBC breast cancer cells (MDA-MB-231) and exosomes derived from high lung metastasis breast cancer cells built in our laboratory (MDA-MB-231-HM) [14][15][16], the invasion capability of MDA-MB-231 was significantly improved. Therefore, we speculate that the key protein carried in exosomes derived from highly metastatic cell lines is one of the relevant factors that promote invasion and distant metastasis. This study used mass spectrometry to screen the differential proteins in exosomes derived from the high lung metastatic cell line MDA-MB-231-HM and the classic TNBC cell line MDA-MB-231, found MMP-1 to be the most different protein. MMPs are a family of zinc-dependent endopeptidases which are crucial to ECM degradation. Several MMPs have been detected in exosomes from tumor cells [17]. MT1-MMP existing in exosomes from tumor cells is involved in activation of proMMP-2 as well as degradation of ECM proteins [18,19]. It was also reported that combined expression of miR-21 and MMP-1 in urinary exosomes detects 95% of breast cancer [20]. We suppose that MMP-1may be a key protein to mediate the transfer of metastatic ability between tumor cells through exosomes.

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 metastasis [14]. 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). 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 20 µl  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 24-36 h 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).

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 100,000×g for 10 h at 4 °C followed by filtration through a 0.22 µm vacuum filtration bottle. Supernatants were collected from 24 to 36 h cell cultures. Exosomes were purified by differential centrifugation at 3000×g for 20 min to remove cell debris and dead cells. Vesicles were pelleted after centrifugation at 16,500×g for 45 min and resuspended in PBS, then centrifuged at 100,000×g for 2 h 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 1500×g for 30 min to obtain cell-free plasma. Plasma was centrifuged at 15,000×g for 30 min. The pelleted vesicles were suspended in PBS and then centrifuged at 100,000×g for 2 h at 4 °C.

Transmission electron microscopy (TEM) and nanosight analysis (NTA) of exosomes
Purified exosomes were prepared on copper TEM grids (3.05 mm; 200 mesh) by negative staining. 10 μl sample was dropped on copper grids and incubated for 3 min. 10 μl 2% uranyl acetate was pipetted on the grid and incubated for 10 min. 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 120 kV Biology Transmission Electron Microscope (Tecnai G2 SpiritBiotwin).
Nanoparticle tracking analysis was conducted by NanoSight NS300. Exosomes in 20 μl PBS were diluted to 1000 μl with DPBS, then loaded with a 1 ml syringe to be tested. Three recordings of 30 s at 37 °C in camera were obtained and processed using NTA software.

Exosome labeling
Exosomes isolated from 50 ml supernatant of about 7 × 10 8 cells were resuspended in PBS and stained with DIO dye for 20 min at room temperature. The labeled exosomes were incubated with 1 × 10 3 MDA-MB-231 cells preseeded on the small discs with medium containing exosome-depleted FBS for 24 h 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).

Mass spectrometry (MS)
Proteins were reduced in 10 mM DTT at 37 °C for 1 h. Protein samples were then allowed to cool to room temperature, and cysteines were blocked via exposure to 30 mM IAA at 37 °C for 30 min in the dark. The extracted protein was then mixed according to groups at equal amounts and then precipitated with acetone overnight. After resuspending the protein in 1 M urea buffer, the sample protein was digested with trypsin overnight. Subsequently, peptides were isotopically labeled with isobaric peptide labeling reagents (Applied Biosystems, Foster City, CA, USA) at room temperature for 2 h. The labeling reaction was then stopped by the addition of water. Next, the samples were separated and identified using a Triple TOF 4600 MS system (AB SCIEX, USA).

Cell proliferation
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 × 10 3 cells per well in quintuplicate wells. At day 1-6 after culture, 10 μl of CCK-8 solution was added to each well and incubated for 2 h at 37 °C, the absorbance of cells was measured at a wavelength of 450 nm 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 10 μl 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 0 h, 24 h and 36 h after scratching.

Chemicals and inhibitors
Vo r a p a x a r ( H Y-1 0 1 1 9 ) wa s o b t a i n e d f r o m MedChemExpress.

Lung colonization study
Six-to eight-week-old nude mice were injected in tail vein with 1 × 10 6 MDA-MB-231 cells. Cell-derived exosomes (15 μg in 100 µl 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 100 μm intervals. Three large lung sections were stained with H&E and tumor nodules were counted and their area measured using the Oly-VIA (Olympus) and ImageJ software. The number of the metastatic nodules were calculated by averaging data from individual sections.

Immunohistochemical staining
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).

Tissue samples
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 4 µM 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.

Enzyme-linked immunosorbent assay (ELISA)
100 μl patient serum, exosomes purified from 250 μl patient serum and 10 ml cell culture media suspended in 100 μl 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.

Statistical analysis
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 logrank 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.

Bioinformatics analysis
The association between MMP1 expression and prognosis in triple-negative breast cancer patients was analyzed 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 5143 breast cancer samples (http:// kmplot. com/ analy sis/).

Isolation and verification of exosomes
In our previously study, MDA-MB-231-HM (MDA-231-HM), a highly pulmonary metastatic variant of parental MDA-MB-231 (MDA-231) cells, was derived within the presented model system by six cycles of the pulmonary metastasis implantation to the mammary fat pad (MFP) [14], which was a valuable model system for the study of the molecular events underlying breast cancer metastasis [15,16]. The differences in pulmonary metastatic potential of the two cell lines make them valuable systems for understanding the molecular mechanisms underlying breast cancer metastasis. Transwell assay showed that migration and invasion capabilities of MDA-231-HM are stronger than MDA-231 cells (Fig. 1A).

MMP-1 in exosomes mediates the transmission of metastasis capability
MDA-231 cells were pretreated with PBS, 231-exo and 231-HM-exo for 24 h and then analyzed by Transwell migration and invasion assay. The results showed that, compared to 231-exo, 231-HM-exo significantly increased the migration and invasion activity of MDA-231 cells. (Fig. 2A). We assume that 231-HM-exo was taken up by MDA-231 cells, releasing its cargo to increase the content of protein or RNA, which directly or indirectly activated downstream signaling pathways and improved the invasion and migration capability of MDA-231 cells in vitro. In order to find the key protein transferred by exosomes that changes the ability of invasion and migration capability, we screened the differential proteins between 231-exo and 231-HM-exo by mass spectrometry. From the heat map we found that MMP-1, HPSE and ADAMTS-1 were upregulated in 231-HM-exo, then we verified them by western blot and found MMP-1 is the protein with the most significant difference in expression (Fig. 2B, C, S1A).
MMP-1 belongs to matrix metalloproteinase (MMP) family, which includes a series of zinc-and calcium-dependent endopeptidases. MMPs are of crucial importance for invasive cancer cells to break extracellular matrix (ECM) barriers and start the metastatic cascade [23,24]. MMP1 has also been shown to be related to invasive phenotype, metastasis and response to chemotherapy in human breast cancer, and is related to poor prognosis [25][26][27]. In addition, we have also verified the presence of MMP-1 in the exosomes derived from MDA-MB-468 and BT549 ( Figure S1B).

MMP-1 binding to PAR1 promotes the metastasis of TNBC possibly through EMT
The function of MMP-1 on ECM degradation has been reported [23,24], which cannot fully explain the enhanced invasion and migration activity both. MMP-1/PAR1 as a key signal to activate downstream signaling pathways to facilitate vascular intravasation and metastatic dissemination has also been proposed in several tumors [28][29][30]. Proteaseactivated receptor 1 (PAR1) is a G protein-coupled receptor that is classically activated by the serine protease thrombin cleavage of the N-terminal outer domain, can also be directly activated by MMP-1 and MMP-9 [31]. PAR1 is also considered as an independent factor for the poor prognosis of tumors and a potential therapeutic target [32].
We found that after ingestion of exosomes with different levels of MMP-1, the level of free MMP-1 secreted by MDA-231 cells into the supernatant was positively correlated with the level of MMP-1 in exosomes (Fig. 4A). Then, we demonstrated through co-immunoprecipitation that MMP-1 can directly interact with PAR1 to stimulate downstream metastasis related pathways (Fig. 4B). The PAR1 irreversible antagonist, Vorapaxar, was added at a concentration gradient to the medium of 231 cells co-cultured with 231-HM-exo and could effectively reverse the effect of 231-HM-exo. Vorapaxar inhibited 58% and 79% of the migration, 45% and 57% of the invasion at 75 μM and 100 μM, respectively (Fig. 4C). We measured the effect of Vorapaxar on cell invasion and migration in the absence of exosome treatment, and the results showed that at a specific concentration of Vorapaxar, the migration and invasion activities were slightly suppressed, which was less than that in the presence of exosomal MMP-1 (Figure S1 C). This strongly explains that MMP-1 can not only degrade collagen to promote invasion, but mainly rely on PAR1 to exert its invasion effect. Inhibiting PAR1 can greatly inhibit this acquired capability of invasion. In order to eliminate that the change in cell proliferation ability brought by PAR1 may interfere the results of migration and invasion assays, we first used CCK-8 assay to detect the proliferation of MDA-231 cells co-cultured with 231-HM-exo and Vorapaxar. Results showed that cell proliferation was independent of 231-HMexo and Vorapaxar (Fig. 4D).
According to previous reports, exosome-associated MMPs are involved in the processes of EMT in some tumors [33][34][35]. The decrease in keratin filaments and the decrease of E-cadherin, as well as the increase of vimentin, fibronectin, N-cadherin, α-SMA and various proteases, resulted in the loss of polarity and acquisition of the mesenchymal phenotype [36,37]. We speculated that PAR1 was activated by MMP-1 and mediated epithelial-mesenchymal transition (EMT) which enhanced the ability of metastasis in TNBC. MDA-231 cells were treated with PBS, 231-HM-exo, 231-HM-shRNA3-exo and sufficient Vorapaxar and Western blot was used to detect the expression of EMT biomarkers. The downregulation of E-cadherin and upregulation of ZEB-1, Vimentin and Slug in MDA-231 cells co-cultured with 231-HM-exo suggested mesenchymal phenotype, while in 231-HM-exo and Vorapaxar group, E-cadherin was upregulated, ZEB-1, Vimentin and Slug were significantly downregulated, suggesting epithelial phenotype. It can be seen that the EMT process promoted by 231-HM-exo can be reversed by PAR1 antagonist (Fig. 4E). Based on the results of in vitro experiments, we propose that after 231-HM-exo is ingested by targeted cells, the secretion of MMP-1 increases, which can directly degrade ECM, and may possibly also cleave and activate PAR1-mediated EMT to promote cell migration and invasion.

Exosomal MMP-1 mediates the transmission of metastasis capability in vivo
To verify the metastasis ability of MDA-231 and MDA-231-HM cells in vivo, we established a human breast cancer lung metastasis model in nude mice. We observed that mice injected with MDA-231-HM cells (n = 3) had significantly more lung metastases than MDA-231 cells (n = 3, p < 0.05) (Fig. 5A), which means that the metastatic ability of MDA-231-HM in vivo is much stronger than that of MDA-231 cells.
To determine the effect of MMP-1 in exosomes on the transfer ability of MDA-231 cells in vivo, We further constructed nude mouse lung metastasis model with MDA-231 cells and treated it with PBS, 231-exo, 231-HM-exo and 231-HM-shMMP1-exo every other day for 2 weeks (Fig. 5B). Metastatic nodules in mice treated with 231HMexo (n = 9) was significantly more than that with PBS (n = 9) Representative images were presented from three independent experiments. Data are shown as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001 and 231-exo (n = 9) (p < 0.001), and which were reduced by 231-HM-shRNA3-exo (n = 9) (p < 0.005) (Fig. 5C-E). A hypothesis consistent with results in vitro can be drawn, exosomes with high levels of MMP-1 can promote metastasis ability of MDA-231 in vivo, which would be weaken by reducing MMP-1 in exosomes.
Conducting immunohistochemical staining for MMP-1 and PAR1 on lung lesions of nude mice, we saw the expression of MMP-1 and PAR1 in lung lesions of mice treated with 231-HM-exo increased, while the expression of 231-HM-shRNA3-exo treated group decreased (Fig. 5F, E).

Plasma exosomal MMP-1 level is elevated in patients with shorter DFS
A total of 134 surgical tissues from TNBC patients from August 2015 to May 2016 were collected and made into tissue microarrays (TMA). The characteristics of patients at baseline are shown in Table 1. The patients were followed up to 2020.8, median follow-up time was 56.8 months. MMP-1 expression was determined by immunohistochemistry and a quarter one fourth of the OD value of MMP-1 (OD = 4.009) was chosen as a cut-off value to define 'high' and 'low' expression (Fig. 6A). In the univariate Kaplan-Meier analysis, TNBC showing high expression of MMP-1 had a significantly shorter disease-free survival (DFS) (logrank p = 0.028) (Fig. 6B). Survival curve retrieved from Kaplan-Meier plotter showed that high level MMP-1 expression is associated with significantly worse DFS (logrank p = 6e−13) in TNBC patients (Fig. 6C). Figure 6B is a TMA made of breast cancer tissue specimens collected in our cancer center, while data in Fig. 6C were acquired and analyzed by Kaplan-Meier Plotter, a public database website. The two figures both show that MMP-1 high expression in tumor tissues is related to shorter DFS.
Blood samples from patients at baseline of post-operation (n = 18) and initial metastasis (n = 30), baseline characteristics are shown in Table 2. TEM and western blot were used to verify the exosomes extracted from serum (Figure S1D, E). The MMP-1 expression level in serum and exosomes purified from serum was detected by ELISA. We found that MMP-1 expression in initial metastasis group was significantly higher than that in pre-operation group, whether in serum (p < 0.0001) or exosomes (p < 0.0001). It  was confirmed that the MMP-1 carried in exosomes was related to the recurrence and systemic metastasis of TNBC (Fig. 6D, E). Furthermore, we distinguished patients with single metastatic site (n = 13) and multiple metastatic sites (n = 17) in the initial metastasis group to research the correlation between MMP-1 and systemic metastasis. The concentration of MMP-1 in the serum has no statistical difference (p = 0.1488) while in exosomes, MMP-1 detected in patients with multiple metastases was significantly higher than that in single metastatic site (p < 0.01), which suggests that for patients with distant recurrence and metastasis, MMP-1 detected in exosomes would be more sensitive than in serum (Fig. 6F, G).
Interestingly, we collected blood samples from several patients (n = 11) before and after first-line chemotherapy and tumor response was evaluated in accordance with the Response Evaluation Criteria in Solid Tumors (RECIST 1.1) guidelines by computed tomography scanning or magnetic resonance imaging after 2 cycles [38].We found that MMP-1 level in exosomes was also related to response to chemotherapy. The expression of MMP-1 in exosomes before and after first-line chemotherapy, measured by ELISA. Efficacy has been assessed as 'stable disease' (n = 4), 'progressive disease' (n = 3) and 'partial response' (n = 4), respectively. Data are shown as the mean ± SD. N.S., no significance, *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001  of patients with partial response (PR) (n = 4) decreased while increased in patients with progressive disease (PD) (n = 3). In patients with stable disease (SD) (n = 4), the expression of MMP-1 in exosomes both increased and decreased, which suggests that MMP-1 in exosomes even has the potential to be an indicator of therapeutic efficacy (Fig. 6H).

Discussion
Many reports demonstrated that exosomes can mediate tumor metastasis by horizontal transfer of bioactive molecules to recipient cells [8,11,[38][39][40]. In this study, we first reported that TNBC cells with high metastasis potential transform those with low metastasis potential via exosomal MMP-1.
To study the role of exosomes in tumor metastasis, we extracted, verified and observed the exosomes taken up by MDA-231 cells. Then mass spectrometry was applied for the most different expressed protein between high and low lung metastasis cell-derived exosomes, which is MMP-1.
Historically, MMPs have been studied for a long time in cancer biology, and accumulated evidence showed that MMPs are related to progression, metastasis, treatment and prognosis of cancer [46][47][48]. As reported, MMPs mediate the degradation of ECM and basement membrane (BM) at all stages of cancer progression, thereby promoting the development of the surrounding microenvironment and distant metastasis [13,28]. However, the fact that MMP-1 is transmitted between cells through exosomes to enhance metastasis ability of low-metastatic cells has not yet been reported.
Unsurprisingly, our study observed that exosomes with reduced MMP-1 content actually affected the invasion and migration ability of MDA-231 in vitro and in vivo. By injection of MDA-231 cells in the tail vein and then treatment by PBS or exosomes for every other day, we established a pulmonary metastasis model in nude mice (Fig. 5B). From the results, we see that the lung metastatic nodules in the 231-HM-exo treatment group increased significantly, and which was significantly reduced after knocking down the MMP-1 in the exosomes. Therefore, we suppose that exosomes with MMP-1 coul;d promote the metastasis of MDA-231 cells in vivo. Hereafter, we explored the mechanism of the influence of exosomal MMP-1. The most widely known function of MMP-1 is to degrade collagen I, II and III in the ECM, but it cannot fully explain the enhanced activity of both invasion and migration. In addition to degrading collagen, MMP-1 can also directly PAR1 by cutting the extracellular N-terminus of PAR1 [26]. PAR1 is a G protein-coupled receptor that is classically activated by the serine protease thrombin cleavage of the N-terminal outer domain. PAR1 activated by MMP-1 provides a link between the extracellular proteolytic activity important for ECM remodeling and the signal transduction leading to cell migration and invasion [31]. It was reported that MMP-1 and MMP-13 cut the N-terminal outer domain of PAR1 at non-canonical sites, activating the G protein signaling pathway [39]. In breast cancer cells, thrombin and MMP-1 activated PAR1dependent phospho-Akt signals [46]. Our research found that MMP-1 can be bonded directly to PAR1, in addition, PAR1 antagonist Vorapaxar can reverse the effect of 231-HM-exo. Furthermore, we found that MMP-1 in the exosomes may activate the EMT process through PAR1, thereby promoting the migration and invasion of cells. We saw that PAR1 in lungs of exosomal MMP-1 treated mice was upregulated, so it was speculated that the exosomal MMP-1 activated PAR1 may also activate the related RNA or protein, which promoted the downstream signal transduction. The expression of PAR1 was upregulated to construct positive feedback for cell migration and invasion. Therefore, it is concluded that free MMP-1 in the supernatant, which sources may be from released MMP-1 from internalized exosomes and production by initial low metastasis capacity MDA-231 cells, binds to the membrane surface receptor PAR1 protein and then promotes the metastasis of the triple-negative breast cancer possibly through EMT.
Through Kaplan-Meier database and TMA of TNBC tissue in our center, we observed that the expression level of MMP-1 is related to DFS of TNBC. There was also significantly upregulated level of MMP-1 in serum and exosomes in patients with metastasis, indicating that exosomes may be more sensitive and accurate in predicting and evaluating distant metastases.
There are still some limitations in this study. In terms of experimental data, first of all, functional experiments data in vitro were confirmed by shRNA3 and shRNA5, but metastases in vivo and its mechanism were verified by only shRNA3. If all data are from both shRNA3 and shRNA5, our conclusions would be more credible. Secondly, in the study of the relationship between the changes in the level of MMP-1 in exosomes and the efficacy of first-line chemotherapy in clinical patients, the number of blood samples was too small to draw a statistical conclusion. In terms of conclusion, the mechanism of EMT caused by the interaction between PAR1 and MMP-1 has not been directly clarified through experiments. We could suppose and prove that the cells go through EMT by western blot assay for changes in EMT-related markers before and after the treatment of exosomes. Finally, the research is currently aimed at TNBC, and ER-positive and HER-2-positive breast cancer cell lines may be included for further study.

Conclusions
In summary, exosomes secreted by cells of high metastasis ability enriched in MMP-1 can transform cells with low metastasis ability in TNBC into more malignant cells, which is possibly mediated EMT via interaction with PAR1. Clinically, higher MMP-1 level in exosomes in patient's blood indicates more chance of occurrence of distant metastasis, along with a certain potential for the evaluation of therapeutic effects.