Ovarian cancer has resulted in over 140 000 deaths reported annually worldwide. This is often attributed to cellular changes in the microenvironment, including increased migration of mesenchymal stem cells (MSCs) and endothelial cells (ECs) to facilitate metastasis. Recently, the ability of exosomes to communicate signals between cells (and promote cancer progression) has been established. In the present study, we explored the effect of exosomes on cells present in the tumour microenvironment. Exosomes were isolated from ovarian cancer cells with different invasive capacity (high = SKOV-3 and low = OVCAR-3) by differential and buoyant density centrifugation and characterised using nanoparticle tracking analysis (NTA), Western blot, and EM. Exosome secretion was positively correlated with invasiveness of releasing cells. Proteomic analyses identified common and unique proteins between exosomes from SKOV-3 and OVCAR-3 with gene ontology analyses revealing that these exosomes are involved in the regulation of cell migration. Since the tumour microenvironment contains multiple cell types, including MSCs and ECs, we examined the effect of these exosomes on MSC and EC migration. Exosomes promoted MSC and EC migration in a time- and concentration-dependent manner. The effect of exosomes isolated from SKOV-3 on cell migration was significantly higher compared with exosomes from OVCAR-3. Thus, we suggest that exosomes from ovarian cancer cells contain a specific set of proteins that are representative of its cell of origin and the invasive capacity.

Introduction

Ovarian cancer is the most lethal gynaecological malignancy with most patients (75%) diagnosed at an advanced stage when the tumour has spread beyond the ovaries [1]. Cancer is a complex disease in which cells with tumorigenic traits (i.e. unregulated cell proliferation and resistance to cell death) require an ability to communicate with neighbouring cells to initiate and facilitate tumour progression. Although malignant cells may be the main driving force of tumorigenesis, their interactions with the tumour microenvironment are critical for progression from a single tumour mass to distant metastases [2]. The tumour microenvironment is a complex milieu with multiple cell types (e.g. endothelial cells (ECs), immune cells and fibroblasts as well as mesenchymal stem cells (MSCs) (which are often recruited from the bone marrow)) [3,4]. During tumour development, neoplastic cancerous cells can modify neighbouring cells or recruit normal cells to create a favourable environment for successful disease progression [2,3]. Some of the cancer cell-mediated modifications include: activation of ECs to promote angiogenesis and vasculogenesis [5], suppression of anti-tumoural immune cells [6] and induction of extracellular matrix deposits and their degradation by fibroblasts [7]. These modifications often aid in processes required for metastasis including cell migration. As multiple distinct cell types mediate metastasis, effective cell-to-cell communication between cancer cells and local/distant microenvironments is essential [8]. Recent studies have highlighted the participation of tissue-specific nanovesicles (i.e. exosomes) in cell-to-cell communication.

Exosomes are small (∼40–120 nm) membranous vesicles of endocytic origin. The biogenesis of exosomes is initiated by the inward budding of multivesicular bodies (MVB), encapsulating cellular proteins and RNA molecules to form intraluminal vesicles [9]. MVBs can either fuse with lysosomes, for degradation, or the plasma membrane, to release the intraluminal vesicles, now termed as exosomes [10]. These nanovesicles have been identified in the plasma of ovarian cancer patients, and their release increases with the progression of disease [11,12]. Recently, Kucharzewska and Belting (2013) [3] demonstrated that exosomes reflect the microenvironmental conditions (e.g. hypoxia) of glioma cells, and promote vasculogenesis which enhances tumour growth. Tumour-derived exosomes containing cellular protein and RNA can mediate the horizontal transfer of these functional molecules to different cell types and ultimately reprogramme recipient cells [13]. It is well established that heterogeneity exists among cells from a single tumour [14], however, tumour-derived exosomes have an ability to propagate oncogenic activity among these cells. High metastatic activity of B16 melanoma cells was transferred to poorly metastatic F1 melanoma cells through the uptake of B16 melanoma-derived exosomes [15]. In addition, human glioma cells horizontally transferred oncogenic epidermal growth factor receptor variant 3 (EGFRvIII) to glioma cells lacking this form of the receptor [16]. The transfer resulted in increased expression of pro-survival genes and reduction in cell-cycle inhibitors, increasing anchorage-independent growth capacity [16]. Exosomes not only facilitate communication among malignant cells but also influence tumour supporting cells such as ECs and MSCs to develop a tumour-promoting microenvironment.

Exosomes have been characterised from the plasma of ovarian cancer patients and cell-conditioned media using a variety of approaches [17]. Proteomic analysis demonstrated that ovarian cancer-derived exosomes may have a role in the progression of disease [18]. Furthermore, it has been shown that breast cancer cell-derived exosomes can contribute to cell differentiation leading to the enhancement of tumour invasiveness [19]. However, the effect of ovarian cancer-derived exosomes on recipient cells (present in the tumour microenvironment) remains to be fully elucidated. The aim of the present study was to test the hypothesis that exosomes from ovarian cancer cells promote cell migration in target cells. Exosomes were isolated from two human epithelial ovarian cancer cell lines, OVACR-3 (exo-OVCAR-3) and SKOV-3 (exo-SKOV-3), and their effects on primary human umbilical vein ECs (HUVEC) and placental MSC (pMSC) in the context of migration were examined. Ovarian cancer cell line-derived exosomes promoted EC and pMSC migration in vitro. The data obtained are consistent with the hypothesis that ovarian cancer-derived exosomes promote cell migration and exosomes might play a role in ovarian cancer progression and metastasis by promoting migration of target cells.

Methods

Cell lines and cell culture

The project was approved by the Human Research Ethics Committees of the Royal Brisbane and Women’s Hospital and the University of Queensland (HREC/09/QRBW/14). Human ovarian cancer cell lines, OVCAR-3 and SKOV-3 were purchased from American Type Culture Collection (ATCC, Rockville, MD). Both cell lines were maintained in RPMI medium (Life Technologies, Carlsbad, California, U.S.A.) supplemented with 10% FBS (PAA Laboratories Pty Ltd., Morningside, Queensland, Australia) and 1% antibiotic–antimycotic (Life Technologies, Carlsbad, California, U.S.A.) under a humidified atmosphere at 37°C with 8% O2 and 5% CO2-balanced N2. Cell viability was routinely tested by Trypan Blue exclusion and Countess® Automated Cell Counter (Life Technologies, Carlsbad, California, U.S.A.). All experimental procedures were conducted within an ISO17025 accredited (National Association of Testing Authorities, Australia) research facility. All data were recorded within 21 Code of Federal Regulation (CFR) part 11 compliant electronic laboratory notebooks (Lab Archives, Carlsbad, CA 92008, U.S.A.). The schematic figure in Supplementary Figure S1 summarises the experimental design used in the present study.

Exosome isolation and purification

Exosomes were isolated and purified from ovarian cancer cell-conditioned media as previously described [20]. In brief, cell-conditioned media was prepared by incubating cells for 24 h in cell culture medium containing FBS depleted of bovine exosomes (ultracentrifugation at 100000×g for 15 h at 4°C followed by filtration of the resulting supernatant through a 0.22-µm filter (Millipore, Billerica, Massachusetts, U.S.A.)). Exosomes were isolated from the cell-conditioned media by differential centrifugation at 300×g for 5 min, 1200×g for 10 min, 10000×g for 30 min and 100000×g for 75 min (Sorvall SureSpin™ 630/36 fixed angle rotor, Thermo Fisher Scientific Ins., Asheville, NC, U.S.A.) to remove live cells, dead cells and cell debris. The resulting pellet was washed in PBS by ultracentrifugation at 100000×g for 70 min. The 100000 ×g pellet was resuspended in 500-μl PBS and layered on the top of a discontinuous iodixanol gradient containing 40% (w/v), 20% (w/v), 10% (w/v) and 5% (w/v) iodixanol (solutions were made by diluting a stock solution of OptiPrep™ (60% (w/v) aqueous iodixanol from Sigma–Aldrich) and centrifuged at 100000×g for 20 h. An exosome-containing fraction (density 1.12–1.19 g/ml) was collected, diluted with PBS and centrifuged at 100000×g for 2 h at 4°C. Finally, the pellet containing the enriched exosome population was resuspended in 100-μl PBS.

Exosome characterisation and analysis

Nanoparticle tracking analysis (NTA) was performed to measure exosome size and particle number using a NanoSight NS500 instrument (NanoSight NTA 2.3 Nanoparticle Tracking and Analysis Release Version Build 0033; Malvern Instruments Ltd, Malvern, Worcestershire, U.K.) as previously described [21,22]. Exosomal protein concentration was determined by DC™ Protein Assay (Bio-Rad, Hercules, California, U.S.A.). The protein profile of exosomes and the originating cells were visualised by SDS/PAGE. Exosomes and cells were lysed with RIPA buffer containing protease inhibitor tablets then separated on a NuPAGE 4–12% Bis/Tris Gel (Life Technologies, Carlsbad, California, U.S.A.). The gel was stained with SimplyBlue™ Safestain (Life Technologies, Carlsbad, California, U.S.A.). For Western blot analysis, exosomal proteins were separated by PAGE, transferred to Immobilon-FL PVDF membrane (Millipore, Billerica, Massachusetts, U.S.A.) and probed with primary rabbit monoclonal antibody, anti-CD63 (1:1000; H-193: sc-15363, Santa Cruz Biotechnology, Dallas, Texas, U.S.A.) and primary mouse monoclonal antibody, anti-CD9 (1:1000, sc-13118, Santa Cruz Biotechnology, Dallas, Texas, U.S.A.). Anti-mouse and anti-rabbit IgG, horseradish peroxide (HRP)-linked antibodies were used as the secondary antibody (1:10000; Santa Cruz Biotechnology, Dallas, Texas, U.S.A.). For EM analysis, exosome pellets were fixed in 3% (w/v) glutaraldehyde and analysed. We performed six independent exosome isolations from approximately 200 million cells each (i.e. Skov-3 and OVCAR-3) to obtain enough exosomes to perform the bioactivity assays. The analysis of the purity of each preparation was evaluated by TEM (6 independent preparations and at least 20 fields per sample), NTA and Western blot.

Mass spectrometry (MS)

Purified exosomes derived from OVCAR-3 and SKOV-3 cells were reduced, alkylated and trypsinised using an in-gel digestion method [23]. Individual exosome samples were separated based on molecular weight using an SDS/PAGE as described in the previous section. The gel was stained using SimplyBlue™ Safestain and 10 or 12 gel fractions were excised from the OVCAR-3 and SKOV-3 exosome lanes respectively. Tryptic peptides were separated using an Eksigent NanoLC system coupled with a ReproSil-Pur Basic-C18-HD, 5-µm column over a 90-min gradient ranging from 2 to 35% (buffer A: 0.1% formic acid (v/v), buffer B: 100% acetonitrile, 0.1% (v/v) formic acid). The resulting peptide samples were processed in an information dependent acquisition (IDA) on an AB Sciex 5600 TripleTOF mass spectrometer with the top 20 precursor ions automatically selected for fragmentation. The mass spectra were processed using the ProteinPilot version 4.5b software and the Paragon™ Algorithm to search against a human SwissProt database (20170906). A global false discovery rate (FDR) of 1% was used as the threshold. The resulting protein lists were further filtered to remove proteins with less than 2 associated peptides (Supplementary Tables S1 and S2).

Bioinformatics and gene ontology analysis of proteomic profiles

A Venn diagram illustrating the shared proteins identified among the OVCAR-3 and SKOV-3 exosomes and the public Vesiclepedia data was prepared using the FunRich version 3.0 software package [24]. Gene ontology analysis was performed on the proteomic profiles of OVCAR-3 and SKOV-3, using the BiNGO application in Cytoscape. The gene ontology and annotation databases used for this analysis were obtained from the Gene Ontology Consortium. Gene ontology networks were merged, and the nodes were annotated using the enhanced Graphics application in Cytoscape. Nodes annotated with a red or blue colour indicate gene ontologies associated with OVCAR-3 or SKOV-3 proteomic profiles respectively.

MSC isolation

A full-term placenta from a healthy pregnancy was collected with informed consent. Human samples were obtained in accordance with the Helsinki Declaration, and the samples were processed under the approval of the written consent statement, and all experimental protocols were by the Human Research Ethics Committees of the Royal Brisbane and Women’s Hospital and the University of Queensland (HREC/09/QRBW/14). pMSCs were isolated from the first trimester placental villi by enzymatic digestion using dispase (2.4 U/ml) and collagenase (240 U/ml; Life Technologies, Carlsbad, California, U.S.A.) as we previously described [25], then cultured in DMEM (Life Technologies, Carlsbad, California, U.S.A.) supplemented with 10% FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Carlsbad, California, U.S.A.) under humidified atmosphere at 37°C with 8% O2 and 5% CO2-balanced N2.

EC isolation

HUVECs were isolated by collagenase digestion as previously described [26] and cultured in Media 199 (Life Technologies, Carlsbad, California, U.S.A.) supplemented with 10% FBS (PAA Laboratories Pty Ltd., Morningside, Queensland, Australia) and antibiotic–antimycotic (Life Technologies, Carlsbad, California, U.S.A.) under humidified atmosphere at 37°C with 8% O2 and 5% CO2-balanced N2.

Effect of exosomes on pMSC and EC migration

Cells (pMSCs or ECs) were grown to confluence on 96-well plates that were coated with attachment factor protein (37°C for 1 h) (Life Technologies, Carlsbad, California, U.S.A.). A cell-free zone was created in each well by generating a wound with a 96-Well WoundMaker™ (Essen BioScience, Ann Arbor, Michigan, U.S.A.). The cells were washed with PBS to remove detached cells and incubated in fresh growth medium supplemented with 2% exosome-depleted FBS and antibiotic–antimycotic in the presence or absence of ovarian cancer cell-derived (either SKOV-3 or OVCAR-3) exosomes (0, 20, 40, 60, 80 or 100 μg protein/ml) under humidified atmosphere at 37°C with 8% O2 and 5% CO2-balanced N2. Wound images were automatically acquired every 4 h over a 48-h period with an automated live-cell imaging system, IncuCyte™ (Essen BioScience, Ann Arbor, Michigan, U.S.A.) and processed and analyzed using the IncuCyte™ 96-Well Cell Migration Software Application Module (Essen BioScience, Ann Arbor, Michigan, U.S.A.). Data were processed and analysed using IncuCyte™ 96-Well Cell Invasion Software Application Module. Data are presented as the Relative Wound Density (RWD, Eizen, v1.0 algorithm). RWD is a representation of the spatial cell density in the wound area relative to the spatial cell density outside of the wound area at every time point (time-curve). Migration assays were performed in the presence of Mitomycin C (100 ng/ml) to minimise any confounding effects of cell proliferation. The rate of wound closure was compared using the half-maximal stimulatory time (ST50) and area under the curve (AUC).

Statistical analysis

Graphical data are represented as mean ± S.E.M. All experiments were performed in triplicates (n=3–6 independent experiments). Statistical significance between two groups was determined by unpaired Student’s ttests and multiple groups were compared using ANOVA in which P<0.05 was considered statistically significant. Post-hoc analyses were used for pairwise comparisons (Bonferroni correction test).

Results

Association between cell migration and exosome release from ovarian cancer cells

SKOV-3 and OVCAR-3 cells are the most common cell lines used for ovarian cancer research and cell authentication of ovarian cancer cell lines were performed with an STR DNA Profiling Analysis. Migration assays for SKOV-3 and OVCAR-3 in the presence of Mitomycin C (100 ng/ml) for 24 h was performed (Figure 1). SKOV-3 cells showed greater cell migration compared with OVCAR-3 cells (Figure 1A,B). The migration capacity of SKOV-3 cells was significantly higher, approximately 1.5-fold, at any point of the time-curve (images were taken every 4 h) for a period of 24 h (Figure 1C).

Ovarian cancer cell migration

Figure 1
Ovarian cancer cell migration

SKOV-3 and OVCAR-3 cells were grown to confluence in complete medium. A wound was made using 96-well WoundMaker and ovarian cancer cell lines migration was measured for 24 h. (A) (a) Wound imaged immediately after wounding; (b) graphical representation from (a) showing the calculation of initial wound width (black) and cell migration (grey) at the midpoint of the experiment. (B) Time course of wound closure for OVACR-3 (open circle) and SKOV-3 (black circle) expressed as RWD (%). (C) Cell migration normalised to values at OVCAR-3. Data are presented as mean ± S.E.M. In (B,C) **P<0.01, ***P<0.001 compared with OVCAR-3.

Figure 1
Ovarian cancer cell migration

SKOV-3 and OVCAR-3 cells were grown to confluence in complete medium. A wound was made using 96-well WoundMaker and ovarian cancer cell lines migration was measured for 24 h. (A) (a) Wound imaged immediately after wounding; (b) graphical representation from (a) showing the calculation of initial wound width (black) and cell migration (grey) at the midpoint of the experiment. (B) Time course of wound closure for OVACR-3 (open circle) and SKOV-3 (black circle) expressed as RWD (%). (C) Cell migration normalised to values at OVCAR-3. Data are presented as mean ± S.E.M. In (B,C) **P<0.01, ***P<0.001 compared with OVCAR-3.

Exosomes were isolated from SKOV-3 and OVCAR-3-conditioned media. Schematic representation of the exosome isolation process is presented in Figure 2A. Protein contents of OVCAR-3 exosomes (exo-OVCAR-3) and SKOV-3 exosomes (exo-SKOV-3) and the originating cells were separated by SDS/PAGE and stained to visualise their protein patterns (Figure 2B). Exo-OVCAR-3 and exo-SKOV-3 were visualised under TEM (Figure 2C). The presence of enriched exosomal proteins, CD63 and CD9 in exosomes isolated from both cell lines were confirmed by Western blot (Figure 2D). Exosomes from both cell lines were identified as small cup-shaped vesicles between 40 and 100 nm in diameter. There was no significant particle size difference between exo-OVCAR-3 (97 ± 26 nm) and exo-SKOV-3 (93 ± 30 nm) as measured by NTA (Figure 2E). However, SKOV-3 released significantly greater amounts of exosomes per milliliter of cell-conditioned media compared with OVCAR-3 cells (Figure 2E). Interestingly, increased exosome release was significantly (P<0.001) correlated with increased cell invasiveness (wound confluence) (Figure 2F).

Association between ovarian cancer cell invasiveness and exosome release

Figure 2
Association between ovarian cancer cell invasiveness and exosome release

Exosome populations were enriched from two different cell lines with varying invasiveness (OVCAR-3 and SKOV-3) and quantified. (A) Flow chart of the exosome purification procedure. (B) SimplyBlue™ SafeStain-stained SDS polyacrylamide gel after separation of 10 μg of total cell lysates (OVCAR-3 and SKOV-3) or exosomes (exo-OVCAR-3 or exo-SKOV-3) from ovarian cancer cells. (C) Representative electron micrograph of exosomes isolated OVCAR-3 and SKOV-3 cells. (D) Representative Western blot for marker enriched in exosomess: CD63 and CD9. (E) Size distribution of exosomes (NTA), isolated from OVCAR-3 and SKOV-3 cells. (F) Association between cell migration and exosomes release from OVCAR-3 and SKOV-3 cells. In (E), data are presented as mean ± S.E.M.

Figure 2
Association between ovarian cancer cell invasiveness and exosome release

Exosome populations were enriched from two different cell lines with varying invasiveness (OVCAR-3 and SKOV-3) and quantified. (A) Flow chart of the exosome purification procedure. (B) SimplyBlue™ SafeStain-stained SDS polyacrylamide gel after separation of 10 μg of total cell lysates (OVCAR-3 and SKOV-3) or exosomes (exo-OVCAR-3 or exo-SKOV-3) from ovarian cancer cells. (C) Representative electron micrograph of exosomes isolated OVCAR-3 and SKOV-3 cells. (D) Representative Western blot for marker enriched in exosomess: CD63 and CD9. (E) Size distribution of exosomes (NTA), isolated from OVCAR-3 and SKOV-3 cells. (F) Association between cell migration and exosomes release from OVCAR-3 and SKOV-3 cells. In (E), data are presented as mean ± S.E.M.

MS identification and gene ontology analysis of OVCAR-3 and SKOV-3 proteomic profiles

A total of 418 and 781 proteins were identified using MS in exosomes derived from SKOV-3 and OVCAR-3, respectively (Supplementary Tables S1 and S2). To determine if these proteins are associated with exosomes, the protein list was analysed using Funrich to search against a publicly available vesicle protein database (Figure 3A). Of the top 104 proteins that have been previously found to be associated with vesicles, a total of 88 and 100 proteins were identified in the SKOV-3 and OVCAR-3 exosomes, respectively.

Venn diagram of proteins identified in SKOV-3 and OVCAR-3 exosomes and gene ontology analysis

Figure 3
Venn diagram of proteins identified in SKOV-3 and OVCAR-3 exosomes and gene ontology analysis

(A) MS was used to identify proteins present in exosomes isolated from SKOV-3 and OVCAR-3 cells. Three hundred and fifteen proteins were common between SKOV-3 and OVCAR-3 exosomes whereas SKOV-3 exosomes had 116 unique proteins. OVCAR-3 exosomes had 450 unique proteins identified. Eighty-six out of the top 104 vesicle-associated proteins (obtained from Vesiclepedia) were present in both SKOV-3 and OVCAR-3 exosomes. (B) Each circle represents a process related to the regulation of cell migration. Nodes annotated with a red or blue colour indicate gene ontologies associated with OVCAR-3 or SKOV-3 proteomic profiles respectively.

Figure 3
Venn diagram of proteins identified in SKOV-3 and OVCAR-3 exosomes and gene ontology analysis

(A) MS was used to identify proteins present in exosomes isolated from SKOV-3 and OVCAR-3 cells. Three hundred and fifteen proteins were common between SKOV-3 and OVCAR-3 exosomes whereas SKOV-3 exosomes had 116 unique proteins. OVCAR-3 exosomes had 450 unique proteins identified. Eighty-six out of the top 104 vesicle-associated proteins (obtained from Vesiclepedia) were present in both SKOV-3 and OVCAR-3 exosomes. (B) Each circle represents a process related to the regulation of cell migration. Nodes annotated with a red or blue colour indicate gene ontologies associated with OVCAR-3 or SKOV-3 proteomic profiles respectively.

Gene ontology analysis revealed that the proteomic profiles of OVCAR-3 and SKOV-3 are involved in the regulation of cell migration (Figure 3B). Interestingly, the proteomic profile of OVCAR-3 cells was enriched for the negative regulation of cell migration (red nodes), indicating that these cells are less migratory. Conversely, the proteomic profile of SKOV-3 was enriched for the positive regulation of cell migration (blue nodes), indicating that these cells are more migratory. Furthermore, there were 12 unique proteins (associated with cell migration) identified in SKOV-3 exosomes compared with OVCAR-3 exosomes (Table 1).

Table 1
Unique SKOV-3 exosomal proteins associated with cell migration
AccessionNameFunctions
sp|P08581|MET_HUMAN Hepatocyte growth factor receptor OS=Homo sapiens GN=MET PE=1 SV=4 Hepatocyte growth factor (HGF) and MET are part of a pathway targeting vascular endothelial growth factor (VEGF) to enhance angiogenesis in cancer cells [34
  Exosomes released by metastatic melonama cells were able to horizontally transfer MET to bone marrow progenitor cells [35
sp|P06756|ITAV_HUMAN Integrin α-V OS=Homo sapiens GN=ITGAV PE=1 SV=2 Higher expression of integrin α-V is associated with increased cell migration [53
sp|Q15262|PTPRK_HUMAN Receptor-type tyrosine-protein phosphatase κ OS=Homo sapiens GN=PTPRK PE=1 SV=2 Abberant glycosylation of PTRPK induces migration of colon cancer cells [36
sp|Q03113|GNA12_HUMAN Guanine nucleotide-binding protein subunit α-12 OS=Homo sapiens GN=GNA12 PE=1 SV=4 Present in exosomes obtained from a hepatocellular carcinoma cell line [54
sp|Q13753|LAMC2_HUMAN Laminin subunit γ-2 OS=Homo sapiens GN=LAMC2 PE=1 SV=2 Overexpression of LAMC2 leads to increased invasion, proliferation and migration of colorectal cancer cells [55
sp|P35443|TSP4_HUMAN Thrombospondin-4 OS=Homo sapiens GN=THBS4 PE=1 SV=2 TSP4 acts in a pro-angiogenic manner in response to TGFβ1 in cultured ECs [56
sp|Q9Y3L5|RAP2C_HUMAN Ras-related protein Rap-2c OS=Homo sapiens GN=RAP2C PE=1 SV=1 Overexpression of RAP2C resulted in osteosarcoma cells gaining increased invasiveness and migratory abilities. Comparatively, knockdown inhibited cell migration and proliferation [57
sp|P29317|EPHA2_HUMAN Ephrin type-A receptor 2 OS=Homo sapiens GN=EPHA2 PE=1 SV=2 Overexpressed in aggressive ovarian cancer cell lines such as SKOV-3. Increased metastatic spread was noted in A2780 orthotopic mice with EPHA2 overexpression [58
  EPHA2 containing exosomes from TIG-3 cells promote cell proliferation through the activation of the Erk pathway [37
K1C16_HUMAN Keratin, type I cytoskeletal 16 OS=Homo sapiens GN=KRT16 PE=1 SV=4 KRT16 has been identified within a transitional cell carcinoma of the bladder, cell line [59
sp|O75340|PDCD6_HUMAN Programmed cell death protein 6 OS=Homo sapiens GN=PDCD6 PE=1 SV=1 Inhibition of PDCD6 through miR-124-3p decreases tumour metastasis in breast cancer [60
sp|P04004|VTNC_HUMAN Vitronectin OS=Homo sapiens GN=VTN PE=1 SV=1 Vitronectin, present in human plasma, had a pro-migratory effect on A549 cells [61
sp|P39060|COIA1_HUMAN Collagen α-1(XVIII) chain OS=Homo sapiens GN=COL18A1 PE=1 SV=5 Exosomes obtained from primary tumour cell lines showed an up-regulation in COL18A1 [38
AccessionNameFunctions
sp|P08581|MET_HUMAN Hepatocyte growth factor receptor OS=Homo sapiens GN=MET PE=1 SV=4 Hepatocyte growth factor (HGF) and MET are part of a pathway targeting vascular endothelial growth factor (VEGF) to enhance angiogenesis in cancer cells [34
  Exosomes released by metastatic melonama cells were able to horizontally transfer MET to bone marrow progenitor cells [35
sp|P06756|ITAV_HUMAN Integrin α-V OS=Homo sapiens GN=ITGAV PE=1 SV=2 Higher expression of integrin α-V is associated with increased cell migration [53
sp|Q15262|PTPRK_HUMAN Receptor-type tyrosine-protein phosphatase κ OS=Homo sapiens GN=PTPRK PE=1 SV=2 Abberant glycosylation of PTRPK induces migration of colon cancer cells [36
sp|Q03113|GNA12_HUMAN Guanine nucleotide-binding protein subunit α-12 OS=Homo sapiens GN=GNA12 PE=1 SV=4 Present in exosomes obtained from a hepatocellular carcinoma cell line [54
sp|Q13753|LAMC2_HUMAN Laminin subunit γ-2 OS=Homo sapiens GN=LAMC2 PE=1 SV=2 Overexpression of LAMC2 leads to increased invasion, proliferation and migration of colorectal cancer cells [55
sp|P35443|TSP4_HUMAN Thrombospondin-4 OS=Homo sapiens GN=THBS4 PE=1 SV=2 TSP4 acts in a pro-angiogenic manner in response to TGFβ1 in cultured ECs [56
sp|Q9Y3L5|RAP2C_HUMAN Ras-related protein Rap-2c OS=Homo sapiens GN=RAP2C PE=1 SV=1 Overexpression of RAP2C resulted in osteosarcoma cells gaining increased invasiveness and migratory abilities. Comparatively, knockdown inhibited cell migration and proliferation [57
sp|P29317|EPHA2_HUMAN Ephrin type-A receptor 2 OS=Homo sapiens GN=EPHA2 PE=1 SV=2 Overexpressed in aggressive ovarian cancer cell lines such as SKOV-3. Increased metastatic spread was noted in A2780 orthotopic mice with EPHA2 overexpression [58
  EPHA2 containing exosomes from TIG-3 cells promote cell proliferation through the activation of the Erk pathway [37
K1C16_HUMAN Keratin, type I cytoskeletal 16 OS=Homo sapiens GN=KRT16 PE=1 SV=4 KRT16 has been identified within a transitional cell carcinoma of the bladder, cell line [59
sp|O75340|PDCD6_HUMAN Programmed cell death protein 6 OS=Homo sapiens GN=PDCD6 PE=1 SV=1 Inhibition of PDCD6 through miR-124-3p decreases tumour metastasis in breast cancer [60
sp|P04004|VTNC_HUMAN Vitronectin OS=Homo sapiens GN=VTN PE=1 SV=1 Vitronectin, present in human plasma, had a pro-migratory effect on A549 cells [61
sp|P39060|COIA1_HUMAN Collagen α-1(XVIII) chain OS=Homo sapiens GN=COL18A1 PE=1 SV=5 Exosomes obtained from primary tumour cell lines showed an up-regulation in COL18A1 [38

Effect of ovarian cancer cell-derived exosomes on pMSCs

The effect of exo-OVCAR-3 and exo-SKOV-3 on pMSC migration was determined. MSCs are involved in vessel formation and angiogenesis, and may play a role in neovascularisation [27]. The effect of exo-SKOV-3 or exo-OVCAR-3 on pMSC cell migration is presented as RWD (percent) over time (Figure 4A,B). The rate of wound closure was significantly increased in the presence of exo-SKOV-3 or exo-OVCAR-3 as measured by ST50 (Table 2) and the effect of exo-SKOV-3 and exo-OVCAR-3 was concentration dependent (Table 2). For all conditions, the relative density increased over time. The area under the curve (AUC) analysis showed that pMSC migration with 40 and 60 µg/ml of exo-SKOV-3 was significantly increased when compared with 40 and 60 µg/ml of exo-OVCAR-3 respectively (Figure 4C). The kinetic parameters showed that for both exo-OVCAR-3 and exo-SKOV-3, the number of hours required to reach the ST50 (rate of wound closure) decreased with increasing exosomal dose/concentration (Table 2). The number of hours required to reach ST50 with 20 µg/ml of exo-OVCAR-3 was 26 ± 0.47 whereas with a dose of 100 µg/ml, it was 14 ± 0.46. For exo-SKOV-3, ST50 at 20 µg/ml was 23 ± 0.54 whereas with a dose of 100 µg/ml, it was 12 ± 0.63. Furthermore, to obtain the ST50, the number of hours required by the exo-SKOV-3 was significantly lower when compared with exo-OVCAR-3 across all concentrations. At a dose of 40 µg/ml, the number of hours for exo-SKOV-3 was 19 ± 0.42 compared with exo-OVCAR-3 which was 22 ± 0.56. However, there was no significant difference between the rates of wound closure when the pMSC cells were not treated with exosomes (control). The EC50 was also higher for exo-OVCAR-3 (59 ± 5.31) when compared with exo-SKOV-3 (38 ± 3.72) (Figure 4D).

The effect of exosomes on MSCs

Figure 4
The effect of exosomes on MSCs

pMSCs were isolated from chorionic villi and were grown to confluence in complete medium (see ‘Methods’ section). A wound was made using 96-well WoundMaker and pMSC migration was measured in the absence (white circles) or the presence (black circles) of exosomes in a time (0–48 h) and concentration (0–100 μg/ml)-dependent manner. (A) Effect of exosomes from SKOV-3 on pMSC migration. (B) The effect of exosomes from OVCAR-3 on pMSC migration. (C) Comparison analysis between the effect (fold change) of exosomes from OVCAR-3 and SKOV-3 on pMSC migration. (D) Dose–response curve for the effect of exosomes on pMSC migration.*P<0.05. In (D), data are presented as a non-linear regression analysis (curve fit) and mean ± S.E.M.

Figure 4
The effect of exosomes on MSCs

pMSCs were isolated from chorionic villi and were grown to confluence in complete medium (see ‘Methods’ section). A wound was made using 96-well WoundMaker and pMSC migration was measured in the absence (white circles) or the presence (black circles) of exosomes in a time (0–48 h) and concentration (0–100 μg/ml)-dependent manner. (A) Effect of exosomes from SKOV-3 on pMSC migration. (B) The effect of exosomes from OVCAR-3 on pMSC migration. (C) Comparison analysis between the effect (fold change) of exosomes from OVCAR-3 and SKOV-3 on pMSC migration. (D) Dose–response curve for the effect of exosomes on pMSC migration.*P<0.05. In (D), data are presented as a non-linear regression analysis (curve fit) and mean ± S.E.M.

Table 2
Kinetic parameters of the effect of ovarian cancer exosomes on target cells
Cell targetpMSCsECs
Exosomes originOVCAR-3SKOV-3P-valuesOVCAR-3SKOV-3P-values
ST50 (hours) Control 39 ± 0.78 38 ± 0.91 0.4131 47 ± 0.71 46 ± 0.62 0.3002 
ST50 (hours) 20 μg/ml 26 ± 0.47 23 ± 0.54 0.0004* 38 ± 0.56 25 ± 0.54 <0.0001* 
ST50 (hours) 40 μg/ml 22 ± 0.56 19 ± 0.42 0.0003* 36 ± 0.66 23 ± 0.63 <0.0001* 
ST50 (hours) 60 μg/ml 20 ± 0.53 18 ± 0.40 0.0064† 31 ± 0.61 19 ± 0.54 <0.0001* 
ST50 (hours) 80 μg/ml 18 ± 0.52 16 ± 0.46 0.0087† 24 ± 0.54 15 ± 0.42 <0.0001* 
ST50 (hours) 100 μg/ml 14 ± 0.46 12 ± 0.63 0.0177‡ 18 ± 0.50 12 ± 0.37 <0.0001* 
EC50 (μg/ml) 59 ± 5.31 38 ± 3.72 0.0038† 54 ± 5.2 46 ± 3.5 0.035‡ 
EC50 (×108 vesicles per ml ± ×1071.96 ± 1.8 1.13 ± 1.0 <0.0001* 1.76 ± 2.6 1.45 ± 2.4 0.040‡ 
Cell targetpMSCsECs
Exosomes originOVCAR-3SKOV-3P-valuesOVCAR-3SKOV-3P-values
ST50 (hours) Control 39 ± 0.78 38 ± 0.91 0.4131 47 ± 0.71 46 ± 0.62 0.3002 
ST50 (hours) 20 μg/ml 26 ± 0.47 23 ± 0.54 0.0004* 38 ± 0.56 25 ± 0.54 <0.0001* 
ST50 (hours) 40 μg/ml 22 ± 0.56 19 ± 0.42 0.0003* 36 ± 0.66 23 ± 0.63 <0.0001* 
ST50 (hours) 60 μg/ml 20 ± 0.53 18 ± 0.40 0.0064† 31 ± 0.61 19 ± 0.54 <0.0001* 
ST50 (hours) 80 μg/ml 18 ± 0.52 16 ± 0.46 0.0087† 24 ± 0.54 15 ± 0.42 <0.0001* 
ST50 (hours) 100 μg/ml 14 ± 0.46 12 ± 0.63 0.0177‡ 18 ± 0.50 12 ± 0.37 <0.0001* 
EC50 (μg/ml) 59 ± 5.31 38 ± 3.72 0.0038† 54 ± 5.2 46 ± 3.5 0.035‡ 
EC50 (×108 vesicles per ml ± ×1071.96 ± 1.8 1.13 ± 1.0 <0.0001* 1.76 ± 2.6 1.45 ± 2.4 0.040‡ 

*P-value<0.001; †P-value<0.01; ‡P-value<0.05

Effect of ovarian cancer cell-derived exosomes on ECs

Previous studies have shown that exosomes activate ECs to support tumour angiogenesis [28–30]. ECs were maintained in growth medium with different concentrations (20, 40, 60, 80 and 100 µg/ml) of either exo-OVCAR-3 or exo-SKOV-3 (Figure 5). Cell cultures were imaged every 4 h over a period of 48 h to determine the RWD for the migration assay. For all conditions, the relative density increased over time (Figure 5A,B). For all exosome concentrations, exo-SKOV-3 significantly increased EC migration compared with exo-OVCAR-3 (Figure 5C,D). The dose–response curve showed that at all doses, a higher dose of exo-OVCAR-3 would be required to elicit a similar response to exo-SKOV-3 (Figure 5D). The ST50 time was significantly lower when ECs were treated with exo-SKOV-3 compared with exo-OVCAR-3 (Table 2). Furthermore, the ST50 time also decreased as the concentration of exosomes increased for both the exo-SKOV-3 and exo-OVCAR-3. The kinetic parameters showed that for both exo-OVCAR-3 and exo-SKOV-3, the number of hours required to reach the ST50 (rate of wound closure) decreased with increasing exosomal dose/concentration (Table 2). The number of hours required to reach ST50 with 20 µg/ml of exo-OVCAR-3 was 38 ± 0.56 h whereas with a dose of 100 µg/ml, it was 18 ± 0.50 h. For the exo-SKOV-3, ST50 at 20 µg/ml was 25 ± 0.54 h whereas with a dose of 100 µg/ml, it was 12 ± 0.37 h. Furthermore, to obtain the ST50, the number of hours required by the exo-SKOV-3 was significantly lower compared with exo-OVCAR-3 across all concentrations. At a dose of 40 µg/ml, the number of hours for exo-SKOV-3 was 23 ± 0.63 h compared with exo-OVCAR-3, which was 36 ± 0.66 h. The effect of exo-SKOV-3 and exo-OVCAR-3 was concentration dependent and the EC50 was also higher for exo-OVCAR-3 (54 ± 5.2 µg/ml) when compared with exo-SKOV-3 (46 ± 3.5 µg/ml).

The effect of exosomes on EC migration

Figure 5
The effect of exosomes on EC migration

ECs were were grown to confluence in complete media (see ‘Methods’ section). A wound was made using 96-well WoundMaker and EC migration was measured in the absence (white circles) or the presence (black circles) of exosomes in a time (0–48 h) and concentration (0–100 μg/ml)-dependent manner. (A) Effect of exosomes from OVCAR-3 on EC migration. (B) The effect of exosomes from SKOV-3 on EC migration. (C) Comparison analysis between the effect (fold change) of exosomes from OVCAR-3 and SKOV-3 on EC migration. (D) Dose–response curve for the effect of exosomes on EC migration. In (D), data are presented as a non-linear regression analysis (curve fit) and mean ± S.E.M.

Figure 5
The effect of exosomes on EC migration

ECs were were grown to confluence in complete media (see ‘Methods’ section). A wound was made using 96-well WoundMaker and EC migration was measured in the absence (white circles) or the presence (black circles) of exosomes in a time (0–48 h) and concentration (0–100 μg/ml)-dependent manner. (A) Effect of exosomes from OVCAR-3 on EC migration. (B) The effect of exosomes from SKOV-3 on EC migration. (C) Comparison analysis between the effect (fold change) of exosomes from OVCAR-3 and SKOV-3 on EC migration. (D) Dose–response curve for the effect of exosomes on EC migration. In (D), data are presented as a non-linear regression analysis (curve fit) and mean ± S.E.M.

Discussion

Ovarian cancer is a broad term for a heterogeneous disease encompassing several cell types. In addition to tumour cells, the tumour microenvironment contains cells from various origins including, but not limited to, ECs, MSCs, fibroblasts, macrophages and other stromal cells [31]. Progression of disease and metastasis requires intricate signalling cascades and cell–cell communication between the multiple cell types. However, the mechanism through which this complex interaction is maintained is currently unclear with only a small number of studies examining the effect of ovarian cancer exosomes on cell migration (Table 3). Therefore, the present study seeks to understand the involvement of exosomes in this network in the tumour microenvironment which ultimately aids metastasis.

Table 3
Studies examining the effect of ovarian cancer derived-exosomes on migration
Exosome releasing cellTarget cellResultsReference
IGROV1 HEK293 IGROV1 cells contain high levels of LIN28 (RNA-binding protein) and treatment of HEK293 cells with IGROV1 exosomes leads to increased invasion and migration [62
CAOV-3 SKOV-3 HUVEC CAOV-3 exosomes enhanced aggressive behaviour in HUVECs [63
SKOV-3 A2780 Macrophages SKOV-3 cells that were treated with the supernatant of SKOV-3 exosome treated macrophages had increased proliferation and migration [64
Exosome releasing cellTarget cellResultsReference
IGROV1 HEK293 IGROV1 cells contain high levels of LIN28 (RNA-binding protein) and treatment of HEK293 cells with IGROV1 exosomes leads to increased invasion and migration [62
CAOV-3 SKOV-3 HUVEC CAOV-3 exosomes enhanced aggressive behaviour in HUVECs [63
SKOV-3 A2780 Macrophages SKOV-3 cells that were treated with the supernatant of SKOV-3 exosome treated macrophages had increased proliferation and migration [64

Tumour progression involves the interaction of multiple cell types and exosomes are known to mediate these interactions as they have an ability to horizontally transfer oncogenic activity between tumour cells, hence altering the characteristics of the recipient cells [32]. Therefore, the proteomic content of exo-OVCAR-3 and exo-SKOV-3 and their effect on the migration capability of different cells found in the tumour microenvironment were tested.

In the present study, we established that exosome release from ovarian cancer cells positively correlated with cell invasiveness suggesting that higher levels of tumour-derived exosomes may reflect tumour invasiveness in ovarian cancer. Furthermore, it has previously been shown that the plasma of patients with ovarian cancer contains greater levels of exosomal proteins compared with plasma from patients with either benign tumours or healthy controls [33]. Additionally, we evaluated the proteomic profile of the SKOV-3 and OVCAR- 3 exosomes and via gene ontology, revealed that SKOV-3 exosomes were positively regulating migration whereas OVCAR-3 exosomes were negatively regulating migration. Gene ontology analysis also identified 12 proteins that were associated with cell migration and only present in SKOV-3 exosomes. Four of these proteins (MET, GNA12, EPHA2 and COIA1) have previously been reported to be present in exosomes [34–38]. Furthermore, literature analysis revealed that all 12 proteins have functions associated with cell proliferation, invasion and migration in different cell types (Table 1). The presence of these proteins in the SKOV-3 exosomes (and absence in OVCAR-3 exosomes) suggests that these proteins may be driving factors of the increased migration capacity noted when cells were treated with SKOV-3 exosomes. Interestingly, we identified discrepancy in the number of proteins present in the exosomes isolated from SKOV-3 compared with OVCAR-3 cells. Ovarian cancer cells with different invasiveness capacity might have different mechanisms associated with the sorting of protein into exosomes which might explain the difference in the number of proteins within exosomes identified in the present study. As a proof of principle, we have calculated the number of proteins associated with exosomes based on the following assumptions (i) exosomes are spherical nanovesicles [39] and (ii) obtaining an estimate of the number of proteins per cell volume, with a major assumption that the contents of an exosome are a scaled-down cell [40]. Exosomes with a radius of 50 nm (i.e. 100-nm vesicles) and 2.7 × 106 proteins/µm3 should theoretically contain 1414 protein molecules per exosome. However, it has been demonstrated that proteins are specifically sorted into vesicles, but the exact mechanism is still to be elucidated [41].

Following the identification of the differential protein profile of the exosomes, we also noted increased secretion of exosomes by invasive cells and it was seen that ovarian cancer cell-derived exosomes increased the migration of MSCs and ECs in a time- and concentration-dependent manner. The ability of a tumour, arising from epithelial tissues, to progress to an advanced stage is dependent on their ability to invade, metastasise and change cell structure [8]. Since tumours contain cells with different invasive potential, the release of exosomes may be a means to transfer invasive capacity from one cell to another resulting in tumour progression [14]. Interestingly, the effect of exosomes on MSC and EC cell migration was significantly higher using exosomes from a highly invasive capacity cell line (i.e. SKOV-3) compared with low invasive capacity cells (i.e. OVCAR-3). This reiterates the importance of exosomes and the relationship between the exosome releasing cell and the effect of these exosomes on the target cell. MSCs and ECs were chosen as target cells as it is well known that they are present in the tumour microenvironment [42,43] where they can mediate activity to facilitate cancer growth and progression.

MSCs have been reported to localise in developing tumours and play a role in tumour stroma formation [44], thus, the effects of exo-OVCAR-3 and exo-SKOV-3 on the migration of pMSCs were investigated. Exo-OVCAR-3 and exo-SKOV-3 increased pMSC migration although there was a significant increase in the fold change when exo-SKOV-3 was used compared with exo-OVCAR-3 at doses of 40 and 60 µg/ml. This may have been due to the lower dose of exosomes being incapable of inducing strong effects and for the highest dosage; the maximum response may have already been reached, thus suggesting that the effect may be dose dependent. Karnoub et al. (2007) [45] have reported that breast cancer cells stimulate migration of MSCs towards them. Stimulated MSCs in turn, secrete chemokine–chemokine (C–C motif) ligand 5 (CCL5) to enhance motility, invasion and metastasis of breast cancer cells [45]. The factor stimulating the MSCs is unknown but exosomes may have a potential role as they are released from tumour cells and mediate cell-to-cell communication. Chowdhury et al. (2015) [46] showed that MSCs treated with prostate cancer cell-derived exosomes differentiated into myofibroblasts with pro-angiogenic capabilities and amplified invasiveness in a 3D co-culture model. Furthermore, Lin et al. (2016) [47] showed that tumour-derived exosomes could be internalised by mesenchymal stromal/stem cells. Exosomes were isolated from the melanoma cell line, B16-F10 and incubated with bone marrow-derived MSCs. The treated MSCs were then injected into mice and it was found that tumour exosomes treated MSCs facilitated tumour growth whereas non-treated MSCs had no significant effect on tumour growth.

The effect of exosomes from ovarian cancer cells on ECs was also determined. Angiogenesis, an important process for tumour progression is initiated by growth factors such as vascular endothelial growth factor (VEGF). VEGF activates ECs resulting in detachment from neighbouring cells thus allowing vessels to sprout and proliferate [5]. Without the process of angiogenesis, tumours are deprived of nutrients and therefore, cannot proliferate and grow [5] and thus, it is essential that the processes involved in angiogenesis and vasculogenesis be understood to target tumour growth and survival. Both exo-OVCAR-3 and exo-SKOV-3 treatment significantly increased EC migration. Furthermore, our laboratory has previously reported that exosomes derived from pMSCs have been found to stimulate human placental microvascular ECs (hPMECs) tube formation [25]. Therefore, the migration capability of ECs is enhanced by both tumour cell-derived exosomes and exosomes from other cells in the tumour microenvironment (pMSCs) to support tumour angiogenesis.

Proteomic analyses performed by Sinha et al. (2014) [48] showed that the three pathways that are enriched in ovarian cancer cell line (OVCAR-3, SKOV-3, OVCAR-5 and OVCAR-433) derived exosomes are ErbB, PI3K and MAPK. These pathways are all involved in the process of cell migration and ultimately cancer development. Furthermore, the different effects that OVCAR-3 and SKOV-3 exosomes had on migration may be attributed to their potential ovarian cancer subtype with SKOV-3 being classified as being more mesenchymal like whereas OVCAR-3 is more epithelial in nature [49]. It is well known that mesenchymal cells are more motile and thus invasive [50,51]. Transcriptomic analysis has also shown that the two cells have differences in the expression of approximately 880 genes which are either up-regulated or down-regulated when comparing the two cell lines [49]. The genes that are up-regulated in SKOV-3 are often related to the mesenchymal phenotype such as SNAIL and TWIST compared with OVCAR-3. Moreover, Lis et al. (2012) [52] showed that when mesenchymal cells were co-cultured with OVCAR-3 and SKOV-3, there was in an increase in gene clusters related to migration and invasion. Therefore, it follows that the gene expression differences between the cell lines and their effects on target cells may also be replicated when exosomes obtained from these cell lines are considered, thus supporting the hypothesis that exosomes are able to influence target cells phenotypically. However, this result has given rise to several questions such as: ‘Which particular genes or cascades are affected in cells upon contact with tumour-derived exosomes?’, ‘How can these exosomes travel to distant sites to promote metastasis?’ and ‘Can these exosomes interact with all types of cells to promote a pro-tumourigenic and pro-metastatic environment in vivo?’ Thus, further research is required to understand the pathways and mechanisms underlying the changes that can be seen in vitro.

Conclusion

The ovarian cancer cell line, SKOV-3, is highly invasive and releases a greater quantity of exosomes compared with the poorly invasive cell line, OVCAR-3. Furthermore, the exosomes released from both the cell lines are able to induce changes in migration capacity of target cells. Tumour cell line-derived exosomes are able to promote migration in non-cancerous cells such as pMSCs and ECs. Exosomes obtained from the highly invasive SKOV-3 cells are able to mediate migration at a faster rate compared with OVCAR-3-derived exosomes. Therefore, exosomes may be facilitating cross-talk between cells in the tumour microenvironment leading to increased cell motility and migration and ultimately to metastasis. However, further research is required to understand the signalling pathways, which are influenced by exosomes leading to cell migration. Finally, we suggest that exosomes released from tumour cells within the ovarian cancer microenvironment play an important role by interacting with neighbouring cells to increase migration which may be a preparatory step in the pre-metastatic niche formation. Although the mechanisms involved in these phenomenon remain to be elucidated, these data suggest that exosomes are capable of engaging in para-cellular interactions (i.e. local cell-to-cell communication between the cell constituents of the tumour microenvironment and contiguous peritoneal tissue) and/or distal interactions (i.e. involving the release of tumour exosomes into biological fluids and their transport to a remote site of action) to modify the phenotype of target cells (e.g. migration capacity) by transferring their contents (e.g. surface and cytoplasmic protein, mRNA and miRNAs) to promote cancer progression.

Clinical perspectives

  • The effect of exosomes from cancer cells on cell migration (MSCs and ECs) in the tumour microenvironment is currently unclear.

  • Release and bioactivity of exosomes on target cells is positively correlated with invasiveness of originating cells and exosomes from tumour cells are able to modulate and change the behaviour of neighbouring cells present within the tumour microenvironment such as MSCs and ECs.

  • The present study has demonstrated that cancer exosomes can influence cell migration, suggesting a potential process through which tumour cells metastasise.

We thank the assistance of Dr Jamie Riches and Dr Rachel Hancock of the Central Analytical Research Facility (CARF), Institute for Future Environments, Queensland University of Technology (QUT) for the electron microscope analyses. We also thank the editorial assistance of Debbie Bullock (UQ Centre for Clinical Research, The University of Queensland).

Competing interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

This work was supported by the Lions Medical Research Foundation [grant number 606833]; the Ovarian Cancer Research Foundation (OCRF) [grant number 020780]; the UQ-Ochsner Seed Fund for Collaborative Research [grant number 610193]; the University of Queensland; the Faculty of Medicine M+BS Emerging Leaders Medical Research Grant; the Fondo Nacional de Desarrollo Científico y Tecnológico [grant number FONDECYT 1170809]; the Ovarian Cancer Research Foundation [grant number 2018001167]; the Research Training Program Scholarship from the University of Queensland, funded by the Commonwealth Government of Australia (to S.S.).

Author contribution

C.S., S.S. and M.K. conceived and designed the study. M.K., S.S., M.A., A.L., C.P. and K.S.-R. performed the experiments. C.S., F.Z. and G.E.R. performed data analysis. C.S., S.S. and M.K. wrote the initial draft of the manuscript. C.S., S.S. and J.D.H. edited the manuscript. V.O. performed the experiments and D.G performed data analysis. All authors reviewed/edited the manuscript and approved the final version.

Abbreviations

     
  • DMEM

    Dulbecco’s modified Eagle medium

  •  
  • EC

    endothelial cell

  •  
  • HUVEC

    human umbilical vein EC

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • MSC

    mesenchymal stem cell

  •  
  • MVB

    multivesicular body

  •  
  • NTA

    nanoparticle tracking analysis

  •  
  • PI3K

    phosphoinositide 3-kinase

  •  
  • pMSC

    placental MSC

  •  
  • RWD

    relative wound density

  •  
  • RIPA

    radioimmunoprecipitation assay

  •  
  • ST50

    half-maximal stimulatory time

  •  
  • VEGF

    vascular endothelial growth factor

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Supplementary data