The PC cell lines Panc-1 and BxPC-3 were purchased from the American Type Culture Collection. Both cell lines were maintained in RPMI 1640 (HyClone; Cytiva) with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37°C under 5% CO₂.

Cells were cultured in 1640 medium supplemented with 10% FBS until they reached 70% confluence. After removing the medium and washing the cells three times with PBS, fresh serum-free 1640 medium (HyClone; Cytiva) was added, and the cells were cultured for another 48 h at 37°C. The medium was aspirated and centrifuged for 10 min at 300 × g to remove the dead cells at room temperature, and then at 9,000 × g for 30 min to remove the cell debris at room temperature. The supernatant was centrifuged at 100,000 × g for 2 h at 4°C, and the pelleted exosomes were resuspended in PBS, then ultracentrifuged at 100,000 × g for 2 h at 4°C. The last two steps were repeated, and the purified EXOs were characterized. The protein concentration of EXOs was determined using a BCA kit (Pierce; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions.

A total of 10 µl purified EXOs (100 µg/ml) was placed on non-glow-discharged carbon-coated grids (300 mesh; Beijing XXBR Technology Co., Ltd.) for 10 min with 4% paraformaldehyde at room temperature and negatively stained with 10 µl 2% uranyl acetate for 1 min at room temperature. The excess solution was removed by wicking onto filter paper, and dried grids were viewed under a FEI/Philips CM12 transmission electron microscope (TEM) operating at 80 KeV (magnification, ×100).

Total protein was extracted from cells using RIPA buffer (cat. no. 89900; Thermo Fisher Scientific, Inc.). The protein concentration was determined using a bicinchoninic acid assay kit (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of cell lysate (WCLs; 20 µg) or exosome lysate (EXOs; 10 µg) were resolved by Tris-Bis gel electrophoresis using a 4–15% gradient gel (Bio-Rad Laboratories, Inc.). The protein bands were transferred to a nitrocellulose membrane and blocked with 5% skimmed milk in PBS with 0.05% Tween-20 (PBST) at room temperature for 2 h. The blots were probed with anti-tumor susceptibility gene 101 protein (Tsg101; cat. no. ab30871; Abcam, 1:800), anti-CD63 (cat. no. ab68418; Abcam; 1:500), anti-Giantin (GM130; cat. no. ab187514; Abcam; 1:1,000) and anti-EphA2 (cat. no. ab73254; Abcam; 1:1,000) antibodies for overnight at 4°C. After washing four times with PBST, the membranes were incubated with the secondary antibody (1:5,000) for 1 h at room temperature. Protein bands were visualized using ECL Western Blotting Detection Reagent (cat. no. G075; Applied Biological Materials, Inc.). The band intensities were quantified by ImageJ software (version 1.8.0; National Institutes of Health).

Panc-1 (1×10⁵) and BxPC-3 (5×10⁵) cells were seeded in 6-well plates and cultured for 24 h to ~100% confluence. The monolayers were gently scratched with 1 ml pipette tips to create a ‘wound’, and the wells were gently washed with 1X PBS to remove the dislodged cells. The cells were cultured for 48 h with 20 µg/ml Panc-1 or Panc-1^EphA2− EXO or 0.5 µg/ml recombinant EphA2 that were added twice (once each at 0 and 24 h) at 37°C. The scratched areas were photographed at 0, 24 and 48 h post-wounding using a confocal microscope (magnification, ×200), and the area was measured using ImageJ (version 1.8.0; National Institutes of Health). Both cell lines were maintained in RPMI 1640 with 1% FBS. The experiment was repeated three times.

The protein extracts were resolved by 15% SDS-PAGE, and the gel was stained with silver nitrate. The appropriate gel pieces were cut and digested, and the resulting peptides were analyzed by positive-ion data-dependent micro-capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an LTQ 2D Linear Ion Trap Mass Spectrometer (Thermo Fisher Scientific, Inc.) as per standard protocol (17). The initial MS scan was followed by eight further MS/MS scans. The proteins were identified by aligning the sequences against the UniProt database (www.uniprot.org) using the Sequest algorithm in Proteomics Browser software (version 2.3.0; Thermo Fisher Scientific, Inc.).

Panc-1 EXOs were labeled with EXO-Red (cat. no. EXOC300A-1; System Biosciences, LLC) according to the manufacturer's instructions. BxPC-3 cells were plated at 2×10⁴ cells/well on 8-well chamber slides for 24 h and supplemented with 20 µg/ml EXO-Red-labeled Panc-1 EXOs. For microscopy studies, cells were washed three times with PBS, incubated with 4% paraformaldehyde for 15 min at 25°C, and incubated in DAPI/PBS solution (1:1,000) for 5 min at 25°C. EXOs were visualized under a laser scanning confocal microscope (magnification, ×40; FV-100; Olympus Corporation).

The plasmid pGLVH1/GFP+Puro, EphA2_shRNA (5′-GAACTTCAACACAGCCTGG-3′) and packaging vector PG-P1-VSVG were prepared by Shanghai GenePharma Co., Ltd., and extracted with high purity and no endotoxins. 293T cells (density, 60–70%) were co-transfected with RNAi-mate (Shanghai GenePharma Co., Ltd.). After 6 h of transfection, they were replaced with a complete culture medium. After 72 h of culture, the supernatant of cells was collected with ultracentrifugation at 40,000 × g for 2 h at 4°C and concentrated to obtain a high titer lentivirus concentrate for infecting target cells.

Panc-1 cells were transduced with lentivirus (1×10⁹ TU/ml; dilution, 1:10) and 5 µg/ml polybrene (Sigma-Aldrich; Merck KGaA) for 24 h at 37°C, then the stable transfectants were selected in the presence of 10 µg/ml puromycin (Invitrogen; Thermo Fisher Scientific, Inc.) for 2 days. Decreased EphA2 protein expression levels were confirmed via western blotting as aforementioned. The stable EphA2-Panc-1 cells were maintained in complete RPMI-1640 supplemented with 2 µg/ml puromycin at 37°C.

Serum samples were obtained from 40 patients with PC (median age, 30–70 years; 36 males and 34 females; 20 patients each at stage I+II and stage III+IV) and 30 healthy controls at the Tianjin Medical University Cancer Institute and Hospital (Tianjin, China) between March 2019 and September 2019. The disease was staged according to the American Joint Committee on Cancer tumor, node, metastasis classification (18). All experiments involving human specimens were performed in accordance with the 1964 Declaration of Helsinki ethical standards and approved by the Research Ethics Committee of Tianjin Medical University Cancer Institute and Hospital (approval no. bc2019112). Written informed consent was obtained from all patients prior to participation.

EXOs were precipitated from 100 µl patient serum, using ExoQuick (System Biosciences, LLC) according to the manufacturer's protocols. EXO pellets were then suspended in PBS and analyzed using an EphA2 ELISA kit (cat. no. DYC3035-2; R&D Systems, Inc.) according to the manufacturer's protocol.

Data are presented as the mean ± standard deviation of three biological repeats. Multiple groups were compared using one- or two-way ANOVA with a post hoc Bonferroni test. All statistical analyses were performed using GraphPad Prism (version 5.0; GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

EXOs released from PC cells were purified and characterized in terms of morphology and exosomal marker expression levels. The distinctive EXO structure was observed via TEM (Fig. 1A), and dynamic light scattering showed a typical size range of 30–100 nm (Fig. 1B). Furthermore, the purified EXOs expressed Tsg101 and CD63, but not the Golgi protein GM130 (Fig. 1C). Thus, these results confirmed the purity of EXO preparation.

Panc-1 cells exhibited significantly higher in vitro migration ability compared with BxPC-3 cells (Figs. 2A and S1). In order to determine whether Panc-1-derived EXOs enhance the migration of BxPC-3 cells, the latter were incubated with EXOs for 48 h. The percentage of migrating BxPC-3 cells increased significantly at 48 h of exposure to Panc-1 EXOs compared with control cells (Figs. 2B and S1). By contrast, EXOs derived from BxPC-3 cells had no effect on migration (Fig. S2). These results indicated that EXOs secreted by metastatic PC cells enhanced the migration of recipient cells, which is likely by transporting relevant factors.

BxPC-3 cells incubated with Panc-1 EXOs exhibited increased expression of Tsg101 (Fig. 2C and D). The internalization of Panc-1 EXOs by BxPC-3 cells was tracked by labeling EXOs with EXO-Red. Recipient cells internalized these EXOs within 10 h (Fig. 2E).

EXOs mediate intercellular communication via horizontal transfer of proteins (19). Therefore, it was hypothesized that EXOs derived from Panc-1 and BxPC-3 cells have distinct proteomes and are enriched in metastasis-associated proteins. In order to validate exosomal protein composition, lysates of purified EXOs derived from Panc-1 and BxPC-3 cells were resolved via SDS-PAGE. The silver-stained gels showed distinct protein profiles of EXOs (Fig. 3A), which was confirmed via LC-MS/MS analysis (Table SI). The expression levels of EphA2 were significantly higher in both the lysates and EXOs of Panc-1 compared with BxPC3 cells (Fig. 3B and C) in terms of the LC-MS/MS score. Taken together, these results indicated that the metastatic effects of Panc-1 EXOs are likely mediated by EphA2.

In order to further validate the metastatic function of EXO_EphA2, a stable EphA2⁻ Panc-1 cell line was generated, which exhibited a 70% decrease in EphA2 expression levels compared with control cells (Fig. 4A). Cell migration was significantly decreased in Panc-1^EphA2− cells compared with Panc-1 cells (Fig. S3). The EXO_EphA2 levels were also significantly decreased in Panc-1^EphA2− cells compared with Panc-1 cells (Fig. 4B). Furthermore, unlike EXOs derived from wild-type Panc-1 cells, Panc-1^EphA2− EXOs did not significantly increase the migration of BxPC-3 compared with control cells. BxPC-3 cells incubated with recombinant EphA2 did not exhibit increased migration, indicating that exosomal delivery is essential for the oncogenic effects of EphA2 (Figs. 4C and S4). These results indicated that EXO_EphA2 mediates migration of PC cells.

Tumor-derived EXOs are easily detectable in circulation, and therefore are promising diagnostic/prognostic markers for cancer. The levels of circulating EXO_EphA2 were significantly higher in the serum of patients with PC compared with those in healthy controls (Fig. 4D). In addition, levels of circulating EXO_EphA2 were significantly elevated in advanced stage (III+IV) patients compared with those in the early stages (I+II). These results indicated that EXO_EphA2 is a potential diagnostic biomarker for PC.