The present study was approved by the Ethics Committee of Huzhou Central Hospital, Zhejiang University (Huzhou, China). Written informed consent was obtained from patients, or from the guardians on behalf of the minors, prior to enrollment in the present study. Patient diagnoses were independently reviewed by two pathologists and classified according to the WHO criteria. A total of 63 patients (age range, 43–85 years) with histologically confirmed PDAC that were treated at Huzhou Central Hospital of Zhejiang University were recruited for this study between May 2009 and December 2014. Of the 63 patient samples collected, 6 were paired fresh PDAC tissues and adjacent normal tissues. Follow-up data were available for all 63 patients. Sera were also collected from three patients that suffered pancreatitis and three patients with PDAC.

The human PDAC cell lines PANC-1 (catalog no. TCHu98), BxPC3 (catalog no. TCHu12) and ASPC-1 (catalog no. TCHu8) were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Capan-1 was purchased from the American Type Culture Collection (catalog no. HTB-79; Manassas, VA, USA). Cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37°C in a humidified atmosphere of 5% CO₂. All cells were free of mycoplasma contamination.

Lentiviral vectors for AEP knockdown (KD) or overexpression (OE) were constructed by Shanghai Hanyin Biotechnology Co., Ltd. (Shanghai, China). Empty vector was used as negative control (NC) for AEP-KD and -OE experiments; AEP-targeted KD sequences and AEP-OE sequences were used as previously described (30). AEP-targeted KD sequences were: KD1, 5′-GATGGTGTTCTACATTGAA-3′, and KD2, 5′-GGGGACTGGTACAGCG TCA-3′. The lentiviral particles were packaged using psPAX2 and pMD2G plasmids (Shanghai Hanyin Biotechnology Co., Ltd.). To obtain stable cells with reduced or overexpressed AEP, lentivirus-containing supernatants (Shanghai Hanyin Biotechnology Co., Ltd.) were added to the PDAC cells, followed by selection with 1 μg/ml puromycin (Shanghai Hanyin Biotechnology Co., Ltd.) for 2 weeks to select stably expressing AEP-KD1, AEP-KD2 or AEP-OE cells (31).

Primary antibodies used in the present study included: Goat anti-human AEP (catalog no. AF2199; R&D Systems; Bio-Techne, Abingdon, UK), rabbit anti-human AKT (catalog no. 4685; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit anti-human phosphorylated (p)-AKT (catalog no. 4060; Cell Signaling Technology, Inc.), rabbit anti-CD63 (catalog no. ab68418; Abcam, Cambridge, UK), rabbit anti-human PI3K (catalog no. 3811; Cell Signaling Technology, Inc.), and rabbit anti-β-actin (catalog no. ab8227; Abcam); and the horseradish peroxidase (HRP)-conjugated donkey anti-goat immunoglobulin G (IgG; catalog no. 705-036-147; Jackson ImmunoResearch, Inc.) the horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (catalog no. 7074; Cell Signaling Technology, Inc.) secondary antibody was also used in the present study.

Tissues were fixed in 4% paraformaldehyde overnight at 4°C, embedded in paraffin and sectioned (6 μm). Immunohistochemical analyses were performed as previously described (32). Goat anti-human AEP antibody (catalog no. AF2199; R&D Systems; Bio-Techne, Abingdon, UK; diluted 1:200 in blocking buffer) was used as primary antibody. Biotin-conjugated donkey anti-goat IgG (catalog no. 705-066-147; Jackson ImmunoResearch, Inc.) was used as secondary antibody. Normal goat IgG (catalog no. AB-108-C; R&D Systems; Bio-Techne) was included as negative control. The proportion of positive protein expressions were evaluated as follows: A score of 0 was indicated if 0% of the tumor cells showed positive staining; 1 if 0–10% of cells were stained; 2, 11–50% stained; 3, 51–75% stained; and 4 if 75–100% stained. The intensity of staining was rated on a scale of 0 to 3: 0, negative; 1, weak; 2, moderate; and 3, strong. The proportion and intensity scores were combined to obtain a total score (range 0–6) and designated 0–3.5 as low expression and 4–6 as high expression.

Total protein was extracted from cells, exosomes and tissue samples using RIPA lysis and extraction buffer (Thermo Fisher Scientific, Inc.). Protein concentration was determined using the bicinchoninic acid protein assay method. Lysates (50 μg per lane) were separated by 8% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat milk for 30 min at 25°C, followed by overnight incubation with primary antibodies (1:500) at 4°C. Subsequently, membranes were incubated with HRP-conjugated secondary antibodies (1:3,000) for 60 min at 25°C. Immunoreactive proteins were visualized with the Immobilon Western Chemiluminescent HRP Substrate (cat. no. WBKLS0500; EMD Millipore, Billerica, MA, USA). Quantity One analysis software version 4.6.9 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to quantify the relative band intensities from western blotting images; actin or CD63 was used for loading controls and for normalization. The assays were conducted in triplicate.

Total RNA was extracted from PDAC cells (2×10⁶) using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer’s instructions. cDNA was reverse transcribed from 1 μg total RNA using the Promega Reverse Transcription System (cat no. A3500; Promega Corporation, Madison, WI, USA). qPCR was performed with the SYBR Premix Ex Taq kit (Takara Biotechnology Co., Ltd., Dalian, China). Primers were purchased from Sangon Biotech Co., Ltd. (Shanghai, China), and the sequences are as follows: AEP, forward 5′-TCA GGGTATGAAACGCAAAGC-3′, reverse 5′-GAGACGATCT TACGCACTGAC-3′; GAPDH, forward 5′-CATGGCCTTCCGTGTTCCTA-3′, reverse 5′-GCGGCACGTCAGATCCA-3′; GAPDH was used as a loading control. Thermocycling conditions comprised initial denaturation at 95°C (5 min), followed by 36 cycles of denaturation at 95°C (10 sec) and annealing/elongation at 60°C (30 sec). Relative mRNA expression levels were calculated using the 2^−ΔΔCq method using the housekeeping gene GAPDH for normalization (33). The assays were conducted in triplicate.

To isolate exosomes, PDAC cells were cultured for 48 h at 37°C and the supernatants of these cells were collected and centrifuged twice (1,000 × g for 10 min at 4°C, and 3,000 × g for 30 min at 4°C) to remove cells and fragments. Subsequently, the exosome isolation reagent from the Total Exosome Isolation kit (Invitrogen; Thermo Fisher Scientific, Inc.) was added to the cell media sample and incubated overnight at 4°C. The precipitated exosomes were recovered by centrifugation at 10,000 × g for 1 h at 4°C. For exosome isolation in sera, the ExoQuick Exosome Precipitation Solution (catalog no. EXOQ5A-1; System Biosciences, Palo Alto, CA, USA) was used to isolate exosomes from serum samples, according to the manufacturer’s instructions. Exosomes were re-suspended in PBS and stored at −80°C. The concentration of exosomes was determined by BCA protein assay. Exosomes (50 ng/rl) were added to 1×10⁵ cells in culture medium for 24 h at 37°C, as previously described (34). The assays were conducted in triplicate.

The exosome suspension was added to an equal volume of 4% paraformaldehyde at 4°C for 30 min and applied to a Formvar/Carbon film-coated transmission electron microscope grid (Alliance Biosystems, Inc., Osaka, Japan). Subsequently, the sample was fixed by incubation with 1% glutaraldehyde for 5 min at 25°C, washed with PBS and contrasted with 1% uranyl acetate for 5 min at 25°C. Samples were embedded in epoxy resin and polymerized at 35°C for 12 h, 45°C for 12 h and 60°C for 24 h. Exosomes were subsequently observed under a Hitachi H-7650 transmission electron microscope (Hitachi, Ltd., Tokyo, Japan).

Cells (1×10⁵) were seeded into the upper chambers of Matrigel-coated Transwell chambers (pore size, 8 μm) in serum-free DMEM. DMEM containing 10% FBS was added to the lower chamber as a chemoattractant. Following incubation for 24 h at 37°C, the upper surfaces of the inserts were gently wiped with a cotton swab and cells that had invaded the lower chambers were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet at 37°C for 30 min. The number of invading cells was counted under an Olympus CKX41 inverted microscope (Olympus Corporation, Tokyo, Japan); five random microscopic fields were analyzed for each insert. The assays were conducted in triplicate.

OS rates were calculated from the date of surgery to the date of death or last follow-up; survival curves were plotted using the Kaplan-Meier method and compared using the log-rank test. Median survival times and hazard ratios (HRs) were shown with 95% confidence intervals (CIs). Data are presented as the mean ± standard deviation. To assess the differences between groups, categorical variables were compared by means of χ² analysis. Analysis of variance tests were followed by two-tailed Dunn’s post-hoc analysis or Tukey’s multiple comparisons test to identify statistically significant differences. Statistical analyses were performed using SPSS software version 15.0 (Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

AEP protein expression levels were analyzed in freshly collected human PDAC tissues (n=6) and adjacent normal tissues (n=6) by western blotting (Fig. 1A). AEP expression levels were notably higher in PDAC tissues compared with expression levels in adjacent normal tissues (Fig. 1A). AEP protein expression levels were also examined by immunohistochemical analysis in the 6 matched tissues as well as the remaining 57 tumoral tissues (Fig. 1B and C, respectively). Consistent with the western blotting results, AEP staining was stronger in tumoral tissues compared with expression levels in the adjacent normal tissues (Fig. 1B). The staining of AEP was revealed to be mainly localized in the cytoplasm in the PDAC tissues (Fig. 1B and C).

According to the expression level score of AEP protein in PDAC samples, all cases were distributed into two groups: A low AEP expression group (n=35), and a high AEP expression group (n=28; Fig. 1C; Table I). Following evaluation of the immunohistochemical staining results, AEP staining levels in the American Joint Committee on Cancer (AJCC) stage II cases were significantly higher compared with staining level in the AJCC stage I cases (P=0.009; Table I). The expression of AEP in PDAC tissues exhibited a strong association with AJCC stage, although no associations were found between AEP expression and other clinicopathological features (Table I).

To assess the relationship between the level of AEP expression with patient prognosis the Kaplan-Meier and log-rank tests were used to evaluate the effects of AEP expression on patient OS. Patients with a high level of AEP expression in tumoral tissues had significantly shorter OS times compared with patients with low AEP expression (n=63; P=0.005; Fig. 1D and Table II). The mean OS time of patients with low AEP expression was 20.29 months (n=35; 95% CI, 14.95–25.63; Table II), whereas the OS time of patients with high AEP expression was 10.11 months (n=28; 95% CI, 5.84–14.37). The log-rank test (univariate analysis) revealed that the patients with low AEP expression had a longer OS time (χ² = 2.536; P=0.005; Table II). Furthermore, multivariate Cox regression analysis was also performed, which indicated that AEP expression was an independent prognostic factor (HR = 2.415; 95% CI, 1.345–4.334; P=0.003; Table III), whereas AJCC stage was not (HR = 1.475; 95% CI, 0.574–3.787; P=0.419).

To examine the functions of AEP in pancreatic adenocarcinoma progression, the expression levels of AEP protein were first examined in several PDAC cell lines by western blotting (Fig. 2A). The results showed that AEP was expressed in all PDAC cell lines. Subsequently, two AEP-KD lentiviral vectors were constructed and used to knock down AEP expression in ASPC-1 cells (Fig. 2B and C). The RT-qPCR and western blotting results demonstrated that AEP was effectively knocked down upon treatment with AEP-KD1 and -KD2 compared with NC-treated cells. The effects of AEP on the invasive ability of PDAC cells were assessed by Matrigel-Transwell invasion assay, which indicated that suppression of AEP expression resulted in reduced invasive ability of ASPC-1 cells compared with NC-treated cells (Fig. 2D and E). AEP-OE BxPC-3 cells were also constructed and verified by RT-qPCR and western blotting (Fig. 3A and B); overexpression of AEP in BxPC3 cells significantly increased the invasive ability of these cells compared with NC-treated cells (Fig. 3C and D). These data suggested that AEP may be crucial for the invasive phenotype of PDAC cells.

AEP was previously reported to be a secreted protein (27), and exosomes are key mediators and modulators of cell-cell communications to promoter tumor metastasis. Therefore, the exosomes secreted by PDAC cells were collected and analyzed. The morphology of exosomes was observed under transmission electron microscopy; exosomes are round in appearance and ~100 nm in diameter (Fig. 4A). Western blotting results demonstrated that the exosomes from each PDAC cell line expressed AEP protein (Fig. 4B). When AEP expression was knocked down in ASPC-1 cells, exosomal AEP protein expression was notably reduced in AEP-KD1-treatd cells compared with AEP-KD2- and NC-treated cells (Fig. 4C). To further determine the putative functions of pancreatic cancer cell-derived exosomal AEP on PDAC metastasis, ASPC-1 cells were cultured with the exosomes isolated from either untreated cells or cells treated with AEP-KD1 or -KD2 and the invasive ability was examined. Results from the Matrigel-Transwell invasion assay indicated that exosomes with a low content of AEP (that is, exosomes isolated from cells treated with either AEP-KD1 or -KD2) exhibited a significantly reduced ability to promote the invasion of PDAC cells compared with cells treated with NC-exosomes (Fig. 4D and E). Conversely, exosomes derived from AEP-overexpressing BxPC3 cells significantly increased the invasive ability of treated PDAC cells compared with cells treated with NC-exosomes (Fig. 4F–H). These results suggested that exosomal AEP may be crucial for the invasive phenotype of PDAC cells.

Exosomes were isolated from the sera of patients with either PDAC or pancreatitis. Western blotting results revealed that AEP was enriched in the exosomes isolated from the sera of patients with PDAC compared with expression levels in patients with pancreatitis (Fig. 5A and B). BxPC3 cells were co-cultured with these isolated exosomes and the invasive ability was examined. Results from the Matrigel-Transwell invasion assays indicated that BxPC3 cells treated with exosomes collected from patients with PDAC (with a high content of AEP) exhibited a significantly higher invasive ability compared with cells treated with exosomes derived from patients with pancreatitis (Fig. 5C and D).

PI3K/AKT signaling is an important survival pathway that is involved in carcinogenesis and malignant cell progression (22–24). Therefore, whether AEP was able to regulate the PI3K/AKT pathway in PDAC cells was investigated. Reduced AEP expression in BxPC3 cells led to decreased expression levels of p-PI3K and p-AKT, but not total PI3K or AKT expression, compared with the respective expression levels in NC-treated cells (Fig. 6A). Furthermore, AEP overexpression in BxPC3 cells resulted in significantly elevated p-PI3K and p-AKT expression levels (Fig. 6B). Cells cultured with exosomes expressing reduced levels of AEP also exhibited significantly reduced p-PI3K and p-AKT expression compared with NC-treated cells (Fig. 6C), whereas cells treated with AEP-OE exosomes exhibited increased expression levels of p-PI3K and p-AKT (Fig. 6D). These results indicated that AEP may be an important element in pancreatic cancer cell invasion and survival by regulating the PI3K/AKT pathway.