Female BALB/c mice (age, 6–8 weeks) were purchased from the Laboratory Animal Center of Southern Medical University (Guangzhou, Guangdong, China). The experiments were conducted according to the guidelines of the China Council on Animal Care. The mice were allowed free access to food and water, and were maintained in specific pathogen-free conditions. The murine breast cancer cell line 4T1, was purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. The murine breast cancer cell line, MA782, was provided by Dr. Yanqing Ding (Southern Medical University). All cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; HyClone Laboratories, Inc., Logan, UT, USA), penicillin (100 units/ml; Sigma-Aldrich, St. Louis, MO, USA) and streptomycin (100 μg/ml; Sigma-Aldrich), at 37°C in a humidified atmosphere containing 5% CO₂. The study was approved by the ethics committee of Southern Medical University, Guangzhou, China.

BL21 E. coli cells and the pET28a⁺ expression vector were purchased from Novagen, Inc. (Madison, WI, USA). The bacterial strains were grown at 37°C in Luria-Bertani (LB) broth or on LB agar plates, and kanamycin was added to the culture medium at a concentration of 50 μg/ml when required. Isopropyl-β-D-thiogalactopyranoside (IPTG, 1 mM) was added to the media as indicated.

Total RNA was extracted from MA782 cells using TRIzol reagent (Life Technologies, Inc., Gaithersburg, MD, USA). Based on the murine Eps8 gene sequence in the Genbank, a pair of PCR primers were designed (amino acids, 440–710). The primer sequences used were as follows: Forward: 5′-GCG GAA TTC CCG ATG CTG AAC TTC ATG-3′, containing an EcoRI site; and reverse: 5′-ATA CTC GAG CCC GAT GGT CAG CCT CTG-3′, containing a XhoI site. The reaction conditions were as follows: 94°C for 5 min, 94°C for 30 sec, 60°C for 45 sec and 72°C for 30 sec (for a total of 35 cycles), and then 72°C for 10 min. Following PCR amplification, the desired fragment was identified by agarose gel electrophoresis. Subsequent to purification, the PCR product and the pET28a vector were digested with EcoRI and XhoI double enzymes, and linked by T4 DNA ligase, at 16°C for 16 h. The sequence was analyzed by the sequencing laboratory of Invitrogen Corporation (Guangzhou, Guangdong, China). Following verification, the recombinant plasmid (pET28a-Eps8) was transformed into BL21 E. coli cells.

BL21 E. coli cells containing pET28a-Eps8 were inoculated in 5 ml LB broth (with 50 μg/ml kanamycin sulfate) and cultured overnight at 37°C. The bacterial culture was grown in 100 ml LB broth to an OD600 of 0.8, and was induced by 1 mM IPTG for 4–5 h at 37°C, harvested by centrifugation (5 min, 8,228 × g at 4°C), and then dispersed into a lysis buffer and sonicated. Nickel-nitrilotriacetic acid (Ni-NTA) agarose was used for 6X His-tagged protein purification, according to the manufacturer's instructions (Qiagen, Hilden, Germany). Endotoxin was removed from the protein using Pierce High Capacity Endotoxin Removal Resin (Thermo Fisher Scientific, Rockford, IL, USA), according to the manufacturer's instructions. The purified protein was boiled for 10 min, and then loaded onto a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and electrophoresed. The samples were transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked by 5% skimmed milk for 1 h, washed three times with phosphate-buffered saline (PBS)-Tween 20 (10 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.05% Tween 20), and incubated with mouse anti-EPS8 monoclonal antibodies (BD Biosciences, San Diego, CA, USA) overnight at 4°C. The membrane was then washed three times with PBS-Tween 20, and incubated with goat anti-mouse immunoglobulin G (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 4 h at 37°C. The immunoreactive bands were detected using the Amersham enhanced chemiluminescence (ECL) detection system and ECL hyperfilm (GE Healthcare, Waukesha, WI, USA), according to the manufacturer's instructions. The FluorChem FC2 imaging system was used to analyze the chemiluminescent blot (Alpha InnoTech Corp., San Leandro, CA, USA).

Bone marrow cells were isolated from the femurs and tibias of 6- to 8-week-old BALB/c mice, and incubated in hypotonic lysis buffer (9.84 g/l NH₄Cl, 1 g/l KHCO₃, 0.1 mM EDTA) for 5 min at 37°C in a water bath, to remove the red blood cells. The cells were washed twice with PBS buffer, and then plated in 6-well plates at a density of 1×10⁶ cells/ml (5×10⁵ cells/well) for 1 h at 37°C, in RPMI-1640 medium with 10% FBS. Non-adherent single cells were gently removed, and the adherent cells were cultured in 2 ml RPMI-1640 with 10% FBS, 50 ng/ml murine rGM-CSF (PeproTech, Inc., Rocky Hill, NJ, USA) and 20 ng/ml murine recombinant interleukin-4 (rIL-4; PeproTech, Inc.) per well for six days. For the induction of maturation, cultures of bone marrow-derived DCs (BM-DCs) were supplemented with 50 ng/ml murine tumor necrosis factor-α (TNF-α; PeproTech, Inc.) for 24 h on the sixth day of the culturing process, prior to use (22,23). The DCs were pulsed on day 7 with either Eps8 protein (50 μg/ml) or chicken egg ovalbumin (OVA; 50 μg/ml; Alpha Diagnostic Intl Inc., San Antonio, TX, USA) for 24 h, and the pulsing was terminated by medium replacement. Different groups of DCs were collected and washed three times with PBS buffer. The DCs were then incubated with the corresponding antibodies on ice for 30 min. Flow cytometric analysis was performed on a fluorescence-activated cell sorter (FACSCalibur flow cytometer; Becton-Dickinson, Mountain View, CA, USA). The DCs were phenotyped with antibodies against complement component 3 receptor 4 subunit (CD11c), cluster of differentiation 80 (CD80), CD86 and major histocompatibility complex class II (MHC-II; BioLegend, San Diego, CA, USA). The negative controls consisted of DCs labeled with mouse Ig.

The spleens were removed from 6- to 8-week-old BALB/c mice under sterile conditions. Subsequently, single-cell suspensions were prepared by pressing the spleens through a sterile 100-mesh metal sieve and filtrated using a 80-μm nylon mesh. The lymphocytes from the suspension of the spleen cells (6 ml) were isolated by Ficoll density gradient centrifugation at 514 × g for 30 min at room temperature. The cells of the middle layer were carefully removed and washed twice with serum-free RPMI-1640 medium. The splenocytes were counted with a hemacytometer and plated in 6-well plates (5×10⁶ cells/well), in 2-ml RPMI-1640 medium with heat-inactivated FBS and 20 ng/ml recombinant mouse IL-2 (24).

Proliferation of the splenocytes was determined by an MTT assay as previously described (25). Splenocytes were harvested and co-cultured with autologous DCs at a ratio of 5:1 in 96-well plates for 72 h, and this ratio had been identified to be optimal (data not shown). The OD570 was read and the stimulation index was calculated by dividing the background mean absorbance from cultures with unstimulated splenocytes by the experimental mean absorbance from cultures with DC-stimulated splenocytes. All experiments were performed in triplicate and repeated three times.

The cytotoxic activity of the splenocytes was assessed by the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega, Madison, WI, USA), according to the manufacturer's instructions. The splenocytes that were co-cultured with autologous DCs served as effector cells, while the 4T1 cells served as target cells. The target cell suspension was adjusted to 5×10⁵ cells/ml and the effector cell suspension was adjusted to 1×10⁷ cells/ml, with RPMI-1640 medium (containing 5% FBS), and the cells were plated in 96-well plates. The effector to target (E/T) cell ratio was 20:1. Wells containing only target cells or only effector cells with culture medium or 0.5% Triton X-100 served as spontaneous or maximal release controls. Following 4 h of incubation at 37°C and 5% CO₂, 50 μl supernatant was analyzed by the Model 680 microplate reader at a reference wavelength of 650 nm and a test wavelength of 450 nm (Bio-Rad, Hercules, CA, USA). The percentage of specific lysis was calculated as follows: Specific lysis (%) = 100 × (experimental release - effector spontaneous release - target spontaneous release) / (target maximum release - target spontaneous release).

The supernatant of the splenocytes was collected at different time points (days 1, 3 and 5) and tested for the presence of IFN-γ by ELISA, according to the manufacturer's instructions (Endogen, Inc., Cambridge, MA, USA). In order to detect the level of IL-12 that was secreted by the DCs, supernatants were collected at the aforementioned time points and analyzed for IL-12 p70, using the ELISA (Biosource, Camarillo, CA, USA).

The experiment was performed on 4- to 6-week-old female BALB/c mice. The BABL/c mice were randomly divided into two groups: The Eps8 group and the PBS group, with 8 healthy mice included in each experimental group. The animals were immunized subcutaneously once a week for three weeks; the mice received 100 μg of either Eps8 protein or PBS mixed with complete Freund's adjuvant for the first immunization, and for the second and third immunizations, the same quantity of either Eps8 protein or PBS was mixed with incomplete Freund's adjuvant. Seven days subsequent to the third immunization, the mice were inoculated subcutaneously with 7×10⁶ 4T1 tumor cells in the right armpit. The mice were monitored each day and the day of the tumor cell inoculation was recorded as day 0.

The animals were vaccinated and then challenged with tumor cells. The antitumor effects were evaluated by measuring the tumor volume, tumor growth inhibition rate and overall survival time. In the tumor protection experiments, the tumor size was determined by the measurement of the shortest (a) and longest (b) diameter using vernier calipers, every 3 days. The volume (V) of the tumors was calculated using the formula: V = (a²b/2) (26). For the comparison of tumor growth inhibition rates, mice were sacrificed by neck dislocation 28 days following inoculation. The tumors were surgically removed and weighed. The inhibition rate was calculated as follows: Inhibition rate (%) = (1 - tumor weight of Eps8 group/tumor weight of PBS group) × 100. For survival analysis, an observation period of 60 days was adopted following the inoculation of the mice with 4T1 tumor cells.

BALB/c mice were grouped and treated as described previously, and sacrificed 28 days following inoculation with tumor cells. The splenocytes were collected as described previously, and incubated with the corresponding antibodies: Fluorescein isothiocyanate (FITC) anti-mouse CD4 mAb and PerCP anti-mouse CD8 mAb (BioLegend). The negative control consisted of splenocytes labeled with the corresponding isotype control antibodies (27). The Mouse Regulatory T cell Staining kit (eBiosciences, Inc., San Diego, CA, USA) was used for detecting the CD4⁺CD25⁺ FoxP3⁺ Treg cells among the splenocytes. Briefly, the splenocytes of the BALB/c mice were surface-stained with FITC anti-mouse CD4 mAb and APC anti-mouse CD25 mAb. Subsequently, the cells were permeabilized using the FoxP3 Staining Buffer set, and stained with rat IgG2a kappa isotype control PE or anti-mouse/rat Foxp3 PE. All samples were run on a FACSCalibur flow cytometer (Becton-Dickinson) and data were analyzed using WinMDI 2.9 software (Joseph Trotter, The Scripps Institute, La Jolla, CA, USA).

The splenocytes were harvested from the tumor-bearing mice that had been immunized with vaccines and cultured as described previously. The viable splenocytes were regarded as the effector cells and the 4T1 cells were used as the target cells. The cytotoxic effect of the splenocytes against the 4T1 cells was measured by the lactate dehydrogenase (LDH) assay and calculated as described previously.

Data are presented as the mean ± standard deviation. The significance of the statistical comparisons was calculated by an analysis of variance (ANOVA) or the Student's t-test using SPSS software, version 13.0 (SPSS, Inc., Chicago, IL, USA). The comparison of the survival data of the two groups was evaluated with a log-rank test of the Kaplan-Meier survival curves. The tumor volumes were analyzed using a repeated measures ANOVA. P<0.05 was considered to indicate a statistically significant difference.

The PCR products were electrophoresed on 1% agarose gels and visualized by ethidium bromide staining under UV light. The results revealed that the fragment was ~801 bp, as expected (Fig. 1A, lanes 1 and 2). Following purification, the Eps8 protein was expressed on the gel with a purity of 90% in the total proteins (Fig. 1B, lanes 4 and 5). In addition, a western blot assay confirmed that the expression product had the antigenicity of Eps8 (Fig. 1C, lanes 1–3).

The DCs were generated from mouse bone marrow cells. On day 7, either TNF-α, Eps8 or OVA were added to the DCs, to induce maturation. Following 8 days of cultivation, unpulsed DCs (the DC group), OVA-pulsed DCs (the DC + OVA group) and Eps8-pulsed DCs (the DC + Eps8 group) were collected and analyzed by flow cytometry (Fig. 2). Compared with the DC and the DC + OVA groups, significant increases in the expression of MHC-II, CD80 and CD86 were observed in the DC + Eps8 group (P<0.05); however, there was no significant difference in CD11c expression among these three groups (P>0.05).

The results demonstrated that Eps8 stimulated the maturation of DCs, and their secretion of IL-12 p70. The IL-12 p70 level peaked at day 3 in all groups of DCs; the highest level was achieved by the DC + Eps8 group, and was significantly higher than the levels observed in the remaining two (control) groups (Fig. 3C). IFN-γ is a significant effector cytokine released by T cells. The ELISA results revealed that Eps8-pulsed DCs enhanced the IFN-γ secretion by the splenocytes; the concentration of IFN-γ was significantly greater in this group, compared with that of the two control groups (Fig. 3D).

The proliferative ability and the cytotoxicity of the splenocytes were also increased by Eps8. The data demonstrated an enhanced proliferation rate (by 3.28±0.34-fold) of splenocytes co-cultured with Eps8-pulsed DCs (Fig. 3A). As demonstrated in Fig. 3B, at an E/T cell ratio of 5:1, the splenocytes that were co-cultured with Eps8-pulsed DCs were able to lyse 54.89±4.99% of the target cells, while the other two (control) groups of splenocytes only exhibited a background lysis of 4T1 cells. In summary, Eps8 possessed the ability to stimulate DC and T-cell activities in vitro.

The 4T1 breast cancer model was used to investigate the effect of the Eps8 vaccine on tumor growth in vivo. The results demonstrated that none (0%) of the mice remained tumor free; however, the tumor growth in the Eps8-immunized mice was inhibited significantly compared with that of the PBS control mice (Fig. 4A). The tumor weights were measured when the mice were sacrificed and the mean tumor weight of the Eps8 group was significantly lower than that of the PBS group (Fig. 4B). In addition, the inhibition rate of tumor growth was calculated to be 33.23%. To determine the immune protection effect of the Eps8 vaccine, mice were inoculated subcutaneously with 4T1 cells following immunization, and the survival time was measured. The results revealed that all mice in the two groups succumbed within 55 days of the inoculation, but the survival time of the tumor-bearing mice that had been immunized with Eps8 was significantly longer than that of the PBS group (Fig. 4C and D). These results suggested that treatment with Eps8 prior to tumor cell inoculation resulted in a significant prophylactic antitumor effect in the 4T1 breast cancer model.

CTL activity was induced by immunization with Eps8. The LDH assay was performed at E/T cell ratios of 10:1, 20:1 and 40:1. T cells isolated from the spleens of the tumor-bearing mice pre-treated with the Eps8 vaccine exhibited greater cytotoxicity against 4T1 cells than those of the PBS group, when the E/T cell ratios were 20:1 and 40:1 (Fig. 5). The results indicated that the killing activity observed may have resulted from Eps8-specific cellular immune responses, which contributed to the observed in vivo antitumor immunity.

The proportions of splenocyte subsets were detected by flow cytometry. Eps8 immunization increased the CD4⁺/CD8⁺ T cell ratio compared with that of the PBS control mice (Fig. 6A). The proportion of CD4⁺CD25⁺ FoxP3⁺ Treg cells in the tumor-bearing mice immunized with the Eps8 vaccine or injected with PBS was also measured by FACS. Cells in the lymphocyte gate were used for analysis and the CD4 gating strategy was used as a secondary gating method (Fig. 6C). As demonstrated in Fig. 6B, the percentage of CD4⁺CD25⁺ FoxP3⁺ Treg cells significantly decreased in the splenocytes of the Eps8-vaccinated mice compared with that of the PBS control mice. Representative results are shown in Fig. 6D and E.