A total of 60 samples were obtained from patients with OS of the extremities who underwent surgery in The First Affiliated Hospital of Nanchang University (Nanchang, China). The examination of pulmonary metastasis was performed using plain films and chest computed tomography (CT) scans at initial diagnosis. None of the patients had a history of previous therapies with antitumor drugs or radiotherapy. There were 14 cases with pulmonary metastasis (23.3%), while 76.7% of cases were without metastasis. The samples were fixed with 10% formalin, embedded in paraffin and then cut into 4-μm sections. Informed consent was obtained from all participants, and the study protocol was approved by the Institutional Ethics Committee (Jiangxi, China).

Histological sections (4-μm) were stained with hematoxylin and eosin (H&E) and examined using IHC. IHC was performed using a streptavidin-peroxidase procedure. Briefly, antigen retrieval was performed by heating the deparaffinized, rehydrated sections in 10 mM citrate buffer (pH 6.0) for 20 min, followed by blocking with 10% goat serum. The sections were then incubated overnight at 4°C with the primary antibody (rabbit anti-Aurora-B monoclonal antibody; Abcam, Cambridge, UK) at a final dilution of 1:500. For the negative controls, the sections were incubated with phosphate-buffered saline (PBS) instead of antibodies. After being washed three times with PBS, the sections were incubated with biotinylated secondary antibody for 40 min and then incubated with horseradish peroxidase (HRP)-conjugated streptavidin for 30 min. The sections were subsequently subjected to chemiluminescent staining and counterstained using hematoxylin. The stained sections were evaluated and scored by two doctors of pathology, in a blind manner and without prior knowledge of the clinical pathological features of the patients. The expression levels of Aurora-B were judged according to staining intensity, following the examination of ≥500 cells in five representative areas, and the intensity scores were recorded as follows: None, 0; weak, 1; moderate, 2; and intense, 3. According to the percentage of tumor cells that were positive for Aurora-B expression, the following percentage scores were assigned: 0% (score 0); >10% (score 1), 11–50% (score 2), 51–80% (score 3), and 81–100% (score 4). The final score was averaged with the scores from the two doctors of pathology; these scores were calculated by adding the intensity score to the percentage score. A final score of <4 was defined as (−), while scores of 4 and 5 were defined as (+) and (++), respectively, and a score of ≥6 was defined as (+++).

The U2-OS human OS cell line was obtained from the American Type Culture Collection (Manassas, VA, USA), and the cells were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM; HyClone™, Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Sigma, St. Louis, MO, USA) in a humidified 37°C incubator containing 5% CO₂.

U2-OS cells were cultured in 96-well tissue culture plates at a cell density of 5,000 cells per well in Minimum Essential Media (MEM; Invitrogen Life Technologies, Carlsbad, CA, USA) containing 10% FBS and 2 mM L-glutamine. Following attachment overnight, the medium was replaced and the cells were incubated with increasing concentrations (0, 5, 10, 50, 100 and 500 nM) of AZD1152-HQPA for 24, 48 and 72 h. Subsequently, MTT assays were performed in triplicate at a wavelength of 490 nm.

U2-OS cells (1×10⁶/ml/well) were seeded in tissue culture plastic dishes and treated with AZD1152-HQPA (100 nM) for two weeks to form colonies. The formed colonies were stained with Giemsa, and the colonies containing >50 cells were counted under an inverted microscope (TE2000; Nikon, Tokyo, Japan). Six independent experiments were performed over multiple days.

U2-OS cells in the exponential growth phase were treated with AZD1152-HQPA at various concentrations (0, 10, 50 and 100 nM) for 24 h. Total protein from the cells was extracted using radioimmunoprecipitation assay lysis buffer containing 60 μg/ml phenylmethylsulfonyl fluoride. Protein concentrations were assessed using a bicinchoninic acid protein assay kit (Boster Biological Technology, Ltd., Wuhan, China). The protein samples were denatured at 100°C for 10 min and then preserved at −20°C for later use. The proteins were separated by SDS-PAGE and transblotted onto polyvinylidene difluoride membranes. The membranes were then probed with rabbit anti-Aurora-B monoclonal antibody (1:500; Abcam) or β-actin antibody (1:2,000; Cell Signaling Technology Inc., Danvers, MA, USA) overnight at 4°C. Following incubation with the appropriate anti-rabbit or anti-mouse HRP-conjugated secondary antibody (1:5,000; Boster Biological Technology, Ltd.) for 1.5 h at room temperature, immunoreactive bands were visualized using chemiluminescence dissolvent (Thermo Fisher Scientific, Inc.) and exposed to X-ray film (Kodak, Rochester, NY, USA). The assessment of the grayscale values was performed using ImageJ (National Institutes of Health, Bethesda, MD, USA). All experiments were repeated six times over multiple days.

The invasion of U2-OS cells was measured using the BD BioCoat™ BD Matrigel™ Invasion Chamber (BD Biosciences, Franklin Lakes, NJ, USA) in accordance with the manufacturer’s instructions. The medium in the lower chamber contained 5% FBS as a source of chemoattractants. Cells were suspended in serum-free medium containing 100 nM AZD1152-HQPA and added to the upper chambers at the same time. The cultures were rinsed with PBS and the medium was replaced with fresh medium alone or medium supplemented with 10% FBS. The cells were then incubated at 37°C for 24 h. Cells that passed through the Matrigel-coated membrane were stained with Diff-Quik (Sysmex Corp., Kobe, Japan) and photographed. Cell migration was quantified using direct microscopic visualization and counting. The values for invasion were obtained by counting three fields per membrane and represented the average of six independent experiments performed over multiple days.

Cell migration was assessed by examining the ability of the cells to move into a cellular space in a two-dimensional in vitro ‘wound healing assay’. In brief, cells were grown to confluence in six-well tissue culture plastic dishes to a density of ~5×10⁶ cells/well. Following treatment with 100 nM AZD1152-HQPA for 24 h, the cells were denuded by dragging a rubber policeman (Fisher Scientific, Hampton, NH, USA) through the center of the plate. Cultures were rinsed with PBS and the medium was replaced with fresh medium alone or medium containing 10% FBS. The cells were then incubated at 37°C for 24 h. Photographs were taken at 0 and 24 h, and the migration distance was measured. The cell migration rate was obtained by counting three fields per area and represented the average of six independent experiments performed over multiple days.

All measurement data are presented as the mean ± standard deviation. Statistical analysis was performed using the independent-samples t-test, and the two-independent-samples test was used for the analysis of the correlation between Aurora-B protein expression levels and pulmonary metastasis. P<0.05 was considered to indicate a statistically significant difference. All analyses were performed using SPSS statistical software version 13.0 (SPSS, Inc., Chicago, IL, USA).

Aurora-B was expressed in the nucleus (Fig. 1), and the positive expression rate was 53.3%. Notably, the positive expression rate of Aurora-B protein in the cases with pulmonary metastasis was 78.6.% (11/14), which was significantly different from that of the cases without pulmonary metastasis 45.7% (21/46). This indicated that Aurora-B may be involved in OS metastasis.

In order to investigate the effect of Aurora-B inhibition on U2-OS cell growth, AZD1152-HQPA, a specific inhibitor of Aurora-B, was used to suppress Aurora-B expression in the U2-OS cells. The cells were treated with various concentrations (0, 5, 10, 50, 100 and 500 nM) of AZD1152-HQPA, and MTT assays were performed to measure the inhibitory effect of AZD1152-HQPA on U2-OS cells proliferation. The results of the MTT assays revealed that AZD1152-HQPA inhibited U2-OS cell proliferation in a dose- and time-dependent manner (Fig. 2A). The IC50 value was 146 nM for 24 h. In the colony formation assays, the results showed that the colony formation rate in the cells treated with 100 nM AZD1152-HQPA was lower than in that in the untreated cells (Fig. 2B). Furthermore, western blot analysis showed that Aurora-B protein expression was downregulated by AZD1152-HQPA in a dose-dependent manner (Fig. 3A). These results showed that Aurora-B inhibition was capable of suppressing U2-OS cell growth in vitro, which suggested that Aurora-B may be a promising target for the treatment of OS.

According to the IC50 value, the appropriate concentration of AZD1152-HQPA for wound healing migration and Transwell invasion assays was determined. To examine the effect of Aurora-B inhibition on the mobility of U2-OS cells, the migration and invasion were measured using wound-healing and Transwell assays, respectively. The cells were treated with 100 nM AZD1152-HQPA for 24 h. In the Transwell invasion assays, the invasion of the cells treated with AZD1152-HQPA was significantly inhibited when compared with that of the untreated cells (75.6±7.4 and 214.5±22.4 cells/high power field, respectively, P<0.05; Fig. 3B). In the wound healing assay, the results showed that the migration rate of the cells treated with AZD1152-HQPA was significantly lower than that of the untreated cells (23.7±5.1 and 75.6±15.3%, respectively, P<0.05; Fig. 3C). These results suggested that Aurora-B inhibition was capable of suppressing U2-OS cell invasion and migration in vitro.