Human NSCLC NCI-H1299 cells (cat. no. CRL-5803; American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI-1640 medium (cat. no. 01-100-1ACS; Biological Industries, Kibbutz Beit Haemek, Israel) supplemented with 10% fetal bovine serum (FBS; cat. no. 04-001-1ACS; Biological Industries), 100 IU/ml penicillin, and 100 µg/ml streptomycin (cat. no. sv30010; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) at 37°C in a humidified atmosphere of 5% CO₂.

The primary antibodies used are as follows: Anti-Op18 (cat. no. 569391; Merck KGaA, Darmstadt, Germany); anti-phospho-Op18-Ser25 (cat. no. ab194752), anti-phospho-stathmin-Ser63 (cat. no. ab76583) and anti-IL-10 (cat. no. ab134742) from Abcam (Cambridge, MA, USA); anti-CDC2 p34 (cat. no. sc-954), anti-caspase-3 (cat. no. sc-7272), anti-ERK (cat. no. sc-93), anti-phospho-ERK (cat. no. sc-7383), anti-β-actin (cat. no. sc-47778) and anti-nuclear factor-κB (NF-κB; cat. no. sc-109) were from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); anti-phospho-Thr161-CDC2 (cat. no. 9114), anti-caspase-8 (cat. no. 9746), anti-caspase-9 (cat. no. 9502) and anti-phospho-Ser536-NF-κB p65 (cat. no. 3031S) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (cat. no. sc-2004) and rabbit anti-mouse IgG (cat. no. sc-358914; Santa Cruz). NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC; cat. no. 5108-96-3, Sigma-Aldrich; Merck KGaA) (10,11) and Taxol (cat. no. sc-201439; Santa Cruz Biotechnology, Inc.) were separately dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C.

RNAi targeting Op18/stathmin was used to silence Op18/stathmin. Small interfering RNA targeting the coding region of Op18/stathmin at 5′-AGAGAAACTGACCCACAAA-3′ (GenBank no. 53305, sequence 374–393) were inserted into the BamH1/HindIII restriction sites of pGCsilencer-U6/Neo/GFP (RNAi; Shanghai GeneChem Co., Ltd., Shanghai, China; sense gaAGAGAAACTGACCCACAAA, antisense TTTGTGGGTCAGTTTCTCTtc). A nonsense sequence not targeting any encoding gene was inserted into pGC-silencer-U6/Neo/GFP-Non (Non) and pGC-silencer-U6/Neo/GFP (Blank) was an empty plasmid. The plasmids (4 µg) were transiently transfected into cells at a transfection efficiency of >80% in 250 µl incomplete RPMI-1640 medium containing 10 µl Lipofectamine^™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA); 4 h later, the medium replaced with complete medium for 24 h. All plasmids were previously constructed by our laboratory (9,12).

Cells were seeded in 96-well plates at 5,000 cells per well and supplemented with different concentrations of anti-IL-10 antibody (0, 0.5 and 2.5 µg/ml; cat. no. ab134742; Abcam). At 24, 48 and 72 h, 10 µl 0.5% MTT was added to the well and incubated at 37°C for 4 h. The medium was then removed and 100 µl DMSO was added and incubated for 10 min. A total of six parallel wells/sample was used. The optical density (OD) value was measured using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) at 490 nm. The relative proliferation ratios were calculated according to the formula: Relative proliferation ratio (%) = (OD_treatment/OD_control) × 100. Cells treated with DMSO were used as a control, and the OD value of the control was set as 1.0.

A total of 2,000 cells per well were seeded in a 6-well plate in the presence of anti-IL-10 antibody (0, 0.5 and 2.5 µg/ml) and incubated for 1–2 weeks. Cell growth was suspended when colonies were observed by the naked eye. The cells were fixed with 100% methanol for 10 min at room temperature and stained with 0.1% crystal violet at room temperature for 30 min. The colonies with >50 cells were counted with an inverted microscope. The relative colony formation efficiency was calculated using the formula: Relative colony formation efficiency (%) = (average colonies/2,000) × 100.

When the confluence of the cells was ~80%, the cells were washed three times with ice-cold phosphate-buffered saline (PBS). A sterilized pipette tip (200 µl) was applied to wipe off the cells along the longitudinal and parallel trails that were marked in the back of the plate. The plate was washed with PBS, and 0, 0.5 and 2.5 µg/ml anti-IL-10 antibody was added. Cell migration was observed with an inverted optical microscope at 0, 12, 24 and 36 h.

The upper Transwell chamber with 8.0 µm pore size of polycarbonate membrane of polystyrene plates (Costar; Corning Incorporated, NY, USA) was pretreated with heated serum-free RPMI-1640 for 30 min, and 800 µl complete medium containing 10% FBS was added to the lower chamber. A total of 10 µl cell suspension (cell density, 2×10⁵ cells/ml with 0.2% FBS of RPMI-1640) was added to the upper chamber at 37°C for 24 h. The cells in the upper Transwell chamber were wiped with a cotton swab, and the cells were fixed with 100% methanol for 10 min and stained with 0.1% crystal violet for 10 min at room temperature. The cells that passed through the membrane were counted in five random visual fields under an inverted microscope. The migrated cells were then imaged and evaluated.

A total of 2.5×10⁵ cells per well were seeded into 6-well plates in the presence of 2.5 µg/ml anti-IL-10 antibody combined with 100 nM Taxol. The cells were cultured for 24 h. The cells were harvested and fixed with 70% ethanol at 4°C overnight for flow cytometric analysis. Cell apoptosis assay was performed using a Fluorescence Activating Cell Sorter (Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China). In brief, cells were resuspended and stained in 500 µl binding buffer with 5 µl Annexin V-fluorescein isothiocyanate and 5 µl propidium following the instruction of Annexin V-fluorescein isothiocyanate Apoptosis Detection kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). In addition, the cells were treated with different concentrations (0, 5 and 10 µM) of PDTC for 24 h.

The cells were treated on ice with a cell lysis buffer (50 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid, 2% sodium dodecyl sulfate, 5 mM dithiothreitol and 10 mM phenylmethylsulfonyl fluoride) for 30 min. The lysates were collected and denatured in boiling water for 8 min and incubated on ice for 5 min. The lysates were sonicated for 30 sec and centrifuged at 4°C for 15 min. The concentration of the protein was measured using BCA Protein Assay reagent (Pierce; Thermo Fisher Scientific Inc.). Total protein (50 µg) was separated by 10–12% SDS-polyacrylamide gel electrophoresis and electrotransferred onto a nitrocellulose membrane. The membrane was blocked for 2 h with 5% non-fat milk at room temperature in PBS with 0.05% Tween-20 and then incubated with primary antibodies, anti-β-actin (1:2,000), anti-Op18 (1:3,000), anti-phospho-Op18-Ser25, anti-phospho-stathmin-Ser63, anti-IL-10, anti-phospho-Thr161-CDC2, anti-CDC2 p34, anti-ERK, anti-phospho-ERK, anti-NF-κB, anti-phospho-Ser536-NF-κB p65, anti-caspase-3, anti-caspase-8 and anti-caspase-9 (all 1:1,000), overnight at 4°C. The membrane was then incubated with HRP-conjugated secondary antibodies (1:2,000-1:3,000) for 1.5 h at room temperature. The membrane was visualized with an enhanced chemiluminescence detection kit (Thermo Fisher Scientific, Inc.).

Cells in the logarithmic growth phase were seeded in 24-well plates. When the cells reached 80% confluence, the media was replaced with fresh RPMI-1640 medium. The cells were incubated in phenol red-free media for 6, 12, 24 and 36 h, while other cells were treated with 0, 5 and 10 µM PDTC in phenol red-free media for 24 h. The supernatant was collected for in vitro detection of autocrine IL-10. The human IL-10 ELISA kit [cat. no. EK1102; Multisciences (Lianke) Biotech Co., Ltd., Hangzhou, China] was used.

Male Kunming mice (4-week-old) were randomly divided into four groups of 15, 30 and 45 day, and parallel controls (8 mice per group). NCI-H1299 cells at the logarithmic growth phase were collected and resuspended in normal saline. A total of 4×10⁶ cells in 0.5 ml were injected intraperitoneally into each mouse. The control mice were injected with the same volume of normal saline. Retro-orbital blood samples were collected from anaesthetized mice at day 0, 15, 30 and 45; the mice were respectively sacrificed at day 15, 30 and 45 at 15 days interval by cervical dislocation for the collection of blood. Flow cytometry (model no. BC-2800vet; Auto Hematology Analyzer; Shenzhen Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China) was performed to sort the leukocytes. Cell counting was performed by Servicebio, Inc. (Woburn, MA, USA).

In addition, part of each blood sample was used to detect IL-10 in the sera by ELISA. The mice were respectively sacrificed at day 15, 30 and 45 at 15 days interval by cervical dislocation for the collection of blood sample, and the tumors were removed from 45-day mice. The 3–5 µm thickness of tissue sections were fixed by 10% formalin overnight and stained with 10% hematoxylin (×5, 10 min) and 1% eosin (1 min) at room temperature and examined by inverted optical microscope. Histopathological examination was performed by Professor Deyun Feng (Department of Pathology, First Xiangya Hospital Affiliated to Central South University, Changsha, China).

Statistical analysis was performed using the SPSS 17.0 statistical software (SPSS, Inc., Chicago, IL, USA). The data are presented as the mean ± standard deviation. Analysis of variance and least significant difference method was applied to perform multiple comparisons between the groups, P<0.05 was considered to indicate a statistically significant difference.

The relative proliferation ratios were 90.32, 85.54 and 82.02% at 24, 48 and 72 h in the presence of 0.5 µg/ml anti-IL-10 antibody, and 78.38, 73.38 and 66.60% at 2.5 µg/ml anti-IL-10 antibody. With the extension of time, the growth curve was steeper for 2.5 µg/ml compared with 0.5 µg/ml. The treatment with anti-IL-10 antibody inhibited cell proliferation in a dose-dependent manner (Fig. 1A). The histograms showed that the relative proliferation ratios of two treated groups were markedly lower compared with the control. In addition, the difference was also significant between two groups that were treated with anti-IL-10 antibody at three time points (P<0.01; Fig. 1B).

The colony formation analysis demonstrated that there were many large colonies with closely adhered cells in the control group. When the cells were exposed to the anti-IL-10 antibody, the number of colonies declined, and the cells became sparse. Only a few of small colonies appeared when treated with 2.5 µg/ml anti-IL-10 (Fig. 1C). The histograms indicated that the colony formation ratios were 11.95, 10.3 and 7.3% at 0, 0.5 and 2.5 µg/ml, respectively. There was no significant difference between the control and the group treated with 0.5 µg/ml anti-IL-10 (P>0.05). The number of colonies was significantly decreased in the group treated with 2.5 µg/ml anti-IL-10 antibody compared with the other two groups (P<0.01, Fig. 1D). The ELISA assays for autocrine IL-10 indicated that the OD values of the supernatants were 0.0845, 0.1115, 0.193 and 0.2285 at 6, 12, 24 and 36 h, respectively. The level of IL-10 reached a peak value at 36 h, which confirmed that the production of autocrine IL-10 was markedly increased with the increase in the duration of incubation in culture. The differences were statistically significant between 36 h and the other time points (Fig. 1E).

The wound healing experiments indicated that there were large blank areas at 12 h in all three groups. By 24 h, the blank areas became narrower, and the blank area was the narrowest in the 0 µg/ml control group. At 36 h, the wound nearly healed fully in the control group and was partially recovered at 0.5 µg/ml anti-IL-10 antibody; however, a large gap remained in the group treated with 2.5 µg/ml anti-IL-10 antibody (Fig. 2A). The Transwell analysis confirmed that the mean numbers of transmembrane cells per visual field were 21.4, 15.4 and 7.8 at 0, 0.5 and 2.5 µg/ml, respectively. Invasion assay by Transwell demonstrated that the cells that invaded through the matrix on Transwell membranes were irregular in shape. The treatment with anti-IL-10 antibody led to the inhibition of cell migration in a 3-D format (Fig. 2B). The histograms indicated that the number of transmembrane cells was decreased in the two IL-10 antibody-treated groups compared with the control. The differences were significant in the group that was exposed to 2.5 µg/ml anti-IL-10 antibody compared with other two other groups (P<0.01; Fig. 2C).

Cell micrographs demonstrated that cells were arranged closely in the three groups, but a small number of floating cells appeared in the treated groups with the addition of anti-IL-10 antibody (indicating that anti-IL-10 antibody promoted cell apoptosis to some extent). Flow cytometric analysis demonstrated that cellular apoptotic rates were 0.96, 1.83 and 3.91% in the 0, 0.5 and 2.5 µg/ml treatment groups, respectively, which was slightly upregulated with the increase of anti-IL-10 antibody (Fig. 3A).

A large number of cells became detached in the three groups treated with Taxol, cells became sparser with anti-IL-10 treatment, and the number of floating cells was highest in the group that received 2.5 µg/ml anti-IL-10 and Taxol. Flow cytometric assays demonstrated that cellular apoptotic ratios were 12.97, 14.40 and 17.55% at 0, 0.5 and 2.5 µg/ml anti-IL-10 antibody treatment, respectively, combined with 100 nM Taxol, which implied that neutralizing autocrine IL-10 promoted the sensitivity of NCI-H1299 cells to Taxol (Fig. 3B).

Western blot analysis demonstrated that anti-IL-10 induced the expression of caspase-3, and cleavage of caspase-8 in NCI-H1299 cells in a dose-dependent manner. However, there was no effect on caspase-9 expression or cleavage (Fig. 3C).

Western blot analysis demonstrated that the addition of anti-IL-10 decreased the expression and the phosphorylation of ERK, and the phosphorylation of CDC2 at Thr161. However, anti-IL-10 did not affect CDC2 expression, which indicated that the activities of ERK and CDC2, which are kinases upstream of Op18/stathmin, were weakened by neutralization of autocrine IL-10 in vitro (Fig. 4A). Similarly, anti-IL-10 antibody reduced the expression of NF-κB and its active subunit p65, which is phosphorylated at Ser536, in a dose-dependent manner (Fig. 4B). Further western blot detection identified that the expression of Op18/stathmin and its phosphorylated forms (Ser25 and Ser63) were gradually reduced with the increase of anti-IL-10 antibody concentration (Fig. 4C and D).

To examine the relevance of the transcription factor, NF-κB and kinases (ERK and CDC2), PDTC, an inhibitor of NF-κB was applied to block NF-κB signaling. The cells grew sparsely in the presence of PDTC, with 10 µM PDTC as the highest concentration. Flow cytometric analysis showed that the apoptotic ratios were 1.34, 13.35 and 16.4% at 0, 5 and 10 µM PDTC, respectively (Fig. 5A).

Western blot analysis demonstrated that PDTC also decreased the expression and phosphorylation of ERK, and the phosphorylation of CDC2 at Thr161 in a concentration-dependent manner. However, PDTC did not exert any effects on CDC2 expression (Fig. 5B).

ELISA assays for autocrine IL-10 demonstrated that the OD values of supernatants were 0.232, 0.21 and 0.168 at 0, 5 and 10 µM PDTC treatment, respectively. The levels of autocrine IL-10 were significantly downregulated at 10 µM PDTC compare with the 0 and 5 µM treatment groups (P<0.01; Fig. 6A). Western blot analysis indicated that PDTC also inhibited IL-10 protein expression in a concentration-dependent manner (Fig. 6B). Similarly, silencing Op18/stathmin by RNAi effectively reduced the expression of NF-κB and IL-10 (Fig. 6C). These findings suggested that blocking NF-κB or silencing Op18/stathmin inhibited the levels of IL-10 protein expression and autocrine release.

To examine whether autocrine IL-10 mediates tumor immune evasion, we collected the blood of mice with xenograft of NCI-H1299 cells on day 15, 30 and 45 for the detection of immune cells by flow cytometry. The average number of neutrophils was 2.800×10⁹/l in the control mice. The number of neutrophils in mice that were inoculated was 2.300×10⁹, 1.750×10⁹ and 1.432×10⁹ on day 15, 30 and 45, respectively. The number of neutrophils was significantly reduced on day 30 and 45 in tumor bearing mice compared with control mice (P<0.01; Fig. 7A). The number of lymphocytes in the four groups of mice was 5.575×10⁹, 4.175×10⁹, 3.925×10⁹ and 2.425×10⁹. The number of lymphocytes was significantly decreased on day 45 compared with the other time points and the control (P<0.01; Fig. 7B). The mean number of monocytes was 0.425×10⁹, 0.325×10⁹, 0.225×10⁹ and 0.200×10⁹ in the control, 15, 30 and 40 days groups. A significant difference in the number of monocytes was observed on day 30 and 45 compared with the control (P<0.01; Fig. 7C). Therefore, the number of neutrophils, lymphocytes and monocytes was all decreased in the blood of mice with xenografts. The levels of IL-10 were low in the sera on day 15 and similar to that of the control, which was gradually increased on day 30 and reached to the peak on day 45 (Fig. 7D).

The images of tumors indicated that the dissected tumors were identical in size in four random samples of mice with NCI-H1299 ×enograft on day 45. Further histopathological examination identified that tumor tissues presented typical lung glandular cavity-like structure, and the cells were heterogeneous in staining of the nuclei, which indicated that these tumors were derived from the lung adenocarcinoma epithelial NCI-H1299 cells that were intraperitoneally injected into the mice (Fig. 7E).