The human NSCLC cell line, NCI-H460, was obtained from the Institute of Basic Medical Sciences at the Chinese Academy of Medical Sciences and Peking Union Medical College (Tianjin, China), and were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Hyclone; GE Healthcare Life Science, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The NCI-H1299 cell line was obtained from American Type Culture Collection (Manassas, VA, USA) and was cultured in RPMI-1640 medium (Hyclone; GE Healthcare Life Science) supplemented with 10% FBS. The cells were incubated in 5% CO₂ at 37°C. DSBs were induced in H460 cells by applying a ¹³⁷Cs Gammacell-40 radiation source (Atomic Energy of Canada Limited, Chalk River, Ontario, Canada) at a photon dose rate of 1.02 Gy/min.

To prepare CM, H460 cells were grown to 80–90% confluence on 100 mm plates (5×10⁶ cells). The cells were then rinsed with phosphate-buffered saline (PBS) and incubated with DMEM/F12 containing 10% FBS. After 48 h, the medium was collected, membrane-filtered (0.22 µm; EMD Millipore, Billerica, MA, USA) and stored at −80°C.

To isolate exosomes, H460 cells were incubated in serum-free DMEM/F12 until they reached 80-90% confluency. Following an additional 48 h of culturing, the medium was collected and then subjected to serial centrifugation at 4°C (300 × g for 10 min; 2,000 × g for 20 min; and 10,000 × g for 30 min). Following filtration of the supernatant (0.22 µm), exosomes were pelleted by ultracentrifugation at 100,000 × g for 2 h at 4°C with a 32Ti rotor (Optima L-100XP Ultracentrifuge; Beckman Coulter, Inc., Brea, CA, USA). The pellet was resuspended in PBS and then subjected to a second ultracentrifugation step (100,000 × g for 2 h at 4°C). The pelleted exosomes were resuspended in 100 µl PBS and stored at −80°C. Bicinchoninic acid protein assays (Beyotime Institute of Biotechnology, Haimen, China) were used to determine total protein concentration for the exosome preparation.

The mixed exosomes were added to a copper net for 2 min, and the residual liquid was removed with filter paper, which was followed by staining with 2% phosphotungstic acid solution (pH 6.4) for 2 min at room temperature. The prepared copper nets were then observed under a transmission electron microscope (HT77000; Hitachi, Ltd., Tokyo, Japan). Images of the exosomes were obtained at ×10 magnification.

To evaluate radiosensitivity, H460 cells were plated in 6-well plates (500 cells/well). The next day, Transwells (0.4 µm; Corning Incorporated, Corning, NY, USA) containing H460 cells (2.5×10⁴ cells/Transwell) were transferred into the 6-well plates. The whole plate was then treated with ¹³⁷Cs γ-radiation (1.02 Gy/min) at variable doses (0, 2 and 4 Gy). In a control group, Transwells containing unirradiated H460 cells (2.5×10⁴ cells) were placed in a 6 well-plate alongside previously plated and irradiated H460 cells (500 cells/well). H1299 cells were plated in 6-well plates (1,000 cells/well) and exposed to 0, 2 or 4 Gy of γ-rays. Transwells containing unirradiated H1299 cells (2.5×10⁴ cells/Transwell) were subsequently added. A second set of experiments was conducted with H460 cells plated in 12-well plates (2.5×10⁴ cells/well) and treated with 0, 2 and 4 Gy irradiation. CM was collected from both irradiated and non-irradiated H460 cells and these samples were filtered (0.22 µm) and added to the appropriate 12-well plates every 48 h for 7 days.

To verify the effect of exosomes on the radiosensitivity of H460 cells, 30 µg exosomes was added to each well of a 6-well plate containing irradiated H460 cells (18).

At 7 days after seeding, all plates were fixed with anhydrous methanol for 20 min and stained with 10% Giemsa stain for 30 min, all at room temperature. The number of colonies was counted under a microscope (Nikon Eclipse TS100; Nikon Corporation, Tokyo, Japan), which a colony consisting of at least 50 cells.

H460 cells were seeded into 96-well plates (1,500 cells/well) and treated with 4 Gy γ-irradiation. After 24 h, the medium was replaced with varying volumes of conditioned medium (50, 100, 150, 200 and 250 µl) and a corresponding volume of fresh medium to achieve a total volume of 300 µl. After 72 h, MTT reagent (Beijing Solarbio Science & Technology, Co., Ltd., Beijing, China) was added to each well. After 4 h, the medium was removed and the cells were lysed with dimethyl sulfoxide to dissolve the formazan present. Absorbance values were measured at 570 nm with a spectrophotometric plate reader (Synergy™ HT; BioTek Instruments, Inc., Winooski, VT, USA).

To investigate the effect of CM on the level of ROS, H460 cells (1×10⁵) were seeded into 60-mm plates, exposed to 4 Gy of γ-rays, and the medium was subsequently replaced with CM or fresh medium. After 24 h, the cells were stained with 5 µM dichlorodihydrofluorescein diacetate (DCFH-DA) from a Reactive Oxygen Species Assay kit (Beyotime Institute of Biotechnology) for 30 min at room temperature and then were washed 3 times with DMEM/F12. Immunofluoresent images were obtained with a fluorescence microscope (EVOS™; Thermo Fisher Scientific). For flow cytometry analysis, the cells were stained as above and then digested into single cell suspensions with 0.25% trypsin and washed 3 times with PBS at room temperature. Single cell suspensions were resuspended in 500 µl PBS, and analyzed by flow cytometry (Mindray BriCyte E6; Mindray, Shenzhen, China).

H460 cells were treated or untreated for 2, 6, 12 or 24 h with CM following exposure to 0 or 4 Gy irradiation. Cell extracts were subsequently collected and lysed with radioimmunoprecipitation assay lysis buffer (CWBio, Beijing, China) at 4°C. Proteins were isolated by centrifugation at 12,000 × g for 10 min at 4°C, then quantified by the bicinchoninic acid assay prior to being separated by 12% SDS-PAGE (30 µg protein per lane). Following transfer of the separated proteins to polyvinylidene difluoride membranes, they were blocked with a 1% BSA solution (Solarbio, China). All of the antibodies were diluted in a 1% bovine serum albumin (BSA) solution (0.1 g BSA in 10 ml tris-buffered saline with Tween-20). Target proteins were probed with antibodies recognizing superoxide dismutase (SOD)-1 (1:1,000; cat. no. ab20926; Abcam, Cambridge, UK), γH2A histone family member X (γH2AX; 1:1,000; cat. no. 9718s; Cell Signaling Technology, Inc., Danvers, MA, USA), Rad51 recombinase (1:1,000; cat. no. sc-398587), cyclin A (1:500; cat. no. sc-271645; both from Santa Cruz Biotechnology, Inc., Dallas, TX, USA), the Mre11-Rad50-Nbs1 (MRN) complex (1:1,000; cat. no. 8344T, MRN Complex Antibody Sampler kit), phosphorylated (p)-ATM (cat. no. 5883T) and ATM (1:1,000; cat. no. 2873T; all from Cell Signaling Technology, Inc.). An analysis of the exosome protein lysates was conducted according to the same method, except that anti-cluster of differentiation 9 (CD9; 1:1,000; cat. no. ab1832-500) and anti-tumor susceptibility gene 101 (TSG101; 1:1,000; cat. no. ab125011; both from Abcam) antibodies were used. Detection of β-tubulin (1:5,000; cat. no. 66240-1-Ig) and β-actin (1:5,000; cat. no. 66009-1-Ig; both from ProteinTech Group, Inc., Chicago, IL, USA) was also performed to provide internal positive controls for the immunoblots. The primary antibodies were incubated with the membranes overnight at 4°C. The secondary antibodies of goat anti-mouse immunoglobulin (Ig)G (H+L), horseradish peroxidase (HRP) conjugate (1:5,000; cat. no. SA00001-1) and goat anti-rat IgG (H+L), HRP conjugate (1:5,000; cat. no. SA00001-15; both from ProteinTech Group, Inc.) were incubated with the membranes at room temperature for 1 h. Bound antibodies were detected with electrochemiluminescence reagents (ProteinTech Group, Inc.) and a ChemiDoc MP Imaging System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Western blot data was analyzed quantitatively with Image Lab 5.2.1 (Bio-Rad Laboratories, Inc.).

H460 cells were treated or untreated with CM with and without irradiation (4 Gy) for 24 h. The cells were then collected and rinsed with PBS three times. The cells were fixed in 70% ethanol overnight at −20°C. The next day, the cells were washed three times with cold PBS and labeled with propidium iodide (PI; Beijing Solarbio Science & Technology, Co., Ltd.) for 20 min at 37°C in the dark. The labeled cells were subsequently detected by flow cytometry (Mindray BriCyte E6) and analyzed with FlowJo v10 software (FlowJo LLC, Ashland, OR, USA).

H460 cells (4.5×10⁴) were seeded into 12-well plates containing coverslips and incubated overnight. The next day, the H460 cells were exposed to 0 or 4 Gy and cultured in normal medium or CM. After 2, 6, 12 and 24 h, the cells were washed 3 times with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. After three additional rinses in PBS, the cell membranes were permeabilized with 0.3% Triton X-100 at room temperature. After 20 min, the cells were rinsed three times with PBS and then incubated in blocking buffer (1% BSA) at room temperature. After 1 h, the cells were incubated with anti-γH2AX antibodies (1:200) at room temperature. After an additional 2 h, the cells were rinsed three times in PBS and then incubated with cyanine 3-conjugated anti-goat/mouse antibodies (1:200; cat. no. SA00009-1; ProteinTech Group, Inc.) at room temperature. After 1 h, the cells were rinsed again in PBS three times and antifade mounting medium containing DAPI (Vector Laboratories, Inc., Burlingame, CA, USA) was used to mount the specimens. Imaging was performed via fluorescence microscopy.

Data are presented as the mean ± standard deviation from at least three independent experiments, with each experiment performed in triplicate. The Student’s t-test and one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test were applied with SPSS 13.0 (SPSS, Inc., Chicago, IL, USA) and P<0.05 was considered to indicate a significant difference between the groups. The intracellular ROS levels were analyzed by Duncan’s multiple range test and other data were analyzed by t-test.

To examine whether autocrine signaling improves the radioresistance of NSCLC cells, H460 colonies were co-cultured with H460 cells contained in Transwells (Fig. 1A). When the cells were incubated with irradiated or unirradiated H460 cells, the number of H460 colonies markedly increased following 2 or 4 Gy irradiation in the co-culture group, compared with in the control group (Fig. 1B and C). These data indicated that the irradiated H460 cells exhibited increased radioresistance following exposure to autocrine factors, and these factors were present independent of whether the added H460 cells had undergone irradiation. To verify whether other NSCLC cell lines also mediate this effect, H1299 cells were tested in the Transwell model. The same effect was observed (Fig. 1D).

Interactions between cells are dynamic and complex. To facilitate studies of these interactions, CM was used to simulate the effect of cell secretions on clonal formation. CM was collected from H460 cells with or without prior irradiation, and the effects of these CM samples on the formation of H460 colonies following irradiation was observed (Fig. 1E). Both non-irradiated and irradiated CM enhanced the radiation resistance of the H460 cells, and these results were consistent with those obtained from the Transwell assays. Consequently, only non-irradiated CM was used as a relevant model in subsequent radioresistance-related studies.

Next, it was postulated that H460-derived CM promotes cell proliferation. When H460 cell viability was measured in MTT assays, the H460 cells that were irradiated and then cultured in CM exhibited greater viability compared with those cells cultured in normal medium (Fig. 1F). Interestingly, the observed effects were dependent on the volume of the CM that was applied: As the volume of CM increased, an initial increase in cell viability was observed, followed by a decrease after 4 Gy irradiation (Fig. 1G, right panel). At the higher volumes of CM, cytotoxicity appeared to be partially induced (Fig. 1G, left panel).

To detect the level of intracellular ROS, cells were stained with the fluorescence indicator, DCFH-DA. Fluorescence intensity (in arbitrary units) measured by flow cytometry demonstrated that the level of ROS increased ~1.25-fold 24 h after H460 cells were irradiated (4 Gy) and then cultured in CM (5,195.87±316.76), compared with in those cells cultured in normal medium (4,152.57±296.77; Fig. 2A and B). The level of intracellular ROS was also directly observed by fluorescence microscopy. In H460 cells cultured in CM after 4 Gy irradiation, the level of intracellular ROS appeared higher than that in the normal-culture irradiated group (Fig. 2C).

The balance of intracellular ROS is regulated by various signaling molecules and signaling pathways (19). Presently, it was hypothesized that SODs may mediate the level of ROS in irradiated H460 cells. On western blot analysis, the H460 cells that were irradiated and then cultured in CM were observed to contain lower levels of SOD1, compared with in those cells cultured in normal medium (Fig. 2D). These results suggest that secretions deriving from H460 cells reduce the degradation of ROS in a SOD1-dependent manner.

To determine whether IR-treated H460 cells undergo cell cycle arrest, H460 cells were treated with 0 or 4 Gy irradiation and then cultured with CM or normal medium. After 24 h, the cells were stained with PI. Flow cytometry detected a greater number of cells in the G2/M phase of the cell cycle among the H460 cells that were administered with 4 Gy irradiation followed by CM, compared with those H460 cells that were irradiated and then cultured in normal medium (Fig. 3A and B). To further characterize the observed cell cycle arrest, the amount of cyclin A in H460 cells with and without irradiation and subsequent culturing in CM was analyzed at various time points (0, 2, 6, 12 and 24 h) after irradiation. These experiments were performed based on previous reports that cyclin A is critical for the S and G2 phases of the cell cycle and is degraded in prometaphase (20,21). When IR-treated H460 cells were co-cultured with the secretions of H460 cells for 2 h, cyclin A was identified to be significantly upregulated compared with in the IR-treated, normal cultured cells at the same time point. Degradation of cyclin A was observed at the subsequent time points assayed (Fig. 3C and D).

One of the earliest responses to DSBs is phosphorylation of histone H2AX at serine 139 (γH2AX) (22). This response has been reported to persist over a longer period of time in radiosensitive cell lines than in radioresistant lines (23). Here, the persistence of γH2AX foci was examined 2, 6, 12 and 24 h after H460 cells were treated with 4 Gy irradiation followed by culture in CM or normal medium. Among the IR-treated, CM-cultured cells, fewer positive cells were detected at the 6 h time point compared with in the population of IR-treated, normal cultured cells (normal medium: 80.13±1.48%; CM: 62.63±2.98; Fig. 4A and B). Detection of γH2AX protein by western blotting further confirmed these results (Fig. 4C). Interestingly, it was also noted that when normal H460 cells were treated with CM, the content of γH2AX appeared to increase with time.

Homologous recombination (HR) and non-homologous end-joining (NHEJ) are the main pathways that mediate the repair of IR-induced DSBs in DNA, with the HR pathway being dominant in the S and G2 phases of the cell cycle (24). To examine HR repair in H460 cells, expression of Rad51 was detected in western blots. In the H460 cells that underwent irradiation and then incubation with CM, a significant increase in Rad51 expression was observed compared with in the control cells at the 6 and 12 h time points following irradiation (Fig. 4D). Additionally, the Rad50, NBS1 and Mre11 proteins (which constitute the MRN complex) exhibited marked increases in expression when cultured in CM for 12 h and/or 24 h after irradiation (Fig. 4D). Meanwhile, it was observed that not only the protein content of p-ATM was increased, but also the total amount of ATM following culture in CM for 24 h after irradiation (Fig. 4E). These data indicate that radiation resistance in H460 cells is achieved with cell cycle arrest in the G2/M phase and DNA repair via HR.

The above experiments suggested that secretions from H460 cells reduce the radiosensitivity of IR-treated H460 cells. To determine whether exosomes contribute to this effect, exosomes were purified from H460 cell medium by differential ultra-centrifugation and then were characterized by western blotting (Fig. 5A). Exosome markers are typically identified by TSG101 and CD9 (25), but the result showed that the purified exosomes expressed the exosomal marker protein TSG101 without CD9. This may be due to the exosomes secreted by H460 cells not expressing CD9 protein. When the purified exosomes were visualized by transmission electron microscopy, round, bi-layer structures were observed and their diameters ranged from 30 to 100 nm (Fig. 5B).

Clonal formation assays were subsequently conducted with H460 cells that were treated with 0 or 4 Gy irradiation and then incubated with or without purified exosomes. The irradiated H460 cells that were cultured with H460 cell secretions in a Transwell system exhibited a significant ~1.5-fold increase in radioresistance (percent survival, 8.47±0.61%) compared with the control cells (5.60±1.31%). The addition of purified exosomes did not increase this effect (8.73±0.42%; Fig. 5C). Taken together, these data suggest that exosomes secreted from H460 cells do not influence the radiosensitivity of H460 cells.