The human lung cancer cell line, NCI-H1975 (L858R/T790M), was obtained from the American Type Culture Collection (Manassas, VA, USA) and authenticated by short-tandem repeat analysis in December 2016. The cells were maintained in RPMI-1640 (cat. no. 22400; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), containing 10% fetal bovine serum (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA; cat. no. SV30087) in a humidified incubator at 37°C with 5% CO₂. Osimertinib (AZD9291) was provided by AstraZeneca (Shanghai, China), and was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany; cat. no. SHBG6226v) for in vitro study and 0.5% Hydroxy propyl methyl cellulose (HPMC; Sigma-Aldrich; Merck KGaA; cat. no. 9004-67-5)/0.1% Tween-80 (cat. no. P1754; Sigma-Aldrich; Merck KGaA) for the in vivo study.

The cells and xenografts were irradiated using the X-cell 160 Irradiator (¹³⁷Cs; Kubtec, Milford, CT, USA) at a dose rate of 140 cGy/min at room temperature with the following protocol: 1.42 min at 2 Gy, 2.85 min at 4 Gy, 4.26 min at 6 Gy, 5.68 min at 8 Gy, 7.10 min at 10 Gy and 14.2 min at 20 Gy, respectively. Cells were treated with Osimertinib at 37°C 1 h prior to irradiation in the combination groups involved in the in vitro study.

Cells were divided into 6 groups: i) Osimertinib alone [at dosages of 0 (control), 0.0001, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1 and 3 µM, respectively); ii) osimertinib with 2 Gy irradiation; iii) osimertinib with 4 Gy irradiation; iv) osimertinib with 6 Gy irradiation; v) osimertinib with 8 Gy irradiation; and vi) osimertinib with 10 Gy irradiation. The proliferation analysis was performed using a tetrazolium-based Cell Titer 96^® Aqueous One Solution Assay (cat. no. G3581; Promega Corporation, Madison, WI, USA), according to the manufacturer's instructions. Briefly, exponentially growing cells were diluted to 2×10⁴/ml and seeded at 100 µl/well into 96-well plates. Following 24 h, cells were treated with irradiation and increasing concentrations of osimertinib. Then, an MTS assay was performed following 3 days. Relative cell viability was expressed as the percentage of untreated control.

Cells in the log phase were plated into 6-well plates with the desired cell density (300 cells/well for 0 Gy, 500 cells/well for 2 Gy, 1,000 cells/well for 4 Gy, 3,000 cells/well for 6 Gy and 5,000 cells/well for 8 Gy, respectively) and pretreated with osimertinib at 10, 30 and 100 nM, or DMSO, respectively. Irradiation was delivered 1 h later. Cells were then maintained for 14 days with osimertinib in RPMI-1640 and stained with 0.1% crystal violet (Sigma-Aldrich; Merck KGaA; cat. no. 5K219R5). Colonies of >50 cells were defined as surviving colonies and the number of colonies was normalized to that of non-irradiated controls. The sensitizer enhancement ratio (SER) for osimertinib treatment was calculated as the ratio of the mean inactivation dose of control cells / the mean inactivation dose of osimertinib-treated cells at the 0.01 survival fraction.

Cells were trypsinized with 0.25% trypsin-EDTA (cat. no. 25200-114; Invitrogen; Thermo Fisher Scientific, Inc.) and the suspended cell pellet was incubated with 70% ethanol (cat. no. 1000927; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) at 4°C. Following the thorough removal of ethanol, the cells were suspended in a proprium iodide (PI) staining solution (cat. no. P3566; Invitrogen; Thermo Fisher Scientific, Inc.) in the dark, at room temperature for 30 min. Flow cytometry was performed using a FACSCanto Cell Analyzer (V07300617; BD Biosciences, Franklin Lakes, NJ, USA).

Cells were cultured on chamber slides and the samples were collected at 2, 24 and 48 h, respectively. Cells were washed with ice-cold Ca²⁺/Mg²⁺-free phosphate buffered saline (PBS), and fixed in 4% formaldehyde (cat. no. SZBF0690v; Sigma-Aldrich; Merck KGaA) for 60 min at room temperature. Following permeabilization in 1% Triton X-100 (cat. no. 057K00161; Sigma-Aldrich; Merck KGaA) and blocking with 5% bovine serum albumin (BSA; cat. no. 12575v; Sigma Aldrich; Merck KGaA)/0.3% Triton™ X-100 in PBS at room temperature for 1 h, the cells were incubated with a fluorescein isothiocyanate-conjugated anti-phospho histone γ-H2A histone family member X (H2AX) primary antibody (dilution, 1:800; cat. no. 2577s; CST Biological Reagents Co., Ltd., Shanghai, China) overnight at 4°C, then incubated with an Alexa 647-conjugated anti-rabbit secondary antibody (dilution, 1:1,000; cat. no. 4412S; CST Biological Reagents Co., Ltd.) for 1 h at room temperature in the dark. Coverslips were mounted using Mounting Medium with DAPI (H-1200; Vector Laboratories, Inc., Burlingame, CA, USA) overnight at 4°C. Images were acquired with an Olympus BX61 laser scanning confocal microscope (7E18988; Olympus Corporation, Tokyo, Japan) using ×60 magnification. Using ≥150 cells from each experiment, the cells were counted and the percentage of cells positive for γ-H2AX was calculated. A positive cell was defined by >5 discrete dots in the nucleus.

Cells were lysed in 2X SDS buffer containing protease and phosphatase inhibitors (cat. no. 1861282; Thermo Fisher Scientific, Inc.), then the protein concentration was determined via a BCA protein assay (cat. no. 34076; Thermo Fisher Scientific, Inc.). Equal amounts of protein (20 µg/well) were loaded for SDS-PAGE using 4–12%-gradient Bis-Tris precast gels (cat. no. 345-0124; Bio-Rad Laboratories, Inc., Hercules, CA, USA), followed by transfer to polyvinylidene difluoride membranes using the iBlot dry transfer system (cat. no. IB21001; Novex; Thermo Fisher Scientific, Inc.). Membranes were blocked in 5% fat-free milk in Tris-buffered saline with 0.1% Tween-20 (TBS-T) for 30 min at room temperature, then incubated at 4°C overnight with the following primary antibodies: Total EGFR (dilution, 1:1,000; cat. no. 4267S; CST Biological Reagents Co., Ltd.), phospho (p)-EGFR (dilution, 1:1,000; Tyr1068l, cat. no. 3777S; or Tyr1173, cat. no. 4407S; CST Biological Reagents Co., Ltd.), total protein kinase B (AKT; dilution, 1:1,000; cat. no. 4691S; CST Biological Reagents Co., Ltd.), phospho-AKT (Ser473; dilution, 1:1,000; cat. no. 12694S; CST Biological Reagents Co., Ltd.), total extracellular signal-regulated kinase (ERK; dilution, 1:1,000; cat. no. 4695S; CST Biological Reagents Co., Ltd.), phospho-ERK (dilution, 1:1,000; Thr202/Tyr204; cat. no. 4370S; CST Biological Reagents Co., Ltd.) and GAPDH (dilution, 1:10,000; cat. no. 2118L; CST Biological Reagents Co., Ltd.). Membranes were then incubated for 1 h at room temperature with a horseradish peroxidase (HRP)-conjugated secondary antibody (dilution, 1:2,000; cat. no. 7074 or 7076; CST Biological Reagents Co., Ltd.). Membranes were visualized using a mixed detection solution (Super Signal West Dura Extended Duration Substrate; cat. no. 34076; Thermo Fisher Scientific, Inc.) for 5 min at room temperature, protected from light. Densitometry of western blots was conducted using a Fujifilm Image Reader LAS-4000 2.1 (Fujifilm Corp., Tokyo, Japan).

Specific pathogen-free immunodeficient female nude mice (n=170; age, 6–8 weeks; weight, 20–25 g) were purchased from Beijing Vital River Laboratory Animal Technology (Beijing, China). Animals had free access to food and water, and were maintained at 21–23°C with 40–70% humidity and a 12-h light/dark cycle. All of the animal studies were approved by the Institutional Animal Care and Use Committee of AstraZeneca (Shanghai, China). NCI-H1975 tumor cells (5×10⁶ cells/0.1 ml) were injected subcutaneously into the left flank of the nude mice to establish the tumor model.

Tumor growth was monitored twice weekly by caliper measurements. When the tumors grew to 0.2–0.4 cm³, the mice were randomized into 6 groups: i) vehicle (no treatment); ii) osimertinib alone (5 mg/kg/day); iii) 2 Gy × 10 F irradiation alone (5 fractions/week from the first day); iv) 2 Gy × 10 F irradiation and osimertinib (5 mg/kg/day); v) 20 Gy × 1 F irradiation alone on the first day; and vi) 20 Gy × 1 F irradiation and osimertinib (5 mg/kg/day). Osimertinib was administered at 5 mg/kg once daily by oral gavage for 31 days. Mice were euthanatized (by excessive CO₂ inhalation) 25 days following the termination of osimertinib treatment to evaluate the persistent inhibitory effect, or when tumor volume ≥1,500 mm³ according to the Animal Welfare of the Institutional Animal Care and Use Committee of AstraZeneca (10). Power analysis was performed whereby group sizes were calculated to enable statistically robust detection of tumor growth inhibition. Tumor growth inhibition from the start of treatment was assessed by comparing the mean change in tumor volume of the control and treatment groups.

For pharmacodynamic studies, mice were randomized when the tumor volumes reached 0.5–0.8 cm³: Mice were treated with either a single dose of osimertinib, IR alone (20 Gy × 1 F), or osimertinib plus IR. Following a single dose, the blood plasma was collected, except for that of the IR 20 Gy × 1 F group due to the absence of osimertinib in the plasma, and the tumors of all groups were harvested 0, 0.5, 1, 2, 4, 7, 16 and 24 h later. The total osimertinib concentration in plasma was detected using the ACQUITY SM Method, as previously described (22). Sections were fixed with 4% formalin for 24 h at room temperature, embedded in paraffin and were then immunohistochemically stained for the phosphorylated forms of EGFR (Tyr1068) and EGFR (Tyr1173).

IHC was performed on 3-µm sections using a Lab Vision autostainer. The tumors were excised as aforementioned for the DMPK/PD assay. The paraffin slides were incubated at 56°C for 30 min then dewaxed and rehydrated in a Leica Autostainer XL and subjected to antigen retrieval (cat. no. S1699; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) for 15 min at room temperature followed by washing under running tap water for 5 min. Then the sections were rinsed in TBS-T, and assessed on a LabVision autostainer. Following incubation with an endogenous peroxidase blocker (3% hydrogen peroxide; cat. no. GTX30967; GeneTex, Inc., Irvine, CA, USA) for 10 min at room temperature, slides were washed twice in TBS-T and blocked with 5% BSA (cat. no. 12575v; Sigma Aldrich; Merck KGaA) at room temperature for 10 min. Sections were then incubated with the following primary antibodies for 60 min at room temperature: p-EGFR Tyr1068 (dilution, 1:200; cat. no. 2234; CST Biological Reagents Co., Ltd.), p-EGFR Tyr1173 (dilution, 1:200; cat. no. 4407; CST Biological Reagents Co., Ltd.), Histone H2AX phospho (ser139; dilution, 1:150; cat. no. 2577; CST Biological Reagents Co., Ltd.) and cleaved caspase-3 (CC3) (Asp175; dilution, 1:200; cat. no. 9661; CST Biological Reagents Co., Ltd.). The sections were then washed twice in TBS-T. The p-EGFR (Tyr1068/Tyr1073)/CC3 slides were incubated with a biotinylated goat anti-rabbit immunoglobulin secondary antibody (cat. no. E0432; dilution, 1:100; Dako; Agilent Technologies, Inc.) and Streptavidin-Peroxidase for 30 min at room temperature. The Histone H2AX phospho slides were incubated with EnVision system-HRP Labeled Polymer Anti-Rabbit for 30 min at room temperature and washed twice in TBS-T. All sections were incubated in diaminobenzidine substrate for 5 min at room temperature and rinsed with tap water. The sections were then counter stained with Mayer's hematoxylin for 5 min at room temperature, dehydrated and cleared with xylene in a Leica XL autostainer, and finally sealed in the ClearVue automated cover slipper. Images were acquired with a laser scanning confocal microscope (Olympus BX61 microscope; Olympus Corporation).

Experiments were performed ≥3 times and data are presented as the mean ± standard error of the mean. Statistical analysis was performed using SPSS 19.0 (IBM Corp., Armonk, NY, USA). One-way analysis of variance (ANOVA) with a Bonferroni post hoc test were conducted for comparisons of the percentage of H2AX positive cells in the immunofluorescence assay, percentage of cells in S and G2/M phase in the flow cytometry assay, the tumor volume 25 days following the final day of osimertinib treatment and the quantitative changes of protein phosphorylation in the western blotting and IHC. Two-way ANOVA with a Bonferroni post hoc test was applied for the comparison of tumor volume in the tumor growth curves. Pearson coefficient-parametric analysis was performed to determine whether there was a correlation between the phosphorylated forms of EGFR (Tyr1068/Tyr1173) and the concentration of osimertinib in the blood plasma. P<0.05 was considered to indicate a statistically significant difference.

To determine the effect of osimertinib combined with irradiation on cell proliferation, the present study performed a cell viability assay 3 days following treatment. As shown in Fig. 1A, the rate of cell proliferation decreased markedly when osimertinib was administrated prior to 2 or 4 Gy irradiation. Proliferation was demonstrated to be inhibited in a dose-dependent manner by CFA in which the SER was >1, indicating that the effect on proliferation inhibition was synergistic when irradiation was combined with osimertinib treatment (Fig. 1B).

To assess the effects of osimertinib on cell cycle arrest following irradiation, cells were treated with 10, 30 or 100 nM osimertinib for 24 h and then irradiated with dose of 2, 6 or 20 Gy due to their different role in clinical practice: 2 Gy was recognized as the conventional fraction; 6 Gy was chosen due to the best inhibiting effect as determined by CFA in Fig. 1; and 20 Gy represented hypofractionated radiotherapy. As shown in Fig. 2, dose-dependent reductions in the G2/M and S phases were demonstrated in the combination treatment group when compared with irradiation alone (DMSO group).

To determine whether the increased radio-sensitivity of cell lines following osimertinib treatment was a product of compromised DNA-break repair, the present study conducted immunofluorescence staining for γ-H2AX in NCI-H1975 cells. It was revealed that the formation of γ-H2AX began to increase in the 2 and 6 Gy groups at 2 (Fig. 3A) and 24 h (Fig. 3B); whilst the formation of γ-H2AX only began to increase at 24 h in the 20 Gy group (Fig. 3C). In addition, osimertinib significantly increased the number of γ-H2AX foci per cell at 48 h following 2 and 6 Gy radiation in a dose-dependent manner (Fig. 3C and D).

Osimertinib was revealed to be a potent inhibitor of EGFR and downstream signaling substrate (p-AKT and p-ERK) phosphorylation in cells with mutant EGFR (10). Therefore, the present study investigated whether AKT/ERK were the main downstream targets of EGFR proteins following treatment with osimertinib combined with radiation. The western blotting results demonstrated that osimertinib inhibits p-EGFR (1068)/p-EGFR (1173)/p-AKT/p-ERK protein expression when treated alone or in combination with IR in a concentration-dependent manner (Fig. 4).

To explore the in vivo activity of osimertinib combined with IR, the present study administered osimertinib during the irradiation of nude mice bearing NCI-H1975 subcutaneous xenografts. As shown in Fig. 5A, the combination treatment exhibited more potent antitumor efficacy when compared with IR or osimertinib alone. At the end of the treatments, the mean residual tumor volume in the IR 2 Gy × 10 F plus osimertinib 5 mg/kg group was lower than that of osimertinib alone or osimertinib plus IR 20 Gy × 1 F groups (Fig. 5A and B). In addition, the tumor complete response rates were 1/7, 3/7 or 4/7 for the osimertinib alone, osimertinib plus IR 20 Gy × 1 F or osimertinib plus IR 2 Gy × 10 F groups, respectively, at the time of treatment termination (day 31). Inhibition of tumor growth was observed for an additional 25 days following treatment termination (Fig. 5A and B). It was demonstrated that radiation of conventional fraction may be more powerful than hypofractionated radiotherapy when combined with osimertinib administration; however, this requires further investigation in future clinical observations. The present study also measured mouse body weight in order to assess the treatment tolerability, and no evident body weight changes were observed (<5% of starting body weight), as shown in Fig. 5C. These results suggested that treatment with IR combined with osimertinib was tolerated well.

To confirm the target and pathway of osimertinib activity when in combination with IR, the present study examined the total osimertinib concentration in the plasma and the phosphorylation level of EGFR in tumor tissues following treatments. In the osimertinib and osimertinib plus IR groups, the expression of p-EGFR (Tyr1068)/p-EGFR (Tyr1173) was negatively associated with the plasma concentration of osimertinib (Fig. 6A and B). As displayed by the representative IHC quantification of p-EGFR (Tyr1068)/p-EGFR (Tyr1173)/γ-H2AX and cleaved caspase-3 (Fig. 6C-G), osimertinib suppressed the activity of p-EGFR (Tyr1068)/p-EGFR (Tyr1173), particularly when administrated combined with IR; while the phosphorylation of EGFR maintained high levels in the IR only group. However, the expression levels of γ-H2AX and cleaved caspase-3 significantly increased in the osimertinib plus IR combination group.