Drugs and Reagents

Commercially available pemetrexed and aumolertinib were both supplied by Hansoh Pharmaceutical Group Co., Ltd. (Shanghai, China). Osimertinib was purchased from AstraZeneca Pharmaceuticals (USA). Cell counting kit-8 was purchased from Beyotime Biotechnology (Beijing, China). Rabbit-monoclonal-antibody against EGFR (ab52894), p-EGFR (ab40815), AKT (ab179463), p-AKT (ab38449), ERK1/2 (ab184699), p-ERK1/2 (ab201015), PARP1 (ab191217), cleaved-PARP1 (ab32064), cleaved-Caspase3 (ab2302), GAPDH (ab8245) and Goat pAb to Rb IgG HRP (ab6721) were purchased from Abcam (USA). Rabbit-monoclonal-antibody against α-SMA, mouse-monoclonal-antibody against CD31, CY2 and CY5- IgG HRP were purchased from Jackson ImmunoResearch (USA). Human HIF-1α, VEGF, TGF-β, ANG, SFLT and Angiostatin primers were synthesized by Introvigen (USA). All other reagents were of analytical grade and commercially available.

Cell lines

Human NSCLC cell lines A549, H1975 and HCC827 were obtained from Cell bank of Chinese Academy of Sciences (Shanghai, China). The above cell lines were all grown in RPMI 1640 (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA), penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37 °C in a humidified atmosphere with 5% CO2. Human umbilical vein endothelial cell (HUVEC) was obtained from Promocell (Heidelberg, Germany) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin at 37 °C in a humidified atmosphere with 5% CO2.

Animals and in vivo treatment

Healthy female Balb/c nude mice (16–18 g and 4–5 weeks of age) were obtained from the Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The mice were maintained under a controlled environment (22–24 ℃, 50–60% humidity, 12-h light/12-h dark cycle) with ad libitum access to standard laboratory food and water. H1975 and HCC827 cells (5 × 106 cells in 100 μl sterile PBS) were injected subcutaneously into the left flank of each mouse, respectively. After tumor formation, the mice bearing H1975 or HCC827 subcutaneous tumor were randomly assigned to different groups.

To compare the difference of the in vivo therapeutic efficacy among different combination strategies, the mice bearing H1975 or HCC827 subcutaneous tumor were randomly assigned to the following four groups over several cycles (4 days per cycle): (a) control group: saline (0.9% w/v, i.p., qd) and CMC-Na (0.5% w/v, i.g., qd); (b) P + A group: concurrent administration of pemetrexed (100 mg/kg, i.p., qd) and aumolertinib (20 mg/kg, i.g., qd) on day 1; (c) P-A group: pemetrexed (100 mg/kg, i.p., qd) administered on day 1 and aumolertinib (20 mg/kg, i.g., qd) on day 2; (d) A-P group: aumolertinib (20 mg/kg, i.g., qd) administered on day 1 and pemetrexed (100 mg/kg, i.p., qd) on day 2. The whole process was repeated five times for HCC827 tumor bearing mice and six times for H1975 tumor bearing mice, respectively. Tumor volume was measured every other day, and the tumor volume was calculated as V = (width* width* length)/2. At the end of the experiment, mice were sacrificed and tumor from each mouse was photted.

To evaluate the in vivo synergistic therapeutic efficacy for P-A sequence treatment, the mice bearing H1975 or HCC827 subcutaneous tumor were randomly assigned to the following five groups and administered over several cycles (4 days per cycle): (a) control group: CMC-Na (0.5% w/v, i.g., qd) for 3 successive days following saline (0.9% w/v, i.p., qd) on day 1; (b) pemetrexed group: CMC-Na (0.5% w/v, i.g., qd) for 3 successive days following pemetrexed administration (100 mg/kg, i.p., qd) on day 1; (c) aumolertinib group: aumolertinib (20 mg/kg, i.g., qd) for 3 successive days following saline (0.9% w/v, i.p., qd) on day 1; (d) P-A group: aumolertinib (20 mg/kg, i.g., qd) for 3 successive days following pemetrexed administration (100 mg/kg, i.p., qd) on day 1; (e) osimertinib group: osimertinib (20 mg/kg, i.g., qd) for 3 successive days following saline (0.9% w/v, i.p., qd) on day 1; the whole process was repeated five times for HCC827 tumor bearing mice and six times for H1975 tumor bearing mice, respectively. Tumor volume was measured every other day, and the tumor volume was calculated as V = (width* width* length)/2. At the end of the experiment, mice were sacrificed. Tumor from each mouse was photted, weighted and then collected for further experiments.

For the pharmacokinetic assay, pemetrexed (100 mg/kg, i.p.) or aumolertinib (20 mg/kg, i.g.) was administered at the next day following repeated cycling drug treatment described above. Blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8,16 and 24 h post drug administration (blood samples were collected no more than 3 times per mouse). At 4 h and 24 h post drug administration, mice were scarified and tumors were collected. The concentrations of pemetrexed and aumolertinib in blood sample or tumor mass were determined by LC–MS/MS.

Synergistic effect of pemetrexed and aumolertinib on cell growth inhibition

A549, HCC827 and H1975 were seeded in 96-well plates (10,000 cells per well) and exposed to serial dilutions of aumolertinib or pemetrexed for 72 h. For A549, the series concentrations of pemetrexed were 0.001, 0.01, 0.05, 0.1, 0.5, 2, 10, 50 μM and the series concentrations of aumolertinib were 1, 2, 5, 10, 20, 50, 100 μM. For HCC827, the series concentrations of pemetrexed were 0.001, 0.01, 0.1, 0.5, 2, 10, 200 μM and the series concentrations of aumolertinib were 0.0003, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1 μM. For H1975, the series concentrations of pemetrexed were 0.001, 0.01, 0.05, 0.5, 2, 10, 200 μM and the series concentrations of aumolertinib were 0.01, 0.1, 1, 10, 20, 50, 100 μM. After treatments, cell viabilities were measured by a CCK-8 Assay Kit (KeyGEN BioTech, Nanjing, China) and quantified relatively to those in wells without drugs. IC50 values were calculated from inhibition curves using GraphPad Prism 8.

Three different combination strategies were designed as follows: (a) P + A: pemetrexed and aumolertinib were co-administered simultaneously for 72 h; (b) P-A: pemetrexed for 24 h previously, and followed by aumolertinib for another 72 h; (c) A-P: aumolertinib for 24 h previously, and followed by pemetrexed for another 72 h. Integration effects of these combination strategies on A549, HCC827 and H1975 were evaluated and compared using combination index (CI). During the experiment, three NSCLC cell lines were treated with series concentrations of pemetrexed and aumolertinib at the ratio of their natural IC50 values, respectively. For A549, the series concentrations of pemetrexed were 1.25 μM (0.25*IC50), 2.5 μM (0.5*IC50), 5 μM (1*IC50), 10 μM (2*IC50), 20 μM (4*IC50), the series concentrations of aumolertinib were 5 μM (0.25*IC50), 10 μM (0.5*IC50), 20 μM (1*IC50), 40 μM (2*IC50), 80 μM (4*IC50). For HCC827, the series concentrations of pemetrexed were 0.038 μM (0.25*IC50), 0.076 μM (0.5*IC50), 0.152 μM (1*IC50), 0.304 μM (2*IC50), 0.608 μM (4*IC50), the series concentrations of aumolertinib were 0.01 μM (0.25*IC50), 0.02 μM (0.5*IC50), 0.04 μM (1*IC50), 0.08 μM (2*IC50), 0.16 μM (4*IC50). For H1975, the series concentrations of pemetrexed were 0.078 μM (0.25*IC50), 0.156 μM (0.5*IC50), 0.312 μM (1*IC50), 0.625 μM (2*IC50), 1.25 μM (4*IC50), the series concentrations of aumolertinib were 0.312 μM (0.25*IC50), 0.625 μM (0.5*IC50), 1.25 μM (1*IC50), 2.5 μM (2*IC50), 5 μM (4*IC50). Cell viabilities were measured by a CCK-8 Assay. Raw data obtained for the effects of monotherapy and different combination strategies were entered in online software ComboSyn (http://www.combosyn.com) to obtain model parameter, CI and concentration-effect plots. In our study, CI < 0.75 indicated synergistic effect, 0.75 < CI < 1.45 indicated additive effect and CI > 1.45 indicated antagonism effect.

Wound healing assay

Cell migration was assessed in a classical wound healing assay with some minor modifications. Briefly, cells were seeded in 6-well plates and the cell layer was gently wounded using a plastic pipette tip after 90%-100% cell confluence. The bottoms of the wells were marked to indicate where the initial images of the wounded area were captured. And the crosses of wounding lines and horizontal lines were observed at different time points (0, 24, 48 h) by Lionheart FXTM Intelligent Live Cell Imaging Analysis System (Bio-Tek Instruments, USA). The wound gaps were measured by Image J software. The migration rate was calculated as follows: migration rate = (wound gap (0 h)—wound gap (48 h)) /wound gap (0 h). Wound gap = wound area/wound length.

Transwell migration and invasion assay

For cell migration assay, 1 × 105 HCC827 or H1975 were added to the upper chambers directly, and for the cell invasion assay, 2 × 105 HCC827 or H1975 were added to the upper chambers pre-coated with Matrigel. After incubation for 18 h, the upper chambers were rinsed with ice-cold PBS, fixed with 4% paraformaldehyde for 10 min and stained with 0.1% crystal violet. Then, the chambers were washed thoroughly in running water and the cells which didn’t migrate through pores were wiped off with cotton swabs. Images were taken with microscope in bright field and the number of cells was measured by Image J software.

Western blot

The immunoblotting assays were compiled as described previously [26]. Cell samples or tumors were lysed on ice with homogenizer in NP40 buffer supplemented with 100 μM Phenylmethanesulfonyl fluoride and 0.1% (v/v) phosphatase inhibitor (Beyotime Biotechnology, China). Protein was extracted by centrifugation (10,000 g, 5 min, 4 ℃). Protein concentrations were determined by the bicinchoninic acid (BCA) Protein Assay. Equal amounts of protein (30 μg) were loaded for each lane, separated by 8%, 10% or 12% SDS-PAGE gel and transferred to PVDF membranes (Bio-Rad, USA). After the transfer, the blots were first saturated by incubation in 5% skim milk (in 10 mM Tris–HCl containing 150 mM sodium chloride and 0.5% Tween 20) for 1 h at 37 ℃ and then incubated overnight at 4 ℃ with antibodies against EGFR (1:1000, Abcam, Cat#ab52894), p-EGFR (1:1000, Abcam, Cat#ab40815), AKT (1:10,000, Abcam, Cat#ab179463), p-AKT (1:500, Abcam, Cat#ab38449), ERK1/2 (1:10,000, Abcam, Cat#ab184699), p-ERK1/2 (1:1000, Abcam, Cat#ab201015), PARP1 (1:1000, Abcam, Cat#ab191217), cleaved-PARP1 (1:1000, Abcam, Cat#ab32064), cleaved-Caspase3 (1:500, Abcam, Cat#ab2302), GAPDH (1:4000, Abcam, ab8245). These blots were further incubated with Goat pAb to Rb IgG HRP (1:10,000, Abcam, ab6721) for 1 h at 37 ℃, developed in ECL solution, and visualized using an enhanced chemiluminescence detection kit and captured using a ChemiDoc XRS − System (Bio-Rad, USA). Signal intensities were normalized to GAPDH. The intensity of the selected band was analyzed using ImageJ.

Immunofluorescence

Xenograft tumor tissues were collected and fixed overnight in 4% paraformaldehyde and then dehydration with 20% and 30% sucrose, respectively. Tumor tissues were cut into 10 μm sections (free-floating) in a cryostat and processed for immunofluorescence as previously described [27]. To determine the vessel branches and calculate the tumor microvascular density, the sections were incubated with anti-CD31 (1:100, BD Biosciences, Franklin Lakes, NJ, USA) and α-SMA (1:100, BD Biosciences, Franklin Lakes, NJ, USA) at 4 ℃ overnight and then incubated with Cy5- or Cy2-conjugated secondary antibody (1:200, Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at 37 ℃. The stained sections were observed with confocal microscope (Olympus FV3000). Cy2 was determined at excitation wavelength 489 nm and emission wavelength 506 nm, Cy5 was determined at excitation wavelength 650 nm and emission wavelength 670 nm. α-SMA+ or CD31+ area was measured by Image J software.

Real-time quantitative PCR

Total RNA of cell samples or tumors was extracted using a High Pure RNA Isolation Kit (RNAiso Plus, Takara, Japan) and reverse transcribed using a PrimeScript RT Regent Kit (Vazyme, Nanjing, China). mRNA expression was assessed by RT-quantitative PCR using a CFX96 real-time detection system (Bio-Rad, USA). The cycling conditions were as follows: 95 ℃ for 10 min, followed by 40 cycles with 95 ℃ for 15 s, 60 ℃ for 30 s, and 72 ℃ for 30 s. Melting curve analysis was performed routinely to verify the specificity of real-time PCR products. Specific mRNA values were calculated after normalization of the results for each sample with those for β-actin mRNA. The data are presented as relative mRNA units with respect to control values. Quantification was performed by the comparative Ct method (2Ct: normalizing cycle threshold (Ct) values with β-actin Ct). The gene-specific primers used in this study are shown in Table S1.

VEGF determination by ELISA

Thirty mg tumor tissue was homogenized with homogenizer in 300 μl pure water. Tumor tissue homogenates were diluted 1:50 in assay diluent solution. The VEGF levels in tumor tissue homogenates and cell supernatants were measured using the human VEGF ELISA kit (ExCell Bio, Shanghai, China) according to the manufacturer’s protocol. Absorbance was measured at 450 nm after the addition of stop solution.

LC–MS/MS-based quantitative analysis of pemetrexed and aumolertinib

The concentrations of pemetrexed and aumolertinib in the plasma, tumor and other tissues were all analyzed on a Shimadzu LC-10AD HPLC system (Kyoto, Japan) coupled to API 4000 (SCIEX, Birmingham, MA, USA). Briefly, plasma and tissue homogenates were protein-precipitated with 3 times volume of ice-cold methanol containing 500 ng/ml osimertinib (Internal Standard, IS). After twice centrifugation (30,000 g, 10 min, 4 °C), the supernatant was injected into the LC–MS/MS system for analysis.

For analysis of pemetrexed and aumolertinib, chromatographic separation was performed on a ZORBAX Eclipse Plus C18 column (150 × 4.6 mm, 5 μm, Agilent, USA) at 40 °C. The mobile phase consisted of solvent A (0.1% acetic acid and 5 mM ammonium acetate) and solvent B (acetonitrile) with the following gradient: 1 min, 1% B; 5 min, 70% B; 8 min, 70% B; 9.5 min, 1% B; 12 min, 1% B. The flow rate was 0.7 ml/min. The mass spectrometer was operated in positive electrospray ionization (ESI) mode. The multiple rection monitoring (MRM) parameters were set as follows: declustering potential set at 80 V for pemetrexed and osimertinib and 70 V for aumolertinib, collision energy set at 27 eV for pemetrexed, 33 eV for aumolertinib and 30 eV for osimertinib, MRM transition set as m/z 428.1 → 281.2 for pemetrexed and m/z 526.5 → 481.3 for aumolertinib and m/z 500.8 → 455.3 for osimertinib.

Clinical retrospective study

We screened patients in Department of Respiratory Medicine (The Affiliated Brain Hospital of Nanjing Medical University) who had received aumolertinib as first-line therapy from April, 2020 to January, 2022. Eligibility for evaluation within the retrospective study was based on the diagnosis of computed tomography, pathologic evaluation and gene detection. Only patients with primary NSCLC harboring EGFR mutant were involved in the study. Overall, 65 patients were submitted, among which 50 patients received aumolertinib monotherapy and 15 patients received combination therapy. For combination therapy, patients first used pemetrexed/cisplatin on day 1 and following aumolertinib on day 8–28 in a 28-day cycle for up to 2 cycles. Tumor response was evaluated by computed tomography scans according to the Response Evaluation Criteria in Solid Tumor Criteria Version 1.1. Complete response (CR) means disappearance of all target lesions. Partial response (PR) means that the longest diameter of target lesion was reduced by at least 30%. Progressive disease (PD) means that the longest diameter of the target lesion increases by at least 20%, or the appearance of new lesion. Stable disease (SD) means that the longest diameter of the target lesion increased to less than PD, or reduced to less than PR. Disease control rate (DCR) = (CR + PR + SD) / total number of cases, and the objective response rate (ORR) = (CR + PR) / total number of cases.

For five representative cases who used the combination of aumolertinib and chemotherapy as neoadjuvant therapy, pathological response was assessed by local pathologists, who measured the percentage of residual viable tumor in primary tumors resected from each patient during surgery. Tumors with < 10% viable tumor cells were considered to have a major pathologic response (MPR) and those with no viable tumor cells were deemed to be complete pathological response (CPR).

Data analysis

For preclinical study, all data are presented as mean ± standard error of mean (SEM). Statistical analyses were performed using GraphPad Prism 8 software. Each continuous variable was analyzed for a normal distribution using the Kolmogorov–Smirnov test, and then statistical analysis was performed using a two-tailed Student’s t-test or one-way ANOVA assay with Dunnett post-hoc test if F was less than 0.05 and there was no significant variance inhomogeneity. Differences were considered significant at *p < 0.05, **p < 0.01, ***p < 0.001.

For clinical study, data are median or n (%). p values were calculated by Mann–Whitney test. Differences were considered significant at *p < 0.05.

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