Cell culture
Two human lung adenocarcinoma cell lines were employed in the present study. PC-9 has an EGFR exon 19 deletion. It was provided by Immuno-Biological Laboratories (Gunma, Japan). NCI-H1975 (H1975) with an exon 21 L858R mutation and secondary exon 20 T790M mutation was purchased from the American Type Culture Collection (Manassas, VA). These cell lines were cultured in RPMI-1640 (FUJIFILM, Osaka, Japan) containing 10% fetal bovine serum (FBS; Biowest, Nuaille, France) and 1% penicillin and streptomycin (FUJIFILM) at 37°C in a 5% CO2 incubator. The cell lines were obtained in 2011 and 2014, expanded and cryopreserved, and one aliquot of each was thawed for this study. No cell line authentication was performed by the authors. All cells were routinely screened for the absence of mycoplasma.
Drugs and cell viability assays
Osimertinib, pemetrexed, and the Polo-like kinase 1 (PLK1) inhibitor volasertib were purchased from Selleck Chemicals (Houston, TX). To assess their sensitivity to drugs in vitro, cells were plated in 96-well tissue culture plates at 5,000 cells/well and incubated at 37℃ for 24 hours. Cells were then incubated with titrated concentrations of drugs (0.001, 0.01, 0.1, 1, 10 μM) or vehicle (DMSO) at 37°C for 72 hours. We estimated cell numbers using the Counting Kit-8 (Dojindo, Kumamoto, Japan). The half maximal inhibitory concentration (IC50) value for the drugs tested was defined as the concentration of osimertinib or pemetrexed or combinations thereof required for a 50% reduction of cell growth. Each experiment was individually performed three times. We calculated the corrected absorbance of each sample and compared it with that of the control group, in accordance with the protocol provided by the manufacturer of the kits.
Western blotting
Protein extraction and Western blotting were performed as previously described 14,15. The membranes were incubated with antibodies against PLK1, TS, EGFR, phosphorylated EGFR (p-EGFR), AKT, p-AKT, ERK, p-ERK, BIM and cleaved PARP, all of which were purchased from Cell Signaling Technology (Danvers, MA). An antibody against GAPDH were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Apoptosis assays
Apoptosis assays were performed using the Annexin Ⅴ-FITC Apoptosis Detection Kit (Nacalai Tesque, Inc, Kyoto, Japan) and measured by flow cytometry. Cells were harvested by treating with trypsin-EDTA, washed with PBS, and centrifuged at 1500 rpm for 3minutes. The cell pellets (5.0 x 105 cells) were resuspended in 100 μL of binding buffer containing 5 μL of Annexin Ⅴ-fluorescein isothiocyanate and 5 μL of propidium iodide, incubated for 15-30 minutes in the dark on ice, after which fluorescence was quantified on a BD FACSVerse flow cytometer. Data were processed with FACSuite software (Becton Dickinson, Franklin Lakes, NJ).
RNA extraction and quantitative real-time reverse transcription-PCR
Total RNA was extracted from cultured cells using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA) as previously described 39. The RNA was reverse-transcribed to cDNA using a ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. The primers, cDNA, and THUNDERBIRD Probe qPCR Mix (Toyobo) were mixed together, and qPCR was performed using the 7500 Fast Real-Time PCR System (Applied Biosystems, San Francisco, CA). GAPDH expression was taken as the standard against which to normalize relative expression of the genes of interest. Plk1 and TS expression was quantified using the TaqMan Gene Expression Assay (Thermo Fisher Scientific) and presented as 2−ΔΔCt values 39.
Microarray analysis
Gene expression microarray analysis was performed using the GeneChip Human Gene 2.0 Sense Target array (Affymetrix, Santa Clara, CA) according to the manufacturer’s protocol. Microarray data have been deposited in NCBI’s Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) and are accessible through a GEO series accession number GSE165029.
Transfection of small-interfering RNA (siRNA)
SiRNA experiments were performed using Silencer Select Negative Control siRNA as the negative control. Pre-Designed Silencer Select siRNAs were used to knock down PLK1 and TS (Thermo Fisher Scientific). SiRNA and Lipofectamine RiMAX Reagent were dissolved in Opti-MEM Media (Thermo Fisher Scientific) at a final concentration of siRNA complexes of 20 nM. Next, we replaced the transfection medium after 6 hours, and finally incubated cells at 37 °C for a further 48 hours.
Tumor cell implantation experiments
Female severe combined immunodeficient (SCID)-Beige mice (CAnN.Cg-Foxn1nu/CrlCrlj) were purchased at 5 weeks of age from Charles River Laboratories Japan Inc., Kanagawa, Japan. A total of 5.0 × 106 PC-9 cells was suspended in 200 μl of Matrigel (CORNING Inc., Corning, NY) / PBS (Nacalai Tesque, Inc) and was injected into the subcutaneous area of 6-week-old mice. When tumor volumes reached an average size of 200 mm3, the mice were divided into four groups (n=6 for each group): vehicle control (per os (p.o.), once a day), pemetrexed (intraperitoneal (i.p.), at 100 mg/kg, twice a week), osimertinib (p.o., at 1.0 mg/kg, once a day), or both osimertinib (p.o., 1.0 mg/kg, once a day) and pemetrexed (intraperitoneal (i.p.), at 100 mg/kg, twice a week). Treatments were administered until the end of the 29-day observation period. Body weights were measured every week. We estimated tumor volumes (V) twice a week by caliper measurements of the width (W) and length (L) of each tumor (W2×L/2). Extraction of tumor proteins for immunohistochemical analysis was performed using samples from mice at the end of the 29-day treatment period. The study protocol was approved by the Ethics Committee on the Use of Laboratory Animals, KAC Co., Ltd (Shiga, Japan) (approval no. 20-0313). All experiments on live vertebrates were carried out in accordance with relevant guidelines and regulations. The study was performed in compliance with the Animal research: Reporting of in vivo experiments (ARRIVE) guidelines.
Immunohistochemical analysis
PLK1 expression was detected in deparaffinized formalin‐fixed, paraffin‐embedded tissue sections (3 μm) using a PLK/PLK1 antibody. Immunohistochemical analysis (IHC) was performed as previously described 40. The sections were stained overnight at 4 °C with rabbit anti-human PLK/PLK1 antibody (ReyBiotech, Norcross, GA) at a final dilution of 1:500. For human specimens, written informed consent was obtained from the patient, and the specimens were collected in accordance with the Declaration of Helsinki 2013. The study protocol was approved by the Ethics Committee Review Board at Nippon Medical School Hospital (approval no. 25-05-299 and B-2020-286).
Statistical analysis
Data were expressed as the mean ± standard error (SE) of three independent experiments and evaluated with Student’s t test. A P value of < 0.05 was considered to be significant.