Materials
We purchased chemicals from Sigma Chemical Company. Anti- mouse and anti-rabbit IgG-horse radish peroxidase conjugate antibody were purchased from Pierce Company (Rockford, IL). Lipofectamine and PlusTM reagent for transfection were purchased from Invitrogen (San Diego, CA). Oligonucleotides, miRNA-mimic, and siRNAs used in this study were purchased from Bioneer Company (Daejon, Korea). Human recombinant CAGE protein was kindly provided by professor Dong Young Kim (Yeungnam University, Korea).
Cell Lines and Cell Culture
Cancer cell lines were cultured in Dulbecco’s modified minimal essential medium (Invitrogen) with 10% fetal bovine serum (Invitrogen).
Generation of Osimertinib-resistant non-small cell lung cancer cell line
Osimertinib resistant non-small cell lung cancer cell line (PC-9/OSI) was generated by culturing PC-9 parental line with sequentially increasing concentrations of osimertinib [14].
Colony Forming Potential and Cellular proliferation
Cells were seeded at a density of 100 cells/35 mm dish. Colonies were stained with 0.01% crystal violet. MTT assays were employed to determine the response to anti-cancer drugs. Viable cell number was determined by trypan blue exclusion assays.
Chemo Invasion Assays
Chemoinvasion assays were performed according to the standard procedures [15]. Differences were considered significant when p < 0.05.
Tumor Spheroid forming Potential
Tumor spheroid forming potential assays were performed according to the standard procedures [15]. The total number of spheres was counted after 7 days by inverted microscopy (Olympus).
Generation of knockout cell line with CRISPR/Cas9 system
Generation of CAGE (NCBI ENTREZ Gene:168400) knockout cell lines was performed according to the standard procedures [15)]. In brief, CRISPR/Cas9-mediated gene editing was performed. A plasmid encoding Cas9 was purchased from Tool Gen. For sgRNA expression, the hU6-sgRNA plasmid that targeted CAGE (5′-AGGCTAATCCAAGAGACCTTGGG-3′) was used (Tool Gen). PC-9/OSI cells were transfected with Cas9, hU6-sgRNA(Cat. No. CP160504), and hygromycin B-resistant reporter plasmid (ToolGen). After 48 h of transfection, cells were treated with hygromycin B (150 µg/ml) three times a week. Hygromycin-resistant colonies were isolated and subjected to immunoblot. Mismatch sensitive nuclease assays was performed to validate correctness of CAGE knockout.
Immunofluorescence Staining
Cells were washed and fixed with 4% paraformaldehyde before permeabilization with Triton X-100. After blocked with goat serum (10%) in 0.1% BSA/ PBS, cells were incubated with anti-LC3 or anti-CAGE at 4oC overnight and then incubated with anti-rabbit Alexa Fluor 488 secondary antibody. After removal of antibodies, cells were stained with DAPI and mounted with mounting medium. The immune fluorescent images were observed and captured using a confocal laser scanning microscopy.
Immunoblot and Immunoprecipitation
Immunoblot was performed according to the standard procedures [13].
To isolate tissue lysates, tissue was frozen in liquid nitrogen and homogenized using lysis buffer. After vortexing and centrifugation at 10,000 X g for 15 min at 4oC, supernatant was used as tissue lysates. For immunoprecipitations, cell lysates or tissue lysates (100–200 µg) were immunoprecipitated with respective primary antibody (0.2 -2 µg) for overnight at 4℃. Twenty µl of Protein A/G PLUS-Agarose (Santa Cruz) was then added and incubation was continued for 1 hour at 4℃. Beads were washed three times with lysis buffer, 2X sample buffer was added. Samples were then denatured (100°C for 5min) and analyzed 10% SDS-PAGE, followed by immunoblot. Band intensity was quantified using Image-J. The detailed information of primary antibodies is described in Supplementary Table S1.
Electron microscopic Observation of Autophagic Process
Cells were treated with the fixing solution (2.5% glutaraldehyde in 0.1 M cacodylate solution (pH 7.0) for 1h), and then mixed with 2% osmium tetroxide for 2 h at 4°C. The samples were dehydrated with a graded acetone series, and embedded into Spurr medium (Electron Microscopy System). The samples were sectioned (60 nm) by using ultra-microtome (RMC MTXL, Arizona, USA). The section was stained with 2% uranyl acetate for 20 min followed by staining with lead citrate for 10 min. The sections were then viewed under a transmission electron microscope (JEM-2100F, Japan) at 200 KV.
In vivo Tumorigenic Potential
Cancer cells (1 × 106) were injected subcutaneously into the dorsal flank area of the BALB/c mice to induce formation of tumors. All animal experiments were performed according to the guidelines of the Korean Council for the Care and Use of Animals in Research and approved by the Institutional Animal Care and Use Committee (IACUC) of Kangwon National University. Tumor volume (0.5 X length X width2) was calculated. For in vivo metastatic assay, PC-9 or PC-9/OSI cells (1 × 106 cells in PBS) were injected intravenously into the tail vein of 4-week-old female athymic nude mice, and the extent of lung metastasis was evaluated. This study was carried out in compliance with the ARRIVE guidelines. For anaesthesia and euthanasia, we follow ARRIVE guidelines. Mice were first anesthetized with intraperitoneal injection of 250 mg/kg tribromoethanol (Avertin, Sigma-Aldrich, USA). Animal euthanasia was performed using CO2 gas at 30–70% displacement rate of the cage volume/min using a flow meter according to the American Veterinary Medical Association (AVMA) euthanasia guideline43.
Immunohistochemical Staining (IHC) of Tumor Tissue
Sections of the paraffin-embedded tissue blocks (4–6 µm-thick) were mounted on positively charged glass slides, and dried in an oven at 56°C for 30 min. The sections were deparaffinized and then rehydrated, and hydrogen peroxide was added to suppress endogenous peroxidase. After treatment with bovine serum albumin (BSA) to block nonspecific binding, the sections were then incubated with primary antibody overnight at 4°C. After washing, biotinylated secondary antibody was added for 1 h. Diaminobenzidine (Vector Laboratories, Inc.) was employed for color development. Mayer’s hematoxylin was used for counterstaining of sections.
Statistical Analysis
Statistical analysis was performed using the GraphPad Prism statistics program (Version 7). All the data were obtained from experiments with adequate sample size and presented as means ± SEM. One–way ANOVA was carried out for comparisons among three or more groups and was followed by Tukey’s post hoc test. Values were considered to be significant at p < 0.05. Student’s t-test was also employed.
Patients and Samples
We collected tissues from 215 patients with pulmonary adenocarcinoma who underwent surgery and had been followed up at Seoul National University Hospital (Seoul, Republic of Korea) from 2001 to 2011. None had received chemotherapy before surgery or had distant metastasis at the time of diagnosis. Clinicopathologic data and pathologic tumor–node– metastasis (TNM) staging from the 8th American Joint Committee on Cancer were obtained from medical and pathologic records. A tissue microarray was constructed from 2-mm diameter cores derived from representative tumor areas of formalin-fixed, paraffin embedded tissue blocks. EGFR, KRAS mutation and ALK translocation status were evaluated as described previously [16 ].
Another cohort of 10 patients who had recurrence or metastatic pADCs with EGFR mutation and were treated with the EGFR-TKI at SNUH was collected for evaluation of CAGE and ATG5 expression. PFS was measured from the first day of inhibitor treatment until the first objective sign of disease progression or death. OS was measured from the date of diagnosis until death from any cause. This study followed the World Medical Association Declaration of Helsinki recommendations and was approved by the Institutional Review Board of Seoul National University Hospital (H-1404-100-572).
IHC and Fluorescence In Situ Hybridization
Immunohistochemistry was performed using the Benchmark XT autostainer (Ventana Medical Systems). The CAGE, ATG5, pBeclin1Ser15, pAMPKɑT172 and p62 immunohistochemistry were evaluated based on the intensity and proportion of cytoplasmic or nuclear staining in tumor cells and the results (H-score) are recorded by multiplying the percentage of positive cells by the intensity. Immunohistochemistry for EGFR and MET was assessed by modified criteria of previous report. Cases were scored into 0 (absent or only focal weak), 1 (weak to moderate in ≤ 40% of tumor cells), 2 (weak to moderate in ≥ 40% of tumor cells), and 3 (strong in ≥ 10% of tumor cells); and then classified into negative (score 0 or 1) and positive (score 2 or 3) for EGFR and MET expression. HER2 expression was scored from 0 to 3 + according to the FDA approved guidelines for the HercepTest and cases with scores of 2 + or 3 + were considered positive for expression. The list of antibodies used in IHC is described in supplementary Table S2.
MET GCN and amplification was estimated using and LSI MET Spectrum Red/CEP7 Spectrum Green probe (Abbott Molecular, DesPlaines, IL, USA), and was counted in at least 100 tumor nuclei. Gene amplification (MET to CEP7 ratio ≥ 2; >15 copies of the MET signals in > 10% of the tumor cells; small gene cluster [4–10 copies] or innumerable tight gene cluster in > 10% of the tumor cells) and high polysomy (≥ 40% of cells displaying ≥ 4 copies of the MET signal) were defined as FISH positivity according to University of Colorado Cancer Center (UCCC) criteria [16].
Statistical analyses of human samples
All statistical analyses were performed using SPSS software (version 25; SPSS, Chicago, IL, USA). Comparisons between variables were performed using the χ2 test, Fisher’s exact test, or Student’s t-test. Cut-off values of continuous variables were determined based on the receiver operating characteristic curve at the highest positive likelihood point for disease-free survival. Survival analysis was performed using the Kaplan–Meier method with log-rank test. Two-sided P-values of < 0.05 were considered statistically significant.