Establishment and Characterization of Three Afatinib-resistant Lung Adenocarcinoma PC-9 Cell Lines Developed with Increasing Doses of Afatinib

Summary

A method for establishing afatinib-resistance cell lines from lung adenocarcinoma PC-9 cells was developed, and resistant cells were characterized. The resistant cells can be used to investigate epidermal growth factor receptor tyrosine kinase inhibitor-resistance mechanisms, applicable for patients with non-small cell lung cancer.

Abstract

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in most countries. The discovery of "oncogenic driver mutations," such as epidermal growth factor receptor (EGFR)-activating mutations, and subsequent development of molecular targeted agents of EGFR tyrosine kinase inhibitors (TKIs) (gefitinib, erlotinib, afatinib, dacomitinib, and osimertinib) have dramatically altered lung cancer treatment in recent decades. However, these drugs are still not effective in patients with non-small cell lung cancer (NSCLC) carrying EGFR-activating mutations. Following acquired resistance, the systemic progression of NSCLC remains a significant obstacle in treating patients with EGFR mutation-positive NSCLC. Here, we present a stepwise dose escalation method for establishing three independent acquired afatinib-resistant cell lines from NSCLC PC-9 cells harboring EGFR-activating mutations of 15-base pair deletions in EGFR exon 19. Methods for characterizing the three independent afatinib-resistance cell lines are briefly presented. The acquired resistance mechanisms to EGFR TKIs are heterogeneous. Therefore, multiple cell lines with acquired resistance to EGFR-TKIs must be examined. Ten to twelve months are required to obtain cell lines with acquired resistance using this stepwise dose escalation approach. The discovery of novel acquired resistance mechanisms will contribute to the development of more effective and safe therapeutic strategies.

Introduction

Five tyrosine kinase inhibitors, targeting epidermal growth factor receptor (EGFR), including gefitinib, erlotinib, afatinib, dacomitinib, and osimertinib are currently available for treating patients with EGFR mutation-positive non-small cell lung cancer (NSCLC). Over the past decade, therapies for such patients have undergone dramatic development with the discovery of new potential EGFR-TKIs. Among patients with lung adenocarcinoma, somatic mutations in EGFR are identified in approximately 50% of Asian and 15% of Caucasian patients¹. The most common mutations in EGFR are an L858R point mutation in EGFR exon 21 and 15 base pair (bp) deletions in EGFR exon 19². In EGFR mutation-positive patients with NSCLC, EGFR-TKIs improve the response rates and clinical outcomes compared to the previous standard of platinum doublet chemotherapy³.

Gefitinib and erlotinib were the first approved small molecule inhibitors and are generally referred to as first-generation EGFR TKIs. These EGFR TKIs block tyrosine kinase activity by competing with ATP and reversibly binding to ATP binding sites⁴. Afatinib is a second-generation EGFR TKI that irreversibly and covalently binds to the tyrosine kinase domain of EGFR and is characterized as a pan-human EGFR family inhibitor⁵.

Despite the dramatical benefit of these therapies in patients with NSCLC, acquired resistance is inevitable. The most common resistance mechanism against first- and second-generation EGFR TKIs is the emergence of the T790M mutation in EGFR exon 20, which is present in 50-70% of tumor samples^6,7,8. Other resistance mechanisms include bypass signals (to MET, IGF1R, and HER2), transformation to small cell lung cancer, and induction of epithelial-to-mesenchymal transition, which occur pre-clinically and clinically⁹. The resistance mechanisms to EGFR TKIs are heterogeneous. By identifying novel resistance mechanisms in preclinical studies, it may be possible to develop novel therapeutics to overcome resistance. Optimal sequence therapies that maximize the clinical benefit to patients must consider the resistance mechanisms and therapeutic target.

It is imperative to choose the right parental cell line, as it is the basis of all the subsequent experiments. The selection strategies begin with clinical relevance; it is necessary to choose a chemotherapy and radiation naïve cell line. Previous chemotherapeutic and/or radiative treatment may induce the alteration of resistance pathways and changes of the expression of drug resistance markers. In this study, PC-9 cells, carrying 15 bp deletions in EGFR exon 19, are employed for the establishment of acquired resistance to afatinib. This cell line was derived from a Japanese NSCLC patient, who did not receive prior chemotherapy and radiation.

Because afatinib is administered orally on a daily basis, continuous in vitro treatment, where the cells are cultured constantly in the presence of afatinib would be clinically relevant. The dose of drugs used in the various steps of the experiment must be optimized for the parental cell line selected. A cytotoxicity assay can be used for determining a suitable drug range, which should be comparable to the pharmacokinetic information of the drug.

Throughout the selection process, the whole population of cells is maintained as a single group; cloning or other separation methods are not used. The cells are first continuously exposed to a low level of the drug. Subsequently, after the cells adapt to grow in the presence of the drug, the dose of the drug is slowly increased to the final optimal dose of drug^10,11. Alternatively, a pulse drug-administration or mutagenesis can be used for selecting resistance cells, which are also performed prior to drug treatment ^12,13. Unfortunately, cases where drug resistance fails to develop are generally not reported. The selection strategies are developed with the aim of trying to mimic the conditions of cancer patients for rebuilding clinically relevant resistance. Sometimes, to identify molecular changes associated with mechanisms of drug resistance, a high drug concentration is used. This model becomes less clinically relevant.

Here, we describe a method for establishing three independent afatinib-resistant cell lines from PC-9 cells harboring 15 bp deletions in EGFR exon 19 as well as the initial characterization of the afatinib-resistant cell lines.

Protocol

1. Establishment of Three Independent Afatinib-resistant PC-9 Cell Lines

1. Determination of the initial afatinib exposure concentration for PC-9 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay
   1. Culture PC-9 cells in growth medium containing fetal bovine serum (10%), penicillin (100 U/mL), and streptomycin (100 µg/mL) in a cell-culture treated 10-cm dish in a 5% CO₂ incubator at 37 °C.
   2. Resuspend PC-9 cells at 4 x 10⁴ cells/mL in growth medium and then seed at 50 µL/well in a 96-well microplate. The final concentration of cells is 2.0 x 10³ cells/50 µL/well. Incubate overnight in a 5% CO₂ incubator at 37°C.
   3. Add 50 µL of afatinib solution at different concentrations: 0, 0.002, 0.006, 0.02, 0.06, 0.2, 0.6, 2, 6, and 20 µM to the wells containing the growth medium (50 µL). The final volume and concentrations of afatinib are 100 µL and 0, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 µM, respectively.
   4. Incubate the 96-well plate for 96 h in a 5% CO₂ incubator at 37 °C.
   5. Add 15 µL of the dye solution (see Table of Materials) to each well and incubate for 4 h in a 5% CO₂ incubator at 37 °C, and then add 100 µL of solubilization/stop-solution (see Table of Materials) to each well and incubate overnight in a 5% CO₂ incubator at 37 °C.
   6. Measure the optical density at 570 nm (OD₅₇₀) using a microplate reader (see Table of Materials). Prepare 6-12 replicates and repeat the experiments at least three times.
   7. Use statistical software (see Table of Materials) to graphically plot these data as a semi-log graph and calculate the IC₅₀ value, which is the drug concentration that reduces response to 50% of its maximum (see Table of Materials).
2. Continuous exposure of PC-9 cells to the irreversible EGFR-TKI, afatinib, by stepwise dose escalation in three independent 10 cm dishes
   1. Culture PC-9 cells in p100 dishes containing 10 mL of growth medium. When the PC-9 cells reach the sub-confluent stage, transfer 1 mL of cell suspension into three new p100 dishes, with 9 mL of growth medium. The 1:10 diluted PC-9 cells become sub-confluent in 3-4 days, with a cell number of approximately 4-5 x 10⁵ cells/mL.
   2. On the next day, add 1/10 of the IC₅₀ value of afatinib into each of the three p100 dishes.
      NOTE: Afatinib is reconstituted in DMSO at stock concentrations of 1 μM, 10 µM, 100 µM, 1 mM, and 5 mM. 1 to 10 µL of afatinib-solution is added into 10 mL of growth medium in the culture, as per the required final concentrations.
   3. When the cells in the afatinib-containing p100 dishes become sub-confluent, mix well by aspiration with a 1 mL pipette and add 1 mL of the cell suspension to 9 mL of fresh growth medium in a new p100 dish. Next, add 10-20% higher concentrations of afatinib to the new culture.
   4. Increase the afatinib concentration of 0.1 nM to 1 μM in the medium by the stepwise dose escalation with the afatinib concentration increased by 10-20% at each step over the period of 10-12 months.
      NOTE: When the afatinib concentration approaches the IC₅₀ value, cell growth becomes quite slow. If the cells are split 1:9, they may not grow, as these cells are killed by higher concentrations of afatinib. Therefore, at higher afanitib concentrations, the cells can be split at a ratio of 1:2. The most resistant cells were grown in afatinib-contained medium for 3-14 days, and the medium was not changed until the resistant cells needed to be passaged.
   5. Culture the afatinib-resistant cells for 2-3 months in 1 µM afatinib-containing growth medium. At an afatinib concentration of 1 µM, 10-12 months are required for developing resistance to afatinib in this model. Perform the MTT assay to confirm that the cells are resistant to afatinib. The three independently established afatinib resistance cell lines were named AFR1, AFR2, and AFR3.

2. Characterization of Three Independent Afatinib-resistant Cells

1. Determination of the growth curve of parental PC-9 cells and establishment of afatinib-resistant cells
   1. Culture the PC-9, AFR1, AFR2, and AFR3 cells in growth medium in a 5% CO₂ incubator at 37 °C.
   2. Resuspend the cells at 5 x 10³ cells/mL with growth medium, and seed 100 µL/well into a 96-well microplate, such that the final concentration of cells is 500 cells/100 µL/well.
      NOTE: The MTT assay is performed to measure the OD₅₇₀ values at 0, 1, 2, 3, 5, and 7 days. Six 96-well microplates are required for each day.
   3. Perform the MTT assay every 24 h and then on days 0, 1, 2, 3, 5, and 7. Measure the OD₅₇₀ values and prepare 6-12 replicates; repeat the experiments at least three times, and graphically plot the results using a statistical software (see Table of Materials).
2. Identification of the genomic DNA alterations in EGFR by real-time PCR
   NOTE: Afatinib is a small molecule inhibitor that targets EGFR tyrosine kinase. The EGFR expression status is determined at the DNA and protein levels.
   1. Genomic DNA is isolated using a DNA purification kit (see Table of Materials) following the manufacturer's instructions. Measure the concentration of the isolated genomic DNA with a spectrophotometer (see Table of Materials) and adjust all genomic DNA samples to 25 ng/ µL.
   2. Amplify 50 ng of genomic DNA, which is equivalent to 2 µL of the 25 ng/µL stocks, using a SYBR Green master mix (see Table of Materials) and analyze the results using a fluorescence-based RT-PCR-detection system (see Table of Materials).
      NOTE: PCR cycling conditions began with an initial denaturation step at 95 °C for 20 s, followed by 40 cycles of 95 °C denaturation for 3 s, 60 °C annealing for 30 s. The specific primer sets are as follows: EGFR F: 5′-CAAGGCCATGGAATCTGTCA-3′, R: 5′-CTGGAATGAGGTGGAGGAACA-3′. Normalization gene LINE-1 F: 5′-AAAGCCGCTCAACTACATGG-3′, R: 5′-TGCTTTGAATGCGTCCCAGAG-3′.
3. Evaluation of the effect of protein alterations on the EGFR level by western blot analysis
   1. Treat the cells with afatinib continuously prior to experiments for 24 h. Wash PC-9, AFR1, AFR2, and AFR3 cells twice with PBS and then seed them in growth media without afatinib. Wash PC-9, AFR1, AFR2, and AFR3 cells twice with 5 mL of ice-cold PBS.
   2. Lyse the cells in RIPA buffer containing 1% protease inhibitor cocktail (see Table of Materials) and phosphatase inhibitor cocktail II and III (see Table of Materials) and incubate this solution at 4 °C for 30 min. Centrifuge the lysates for 10 min at 100 x g and 4 °C and collect the cleared lysates.
   3. Determine protein concentrations using the bicinchoninic acid assay (see Table of Materials), adjust all protein samples to 0.5 or 1 µg/µL using 4x sample buffer (500 mM Tris (PH 6.8), 40% glycerol, 8% SDS, 20% H₂O, 0.02% bromophenol blue) and boil at 96 °C for 5 min. Store these protein samples at -80 °C until western blot analysis is performed.
   4. Separate equal amounts of protein samples, preferably 20-30 µL, by 8% SDS-page and transfer the proteins to a polyvinylidene fluoride (PVDF) membrane.
      NOTE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is commonly used in the lab for the separation of proteins based on their molecular weight.
      1. Clean glass plates with ethanol and assemble the glass plate and spacers. Prepare 8% poly-acrylamide gels containing 1.5 M Tris-HCl, pH 8.8, 40% Bis-acrylamide, 10% SDS, 10% APS, and TEMED. Polymerize for 30 min at room temperature.
      2. Subsequently, prepare a stacking gel containing 0.5 M Tris-HCl, pH 6.8, 40% bis-acrylamide, 10% SDS, 10% APS, and TEMED. Add the stacking gel solution, insert the comb, and polymerize the gel for 20-30 min at room temperature.
      3. Place the gels in the electrophoresis apparatus and fill the tank with running buffer (0.25 M Tris, 1.92 M glycine, and 1% SDS). Load equal amount of protein samples (20-30 µL) and run the gel at 180 V. Stop electrophoresis once the dye front flows out of the gel, after approximately 60 min.
      4. Wash the gel for 1-2 min with TBST and then transfer the proteins on to a PVDF membrane by semi-dry blotting (see Table of Materials) for 1.5 h at a constant current of 300 mA.
   5. Block the membranes with 5% of nonfat dry milk (see Table of Materials) diluted with TBST solution (see Table of Materials) for 1 h at room temperature, and then probe the membranes with anti-EGFR, anti-phospho-EGFR (Y1068), anti-HER2, anti-HER3, anti-MET, and anti-actin antibodies (diluted 1:3,000 in TBST) (see Table of Materials) at 4 °C overnight.
   6. Wash the membranes with TBST three times for 10 min, and then expose the membranes to the secondary antibody (diluted 1:200 in TBST) for 1-1.5 h at room temperature. Wash the membranes five times with TBST for 10 min at room-temperature, expose them to the ECL solution (see Table of Materials), and visualize the signals using films.
4. Analysis of EGFR mutations by sequencing
   1. Amplify genomic DNA using specific primers for EGFR exons 19-21. The PCR cycling conditions begin with an initial denaturation step at 94 °C for 1 min, followed by 30 cycles of denaturation at 98 °C for 10 s, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min.
      NOTE: The specific primers for EGFR exon 19: F: 5′-GCAATATCAGCCTTAGGTGCGGCTC-3′ R: 5′-CATAGAAAGTGAACATTTAGGATGTG-3′, exon 20: F: 5′-CCATGAGTACGTATTTTGAAACTC-3′, R: 5′-CATATCCCCATGGCAAACTCTTGC-3′, and exon 21: F: 5′-ATGAACATGACCCTGAATTCGG-3′, R: 5′-GCTCACCCAGAATGTCTGGAGA-3′.
   2. Purify the amplified PCR products using a PCR purification kit (see Table of Materials) and sequence the amplicons.

Results

The schema for establishing three afatinib-resistance cell lines from PC-9 cells using a stepwise dose-escalation procedure is shown in Figure 1. Figure 2 shows a decrease in cell proliferation of parental PC-9 cells as the concentration of afatinib is increased, indicating that PC-9 cells are sensitive to afatinib exposure. Figure 3 shows the afatinib-resistance of the three cell lines. None of the ...

Discussion

Here, we described a method for establishing three independent afatinib-resistant cell lines and characterized these cells by comparison to parental PC-9 cells. By stepwise dose escalation exposure, the parental PC-9 cells acquired resistance to afatinib over a period of 10-12 months. Clinically, the resistance mechanisms to EGFR TKIs are heterogeneous, and therefore, after the initial treatment with afatinib, PC-9 cells were divided into three independent p100 dishes and exposed further to afatinib. Initially, cell grow...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
afatinib	Selleck	S1011
anti-EGFR monoclonal antibody	cell signaling technology	4267S
bicinchoninc acid assay	sigma	B9643
cell-culture treated 10 cm dish	Violamo	2-8590-03
CELL BANKER1	TakaRa	CB011	cryopreservation media
CellTiter 96	Promega	G4100	Non-Radioactive Cell Proliferation Assay; Dye solution and Solubilization/Stop solution
DMSO	Wako	043-07216
ECL solution	Perkin Elmer	NEL105001EA
FBS	gibco	26140-079
GeneAmp 5700	Applied Biosystems		fluorescence-based RT-PCR-detection system
GraphPad Prism v.7 software	GraphPad, Inc.		a statistical software
NanoDrop Lite spectrophotometer	Thermo		spectrophotometer
Nonfat dry milk	cell signaling technology	9999S
Pen Strep	gibco	15140-163
phosphatase inhibitor cocktail 2	sigma	P5726
phosphatase inhibitor cocktail 3	sigma	P0044
Powerscan HT microplate reader	BioTek
Power SYBR Green master mix	Applied Biosystems		SYBR Green master mix
protease inhibitor cocktail	sigma	P8340
QIAamp DNA Mini kit	Qiagen	51306	DNA purification kit
QIAquick PCR Purification Kit	QIAGEN		PCR purification kit
RPMI-1640	Wako	189-02025	with L-Glutamine and Phenol Red
TBST powder	sigma	T9039
Trans-Blot SD Semi-Dry Electrophoretic Transfer cell	Bio-Rad		semi-dry t4ransfer apparatus
96 well microplate	Thermo	130188