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.
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.
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 samples6,7,8. 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 drug10,11. 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.
1. Establishment of Three Independent Afatinib-resistant PC-9 Cell Lines
2. Characterization of Three Independent Afatinib-resistant Cells
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 three afatinib-resistant cell lines, AFR1, AFR2, and AFR3, showed suppression of cell proliferation under afatinib exposure. Figure 4 shows the cell-proliferation curves for PC-9, AFR1, AFR2, and AFR3 cells. The three afatinib-resistant cell lines exhibited significantly slower growth than the parental PC-9 cells. Figure 5 shows the expression levels of EGFR gDNA in PC-9 and the three afatinib-resistant cells, which indicate that afatinib-resistant cells expressed significantly higher levels of EGFR gDNA than the parental PC-9 cells. Figure 6 shows the protein expression of EGFR in PC-9 and afatinib-resistant cells. At comparable gDNA expression levels, EGFR protein expression was higher in resistant cells than in parental PC-9 cells. Figure 7 shows that the sequencing results of EGFR exons 19 and 20 in PC-9, AFR1, AFR2, and AFR3 cells. PC-9 cells showed 15 bp deletions in EGFR exon 19 and wild-type EGFR in exon 20. However, AFR1 and AFR2 cells exhibited amplification of wild-type EGFR exon 19. AFR3 cells contained 15 bp deletions in EGFR exon 19 as in PC-9 cells, but the point mutation T790M was observed in EGFR exon 20.
Figure 1: Schema of the process used to establish three afatinib-resistant cell lines from PC-9. First, PC-9 cells were separated into three p100 dishes and exposed to afatinib at 1/10 of the IC50 value. Next, afatinib concentrations in the growth medium were increased by stepwise dose escalation to 1 µM. After 10-12 months, three independent afatinib-resistant cell lines were established and named AFR1, AFR2, and AFR3. Please click here to view a larger version of this figure.
Figure 2: Parental PC-9 cells are sensitive to the irreversible EGFR TKI, afatinib. Cells were seeded into a 96-well microplate at 2 x 103 cells/well/50 µL of growth medium, and preincubated overnight. The cells were treated with the indicated concentrations of afatinib for 96 h. An MTT assay was performed, OD570 values were measured using a microplate reader (see Table of Materials) and expressed as a percentage of the value obtained for the control cells. Data are presented as mean ± SEM of values from 6-12 replicate wells. Please click here to view a larger version of this figure.
Figure 3: Established cells exhibited resistance to irreversible EGFR TKI, afatinib. Cells were seeded into a 96-well microplate at 2 x 103 cells/well/50 µL of growth medium and preincubated overnight. The cells were treated with the indicated concentrations of afatinib for 96 h. An MTT assay was performed, OD570 values were measured using a microplate reader (see Table of Materials) and expressed as a percentage of the value obtained for the control cells. Data are presented as mean ± SEM of values from 6-12 replicate wells. Please click here to view a larger version of this figure.
Figure 4: Afatinib-resistant cell lines showed slower proliferation than parental PC-9 cells. Cells were seeded into 96-well microplates at 5 x 102 cells/100 µL/well. MTT assay was performed, and OD570 values were measured on days 0, 1, 2, 3, 5, and 7 using a microplate reader (see Table of Materials) and expressed as a percentage of the value obtained for the control cells. Data are presented as mean ± SEM of values from 6-12 replicate wells. Please click here to view a larger version of this figure.
Figure 5: Gene copy number of EGFR was elevated in afatinib-resistant cells. The elevation of the EGFR gene copy number was measured by quantitative PCR of genomic DNA isolated from PC-9, AFR1, AFR2, and AFR3 cells. Please click here to view a larger version of this figure.
Figure 6: Basal level of EGFR protein was increased in afatinib-resistant cells. Western blot analysis of phospho-EGFR, EGFR, HER2, HER3, and MET expression in PC-9, AFR1, AFR2, and AFR3 cells. β-Actin was used as a loading control. Please click here to view a larger version of this figure.
Figure 7: DNA sequence reads in EGFR exons 19 and 20. Genomic DNA of PC-9, AFR1, AFR2, and AFR3 was amplified with specific primers for EGFR exon 19 and 21, and purified for sequencing. Please click here to view a larger version of this figure.
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 growth was not significantly slowed, but as the drug concentration approached the IC50 value, cell proliferation was slowed. This is a critical step for obtaining cells with acquired resistance to inhibitors. The proliferating cells should be split and transferred to new p100 dishes at a ratio of 1:10 or 1:5. When PC-9 cells were cultured in a p100 dish, some adherence was observed, but most cells grew in suspension. As the concentration of afatinib was increased, the cells adhered to the bottom of the tissue culture treated dishes. If most cells are adherent, they can be detached with a cell-scraper. The final concentration of afatinib was 1 µM, which is about 5 times the maximum serum concentration (Cmax)14. To obtain clear differences between parental and resistance clones, the final concentration was set to be higher than Cmax.
One serious concern during the procedure is bacterial contamination, even though RPMI-1640 contains penicillin and streptomycin. To avoid this, two p100 dishes containing fresh growth medium can be prepared when the cells are split. When the cells reach the sub-confluent stage, the cells in one p100 dish can be further split, while the cells in the other p100 dish can be stored at -80 °C in cryopreservation medium (see Table of Materials) as a backup, such that if one line is contaminated, the stored line can be used.
It would be difficult to completely reproduce the acquisition of afatinib resistance in humans using cell culture. The emergence of the T790M mutation in EGFR exon 20 was reported as the dominant cause of resistance to afatinib. In our report, one resistant clone contained the T790M mutation11. Furthermore, the increase in wild-type EGFR, such as in AFR1 and AFR2 cells, has been reported by us and other groups15,16. The loss of the EGFR mutation and increase in wild type EGFR is also reported in clinical samples from patients with acquired resistance to EGFR-TKIs 17,18. Therefore, in vitro studies of our current model may reflect the molecular profiles of clinical specimens with acquired resistance.
This stepwise dose escalation method is considered the most reliable for obtaining acquired resistant cells lines. However, initial high-dose afatinib exposure in cultured cells would likely better reflect the effects of afatinib treatment in patients with cancer, although establishing resistant cells is more difficult. Not only floating cell lines, such as PC-9 but also adherent cell lines, such as HCC827, 11-18, or HCC4006, can be employed for this method. This stepwise dose escalation method is also useful for establishing clones resistant to other inhibitors, using other cell lines, representing other types of cancer.
Exposure of parental cells to mutagenic agents, such as N-ethyl-N-nitrosourea, followed by selection of cells resistant to afatinib or osimertinib treatment has been reported to enable rapid acquisition of resistant clones 19,20. However, this artificial method tends to cause specific base substitutions, such as GC to AT transitions and AT to TA transversions. Moreover, EGFR TKI is not a mutagenic agent in patients with NSCLC. Therefore, the method of stepwise dose escalation is more representative than using mutagenic agents.
Although EGFR TKIs are initially effective, cells eventually develop resistance to such single-target drugs, making it difficult to cure cancer. Inhibitors that target multiple molecules are therefore essential to develop. To this end, it is necessary to obtain cells with acquired resistance to multi-target inhibitors and evaluate the mechanisms underlying drug resistance.
The authors have nothing to disclose.
We thank the member of the Advanced Cancer Translational Research Institute for their thoughtful comments and Editage for their assistance with English language editing. This work was supported by JSPS KAKENHI (grant number: 16K09590 to T.Y.).
afatinib | Selleck | S1011 | |
anti-EGFR monoclonal antibody | cell signaling technology | 4267S | |
bicinchoninc acid assay | sigma | B9643 | |
cell-culture treated 10cm 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 |