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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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) d....

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 .......

Representative 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.......

Acknowledgements

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.).

....

Materials

NameCompanyCatalog NumberComments
afatinibSelleckS1011
anti-EGFR monoclonal antibodycell signaling technology4267S
bicinchoninc acid assaysigmaB9643
cell-culture treated 10cm dishViolamo2-8590-03
CELL BANKER1 TakaRaCB011cryopreservation media
CellTiter 96Promega G4100 Non-Radioactive Cell Proliferation Assay; Dye solution and Solubilization/Stop solution
DMSOWako043-07216
ECL solutionPerkin ElmerNEL105001EA
FBSgibco26140-079
GeneAmp 5700Applied Biosystemsfluorescence-based RT-PCR-detection system 
GraphPad Prism v.7 software GraphPad, Inc.a statistical software
NanoDrop Lite spectrophotometerThermospectrophotometer
Nonfat dry milkcell signaling technology9999S
Pen Strepgibco15140-163
phosphatase inhibitor cocktail 2sigmaP5726
phosphatase inhibitor cocktail 3sigmaP0044
Powerscan HT microplate readerBioTek
 Power SYBR Green master mix Applied BiosystemsSYBR Green master mix
protease inhibitor cocktailsigmaP8340
QIAamp DNA Mini kitQiagen51306DNA purification kit
QIAquick PCR Purification KitQIAGENPCR purification kit
RPMI-1640 Wako189-02025with L-Glutamine and Phenol Red
TBST powdersigmaT9039
Trans-Blot SD Semi-Dry Electrophoretic Transfer cellBio-Radsemi-dry t4ransfer apparatus
96 well microplateThermo130188

References

  1. Chan, B. A., Hughes, B. G. Targeted therapy for non-small cell lung cancer: current standards and the promise of the future. Translational Lung Cancer Research. 4 (1), 36-54 (2015).
  2. Mitsudomi, T., Yatabe, Y.

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Afatinib ResistanceLung AdenocarcinomaPC 9 Cell LinesAcquired ResistanceStepwise Dose EscalationMTT AssayCell CultureSubconfluencyDose dependent Resistance

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