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) 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 .......

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

Name	Company	Catalog Number	Comments
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