Establishing Dual Resistance to EGFR-TKI and MET-TKI in Lung Adenocarcinoma Cells In Vitro with a 2-step Dose-escalation Procedure

Summary

An in vitro method for establishing dual resistance to an EGFR-TKI and a MET-TKI in cancer cells is described. This method is useful for developing treatments for patients with EGFR-mutations, who exhibit disease progression despite EGFR-TKI treatment with MET-amplification. It can also be modified for inhibitors targeting other molecules.

Abstract

Drug resistance is a major challenge in cancer therapy. The generation of resistant sublines in vitro is necessary for discovering novel mechanisms to overcome this challenge. Here, a 2-step dose-escalation method for establishing dual-resistance to an epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI), gefitinib, and a MET-TKI, PHA665752, is described. This method is based on simple stepwise dose-escalation of inhibitors for inducing acquired resistance in cell lines. The alternate method for generating resistant sublines involves exposing the cells to high concentrations of the inhibitor in one step. The stepwise dose-escalation method has a higher possibility of successfully inducing acquired resistance than this method. Activating EGFR mutations are biomarkers of a response to treatment with EGFR-TKI, which is an applied first-line treatment for non-small cell lung cancers (NSCLC) that harbor these mutations. However, despite reports of effective responses, the use of EGFR-TKI is limited because tumors inevitably acquire resistance. The major mechanisms behind EGFR-TKI resistance include a secondary mutation at the gatekeeper site, T790M in exon 20 of EGFR, and a bypass signal of MET. Thus, a potential solution for this issue would be a combination of EGFR-TKI and MET-TKI. This combined treatment has been shown to be effective in an in vitro study model. Acquired gefitinib-resistance was established through MET-amplification by stepwise dose-escalation of gefitinib for 12 months, and a cell line named PC-9MET1000 was generated in a previous study. To further investigate the mechanisms of acquired MET-TKI and EGFR-TKI resistance, a MET-TKI, PHA665752, was administered to these cells with stepwise dose-escalation in the presence of gefitinib for 12 months. This protocol has also been successfully applied for a number of combination therapies to establish acquired resistance to other inhibitor molecules.

Introduction

Oncogenic mutations in the epidermal growth factor receptor (EGFR) are found in a subset of patients with non-small cell lung cancer (NSCLC) and are important predictive biomarkers of this disease^1,2. These activating EGFR mutations most commonly occur as either an in-frame deletion in exon 19 (delE746-A750) or a point mutation replacing leucine with arginine at codon 858 of exon 21 (L858R) in the EGFR tyrosine kinase domain^3,4. Treatment with EGFR-tyrosine kinase inhibitors (TKIs) is a clinically effective therapy for patients with NSCLC harboring EGFR mutations. However, this therapy is limited by the inevitable acquisition of resistance to EGFR-TKIs such as gefitinib and erlotinib. The most common acquired resistance occurs through a secondary T790M mutation in exon 20 of EGFR, which was detected in approximately 60% of patients who acquired EGFR-TKI (gefitinib or erlotinib) resistance. Other molecular mechanisms associated with acquired resistance to EGFR-TKIs are activation of bypass signaling caused by MET amplification, transformation of small-cell lung cancers, and epithelial-to-mesenchymal transition^5,6. The receptor for the hepatocyte growth factor, encoded by the MET gene, which is dysregulatedin many tumors, has been reported to be an important candidate for targeted therapies^7,8.

Strategies for overcoming the acquired resistance to EGFR-TKIs are now undergoing clinical evaluation. Preclinical and clinical studies have shown that third-generation EGFR inhibitors such as osimertinib are effective for patients with the T790M mutation⁹. In addition, the growth of EGFR-mutant cancers with MET amplification can be inhibited by combined treatment with EGFR- and MET-TKIs^6,10. Clinical trials assessing a combination of MET and EGFR inhibitors in patients with acquired resistance to gefitinib and erlotinib are underway now¹¹.

To study the molecular mechanism of acquired resistance to chemotherapeutic agents, cell lines that initially respond to inhibitors are treated continuously with these inhibitors. Two methods are available to establish acquired resistance in cell lines. One is stepwise dose-escalation, and the other is high-concentration exposure. The stepwise escalation method has been more commonly used, because it has a higher success rate. The cells are first exposed to low concentrations of the inhibitors, nearly a tenth of the half-maximal inhibitory concentration (IC₅₀) value, and the concentration is then gradually increased by 10-30% until the target concentration is achieved. The high-concentration exposure method involves exposing the cells to the final dose of inhibitor in the culture medium, which is more than the IC₅₀ value, so the parental cells are almost completely killed. This high-concentration exposure method has much a lower success rate than the stepwise escalation method.

In a previous study, combined treatment with EGFR-TKI and MET-TKI was shown to be effective in an in vitro system. Acquired resistance to an EFGR-TKI, gefitinib, was established through stepwise escalation for 12 months, along with MET-amplification, and thus, a gefitinib-resistant cell line called PC-9MET1000 was generated¹².

The protocol presented here is a two-step dose-escalation procedure for obtaining EGFR-mutated NSCLC PC-9 cells with dual resistance to the EGFR-TKI, gefitinib, and a MET-TKI, PHA665752 (Figure 1). This method for establishing acquired resistant in cell lines is relatively easy to perform, with a high success rate. The described protocol can be modified for application with inhibitors of other molecular target drugs and cytotoxic agents as well.

Protocol

1. Establish Gefitinib-resistant Cells

1. Determine the initial gefitinib concentration by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay.
   1. Culture PC-9 cells in growth medium (RPMI-1640 medium with 10% FBS and 1% antibiotics (penicillin: 100 U/mL and streptomycin: 100 µg/mL)), in a 5% CO₂ incubator at 37 °C.
   2. Adjust the cells to 1.0 × 10⁵ cells/mL using an automated cell counter (see Table of materials) and seed 50 µL of this in each well of a 96-well plate using the growth medium; the final concentration of cells should be 5.0 × 10³ cells/well.
   3. Incubate the plate overnight at 37 °C.
   4. Add 50 µL of gefitinib (at 2X final concentration) at different concentrations: 0, 0.002, 0.006, 0.02, 0.06, 0.2, 0.6, 2, 6, and 20 µM to these wells such that the final volume in each well is 100 µL.
   5. Incubate the plate for 72 h at 37 °C.
   6. Add 15 µL of the dye solution (see Table of Materials) to each well (use final 13% concentration of the dye solution), and incubate for 4 h at 37 °C.
   7. Add 100 µL of the solubilization/stop solution (see Table of Materials) for solubilizing the formazan produced by the dye solution (see Table of Materials) in each well, and incubate it overnight at 37 °C.
   8. Measure the optical density at 570 nm (OD₅₇₀) using a microplate reader for obtaining the IC₅₀ value by generating a cell-proliferation curve.
   9. Use a statistical software (see Table of Materials) to graphically display the data, and calculate the IC₅₀ value (Figure 2).
   10. Prepare 6-12 replicates and repeat the experiments at least three times.
2. Continuous stepwise dose-escalation of gefitinib for treatment of PC-9 cells.
   1. Culture PC-9 cells (1 mL in sub-confluent condition) in p100 dishes with 10 mL of growth medium in a 5% CO₂ incubator at 37 °C.
   2. Add one-tenth of the IC₅₀ value of gefitinib into the p100 dish.
      1. When the cells in the culture medium become sub-confluent, add 1-2 mL of cells to 10 mL of fresh growth medium using a 1-mL pipet with 10-30% higher concentration of gefitinib in the same culture conditions.
   3. Increase the gefitinib concentration progressively by 10-30% with each split until it reaches 1 µM; this might take about 6-12 months.
      NOTE: When the concentration of gefitinib reaches the IC₅₀ value, the cell proliferation becomes considerably slower, and so, 2-4 mL of cells may be added to 10 mL of the fresh growth medium with higher concentration of gefitinib; reducing the increase in the concentration of gefitinib might solve the problem. Moreover, it would be ideal to store the remaining cells at -80 °C, and reuse them at the previous concentration. As PC-9 cells are in suspension, it is unnecessary to use trypsin for passaging; however, during the process of increasing gefitinib concentration, the cells may attach to the bottom of culture dish. In such a situation, a cell-scraper can be used for detaching the adherent cells.
   4. Culture the gefitinib-resistant PC-9 cells in the growth medium with 1 µM of gefitinib.
   5. Perform the MTT assay to confirm the gefitinib-resistance of the cells¹³; these gefitinib-resistant cells were named PC-9MET1000 (designated as MET1000 cells), because they exhibited increased protein and RNA levels of MET (Figure 3A, Figure 4A, B).

2. Establish Dual-resistance to Gefitinib and PHA665752

1. Determine the initial concentration of PHA665752 by MTT assay.
   1. Culture the MET1000 cells in p100 dishes with 10 mL of growth medium and 1 µM gefitinib in a 5% CO₂ incubator at 37 °C.
   2. Seed the cells into a 96-well plate at 5.0 × 10³ cells/well with 50 µL of the growth medium in each well. Adjust the final concentration of the cells to 1.0 × 10⁵ cells/mL using an automated cell counter (see Table of Materials).
   3. Incubate the plate overnight at 37 °C.
   4. Add 50 µL of PHA665752, a MET-TKI (at 2X final concentration), to the cells in different concentrations: 0, 0.002, 0.006, 0.02, 0.06, 0.2, 0.6, 2, 6, and 20 µM including 2 µM gefitinib (at 2X final concentration); the final volume in each well should be 100 µL.
   5. Incubate the plate for 72 h at 37 °C.
   6. Add 15 µL of the dye solution (see Table of Materials) to each well, and incubate for 4 h at 37 °C.
   7. Add 100 µL of the solubilization/stop Solution for solubilizing the formazan produced by the dye solution (see Table of Materials) to each well, and again incubate overnight at 37 °C.
   8. Measure the optical density at 570 nm (OD₅₇₀) using a microplate reader for obtaining the IC₅₀ value by generating a cell-proliferation curve.
   9. Use a statistical software (see Table of Materials) to graphically display the data, and calculate the IC₅₀ value of PHA665752 in the presence of gefitinib (Figure 3B).
   10. Prepare 6-12 replicates and repeat the experiments at least three times.
2. Establish the dual-resistance in MET1000 cells to gefitinib and PHA665752.
   1. Culture the MET1000 cells in p100 dishes with 10 mL of growth medium, and 1 µM gefitinib in a 5% CO₂ incubator at 37 °C.
   2. Add one-tenth of the IC₅₀ value of PHA665752 into the growth medium in the presence of 1 µM of gefitinib with the same culture conditions.
   3. Increase the PHA665752 concentration progressively by 10-30% with each split until it reaches 1 µM. This might take about 6-12 months.
      NOTE: When the concentration of PHA665752 reaches the IC₅₀ value, the cell proliferation becomes considerably slower, and so, 2-4 ml of cells may be added using a 1-mL pipet to 10 mL of the fresh growth medium with higher concentration of PHA665752; reducing the increase in the concentration of PHA665752 might solve the problem. Moreover, it would be ideal to store the remaining cells at -80 °C, and reuse them at the previous concentration. It is unnecessary to use trypsin for passaging; however, during the increase of PHA665752 concentration, the cells may attach to the bottom of the culture dish. In such situations, a cell-scraper can be used to detach the cells.
   4. Finally, culture the PC-9 cells resistant to both gefitinib and PHA665752 in the growth medium with 1 µM each of gefitinib and PHA665752 in a 5% CO₂ incubator at 37 °C.
   5. Perform the MTT assay to confirm that the cells are dual-resistant to gefitinib and PHA665752¹³, and that they are not sensitive to PHA665752 in the presence of gefitinib; these dual-resistance cells were named PC-9DR (designated as DR cells) (Figure 5).

Results

The overview of the protocol presented in this manuscript is shown in Figure 1, including the induction of acquired dual-resistance to gefitinib and PHA665752 in PC-9 cells by a 2-step dose-escalation procedure. Figure 2 shows a decrease in the proliferation of parental PC-9 cells as the concentration of gefitinib increases. This indicates that the PC-9 cells became sensitive to gefitinib after the dose-escalation procedure was p...

Discussion

The protocol described here can be used for the establishment of acquired dual-resistance in cancer cells using a 2-step dose-escalation method in vitro. Many research papers have reported that PC-9 cells developed EGFR-TKI (gefitinib or erlotinib) resistance through T790M EGFR mutations^15,16. However, we did not detect T790M mutations in exon 20 of our PC-9MET1000 cells by direct sequencing (Figure 4C). Moreover, MET amplif...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RPMI-1640	Wako	189-02025	with L-Glutamine and Phenol Red
CellTiter 96	Promega	G4100	Non-Radioactive Cell Proliferation Assay
FBS	gibco	26140-079
Pen Strep	gibco	15140-163
gefitinib	Selleck	S1025	stocked 10 mM/EMSO at -20ºC
PHA665752	Selleck	S1070	stocked 10 mM/EMSO at -20ºC
96-well plate	IWAKI	860-096	flat bottom with lid, low evaporation type
TC10 Automated Cell Counter	BIO RAD	#1450010
CellTiter 96 Non-Radioactive Cell Proliferation Assay	Promega	G4100	Dye Solution and Solubilization/Stop Solution
Powerscan HT microplate reader	BioTek
GraphPad Prism v.5.0 software	GraphPad Inc.
PC-9	Riken BioResource Center	RCB4455
P100 dish	FALCON	353003