Name: establishment and characterization of three afatinib-resistant lung adenocarcinoma pc-9 cell lines developed with increasing doses of afatinib
Uploaded: 2019-06-26T13:30:00
Description: Watch this Scientific Journal Video about Establishment and Characterization of Three Afatinib-resistant Lung Adenocarcinoma PC-9 Cell Lines Developed with Increasing Doses of Afatinib at JoVE.com

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.).

1. Establishment of Three Independent Afatinib-resistant PC-9 Cell Lines
<ol>
	<li>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
	<ol>
		<li>Culture PC-9 cells in growth medium containing fetal bovine serum (10%), penicillin (100 U/mL), and streptomycin (100 &#181;g/mL) in a cell-culture treated 10-cm dish in a 5% CO2 incubator at 37 &#176;C.</li>
		<li>Resuspend PC-9 cells at 4 ...

<ol>
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J., Shen, D. W., Garfield, S., Gottesman, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mislocalization+of+membrane+proteins+associated+with+multidrug+resistance+in+cisplatin-resistant+cancer+cell+lines.">Mislocalization of membrane proteins associated with multidrug resistance in cisplatin-resistant cancer cell lines.</a> Cancer Research. 63 (18), 5909-5916 (2003).</li><li>Shen, D. W., Akiyama, S., Schoenlein, P., Pastan, I., Gottesman, M. 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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...

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 na&#239;ve 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&#160;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.

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	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:221px;">afatinib</td>
			<td style="width:187px;">Selleck</td>
			<td style="width:353px;">S1011</td>
			<td style="width:359px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:221px;">anti-EGFR monoclonal antibody</td>
			<td style="width:187px;">cell signaling technology</td>
			<td>4267S</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:221px;">bicinchoninc acid assay</td>
			<td style="width:187px;">sigma</td>
			<td>B9643</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:221px;">cell-culture treated 10cm dish</td>
			<td style="width:187px;">Violamo</td>
			<td style="width:353px;">2-8590-03</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:221px;">CELL BANKER1&#160;</td>
			<td style="width:187px;">TakaRa</td>
			<td style="width:353px;">CB011</td>
			<td>cryopreservation media</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:221px;">CellTiter 96</td>
			<td style="width:187px;">Promega</td>
			<td style="width:353px;">&#160;G4100</td>
			<td style="width:359px;">&#160;Non-Radioactive Cell Proliferation Assay; Dye solution and Solubilization/Stop solution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:221px;">DMSO</td>
			<td style="width:187px;">Wako</td>
			<td style="width:353px;">043-07216</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:221px;">ECL solution</td>
			<td style="width:187px;">Perkin Elmer</td>
			<td>NEL105001EA</td>
			<td style="width:359px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:221px;">FBS</td>
			<td style="width:187px;">gibco</td>
			<td style="width:353px;">26140-079</td>
			<td style="width:359px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:221px;">GeneAmp 5700</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td>fluorescence-based RT-PCR-detection system&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">GraphPad Prism v.7 software&#160;</td>
			<td>GraphPad, Inc.</td>
			<td></td>
			<td>a statistical software</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">NanoDrop Lite spectrophotometer</td>
			<td>Thermo</td>
			<td></td>
			<td>spectrophotometer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:221px;">Nonfat dry milk</td>
			<td style="width:187px;">cell signaling technology</td>
			<td>9999S</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:221px;">Pen Strep</td>
			<td style="width:187px;">gibco</td>
			<td style="width:353px;">15140-163</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:221px;">phosphatase inhibitor cocktail 2</td>
			<td style="width:187px;">sigma</td>
			<td>P5726</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:221px;">phosphatase inhibitor cocktail 3</td>
			<td style="width:187px;">sigma</td>
			<td>P0044</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:221px;">Powerscan HT microplate reader</td>
			<td>BioTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:221px;">&#160;Power SYBR Green master mix&#160;</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td>SYBR Green master mix</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:221px;">protease inhibitor cocktail</td>
			<td style="width:187px;">sigma</td>
			<td>P8340</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:221px;">QIAamp DNA Mini kit</td>
			<td style="width:187px;">Qiagen</td>
			<td>51306</td>
			<td>DNA purification kit</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:221px;">QIAquick PCR Purification Kit</td>
			<td>QIAGEN</td>
			<td></td>
			<td>PCR purification kit</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:221px;">RPMI-1640&#160;</td>
			<td style="width:187px;">Wako</td>
			<td style="width:353px;">189-02025</td>
			<td>with L-Glutamine and Phenol Red</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:221px;">TBST powder</td>
			<td style="width:187px;">sigma</td>
			<td>T9039</td>
			<td></td>
		</tr>
		<tr height="56">
			<td height="56" style="height:56px;width:221px;">Trans-Blot SD Semi-Dry Electrophoretic Transfer cell</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>semi-dry t4ransfer apparatus</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:221px;">96 well microplate</td>
			<td style="width:187px;">Thermo</td>
			<td>130188</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment and characterization of three afatinib-resistant lung adenocarcinoma pc-9 cell lines developed with increasing doses of afatinib

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.

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 ...

Watch this Scientific Journal Video about Establishment and Characterization of Three Afatinib-resistant Lung Adenocarcinoma PC-9 Cell Lines Developed with Increasing Doses of Afatinib at JoVE.com

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

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 ...

The study of novel acquired resistance mechanisms will contribute to the development of more effective and safe therapeutic strategies in patients with cancer agent resistant and resectable cancer. Unfortunately, cases in which drug resistance failed to develop are generally not reported. This stepwise dose escalation method is considered the most reliable technique for obtaining acquired-resistance cells.To determine the appropriate afatinib-resistance concentration, culture PC-9 cells in growth medium in a 10-centimeter cell culture-treated dish at 37 degrees Celsius and 5%carbon dioxide. Resuspend the PC-9 cells at a four-times-10-to-the-four cells per milliliter of growth medium concentration, and seat the cells at 50 microliters of cell suspension per well in a 96-well microplate. After an overnight incubation in the cell culture incubator, treat the cells with 50 microliters of afatinib solution per well, at six replicates of the indicated concentrations, and after a 96-hour incubation in the cull culture incubator, add 15 microliters of MTT dye to each well.After four hours in the cell culture incubator, add 100 microliters of solubilization/stop solution to each well, and return the plate to the incubator overnight. The next morning, measure the optical density at 570 nanometers on a microplate reader. For continuous afatinib exposure, culture PC-9 cells in P-100 dishes containing 10 milliliters of growth medium until the cells reach subconfluency.Transfer the subconfluent cultures into three new P-100 dishes of nine milliliters of growth medium per initial culture dish, and return the cells to the cell culture incubator. The next morning, add 0.1 nanomolar, or 1/10 of the half-maximal inhibitory concentration of afatinib, to each set of three culture dishes. When the afatinib-treated cells become subconfluent, use a one-milliliter pipette to thoroughly mix the cultures, and transfer one milliliter of cells from each dish to nine milliliters of fresh growth medium in new P-100 dishes.Add 10%to 20%higher concentrations of afatinib to the new cultures, increasing the afatinib concentration by stepwise dose escalation each time the cultures reach subconfluency over a 10-to 12-month period. When an afatinib concentration of one micromolar is reached, perform the MTT assay as demonstrated to confirm that the cells have developed afatinib-resistance. To determine the growth curve for the cell lines, culture parental PC-9 and the three afatinib-resistant cell lines in growth medium in the cell culture incubator for 24 hours.Resuspend the cells from each culture at a five-times-10-to-the-third cells per milliliter of growth medium concentration, and seat 12 100-microliter replicates of each cell population per well in a 96-well microplate. Then perform the MTT assay on days zero, one, two, three, five, and seven of culture as demonstrated, and plot the results using an appropriate statistical analysis software program. To identify alterations in epidermal growth factor receptor, or EGFR genomic DNA expression, by reverse transcriptase polymerase chain analysis, isolate genomic DNA from the cell culture of interest using a DNA purification kit according to the manufacturer's instructions.Measure the concentration of 25-nanogram-microliter concentrations of the isolated genomic DNA on a spectrophotometer. Then amplify 50 nanograms of genomic DNA using a SYBR green master mix, and analyze the results using a fluorescence-based reverse transcriptase polymerase chain reaction detection system. To identify alterations in EGFR genomic DNA expression by sequencing, amplify the genomic DNA using specific primers for EGFR exons 19 to 21.Then purify the amplified PCR products using a PCR purification kit according to the manufacturer's instructions, and sequence the amplicons. To identify alterations in epidermal growth factor receptor protein expression by western blot analysis, treat the cells with afatinib for 24 hours. After 24 hours, wash the cell cultures two times with five milliliters of ice-cold PBS per culture per wash.Lyse the cells in radioimmunoprecipitation assay buffer, supplemented with 1%protease inhibitor cocktail and phosphatase inhibitors cocktail two and three for 30 minutes at four degrees Celsius. At the end of the incubation, collect the lysates by centrifugation. Use the bicinchoninic acid assay to determine the protein concentration of the lysate samples.Adjust the protein samples to 0.5, or one microgram per microliter concentrations, in 4-X sample buffer, and boil the samples at 96 degrees Celsius for five minutes. For western blot analysis of the samples, assemble ethanol-cleaned glass plates and spacers, and load the mold with an 8%polyacrylamide gel with the appropriate experimental supplements. While the gel is polymerizing, add stacking gel solution to an appropriate mold, insert the comb, and allow the stacking gel to polymerize for 20 to 30 minutes at room temperature.When both gels have polymerized, place them in the electrophoresis apparatus and fill the tank with running buffer. Then load one 20-to 30-microliter volume of each protein sample into each well, and run the gel at 180 volts. Stop the electrophoresis after approximately 60 minutes, once the dye front flows out of the gel, and wash the gel with tris-buffered saline, supplemented with Tween, for one to two minutes.Transfer the proteins onto a polyvinylidene fluoride membrane by semi-dry blotting for one and 1/2 hours at a constant current of 300 milliamps. Block the membrane with 5%nonfat dry milk diluted with tris-buffered saline plus Tween solution for one hour at room temperature. Next, probe the membrane with the appropriate antibodies of interest at four degrees Celsius overnight.The next morning, wash the membranes with three 10-minute washes of fresh tris-buffered saline per wash before exposing the membrane to the appropriate secondary antibody for one to one and 1/2 hours at room temperature. Then wash the membrane five times with fresh tris-buffered saline per wash and expose the membrane to enhanced chemiluminescence solution for signal visualization using films. Here, the decrease observed in the cell proliferation of parental PC-9 cells in response to increasing concentrations of afatinib is illustrated, confirming that PC-9 cells are sensitive to afatinib exposure.However, none of the three afatinib-resistant cell lines demonstrate a suppression of cell proliferation under afatinib exposure. Afatinib-resistant cell lines exhibit significantly slower growth curves than parental PC-9 cells. Afatinib-resistant cells express significantly higher levels of EGFR genomic DNA and EGFR protein expression than do parental PC-9 cells.EGFR sequencing demonstrates that PC-9 cells exhibit 15 base pair deletions in EGFR exon 19, and wild type EGFR in exon 20. Afatinib-resistant one and two cell line cells, however, exhibit an amplification of wild type EGFR exon 19. Afatinib-resistant cell line three cells contain the same 15 base pair deletions in EGFR exon 19 as in PC-9 cells, but a point mutation in T790M is observed in EGFR exon 20.Cell growth can become quite slow when the inhibitor concentration is close to IC-50 value. Do not discuss culture, as the cell will continue to proliferate gradually. These strategies were developed to mimic the conditions under which cancer patients develop clinically-relevant resistance to assess acquired resistance mechanisms and to develop safe and effective therapeutic strategies.

Research

JoVE Journal

Cancer Research

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

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

Drug resistance is a major challenge in cancer therapy. The generation of resistant sublines in vitro is necessary for discovering novel mechanisms to ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

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Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

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Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Radiosensitivity of Cancer Stem Cells in Lung Cancer Cell Lines

The presence of cancer stem cells (CSCs) has been associated with relapse or poor outcomes after radiotherapy. Studying radioresistant CSCs may provide ...

Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids

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Isolation and Characterization of Patient-derived Pancreatic Ductal Adenocarcinoma Organoid Models

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the ...

Analysis of Side Population in Solid Tumor Cell Lines

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great ...

Subculture and Cryopreservation of Esophageal Adenocarcinoma Organoids: Pros and Cons for Single Cell Digestion

The lack of suitable translational research models reflecting primary disease to explore tumorigenesis and therapeutic strategies is a major obstacle in ...