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 x 104 cells/mL in growth medium and then seed at 50 &#181;L/well in a 96-well microplate. The final concentration of cells is 2.0 x 103 cells/50 &#181;L/well. Incubate overnight in a 5% CO2 incubator at 37&#176;C.</li>
		<li>Add 50 &#181;L of afatinib solution at different concentrations: 0, 0.002, 0.006, 0.02, 0.06, 0.2, 0.6, 2, 6, and 20 &#181;M to the wells containing the growth medium (50 &#181;L). The final volume and concentrations of afatinib are 100 &#181;L and 0, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 &#181;M, respectively.</li>
		<li>Incubate the 96-well plate for 96 h in a 5% CO2 incubator at 37 &#176;C.</li>
		<li>Add 15 &#181;L of the dye solution (see Table of Materials) to each well and incubate for 4 h in a 5% CO2 incubator at 37 &#176;C, and then add 100 &#181;L of solubilization/stop-solution (see Table of Materials) to each well and incubate overnight in a 5% CO2 incubator at 37 &#176;C.</li>
		<li>Measure the optical density at 570 nm (OD570) using a microplate reader (see Table of Materials). Prepare 6-12 replicates and repeat the experiments at least three times.</li>
		<li>Use statistical software (see Table of Materials) to graphically plot these data as a semi-log graph and calculate the IC50 value, which is the drug concentration that reduces response to 50% of its maximum (see Table of Materials).</li>
	</ol>
	</li>
	<li>Continuous exposure of PC-9 cells to the irreversible EGFR-TKI, afatinib, by stepwise dose escalation in three independent 10 cm dishes
	<ol>
		<li>Culture PC-9 cells in p100 dishes containing 10 mL of growth medium. When the PC-9 cells reach the sub-confluent stage, transfer 1 mL of cell suspension into three new p100 dishes, with 9 mL of growth medium. The 1:10 diluted PC-9 cells become sub-confluent in 3-4 days, with a cell number of approximately 4-5 x 105 cells/mL.</li>
		<li>On the next day, add 1/10 of the IC50 value of afatinib into each of the three p100 dishes. 
		NOTE: Afatinib is reconstituted in DMSO at stock concentrations of 1 &#956;M, 10 &#181;M, 100 &#181;M, 1 mM, and 5 mM. 1 to 10 &#181;L of afatinib-solution is added into 10 mL of growth medium in the culture, as per the required final concentrations.</li>
		<li>When the cells in the afatinib-containing p100 dishes become sub-confluent, mix well by aspiration with a 1 mL pipette and add 1 mL of the cell suspension to 9 mL of fresh growth medium in a new p100 dish. Next, add 10-20% higher concentrations of afatinib to the new culture.</li>
		<li>Increase the afatinib concentration of 0.1 nM to 1 &#956;M in the medium by the stepwise dose escalation with the afatinib concentration increased by 10-20% at each step over the period of 10-12 months. 
		NOTE: When the afatinib concentration approaches the IC50 value, cell growth becomes quite slow. If the cells are split 1:9, they may not grow, as these cells are killed by higher concentrations of afatinib. Therefore, at higher afanitib concentrations, the cells can be split at a ratio of 1:2. The most resistant cells were grown in afatinib-contained medium for 3-14 days, and the medium was not changed until the resistant cells needed to be passaged.</li>
		<li>Culture the afatinib-resistant cells for 2-3 months in 1 &#181;M afatinib-containing growth medium. At an afatinib concentration of 1 &#181;M, 10-12 months are required for developing resistance to afatinib in this model. Perform the MTT assay to confirm that the cells are resistant to afatinib. The three independently established afatinib resistance cell lines were named AFR1, AFR2, and AFR3.</li>
	</ol>
	</li>
</ol>
2. Characterization of Three Independent Afatinib-resistant Cells
<ol>
	<li>Determination of the growth curve of parental PC-9 cells and establishment of afatinib-resistant cells
	<ol>
		<li>Culture the PC-9, AFR1, AFR2, and AFR3 cells in growth medium in a 5% CO2 incubator at 37 &#176;C.</li>
		<li>Resuspend the cells at 5 x 103 cells/mL with growth medium, and seed 100 &#181;L/well into a 96-well microplate, such that the final concentration of cells is 500 cells/100 &#181;L/well. 
		NOTE: The MTT assay is performed to measure the OD570 values at 0, 1, 2, 3, 5, and 7 days. Six 96-well microplates are required for each day.</li>
		<li>Perform the MTT assay every 24 h and then on days 0, 1, 2, 3, 5, and 7. Measure the OD570 values and prepare 6-12 replicates; repeat the experiments at least three times, and graphically plot the results using a statistical software (see Table of Materials).</li>
	</ol>
	</li>
	<li>Identification of the genomic DNA alterations in EGFR by real-time PCR 
	NOTE: Afatinib is a small molecule inhibitor that targets EGFR tyrosine kinase. The EGFR expression status is determined at the DNA and protein levels.
	<ol>
		<li>Genomic DNA is isolated using a DNA purification kit (see Table of Materials) following the manufacturer&#39;s instructions. Measure the concentration of the isolated genomic DNA with a spectrophotometer (see Table of Materials) and adjust all genomic DNA samples to 25 ng/ &#181;L.</li>
		<li>Amplify 50 ng of genomic DNA, which is equivalent to 2 &#181;L of the 25 ng/&#181;L stocks, using a SYBR Green master mix (see Table of Materials) and analyze the results using a fluorescence-based RT-PCR-detection system (see Table of Materials). 
		NOTE: PCR cycling conditions began with an initial denaturation step at 95 &#176;C for 20 s, followed by 40 cycles of 95 &#176;C denaturation for 3 s, 60 &#176;C annealing for 30 s. The specific primer sets are as follows: EGFR F: 5&#8242;-CAAGGCCATGGAATCTGTCA-3&#8242;, R: 5&#8242;-CTGGAATGAGGTGGAGGAACA-3&#8242;. Normalization gene LINE-1 F: 5&#8242;-AAAGCCGCTCAACTACATGG-3&#8242;, R: 5&#8242;-TGCTTTGAATGCGTCCCAGAG-3&#8242;.</li>
	</ol>
	</li>
	<li>Evaluation of the effect of protein alterations on the EGFR level by western blot analysis
	<ol>
		<li>Treat the cells with afatinib continuously prior to experiments for 24 h. Wash PC-9, AFR1, AFR2, and AFR3 cells twice with PBS and then seed them in growth media without afatinib. Wash PC-9, AFR1, AFR2, and AFR3 cells twice with 5 mL of ice-cold PBS.</li>
		<li>Lyse the cells in RIPA buffer containing 1% protease inhibitor cocktail (see Table of Materials) and phosphatase inhibitor cocktail II and III (see Table of Materials) and incubate this solution at 4 &#176;C for 30 min. Centrifuge the lysates for 10 min at 100 x g and 4 &#176;C and collect the cleared lysates.</li>
		<li>Determine protein concentrations using the bicinchoninic acid assay (see Table of Materials), adjust all protein samples to 0.5 or 1 &#181;g/&#181;L using 4x sample buffer (500 mM Tris (PH 6.8), 40% glycerol, 8% SDS, 20% H2O, 0.02% bromophenol blue) and boil at 96 &#176;C for 5 min. Store these protein samples at -80 &#176;C until western blot analysis is performed.</li>
		<li>Separate equal amounts of protein samples, preferably 20-30 &#181;L, by 8% SDS-page and transfer the proteins to a polyvinylidene fluoride (PVDF) membrane. 
		NOTE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is commonly used in the lab for the separation of proteins based on their molecular weight.
		<ol>
			<li>Clean glass plates with ethanol and assemble the glass plate and spacers. Prepare 8% poly-acrylamide gels containing 1.5 M Tris-HCl, pH 8.8, 40% Bis-acrylamide, 10% SDS, 10% APS, and TEMED. Polymerize for 30 min at room temperature.</li>
			<li>Subsequently, prepare a stacking gel containing 0.5 M Tris-HCl, pH 6.8, 40% bis-acrylamide, 10% SDS, 10% APS, and TEMED. Add the stacking gel solution, insert the comb, and polymerize the gel for 20-30 min at room temperature.</li>
			<li>Place the gels in the electrophoresis apparatus and fill the tank with running buffer (0.25 M Tris, 1.92 M glycine, and 1% SDS). Load equal amount of protein samples (20-30 &#181;L) and run the gel at 180 V. Stop electrophoresis once the dye front flows out of the gel, after approximately 60 min.</li>
			<li>Wash the gel for 1-2 min with TBST and then transfer the proteins on to a PVDF membrane by semi-dry blotting (see Table of Materials) for 1.5 h at a constant current of 300 mA.</li>
		</ol>
		</li>
		<li>Block the membranes with 5% of nonfat dry milk (see Table of Materials) diluted with TBST solution (see Table of Materials) for 1 h at room temperature, and then probe the membranes with anti-EGFR, anti-phospho-EGFR (Y1068), anti-HER2, anti-HER3, anti-MET, and anti-actin antibodies (diluted 1:3,000 in TBST) (see Table of Materials) at 4 &#176;C overnight.</li>
		<li>Wash the membranes with TBST three times for 10 min, and then expose the membranes to the secondary antibody (diluted 1:200 in TBST) for 1-1.5 h at room temperature. Wash the membranes five times with TBST for 10 min at room-temperature, expose them to the ECL solution (see Table of Materials), and visualize the signals using films.</li>
	</ol>
	</li>
	<li>Analysis of EGFR mutations by sequencing
	<ol>
		<li>Amplify genomic DNA using specific primers for EGFR exons 19-21. The PCR cycling conditions begin with an initial denaturation step at 94 &#176;C for 1 min, followed by 30 cycles of denaturation at 98 &#176;C for 10 s, annealing at 55 &#176;C for 30 s, and extension at 72 &#176;C for 1 min. 
		NOTE: The specific primers for EGFR exon 19: F: 5&#8242;-GCAATATCAGCCTTAGGTGCGGCTC-3&#8242; R: 5&#8242;-CATAGAAAGTGAACATTTAGGATGTG-3&#8242;, exon 20: F: 5&#8242;-CCATGAGTACGTATTTTGAAACTC-3&#8242;, R: 5&#8242;-CATATCCCCATGGCAAACTCTTGC-3&#8242;, and exon 21: F: 5&#8242;-ATGAACATGACCCTGAATTCGG-3&#8242;, R: 5&#8242;-GCTCACCCAGAATGTCTGGAGA-3&#8242;.</li>
		<li>Purify the amplified PCR products using a PCR purification kit (see Table of Materials) and sequence the amplicons.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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.

<table border="0" cellpadding="0" cellspacing="0" style="width:1120px;" width="1121">
	<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 ...

establishment-characterization-three-afatinib-resistant-lung

Research

JoVE Journal

Cancer Research

7.8K Views.  Showa University. 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.

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

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 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.

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

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.

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

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

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, including genetically engineered models as well as transplantation models. However, genetically engineered mouse models are time-consuming and expensive, whereas some orthotopic transplantation models are difficult to reproduce. Here, a non-invasive intratracheal delivery method of lung tumor cells as an alternative orthotopic transplantation model is described. The use of mouse lung adenocarcinoma cells and syngeneic graft recipients allows studying tumorigenesis under the presence of a fully active immune system. Furthermore, genetic manipulations of tumor cells before transplantation makes this model an attractive time-saving approach to study the impact of genetic factors on tumor growth and tumor cell gene expression profiles under physiological conditions. Using this model, we show that lung adenocarcinoma cells express increased levels of the T-cell suppressor programmed death-ligand 1 (PD-L1) when grown in their natural environment as compared to cultivation in vitro.

Here we describe a minimally invasive syngeneic orthotopic transplantation model of mouse lung adenocarcinoma cells as a time- and cost-reducing model to study non-small cell lung cancer.

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

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

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. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

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 clues to overcoming radioresistance. Voltage-gated calcium channel &#945;2&#948;1 subunit isoform 5 has been reported as a marker for radioresistant CSCs in non-small cell lung cancer (NSCLC) cell lines. Using calcium channel &#945;2&#948;1 subunit as an example of a CSC marker, methods to study the radiosensitivity of CSCs in NSCLC cell lines are presented. CSCs are sorted with putative markers by flow cytometry, and the self-renewal capacity of sorted cells is evaluated by sphere formation assay. Colony formation assay, which determines how many cells lose the ability to generate descendants forming the colony after a certain dose of radiation, is then performed to assess the radiosensitivity of sorted cells. This manuscript provides initial steps for studying the radiosensitivity of CSCs, which establishes the basis for further understanding of the underlying mechanisms.

The presence of cancer stem cells have been associated with relapse or poor outcomes after radiotherapy. This manuscript describes the methods to study the 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

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited because very few patient derived SBNET cell lines have been generated. Well-differentiated SBNET cells are slow growing and are hard to propagate. The few cell lines that have been established are not readily available, and after time in culture may not continue to express characteristics of NET cells. Generating new cell lines could take many years since SBNET cells have a long doubling time and many enrichment steps are needed in order to eliminate the rapidly dividing cancer-associated fibroblasts. To overcome these limitations, we have developed a protocol to culture SBNET cells from surgically removed tumors as spheroids in extracellular matrix (ECM). The ECM forms a 3-dimensional matrix that encapsulates SBNET cells and mimics the tumor micro-environment for allowing SBNET cells to grow. Here, we characterized the growth rate of SBNET spheroids and described methods to identify SBNET markers using immunofluorescence microscopy and immunohistochemistry to confirm that the spheroids are neuroendocrine tumor cells. In addition, we used SBNET spheroids for testing the cytotoxicity of rapamycin.

Neuroendocrine tumors (NETs) originate from neuroendocrine cells of the neural crest. They are slow growing and challenging to culture. We present an alternative strategy to grow NETs from the small bowel by culturing them as spheroids. These spheroids have small bowel NET markers and can be used for drug testing.

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited ...

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 3-dimensional (3D) modeling of this disease have been described. Patient-derived organoid (PDO) models can be isolated from both surgical specimens as well as small biopsies and form rapidly in culture. Importantly, organoid models preserve the pathogenic genetic alterations detected in the patient&#39;s tumor and are predictive of the patient&#39;s treatment response, thus enabling translational studies. Here, we provide comprehensive protocols for adapting tissue culture workflow to study 3D, matrix embedded, organoid models. We detail methods and considerations for isolating and propagating primary PDAC organoids. Furthermore, we describe how bespoke organoid media is prepared and quality controlled in the laboratory. Finally, we describe assays for downstream characterization of the organoid models such as isolation of nucleic acids (DNA and RNA), and drug testing. Importantly we provide critical considerations for implementing organoid methodology in a research laboratory.

Patient-derived organoid cultures of pancreatic ductal adenocarcinoma are a rapidly established 3-dimensional model that represent epithelial tumor cell compartments with high fidelity, enabling translational research into this lethal malignancy. Here, we provide detailed methods to establish and propagate organoids as well as to perform relevant biological assays using these 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 significance for tumor research. Currently, several techniques are used for the identification and purification of CSCs from tumor tissues and tumor cell lines. Separation and analysis of side population (SP) cells are two of the commonly used methods. The methods rely on the ability of CSCs to rapidly expel fluorescent dyes, such as Hoechst 33342. The efflux of the dye is associated with the ATP-binding cassette (ABC) transporters and can be inhibited by ABC transporter inhibitors. Methods for staining cultured tumor cells with Hoechst 33342 and analyzing the proportion of their SP cells by flow cytometry are described. This assay is convenient, fast, and cost-effective. Data generated in this assay can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on the stemness properties of tumor cells.

A convenient, fast, and cost-effective method to measure the proportion of side population cells in solid tumor cell lines is presented.

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 esophageal adenocarcinoma (EAC). Patient-derived organoids (PDOs) have recently emerged as a remarkable preclinical model in a variety of cancers. However, there are still limited protocols available for developing EAC PDOs. Once the PDOs are established, the propagation and cryopreservation are essential for further downstream analyses. Here, two different methods have been standardized for EAC PDOs subculture and cryopreservation, i.e., with and without single cell digestion. Both methods can reliably obtain appropriate cell viability and are applicable for a diverse experimental setup. The current study demonstrated that subculturing EAC PDOs with single cell digestion is suitable for most experiments requiring cell number control, uniform density, and a hollow structure that facilitates size tracking. However, the single cell-based method shows slower growth in culture as well as after re-cultivation from frozen stocks. Besides, subculturing with single cell digestion is characterized by forming hollow structures with a hollow core. In contrast, processing EAC PDOs without single cell digestion is favorable for cryopreservation, expansion, and histological characterization. In this protocol, the advantages and disadvantages of subculturing and cryopreservation of EAC PDOs with and without single cell digestion are described to enable researchers to choose an appropriate method to process and investigate their organoids.

This protocol describes the methods of subculture and cryopreservation of esophageal adenocarcinoma organoids with and without single cell digestion to enable researchers to choose appropriate strategies based on their experimental design.

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