Name: establishing dual resistance to egfr-tki and met-tki in lung adenocarcinoma cells in vitro with a 2-step dose-escalation procedure
Uploaded: 2017-08-11T14:00:00
Description: Watch this Scientific Journal Video about Establishing Dual Resistance to EGFR-TKI and MET-TKI in Lung Adenocarcinoma Cells In Vitro with a 2-step Dose-escalation Procedure at JoVE.com

We thank the members of the Institute of Molecular Oncology for their thoughtful comments and helpful advice, and Editage for their assistance with English language editing. This work was supported by the MEXT-supported Program for the Strategic Research Foundation at Private Universities (2012-2016) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Tohru Ohmori received research funding (2015-2016) from Boehringer-Ingelheim (Ingelheim, Germany).

1. Establish Gefitinib-resistant Cells
<ol>
	<li>Determine the initial gefitinib concentration by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay.
	<ol>
		<li>Culture PC-9 cells in growth medium (RPMI-1640 medium with 10% FBS and 1% antibiotics (penicillin: 100 U/mL and streptomycin: 100 &#181;g/mL)), in a 5% CO2 incubator at 37 &#176;C.</li>
		<li>Adjust the cells to 1.0 &#215; 105 cells/mL using an automated cell counter (see Table of materials<...

<ol>
	<li>Lynch, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activating+mutations+in+the+epidermal+growth+factor+receptor+underlying+responsiveness+of+non-small-cell+lung+cancer+to+gefitinib.">Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib.</a> N Engl J Med. 350 (21), 2129-2139 (2004).</li><li>Paez, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGFR+mutations+in+lung+cancer:+correlation+with+clinical+response+to+gefitinib+therapy.">EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy.</a> Science. 304 (5676), 1497-1500 (2004).</li><li>Pao, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGF+receptor+gene+mutations+are+common+in+lung+cancers+from+"never+smokers"+and+are+associated+with+sensitivity+of+tumors+to+gefitinib+and+erlotinib.">EGF receptor gene mutations are common in lung cancers from "never smokers" and are associated with sensitivity of tumors to gefitinib and erlotinib.</a> Proc Natl Acad Sci U S A. 101 (36), 13306-13311 (2004).</li><li>Mitsudomi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+of+the+epidermal+growth+factor+receptor+gene+predict+prolonged+survival+after+gefitinib+treatment+in+patients+with+non-small-cell+lung+cancer+with+postoperative+recurrence.">Mutations of the epidermal growth factor receptor gene predict prolonged survival after gefitinib treatment in patients with non-small-cell lung cancer with postoperative recurrence.</a> J Clin Oncol. 23 (11), 2513-2520 (2005).</li><li>Sequist, L. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotypic+and+histological+evolution+of+lung+cancers+acquiring+resistance+to+EGFR+inhibitors.">Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors.</a> Sci Transl Med. 3 (75), 75ra26 (2011).</li><li>Engelman, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MET+amplification+leads+to+gefitinib+resistance+in+lung+cancer+by+activating+ERBB3+signaling.">MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling.</a> Science. 316 (5827), 1039-1043 (2007).</li><li>Schmidt, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+mutations+of+the+MET+proto-oncogene+in+papillary+renal+carcinomas.">Novel mutations of the MET proto-oncogene in papillary renal carcinomas.</a> Oncogene. 18 (14), 2343-2350 (1999).</li><li>Olivero, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+mutation+in+the+ATP-binding+site+of+the+MET+oncogene+tyrosine+kinase+in+a+HPRCC+family.">Novel mutation in the ATP-binding site of the MET oncogene tyrosine kinase in a HPRCC family.</a> Int J Cancer. 82 (5), 640-643 (1999).</li><li>Janne, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AZD9291+in+EGFR+inhibitor-resistant+non-small-cell+lung+cancer.">AZD9291 in EGFR inhibitor-resistant non-small-cell lung cancer.</a> N Engl J Med. 372 (18), 1689-1699 (2015).</li><li>Bean, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MET+amplification+occurs+with+or+without+T790M+mutations+in+EGFR+mutant+lung+tumors+with+acquired+resistance+to+gefitinib+or+erlotinib.">MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib.</a> Proc Natl Acad Sci U S A. 104 (52), 20932-20937 (2007).</li><li>. NCT02468661 - ClinicalTrials.gov Available from: <a target="_blank" href="https://clinicaltrials.gov/ct2/show/NCT02468661">https://clinicaltrials.gov/ct2/show/NCT02468661</a> (2017)</li><li>Yamaoka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquired+Resistance+Mechanisms+to+Combination+Met-TKI/EGFR-TKI+Exposure+in+Met-Amplified+EGFR-TKI-Resistant+Lung+Adenocarcinoma+Harboring+an+Activating+EGFR+Mutation.">Acquired Resistance Mechanisms to Combination Met-TKI/EGFR-TKI Exposure in Met-Amplified EGFR-TKI-Resistant Lung Adenocarcinoma Harboring an Activating EGFR Mutation.</a> Mol Cancer Ther. 15 (12), 3040-3054 (2016).</li><li>Mosmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+colorimetric+assay+for+cellular+growth+and+survival:+application+to+proliferation+and+cytotoxicity+assays.">Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.</a> J Immunol Methods. 65 (1-2), 55-63 (1983).</li><li>Cifone, M. A., Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+patterns+of+anchorage-independent+growth+with+in+vivo+behavior+of+cells+from+a+murine+fibrosarcoma.">Correlation of patterns of anchorage-independent growth with in vivo behavior of cells from a murine fibrosarcoma.</a> Proc Natl Acad Sci U S A. 77 (2), 1039-1043 (1980).</li><li>Ercan, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplification+of+EGFR+T790M+causes+resistance+to+an+irreversible+EGFR+inhibitor.">Amplification of EGFR T790M causes resistance to an irreversible EGFR inhibitor.</a> Oncogene. 29 (16), 2346-2356 (2010).</li><li>Ogino, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+epidermal+growth+factor+receptor+T790M+mutation+during+chronic+exposure+to+gefitinib+in+a+non+small+cell+lung+cancer+cell+line.">Emergence of epidermal growth factor receptor T790M mutation during chronic exposure to gefitinib in a non small cell lung cancer cell line.</a> Cancer Res. 67 (16), 7807-7814 (2007).</li><li>Ando, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+sensitivity+to+tumor+necrosis+factor+alpha+in+non-small+cell+lung+cancer+cells+with+acquired+resistance+to+gefitinib.">Enhancement of sensitivity to tumor necrosis factor alpha in non-small cell lung cancer cells with acquired resistance to gefitinib.</a> Clin Cancer Res. 11 (24 Pt 1), 8872-8879 (2005).</li><li>Suda, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+and+complementary+role+of+MET+amplification+and+EGFR+T790M+mutation+in+acquired+resistance+to+kinase+inhibitors+in+lung+cancer.">Reciprocal and complementary role of MET amplification and EGFR T790M mutation in acquired resistance to kinase inhibitors in lung cancer.</a> Clin Cancer Res. 16 (22), 5489-5498 (2010).</li><li>Shien, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquired+resistance+to+EGFR+inhibitors+is+associated+with+a+manifestation+of+stem+cell-like+properties+in+cancer+cells.">Acquired resistance to EGFR inhibitors is associated with a manifestation of stem cell-like properties in cancer cells.</a> Cancer Res. 73 (10), 3051-3061 (2013).</li><li>Sagawa, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+three+cisplatin-resistant+endometrial+cancer+cell+lines+using+two+methods+of+cisplatin+exposure.">Establishment of three cisplatin-resistant endometrial cancer cell lines using two methods of cisplatin exposure.</a> Tumour Biol. 32 (2), 399-408 (2011).</li><li>Ritter, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+breast+cancer+cells+selected+for+resistance+to+trastuzumab+in+vivo+overexpress+epidermal+growth+factor+receptor+and+ErbB+ligands+and+remain+dependent+on+the+ErbB+receptor+network.">Human breast cancer cells selected for resistance to trastuzumab in vivo overexpress epidermal growth factor receptor and ErbB ligands and remain dependent on the ErbB receptor network.</a> Clin Cancer Res. 13 (16), 4909-4919 (2007).</li></ol>

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 mutations15,16. However, we did not detect T790M mutations in exon 20 of our PC-9MET1000 cells by direct sequencing (Figure 4C). Moreover, MET amplif...

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 disease1,2. 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 domain3,4. 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 transition5,6. 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 therapies7,8.
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-TKIs6,10. 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.

<table width="970">
	<tbody>
		<tr height="21">
			<td height="21" style="width:365px;height:21px;">RPMI-1640</td>
			<td style="width:177px;">Wako</td>
			<td style="width:119px;">189-02025</td>
			<td style="width:309px;">with L-Glutamine and Phenol Red</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTiter 96</td>
			<td style="width:177px;">Promega</td>
			<td style="width:119px;">G4100</td>
			<td>Non-Radioactive Cell Proliferation Assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:365px;height:21px;">FBS</td>
			<td style="width:177px;">gibco</td>
			<td style="width:119px;">26140-079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:365px;height:21px;">Pen Strep</td>
			<td style="width:177px;">gibco</td>
			<td style="width:119px;">15140-163</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:365px;height:21px;">gefitinib</td>
			<td style="width:177px;">Selleck</td>
			<td style="width:119px;">S1025</td>
			<td>stocked 10 mM/EMSO at -20&#186;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:365px;height:21px;">PHA665752</td>
			<td style="width:177px;">Selleck</td>
			<td style="width:119px;">S1070</td>
			<td>stocked 10 mM/EMSO at -20&#186;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:365px;height:21px;">96-well plate</td>
			<td style="width:177px;">IWAKI</td>
			<td style="width:119px;">860-096</td>
			<td>flat bottom with lid, low evaporation type</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:365px;height:21px;">TC10 Automated Cell Counter</td>
			<td style="width:177px;">BIO RAD</td>
			<td>#1450010</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="width:365px;height:26px;">CellTiter 96 Non-Radioactive Cell Proliferation Assay</td>
			<td style="width:177px;">Promega</td>
			<td style="width:119px;">G4100</td>
			<td style="width:309px;">Dye Solution and Solubilization/Stop Solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Powerscan HT microplate reader</td>
			<td>BioTek</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="width:365px;height:26px;">GraphPad Prism v.5.0 software</td>
			<td style="width:177px;">GraphPad Inc.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="width:365px;height:26px;">PC-9</td>
			<td style="width:177px;">Riken BioResource Center</td>
			<td style="width:119px;">RCB4455</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:365px;height:21px;">P100 dish</td>
			<td style="width:177px;">FALCON</td>
			<td align="right" style="width:119px;">353003</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

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...

Watch this Scientific Journal Video about Establishing Dual Resistance to EGFR-TKI and MET-TKI in Lung Adenocarcinoma Cells In Vitro with a 2-step Dose-escalation Procedure at JoVE.com

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.

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 overall goal of this experiment is to discover novel mechanisms to overcome drug resistance. This method can help answer key questions in the cancer research field such as those about graft resistance. The main advantage of this technique is that the cells resistant to true inhibitors can be established.To begin, plate PC-9 cells in growth medium and culture the cells at 37 degrees Celsius in a 5%CO2 incubator. Use an automated cell counter to count the cells and adjust the concentration to 1.0 times 10 to the fifth cells per milliliter in growth medium. Then, seed 50 microliters of the cells into each well of a 96-well plate.Incubate the plate overnight at 37 degrees Celsius. The following day, add 50 microliters of Gefitinib at different concentrations so that the final volume in each well is 100 microliters. Incubate the plate at 37 degrees Celsius for 72 hours.Next, add 15 microliters of dye solution to each well and incubate the plate at 37 degrees Celsius for four hours. Add 100 microliters of the solubilization stop solution to each well for solubilizing the formazan produced by the dye solution and incubate the plate at 37 degrees Celsius overnight. Using a microplate reader, measure the optical density at 570 nanometers and use statistical software to generate a cell proliferation curve to obtain the IC50 value.Plate one milliliter of PC-9 cells in P100 dishes with 10 milliliters of growth medium and incubate the cells in a 5%CO2 incubator at 37 degrees Celsius. Add 1/10 the IC50 value of Gefitinib into the P100 dish. When the cells in the culture medium become subconfluent, use a one milliliter pipette to add one to two milliliters of cells to 10 milliliters of fresh growth medium with a 10 to 30%higher concentration of Gefitinib and culture the cells under the same conditions as just demonstrated.Increase the Gefitinib concentration progressively by 10 to 30%with each split until it reaches one micromolar. This may take about six to 12 months. After the cells have been cultured with one micromolar of Gefitinib, perform the MTT assay to confirm that the cells are Gefitinib resistant by the increased protein and RNA levels of MET.To determine the initial concentration of PHA-665752 using the MTT assay, plate the MET1000 cells in P100 dishes with 10 milliliters of growth medium and one micromolar Gefitinib and incubate the cultures in a 5%CO2 incubator at 37 degrees Celsius. After adjusting the final concentration of the cells to 1.0 times 10 to the fifth cells per milliliter, seed the cells in 50 microliters of the growth medium in a 96-well plate at 5.0 times 10 to the third cells per well. Incubate the plate overnight.After the incubation period, add 50 microliters of PHA-665752 at different concentrations in the presence or absence of two micromolar Gefitinib so that the final volume in each well is 100 microliters. Incubate the plate at 37 degrees Celsius for 72 hours. Next, add 15 microliters of the dye solution to each well and incubate the plate at 37 degrees Celsius for four hours.Add 100 microliters of the solubilization stop solution to each well for solubilizing the formazan produced by the dye and again incubate the plate at 37 degrees Celsius overnight. Measure the optical density of the cells at 570 nanometers and use statistical software to obtain the IC50 value of PHA-665752 in the presence of Gefitinib by generating a cell proliferation curve. To establish dual resistance in MET1000 cells to Gefitinib and PHA-665752, culture the MET1000 cells in P100 dishes with 10 milliliters of growth medium and one micromolar Gefitinib.Add 1/10 of the IC50 value of PHA-665752 into the growth medium in the presence of one micromolar of Gefitinib and culture the cells under the same conditions. Increase the PHA-665752 concentration progressively by 10 to 30%with each split until it reaches one micromolar. This might take about six to 12 months.Finally, perform the MTT assay to confirm that the cells are resistant to Gefitinib and PHA-665752 and that they are not sensitive to PHA-665752 in the presence of Gefitinib. These dual resistant cells are referred to as PC-9DR. This graph illustrates the results of an MTT assay where a decrease was seen in the proliferation of parental PC-9 cells as the concentration of Gefitinib increased, indicating that the PC-9 cells are sensitive to Gefitinib after the dose escalation procedure was performed.As seen here, PC-9MET1000 cells exhibit resistance to Gefitinib even at high concentrations, but are sensitive to a MET-TKI PHA-665752 in the presence of Gefitinib. PC-9 cell proliferation was suppressed at higher concentrations of Gefitinib, indicating that they were sensitive to Gefitinib. Amplified levels of MET protein and RNA in PC-9MET1000 and PC-9DR cells are demonstrated here and no acquired mutations are present in EGFR and MET.Therefore, the emergence of bypass signaling is highly suggested as the cause of the acquired resistance to EGFR-TKI and/or MET-TKI. In this experiment, the MTT assay was used to show resistance of PC-9DR cells to PHA-665752 in the presence of one micromolar Gefitinib. Although proliferation of PC-9MET1000 cells was inhibited by treatment with Gefitinib and PHA-665752, PC-9DR cells survived the treatment, indicating that they had acquired dual resistance to Gefitinib and PHA-665752.Once mastered, each procedure can be performed in 30 to 60 minutes. While attempting this procedure, it is important to remember to seed equal volumes of the viable cells into each well of the 96 plate for the MTT assay. After its development, this technique paved the way for the researchers in the field of cancer research to explore novel resistant mechanisms to cancer agent in all kinds of cancers.After watching this video, you should have a good understanding of how to obtain resistant cell rise to cancer agent in cancer therapy through the procedure. Don't forget that working with the solubilization stop solution in the MTT assay can be extremely hazardous and precautions such as wearing gloves should always be taken while performing this procedure.

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Cancer Research

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell&#39;s arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human central nervous system (CNS). However, both the cytological origin and the evolutionary process of HBs (including neovascularization) remain controversial, and anti-angiogenesis for VHL-HBs, based on classic HB angiogenesis, have produced disappointing results in clinical trials. One major obstacle to the successful clinical translation of anti-vascular treatment is the lack of a thorough understanding of neovascularization in this vascular tumor. In this article, we present a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in HBs, as well as its role in HBs. With this procedure, researchers can accurately understand the complexity of HB neovascularization and identify the function of this common form of angiogenesis in HBs. These protocols can be used to evaluate the most promising anti-vascular therapy for tumors, which has high translational potential either for tumors treatment or for aiding in the optimization of the anti-angiogenic treatment for HBs in future translations. The results highlight the complexity of HB neovascularization and suggest that this common form angiogenesis is only a complementary mechanism in HB neovascularization.

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

This paper presents a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in hemangioblastomas (HBs) and its role in HBs. The results highlight the complexity of HB-neovascularization and suggest that this common form of angiogenesis is only a complementary mechanism in the HB-neovascularization.

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human ...

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 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.

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 ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. The leading cause of breast cancer related deaths is the metastatic burden. Therefore, preclinical models for breast cancer need to analyze metastatic burden to be clinically relevant. The 4T1 breast cancer model provides a spontaneously-metastasizing, quantifiable mouse model for stage IV human breast cancer. However, most 4T1 protocols quantify the metastatic burden by manually counting stained colonies on tissue culture plates. While this is sufficient for tissues with lower metastatic burden, human error in manual counting causes inconsistent and variable results when plates are confluent and difficult to count. This method offers a computer-based solution to human counting error. Here, we evaluate the protocol using the lung, a highly metastatic tissue in the 4T1 model. Images of methylene blue-stained plates are acquired and uploaded for analysis in Fiji-ImageJ. Fiji-ImageJ then determines the percentage of the selected area of the image that is blue, representing the percentage of the plate with metastatic burden. This computer-based approach offers more consistent and expeditious results than manual counting or histopathological evaluation for highly metastatic tissues. The consistency of Fiji-ImageJ results depends on the quality of the image. Slight variations in results between images can occur, thus it is recommended that multiple images are taken and results averaged. Despite its minimal limitations, this method is an improvement to quantifying metastatic burden in the lung by offering consistent and rapid results.

We describe a more consistent and expeditious method to quantify lung metastasis in the 4T1 breast cancer model by using Fiji-ImageJ.

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. ...