The authors wish to thank Dr. Veronica Zanoni for editing the manuscript.

NOTE: This protocol has been specifically adjusted for the analysis of the mechanisms underlying acquired resistance to trastuzumab of HER-positive gastric cancer cell lines. All laboratory instruments needed are reported in the Table of Materials.
1. Generation of Targeted Therapy Resistant Subclones
NOTE: This is the most critical and difficult to standardize step in the protocol due to the fact that different cell lines may exhibit different sensit...

<ol>
	<li>Gerber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+therapies:+a+new+generation+of+cancer+treatments.">Targeted therapies: a new generation of cancer treatments.</a> Am Fam Physician. 77 (3), 311-319 (2008).</li><li>Zhang, J., Yang, P. L., Gray, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+cancer+with+small+molecule+kinase+inhibitors.">Targeting cancer with small molecule kinase inhibitors.</a> Nat Rev Cancer. 9 (1), 28-39 (2009).</li><li>Nelson, A. L., Dhimolea, E., Reichert, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+trends+for+human+monoclonal+antibody+therapeutics.">Development trends for human monoclonal antibody therapeutics.</a> Nat Rev Drug Discov. 9 (10), 767-774 (2010).</li><li>Chabner, B. A., Roberts, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timeline:+Chemotherapy+and+the+war+on+cancer.">Timeline: Chemotherapy and the war on cancer.</a> Nat Rev Cancer. 5 (1), 65-72 (2005).</li><li>Gilbert, L. A., Hemann, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapeutic+resistance:+surviving+stressful+situations.">Chemotherapeutic resistance: surviving stressful situations.</a> Cancer Res. 71 (15), 5062-5066 (2011).</li><li>Izar, B., Rotow, J., Gainor, J., Clark, J., Chabner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetics,+clinical+indications,+and+resistance+mechanisms+in+molecular+targeted+therapies+in+cancer.">Pharmacokinetics, clinical indications, and resistance mechanisms in molecular targeted therapies in cancer.</a> Pharmacol Rev. 65, 1351-1395 (2013).</li><li>Ellis, L. M., Hicklin, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+Targeted+Therapies:+Refining+Anticancer+Therapy+in+the+Era+of+Molecular+Oncology.">Resistance to Targeted Therapies: Refining Anticancer Therapy in the Era of Molecular Oncology.</a> Clin Cancer Res. 15 (24), 7471-7478 (2009).</li><li>Asi&#263;, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dominant+mechanisms+of+primary+resistance+differ+from+dominant+mechanisms+of+secondary+resistance+to+targeted+therapies.">Dominant mechanisms of primary resistance differ from dominant mechanisms of secondary resistance to targeted therapies.</a> Crit Rev Oncol Hematol. 97, 178-196 (2016).</li><li>Arienti, C., 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=Preclinical+evidence+of+multiple+mechanisms+underlying+trastuzumab+resistance+in+gastric+cancer.">Preclinical evidence of multiple mechanisms underlying trastuzumab resistance in gastric cancer.</a> Oncotarget. 7 (14), 18424-18439 (2016).</li><li>Tanner, 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=Amplification+of+HER-2+in+gastric+carcinoma:+association+with+Topoisomerase+IIalpha+gene+amplification,+intestinal+type,+poor+prognosis+and+sensitivity+to+trastuzumab.">Amplification of HER-2 in gastric carcinoma: association with Topoisomerase IIalpha gene amplification, intestinal type, poor prognosis and sensitivity to trastuzumab.</a> Ann Oncol. 16 (2), 273-278 (2005).</li><li>Bang, Y. 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=Trastuzumab+in+combination+with+chemotherapy+versus+chemotherapy+alone+for+treatment+of+HER2-positive+advanced+gastric+or+gastro-oesophageal+junction+cancer+(ToGA):+a+phase+3,+open-label,+randomised+controlled+trial.">Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial.</a> Lancet. 376 (9742), 687-697 (2010).</li><li>Shimoyama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+trastuzumab+and+lapatinib+inefficiency+in+gastric+cancer:+Future+steps+(Review).">Unraveling trastuzumab and lapatinib inefficiency in gastric cancer: Future steps (Review).</a> Mol Clin Oncol. 2 (2), 175-181 (2014).</li><li>Kelly, C. M., Janjigian, Y. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genomics+and+therapeutics+of+HER2-positive+gastric+cancer-from+trastuzumab+and+beyond.">The genomics and therapeutics of HER2-positive gastric cancer-from trastuzumab and beyond.</a> J Gastrointest Oncol. 7 (5), 750-762 (2016).</li><li>Wong, S. S., 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=Genomic+landscape+and+genetic+heterogeneity+in+gastric+adenocarcinoma+revealed+by+whole-genome+sequencing.">Genomic landscape and genetic heterogeneity in gastric adenocarcinoma revealed by whole-genome sequencing.</a> Nat Commun. 5, 5477 (2014).</li><li>Oshima, 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=Lapatinib+sensitivities+of+two+novel+trastuzumab-resistant+HER2+gene-amplified+gastric+cancer+cell+lines.">Lapatinib sensitivities of two novel trastuzumab-resistant HER2 gene-amplified gastric cancer cell lines.</a> Gastric Cancer. 17 (3), 450-462 (2014).</li><li>Pignatta, S., 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=Prolonged+exposure+to+(R)-bicalutamide+generates+a+LNCaP+subclone+with+alteration+of+mitochondrial+genome.">Prolonged exposure to (R)-bicalutamide generates a LNCaP subclone with alteration of mitochondrial genome.</a> Mol Cell Endocrinol. 382 (1), 314-324 (2014).</li></ol>

While personalized and targeted therapies have elicited growing enthusiasm, these so-called &#39;smart&#39; therapies face the same major hurdle as traditional chemotherapy drugs, i.e., the rapid and unpredictable acquisition of drug resistance. To improve the results of targeted therapy, drug-resistance problems must be solved through a better understanding of the mechanisms that cause them. Here, we presented a widely accessible in vitro methodology to investigate the mechanisms of acquired resistance...

Following up on the crucial discoveries in molecular and cellular biology, a number of novel synthesized anticancer molecules have been developed to selectively target oncogenic signaling pathways in a broad array of tumor types. In particular, two categories of targeted therapeutic agents with unique properties in oncology, namely synthetic chemical compounds and recombinant monoclonal antibodies, are known to have achieved clinical successes and dramatically altered cancer care in recent decades1,2,3.
Nevertheless, despite their impressive initial response to treatment, the majority of cancer patients have developed drug resistance to all targeted therapeutic agents, both monoclonal antibodies and kinase inhibitors. As a consequence, drug resistance, the major hurdle for classic anti-cancer medicines4, is still a great challenge for emerging targeted therapies5,6.
Resistance to targeted therapy may be intrinsic (i.e., primary) or acquired (i.e., secondary). Intrinsic resistance describes a de novo lack of response to therapy, whereas secondary resistance occurs after a period of response to drug treatment7. The latter is caused by outbreaks of small cohorts of tumor cells within the bulk of the primitive tumor or nestled in distal cryptic anatomical niches, exhibiting up to 90% resistance to one or more targeted therapeutic agents. Molecular mechanisms underlying a resistance development to targeted therapy mainly result from target gene mutations and redundant activation of other pro-survival signaling pathways, whose understanding is still far from complete8.
In this work, we proposed a time- and cost-effective approach to investigate in vitro mechanisms of acquired resistance to targeted therapeutic agents, starting from the generation of drug-resistant cellular subclones to the description of silencing procedures used for restoring the sensitivity to the inhibitor, a crucial tool used for testing and validating the working hypotheses. In particular, our approach was used to investigate, in gastric cancer, the resistance mechanisms to trastuzumab (e.g., Herceptin), a humanized monoclonal antibody targeting the extracellular domain of HER2 protein9. Trastuzumab + chemotherapy is widely accepted as the standard first-line treatment for HER2-positive metastatic breast cancer. Thanks to recent preclinical studies showing that anti-HER2 therapies have significant activity in both in vitro and in vivo HER2-positive gastric cancer models10,11, molecular drugs targeting HER2 have been extensively examined in clinical trials, some of which are still ongoing, on patients with gastroesophageal cancer12,13,14,15.
These studies have emphasized the rising number of patients experiencing resistance to trastuzumab16, similarly to what is observed for other targeted therapeutic inhibitors. In particular, the response rate of trastuzumab was 47%, and the median overall survival for patients on trastuzumab + chemotherapy was only 2.7 months longer than for patients on chemotherapy alone17. This suggested that while primary trastuzumab resistance is prevalent, secondary trastuzumab resistance is inevitable. For this reason, there is urgent need to clarify the mechanisms causing HER2-targeted therapy resistance in gastric cancer, which is even more problematic given the intra-tumoral genetic heterogeneity of this neoplasia18.
In addition, mechanisms of resistance to trastuzumab in gastric cancer have proven challenging to explain, partially due to the difficulty in obtaining reliable preclinical models representative of this condition. Oshima et al. reported an in vivo selection method of trastuzumab-resistant gastric cancer cell lines, consisting of a repeated culture of a small residual peritoneal metastasis after treatment with trastuzumab19. However, the need of an enclosure, of specific animal handling expertise, and of the approval of the Animal Ethics Committee contributed to making it a time- and cost-consuming method. The approach we describe in this work is simple and widely applicable, and could be easily extended to investigate the resistance mechanisms to other therapeutic drugs in different tumor histotypes20.

<table>
	<tbody>
		<tr>
			<td>Trizol Reagent</td>
			<td>Invitrogen</td>
			<td>15596026</td>
			<td>TRIzol Reagent is a reagent for isolating high-quality total RNA or simultaneously isolating RNA, DNA, and protein from a variety of biological samples</td>
		</tr>
		<tr>
			<td>ISCRIPT CDNA SYNTHESIS KIT</td>
			<td>Bio-Rad</td>
			<td>1708891</td>
			<td>The iScript cDNA synthesis kit is a sensitive and easy-to-use first-strand cDNA synthesis kit for gene expression analysis using real-time qPCR.</td>
		</tr>
		<tr>
			<td>TaqMan gene expression assay</td>
			<td>Life Technologies</td>
			<td>4331182</td>
			<td>Applied Biosystems TaqMan Gene Expression Assays consist of a pair of unlabeled PCR primers and a TaqMan probe with an Applied Biosystems FAM or VIC dye label on the 5&#8217; end and minor groove binder (MGB) and nonfluorescent quencher (NFQ) on the 3&#8217; end.</td>
		</tr>
		<tr>
			<td>M-PER Mammalian Protein Extraction Reagent</td>
			<td>Thermo Scientific</td>
			<td>78501</td>
			<td>Thermo Scientific M-PER Mammalian Protein Extraction Reagent is designed to provide highly efficient total soluble protein extraction from cultured mammalian cells.</td>
		</tr>
		<tr>
			<td>Halt Protease and Phosphatase Inhibitor Cocktail (100X)</td>
			<td>Thermo Scientific</td>
			<td>78440</td>
			<td>Thermo Scientific Halt Protease and Phosphatase Inhibitor Cocktail (100X) provides the convenience of a single solution with full protein sample protection for cell and tissue lysates.</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td>The Thermo Scientific Pierce BCA Protein Assay Kit is a two-component, high-precision, detergent-compatible assay reagent set to measure (A562nm) total protein concentration compared to a protein standard.</td>
		</tr>
		<tr>
			<td>4x Laemmli Sample Buffer</td>
			<td>Bio-Rad</td>
			<td>1610747</td>
			<td>Use 4x Laemmli Sample Buffer for preparation of samples for SDS PAGE. For reduction of samples, add a reducing agent such as 2-mercaptoethanol to the buffer prior to mixing with the sample.</td>
		</tr>
		<tr>
			<td>4&#8211;20% Mini-PROTEAN TGX Precast Protein Gels</td>
			<td>Bio-Rad</td>
			<td>456-1094</td>
			<td>Long-life TGX (Tris-Glycine eXtended) Gels have a novel formulation and can be used for both standard denaturing protein separations as well as native electrophoresis.</td>
		</tr>
		<tr>
			<td>Precision Plus Protein WesternC Blotting Standards</td>
			<td>Bio-Rad</td>
			<td>1610376</td>
			<td>Precision Plus Protein Prestained Standards are available in Dual Color, All Blue, Kaleidoscope, and Dual Xtra formats. All four have the same gel migration patterns, with 3 high-intensity reference bands (25, 50, and 75 kD).</td>
		</tr>
		<tr>
			<td>10x Tris/Glycine/SDS</td>
			<td>Bio-Rad</td>
			<td>1610732</td>
			<td>10x Tris/glycine/SDS is a premixed running buffer for separating protein samples by SDS-PAGE.</td>
		</tr>
		<tr>
			<td>Trans-Blot Turbo Mini PVDF Transfer Packs</td>
			<td>Bio-Rad</td>
			<td>1704156</td>
			<td>Trans-Blot Turbo Mini PVDF Transfer Packs for fast, efficient transfer of proteins from mini gels using the Trans-Blot Turbo Transfer System. Each vacuum sealed, ready-to-use transfer pack contains two buffer-saturated ion reservoir stacks and a prewetted PVDF membrane.</td>
		</tr>
		<tr>
			<td>Precision Protein StrepTactin-HRP Conjugate</td>
			<td>Bio-Rad</td>
			<td>1610381</td>
			<td>StrepTactin-Horseradish Peroxidase (HRP) Conjugate for chemiluminescent or colorimetric detection of Precision Plus Protein Unstained Protein Standards or Precision Plus Protein WesternC Protein Standards on western blots.</td>
		</tr>
		<tr>
			<td>Immun-Star WesternC solution</td>
			<td>Bio-Rad</td>
			<td>5572</td>
			<td>Immun-Star WesternC solution, a method of protein detection , detects mid-femtogram amounts of protein.</td>
		</tr>
		<tr>
			<td>Universal Negative Control</td>
			<td>Invitrogen</td>
			<td>12935300</td>
			<td>Negative Control siRNA has no significant sequence similarity to mouse, rat, or human gene sequences. The control has also been tested in cell-based screens and proven to have no significant effect on cell proliferation, viability, or morphology.</td>
		</tr>
		<tr>
			<td>opti-MEM Glutamax Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>51985026</td>
			<td>Opti-MEM Medium is an improved Minimal Essential Medium (MEM) that allows for a reduction of Fetal Bovine Serum supplementation by at least 50% with no change in growth rate or morphology.</td>
		</tr>
		<tr>
			<td>Silencer Select Pre-Designed &#38; Validated siRNA</td>
			<td>Ambion</td>
			<td>4390824</td>
			<td>RNA interference (RNAi) is the best way to effectively knock down gene expression to study protein function in a wide range of cell types.</td>
		</tr>
		<tr>
			<td>Lipofectamine RNAiMAX Transfection Reagent</td>
			<td>Invitrogen</td>
			<td>13778075</td>
			<td>Lipofectamine RNAiMAX Transfection Reagent offers an advanced, efficient solution for siRNA delivery. No other siRNA specific transfection reagent provides such easy and efficient siRNA delivery in a wide variety of cell lines including common cell types, stem cells and primary cells, as well as traditionally hard-to-transfect cell types.</td>
		</tr>
		<tr>
			<td>Mouse anti-IQGAP1</td>
			<td>Invitrigen</td>
			<td>33-8900</td>
			<td>working concentration: 1:400 in 5% milk solution</td>
		</tr>
		<tr>
			<td>goat anti-mouse IgG-HRP: sc-2005</td>
			<td>Santa Cruz</td>
			<td>sc-2005</td>
			<td>working concentration: 1:10000 in 5% milk solution</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma Aldrich</td>
			<td>P 7626</td>
			<td>this is an inhibitor of serine proteases and acetylcholinesterase</td>
		</tr>
		<tr>
			<td>Thermocycler for RT-PCR</td>
			<td>MJ Research</td>
			<td>MJ Research PTC-200 Thermal Cycler</td>
			<td>it allows temperature homogeneity and a ramping speed of up to 3&#176;C/sec. The protocol can be performed also with other thermalcyclers</td>
		</tr>
		<tr>
			<td>Real Time RT-PCR instrument</td>
			<td>Applied Biosystems</td>
			<td>7500 RT-PCR system</td>
			<td>This is a five-color platform that uses fluorescence-based polymerase chain reaction (PCR) reagents to provide a relative quantification using comparative CT assay type. The protocol can be performed also with other thermalcyclers</td>
		</tr>
		<tr>
			<td>Microvolume Spectrophotometers</td>
			<td>Thermo Scientific</td>
			<td>Nanodrop</td>
			<td>It provides an accurate and fast acid nucleid measurement using little volume of sample</td>
		</tr>
		<tr>
			<td>Blotting system</td>
			<td>Bio-Rad</td>
			<td>Trans-Blot Turbo system</td>
			<td>It perfoms a semy-dry transfer in a few minutes</td>
		</tr>
		<tr>
			<td>Image acquisition system and gel documentation</td>
			<td>Bio-Rad</td>
			<td>Chemidoc image system</td>
			<td>It is easy to use for chemiluminescent detection</td>
		</tr>
	</tbody>
</table>

looking for driver pathways of acquired resistance to targeted therapy: drug resistant subclone generation and sensitivity restoring by gene knock-down

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more impressive clinical efficacy and minimized adverse effects than traditional treatments, drug resistance has become the main limitation to their benefits. Several preclinical in vitro/in vivo models of acquired resistance to targeted agents in clinical practice have been developed mainly by using two strategies: i) genetic manipulation for modeling genotypes of acquired resistance, and ii) in vitro/in vivo selection of resistant models. In the present work, we propose a unifying framework, for investigating the underlying mechanisms responsible for acquired resistance to targeted therapeutic agents, starting from the generation of drug-resistant cellular subclones to the description of silencing procedures used for restoring the sensitivity to the inhibitor. This simple time- and cost-effective approach is widely applicable, and could be easily extended to investigate resistance mechanisms to other targeted therapeutic drugs in different tumor histotypes.

Figure 1 outlines the framework of the experimental procedure. Three gastric cancer cell-lines expressing a high level of HER2 and sensitive to trastuzumab (IC50 &#60; peak plasma level of drug, Figure 2A, left graph) were grown in culture medium containing the targeted therapeutic agent. A dose 10-fold lower than the peak plasma concentration of trastuzumab (10 &#181;g/mL) was set as the starting dose, and gradually i...

Watch this Scientific Journal Video about Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down at JoV

Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down

This is a time- and cost-effective in vitro protocol investigating the mechanisms of acquired resistance to targeted therapeutic agents, which is a highly unmet medical need in cancer management.

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more ...

The overall goal of this preclinical platform is to provide a standard procedure to identify and characterize the mechanisms driving acquired resistance to targeted therapies. This method can help to implement genome drive in oncology, in particular, address key questions about tumor heterogeneity and the multiple mechanisms responsible for acquired resistance to targeted therapy. The main advantage of this technique is the time and cost-effective approach which allows the in-vitro investigation of resistance mechanisms to targeted therapy.The implication of this technique extend to cancer therapy, as lung resistance is still challenging for emerging targeted therapies. This method can provide insight into the mechanism involved in targeted therapy resistance, but it can also be used to study the efficacy of conventional chemotherapeutic agents. Generally, operators who use this method will stagger with a generation of drug-resistant subclones, the step most difficult to standardize.We first conceived the idea for this method when we characterized the acquired resistant mechanism to trastuzumab in HER-positive gastric cancer cell lines. Visual demonstration of this method is critical as several techniques are needed for the generation and validation of in-vitro drug resistant models including restoring sensitivity by gene knock-down. To begin this procedure, seed NCI N87 cells into 25 square centimeter culture flasks.Add 10 micrograms per milliliter of trastuzumab to the culture medium in each flask. Expose the cells to this starting dose until they resume growing. After cell growth has resumed in approximately two weeks, expose cells to a one hundred microgram per milliliter concentration dose of trastuzumab.Continue gradually increasing the trastuzumab concentration waiting until the cells resume growth before each exposure, until a concentration of 400 micrograms per milliliter is reached. A cell line may exhibit a different sensitivity to different drugs. One solution to this problem is to expose cells to gradually increasing drug doses, starting from a dose about tenfold the hour the plasma peak concentration until the cells keep growing.Next, seed cells into 24 multi-well culture plates. Expose the seeded cells to increasing target therapy inhibitor concentrations from 100 micrograms per milliliter to 400 micrograms per milliliter. Incubate in a CO2 incubator at 37 degrees Celsius for 15 days.After this, remove the medium and rinse the cells with 10 milliliters of PBS-1X. Remove the PBS-1X and add between two and three milliliters of fixation solution. After 20 minutes, remove the fixation solution and add two milliliters of 0.5%crystal violet solution to each well.Incubate at room temperature for two hours. Then, use a pipette to carefully aspirate the crystal violet solution. Immerse the plates in tap water to rinse off any remaining residues.Air dry at room temperature for up to two days. Next, use an inverted microscope at 4X magnification to count colonies containing more than 50 cells. Calculate the relative resistance IC50 ratio as outlined in the text protocol.To begin, prepare a single-cell suspension by trypsinization as outlined in the text protocol. Using a hemocytometer and Trypan Blue staining, count the cells. Then, seed 2.5 times 10-to-the-five cells onto a T-25 flask for each condition.Next, dilute 12.5 microliters of each gene-targeting siRNA oligonucleotide or negative control siRNA oligonucleotide in Minimal Essential Transfection Medium. Add a cationic liposome transfection reagent at a 1:1 ratio with the oligonucleotides. Incubate at room temperature for 20 minutes.After this, add 700 microliters of the transfection mix to each T-25 flask. Incubate at 37 degrees Celsius for one to three days. Use RT-PCR and Western Blot analysis to verify gene knock-down.Next, seed cells into 24 multi-well culture plates. Expose the cells to increasing concentrations of target therapy inhibitor from 100 micrograms per milliliter to 400 micrograms per milliliter. For the efficacy of gene knock-down, siRNA oligonucleotides must be specific for reducing target expression.Designed tools and the measure of siRNA was the same target gene are useful for this purpose. Incubate in a CO2 incubator at 37 degrees Celsius for 15 days. Remove the medium and rinse the cells with 10 milliliters of PBS-1X.After removing the PBS, add two to three milliliters of fixation solution. Then, remove the fixation solution and add 0.5%crystal violet solution. Incubate at room temperature for two hours.Using a pipette, carefully aspirate the crystal violet solution. Next, immerse the plates in tap water. Air dry at room temperature for up to two days.After this, use an inverted microscope at 4X magnification to count the colonies containing more than 50 cells. In this study, three gastric cancer cell lines which express a high level of HER2 and are sensitive to trastuzumab are grown in a culture medium containing the targeted therapeutic agents. From this, three subclones characterized by a stable resistant phenotype are obtained.Of the subclones, HR NCI N87 is seen to be the most resistant reaching a Relative Resistance IC50 of 28.57. Analysis of protein and gene expression supports the hypothesis that the gene IQGAP1 plays a pivotal role in the resistance phenotype of the HR NCI N87 subline. As shown here, IQGAP1 protein level is fivefold tier in the resistant subclone than in the parental cells.And IQGAP1 gene expression is 2.5-fold tier in the HR NCI N87 subline than in the original subclone. IQGAP1 gene silencing is then performed to evaluate its role in the development of the trastuzumab resistance phenotype. A clonogenic assay further confirms the hypothesis of IQGAP's importance.As seen here, the gene silencing restores the cell's sensitivity to trastuzumab, lowering the IC50 value to seven micrograms per milliliter. What I think of this procedure is important to keep in mind that it is not generally easy to obtain drug-resistant cell subclones. And that this method holds a potential failure risk.Through this procedure it is possible to obtain several cells of clones characterized by different mechanisms driving acquired resistance to target therapies. The development of standard procedure for the in-vitro modeling of target therapy resistance should allow an oncology researcher to investigate novel drug-enabled target to overcome drug resistance which is greatest criticism of the latest generation therapies.

looking-for-driver-pathways-acquired-resistance-to-targeted-therapy

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Cancer Research

7.1K vistas.  Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS. The overall goal of this preclinical platform is to provide a standard procedure to identify and characterize the mechanisms driving acquired resistance to targeted therapies. This method can help to implement genome drive in oncology, in particular, address key questions about tumor heterogeneity and the multiple mechanisms responsible for acquired resistance to targeted therapy. The main advantage of this technique is the time and cost-effective approach which allows the in-vitro investigat...

Video: Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down