We are grateful for the generous financial support of the Lawrence J. Ellison Institute of Transformative Medicine of USC (a gift to David Agus). We appreciate the support of Autumn Beemer and Lisa Flashner which lead to the generation and publication of this manuscript. We thank Laura Ng for her administrative support.

1. Protein Extraction
<ol>
	<li>Plate 5 x 106 cells on a 10 mm tissue culture plate (or flask) in a tissue culture hood. Count the cells at the time of plating with an automatic cell counter or hemocytometer. Alternatively, estimate the cell count.</li>
	<li>Rinse the cells grown on the 10 mm plate (or flask) thoroughly 3x with 10 mL of phosphate-buffered saline (PBS) (pH = 7.4). Make sure to remove all PBS before adding the lysis buffer. 
	NOTE: The lysis buffer is usually provided with the kit. If not...

<ol>
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The authors have nothing to disclose.

Approaches that combine many biological readouts are inherently more accurate representations of the cellular machinery in an experiment performed. The advent of phospho-antibody arrays enables a rapid characterization of the pattern of modifications which may be more informative than the modification status of any single protein. The general workflow for the application of phospho-antibody arrays is based on a modification in serine, threonine, or tyrosine. This example focused on characterizing the changes associated w...

The clinical implementation of the targeted tyrosine kinase inhibitors (TKI) has transformed cancer treatment by providing physicians with effective tools to target the specific proteins that drive neoplastic transformation. These compounds inhibit or block the phosphorylation of proteins targeted by tyrosine kinases1,2. TKIs were developed in part because genetic alterations in various key signaling genes are sufficient to drive cancer initiation and progression [e.g.,epidermal growth factor receptor (EGFR), proto-oncogene tyrosine-protein kinase Src (SRC), BCR-ABL, and human epidermal growth factor receptor 2 (HER2)]3,4. The impact of TKIs on the cell cycle5 and the molecular signaling pathways6 represents a transformation from the untargeted to the molecularly guided cancer treatment. The key advantage of TKIs versus chemotherapy is the increased response rates and the lower risk of toxicity to healthy cells7. As a result, there has been increasing attention on the research and development of novel TKIs.
Access to the genomic sequencing results started with the Human Genome Project8,9,10 and continues today with various next-generation (NextGen) cancer sequencing efforts [e.g., The Cancer Genome Atlas (TCGA)11,12]. This has inspired many experimental methodologies that provide simultaneous information on thousands of genes and/or provide unbiased snapshots of genes or proteins modulated by biological perturbations13. Since the regulation of the cellular function occurs at multiple levels, from the transcription of genes to the post-translational modification of proteins and their activity, a complete understanding of the events controlling the cellular function will ultimately require an integration of data from various biological readouts. The ability to monitor the messenger RNA (mRNA) levels of thousands of genes with a single-cell gene resolution has increased the ability to make inferences about the gene function and interactions on a whole-genome scale. However, the interpretation of gene expression arrays will always be inherently incomplete without the integration of other levels of regulation: namely, the protein expression levels, the protein modification states, and the protein post-translational modifications (phosphorylation, ubiquitylation, methylation, etc.). Here, we describe the utility of antibody arrays as means to interrogate post-translational modifications of important signaling components as a function of various conditions in a single experiment14,15,16.
Phospho-antibody arrays can be employed to distinguish and analyze changes in the signal transduction pathways16. These can arise from a genetic modification or treatments of cell lines with kinase inhibitors, chemotherapeutics, stress caused by glucose deprivation, hypoxia, or serum starvation. Of note, drug resistance or a specific gene up- or downregulation can also cause changes in the signal transduction pathways17.
Drug resistance, for example, can arise from mutations of the drug target to avoid sensitivity. In lung cancer, known EGFR mutations render the cancer insusceptible to certain TKIs, but more susceptible to others. Alternative signaling pathways can be activated upon mutation17. As a broader application for the identification of the signal transduction pathways involved in resistance and hypoxia, etc., phospho-antibody arrays provide more insight into, and consequently understanding of, the mechanisms involved.
Technologies that permit an assessment of protein modifications represent an important component of systems biology because they often serve a regulatory function, such as modulating the activity of an enzyme or the physical interactions between proteins. The importance of post-translational modifications is illustrated by the role of protein phosphorylation in nearly all extracellular-triggered signal transduction pathways18. Traditionally, the identification of kinases or of the phosphorylation status of proteins could also be determined by Western blot analysis, especially if the researcher is interested in only 1&#8211;5 targets. However, Western blots are very selective and can be biased toward prior knowledge and might miss important targets as a result. Antibody array(s) provide a medium-throughput readout of multiple targets by embedding various capture antibodies [pan-specific phosphorylated tyrosine(s), anti-ubiquitin, etc.] on a solid matrix (e.g., glass or nitrocellulose). Secondary antibodies provide information on specific proteins in a sandwich-based ELISA format (Figure 1). This assay becomes more powerful and pertinent as more targets are of interest or the prior knowledge is restricted15. The phospho-arrays are more broadly employable as they can compare phosphorylation as well as general amounts of protein of a wider variety of targets in one experiment and provide a significantly improved quantification over mass spectrometry. This technique is not applicable for the identification of new or previously unknown phosphorylation sites.
Large-scale mass spectrometry-based proteomics could be employed to identify specific phosphorylation sites of proteins19. Although this technique can enumerate thousands of post-translational events, it requires expensive instrumentation, dedicated experimental pipelines, and a computational expertise that are beyond the reach of most researchers.
Antibody arrays provide a simultaneous readout on various protein readouts16. These may be changes in protein ubiquitylation (ubiquitin array) or phosphorylation. The key advantage of this array technology is that it provides important feedback on the biological state of various biological pathways associated with important cell parameters (protein of 53 kDa, p53, receptor tyrosine kinases, and intracellular pathways) simultaneously. In addition, it is possible to combine various array types to increase the penetration of an assay (e.g., apoptosis and ubiquitin and phosphorylation). This ability to combine multiple arrays to assess various post-translational alterations in several samples simultaneously in a time- and cost-effective manner that does not require specific instrumentation or expertise is of a significant advantage in the case of antibody arrays.

<table border="0" cellpadding="0" cellspacing="0" width="1045">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Odysee SA Imager</td>
			<td style="width:195px;">Li-Cor Biosciences</td>
			<td style="width:119px;"></td>
			<td style="width:364px;">Fluorescent Imager</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">1.5 ml tube</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:119px;">22363212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Cell Scraper</td>
			<td style="width:195px;">Falkon (Corning)</td>
			<td style="width:119px;">353085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Dulbeco&#39;s Phosphate buffered Saline (PBS)</td>
			<td style="width:195px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td>wash buffer for protein extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Tissue Culture dish 100mm</td>
			<td style="width:195px;">TRP</td>
			<td style="width:119px;">93100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">ICC Insulin syringe U100</td>
			<td style="width:195px;">Becton Dickinson</td>
			<td style="width:119px;">329412</td>
			<td>27G5/8,&#160; 1ml for needle treatment of protein samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Protein Profiler ARRAY&#160;</td>
			<td style="width:195px;">R&#38;D</td>
			<td style="width:119px;">ARY003B</td>
			<td>Human phospho MAPK array</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Protein Profiler ARRAY&#160;</td>
			<td style="width:195px;">R&#38;D</td>
			<td style="width:119px;">ARY002B</td>
			<td>Human phospho kinase array</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Centrifuge Eppendorf 5430R</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td>Table top centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Pierce BCA protein assay kit</td>
			<td style="width:195px;">Thermo Fisher</td>
			<td style="width:119px;">23225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">SpectraMAX M2</td>
			<td style="width:195px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td>Absorbance reader for protein quantification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">IRDye 800CW Streptavidin</td>
			<td style="width:195px;">Li-Cor Biosciences</td>
			<td style="width:119px;">925-32230</td>
			<td>Streptavidin conjugate for fluorescent detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">LabGard ES Class II, Type A2 biosafety cabinet</td>
			<td style="width:195px;">NuAire</td>
			<td style="width:119px;">NU-425-400</td>
			<td>Tissue culture hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">TC20 automated cell counter</td>
			<td style="width:195px;">Bio-Rad</td>
			<td style="width:119px;">1450102</td>
			<td>Cell counter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Halt Protease &#38; Phosphatase Inhibitor Cocktail (100X)</td>
			<td style="width:195px;">Thermo Fisher</td>
			<td style="width:119px;">78446</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">RIPA buffer</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">R0278</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Sonic Dismembrator</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">F60</td>
			<td>sonicator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Rocking platform shaker</td>
			<td style="width:195px;">VWR</td>
			<td style="width:119px;">10860-780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">ImageJ</td>
			<td style="width:195px;">NIH open source</td>
			<td style="width:119px;"></td>
			<td>https://imagej.net/Welcome</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SAS Institutie JMP&#174; 12.1.0 (64-bit)</td>
			<td style="width:195px;">Microsoft</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of resistance to tyrosine kinase inhibitors by an interrogation of signal transduction pathways by antibody arrays

Cancer patients with an aberrant regulation of the protein phosphorylation networks are often treated with the tyrosine kinase inhibitors. Response rates approaching 85% are common. Unfortunately, patients often become refractory to the treatment by altering their signal transduction pathways. An implementation of the expression profiling with microarrays can identify the overall mRNA-level changes, and proteomics can identify the overall changes in protein levels or can identify the proteins involved, but the activity of the signal transduction pathways can only be established by interrogating post-translational modifications of the proteins. As a result, the ability to identify whether a drug treatment is successful or whether resistance arose, or the ability to characterize any alterations in the signaling pathways, is an important clinical challenge. Here, we provide a detailed explanation of antibody arrays as a tool which can identify system-wide alterations in various post-translational modifications (e.g., phosphorylation). One of the advantages of using antibody arrays includes their accessibility (an array does not require either an expert in proteomics or costly equipment) and speed. The availability of arrays targeting a combination of post-translational modifications is the primary limitation. In addition, unbiased approaches (phosphoproteomics) may be more suitable for the novel discovery, whereas antibody arrays are ideal for the most widely characterized targets.

To investigate the effect of TKI resistance on signal transduction pathways in cell lines, four samples were analyzed. One control sample (H3255r1) and 3 TKI-resistant cell lines (H3255r2-4) (Figure 2) were related to the spot antibody template (Figure 3). All 4 samples were prepared using this protocol. Six phosphoproteins with differential activity were chosen for the demonstration of the analysis of the antibody arrays (<stron...

Watch this Scientific Journal Video about Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays at JoVE.com

Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays

Here, we present a protocol for antibody arrays to identify alterations in signaling pathways in various cellular models. These changes, caused by drugs/hypoxia/ultra-violet light/radiation, or by overexpression/downregulation/knockouts, are important for various disease models and can indicate whether a therapy will be effective or can identify mechanisms of drugs resistance.

Cancer patients with an aberrant regulation of the protein phosphorylation networks are often treated with the tyrosine kinase inhibitors. Response rates ...

This method can help answer key questions about perturbations in signaling networks that can cause cancer growth or the development of resistance to specific therapies. The main advantage of this technique is that it can provide rapid and reliable feedback on the phosphorylation state of multiple networks that are frequently deregulated in cancer. The implication of this technique extends towards therapy of drug-resistant cancers because identification of alterations and post-translational modifications can elucidate changes in signal transduction pathway activity.While this technique is used on phosphorylation, it can also be used to look at other post-translational modifications such as glycosylation and ubiquitylation. Begin by thoroughly rinsing overnight cultured cells three times with 10 milliliters of PBS taking care to remove all of the PBS after the last wash. Next, add the appropriate volume of lysis buffer from the protein assay kit and use a cell scraper to detach the cells.Triturate the resulting cell solution approximately 10 times before transferring at least one times 10 to the seventh cells to a 1.5 milliliter tube. Incubate the cells for 30 minutes at four degrees Celsius with shaking. Then load the lysate into a syringe equipped with a 27 gauge needle and aspirate and dispense the sample 10 times to shear the cell membranes.When the cell membranes have been completely disrupted, centrifuge the lysate and transfer the supernatant to a new 1.5 milliliter tube for subsequent total protein quantification according to standard protocols. To perform a human phosphokinase assay, first use flat-tipped tweezers to place one array membrane into each well of a four-well multi-tray containing two milliliters of freshly prepared array buffer one per well with the numbers of the membranes facing up. Upon submersion, the dye on the membrane will disappear.Cover the tray with a lid and incubate the membranes on a rocking platform shaker for 60 minutes at room temperature. During this blocking incubation, dilute each lysate sample to a 50 to 100 micrograms of protein concentration in a maximum volume of 334 microliters of lysis buffer and bring the final volume of extracted protein sample up to 1.5 milliliters with fresh array buffer. Next, carefully aspirate the array buffer from each well of the multi-tray and incubate the membranes with 1.5 milliliters of protein sample per well overnight at two to eight degrees Celsius on a rocking platform shaker.The next morning, carefully transfer each array into individual plastic containers containing 20 milliliters of wash buffer for three 10-minute washes on a rocking platform at room temperature. After the last wash, add 20 microliters of the appropriate reconstituted antibody cocktail to two milliliters of array buffer, two per well, and carefully blot the lower edge of each membrane on a paper towel to remove any excess wash buffer. Then place each array back into the appropriate well of the tray of antibody and cover the tray for a two-hour incubation at room temperature on a rocking platform.At the end of the incubation, carefully transfer each array into a new eight by 11 centimeter plastic container containing 20 milliliters of wash buffer for three 10-minute washes as just demonstrated. After the last wash, add one milliliter of an appropriate streptavidin horse radish peroxidase solution to each well of the multi-well tray and return the membranes to their appropriate wells for a 30-minute incubation at room temperature. At the end of the incubation, place each membrane between two pieces of five millimeter filter paper to remove any excess buffer and incubate the dried membranes with an appropriate horse radish peroxidase detection solution for three minutes.Then place the membranes into individual clear plastic sheet protectors for subsequent imaging. To assess the receptor tyrosine kinase expression in each sample, open the images in an appropriate image processing program such as Image J and select Edit and Invert from the appropriate dropdown menus to invert the black spots on the white background of the image to white spots on a black background. Next, use the Oval tool to encircle one pair of white dots on the dot blot and select Analyze and Measure to automatically open a new window displaying the values for the selected area.Then place the point of the mouse arrow into the center of the oval and drag the circular area around the next dot area until all the spots of interest have been measured. In this representative experiment, the effects of tyrosine kinase inhibitor resistance on signal transduction pathways was analyzed in one control in three tyrosine kinase inhibitor resistant cell lines. Six phospho proteins with differential activity were chosen and the relative intensity was determined for several candidate proteins in each cell line.As demonstrated by the heat map, a semi-quantitative readout on the changes in the protein phosphorylation of important signal transduction proteins could then be generated. While attempting this procedure, it's important to remember to start with healthy and actively dividing cells as stressors may induce changes in signal transduction pathway activity. Following this procedure, other methods such as digital PCR and immunoblotting can be used to answer additional questions such as do changes in phosphorylation lead to the activation of transcription factors?After its development, this technique has paved the way for researchers in cancer biology to explore fundamental changes in the post-translational modification of proteins leading to the advent of personalized medicine for patients with cancer. Don't forget that working with cancer cell lines is extremely hazardous so be sure to use aseptic techniques, caution when disposing of sharps and wear a lab coat at all times.

assessment-resistance-to-tyrosine-kinase-inhibitors-an-interrogation

Research

JoVE Journal

Cancer Research

7.8K Görüntüleme.  University of Southern California. Bu yöntem, kanser büyümesine veya belirli tedavilere karşı direnç gelişimine neden olabilecek sinyal ağlarındaki tedirginliklerle ilgili anahtar soruları yanıtlamaya yardımcı olabilir. Bu tekniğin en büyük avantajı, kanserde sıklıkla deregüle olan birden fazla anın fosforilasyon durumu hakkında hızlı ve güvenilir geri bildirim sağlayabilmektir. Bu tekniğin anlamı, değişikliklerin ve çeviri sonrası değişikliklerin tanımlanması sinyal transdüksiyon yolu akti...