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, then use a radioimmunoprecipitation assay buffer (RIPA) or any other cell lysis buffer containing a cocktail of protease and phosphatase inhibitors.</li>
	<li>Lyse 1 x 107 cells/mL of cells in the lysis buffer by adding the correct amount of buffer to the cells and scraping the cells with a cell scraper into the lysis buffer. (e.g., for HeLa cells and MiaPaCa-2, use 600 &#181;L per 10 cm plate).</li>
	<li>Pipette the lysate up and down (approximately 10x) and transfer it to a new 1.5 mL tube.</li>
	<li>Incubate the lysates for 30 min at 4 &#176;C, preferably on a rocker/shaker. Press the lysate through a syringe with a 27 G needle for 10x to ensure a proper disruption of the cell membrane(s). Alternatively, sonicate the lysate. Store the lysates at -20 &#176;C or use them immediately.</li>
	<li>Centrifuge the lysate at 14,000 x g at 4 &#176;C for 15 min and transfer the supernatant to a clean 1.5 mL tube.</li>
	<li>Quantitate the amount of total protein by bicinchoninic acid assay (BCA)20 or an equivalent such as Lowry or Bradford and continue with at least 50&#8211;400 &#956;g. Use the proteins immediately or aliquot and freeze/store them at -70 &#176;C (avoid multiple freeze-thaw cycles).</li>
</ol>
2. Human Phosphokinase Array
<ol>
	<li>Bring all reagents to room temperature before starting (for approximately 1 h). 
	NOTE: All reagents and plastic wear are included in the kit.</li>
	<li>Prepare all the reagents fresh (array buffers) before starting the procedure following the manufacturer&#39;s instructions (depending on the choice of targets/arrays, the instructions might vary slightly).</li>
	<li>Reconstitute the detection antibody cocktails in 100 &#956;L of deionized water or follow the manufacturer&#39;s instructions should they differ for the 1.5 mL test tube provided.</li>
	<li>Prepare 1x wash buffer by diluting 40 mL of 25x wash buffer in 960 mL of deionized water and mix them by inverting. 
	NOTE: Crystals dissolve at room temperature. The buffer may turn yellow over time but will still work.</li>
	<li>Pipette 1 mL of array buffer 1 into each well of an 8-well multi-tray (or 2 mL in a 4-well multi-tray).</li>
	<li>With flat-tip tweezers, remove the array membranes between the protective sheets and place them into the wells. Make sure the numbers on the membrane are facing upwards. 
	NOTE: Upon submersion, the dye on the membrane will disappear.</li>
	<li>Cover the tray with a lid and incubate it on a rocking platform shaker for 60 min at room temperature. 
	NOTE: This is the membrane blocking step.</li>
	<li>During the incubation period, prepare the protein samples. Add 50&#8211;100 &#956;g of total protein. Dilute the lysate, having a maximum volume of 334 &#956;L, with lysis buffer to a final volume of 1 mL. 
	NOTE: 50&#8211;100 &#956;g of total protein usually suffices.</li>
	<li>Aspirate array buffer 1 carefully and incubate the membranes with 1 mL of the samples overnight at 2&#8211;8 &#176;C on a rocking platform shaker. 
	NOTE: Do not touch/scratch the membranes.</li>
	<li>The next day, wash the array by carefully removing each array and placing it into individual plastic containers (approximately 8 x 11 cm2) with 20 mL of 1x wash buffer. Wash the membranes 3 x 10 min on a rocking platform in 1x washing buffer at room temperature.</li>
	<li>Pipette 20 &#956;L of the reconstituted antibody cocktail from step 2.3 to 1 mL of 1x array buffer 2. Add 1 mL of this solution to each 8-well to be used.</li>
	<li>Carefully remove the membranes from the wash trays. Blot the lower edge onto the paper towels to remove any excess wash buffer and transfer them back into the tray containing the antibody cocktails. Cover the tray with the lid and incubate it for 2 h at room temperature on a rocking platform. Thoroughly rinse the used trays with dH2O and dry them for later usage.</li>
	<li>Carefully remove each array and place them back into the clean individual plastic containers (approximately 8 x 11 cm2) with 20 mL of 1x wash buffer. Wash them 3 x 10 min with the wash buffer on a rocking platform at room temperature.</li>
	<li>Dilute Streptavidin-HRP (provided with the kit) or streptavidin-fluorescent dye (for a more quantitative detection) 1:1,000 in 1x array buffer 2 in a 15 mL test tube.</li>
	<li>Return the membranes into the 8-well dishes containing the HP solution and incubate them for 30 min at room temperature on a rocking platform (if using fluorescence, wrap the tray in aluminum foil to avoid any light exposure).</li>
	<li>Remove the excess buffer by placing the membrane in between 2 pieces of 5 mm of 3 M paper. For imaging with an X-ray film/chemiluminescent imager, incubate the dried membranes with an HRP detection solution (mix the two chemiluminescent solutions 1:1) for 3 min and place the membrane into a clear plastic sheet protector. 
	NOTE: Dried membrane(s) may also be placed in a fluorescent imager to detect the fluorescent signal; minimize the light exposure to the membrane before imaging. For a quantification of the X-ray film or imager files, avoid overexposure. Most chemiluminescent/fluorescent imagers have a function to avoid a saturation of the signal.</li>
</ol>
3. Data Analysis
<ol>
	<li>Download ImageJ, an image processing program21, and install the software.</li>
	<li>Open ImageJ by double-clicking on the &#39;ImageJ icon&#39;. Select the image to be analyzed by clicking &#39;File&#39;&#160;in the menu bar, then select &#39;Open&#39;&#160;from the drop-down menu and browse the computer files for the image of interest. Click the file to open it.</li>
	<li>Invert the picture for the analysis (black spots on the white background to white spots on a black background) by clicking &#39;Edit&#39;&#160;in the menu bar and selecting &#39;Invert&#39;&#160;from the drop-down menu.</li>
	<li>Select the &#39;oval icon&#39; from the toolbar (the second option in the ImageJ toolbar). Draw a circle/oval by clicking on the picture (black cross) and moving the mouse. Adjust the size/shape of the circle/oval to encircle the dots on the dot blot.</li>
	<li>Click &#39;Analyze&#39;&#160;in the menu bar and then select &#39;Measure&#39;&#160;from the drop-down menu to automatically open a new window with the values of the selected area (area, mean, minimum, and maximum).</li>
	<li>Move the circular area by dragging the circle from the center (put the point of the arrow right in the middle) and place it around the next measurement (dot) area. Measure all spots of interest, the positive control, the negative control, and the background for the analysis. 
	NOTE: The negative control is the PBS, the background is a part of the membrane without any spot (any part will do), and the positive control is a control provided by the manufacturer.</li>
	<li>Select the PBS negative control spots and subtract the value from each spot. Average the intensity for each pair of duplicate spots representing each receptor tyrosine kinase (RTK). Each average intensity can be related to the control to calculate the fold change.</li>
</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><tbody><tr><td>Odysee SA Imager</td><td>Li-Cor Biosciences</td><td></td><td>Fluorescent Imager</td></tr><tr><td>1.5 mL tube</td><td>Eppendorf</td><td>22363212</td><td></td></tr><tr><td>Cell Scraper</td><td>Falkon (Corning)</td><td>353085</td><td></td></tr><tr><td>Dulbeco&#039;s Phosphate buffered Saline (PBS)</td><td>Corning</td><td>21-031-CV</td><td>wash buffer for protein extraction</td></tr><tr><td>Tissue Culture dish 100 mm</td><td>TRP</td><td>93100</td><td></td></tr><tr><td>ICC Insulin syringe U100</td><td>Becton Dickinson</td><td>329412</td><td>27 G5/8,&amp;nbsp; 1 mL for needle treatment of protein samples</td></tr><tr><td>Protein Profiler ARRAY&amp;nbsp;</td><td>R&amp;amp;D</td><td>ARY003B</td><td>Human phospho MAPK array</td></tr><tr><td>Protein Profiler ARRAY&amp;nbsp;</td><td>R&amp;amp;D</td><td>ARY002B</td><td>Human phospho kinase array</td></tr><tr><td>Centrifuge Eppendorf 5430R</td><td>Eppendorf</td><td></td><td>Table top centrifuge</td></tr><tr><td>Pierce BCA protein assay kit</td><td>Thermo Fisher</td><td>23225</td><td></td></tr><tr><td>SpectraMAX M2</td><td>Molecular Devices</td><td></td><td>Absorbance reader for protein quantification</td></tr><tr><td>IRDye 800CW Streptavidin</td><td>Li-Cor Biosciences</td><td>925-32230</td><td>Streptavidin conjugate for fluorescent detection</td></tr><tr><td>LabGard ES Class II, Type A2 biosafety cabinet</td><td>NuAire</td><td>NU-425-400</td><td>Tissue culture hood</td></tr><tr><td>TC20 automated cell counter</td><td>Bio-Rad</td><td>1450102</td><td>Cell counter</td></tr><tr><td>Halt Protease &amp;amp; Phosphatase Inhibitor Cocktail (100x)</td><td>Thermo Fisher</td><td>78446</td><td></td></tr><tr><td>RIPA buffer</td><td>Sigma</td><td>R0278</td><td></td></tr><tr><td>Sonic Dismembrator</td><td>Fisher Scientific</td><td>F60</td><td>sonicator</td></tr><tr><td>Rocking platform shaker</td><td>VWR</td><td>10860-780</td><td></td></tr><tr><td>ImageJ</td><td>NIH open source</td><td></td><td>https://imagej.net/Welcome</td></tr><tr><td>SAS Institutie JMP&amp;reg; 12.1.0 (64-bit)</td><td>Microsoft</td><td></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...

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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 ...

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

Research

JoVE Journal

Cancer Research

7.9K Views.  University of Southern California. Deze methode kan helpen bij het beantwoorden van belangrijke vragen over verstoringen in signalering netwerken die kankergroei of de ontwikkeling van resistentie tegen specifieke therapieën kan veroorzaken. Het belangrijkste voordeel van deze techniek is dat het snelle en betrouwbare feedback kan geven over de fosforylatietoestand van meerdere netwerken die vaak worden gedereguleerd bij kanker. De implicatie van deze techniek strekt zich uit tot therapie van resistente kankers, omdat identif...

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

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.

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 ...

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, a small population of cells does not respond to TKI treatment, resulting in minimal residual disease (MRD); even the most potent TKIs fail to eradicate these cells. These MRD cells serve as a reservoir to develop resistance to therapy. Why TKI treatment is ineffective against MRD cells is not known. Growth factor signaling is implicated in supporting the survival of MRD cells during TKI treatment, but a mechanistic understanding is lacking. Recent studies demonstrated that an elevated c-Fos and Dusp1 expression as a result of convergent oncogenic and growth factor signaling in MRD cells mediate TKI resistance. The genetic and chemical inhibition of c-Fos and Dusp1 renders CML exquisitely sensitive to TKIs and cures CML in both genetic and humanized mouse models. We identified these target genes using multiple microarrays from TKI-sensitive and -resistant cells. Here, we provide methods for target validation using in vitro and in vivo mouse models. These methods can easily be applied to any target for genetic validation and therapeutic development.

Here, we describe protocols for the genetic and chemical validation of c-Fos and Dusp1 as a drug target in leukemia using in vitro and in vivo genetic and humanized mouse models. This method can be applied to any target for genetic validation and therapeutic development.

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, ...

The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment resistance is thought to be the molecular complexity of the disease 1. Targeted therapy such as the tyrosine kinase inhibitor (TKI)-sunitinib have been utilized, but only 40% of patients will respond, with the overwhelming majority of these patients relapsing within 1 year 2. As such the question of intrinsic and acquired resistance in renal cell cancer patients is highly relevant 3.
In order to study resistance to TKIs, with the ultimate goal of developing effective, personalized treatments, sequential tissue after a specific period of targeted therapy is required, an approach which had proved successful in chronic myeloid leukaemia 4. However the application of such a strategy in renal cell carcinoma is complicated by the high level of both inter- and intratumoral heterogeneity, which is a feature of renal cell carcinoma5,6 as well as other solid tumors 7. Intertumoral heterogeneity due to transcriptomic and genetic differences is well established even in patients with similar presentation, stage and grade of tumor. In addition it is clear that there is great morphological (intratumoral) heterogeneity in RCC, which is likely to represent even greater molecular heterogeneity. Detailed mapping and categorization of RCC tumors by combined morphological analysis and Fuhrman grading allows the selection of representative areas for proteomic analysis.
Protein based analysis of RCC8 is attractive due to its widespread availability in pathology laboratories; however, its application can be problematic due to the limited availability of specific antibodies 9. Due to the dot blot nature of the Reverse Phase Protein Arrays (RPPA), antibody specificity must be pre-validated; as such strict quality control of antibodies used is of paramount importance. Despite this limitation the dot blot format does allow assay miniaturization, allowing for the printing of hundreds of samples onto a single nitrocellulose slide. Printed slides can then be analyzed in a similar fashion to Western analysis with the use of target specific primary antibodies and fluorescently labelled secondary antibodies, allowing for multiplexing. Differential protein expression across all the samples on a slide can then be analyzed simultaneously by comparing the relative level of fluorescence in a more cost-effective and high-throughput manner.

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

RPPA enables the protein expression of hundreds of samples, printed on nitrocellulose slides to be interrogated simultaneously, using fluorescently labelled antibodies. This technique has been applied to study the effect of drug treatment heterogeneity within clear cell renal carcinoma.

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

Medicine

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.

A detailed protocol for the generation of self-assembled human protein microarrays for the screening of kinase inhibitors is presented.

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...

Biochemistry

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

Time-resolved F&#246;rster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in mediating cellular responses to cytokines and growth factors. STAT proteins are activated by tyrosine phosphorylation mediated mainly by JAKs. The abnormal activation of STAT signaling pathways is implicated in many human diseases, especially cancer and immune-related conditions. Therefore, the ability to monitor STAT protein phosphorylation within the native cell signaling environment is important for both academic and drug discovery research. The traditional assay formats available to quantify phosphorylated STAT proteins include western blotting and the enzyme-linked immunosorbent assay (ELISA). These heterogeneous methods are labor-intensive, low-throughput, and often not reliable (specific) in the case of western blotting. Homogeneous (no-wash) methods are available but remain expensive. 
Here, detailed protocols are provided for the sensitive, robust, and cost-effective measurement in a 384-well format of endogenous levels of phosphorylated STAT1 (Y701), STAT3 (Y705), STAT4 (Y693), STAT5 (Y694/Y699), and STAT6 (Y641) in cell lysates from adherent or suspension cells using the novel THUNDER time-resolved F&#246;rster resonance energy transfer (TR-FRET) platform. The workflow for the cellular assay is simple, fast, and designed for high-throughput screening (HTS). The assay protocol is flexible, uses a low-volume sample (15 &#181;L), requires only one reagent addition step, and can be adapted to low-throughput and high-throughput applications. Each phospho-STAT sandwich immunoassay is validated under optimized conditions with known agonists and inhibitors and generates the expected pharmacology and Z'-factor values. As TR-FRET assays are ratiometric and require no washing steps, they provide much better reproducibility than traditional approaches. Together, this suite of assays provides new cost-effective tools for a more comprehensive analysis of specific phosphorylated STAT proteins following cell treatment and the screening and characterization of specific and selective modulators of the JAK/STAT signaling pathway.

Time-resolved F&#246;rster resonance energy transfer cell-based assay protocols are described for the simple, specific, sensitive, and robust quantification of endogenous phosphorylated signal transducer and activator of transcription (STAT) 1/3/4/5/6 proteins in cell lysates in a 384-well format.

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in mediating cellular responses to ...

Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of action of the antibodies is antibody-dependent cellular cytotoxicity (ADCC), where the antibody binds to the cancer cells and engages the cellular component of the immune system, e.g., natural killer (NK) cells, to kill the tumor cells. The effectiveness of these therapies could be improved by identifying adjuvant compounds that increase the sensitivity of the cancer cells or the potency of the immune cells. In addition, undiscovered drug interactions in cancer patients co-medicated for previous conditions or cancer-associated symptoms may determine the success of the antibody therapy; therefore, such unwanted drug interactions need to be eliminated. With these goals in mind, we created a cancer ADCC model and describe here a simple protocol to find ADCC-modulating drugs. Since 3D models such as cancer cell spheroids are superior to 2D cultures in predicting in vivo responses of tumors to anticancer therapies, spheroid co-cultures of EGFP-expressing HER2+ JIMT-1 breast cancer cells and the NK92.CD16 cell lines were set up and induced with Trastuzumab, a monoclonal antibody clinically approved against HER2-positive breast cancer. JIMT-1 spheroids were allowed to form in cell-repellent U-bottom 96-well plates. On day 3, NK cells and Trastuzumab were added. The spheroids were then stained with Annexin V-Alexa 647 to measure apoptotic cell death, which was quantitated in the peripheral zone of the spheroids with an automated microscope. The applicability of our assay to identify ADCC-modulating molecules is demonstrated by showing that Sunitinib, a receptor tyrosine kinase inhibitor approved by the FDA against metastatic cancer, almost completely abolishes ADCC. The generation of the spheroids and image acquisition and analysis pipelines are compatible with high-throughput screening for ADCC-modulating compounds in cancer cell spheroids.

Here, we present a method to identify compounds that modulate the ADCC mechanism, an important cancer cell-killing mechanism of antitumor antibodies. The cytotoxic effect of NK cells is measured in breast cancer cell spheroids in the presence of Trastuzumab. Image analysis identifies live and dead killer and target cells in spheroids.

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of ...

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings.

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...

Immunology and Infection

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Biology

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

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

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