Name: assessment of resistance to tyrosine kinase inhibitors by an interrogation of signal transduction pathways by antibody arrays
Uploaded: 2018-09-19T13:00:00
Description: 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

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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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.

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	<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

Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the gene of interest in hematopoietic cells and can be used to study the gene's role in normal and/or malignant hematopoiesis. This protocol provides a detailed description on how to perform transduction-transplantation using calreticulin (CALR) mutations recently identified in myeloproliferative neoplasm (MPN) as an example. In this protocol whole bone marrow cells from 5-flurouracil (5-FU) treated donor mice are transduced with a retrovirus encoding mutant CALR and transplanted into lethally irradiated syngeneic hosts. Donor cells expressing mutant CALR are marked with green fluorescent protein (GFP). Transplanted mice develop an MPN phenotype including elevated platelets in the peripheral blood, expansion of megakaryocytes in the bone marrow, and bone marrow fibrosis. We provide a step-by-step account of how to generate retrovirus, calculate viral titer, transduce whole bone marrow cells, and transplant into irradiated recipient mice.

This manuscript provides a description of the methodology used to establish transduction-transplantation mouse models. A detailed account is given of technical errors to avoid when performing bone marrow transplants. A clear understanding should be gained of the importance of high viral titer, transfection/transduction, and irradiation.

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the ...

Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane resistance in cancerous cells drastically reduces the effectiveness of the drugs' long-term usage. Accumulated evidence suggests that the mechanisms underlying taxane resistance include both general mechanisms, such as the development of multidrug resistance due to the overexpression of drug-efflux proteins, and taxane-specific mechanisms, such as those that involve microtubule dynamics. 
Because taxanes target cell microtubules, measuring microtubule dynamic instability is an important step in determining the mechanisms of taxane resistance and provides insight into how to overcome this resistance. In the experiment, an in vivo method was used to measure microtubule dynamic instability. GFP-tagged &#945;-tubulin was expressed and incorporated into microtubules in MCF-7 cells, allowing for the recording of the microtubule dynamics by time lapse using a sensitive camera. The results showed that, as opposed to the non-resistant parental MCF-7CC cells, the microtubule dynamics of docetaxel-resistant MCF-7TXT cells are insensitive to docetaxel treatment, which causes the resistance to docetaxel-induced mitotic arrest and apoptosis. This paper will outline this in vivo method of measuring microtubule dynamic instability.

In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells.

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane ...

Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a common mechanism of disease progression and a prognostic-determinant feature of malignant tumors. In prostate cancer (PC), resistance to MTAs such as the taxane Docetaxel dictates treatment failure as well as progression towards lethal stages of disease that are defined by a poor prognosis and high mortality rates. Though studied for decades, the array of mechanisms contributing to acquired resistance are not completely understood, and thus pose a significant limitation to the development of new therapeutic strategies that could benefit patients in these advanced stages of disease. In this protocol, we describe the generation of Docetaxel-resistant prostate cancer cell lines that mimic lethal features of late-stage prostate cancer, and therefore can be used to study the mechanisms by which acquired chemoresistance arises. Despite potential limitations intrinsic to a cell based model, such as the loss of resistance properties over time, the Docetaxel-resistant cell lines produced by this method have been successfully used in recent studies and offer&#160;the opportunity to advance our molecular understanding of acquired chemoresistance in lethal prostate cancer.

Resistance to cancer therapies contributes to disease progression and death. Determining the mechanistic underpinnings of resistance is crucial for improving therapeutic response. This manuscript details the protocol to generate taxane-resistant cell models of prostate cancer (PC) to help dissecting the pathways involved in progression to Docetaxel resistance in PC patients.

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a ...

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 ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines respond to therapeutic compounds in 2D (dimensional) monolayer cell cultures, 3D culture systems were developed to understand the efficacy of drugs in more physiologically relevant models. In recent years, a paradigm shift was observed in pre-clinical research to validate the potency of new molecules in 3D culture systems, more precisely mimicking the tumor microenvironment. These systems characterize the disease state in a more physiologically relevant manner and help to gain better mechanistic insight and understanding of the pharmacological potency of a given molecule. Moreover, with the current trend in improving in vivo cancer models, zebrafish has emerged as an important vertebrate model to assess in vivo tumor formation and study the effect of therapeutic agents. Here, we investigated the therapeutic efficacy of hydroxycoumarin OT48 alone or in combination with BH3 mimetics in lung cancer cell line A549 by using three different 3D culture systems including colony formation assays (CFA), spheroid formation assay (SFA) and in vivo zebrafish xenografts.

Here, we present the preclinical screening of anticancer coumarins using 3D culture and zebrafish.

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines ...

Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy against cancer. Here we provide a protocol to identify a rational combination at the preclinical step. First, we describe a cell-based assay to assess the synergism between anticancer mAb and cytotoxic drugs, that uses the combination index equation of Chou and Talalay1. This includes the measurement of tumor cell drug- and antibody-sensitivity using an MTT assay, followed by an automated computer analysis to calculate the combination index (CI) values. CI values of &lt;1 indicate synergism between tested mAbs and cytotoxic agents1. To corroborate the in vitro findings in vivo, we further describe a method to assess the combination regimen efficacy in a xenograft tumor model. In this model, the combined regimen significantly delays tumor growth, which results in a significant extended survival in comparison to single-agent controls. Importantly, the in vivo experimentation reveals that the combination regimen is well tolerated. This protocol allows the effective evaluation of anticancer drug combinations in preclinical models and the identification of rational combination to evaluate in clinical trials.

This protocol describes how to assess synergism between an anticancer antibody and antineoplastic drugs in preclinical models by using the combination index equation of Chou and Talalay.

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy ...

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in tumorigenesis, can be directed to cancer biomarkers enabling tumor detection and characterization, and can be used for cancer therapy as mAbs or antibody-drug conjugates to activate immune effector cells, to inhibit signaling pathways, or directly kill cells carrying the specific antigen. Despite clinical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be impaired by the complexity and heterogeneity of the tumor microenvironment. Thus, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate with the living tumor microenvironment. Here, we describe In Vivo Immunofluorescence Localization (IVIL) as a new approach to study interactions of antibody-based therapeutics and diagnostics in the in vivo physiological and pathological conditions. In this technique, a therapeutic or diagnostic antigen-specific antibody is intravenously injected in&#160;vivo and localized ex vivo with a secondary antibody in isolated tumors. IVIL, therefore, reflects the in vivo biodistribution of antibody-based drugs and targeting agents. Two IVIL applications are described assessing the biodistribution and accessibility of antibody-based contrast agents for molecular imaging of breast cancer. This protocol will allow future users to adapt the IVIL method for their own antibody-based research applications.

The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining methods.

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and invasive potential of cancer cells, and consequently the metastatic spread of disease, are being discovered and considered as complementary treatments in highly invasive cancers such as gliomas. Thus, generating data enabling the detailed analyses of cells in a 3D environment following the addition of a drug is required. The methodology described here, combining spheroid invasion assays with high-resolution image capture and data analysis by confocal laser scanning microscopy (CLSM), enabled detailed characterization of the effects of the potential anti-migratory inhibitor MI-192 on glioma cells. Spheroids were generated from cell lines for invasion assays in low adherent 96-well plates and then prepared for CLSM analysis. The described workflow was preferred over other commonly used spheroid-generating techniques due to both ease and reproducibility. This, combined with the enhanced image resolution attained by confocal microscopy compared to conventional wide-field approaches, allowed the identification and analysis of distinct morphological changes in migratory cells in a 3D environment following treatment with the migrastatic drug MI-192.

The effects of migrastatic inhibitors on glioma cancer cell migration in three-dimensional (3D) invasion assays using a histone deacetylase (HDAC) inhibitor are characterized by high-resolution confocal microscopy.

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and ...

Assays for Validating Histone Acetyltransferase Inhibitors

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene expression. KATs, such as CBP/p300, are under intense investigation as therapeutic targets due to their critical role in tumorigenesis of diverse cancers. The development of novel small molecule inhibitors targeting the histone acetyltransferase (HAT) function of KATs is challenging and requires robust assays that can validate the specificity and potency of potential inhibitors.
This article outlines a pipeline of three methods that provide rigorous in vitro validation for novel HAT inhibitors (HATi). These methods include a test tube HAT assay, Chromatin Hyperacetylation Inhibition (ChHAI) assay, and Chromatin Immunoprecipitation-quantitative PCR (ChIP-qPCR). In the HAT assay, recombinant HATs are incubated with histones in a test tube reaction, allowing for acetylation of specific lysine residues on the histone tails. This reaction can be blocked by a HATi and the relative levels of site-specific histone acetylation can be measured via immunoblotting. Inhibitors identified in the HAT assay need to be confirmed in the cellular environment.
The ChHAI assay uses immunoblotting to screen for novel HATi that attenuate the robust hyperacetylation of histones induced by a histone deacetylase inhibitor (HDACi). The addition of an HDACi is helpful because basal levels of histone acetylation can be difficult to detect via immunoblotting.
The HAT and ChHAI assays measure global changes in histone acetylation, but do not provide information regarding acetylation at specific genomic regions. Therefore, ChIP-qPCR is used to investigate the effects of HATi on histone acetylation levels at gene regulatory elements. This is accomplished through selective immunoprecipitation of histone-DNA complexes and analysis of the purified DNA through qPCR. Together, these three assays allow for the careful validation of the specificity, potency, and mechanism of action of novel HATi.

Inhibitors of histone acetyltransferases (HATs, also known as lysine acetyltransferases), such as CBP/p300, are potential therapeutics for treating cancer. However, rigorous methods for validating these inhibitors are needed. Three in vitro methods for validation include HAT assays with recombinant acetyltransferases, immunoblotting for histone acetylation in cell culture, and ChIP-qPCR.