This work was supported by National Institutes of Health Grant 1R21CA195422 (to L. T.), Epstein Family Foundation Award (to N. D. P. C.), and NCI Cancer Center Support Grant P30CA030199. Additionally, this project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Chemical Biology Consortium Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

1. Preparation of cell culture and reagents
<ol>
	<li>Formulate a 500 mL bottle of growth media with 10% fetal bovine serum, 1x antibiotic/antimicotic, 20 mM HEPES, and 1 mM sodium pyruvate. Store at 4 &#176;C.</li>
	<li>Thaw cellular thermal shift reagents (EA reagent, lysis buffer, and substrate) from frozen original stock bottles.</li>
	<li>Dispense reagents and buffer as 2 mL aliquots and store at -20 &#176;C. 
	NOTE: Avoid freeze/thaw for reproducibility and use only that volume of reagent required for the assay procedure.</li>
</ol>
2. Growth and maintenance of HEK293T cells
<ol>
	<li>Obtain low passage adherent HEK293T cells from cryo storage.</li>
	<li>Maintain HEK293T cells in growth media at 37 &#176;C, 5% CO2.</li>
	<li>Split cells every 4 days in a 1:14 ratio. 
	NOTE: For best performance, HEK293T cells are not allowed to passage greater than 25 times.</li>
</ol>
3. HEK293T cell preparation for transient transfection
<ol>
	<li>Detach HEK293T cells from 16 mL plates using 3 mL of the cell detachment reagent.</li>
	<li>Dilute with 12 mL of growth media. Collect cells by centrifugation at 1,400 x g for 4 min.</li>
	<li>Resuspend the pellet in 10 mL growth media. Measure the concentration and viability of cells using trypan blue and a cell counter.</li>
	<li>Plate 7.0 x 105 exponentially growing HEK293T cells per well in a 6 well cell culture plate approximately 24 h before transfection. 
	NOTE: Reserve one well for each plasmid to be transfected.</li>
	<li>Incubate for 24 h at 37 &#176;C, 5% CO2.</li>
</ol>
4. Transfection of HEK293T cells
<ol>
	<li>From a purified plasmid stock of pICP-ePL-N-SHP2-WT or pICP-ePL-N-SHP2-E76K (~200 ng/&#181;L) dilute 2 &#181;g of plasmid DNA into 200 &#181;L of transfection buffer.</li>
	<li>Vortex for 10 s and centrifuge at 1,400 x g for 4 min.</li>
	<li>Add 4 &#181;L of transfection reagent to the diluted DNA. Vortex for 10 s and centrifuge at 1,400 x g for 4 min.</li>
	<li>Incubate at 23 &#176;C for 10 min.</li>
	<li>Remove the 6 well plate containing growing HEK293T cells from incubator. Add the transfection mix to the attached cells in the 6-well plate. Incubate for 24 h at 37 &#176;C, 5% CO2.</li>
</ol>
5. Preparation of assay plates
<ol>
	<li>Prepare 10 mM stock solutions in DMSO of compounds to be tested.</li>
	<li>Dispense inhibitor solutions into a 384-well low dead volume source plate for immediate use.</li>
	<li>Spot the desired volume of inhibitors or vehicle (DMSO) using a liquid handler into 384-well real-time PCR plates at a target final volume of less than 0.5% DMSO (v/v). Seal the plate using a plate sealer with inert gas purging. 
	NOTE: For inhibitor dose-response assays, make sure to backfill DMSO accordingly so that equal amounts of DMSO are used for each inhibitor concentration. For best results, store plates at 23 &#176;C and use plates within 24 h.</li>
</ol>
6. Transfected cell detachment and preparation
<ol>
	<li>Preincubate growth media and cell detachment reagent in a 37 &#176;C water bath. Remove transfected cells from the incubator. Gently aspirate media from the wells of the plate. 
	NOTE: Use a new aspirator for every well that contains a different transfected plasmid.</li>
	<li>Add 0.3 mL of the cell detachment reagent. Gently rock the plate back and forth to thoroughly cover the surface of the plate bottom. Incubate at 23 &#176;C for 2 min.</li>
	<li>Add 1 mL of growth media to each well. Gently pipette media and cells in the well and transfer to a 15 mL Falcon centrifuge tube. Collect cells by centrifugation at 1,400 x g for 4 min.</li>
	<li>Gently but thoroughly aspirate off the media. 
	NOTE: Residual phenol red in the cell detachment reagent can interfere with the assay.</li>
	<li>Carefully resuspend cell pellet in 2 mL of growth media. Measure the concentration and viability of cells using trypan blue and a cell counter. 
	NOTE: Cell viability should be &#62; 90% for best results.</li>
	<li>Dilute cells to a concentration of 125 cells/&#181;L. For example, for a 5 &#181;L assay this is ~625 cells/&#181;L. For optimal viability keep cells in suspension for no more than 2 h.</li>
</ol>
7. Incubation of cells with SHP2 inhibitors
<ol>
	<li>Dispense cells into a sterile single channel solution trough.</li>
	<li>Centrifuge 384-well real-time PCR plate that has been pre-prepared by SHP2 inhibitor deposition at 2,500 x g for 5 min at 23 &#176;C.</li>
	<li>Remove seal from the compound plate. Using a 125 &#181;L multichannel pipette, add 5 &#181;L of the diluted cells to desired wells.</li>
	<li>Centrifuge the plate at 42 x g for 30 s without a lid on the plate. Attach a lid seal to the plate after the plate is spun down and incubate the assay plate at 37 &#176;C, 5% CO2 for 1 h.</li>
</ol>
8. Preparation of chemiluminescent reagent master mix
<ol>
	<li>Remove the necessary quantity of detection reagents (EA reagent, lysis buffer, and substrate) from -20 &#176;C storage after plating cells. Thaw reagents at 23 &#176;C. 
	NOTE: For convenience in transfer, prepare a volume that is 1.5x the total volume of cells that have been deposited to the plate. Do not reuse reagents after use.</li>
	<li>Prepare a master mix of the reagents using condition EA-10, which consists of component and volume fraction as follows: EA reagent (0.17), lysis buffer (0.17), substrate (0.67). 
	NOTE: Reagent ratios are based on optimization experiments performed as specified in the supplier&#39;s manual.</li>
</ol>
9. Isothermal or thermal profile gradient heat pulse
<ol>
	<li>Program the gradient capable thermocycler for the delivery of heat pulse.
	<ol>
		<li>For thermal profile gradient experiments, preprogram the thermocycler with the following example specifications:</li>
		<li>Heat pulse: 3 min desired melting temperature (vertical or horizontal gradient +/- 15 &#176;C; example 38-68 &#176;C spread across 24 wells yields temperature increments of 1.25 &#176;C).</li>
	</ol>
	</li>
	<li>Equilibration recovery step: 3 min 20 &#176;C with ramp speed = 1 &#176;C/s
	<ol>
		<li>For isothermal experiments set up the protocol set up as follows:</li>
		<li>Heat pulse: 3 min 55 &#176;C. Equilibration recovery step: 3 min 20 &#176;C with ramp speed = 1&#176;C/s 
		NOTE: For increased reproducibility, as it can be difficult to place the plate precisely at the time the thermocycler reaches its desired temperature, set up the program to count down 15 s before beginning the 3 min pulse. For the thermocycler used in this experiment see Table of Materials, the lid is kept up during the heat pulse.</li>
	</ol>
	</li>
</ol>
10. Lysis detection and measurement
<ol>
	<li>Supplement assay plates with lysis detection master mix by addition of equal volumes (5 &#181;L) to each well to be analyzed using a multichannel pipette.</li>
	<li>Centrifuge the plate 42 x g for 30 s. Store at 23 &#176;C in darkness for 30-60 min.</li>
	<li>Measure chemiluminescence using a microplate reader capable of detecting luminescence in 384-well format. Perform luminescence measurement with the plate type and integration time optimized (integration time 1000 ms, settle time 0 s). Measure values as counts/s and output for further analysis.</li>
</ol>
11. Data analysis
<ol>
	<li>Analyze the luminescence data using a scientific 2D graphing and statistics software.</li>
	<li>Generate thermal profiles by analyzing the normalized luminescence data (normalized to the vehicle control) using nonlinear regression and a Boltzmann sigmoidal model. 
	NOTE: In thermal profile experiments, the calculated V50, the temperature that is the halfway point between bottom and top of the curve, defines the melting temperature of SHP2. In isothermal experiments, EC50 defines the concentration of the inhibitor that gives half-maximal response.</li>
</ol>

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The authors declare that they have no conflicts of interest with the contents of this article.

We have presented a target engagement assay that can confirm direct binding of small molecules to the SHP2 phosphatase in cells. The assay can discriminate between low and high affinity inhibitors and, importantly, confirm a lack of potency by the allosteric inhibitors of the SHP099-type for the GOF oncogenic SHP2-E76K mutant. A strength of this miniaturized assay is its ability to be integrated into a SHP2 inhibitor screening campaign. The ability of the assay to confirm intracellular binding to SHP2 by unknown chemical...

Tyrosine phosphorylation plays an important role in signal transduction in cells1,2. This post-translational modification is catalyzed by protein tyrosine kinases (PTKs) and reversed by protein tyrosine phosphatases (PTPs). Therefore, aberrant PTK or PTP function leads to many inherited or acquired human diseases3,4,5,6. The Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2) is a widely expressed non-receptor type PTP encoded by the proto-oncogene PTPN117 and is a key regulator of numerous physiological processes that involve signal transduction by activation of the Ras/Raf/ERK, PI3K/Akt, or JAK/STAT signaling pathways8. Normally, SHP2 activity is tightly regulated in order to prevent aberrant signaling. Under basal conditions SHP2 is autoinhibited by its N-terminal SH2 domain, which blocks access to the active site within the catalytic phosphatase domain (Figure 1A)9,10. Upon cell activation, tyrosine phosphorylated binding proteins recruit SHP2, causing it to adopt its active conformation, in which the active site is now accessible to its substrates. In many cancers SHP2 activity is elevated. Somatic gain-of-function (GOF) mutations in PTPN11 have been identified mainly in leukemias and prevent binding of the N-SH2 domain to the phosphatase domain, resulting in constitutively active SHP2 (Figure 1B)11. Germline GOF mutations in PTPN11 are responsible for ~50% of cases of Noonan syndrome, a developmental disorder with an increased risk of malignancy12. In solid tumors, where PTPN11 mutations are rare, greater levels of phosphorylated binding proteins lead to enhanced SHP2 activity (Figure 1C). SHP2 is also important for immune checkpoint signaling, as checkpoint receptors such as BTLA or PD-1 recruit SHP2 to dephosphorylate key signaling molecules, preventing immune cell activation13,14,15.
Targeting PTPs with small molecules has been a challenge, because the active site of PTPs is highly conserved and highly charged; inhibitors that target the active site are often potent, but exhibit poor selectivity and oral bioavailability16,17,18,19,20,21,22. Indeed, many reported SHP2 inhibitors suffer from poor selectivity and lack of efficacy in cells23. Recently, allosteric inhibitors of SHP2 with good potency and excellent selectivity have been reported (e.g., SHP09924 and RMC-455025) and have sparked renewed interest in SHP2 inhibitors. Compounds based on SHP099 and RMC-4550 are currently in phase I clinical trials to treat solid tumors with receptor tyrosine kinase (RTK) pathway activation26,27. While groundbreaking, these compounds are ineffective against many of the SHP2 oncogenic mutants that drive leukemogenesis in a significant number of blood cancer patients28,29,30. This lack of potency of SHP099-like compounds toward the SHP2 oncogenic variants stems from their unique allosteric mechanism, as they inhibit SHP2 activity by binding and stabilizing the inactive, closed conformation, which is disrupted in SHP2 mutants. Further, based on a recent report31, adaptive resistance mechanisms in patients treated with SHP099-like inhibitors are quite conceivable. Consequently, the development of next generation SHP2 inhibitors that target its active, open state is a subject of intense research.
The characterization of novel SHP2 inhibitors in cells is an essential aspect of the lead optimization process. Critically, proven target engagement of the inhibitor under physiological conditions provides an additional level of confidence that resources for medicinal chemistry are efficiently deployed on compounds with promising cellular efficacy. In the past, several methods to assess the binding of small molecule inhibitors to their targets have been developed, primarily for protein kinases32. To develop a SHP2 cellular target engagement assay, we utilized a cellular thermal shift assay33. This assay, similar to the in vitro thermal shift (PTS) assay for proteins34, monitors the target protein thermal stability, which is typically altered by the binding of small molecules. The original assay is a low throughput assay that utilizes antibodies to quantify target protein levels. Alternatively, we chose a recently reported variant of the thermal shift assay that utilizes a &#946;-galactosidase enzyme fragment complementation (EFC) assay (Figure 2)35. For these experiments, the protein of interest is expressed in cells as an N- or C-terminal fusion protein carrying an enhanced ProLabel tag (ePL, a 42 amino acid fragment of &#946;-galactosidase). Cells are then transferred to 384-well PCR compatible plates and incubated with compounds of interest. A thermocycler is utilized to apply a temperature gradient to the cells, whose proteins will denature and aggregate as the temperature increases based upon their thermal stability. The ability of a candidate compound to bind and stabilize the protein of interest will result in an increased thermal stability of that protein. Therefore, following the lysis of the cells, those tagged proteins that have been stabilized by a candidate compound will remain in solution at higher temperatures than tagged proteins of cells incubated with vehicle control. Reporter enzyme acceptor (EA) is able to complement the soluble ePL-tagged proteins, resulting in detectable &#946;-galactosidase activity using a luminescence substrate.
We recently developed a robust cellular thermal shift assay for wild-type SHP2 (SHP2-WT) and a frequent SHP2 oncogenic variant (SHP2-E76K) in a miniaturized 384-well format36. Here, we report a detailed protocol of this assay, which reliably detects target engagement by SHP2 inhibitors in cells and demonstrates a high degree of correlation between inhibitor potency and cellular thermal shift data. The general assay workflow is illustrated in Figure 3. Our platform uses N-terminally tagged full-length ePL-SHP2 fusion proteins. For the generation of the corresponding pICP-ePL-N-SHP2-WT and pICP-ePL-N-SHP2-E76K expression plasmids, please refer to our recent publication36. This assay can be performed using a thermal gradient to establish SHP2 thermal profiles and determine SHP2 melting temperatures in the presence or absence of inhibitor. Once thermal profiles have been established, it can also be performed under isothermal conditions, allowing inhibitor dose-response assessment. Both types of experiments are described below.

<table>
	<tbody>
		<tr>
			<td>384-well gradient equipped thermocycler</td>
			<td>Eppendorf AG</td>
			<td>X50h</td>
			<td></td>
		</tr>
		<tr>
			<td>384-well low dead volume microplate Echo qualified</td>
			<td>Beckman Coulter, Inc.</td>
			<td>LP-0200</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well cell culture plates</td>
			<td>Greiner Bio-One</td>
			<td>657 160</td>
			<td>Sterile with lid</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (Anti Anti) 100 X</td>
			<td>Thermo Fisher Scientific</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counter</td>
			<td>Thermo Fisher Scientific</td>
			<td>Countess II FL</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium 1X + GlutaMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>10566-016</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Echo acoustic liquid handler</td>
			<td>Beckman Coulter, Inc.</td>
			<td>Echo 550</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic multichannel pipette</td>
			<td>Thermo Fisher Scientific</td>
			<td>E1 ClipTip</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>26140-079</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>HEPES buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630-680</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>InCell Pulse starter kit</td>
			<td>Eurofins DiscoverX Corp.</td>
			<td>94-4007</td>
			<td>Components include EA buffer, lysis buffer, and substrate</td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Tecan Trading AG</td>
			<td>Spark</td>
			<td></td>
		</tr>
		<tr>
			<td>Single channel solution trough</td>
			<td>Thermo Fisher Scientific</td>
			<td>S253012005</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Thermo Fisher Scientific</td>
			<td>11360-010</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>Thermal microplate sealer</td>
			<td>Agilent Technologies, Inc.</td>
			<td>PlateLoc</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection reagents</td>
			<td>Polyplus Transfection</td>
			<td>jetPRIME</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Thermo Fisher Scientific</td>
			<td>T10282</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>12605-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Twin.tec 384 real-time PCR plates</td>
			<td>Eppendorf AG</td>
			<td>30132734</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing cellular target engagement by shp2 (ptpn11) phosphatase inhibitors

The Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 proto-oncogene, is a key mediator of receptor tyrosine kinase (RTK)-driven cell signaling, promoting cell survival and proliferation. In addition, SHP2 is recruited by immune check point receptors to inhibit B and T cell activation. Aberrant SHP2 function has been implicated in the development, progression, and metastasis of many cancers. Indeed, small molecule SHP2 inhibitors have recently entered clinical trials for the treatment of solid tumors with Ras/Raf/ERK pathway activation, including tumors with some oncogenic Ras mutations. However, the current class of SHP2 inhibitors is not effective against the SHP2 oncogenic variants that occur frequently in leukemias, and the development of specific small molecules that target oncogenic SHP2 is the subject of current research. A common problem with most drug discovery campaigns involving cytosolic proteins like SHP2&#160;is that the primary assays that drive chemical discovery are often in vitro assays that do not report the cellular target engagement of candidate compounds. To provide a platform for measuring cellular target engagement, we developed both wild-type and mutant SHP2 cellular thermal shift assays. These assays reliably detect target engagement of SHP2 inhibitors in cells. Here, we provide a comprehensive protocol of this assay, which provides a valuable tool for the assessment and characterization of SHP2 inhibitors.

The thermal gradient experiment for SHP2-WT resulted in a sigmoidal cellular thermal profile with a narrow melting transition that is typical and consistent for a folded protein (Figure 4A). SHP2 consists of three independent domains: two SH2 domains and the catalytic domain (Figure 1). In the autoinhibited closed conformation these domains self-associate; the melting transition that was observed in the thermal profile experiment presumably reflected this state ...

Watch this Scientific Journal Video about Assessing Cellular Target Engagement by SHP2 PTPN11 Phosphatase Inhibitors at JoVE.com

Assessing Cellular Target Engagement by SHP2 PTPN11 Phosphatase Inhibitors

The ability to assess target engagement by candidate inhibitors in intact cells is crucial for drug discovery. This protocol describes a 384 well format cellular thermal shift assay that reliably detects cellular target engagement of inhibitors targeting either wild-type SHP2 or its oncogenic variants.

Assessing Cellular Target Engagement by SHP2 (PTPN11) Phosphatase Inhibitors

The Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 proto-oncogene, is a key mediator of receptor tyrosine kinase ...

assessing-cellular-target-engagement-shp2-ptpn11-phosphatase

Research

JoVE Journal

Cancer Research

6.1K Views.  Sanford Burnham Prebys Medical Discovery Institute. The ability to assess target engagement by candidate inhibitors in intact cells is crucial for drug discovery. This protocol describes a 384 well format cellular thermal shift assay that reliably detects cellular target engagement of inhibitors targeting either wild-type SHP2 or its oncogenic variants.

Video: Assessing Cellular Target Engagement by SHP2 PTPN11 Phosphatase Inhibitors

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

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.

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 ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

A Spheroid Killing Assay by CAR T Cells

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric antigen receptor (CAR) redirected T cells obtained the most spectacular results, in particular with pediatric B-acute lymphoblastic leukemia (B-ALL). Classical validation methods of CAR T cells rely on the use of specificity and functionality assays of the CAR T cells against target cells in suspension and in xenograft models. Unfortunately, observations made in vitro are often decoupled from results obtained in vivo and a lot of effort and animals could be spared by adding another step: the use of 3D culture. The production of spheroids out of potential target cells that mimic the 3D structure of the tumor cells when they are engrafted into the animal model represents an ideal alternative. Here, we report an affordable, reliable and easy method to produce spheroids from a transduced colorectal cell line as a validation tool for adoptive cell therapy (exemplified here by CD19 CAR T cells). This method is coupled with an advanced live imaging system that can follow spheroid growth, effector cells cytotoxicity and tumor cell apoptosis.

This protocol is designed to assess immunotherapeutic redirected T-cell (CAR T-cell) cytotoxicity against 3D structured cancerous cells (spheroids) in real time.

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

Using Microarrays to Interrogate Microenvironmental Impact on Cellular Phenotypes in Cancer

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth factors and matrix-associated proteins in the microenvironment in vivo. Furthermore, readily available reagents for the modeling of microenvironments in vitro typically utilize complex mixtures of proteins that are incompletely defined and suffer from batch to batch variability. The microenvironment microarray (MEMA) platform allows for the assessment of thousands of simple combinations of microenvironment proteins for their impact on cellular phenotypes in a single assay. The MEMAs are prepared in well plates, which allows the addition of individual ligands to separate wells containing arrayed extracellular matrix (ECM) proteins. The combination of the soluble ligand with each printed ECM forms a unique combination. A typical MEMA assay contains greater than 2,500 unique combinatorial microenvironments that cells are exposed to in a single assay. As a test case, the breast cancer cell line MCF7 was plated on the MEMA platform. Analysis of this assay identified factors that both enhance and inhibit the growth and proliferation of these cells. The MEMA platform is highly flexible and can be extended for use with other biological questions beyond cancer research.

The purpose of the method presented here is to show how microenvironment microarrays (MEMA) can be fabricated and used to interrogate the impact of thousands of simple combinatorial microenvironments on the phenotype of cultured cells.

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth ...

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental disorders. A characterization of disease-related RAF mutants will help us select appropriate therapeutic strategies for treating these diseases. Recent studies have shown that RAF family kinases have both catalytic and allosteric activities, which are tightly regulated by dimerization. Here, we constructed a set of practical and feasible methods to determine the catalytic and allosteric activities and the relative dimer affinity/stability of RAF family kinases and their mutants. Firstly, we amended the classical in vitro kinase assay by reducing the detergent concentration in buffers, utilizing a gentle quick wash procedure, and employing a glutathione S-transferase (GST) fusion to prevent RAF dimers from dissociating during purification. This enables us to measure the catalytic activity of constitutively active RAF mutants appropriately. Secondly, we developed a novel RAF co-activation assay to evaluate the allosteric activity of kinase-dead RAF mutants by using N-terminal truncated RAF proteins, eliminating the requirement of active Ras in current protocols and thereby achieving a higher sensitivity. Lastly, we generated a unique complementary split luciferase assay to quantitatively measure the relative dimer affinity/stability of various RAF mutants, which is more reliable and sensitive compared to the traditional co-immunoprecipitation assay. In summary, these methods have the following advantages: (1) user-friendly; (2) able to carry out effectively without advanced equipment; (3) cost-effective; (4) highly sensitive and reproducible.

In this article, we presented a set of practical and feasible methods for characterizing disease-related mutants of RAF family kinases, which include in vitro kinase assay, RAF co-activation assay, and complementary split luciferase assay.

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental ...

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 ...

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few decades, the field of antibody-drug conjugates, bispecific antibodies, chimeric antigen receptors (CAR) and cancer immunotherapy has emerged as the most promising area of basic and therapeutic investigations. With numerous successful human trials targeting immune checkpoint receptors and CAR-T cells in leukemia and melanoma at a breakthrough pace, it is highly exciting times for oncologic therapeutics derived from variations of antibody engineering. Regrettably, a significantly large numbers of antibody and CAR based therapeutics have also proven disappointing in human trials of solid cancers because of the limited availability of immune effector cells in the tumor bed. Importantly, nonspecific distribution of therapeutic antibodies in tissues other than tumors also contribute to the lack of clinical efficacy, associated toxicity and clinical failure. As faithful translation of preclinical studies into human clinical trails are highly relied on mice tumor xenograft efficacy and safety studies, here we highlight a method to test the tumor and general tissue distribution of therapeutic antibodies. This is achieved by labeling the protein-A purified antibody with near Infrared fluorescent dye followed by live imaging of tumor bearing mice.

Here we present a protocol to study the in vivo localization of antibodies in mice tumor xenograft models.

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few ...

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.