The authors would like to acknowledge the Hairy Cell Leukemia Fellowship for support of Yuan Jimin. This work was supported by Asia Fund Cancer Research (AFCR2017/2019-JH), Duke-NUS Khoo Bridge Funding Award (Duke-NUS-KBrFA/2018/0014), NCCRF bridging grant (NCCRF-YR2018-JUL-BG4), NCCRF pilot grant (NCCRF-YR2017-JUL-PG3), and SHF Academic Medicine Research Grant (AM/TP011/2018).

1. In Vitro Kinase Assay for Measuring the Catalytic Activity of RAF Mutants
<ol>
	<li>Construct vectors encoding RAF mutants (Figure 1A) with FLAG(DYKDDDDK) tag at C-terminus by using Gibson Assembly or traditional molecular cloning methods.
	<ol>
		<li>Introduce the FLAG tag and mutations into the RAF coding sequences by PCRs, and then insert whole sequences into pCDNA3.1(+) vector by using Gibson assembly or T4 DNA ligation and following the manufacture&#8217;s protocols. Use the following conditions for PCR reactions: (1) 95 &#176;C, 2 min; (2) 95 &#176;C, 30 s; (3) 59 &#176;C, 30 s; (4) 68 &#176;C, 3 min; (5) 20 cycles of (2 to 4); (6) 4 &#176;C hold. 
		NOTE: The PCR primers for cloning: 5- AAATTAATACGACTCACTATAGGGAGACCC-3 and 5-CAGCGGGTTTAAACGGGCCCTCTA-3.</li>
		<li>Insert the GST coding sequence upstream of RAF mutant coding sequences to generate vectors encoding GST-fused RAF mutants by using same methods as described in step 1.1.1.</li>
		<li>Validate all vectors by DNA sequencings before transfection.</li>
	</ol>
	</li>
	<li>Plate 293T cells in 6-well plates at a density of 5 x 105 cells/well one day before transfection. When the cell density reaches 80~90% confluence on the second day, transfect with vectors encoding FLAG-tagged RAF kinases or their mutants from step 1.1 into cells by following the manufacture&#8217;s protocol of transfection reagents (Table of Materials).</li>
	<li>Replace the culture medium 24 h after transfection.</li>
	<li>Aspirate the culture medium 48 h after transfection and add 400 &#181;L/well of lysis buffer (25 mM Tris&#183;HCl, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.25% NP-40, pH 7.2) supplemented with protease and phosphatase inhibitors to lyse cells on ice. 
	NOTE: The concentration of NP-40 in lysis buffer is critical for detecting the catalytic activity of RAF mutants with moderate dimer affinity/stability in vitro. A high concentration of detergent or a strong detergent in lysis buffer may break RAF dimers and thereby kill the catalytic activity of RAF kinases or their mutants.</li>
	<li>Transfer the cell lysates to a 1.5 mL tube, and spin down by 12,000 x g for 10 min at 4 &#176;C to deplete cell debris.</li>
	<li>Transfer 300 &#181;L per sample of clean whole cell lysates to 1.5 mL tubes, add 20 &#181;L per sample of anti-FLAG affinity beads, and rotate in a cold room (4 &#176;C) for 1 h. Also take 40 &#181;L per sample of clean whole cell lysate aside for detecting the expression and activity (phospho-ERK1/2) of RAF mutants by immunoblots as described below.</li>
	<li>Wash the anti-FLAG beads once with lysis buffer, then once with kinase reaction buffer (20 mM HEPES, 10 mM MgCl2, 0.5 mM Na3VO4, 0.5 mM DTT, pH 7.2), and add 20 &#181;L of kinase reaction mixture (2 &#956;g of MEK1(K97A) and 100 &#181;M ATP in 20 &#181;L of kinase reaction buffer) per sample. 
	NOTE: The bead washing should be completed gently and quickly, the residual buffer should be aspirated completely before adding kinase reaction mixture, and all operations at this step should be carried out at 4 &#176;C in a cold room.</li>
	<li>Incubate the kinase reactions at room temperature (25 &#176;C) for 10 min, and flip the tubes containing kinase reactions with fingers every other minute during incubation.</li>
	<li>Add 5 &#181;L of 5x SDS sample buffer (375 mM Tris&#183;HCl, 9% sodium dodecyl sulfate (SDS), 50% Glycerol, 0.03% Bromophenol Blue) per sample to stop kinase reactions, and then heat the samples at 90 &#176;C for 5 min.</li>
	<li>Run the samples in 9~12% polyacrylamide gel electrophoresis (PAGE) with 0.1% SDS, transfer the proteins to nitrocellulose membrane, and detect the levels of phospho-MEK and RAF mutants in samples by immunoblots. 
	NOTE: The phospho-MEK can also be quantified by using &#947;32P-ATP incorporation. Briefly, 10 &#181;M &#947;32P-ATP is added to the kinase reaction buffer, and the amount of phosphorylated MEK is then quantified after PAGE separation by using standard autoradiography, phosphorimaging, or liquid scintillation counting techniques as appropriate.</li>
</ol>
2. RAF Co-activation Assay for Evaluating the Allosteric Activity of Kinase-dead RAF Mutants
<ol>
	<li>Construct vectors encoding the RAF receiver (CRAF kinase domain with unphosphorylatable NtA motif, AAFF) or the kinase-dead RAF activators (RAF kinase domain with phosphorylation-mimicked NtA motif, SSDD, DDEE or DGEE) (Figure 1A) as described in step 1.1.</li>
	<li>Transfect 293T cells with two vectors encoding both the RAF receiver and the kinase-dead RAF activator or a single vector encoding one of proteins as described in steps 1.2 and 1.3.</li>
	<li>Replace the culture medium at 24 h after transfection, and harvest 293T transfectants at 48 h to prepare the whole cell lysates as described in steps 1.4 and 1.5.</li>
	<li>Mix the clean whole cell lysate with 5x SDS sample buffer quickly at room temperature (25 &#176;C) and then boil at 90 &#176;C for 5 min.</li>
	<li>Run the boiled whole cell lysate samples in 9~12% PAGE with 0.1% SDS, transfer the proteins to nitrocellulose membrane, and detect the levels of phospho-ERK1/2 and control proteins by immunoblots.</li>
</ol>
3. Complimentary Split Luciferase Assay for Measuring the Relative Dimer Affinity/Stability of RAF Mutants
<ol>
	<li>Construct vectors encoding FLAG-tagged RAF mutants fused to the N-terminus of Nluc (N-terminus of firefly luciferase, aa2-416) or the C-terminus of Cluc (C-terminus of firefly luciferase, aa398-550) as described in step 1.1.</li>
	<li>Transfect 293T cells with a pair of vectors encoding different Nluc-RAF mutants and Cluc-RAF mutants as described in step 1.2.</li>
	<li>At 24 h after transfection, replate 293T cell transfectants into Krystal black image plates at the cell density of 2x105 per well with color-free medium (i.e., DMEM without phenol red).</li>
	<li>24 h later, add D-luciferin (0.2 mg/mL) to 293T cell transfectants, incubate for 30 min, and measure the luciferase signals by using a multi detection system (Table of Materials).</li>
	<li>After measuring the luciferase signals, aspirate the medium and lyse 293T transfectants with lysis buffer to prepare the whole cell lysates as described in steps 1.4 and 1.5.</li>
	<li>Run the whole cell lysate samples in 9~12% PAGE with 0.1% SDS and detect the expression levels of Nluc-RAF mutants and Cluc-RAF mutants by anti-FLAG immunoblot as described in step 2.5. The relative expression levels of both Nluc-RAF mutant and Cluc-RAF mutant in 293T transfectants are quantified by using image J from their immunoblots.</li>
	<li>Normalize the luciferase signals of 293T cell transfectants according to the expression levels of Nluc-RAF mutants and Cluc-RAF mutants. Briefly, this is achieved by dividing the raw luciferase signal by the relative expression levels of Nluc-RAF mutants and Cluc-RAF mutants from step 3.6.</li>
</ol>

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The authors declare that they have no competing financial interests.

In this article, we presented three methods for characterizing disease-related RAF mutants, which include in vitro kinase assay, RAF co-activation assay, and complimentary split luciferase assay. Since RAF kinases have both catalytic activity and allosteric activity, various RAF mutants can activate the downstream signaling through two distinct mechanisms13,14,16,17,<sup class="xref"...

The RAF family kinases are a key component of RAS/RAF/MEK/ERK signaling cascade, which transmit a signal from RAS to activate mitogen-activated protein kinase (MEK)1,2,3,4. This family of kinases plays a crucial role in cell growth, survival and differentiation, and their alterations induce many diseases, notably cancer5,6,7,8. Recently, genomic sequencings have identified many disease-related RAF mutants that exhibit different properties in the signal transmission of RAS/RAF/MEK/ERK cascade9,10,11. A careful characterization of RAF mutants will help us understand the molecular mechanisms of how RAF mutants alter the signal output of RAS/RAF/MEK/ERK cascade, eventually select appropriate approaches for treating various RAF mutant-driven diseases.
The RAF family kinases include three members, CRAF, BRAF, and ARAF, which have similar molecular structures but different abilities to activate downstream signaling1,2,3,4. Among these paralogs, BRAF has the highest activity by virtue of its constitutively phosphorylated NtA (N-terminal acidic) motif12,13,14, while ARAF has the lowest activity arising from its non-canonical APE motif15. This may explain the different mutation frequencies of RAF paralogs in diseases: BRAF&#62;CRAF&#62;ARAF. Moreover, within the same RAF paralog, mutations in different sites may trigger downstream signaling in distinct manners, which adds another layer of complexity to the regulation of RAF family kinases. Recent studies have demonstrated that all RAF kinases have both catalytic and allosteric activities13,14,16,17,18. Constitutively active RAF mutants turn on the downstream signaling directly by phosphorylating MEK, whereas kinase-dead RAF mutants can transactivate their wild-type counterparts through side-to-side dimerization and activate MEK-ERK signaling16,19,20. The dimer affinity/stability is a key parameter that not only determines the allosteric activity of kinase-dead RAF mutants but also affects the catalytic activity of constitutively active RAF mutants15,21,22. The kinase-dead RAF mutants with high dimer affinity/stability can transactivate the endogenous wild-type RAFs directly15, while those with intermediate dimer affinity/stability requires a coordination of active Ras or an elevated level of wild-type RAF molecules to function13,15,20,21,23. Similarly, constitutively active RAF mutants phosphorylate MEK in a dimer-dependent manner, and those with low dimer affinity/stability lose their catalytic activity in vitro upon immunoprecipitation that breaks the weak RAF dimers15,21,22. The dimer affinity/stability also determines the sensitivity of RAF mutants to their inhibitors, and positively correlates to the resistance of RAF inhibitors24. Therefore, to characterize disease-related RAF mutants, it is necessary to measure their catalytic and allosteric activities, and dimer affinity/stability.
In recent years, our laboratory and others have developed various methods to characterize RAF family kinases and their mutants. According to our laboratory and others&#39;&#160;experience, we think that the following three assays have advantages in defining disease-related RAF mutants: (1) the in vitro kinase assay that can be carried out with ease to detect the catalytic activity of constitutively active RAF mutants15; (2) the RAF co-activation assay that is a reliable and convenient method to measure the allosteric activity of kinase-dead RAF mutants13,15,21,22,23,25; (3) the complimentary split luciferase assay that has much higher sensitivity in measuring the relative dimer affinity/stability of RAF mutants in contrast to the traditional co-immunoprecipitation assay, and is able to carry out without advanced equipment in contrast to the quantitative analytic methods such as SPR (Surface Plasmon Resonance) analysis15,22. Combining these three assays, we can understand easily how a disease-related RAF mutant alters the downstream signaling and thereby utilize an appropriate therapeutic strategy to treat the disease caused by this RAF mutation.

<table border="0" cellpadding="0" cellspacing="0" style="width:660px;" width="660">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">anti-phosphoERK1/2</td>
			<td style="width:197px;">Cell Signaling Technologies</td>
			<td style="width:104px;">4370</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">anti-phosphoMEK1/2</td>
			<td style="width:197px;">Cell Signaling Technologies</td>
			<td style="width:104px;">9154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">anti-ERK1/2</td>
			<td style="width:197px;">AB clonal</td>
			<td style="width:104px;">A0229</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">anti-MEK1/2</td>
			<td style="width:197px;">Cell Signaling Technologies</td>
			<td style="width:104px;">9124</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">anti-FLAG(mouse)</td>
			<td style="width:197px;">Sigma-Aldrich</td>
			<td style="width:104px;">F3165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">anti-HA</td>
			<td style="width:197px;">Novus Biologicals</td>
			<td style="width:104px;">MAB6875</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">anti-FLAG(Rabbit)</td>
			<td style="width:197px;">Cell Signaling Technologies</td>
			<td style="width:104px;">14793</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">anti-&#946;-actin</td>
			<td style="width:197px;">Sigma-Aldrich</td>
			<td style="width:104px;">A2228</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">anti-FLAG beads(M2)</td>
			<td style="width:197px;">Sigma-Aldrich</td>
			<td style="width:104px;">A4596</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">HRP-conjugated anti-mouse IgG</td>
			<td style="width:197px;">Jackson Laboratories</td>
			<td style="width:104px;">115-035-003</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">HRP-conjugated anti-Rabbit IgG</td>
			<td style="width:197px;">Jackson Laboratories</td>
			<td style="width:104px;">111-035-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">pcDNA3.1(+)</td>
			<td style="width:197px;">In vitrogen</td>
			<td style="width:104px;">V79020</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Gibson Assembly Cloning&#160; Kit</td>
			<td style="width:197px;">New England Biolabs</td>
			<td style="width:104px;">E5510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">T4 DNA ligase</td>
			<td style="width:197px;">New England Biolabs</td>
			<td style="width:104px;">M0202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">lipofectamine 2000</td>
			<td style="width:197px;">Invitrogen</td>
			<td align="right" style="width:104px;">11668019</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Fugene 6</td>
			<td style="width:197px;">Roche</td>
			<td style="width:104px;">11 814 443 001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">DMEM w/o phenol red</td>
			<td style="width:197px;">Invitrogen</td>
			<td style="width:104px;">21063-029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-luciferin&#160;</td>
			<td style="width:197px;">GoldBio</td>
			<td style="width:104px;">LUCK-100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">6xhis-tagged MEK1 (K97A)</td>
			<td style="width:197px;">&#160;prepared in our previous studies</td>
			<td style="width:104px;">N.A.</td>
			<td>Reference 15.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">GloMax-Multi Detection System.</td>
			<td style="width:197px;">Promega</td>
			<td style="width:104px;">E7041</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The RAF family kinases have both catalytic and allosteric activities, which enable their disease-related mutants to turn on the downstream signaling through different mechanisms13,14,16,17,18. The constitutively active RAF mutants directly phosphorylate their substrates, while the kinase-dead RAF mutants fulfill their function through transactivating wild-type...

Watch this Scientific Journal Video about Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods at JoVE.com

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

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 ...

characterize-disease-related-mutants-raf-family-kinases-using-set

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

5.9K Views.  National Cancer Centre Singapore. 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.

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

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without the ability to target a specific or well-delineated tumor volume. The delivery of radiation was achieved using fixed radiation sources or linear accelerators producing megavoltage (MV) X-rays. These devices are unable to achieve sub-millimeter precision required for small animals. Furthermore, the high doses delivered to healthy surrounding tissue hamper response assessment. To increase the translation between small animal studies and humans, our goal was to mimic the treatment of human glioblastoma in a rat model. To enable a more accurate irradiation in a preclinical setting, recently, precision image-guided small animal radiation research platforms were developed. Similar to human planning systems, treatment planning on these micro-irradiators is based on computed tomography (CT). However, low soft-tissue contrast on CT makes it very challenging to localize targets in certain tissues, such as the brain. Therefore, incorporating magnetic resonance imaging (MRI), which has excellent soft-tissue contrast compared to CT, would enable a more precise delineation of the target for irradiation. In the last decade also biological imaging techniques, such as positron emission tomography (PET) gained interest for radiation therapy treatment guidance. PET enables the visualization of e.g., glucose consumption, amino-acid transport, or hypoxia, present in the tumor. Targeting those highly proliferative or radio-resistant parts of the tumor with a higher dose could give a survival benefit. This hypothesis led to the introduction of the biological tumor volume (BTV), besides the conventional gross target volume (GTV), clinical target volume (CTV), and planned target volume (PTV).
At the preclinical imaging lab of Ghent University, a micro-irradiator, a small animal PET, and a 7 T small animal MRI are available. The goal was to incorporate MRI-guided irradiation and PET-guided sub-volume boosting in a glioblastoma rat model.

In the past, small animal irradiation was usually performed without the ability to target a well-delineated tumor volume. The goal was to mimic the treatment of human glioblastoma in rats. Using a small animal irradiation platform, we performed MRI-guided 3D conformal irradiation with PET-based sub-volume boosting in a preclinical setting.

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without ...

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, ...

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 ...

Detection of Protease Activity by Fluorescent Peptide Zymography

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using electrophoresis through a resolving gel embedded with a degradable substrate. This method differs from traditional gel zymography in that a quenched fluorogenic peptide is covalently incorporated into the resolving gel instead of full length proteins, such as gelatin or casein. Use of the fluorogenic peptides enables direct detection of proteolytic activity without additional staining steps. Enzymes within the biological samples cleave the quenched fluorogenic peptide, resulting in an increase in fluorescence. The fluorescent signal in the gels is then imaged with a standard fluorescent gel scanner and quantified using densitometry. The use of peptides as the degradable substrate greatly expands the possible proteases detectable with zymographic techniques.

Here, we present a detailed protocol for a modified zymographic technique in which fluorescent peptides are used as the degradable substrate in place of native proteins. Electrophoresis of biological samples in fluorescent peptide zymograms enables detection of a wider range of proteases than previous zymographic techniques.

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using ...

A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

Neuroendocrine tumors are a rare form of cancer that arise from neuroendocrine cells and can be present at almost any location throughout the body. Although heterogeneous in presentation, a common denominator among these tumors is the overexpression of somatostatin receptors. 68Ga-DOTATATE is a somatostatin analog labeled with the positron emitter gallium-68 (68Ga). For well-differentiated neuroendocrine tumors, 68Ga-DOTATATE positron emission tomography (PET)/computed tomography (CT) imaging is used for diagnosis, determination of disease burden, and therapy selection.
This protocol details the radiolabeling of 68Ga-DOTATATE, quality control, patient preparation, and subsequent PET/CT imaging. Radiolabeling of 68Ga-DOTATATE is performed with a fully automated labeling module coupled to a germanium-68 (68Ge)/68Ga generator. Quality control of the final product evaluates radiochemical purity with instant thin-layer chromatography and solid-phase chromatography, and pH prior to patient injection. Periodic quality control is performed to determine 68Ge breakthrough, sterility, and (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) content. Patient preparation includes patient instructions, a protocol for 68Ga-DOTATATE during treatment with somatostatin analogs, and intravenous administration of the radiopharmaceutical. For PET/CT imaging, the acquisition and reconstruction settings are described. For each step, radiation safety will be highlighted, as well as time constrictions due to the short half-life of 68Ga.
Fully automated in-house production and quality control of 68Ga-DOTATATE leads to very high success rates (95%) and produces two to four patient dosages per batch, depending on the yield of the generator. In conclusion, 68Ga-DOTATATE PET/CT imaging is a noninvasive and fast method of providing information on the tumor burden of neuroendocrine tumors (NETs) while also assisting in diagnosis and therapy selection.

A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

Well-differentiated neuroendocrine tumors overexpress somatostatin receptors which can be utilized for diagnostic imaging with the radiolabeled somatostatin analog 68Ga-DOTATATE. This protocol details the radiolabeling of 68Ga-DOTATATE, quality control, patient preparation, and subsequent PET/CT imaging. Radiation safety and time constrictions due to the short half-life of 68Ga are taken into account.

Neuroendocrine tumors are a rare form of cancer that arise from neuroendocrine cells and can be present at almost any location throughout the body. ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells

Formation of the eIF4F complex has been shown to be the key downstream node for the convergence of signalling pathways that often undergo&#160;oncogenic activation in humans. eIF4F is a cap-binding complex involved in the mRNA-ribosome recruitment phase of translation initiation. In many cellular and pre-clinical model of cancers, the deregulation of eIF4F leads to increased translation of specific mRNA subsets that are involved in cancer proliferation and survival. eIF4F is a hetero-trimeric complex built from the cap-binding subunit eIF4E, the helicase eIF4A and the scaffolding subunit eIF4G. Critical for the assembly of active eIF4F complexes is the protein-protein interaction between eIF4E and eIF4G proteins. In this article, we describe a protocol to measure eIF4F assembly that monitors the status of eIF4E-eIF4G interaction in live cells. The eIF4e:4G cell-based protein-protein interaction assay also allows drug induced changes in eIF4F complex integrity to be accurately and reliably assessed. We envision that this method can be applied for verifying the activity of commercially available compounds or for further screening of novel compounds or modalities that efficiently disrupt formation of eIF4F complex.

Here, we present a protocol to measure eIF4E-eIF4G interaction in live cells that would enable the user to evaluate drug induced perturbation of eIF4F complex dynamics in screening formats.

Formation of the eIF4F complex has been shown to be the key downstream node for the convergence of signalling pathways that often undergo&#160;oncogenic ...

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and treatment response to targeted inhibitors in cancer. Pioneering work in gastrointestinal and pancreatic cancers has highlighted the promise of patient-derived organoids (PDOs) as a patient-proximate culture system, with an increasing number of models emerging. Similarly, work in other cancer types has focused on establishing organoid models and optimizing culture protocols. Notably, 3D cancer organoid models maintain the genetic complexity of original tumor specimens and thus translate tumor-derived sequencing data into treatment with genetically informed targeted therapies in an experimental setting. Further, PDOs might foster the evaluation of rational combination treatments to overcome resistance-associated adaptation of tumors in the future. The latter focuses on intense research efforts in non-small-cell lung cancer (NSCLC), as resistance development ultimately limits the treatment success of targeted inhibitors. An early assessment of therapeutically targetable mechanisms using NSCLC PDOs could help inform rational combination treatments. This manuscript describes a standardized protocol for the cell culture plate-based assessment of drug sensitivities to targeted inhibitors in NSCLC-derived 3D PDOs, with potential adaptability to combinational treatments and other treatment modalities.

This protocol describes a standardized evaluation of drug sensitivities to targeted signaling inhibitors in NSCLC patient-derived organoid models.

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and ...