We thank Dr. Swathi Arur (MD Anderson Cancer Center) for providing the let-60(n1046). We also thank Dr. David Reiner (Texas A&#38;M Health Science Center Institute of Biosciences &#38; Technology in Houston) for the lin-1 strain. Finally, we thank Dr. Danielle Garsin and her lab (The University of Texas, McGovern Medical School) for providing some of the reagents. Some worm strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). This Research was supported by the Cancer Prevention and Research Institute of Texas (CPRIT) grant RP200047 to JF Hancock.

1. Nematode growth medium (NGM) plate preparation
<ol>
	<li>Add 2.5 g of peptone and 3 g of NaCl to 970 mL of deionized water contained in a 2 L Erlenmeyer flask. Stir contents using a magnetic stir bar. Thereafter, add 20 g of agar to the flask. Autoclave the contents of the flask at 121 &#176;C and a pressure of 15 lb/in2 for 30 min. After sterilization, place the flask on a stir plate and allow the medium to cool until the temperature reaches 50 &#176;C.</li>
	<li>To prepare the NGM plates add the following reagents to the cooled medium: 25 mL of 1 M potassium phosphate buffer (pH = 6.0), 1 mL of 1 M MgSO4, 1 mL of 1 M CaCl2, 1 mL of (5 mg/mL in 95% ethanol) cholesterol, 1 mL of (10% v/w in ethanol) nystatin, and 1 mL of 25 mg/mL streptomycin.</li>
	<li>Under a laminar flow, pour the cooled medium into 60 mm x 15 mm sterile Petri dishes. Let the plates solidify for 2 h. These plates can be kept for 1 month at 4 &#176;C.</li>
</ol>
2. Propagation of C. elegans
<ol>
	<li>Spot 100 &#956;L of overnight grown E. coli OP50 onto the center of each NGM plate and allow the plates to dry for 24 h in a laminar hood. Subsequently, the plates can be stored in polystyrene container. 
	NOTE: The E. coli OP50 culture is grown in Luria-Bertani (LB) media at 37 &#176;C media prior to seeding of the plates. The culture can be stored at 4 &#176;C for 1&#8211;2 months and used for periodic seeding of plates.</li>
	<li>Using a sterile worm pick, gather 10&#8211;12 gravid adult worms from a previously grown plate under a dissecting microscope. Transfer these worms to a fresh NGM plate seeded with E. coli OP50 and incubate the plate for 24 h at 20 &#176;C.</li>
	<li>After 24 h, using a sterile worm-pick remove adult worms from the plates. Incubate the plates at 20 &#176;C for ~ 3 days. Embryos will develop into gravid adult worms.</li>
</ol>
3. Preparation of a synchronous C. elegans culture
<ol>
	<li>Collect gravid adult worms into a 15 mL conical tube by washing 2&#8211;4 plates with M9W.
	<ol>
		<li>Preparation of M9W: Dissolve 5 g of NaCl, 6 g of Na2HPO4, and 3 g of KH2PO4 in deionized water to a final volume of 1 L. Autoclave the solution at 121 &#176;C at a pressure of 15 lb/in2 for 30 min. Add 1 mL of 1 M MgSO4 to the cooled solution.</li>
	</ol>
	</li>
	<li>Pellet the worms by centrifuging the tube at 450 x g for 1 min. Decant the supernatant without disturbing the worm pellet.</li>
	<li>Prepare worm lysis solution by combining 400 &#956;L of 8.25% sodium hypochlorite (household bleach) and 100 &#956;L of 5 N NaOH. Add this solution to the worm pellet and flick the tube to mix contents of the tube. Prevent overbleaching of the embryos in the tube by observing the lysis of the worms under a dissecting microscope.</li>
	<li>Add 10 mL of M9W to the conical tube to dilute the lysis mix when 70% of the adult worms have lysed.</li>
	<li>Centrifuge the tube at 450 x g for 1 min. Replace the supernatant with 10 mL of M9W.</li>
	<li>Repeat step 3.5 two times.</li>
	<li>After completing the washing steps, add 3&#8211;5 mL of M9W to resuspend the egg pellet. Rotate the tube overnight at a speed of 18 rpm on a tube rotator at room temperature (RT).</li>
	<li>After overnight incubation, remove the tube from the rotator, pellet the L1 larvae by centrifuging the tube at 450 x g for 1 min. Aspirate the M9W until 250 &#956;L is left in the tube.</li>
	<li>Shake the tube to resuspend the larvae. Add three 5 &#956;L drops containing the larvae onto a Petri dish lid. Using a dissecting microscope count the number of worms in each drop and determine the number of worms in 1 &#956;L.</li>
</ol>
4. Preparation of drug assays
NOTE: The steps in this assay are shown in Figure 1.
<ol>
	<li>Grow 30 mL of E. coli OP50 in a 50 mL conical tube at 37 &#176;C overnight in an orbital shaker at 150 rpm.</li>
	<li>Spin overnight grown E. coli OP50 culture at 4,000 x g for 10 min to pellet the cells. Remove the supernatant and resuspend the pellet in 3 mL of M9W to concentrate the culture.</li>
	<li>Prior to preparing the working solutions for the drug assay, add 0.1 mL of (5 mg/mL in 95% ethanol) cholesterol into 100 mL of M9W.</li>
	<li>Prepare working solutions of each experimental drug by diluting each drug plus vehicle (dimethyl sulfoxide; DMSO) in 4.8 mL M9W supplemented with cholesterol to obtain the appropriate concentrations. Dissolve DMSO in 4.8 mL of M9W supplemented with cholesterol to prepare the vehicle control.</li>
	<li>Add 200 &#956;L of concentrated E. coli OP50 culture to each tube containing the vehicle control or drugs and vortex tubes to ensure they are mixed.</li>
	<li>To perform the experiment, add 2 mL of each working drug solution or vehicle control to each well in a 12-well tissue culture plate. Test each drug concentration and vehicle control in duplicate.</li>
	<li>Add ~100 L1 larvae per well (see synchronous C. elegans culture steps) using a sterile micropipette. Limit the volume of worms to 10 &#956;L.</li>
	<li>Incubate the plates at 20 &#176;C.</li>
	<li>Supplement wells with 50 &#956;L of 10x concentrated E. coli OP50 on day 3 of the assay if needed.</li>
</ol>
5. Agarose pad preparation for microscopy
<ol>
	<li>Prepare a 2% w/v solution of agarose by dissolving 0.1 g of agarose in 5 mL of deionized water. Heat the contents in a microwave to dissolve the agarose. 20 slides can be prepared from 5 mL of agarose solution.</li>
	<li>Place strips of lab tape along two glass slides. The tape will act as spacers limiting the thickness of the agarose pads. Thereafter, place a third clean glass slide between the taped slides.</li>
	<li>To make an agarose pad, spot 100 &#956;L of molten agarose onto the center of the clean slide. Place another clean glass slide across and on top of the agarose and gently press down to form a pad. Remove the top slide when the pad has solidified.</li>
</ol>
6. Observation of the Muv phenotype in the let-60, let-23 and lin-1 strains
NOTE: Only candidate drugs that suppress the Muv phenotypes in let-23 and let-60 strains will be assayed using the lin-1 strain to determine if the inhibition occurs at the level of RAS or EGFR.
<ol>
	<li>When the appropriate stage of the life cycle is reached (3 days for the let-60 and let-23 strains, 5 days for the lin-1 strain), remove the plates from the incubator. and collect the worms in 15 mL conical tubes using a pipette.</li>
	<li>Centrifuge the tubes at 450 x g for 1 min. Remove M9W without disturbing the worm pellet and add 5 mL of fresh M9W.</li>
	<li>Repeat step 6.2 two times.</li>
	<li>Without disturbing the worm pellet, remove all remaining M9W by aspiration. Thereafter, add 500 &#956;L of 2 mM sodium azide or 2 mM tetramisole hydrochloride. Allow tubes to incubate at RT for 15 min. 2 mM sodium azide or 2 mM tetramisole hydrochloride will anesthetize the worms for imaging. 
	CAUTION: Make sure to wear personal protective equipment (PPE) when handling sodium azide. The solution must be prepared under a chemical hood.</li>
	<li>Add 10 &#956;L of the anesthetized worm suspension onto the center of an agarose pad. Place a #1.5 coverslip gently over the worm suspension. If needed, fix the coverslip with some nail polish to prevent drying out.</li>
	<li>Use a DIC microscope to observe the let-60(n1046), let-23(sa62) and lin-1(sy254) strains. Image the worms at the 10x and 20x magnifications.</li>
	<li>For let-60(n1046) and let-23(sa62), score the adult worms based on the presence or absence of the Muv phenotype. While for the lin-1 strain, count the number VPCs that adopted 1&#176; or 2&#176; cell fates on the ventral side of L4 larvae using a high resolution DIC microscope.</li>
	<li>After scoring the micrographs for each strain and drug treatment, perform Student&#8217;s t-test to determine the statistical differences between the treatments.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+levels+of+transforming+growth+factor+alpha+and+epidermal+growth+factor+receptor+messenger+RNA+are+early+markers+of+carcinogenesis+in+head+and+neck+cancer.">Elevated levels of transforming growth factor alpha and epidermal growth factor receptor messenger RNA are early markers of carcinogenesis in head and neck cancer.</a> Cancer Research. 53 (15), 3579-3584 (1993).</li><li>Sasahira, T., Kirita, T., Kuniyasu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+of+molecular+pathobiology+in+oral+cancer:+a+review.">Update of molecular pathobiology in oral cancer: a review.</a> International Journal of Clinical Oncology. 19 (3), 431-436 (2014).</li><li>Stransky, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mutational+landscape+of+head+and+neck+squamous+cell+carcinoma.">The mutational landscape of head and neck squamous cell carcinoma.</a> Science. 333 (6046), 1157-1160 (2011).</li><li>Bos, J. 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The authors declare no competing financial interests.

The assays we describe using the worm are simple and inexpensive to identify inhibitors of EGFR and RAS function. C. elegans is an attractive model for drug discovery because it is easy to grow in the lab due to the short life cycle (3 days at 20 &#176;C) and the ability to generate large numbers of larvae. More importantly, the EGFR-RAS-ERK MAPK pathway is evolutionarily and functionally conserved with mammals providing a genetically tractable system to analyze the effects of EGFR and RAS inhibitors. Further, t...

The cellular pathways that regulate developmental events within organisms are highly conserved among all metazoans. One such pathway is the EGFR-RAS-ERK mitogen activated protein kinase (MAPK) signaling cascade which is a critical pathway that governs cell proliferation, differentiation, migration and survival1,2. Defects in this signaling pathway can lead to pathological or disease states such as cancer. The epidermal growth factor receptor (EGFR) has shown to be highly expressed in human tumors, including 50% of oral squamous cell carcinomas, and contributes to the development of malignant tumors3,4,5. Whereas mutations in the three RAS isoforms H-, K- and N-RAS are major drivers for malignant transformation in multiple human cancers. Amongst these three RAS isoforms, oncogenic mutations in K-RAS are most prevalent6,7,8. For EGFR and RAS to function, they must localize to the plasma membrane (PM). Preventing the localization of these molecules to the PM can completely abrogate the biological activity of this signal pathway9,10. Hence the inhibition of the localization of these proteins to the PM is a therapeutic strategy to block the downstream signaling and the resulting adverse outcomes. Using a high-content screening assay, fendiline, an L-type calcium channel blocker, was identified as an inhibitor of K-RAS activity11. Nanoclustering of K-RAS to the inner leaflet of the PM is significantly reduced in the presence of fendiline. Furthermore, K-RAS is redistributed from the plasma membrane to the endoplasmic reticulum (ER), Golgi apparatus, endosomes, and cytosol. More importantly, the proliferation of pancreatic, colon, lung, and endometrial cancer cell lines expressing oncogenic mutant K-RAS is blocked by the inhibition of downstream signaling by fendiline11. These data suggest fendiline functions as a specific K-RAS anticancer therapeutic that causes the mis-localization of the RAS protein to the PM.
The nematode Caenorhabditis elegans has been extensively studied in the context of development. Many of the signal pathways that govern development in the worm are evolutionary and functionally conserved. For example, the EGFR mediated activation of RAS and the subsequent activation of the ERK MAPK signal cascade is conserved in the worm12. The cascade is represented by the following proteins: LET-23 &#62; LET-60 &#62; LIN-45 &#62; MEK-2 &#62; MPK-1. LET-60 is homologous to RAS, while LET-23 is homologous to EGFR. In the worm, this pathway regulates the development of the vulva13. The vulva is an epithelial aperture on the ventral body wall of the worm that allows fertilized eggs to be laid. The formation of the vulva in the worm is dependent on the exposure of the vulval precursor cells (VPC) to a gradient of activation of the EGFR-RAS-MAPK signal cascade. During the normal development, the proximal VPCs receive strong signals from the gonadal anchor cells to differentiate into 1&#176; and 2&#176; cell fates which give rise to a functional vulva12. Whereas distal VPCs differentiate into 3&#176; cell fates that fuse to the hypodermal syncytium and do not form vulva due to depleted signaling. In the absence of signaling, all VPCs differentiate into 3&#176; cell fates resulting in the formation of no vulva. However, constitutive signaling leads to the formation one or more non-functional vulva due to the induction of all VPCs to assume&#160;1&#176; and 2&#176; cell fates.
Mutations that cause defective or excessive vulval induction have been identified for many of the genes that encode for proteins representing this pathway. Defective vulval induction results in a vulvaless (Vul) phenotype, while excessive vulval induction results in a multivulva (Muv) phenotype that is represented by the development of numerous nonfunctional ectopic pseudovulvae throughout the ventral body wall. The Muv phenotype expressed by the let-60(n1046) strain is due to a gain of function mutation in RAS, while in the let-23(sa62) strain it is due an activating mutation in EGFR14,15. The strong Muv phenotype in these mutant strains has been shown to be perturbed by pharmacological interventions as demonstrated by the treatment of let-60(n1046) worms with the MEK-1 inhibitor U012616,17. Interestingly, we have shown that R-fendiline and inhibitors that affect sphingomyelin metabolism suppress the Muv phenotype in the worm18. To demonstrate these inhibitors block let-60 signaling at the level of RAS, the lin-1 null strain has been utilized17. Lin-1 is an Ets-like inhibitory transcription factor that functions as a repressor in the development of the vulva19. Strong reversion of the Muv phenotype in let-60(n1046) worms and no effect on lin-1 null worms suggest that these inhibitions occur at the level of RAS.
In this protocol, we demonstrate the use of C. elegans as a model to identify inhibitors of RAS and EGFR proteins. Using a liquid-based assay, we demonstrate the inhibitory effects of R-fendiline by suppressing the Muv phenotypes in the let-60(n1046) and let-23(sa62) mutant strains of C. elegans. This assay validates the use of C. elegans as a tool in the initial phase of drug discovery for anticancer therapeutics.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Media and chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose&#160;</td>
			<td>Millipore Sigma&#160;</td>
			<td>A9539-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Peptone&#160;</td>
			<td>Fisher Scientific</td>
			<td>DF0118-17-0</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Difco Agar&#160;</td>
			<td>Fisher Scientific</td>
			<td>DF0145-17-0</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Difco LB Broth</td>
			<td>Fisher Scientific</td>
			<td>DF0446-17-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP510-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Fisher Scientific</td>
			<td>ICN10138201</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Fisher Scientific</td>
			<td>BP213-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Nystatin</td>
			<td>Acros organics</td>
			<td>AC455500050</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Dibasic</td>
			<td>Fisher Scientific</td>
			<td>BP363-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium pPhosphate Monobasic</td>
			<td>Fisher Scientific</td>
			<td>BP362-500</td>
			<td></td>
		</tr>
		<tr>
			<td>R-Fendiline</td>
			<td></td>
			<td></td>
			<td>Commercially Synthesized (Pharmaceutical grade)</td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Millipore Sigma&#160;</td>
			<td>S2002-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride&#160;</td>
			<td>Fisher Scientific</td>
			<td>BP358-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Fisher Scientific</td>
			<td>SS266-1</td>
			<td></td>
		</tr>
		<tr>
			<td>8.25% Sodium Hypochlorite&#160;</td>
			<td></td>
			<td></td>
			<td>Bleach</td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic&#160;</td>
			<td>Fisher Scientific</td>
			<td>BP332-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin Sulfate&#160;</td>
			<td>Fisher Scientific</td>
			<td>BP910-50</td>
			<td></td>
		</tr>
		<tr>
			<td>(&#8722;)-Tetramisole Hydrochloride</td>
			<td>Millipore Sigma&#160;</td>
			<td>L9756</td>
			<td></td>
		</tr>
		<tr>
			<td>UO126 (MEK inhibitor)</td>
			<td>Millipore Sigma&#160;</td>
			<td>19-147</td>
			<td></td>
		</tr>
		<tr>
			<td>Consumables&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15mL Conical Sterile Polypropylene Centrifuge Tubes&#160;</td>
			<td>Fisher Scientific</td>
			<td>12-565-269</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL Conical Sterile Polypropylene Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>12-565-271</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Polystyrene Serological Pipettes 10mL</td>
			<td>Fisher Scientific</td>
			<td>07-200-574</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Polystyrene Serological Pipettes 25mL</td>
			<td>Fisher Scientific</td>
			<td>07-200-575</td>
			<td></td>
		</tr>
		<tr>
			<td>No. 1.5&#160; 18 mm X 18 mm Cover Slips</td>
			<td>Fisher Scientific</td>
			<td>12-541A</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish with Clear Lid (60 x 15 mm)</td>
			<td>Fisher Scientific</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes with Clear Lid (100X15mm)</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>Plain Glass Microscope Slides (75 x 25 mm)</td>
			<td>Fisher Scientific</td>
			<td>12-544-4</td>
			<td></td>
		</tr>
		<tr>
			<td>12- Well Tissue Culture Plates</td>
			<td>Fisher Scientific</td>
			<td>50-197-4804</td>
			<td></td>
		</tr>
		<tr>
			<td>Software&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>Graphpad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli OP50</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Worm Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Strain</td>
			<td>Genotype</td>
			<td>Transgene</td>
			<td>Source</td>
		</tr>
		<tr>
			<td>MT2124 &#160;</td>
			<td>let-60(n1046) IV.</td>
			<td></td>
			<td>CGC</td>
		</tr>
		<tr>
			<td>MT7567</td>
			<td>lin-1(sy254) IV.</td>
			<td></td>
			<td>CGC</td>
		</tr>
		<tr>
			<td>PS1839</td>
			<td>let-23(sa62) II.</td>
			<td></td>
			<td>CGC</td>
		</tr>
	</tbody>
</table>

identification of egfr and ras inhibitors using caenorhabditis elegans

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in several diseases including cancer. The free-living nematode C. elegans possesses an evolutionary and functionally conserved EGFR-RAS-ERK MAP signal cascade which is central for the development of the vulva. Gain of function mutations in RAS homolog LET-60 and EGFR homolog LET-23 induce the generation of visible nonfunctional ectopic pseudovulva along the ventral body wall of these worms. Previously, the multivulval (Muv) phenotype in these worms has been shown to be inhibited by small chemical molecules. Here we describe a protocol for using the worm in a liquid-based assay to identify inhibitors that abolish the activities of EGFR and RAS proteins. Using this assay, we show R-fendiline, an indirect inhibitor of K-RAS, suppresses the Muv phenotype expressed in the let-60(n1046) and let-23(sa62) mutant worms. The assay is simple, inexpensive, is not time consuming to setup, and can be used as an initial platform for the discovery of anticancer therapeutics.

We first demonstrate that R-fendiline is able to suppress the Muv phenotype in the let-60(n1046) mutant strain compared to the DMSO treated worms. Our data shows that R-fendiline is able to block the Muv phenotype in the let-60(n1046) in a dose-dependent manner (Figure 2A,B). However, non-reversal of the Muv phenotype was observed in the lin-1 null mutant strain in response to increasing concentrations of R-fendiline (Figure 2B...

Watch this Scientific Journal Video about Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans at JoVE.com

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The genetically tractable nematode Caenorhabditis elegans can be used as a simple and inexpensive model for drug discovery. Described here is a protocol to identify anticancer therapeutics that inhibit the downstream signaling of RAS and EGFR proteins.

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in ...

identification-of-egfr-and-ras-inhibitors-using-caenorhabditis-elegans

Research

JoVE Journal

Cancer Research

2.9K Views.  University of Texas Health Science Center. The genetically tractable nematode Caenorhabditis elegans can be used as a simple and inexpensive model for drug discovery. Described here is a protocol to identify anticancer therapeutics that inhibit the downstream signaling of RAS and EGFR proteins.

Video: Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

Sectioning Mammary Gland Whole Mounts for Lesion Identification

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and genetic alterations. Mammary gland whole mounts are an inexpensive method to capture the progression of morphological changes that may arise after exposure. However, in later life, when abnormalities are more prone to develop, sole reliance on this one method may not always provide enough information to make a proper diagnosis of the abnormality. Historically, in chemical test guideline studies, a single mammary gland is removed at necropsy and prepared as a hematoxylin and eosin (H&#38;E)-stained section. The incorporation of contralateral mammary whole-mount collection and analysis decreases the likelihood of a false-negative assessment. Evaluation of the whole mount is limited by the presence of one or two entire mammary glands on a slide, and in some cases, the abnormalities observed in the whole mount are not uniformly represented in the H&#38;E section.&#160; The goal of this study was to develop a protocol for converting coverslipped mammary whole mounts to H&#38;E-stained sections so that lesions that would otherwise have been missed or that are difficult to diagnose can be identified. Here, we detail a method to produce a high-quality, paraffin-embedded H&#38;E section from a mammary gland that was initially prepared as a whole mount. In comparison to a tissue that was intentionally prepared for H&#38;E sectioning, the whole mount requires additional preparation for tissue removal and processing. However, this method is considered inexpensive, as it requires common lab reagents and little additional time. As a result, this method can provide invaluable information on how chemical and environmental exposures alter normal mammary development, as well as display changes that occur because of genetic modifications.

We developed a method to successfully remove, process, section, and stain, for histopathological evaluation, mammary tissue that had originally been fixed on slides as whole mounts. This method may promote the collection and evaluation of mammary gland whole mounts in reproductive and developmental test guideline studies.

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and ...

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

Metastatic cancer accounts for 90% of deaths in patients with solid tumors. There is an urgent need to better understand the drivers of cancer metastasis and to identify novel therapeutic targets. To investigate molecular events that drive the progression from primary cancer to metastasis, we have developed a bitransgenic mouse model, RIP-Tag; RIP-tva. In this mouse model, the rat insulin promoter (RIP) drives the expression of the SV40 T antigen (Tag) and the receptor for subgroup A avian leukosis virus (tva) in pancreatic &#946; cells. The mice develop pancreatic neuroendocrine tumors with 100% penetrance through well-defined stages that are similar to human tumorigenesis, with stages including hyperplasia, angiogenesis, adenoma, and invasive carcinoma. Because RIP-Tag; RIP-tva mice do not develop metastatic disease, genetic alterations that promote metastasis can be identified easily. Somatic gene transfer into tva-expressing, proliferating pancreatic &#946; premalignant lesions is achieved through intracardiac injection of avian retroviruses harboring the desired genetic alteration. A titer of &#62;1 x 108 infectious units per ml is considered appropriate for in vivo infection. In addition, avian retroviruses can infect cell lines derived from tumors in RIP-Tag; RIP-tva mice with high efficiency. The cell lines can also be used to characterize the metastatic factors. Here we demonstrate how to utilize this mouse model and cell lines to assess the functions of candidate genes in tumor metastasis.

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

We present a protocol to demonstrate a novel somatic gene transfer system utilizing RIP-Tag; RIP-tva mouse model to study the function of genes in metastasis. The avian retroviruses are delivered intracardiacally to ensure gene transfer into pre-malignant, noninvasive lesions of pancreatic &#946; cells in adult mice.

Metastatic cancer accounts for 90% of deaths in patients with solid tumors. There is an urgent need to better understand the drivers of cancer metastasis ...

Establishing Dual Resistance to EGFR-TKI and MET-TKI in Lung Adenocarcinoma Cells In Vitro with a 2-step Dose-escalation Procedure

Drug resistance is a major challenge in cancer therapy. The generation of resistant sublines in vitro is necessary for discovering novel mechanisms to overcome this challenge. Here, a 2-step dose-escalation method for establishing dual-resistance to an epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI), gefitinib, and a MET-TKI, PHA665752, is described. This method is based on simple stepwise dose-escalation of inhibitors for inducing acquired resistance in cell lines. The alternate method for generating resistant sublines involves exposing the cells to high concentrations of the inhibitor in one step. The stepwise dose-escalation method has a higher possibility of successfully inducing acquired resistance than this method. Activating EGFR mutations are biomarkers of a response to treatment with EGFR-TKI, which is an applied first-line treatment for non-small cell lung cancers (NSCLC) that harbor these mutations. However, despite reports of effective responses, the use of EGFR-TKI is limited because tumors inevitably acquire resistance. The major mechanisms behind EGFR-TKI resistance include a secondary mutation at the gatekeeper site, T790M in exon 20 of EGFR, and a bypass signal of MET. Thus, a potential solution for this issue would be a combination of EGFR-TKI and MET-TKI. This combined treatment has been shown to be effective in an in vitro study model. Acquired gefitinib-resistance was established through MET-amplification by stepwise dose-escalation of gefitinib for 12 months, and a cell line named PC-9MET1000 was generated in a previous study. To further investigate the mechanisms of acquired MET-TKI and EGFR-TKI resistance, a MET-TKI, PHA665752, was administered to these cells with stepwise dose-escalation in the presence of gefitinib for 12 months. This protocol has also been successfully applied for a number of combination therapies to establish acquired resistance to other inhibitor molecules.

Establishing Dual Resistance to EGFR-TKI and MET-TKI in Lung Adenocarcinoma Cells In Vitro with a 2-step Dose-escalation Procedure

An in vitro method for establishing dual resistance to an EGFR-TKI and a MET-TKI in cancer cells is described. This method is useful for developing treatments for patients with EGFR-mutations, who exhibit disease progression despite EGFR-TKI treatment with MET-amplification. It can also be modified for inhibitors targeting other molecules.

Drug resistance is a major challenge in cancer therapy. The generation of resistant sublines in vitro is necessary for discovering novel mechanisms to ...

Identification of OTX1 and OTX2 As Two Possible Molecular Markers for Sinonasal Carcinomas and Olfactory Neuroblastomas

OTX homeobox (HB) genes are expressed during embryonic morphogenesis and during the development of olfactory epithelium in adult organisms. Mutations occurring in these genes are often related to tumorigenesis in human. No data are available today regarding the possible correlation between OTX genes and tumors of the nasal cavity. The aim of this work is to understand if OTX1 and OTX2 can be considered as molecular markers in the development of nasal tumors. We selected nasal and sinonasal adenocarcinomas to investigate the expression of OTX1 and OTX2 genes through immunohistochemical and real-time PCR analyses.Both OTX1 and OTX2 were absent in all the samples of sinonasal Intestinal-Type Adenocarcinomas (ITACs). OTX1 mRNA was identified only in Non-Intestinal Type Adenocarcinomas (NITACs) while OTX2 mRNA was expressed only in Olfactory Neuroblastomas (ONs). We have demonstrated that the differential gene expression for both OTX1 and OTX2 genes might be a useful molecular marker to distinguish the different types of sinonasal tumors.

Homeobox genes are regulatory genes often associated with tumors in the adult organisms. We investigated their comparative expression by immunohistochemical and real-time PCR analysis, in normal and inflammatory nasal mucosae and in sinonasal neoplasms in order to use them as possible diagnostic and therapeutic targets.

OTX homeobox (HB) genes are expressed during embryonic morphogenesis and during the development of olfactory epithelium in adult organisms. Mutations ...

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 ...

Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate cancer (PCa). Understanding the anatomy and histology of the mouse prostate is important for the efficient use and proper characterization of such animal models. The mouse prostate has four distinct pairs of lobes, each with their own characteristics. This article demonstrates the proper method of dissection and identification of mouse prostate lobes for disease analysis. Post-dissection, the prostate cells can be further cultured in vitro for mechanistic understanding. Since mouse prostate primary cells tend to lose their normal characteristics when cultured in vitro, we outline here a method for isolating the cells and growing them as 3D spheroid cultures, which is effective for preserving the physiological characteristics of the cells. These 3D cultures can be used for analyzing cell morphology and behavior in near-physiological conditions, investigating altered levels and localizations of key proteins and pathways involved in the development and progression of a disease, and looking at responses to drug treatments.

Genetically engineered mice are useful models for investigating prostate cancer mechanisms. Here we present a protocol to identify and dissect prostate lobes from a mouse urogenital system, differentiate them based on histology, and isolate and culture the primary prostate cells in vitro as spheroids for downstream analyses.

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate ...

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 ...

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.

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene ...

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.

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.

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