Name: detection of protease activity by fluorescent peptide zymography
Uploaded: 2019-01-20T14:45:00
Description: Watch this Scientific Journal Video about Detection of Protease Activity by Fluorescent Peptide Zymography at JoVE.com

Funding provided by The Ohio State University College of Engineering, Biomedical Engineering Department, and the Comprehensive Cancer Center - Arthur G. James Cancer Hospital and Richard J. Solove Research Institute.

1. Preparation of the Resolving Gel Layer
<ol>
	<li>Prepare a 10% polyacrylamide resolving gel solution as per Table 1. Add the Tetramethylethylenediamine (TEMED) and Ammonium Persulfate (APS) immediately prior to pouring the gel as their addition initiates the polymerization reaction.</li>
	<li>Fill an empty 1.5 mm mini-gel cassette half way (5 mL) with the 10% resolving gel solution.</li>
	<li>Add a thin layer of isopropanol (~500 &#181;L) to the top of the polyacrylamide gel to produce a level gel a...

<ol>
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K. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Aspartic+Proteinase+Activities+Using+Gel+Zymography.">Detection of Aspartic Proteinase Activities Using Gel Zymography.</a> Zymography. , 43-52 (2017).</li><li>van Beurden, P. A. M. S. n. o. e. k. -., Vonden Hoff, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zymographic+techniques+for+the+analysis+of+matrix+metalloproteinases+and+their+inhibitors.">Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors.</a> BioTechniques. 38 (1), 73-83 (2005).</li><li>Oliver, G. W., Stetler-Stevenson, W. G., Kleiner, D. E., Zymography, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zymography,+Casein+Zymography,+and+Reverse+Zymography:+Activity+Assays+for+Proteases+and+their+Inhibitors.">Zymography, Casein Zymography, and Reverse Zymography: Activity Assays for Proteases and their Inhibitors.</a> Proteolytic Enzymes. Springer Lab Manual. , 63-76 (1999).</li><li>Deshmukh, A. A., Weist, J. L., Leight, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+proteolytic+activity+by+covalent+tethering+of+fluorogenic+substrates+in+zymogram+gels.">Detection of proteolytic activity by covalent tethering of fluorogenic substrates in zymogram gels.</a> BioTechniques. 64 (5), 203-210 (2018).</li><li>Yasothornsrikul, S., Hook, V. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+proteolytic+activity+by+fluorescent+zymogram+in-gel+assays.">Detection of proteolytic activity by fluorescent zymogram in-gel assays.</a> BioTechniques. 28 (6), 1172-1173 (2000).</li><li>Leight, J. L., Alge, D. L., Maier, A. J., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+matrix+metalloproteinase+activity+in+3D+cellular+microenvironments+using+a+fluorogenic+peptide+substrate.">Direct measurement of matrix metalloproteinase activity in 3D cellular microenvironments using a fluorogenic peptide substrate.</a> Biomaterials. 34 (30), 7344-7352 (2013).</li><li>Leight, J. L., Tokuda, E. Y., Jones, C. E., Lin, A. J., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifunctional+bioscaffolds+for+3D+culture+of+melanoma+cells+reveal+increased+MMP+activity+and+migration+with+BRAF+kinase+inhibition.">Multifunctional bioscaffolds for 3D culture of melanoma cells reveal increased MMP activity and migration with BRAF kinase inhibition.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (17), 5366-5371 (2015).</li><li>Ren, Z., Chen, J., Khalil, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zymography+as+a+Research+Tool+in+the+Study+of+Matrix+Metalloproteinase+Inhibitors.">Zymography as a Research Tool in the Study of Matrix Metalloproteinase Inhibitors.</a> Methods in Molecular Biology. 1626, 79-102 (2017).</li><li>Nagase, H., Fields, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+matrix+metalloproteinase+specificity+studies+using+collagen+sequence-based+synthetic+peptide.">Human matrix metalloproteinase specificity studies using collagen sequence-based synthetic peptide.</a> Biopolymers. 40 (4), 399-416 (1996).</li><li>Mucha, A., 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=Membrane+Type-1+Matrix+Metalloprotease+and+Stromelysin-3+Cleave+More+Efficiently+Synthetic+Substrates+Containing+Unusual+Amino+Acids+in+Their+P1&#8242;+Positions.">Membrane Type-1 Matrix Metalloprotease and Stromelysin-3 Cleave More Efficiently Synthetic Substrates Containing Unusual Amino Acids in Their P1&#8242; Positions.</a> Journal of Biological Chemistry. 273 (5), 2763-2768 (1998).</li><li>Thimon, V., Belghazi, M., Labas, V., Dacheux, J. -. L., Gatti, J. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-+and+two-dimensional+SDS-PAGE+zymography+with+quenched+fluorogenic+substrates+provides+identification+of+biological+fluid+proteases+by+direct+mass+spectrometry.">One- and two-dimensional SDS-PAGE zymography with quenched fluorogenic substrates provides identification of biological fluid proteases by direct mass spectrometry.</a> Analytical Biochemistry. 375 (2), 382-384 (2008).</li><li>Sun, X., Salih, E., Oppenheim, F. G., Helmerhorst, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-based+mass+spectrometric+characterization+of+proteases+and+inhibitors+in+human+saliva.">Activity-based mass spectrometric characterization of proteases and inhibitors in human saliva.</a> Proteomics. Clinical Applications. 3 (7), 810-820 (2009).</li><li>Yu, W., Woessner, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparin-Enhanced+Zymographic+Detection+of+Matrilysin+and+Collagenases.">Heparin-Enhanced Zymographic Detection of Matrilysin and Collagenases.</a> Analytical Biochemistry. 293 (1), 38-42 (2001).</li></ol>

Current zymographic techniques rely on the incorporation of native substrates into polyacrylamide gels for the detection of proteolysis. While these techniques have garnered widespread use, they are still limited in the number of proteases they can detect. Here, a protocol was described in which fluorescent, protease-degradable peptides are incorporated into the polyacrylamide resolving gel. Covalent coupling using an azido-PEG3-maleimide linker molecule enables the separation and detection of a wider variety of protease...

Gel zymography is a biological technique used to measure proteolytic activity within biological samples, such as body fluids or cell culture media1,2,3. The samples are separated by their molecular weights with electrophoresis through a polyacrylamide gel embedded with a degradable substrate. Common degradable substrates include gelatin, casein, collagen and elastin, which have been used to measure the activity of matrix metalloproteinases (MMPs) -1, -2, -3, -7, -8, -9, and -11, in addition to a variety of cathepsins1,2,4,5,6,7,8. After electrophoresis, the enzymes are renatured and allowed to degrade the protein within the gel. In traditional gel zymography, the gel is stained with a protein dye, such as Coomassie&#160;Blue, and protease activity is detected as a loss of signal, i.e., white bands (degradation of protein) on a dark blue background.
Here, we describe a protocol for an alternative method of gel zymography, in which the degradable substrate is a short, fluorogenic peptide covalently incorporated into the polyacrylamide gel (Figure 1). The substitution of synthetic peptides as the degradable substrates enables detection of a wider range of proteases as compared to traditional gel zymography with native proteins9. Covalent linkage of the fluorogenic peptide prevents peptide diffusion and migration during gel electrophoresis observed with previous methods9,10. Furthermore, the use of a fluorogenic substrate enables direct detection of protease activity without additional staining and de-staining steps. The overall goal of this method is the detection of protease activity in biological samples via the covalent incorporation of fluorogenic peptides in zymogram gels.

<table>
	<tbody>
		<tr>
			<td>1.5 mm Empty Gel Cassettes</td>
			<td>ThermoFisher Scientific</td>
			<td>NC2015</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mm, 10 well Empty Gel Cassette Combs</td>
			<td>ThermoFisher Scientific</td>
			<td>NC3510</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Phosphate Buffered Saline</td>
			<td>Fisher Scientific</td>
			<td>10-010-049</td>
			<td></td>
		</tr>
		<tr>
			<td>20% SDS Solution</td>
			<td>Ambion</td>
			<td>AM9820</td>
			<td></td>
		</tr>
		<tr>
			<td>3x Zymography Sample Buffer</td>
			<td>Bio-Rad</td>
			<td>1610764</td>
			<td></td>
		</tr>
		<tr>
			<td>40% (w/v) Acrylamide/Bis (19:1)</td>
			<td>Ambion</td>
			<td>AM9022</td>
			<td></td>
		</tr>
		<tr>
			<td>6 Well Tissue Culture Plates</td>
			<td>ThermoFisher Scientific</td>
			<td>087721B</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-2 Centrifugal Filter Unit (10 kDa MWCO)</td>
			<td>Sigma-Aldrich</td>
			<td>UFC201024</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammounium Persulfate</td>
			<td>Sigma-Aldrich</td>
			<td>A3678</td>
			<td></td>
		</tr>
		<tr>
			<td>Azido-PEG3-Maleimide Kit</td>
			<td>Click Chemistry Tools</td>
			<td>AZ107</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>ThermoFisher Scientific</td>
			<td>BP510100</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Fisher Scientific</td>
			<td>BP231</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>A416P</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro BCA Protein Assay Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>23235</td>
			<td></td>
		</tr>
		<tr>
			<td>N N N&#39; N&#39;-Tetramethylethylenediamine (TEMED)</td>
			<td>Sigma-Aldrich</td>
			<td>T9281</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerPac Basic Power Supply</td>
			<td>Bio-Rad</td>
			<td>1645050</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Plus Protein Dual Color Standard</td>
			<td>Bio-Rad</td>
			<td>161-0374</td>
			<td></td>
		</tr>
		<tr>
			<td>PrecisionGlide Hypodermic Needles</td>
			<td>Fisher Scientific</td>
			<td>14-826</td>
			<td></td>
		</tr>
		<tr>
			<td>Round Bottom Flask (100 mL)</td>
			<td>Fisher Scientific</td>
			<td>50-873-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Septum Rubber Stopper</td>
			<td>Fisher Scientific</td>
			<td>50-872-546</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Slip Tip Syringe (1 mL)</td>
			<td>Fisher Scientific</td>
			<td>14-823-434</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma hydrochlroide</td>
			<td>Sigma-Aldrich</td>
			<td>T5941</td>
			<td></td>
		</tr>
		<tr>
			<td>Typhoon 9410 Molecular Imager</td>
			<td>GE Amersham</td>
			<td>8149-30-9410</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>208086</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Using the method described here, two fluorescent protease-degradable peptides were incorporated into polyacrylamide gels: GGPQG&#8595;IWGQK(PEG)2C (abbreviated as QGIW throughout the text and figures) and GPLA&#8595;CpMeOBzlWARK(PEG)2C (abbreviated as LACW throughout the text and figures). &#8595; indicates the site of cleavage. QGIW is a collagen-I derived sequence designed to detect cellular collagenases14. LACW is a sequence that...

Watch this Scientific Journal Video about Detection of Protease Activity by Fluorescent Peptide Zymography at JoVE.com

Detection of Protease Activity by Fluorescent Peptide Zymography

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 ...

Current zymographic techniques detect a limited number of proteases. Fluorescent peptide zymography uses fluorescent peptides as the degradable substrate enabling measurement of a greater range of proteases. The main advantage of this method is it's modular design.The sequence of the fluorescent peptide determines which proteases are detected and the fluorescent peptide can easily be swapped with a peptide of a different sequence, an ability to detect other proteases. This technique can help inform the design of peptides'use as degradable cross-linkers in engineered biomaterials such as those used for drug delivery or tissue engineering applications. Incorporation of this peptide into this technique can identify tissue secreted proteases that are responsible for biomaterial degradation.Demonstration of this technique is important in order to visualize the multi-layer approach that is unique compared to other zymography techniques. Demonstrating this technique will be, Ameya Deshmukh, a graduate student from my laboratory. To begin, prepare a 10%polyacrylamide resolving gel solution as outlined in table one of the text protocol.Immediately prior to pouring the gel, add the TEMED and APS. Then, fill and empty 1.5 millimeter mini gel cassette halfway with the resolving gel solution. Add a thin layer of isopropanol to the top of the polyacrylamide gel to produce a level gel and prevent bubbles.Use the leftover polyacrylamide solution to track the progress of the polymerization reaction. When the polyacrylamide in the tube has completely solidified the reaction is complete. While the first resolving gel layer is polymerizing, retrieve the Azido-PEG3-Maleimide kit from storage at 20 degrees Celsius and allow the components to reach room temperature.Then, dissolve the components of vial two in the manufacturer recommended volume of DMSO. And vortex for 30 seconds to ensure the liquids are well-mixed. Transfer the contents of vial one into a clean, dry 100 millileter round bottom flask that contains a stir bar.Immediately insert a rubber septum stopper with a diaphragm that can be punctured with a syringe into the mouth of the flask. Next, insert two 18-gauge syringe needles into the diaphragm. Connect one of the syringe needles to an inert gas tank and allow the gas to fill the round bottom flask for three minutes.Then, shut off the inert gas and detach it from the needle. Using a syringe, inject the full contents of vial two into the flask. Remove both of the needles and the syringe.Allow the components to mix for 30 minutes at room temperature while stirring. After this remove the rubber septum stopper and transfer the contents to a clean five milliliter centrifuge tube. Once the first resolving gel layer has polymerized pour off the isopropanol layer.Pipet one milliliter of de-ionized water on top of the gel and then pour the water off to rinse the gel. Retrieve the thiol functionalized fluorescent peptide from storage at 80 degrees Celsius. Allow it to thaw at room temperature.Prepare a 10%resolving gel solution containing the Azido-PEG3-Maleimide linker molecule and the fluorescent peptide as outlined in table one of the text protocol. Immediately prior to pouring the gel add the TMED and APS. Next, fill half of the remaining portion of the gel cassette with the peptide resolving gel solution.Add a thin layer of isopropanol to the top of the polyacrylamide gel to produce a level gel and prevent bubbles. Use the leftover polyacrylamide solution to track the progress of the polymerization reaction. After the reaction is complete, pour off the isopropanol layer.Pipet one milliliter of de-ionized water on top of the gel and then pour the water off to rinse the gel. If not using the gels immediately, store them by immersing them in a plastic box filled with 100 milliliters of 1x PBS at four degrees Celsius. Wrap the box in aluminum foil to prevent photobleaching.First, prepare a 5%stacking gel solution as outlined in table one of the text protocol. Immediately prior to pouring the gel add the TMED and APS. Fill the remaining empty portion of the gel cassette with the stacking gel solution.Quickly insert a 1.5 millimeter gel comb into the stacking gel layer making sure no bubbles remain trapped under the wells. Use the leftover polyacrylamide solution to track the progress of the polymerization reaction. When the reaction is complete, gently remove the comb and the tape from the back of the gel cassette.To begin, dissolve samples in conventional zymography sample buffer. Add 400 milliliter os 1X tris glycene SDS running buffer to the gel apparatus. Load up to 35 microliters of sample per well.Run the samples at 120 volts at four degrees Celsius for one and a half hours or until the molecular weight standards indicate that the proteases of interest are within the peptide-resolving gel layer. After this, remove the gels from the plastic cassette. Wash the gels three times in re-naturing buffer at room temperature under gentle agitation.With each wash, lasting 10 minutes. Transfer gels to a developing buffer solution for 15 minutes. Then replace the solution with fresh developing buffer and incubate at 37 degrees Celsius under gentle agitation for 24 hours.After this, use a fluorescent gel scanner imager with the appropriate excitation and emission filters to image the gels. In this study to fluorescent protease degradable peptides are incorporated into polyacrylamide gels. QGIW is a collagen one derived sequence designed to detect cellular collagenesis, while LACW is a sequence that has been optimized for the detection of MMP-14 and MMP-11.Fluorescent imaging reveals numerous bands within the LACW peptide gels, While only a single band is seen in the QGIW gels. When compared to gelatin zymography, LACW gels are able to detect more proteolytic bands, which demonstrates the ability of peptide zymography to detect a wider range of proteases present within biological samples than traditional methods using native substrates. Peptide zymography cells are then incubated in development buffer containing either GM6001, a broad spectrum MMP-inhibitor, or E64, a general cathepsin inhibitor.Treatment of the LACW peptide gels with GM6001, decreases the intensity of the bands. While treatment with E64 has no discernible effect. Treatment of the QGIQ peptide gels with GM6001 results in complete ablation of the previously seen bands.While E64 has no effect. Gelatin zymography is conducted using purified, activated MMP-9 to compare the sensitivity of peptide zymography to the current gold standard. LACW gels are able to detect the smallest concentrations of MMP-9.Proteases require specific conditions to re-nature and become activated. Carefully prepare the buffer solutions to ensure that the composition and PH are correct. This method can be complemented by existing techniques to verify the identity and perform further analysis of the measured proteases.This includes western blotting, real-time PCR, and mass spectrometry. Be cautious when handling polyacrylamide. Unpolymerized solution is considered a potent neurotoxin and appropriate personal protective equipment should always be worn when handling it.

detection-of-protease-activity-by-fluorescent-peptide-zymography

Research

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Cancer Research

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

GnRH analogues are effective targeting moieties and able to deliver anticancer agents selectively into malignant tumor cells which highly express GnRH receptors. However, the quantitative analysis of GnRH analogues&#39; cellular uptake and the investigated cell types in GnRH-based drug delivery systems are currently limited. Previously introduced, selectively labeled fluorescent GnRH I, -II and -III derivatives provide great detectability, and they have suitable chemical properties for reproducible and robust experiments. We also found that the appropriate up-to-date methods with these labeled GnRH analogues could offer novel information about the GnRH-based drug delivery systems. This manuscript introduces some simple and fast experiments regarding the cellular uptake of [D-Lys6(FITC)]-GnRH-I, [D-Lys6(FITC)]-GnRH-II and [Lys8(FITC)]-GnRH-III on the EBC-1 (lung), the BxPC-3 (pancreas) and on the Detroit-562- (pharynx) malignant tumor cells. In parallel with these GnRH-FITC conjugates, the cell surface level of GnRH-I receptors was also examined on these cell lines before and after the GnRH treatment by confocal laser scanning microscopy. The cellular uptake of GnRH-FITC conjugates was quantified by fluorescence-activated cell sorting. In these experiments minor differences among GnRH analogues and major differences among cell types was observed. The significant differences among cell lines are correlated with their distinct level of cell surface GnRH-I receptors. The introduced experiments contain practical methods to visualize, quantify and compare the uptake efficiency of GnRH-FITC conjugates in a time- and concentration-dependent manner on various adherent cell cultures. These results could predict the drug targeting efficiency of GnRH conjugates on the given cell culture, and offer a good basis for further experiments in the examination of GnRH-based drug delivery systems.

In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

Selectively labeled fluorescent GnRH-I, -II and -III derivatives are reliable tools for tracking and quantifying their cellular uptake. This manuscript introduces experiments to visualize, quantify and compare the uptake efficiency of these GnRH conjugates in various cell lines.

GnRH analogues are effective targeting moieties and able to deliver anticancer agents selectively into malignant tumor cells which highly express GnRH ...

High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this clinical challenge, tumor xenograft mouse models using long-established human carcinoma cell lines and many transgenic mouse models with tumors have been developed as preclinical models. They partially mimic the features of human carcinomas, but often fail to recapitulate the key aspects of human malignancies including invasion and metastasis. Thus, alternative models that better represent the malignant progression in human CRC have long been awaited.
We herein show generation of patient-derived tumor xenografts (PDXs) by subcutaneous implantation of small CRC fragments surgically dissected from a patient. The colon PDXs develop and histopathologically resemble the CRC in the patient. However, few spontaneous micrometastases are detectable in conventional cross-sections of affected distant organs in the PDX model. To facilitate the detection of metastatic dissemination into distant organs, we extracted the tumor organoid cells from the colon PDXs in culture and infected them with GFP lentivirus prior to injection into highly immunodeficient NOD/Shi-scid IL2R&#947;null (NOG) mice. Orthotopically injected PDX-derived CRC organoid cells consistently form primary tumors positive for GFP in recipient mice. Moreover, spontaneously developing micrometastatic colonies expressing GFP are notably detected in the lungs of these mice by fluorescence microscopy. Moreover, intrasplenic injection of CRC organoids frequently produces hepatic colonization. Taken together, these findings indicate GFP-labelled PDX-derived CRC organoid cells to be visually detectable during a multistep process termed the invasion-metastasis cascade. The described protocols include the establishment of PDXs of human CRC and 3D culture of the corresponding CRC organoid cells transduced by GFP lentiviral particles.

To allow highly sensitive detection of the disseminating human colorectal cancer (CRC) cells colonizing tissues, we herein show a protocol for efficient transduction of green fluorescent protein (GFP) lentiviral particles into PDX-derived CRC organoid cells prior to their injection into recipient mice, with stereo-fluorescence microscopic observation.

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this ...

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 ...

Evaluation of Colorectal Cancer Risk and Prevalence by Stool DNA Integrity Detection

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a reliable probability of the presence of pre-neoplastic or neoplastic colorectal lesions. This method, called fluorescence long DNA (FL-DNA), is a fast, non-invasive procedure that is an improvement upon the primary prevention system. This method is based on evaluation of fecal DNA integrity by quantitative amplification of specific targets of genomic DNA. In particular, the evaluation of DNA fragments longer than 200 bp allows for detection of patients with colorectal lesions with very high specificity. However, this system and all currently available stool DNA tests present some general issues that need to be addressed (e.g., the frequency at which tests should be carried out and optimal number of stool samples collected at each timepoint for each individual). However, the main advantage of FL-DNA is the possibility to use it in association with a test currently used in the CRC screening program, known as the immunochemical-based fecal occult blood test (iFOBT). Indeed, both tests can be performed on the same sample, reducing costs and achieving a better prediction of the eventual presence of colorectal lesions.

The presented diagnostic FL-DNA kit is a time-saving and user-friendly method to determine the reliable probability of the presence of colorectal cancer lesions.

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a ...

Detection of Retrotransposition Activity of Hot LINE-1s by Long-Distance Inverse PCR

Long interspersed nuclear elements 1 (LINE-1s) are the only family of mobile genetic elements in the human genome that can move autonomously. They do so by a process called retrotransposition wherein they transcribe to form an mRNA intermediate which is then consequently inserted into the genome by reverse transcription. Despite being silent in normal cells, LINE-1s are highly active in different epithelial tumors. De novo LINE-1 insertions can potentially drive tumorigenesis, and hence it is important to systematically study LINE-1 retrotransposition in cancer. Out of ~150 retrotransposition-competent LINE-1s present in the human genome, only a handful of LINE-1 loci, also referred to as &#34;hot&#34; LINE-1s, account for the majority of de novo LINE-1 insertion in different cancer types. We have developed a simple polymerase chain reaction (PCR)-based method to monitor retrotransposition activity of these hot LINE-1s. This method, based on long-distance inverse (LDI)-PCR, takes advantage of 3&#180; transduction, a mechanism by which a LINE-1 mobilizes its flanking non-repetitive region, which can subsequently be used to identify de novo LINE-1 3&#180; transduction events stemming from a particular hot LINE-1.

This article outlines a simple PCR-based assay to monitor the activity of an active LINE-1 retrotransposon and to map de novo retrotranspositions in a given genome. Using the MCF7 cell line, we demonstrate herein how this method can be applied to detect activity of a LINE-1 located at 22q12.1.

Long interspersed nuclear elements 1 (LINE-1s) are the only family of mobile genetic elements in the human genome that can move autonomously. They do so ...

Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples from cancer patients often directly influences diagnosis, prognosis, and/or therapy selection. As a result, the accurate detection of gene fusions has become a critical component of clinical management for many disease types. Until recently, clinical gene fusion detection was predominantly accomplished through the use of single-gene assays. However, the ever-growing list of gene fusions with clinical significance has created a need for assessing fusion status of multiple genes simultaneously. Next generation sequencing (NGS)-based testing has met this demand through the ability to sequence nucleic acid in massively parallel fashion. Multiple NGS-based approaches that employ different strategies for gene target enrichment are now available for use in clinical molecular diagnostics, each with its own strengths and weaknesses. This article describes the use of anchored multiplex PCR (AMP)-based target enrichment and library preparation followed by NGS to assess for gene fusions in clinical solid tumor specimens. AMP is unique among amplicon-based enrichment approaches in that it identifies gene fusions regardless of the identity of the fusion partner. Detailed here are both the wet-bench and data analysis steps that ensure accurate gene fusion detection from clinical samples.

This article details the use of an anchored multiplex polymerase chain reaction-based library preparation kit followed by next-generation sequencing to assess for oncogenic gene fusions in clinical solid tumor samples. Both wet-bench and data analysis steps are described.

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining&#160;propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...

Far-Red Fluorescent Senescence-Associated &#946;-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge rapidly in tumor cells as a response to damage induced by various cancer treatments. Tumor cell senescence is generally considered undesirable, as senescent cells become resistant to death and block tumor remission while exacerbating tumor malignancy and treatment resistance. Therefore, the identification of senescent tumor cells is of ongoing interest to the cancer research community. Various senescence assays exist, many based on the activity of the well-known senescence marker, senescence-associated beta-galactosidase (SA-&#946;-Gal). 
Typically, the SA-&#946;-Gal assay is performed using a chromogenic substrate (X-Gal) on fixed cells, with the slow and subjective enumeration of "blue" senescent cells by light microscopy. Improved assays using cell-permeant, fluorescent SA-&#946;-Gal substrates, including C12-FDG (green) and DDAO-Galactoside (DDAOG; far-red), have enabled the analysis of living cells and allowed the use of high-throughput fluorescent analysis platforms, including flow cytometers. C12-FDG is a well-documented probe for SA-&#946;-Gal, but its green fluorescent emission overlaps with intrinsic cellular autofluorescence (AF) that arises during senescence due to the accumulation of lipofuscin aggregates. By utilizing the far-red SA-&#946;-Gal probe DDAOG, green cellular autofluorescence can be used as a secondary parameter to confirm senescence, adding reliability to the assay. The remaining fluorescence channels can be used for cell viability staining or optional fluorescent immunolabeling.
Using flow cytometry, we demonstrate the use of DDAOG and lipofuscin autofluorescence as a dual-parameter assay for the identification of senescent tumor cells. Quantitation of the percentage of viable senescent cells is performed. If desired, an optional immunolabeling step may be included to evaluate cell surface antigens of interest. Identified senescent cells can also be flow cytometrically sorted and collected for downstream analysis. Collected senescent cells can be immediately lysed (e.g., for immunoassays or 'omics analysis) or further cultured.

A protocol for fluorescent, flow cytometric quantification of senescent cancer cells induced by chemotherapy drugs in cell culture or in murine tumor models is presented. Optional procedures include co-immunostaining, sample fixation to facilitate large batch or time point analysis, and the enrichment of viable senescent cells by flow cytometric sorting.

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge ...