Name: potentiation of anticancer antibody efficacy by antineoplastic drugs: detection of antibody-drug synergism using the combination index equation
Uploaded: 2019-01-19T13:30:00
Description: Watch this Scientific Journal Video about Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation at JoVE.com

Grant support: Fondation de Projet de L&#39;Universit&#233; de Nantes, les Bagouz&#39; &#224; Manon, La Ligue contre le Cancer comit&#233; de Loire-Atlantique, comit&#233; du Morbihan, and comit&#233; de Vend&#233;e, une rose pour S.A.R.A.H, L&#39;Etoile de Martin and la Soci&#233;t&#233; Fran&#231;aise de Lutte contre les Cancers et les leuc&#233;mies de L&#39;Enfant et de L&#39;adolescent (SFCE).&#160;M.B. and J.F. are supported by La Ligue Contre Le Cancer. The authors thank the UTE-facility of the Structure F&#233;d&#233;rative de Recherche Fran&#231;ois Bonamy. The authors also thank Dr. S. Suzin (Inserm, Paris) for providing the IMR5 cells and Ms. H. Est&#233;phan for her technical assistance.

Animal housing and experimental procedure were approved by the French Government (agreements #C44-278 and #APAFIS 03479.01). Animal care and procedures were conducted under directive EU 2010/63/EU and French Law #2013-118 on the protection of animals used for scientific purposes.
1. Evaluation of the Drug Interaction Between mAb 8B6 and Topotecan In Vitro
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	<li>96-well sample preparation 
	CAUTION: Consult the institution&#39;s Health and Safety committee a...

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Otol Neurotol 2012: 33;291-6.</a> Otol Neurotol. 34 (4), 777-778 (2013).</li><li>Choi, B., 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=Sensitization+of+lung+cancer+cells+by+altered+dimerization+of+HSP27.">Sensitization of lung cancer cells by altered dimerization of HSP27.</a> Oncotarget. 8 (62), 105372-105382 (2017).</li><li>Weiner, L. M., Surana, R., Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies:+versatile+platforms+for+cancer+immunotherapy.">Monoclonal antibodies: versatile platforms for cancer immunotherapy.</a> Nature Reviews Immunology. 10 (5), 317-327 (2010).</li><li>Mellor, J. D., Brown, M. P., Irving, H. R., Zalcberg, J. 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W., 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=Characterization+of+a+proapoptotic+antiganglioside+GM2+monoclonal+antibody+and+evaluation+of+its+therapeutic+effect+on+melanoma+and+small+cell+lung+carcinoma+xenografts.">Characterization of a proapoptotic antiganglioside GM2 monoclonal antibody and evaluation of its therapeutic effect on melanoma and small cell lung carcinoma xenografts.</a> Cancer Research. 65 (14), 6425-6434 (2005).</li><li>Nakamura, K., 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=Apoptosis+induction+of+human+lung+cancer+cell+line+in+multicellular+heterospheroids+with+humanized+antiganglioside+GM2+monoclonal+antibody.">Apoptosis induction of human lung cancer cell line in multicellular heterospheroids with humanized antiganglioside GM2 monoclonal antibody.</a> Cancer Research. 59 (20), 5323-5330 (1999).</li><li>Cochonneau, D., 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=Cell+cycle+arrest+and+apoptosis+induced+by+O-acetyl-GD2-specific+monoclonal+antibody+8B6+inhibits+tumor+growth+in+vitro+and+in+vivo.">Cell cycle arrest and apoptosis induced by O-acetyl-GD2-specific monoclonal antibody 8B6 inhibits tumor growth in vitro and in vivo.</a> Cancer Letters. 333 (2), 194-204 (2013).</li><li>Chou, T. 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To predict the effect of drug interactions, three methods can be used: the isobologram methodology17, the nonlinear mixture model18, and the combination index1. Combination index analysis is the most commonly used because its application is simplified by the availability of a user-friendly computer program. For this purpose, we first characterized the dose-effect response of each agent used alone or in combination, by performing an MTT assay<sup clas...

The conventional approach in the treatment of a large number of different types of cancer was based on monotherapy. Even if it is still used in many cases, this method met several obstacles leading to opting for combined therapies2. Particularly, cancer cells are more susceptible to develop resistance when treated with a single drug by inducing alternative survival mechanisms3, resulting in therapeutic failure in patients4. Moreover, in monotherapy, drugs are usually administrated at a high dose. This situation often results in the occurrence of strong dose-dependent side effects that can be intolerable and force physicians to stop the treatment2. For these reasons, the association of anticancer molecules is now preferred to monotherapy.
Ideal drug combinations would be those that act in synergy against tumor cells, without increased toxicity against normal cells. Synergism refers to the interaction of two or more drugs that produces a therapeutic effect greater than the sum of each individual drug acting separately. Such interactions may result in enhanced clinical therapeutic efficacy2. It limits treatment resistance, increases efficacy, and can also reduce toxicity2. In fact, the dosage of each drug can be reduced to lower their side effects by targeting different pathways. In addition, one of the molecules can also serve as a sensitizing agent against cancer cells. The effect of the second drug may be enhanced on sensitized cells and fewer dosages can be used5.
Combined therapy can include two or more chemotherapeutic drugs and/or biologics, such as monoclonal antibodies6. These mAbs specifically target cells expressing a cell surface antigen of interest and are able to kill tumor cells through immunological pathways including antibody-dependent cell-mediated cytotoxicity (ADCC), with the involvement of immune effector cells7, and complement-dependent cytotoxicity (CDC)6. They can also act via a non-immunological mechanism mediated by apoptosis8,9,10,11. In this case, the induction of the process of programmed cell death may sensitize cancer cells, weaken their function, and make the associated chemotherapeutic drug more effective at a lower dosage. As such, proapoptotic mAb are good candidates for designing combination regimens with antineoplastic drugs.
Different mathematical models have been described to assess drug synergism; one of them is based on the combination index method1. This method is based on the median-effect principle developed by Chou1. The median-effect equation correlates the drug dose and drug effect as follows.
<img alt="Equation 1" src="/files/ftp_upload/58291/58291eq1.jpg" />
Here, D is the drug dose; Dm is the median-effect dose; Fa is the fraction affected by the dose; m is an exponent that signifies the shape of the dose-effect plot1. The median-effect dose is used to calculate the dose Dx of a drug that inhibits or kills &#34;x&#34; percent of cells. The CI value is then calculated to assess the additive effect of the drug combination, as follows1.
<img alt="Equation 2" src="/files/ftp_upload/58291/58291eq2.jpg" style="font-size: 14px;" />
A CI value of 1 indicates an additive effect and a CI value of &#60;1 indicates a synergistic effect, while a CI value of &#62;1 indicates antagonism1. The application of this method is further facilitated by the availability of a computer program, CompuSyn, that determines synergism and antagonism at all doses or effect levels simulated automatically12.
Our group has developed the mAb 8B6 specific for O-acetyl-GD2 ganglioside (OAcGD2) neuroblastoma antigen13 and further demonstrated that this mAb is able to induce cell death with attributes of apoptosis11. To test whether mAb 8B6 can sensitize neuroblastoma cells to the antineoplastic agent topotecan, we adapted the above-mentioned method developed by Chou1. First, we determine the effective dose 50 (ED50) values of mAb 8B6 and topotecan. Next, the neuroblastoma cells with equipotent ratios of the two compounds based on ED50 values are exposed to determine the CI values using the above-mentioned simulation software. This method allows us to demonstrate synergism between mAb 8B6 and topotecan in vitro. Next, we describe a protocol to further assess the potency and the safety of this combination regimen in vivo. This protocol can be easily applied to select potent and safe anticancer mAb and chemotherapeutic agent combinations in preclinical studies. A schematic representation of this study is provided in Figure 1.

<table border="0" cellpadding="0" cellspacing="0" style="width:1389px;" width="1390">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Cell Proliferation kit (MTT)</td>
			<td style="width:161px;">Roche</td>
			<td style="width:132px;">11-465-007-001</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">CompuSyn software</td>
			<td style="width:161px;">ComboSyn</td>
			<td style="width:132px;"></td>
			<td style="width:852px;">Combosyn can be downloaded for free at http://www.combosyn.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Electric shaver</td>
			<td style="width:161px;">Bioseb</td>
			<td style="width:132px;">BIO-1556</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Fetal calf serum</td>
			<td style="width:161px;">Eurobio</td>
			<td style="width:132px;">CVFSVFF00-01</td>
			<td style="width:852px;">10% heat-inactivated fetal calf serum in RPMI 1640</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Firefox&#160;</td>
			<td style="width:161px;">Mozilla Corporation</td>
			<td style="width:132px;"></td>
			<td style="width:852px;">Firefox can be downloaded for free at http://www.mozilla.org/en-US/firefox/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Heat lamp</td>
			<td style="width:161px;">Verre&#38;Quartz</td>
			<td style="width:132px;">4003/1R</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Human neuroblastoma IMR-5 cell line</td>
			<td style="width:161px;">Accegen Biotechnology</td>
			<td style="width:132px;">ABC-TC0450</td>
			<td style="width:852px;">IMR-5 is a clone of the human neuroblastoma cell line IMR32 5459762. IMR-5 cells were generously provided by Dr. Santos Susin (U.872, Paris, France)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">L-glutamine</td>
			<td style="width:161px;">Gibco</td>
			<td style="width:132px;">25030-024</td>
			<td style="width:852px;">2 mM in RPMI 1640</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Lysis solution&#160;</td>
			<td style="width:161px;">Roche&#160;</td>
			<td style="width:132px;">11-465-007-001</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">mAb 8B6</td>
			<td style="width:161px;">University of Nantes</td>
			<td style="width:132px;">N/A</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Matrigel</td>
			<td style="width:161px;">Corning</td>
			<td style="width:132px;">354248</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Multiskan FC</td>
			<td style="width:161px;">Thermofischer Scientific&#160;</td>
			<td style="width:132px;">N08625</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Needle 21G 1 &#189;&#160;</td>
			<td style="width:161px;">BD Microlance</td>
			<td style="width:132px;">304432</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Needle 25G 1</td>
			<td style="width:161px;">Terumo</td>
			<td style="width:132px;">NN-2525R</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">NSG mice</td>
			<td style="width:161px;">Charles River Laboratories</td>
			<td style="width:132px;">5557</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Nunc MicroWell 96-well microplates</td>
			<td style="width:161px;">Thermofisher</td>
			<td style="width:132px;">167008</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">PBS</td>
			<td style="width:161px;">VWR</td>
			<td style="width:132px;">L182-10</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">PBS, 0,05% EDTA</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td>E9884</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">PC that runs windows 7</td>
			<td style="width:161px;">Microsoft</td>
			<td style="width:132px;"></td>
			<td style="width:852px;">Windows 7 can be purchased at http://www.microsoft.com/en-gb/software-download/windows7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Penicillin-Streptomycin</td>
			<td style="width:161px;">Gibco</td>
			<td style="width:132px;">15140-122</td>
			<td style="width:852px;">100 units/mL penicillin and 100 mg/mL streptomycin in RPMI 1640</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Reagent reservoir</td>
			<td style="width:161px;">Thermofischer Scientific</td>
			<td style="width:132px;">8094</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Rodent restrainer</td>
			<td style="width:161px;">Bioseb</td>
			<td style="width:132px;">TV-150-SM</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">RPMI 1640</td>
			<td style="width:161px;">Gibco</td>
			<td style="width:132px;">31870-025</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Syringe 1 mL</td>
			<td style="width:161px;">Henke Sass Wolf</td>
			<td style="width:132px;">5010.200V0</td>
			<td style="width:852px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Topotecan</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:132px;">T2705</td>
			<td style="width:852px;"></td>
		</tr>
	</tbody>
</table>

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.

The representative results and figures are adapted with permission from earlier published work14.
Anti-OAcGD2 mAb 8B6 Synergistically Enhances the Inhibitory Effects of Topotecan on Neuroblastoma Cell Line Growth:
To establish the drug and the antibody concentrations to be used for assessing synergism between topotecan and mAb 8...

Watch this Scientific Journal Video about Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation at JoVE.com

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

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 ...

Immunotherapy combined with chemotherapy represents an innovative approach to develop efficient therapy in cancer.The main aims are to achieve synergistic, therapeutic effects, dose and toxicity reduction, and to minimize or delay the induction of drug resistance.However, for practical and ethical reasons, it's not possible to determine synergism in patients.Thus, prior to direct combination in clinical trials, Prenicaptor combination studies, in vitro, and/or in animals, should be carried out to obtain the rationale for clinical studies.A simple robust method for the quantification of synergism between drugs is based on combination index equation developed by Shouan Talale, as illustrated in this video.Using this method, and its computerized simulation, we evidence here, the synergism between Monoclonal Antibody 8B6, which is specific for the O-Acetyl-GD2 Ganglioside neuroblastoma antigen and the chemotherapeutic drug Topotecan on neuroblastoma cells.Briefly, after determining the effective dose 50 values of each compound in human IMO, 5 neuroblastoma cell line, these cells were intubated with equitable ratios of the two compounds, and the combination index values were calculated using automated computer simulation.We next confirmed these results in Vivo, in an IMO, 5 tumor xenographed model.We grew IMR-5 cells in a T75 flask, observed them under a microscope, to check cell confluency.Asperate cell medium from the flask, wash with 5 mL of PBS.Add 3 mL of 0.05%EDTA/PBS solution.Return the flask to the incubator for 3 minutes.Examine cell culture under a microscope for cell detachment.Add 10 mL of complete cell medium to the flask, and transfer the cell suspension into a sterile, 15 mL conical tube.Centrifuge for five minutes at 300 g.Count cells using a hemocytometer.Remove and discard the supernatant.Re suspend cells in in Complete Growth Medium.Adjust the medium volume to obtain final concentration of 100, 000 cells/mL.Seed 84 wells, 96 well culture plate with 10, 000 cells each, which is 100 microliters of cell suspension.Incubate cells for 18 hours in the Cell Incubator.Dilute monoclonal antibody in 500 microliters of Complete Growth Medium to obtain an antibody working solution with a monoclonal antibody concentration 240 micro-grams per mL.Perform 5 two-fold serial delusions as indicated in the protocol.Dilute the drug in 500 microliters of Complete Growth Medium to obtain a drug working solution with a final concentration 120 nanomolar.Perform 5, two-fold serial delusions as indicated in the protocol.Dilute the same way the drug plus monoclonal anibodies solutions.To arrive at the final concentration, transfer 50 microliters of each drug solutions in the corresponding wells as shown on this experimental layout.Incubate the cells for 72 hours in the incubator.Add 10 microliters of MTT reagent solution into each well.Incubate at 37 degrees Celsius for 4 hours.Add 100 microliters of lycine solution into each well using a multichannel pipette and mix thoroughly by pipetting.Incubate at 37 degrees Celsius for 4 hours in a humidified chamber.Rid the absorbents at 570 and 620 nanometers using a spectrophotometer.Calculate the cell viability as follows.Calculate the fraction affected values using the following equation.Run the simulation software, click on the new experiment button to obtain the main window.Type the name of the experiment.Click new single drug.Type the name.Type the abbreviation.Type the drug concentration unit.Enter data 1 dose and FA value.Press Enter.Repeat this step until all data points are entered.Click Finished.Follow the same steps to enter Monoclonal antibody data points.Click New Drug Combo.Select drug and monoclonal antibodies.Select Constant Ratio.Click OK.Type the name.Type the abbreviation.Type the drug monoclonal antibody ratio.Enter data 1 dose.Press Enter.The program will automatically calculate the doses of Monoclonal Antibody 8B6 and combo.Enter data 1 FA value.Press Enter.Repeat this step until all data points are entered.Click Finished.Then, click Generate Report.Select drug and monoclonal antibody and then click OK.Select Combo, then click OK.Select Header, CI Table, and Summary Table, then click OK.Type the file name and the analysis and click save to generate the report.The report automatically opens in one's default web browser.The report contains a summary table section that includes title, date, final name, description note, m, Dm, and r parameters, effective dose 50 for either agent used as monotherapy or in combination, and the combination index table for each combination at the effective dose 50, 75, 90, and 95.A combination index value lesser than 1 indicates synergism.A value equals to one indicates additivity.A value greater than 1 indicates antagonism.Thaw basement membrane matrix overnight by submerging the vial in ice in 4 degrees C refrigerator before use.All culture wear, or media coming in contact with the basement membrane metric should be prechilled.Harvest the culture IMR5.Transfer the cells to a 15 mL conical tube and centrifuge at 300 g for 5 minutes.Discard the supernatant.Wash cells with 15 mL ice cold PBS twice.Prepare a cell suspension for 5 million cells per mL in ice-cold PBS.Transfer the cell suspension into 1.5 mL microtube.Swivel basement membrane matrix vial.Add 1 volume of the basement membrane matrix reagent, mixed by pipetting to obtain cell suspension of 2.5 million cells per mL.Keep the cell suspension on ice.Shave the flank of where the injection will be done.Mix cells and draw carefully the cell suspension into a 1 mL syringe mounted with a 21 g needle.Check to be sure there are no air bubbles in the syringe.Disinfect the inoculation area of the mice with an antiseptic solution.Gently squeeze the mouse skin on the flank between fingers at the injection site.Insert the needle exactly into the skinfold.Do not place the needle deep into the tissue to insure subcutaneous injection.Inject 100 microliters of IMR5 cell suspension subcutaneously.Rotate the syringe to prevent liquid spread and withdraw the needle.Monitor mice for body weight and tumor growth.Measure the length and width of the tumor with a caliber.Calculate the tumor volume using this formula.Start therapy when tumors have reached an average volume of 50 to 60 mm

potentiation-anticancer-antibody-efficacy-antineoplastic-drugs

Research

JoVE Journal

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 ...

Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. Photosensitizers selectively accumulate in tumor tissue and lead to tumor cell death in the presence of oxygen and the proper wavelength of light.
To increase the therapeutic effect of PDT, we developed both photosensitizer- and anticancer agent-loaded lung cancer-targeted nanoparticles. Both enhanced permeability and retention (EPR) effect-based passive targeting and hyaluronic-acid-CD44 interaction-based active targeting were applied. CD44 is a well-known hyaluronic acid receptor that is often introduced as a biomarker of non-small cell lung cancer. 
In addition, a combination of PDT and chemotherapy is adopted in the present study. This combination concept may increase anticancer therapeutic effects and reduce adverse reactions. 
We chose hypocrellin B (HB) as a novel photosensitizer in this study. It has been reported that HB causes higher anticancer efficacy of PDT compared to hematoporphyrin derivatives1. Paclitaxel was selected as the anticancer drug since it has proven to be a potential treatment for lung cancer2. 
The antitumor efficacies of photosensitizer (HB) solution, photosensitizer encapsulated hyaluronic acid-ceramide nanoparticles (HB-NPs), and both photosensitizer- and anticancer agent (paclitaxel)-encapsulated hyaluronic acid-ceramide nanoparticles (HB-P-NPs) after PDT were compared both in vitro and in vivo. The in vitro phototoxicity in A549 (human lung adenocarcinoma) cells and the in vivo antitumor efficacy in A549 tumor-bearing mice were evaluated.
The HB-P-NP treatment group showed the most effective anticancer effect after PDT. In conclusion, the HB-P-NPs prepared in the present study represent a potential and novel photosensitizer delivery system in treating lung cancer with PDT.

Photodynamic therapy (PDT) is an alternative choice for lung cancer treatment. To increase the therapeutic effect of PDT, lung cancer-targeted nanoparticles combined with chemotherapy were developed. Both in vitro and in vivo anticancer efficacies of PDT with prepared nanoparticles were evaluated.

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. ...

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may be classified as either clinically functional or nonfunctional PA (FPA and NFPA). NFPA is difficult for early stage diagnosis and therapy due to barely elevating hormones in the blood compared to FPA. Our long-term goal is to use proteomics methods to discover reliable biomarkers for clarification of PA molecular mechanisms and recognition of effective diagnostic, prognostic markers and therapeutic targets. Effective two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) methods were presented here to analyze human PA proteomes, including preparation of samples, 2D gel electrophoresis, protein visualization, image analysis, in-gel trypsin digestion, peptide mass fingerprint (PMF), and tandem mass spectrometry (MS/MS). 2-Dimensional gel electrophoresis matrix-assisted laser desorption/ionization mass spectrometry PMF (2DE-MALDI MS PMF), 2DE-MALDI MS/MS, and 2DE-liquid chromatography (LC) MS/MS procedures have been successfully applied in an analysis of NFPA proteome. With the use of a high-sensitivity mass spectrometer, many proteins were identified with the 2DE-LC-MS/MS method in each 2D gel spot in an analysis of complex PA tissue to maximize the coverage of human PA proteome.

Here, we present a two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) to separate and identify human pituitary adenoma tissue proteome, which presents a good and reproducible 2DE pattern. Many proteins are observed in each 2DE spot when analyzing complex cancer proteome with the use of high-sensitivity MS.

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may ...

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

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

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

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

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

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

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

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

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

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 ...

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 ...

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 ...

Preparation of Mitochondria from Ovarian Cancer Tissues and Control Ovarian Tissues for Quantitative Proteomics Analysis

Ovarian cancer is a common gynecologic cancer with high mortality but unclear molecular mechanism. Most ovarian cancers are diagnosed in the advanced stage, which seriously hampers therapy. Mitochondrial changes are a hallmark of human ovarian cancers, and mitochondria are the centers of energy metabolism, cell signaling, and oxidative stress. In-depth insights into the changes of the mitochondrial proteome in ovarian cancers compared to control ovarian tissue will benefit in-depth understanding of the molecular mechanisms of ovarian cancer, and the discovery of effective and reliable biomarkers and therapeutic targets. An effective mitochondrial preparation method coupled with an isobaric tag for relative and absolute quantification (iTRAQ) quantitative proteomics are presented here to analyze human ovarian cancer and control mitochondrial proteomes, including differential-speed centrifugation, density gradient centrifugation, quality assessment of mitochondrial samples, protein digestion with trypsin, iTRAQ labeling, strong cation exchange fractionation (SCX), liquid chromatography (LC), tandem mass spectrometry (MS/MS), database analysis, and quantitative analysis of mitochondrial proteins. Many proteins have been successfully identified to maximize the coverage of the human ovarian cancer mitochondrial proteome and to achieve the differentially expressed mitochondrial protein profile in human ovarian cancers.

This article presents a protocol of differential-speed centrifugation in combination with density gradient centrifugation to separate mitochondria from human ovarian cancer tissues and control ovarian tissues for quantitative proteomics analysis, resulting in a high-quality mitochondrial sample and high-throughput and high-reproducibility quantitative proteomics analysis of a human ovarian cancer mitochondrial proteome.

Ovarian cancer is a common gynecologic cancer with high mortality but unclear molecular mechanism. Most ovarian cancers are diagnosed in the advanced ...

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 ...