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
<ol>
	<li>96-well sample preparation 
	CAUTION: Consult the institution&#39;s Health and Safety committee a...

<ol>
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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. R., Dobrovic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+review+of+the+role+of+Fc+gamma+receptor+polymorphisms+in+the+response+to+monoclonal+antibodies+in+cancer.">A critical review of the role of Fc gamma receptor polymorphisms in the response to monoclonal antibodies in cancer.</a> Journal of Hematology &amp; Oncology. 6, 1 (2013).</li><li>Kowalczyk, 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=The+GD2-specific+14G2a+monoclonal+antibody+induces+apoptosis+and+enhances+cytotoxicity+of+chemotherapeutic+drugs+in+IMR-32+human+neuroblastoma+cells.">The GD2-specific 14G2a monoclonal antibody induces apoptosis and enhances cytotoxicity of chemotherapeutic drugs in IMR-32 human neuroblastoma cells.</a> Cancer Letters. 281 (2), 171-182 (2009).</li><li>Retter, M. 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. C., Martin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> CompuSyn for drug combinations: PC software and user&#8217;s guide: a computer program for quantitation of synergism and antagonism in drug combinations, and the determination of IC50 and ED50 and LD50 values. , (2005).</li><li>Alvarez-Rueda, 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=A+monoclonal+antibody+to+O-acetyl-GD2+ganglioside+and+not+to+GD2+shows+potent+anti-tumor+activity+without+peripheral+nervous+system+cross-reactivity.">A monoclonal antibody to O-acetyl-GD2 ganglioside and not to GD2 shows potent anti-tumor activity without peripheral nervous system cross-reactivity.</a> PLoS One. 6 (9), e25220 (2011).</li><li>Faraj, S., 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=Neuroblastoma+chemotherapy+can+be+augmented+by+immunotargeting+O-acetyl-GD2+tumor-associated+ganglioside.">Neuroblastoma chemotherapy can be augmented by immunotargeting O-acetyl-GD2 tumor-associated ganglioside.</a> Oncoimmunology. 7 (1), e1373232 (2017).</li><li>Ishikawa, F., 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=Development+of+functional+human+blood+and+immune+systems+in+NOD/SCID/IL2+receptor+{gamma}+chain(null)+mice.">Development of functional human blood and immune systems in NOD/SCID/IL2 receptor {gamma} chain(null) mice.</a> Blood. 106 (5), 1565-1573 (2005).</li><li>Ullman-Cullere, M. 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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

11.9K Views.  Centre de Recherche en Cancérologie et Immunologie Nantes Angers (CRCINA), Institut national de la santé et de la recherche médicale (INSERM), Université d'Angers, Université de Nantes, Nantes, France. 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 ou...