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
