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

The overall goal of this experiment is to discover novel mechanisms to overcome drug resistance. This method can help answer key questions in the cancer research field such as those about graft resistance. The main advantage of this technique is that the cells resistant to true inhibitors can be established.

To begin, plate PC-9 cells in growth medium and culture the cells at 37 degrees Celsius in a 5%CO2 incubator. Use an automated cell counter to count the cells and adjust the concentration to 1.0 times 10 to the fifth cells per milliliter in growth medium. Then, seed 50 microliters of the cells into each well of a 96-well plate.

Incubate the plate overnight at 37 degrees Celsius. The following day, add 50 microliters of Gefitinib at different concentrations so that the final volume in each well is 100 microliters. Incubate the plate at 37 degrees Celsius for 72 hours.

Next, add 15 microliters of dye solution to each well and incubate the plate at 37 degrees Celsius for four hours. Add 100 microliters of the solubilization stop solution to each well for solubilizing the formazan produced by the dye solution and incubate the plate at 37 degrees Celsius overnight. Using a microplate reader, measure the optical density at 570 nanometers and use statistical software to generate a cell proliferation curve to obtain the IC50 value.

Plate one milliliter of PC-9 cells in P100 dishes with 10 milliliters of growth medium and incubate the cells in a 5%CO2 incubator at 37 degrees Celsius. Add 1/10 the IC50 value of Gefitinib into the P100 dish. When the cells in the culture medium become subconfluent, use a one milliliter pipette to add one to two milliliters of cells to 10 milliliters of fresh growth medium with a 10 to 30%higher concentration of Gefitinib and culture the cells under the same conditions as just demonstrated.

Increase the Gefitinib concentration progressively by 10 to 30%with each split until it reaches one micromolar. This may take about six to 12 months. After the cells have been cultured with one micromolar of Gefitinib, perform the MTT assay to confirm that the cells are Gefitinib resistant by the increased protein and RNA levels of MET.

To determine the initial concentration of PHA-665752 using the MTT assay, plate the MET1000 cells in P100 dishes with 10 milliliters of growth medium and one micromolar Gefitinib and incubate the cultures in a 5%CO2 incubator at 37 degrees Celsius. After adjusting the final concentration of the cells to 1.0 times 10 to the fifth cells per milliliter, seed the cells in 50 microliters of the growth medium in a 96-well plate at 5.0 times 10 to the third cells per well. Incubate the plate overnight.

After the incubation period, add 50 microliters of PHA-665752 at different concentrations in the presence or absence of two micromolar Gefitinib so that the final volume in each well is 100 microliters. Incubate the plate at 37 degrees Celsius for 72 hours. Next, add 15 microliters of the dye solution to each well and incubate the plate at 37 degrees Celsius for four hours.

Add 100 microliters of the solubilization stop solution to each well for solubilizing the formazan produced by the dye and again incubate the plate at 37 degrees Celsius overnight. Measure the optical density of the cells at 570 nanometers and use statistical software to obtain the IC50 value of PHA-665752 in the presence of Gefitinib by generating a cell proliferation curve. To establish dual resistance in MET1000 cells to Gefitinib and PHA-665752, culture the MET1000 cells in P100 dishes with 10 milliliters of growth medium and one micromolar Gefitinib.

Add 1/10 of the IC50 value of PHA-665752 into the growth medium in the presence of one micromolar of Gefitinib and culture the cells under the same conditions. Increase the PHA-665752 concentration progressively by 10 to 30%with each split until it reaches one micromolar. This might take about six to 12 months.

Finally, perform the MTT assay to confirm that the cells are resistant to Gefitinib and PHA-665752 and that they are not sensitive to PHA-665752 in the presence of Gefitinib. These dual resistant cells are referred to as PC-9DR. This graph illustrates the results of an MTT assay where a decrease was seen in the proliferation of parental PC-9 cells as the concentration of Gefitinib increased, indicating that the PC-9 cells are sensitive to Gefitinib after the dose escalation procedure was performed.

As seen here, PC-9MET1000 cells exhibit resistance to Gefitinib even at high concentrations, but are sensitive to a MET-TKI PHA-665752 in the presence of Gefitinib. PC-9 cell proliferation was suppressed at higher concentrations of Gefitinib, indicating that they were sensitive to Gefitinib. Amplified levels of MET protein and RNA in PC-9MET1000 and PC-9DR cells are demonstrated here and no acquired mutations are present in EGFR and MET.

Therefore, the emergence of bypass signaling is highly suggested as the cause of the acquired resistance to EGFR-TKI and/or MET-TKI. In this experiment, the MTT assay was used to show resistance of PC-9DR cells to PHA-665752 in the presence of one micromolar Gefitinib. Although proliferation of PC-9MET1000 cells was inhibited by treatment with Gefitinib and PHA-665752, PC-9DR cells survived the treatment, indicating that they had acquired dual resistance to Gefitinib and PHA-665752.

Once mastered, each procedure can be performed in 30 to 60 minutes. While attempting this procedure, it is important to remember to seed equal volumes of the viable cells into each well of the 96 plate for the MTT assay. After its development, this technique paved the way for the researchers in the field of cancer research to explore novel resistant mechanisms to cancer agent in all kinds of cancers.

After watching this video, you should have a good understanding of how to obtain resistant cell rise to cancer agent in cancer therapy through the procedure. Don't forget that working with the solubilization stop solution in the MTT assay can be extremely hazardous and precautions such as wearing gloves should always be taken while performing this procedure.