- Tyrosine Kinase Inhibitors, TKI, blocks the kinase receptor from binding to ATP, preventing the phosphorylation. Begin with plating cancer cells in growth media. Allow them to reach favorable density, and transfer the cells to multiple wells. After overnight growth, dispense different drug concentrations in multiple wells and let the cells grow for the desired duration. Add MTT, a water soluble dye, to measure cell viability. Mitochondrial enzymes convert MTT to insoluble purple formazan inside living cells.
Stop the reaction by adding a suitable solubilizing agent and measure formazan spectrophotometrically. This method is called the MTT assay. Plot a graph of living cells indicated by formazan against drug concentrations. Calculate inhibitory concentration, IC50, by finding drug concentration that kills 50% of cells using the software.
Next, grow cancer cells in the growth medium and distribute them in three different plates. After the cells reach subconfluency add 1/10 dilution of IC50 concentration. Allow the cells to grow and perform the MTT assay. Repeat this process with increasing drug dosage. Cancerous cells slowly develop resistance to TKIs due to mutations in the protein sequence.
The following protocol shows the development of afatinib resistance in PC9 cells by dose escalation.
- To determine the appropriate afatinib resistance concentration, culture PC9 cells in growth medium in a 10 centimeter cell culture treated dish at 37 degrees Celsius and 5% carbon dioxide. Resuspend the PC9 cells at a 4 times 10 to the 4 cells per milliliter of growth medium concentration and seed the cells at 50 microliters of cells suspension per well in a 96 well microplate. After an overnight incubation in the cell culture incubator, treat the cells with 50 microliters of afatinib solution per well at six replicates of the indicated concentrations. And after a 96 hour incubation in the cell culture incubator, add 15 microliters of MTT dye to each well.
After four hours in the cell culture incubator, add 100 microliters of solubilization stop solution to each well and return the plate to the incubator overnight. The next morning, measure the optical density at 570 nanometers on a microplate reader. For continuous afatinib exposure, culture PC9 cells in P100 dishes containing 10 milliliters of growth medium until the cells reach subconfluency.
Transfer the subconfluent cultures into three new P100 dishes of 9 milliliters of growth medium per initial culture dish and return the cells to the cell culture incubator. The next morning, add 0.1 nanomolar or 1/10 of the half maximal inhibitory concentration of afatinib to each set of three culture dishes. When the afatinib treated cells become subconfluent, use a 1 milliliter pipette to thoroughly mix the cultures and transfer 1 milliliter of cells from each dish to 9 milliliters of fresh growth medium in new P100 dishes.
Add 10% to 20% higher concentrations of afatinib to the new cultures, increasing the afatinib concentration by stepwise dose escalation each time the cultures reach subconfluency over a 10 to 12 month period. When an afatinib concentration of 1 micromolar is reached, perform the MTT assay as demonstrated to confirm that the cells have developed afatinib resistance.