Establishment and Characterization of Three Afatinib-resistant Lung Adenocarcinoma PC-9 Cell Lines Developed with Increasing Doses of Afatinib

The study of novel acquired resistance mechanisms will contribute to the development of more effective and safe therapeutic strategies in patients with cancer agent resistant and resectable cancer. Unfortunately, cases in which drug resistance failed to develop are generally not reported. This stepwise dose escalation method is considered the most reliable technique for obtaining acquired-resistance cells.

To determine the appropriate afatinib-resistance concentration, culture PC-9 cells in growth medium in a 10-centimeter cell culture-treated dish at 37 degrees Celsius and 5%carbon dioxide. Resuspend the PC-9 cells at a four-times-10-to-the-four cells per milliliter of growth medium concentration, and seat the cells at 50 microliters of cell 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 cull 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 PC-9 cells in P-100 dishes containing 10 milliliters of growth medium until the cells reach subconfluency.

Transfer the subconfluent cultures into three new P-100 dishes of nine 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 one-milliliter pipette to thoroughly mix the cultures, and transfer one milliliter of cells from each dish to nine milliliters of fresh growth medium in new P-100 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 one micromolar is reached, perform the MTT assay as demonstrated to confirm that the cells have developed afatinib-resistance. To determine the growth curve for the cell lines, culture parental PC-9 and the three afatinib-resistant cell lines in growth medium in the cell culture incubator for 24 hours.

Resuspend the cells from each culture at a five-times-10-to-the-third cells per milliliter of growth medium concentration, and seat 12 100-microliter replicates of each cell population per well in a 96-well microplate. Then perform the MTT assay on days zero, one, two, three, five, and seven of culture as demonstrated, and plot the results using an appropriate statistical analysis software program. To identify alterations in epidermal growth factor receptor, or EGFR genomic DNA expression, by reverse transcriptase polymerase chain analysis, isolate genomic DNA from the cell culture of interest using a DNA purification kit according to the manufacturer's instructions.

Measure the concentration of 25-nanogram-microliter concentrations of the isolated genomic DNA on a spectrophotometer. Then amplify 50 nanograms of genomic DNA using a SYBR green master mix, and analyze the results using a fluorescence-based reverse transcriptase polymerase chain reaction detection system. To identify alterations in EGFR genomic DNA expression by sequencing, amplify the genomic DNA using specific primers for EGFR exons 19 to 21.

Then purify the amplified PCR products using a PCR purification kit according to the manufacturer's instructions, and sequence the amplicons. To identify alterations in epidermal growth factor receptor protein expression by western blot analysis, treat the cells with afatinib for 24 hours. After 24 hours, wash the cell cultures two times with five milliliters of ice-cold PBS per culture per wash.

Lyse the cells in radioimmunoprecipitation assay buffer, supplemented with 1%protease inhibitor cocktail and phosphatase inhibitors cocktail two and three for 30 minutes at four degrees Celsius. At the end of the incubation, collect the lysates by centrifugation. Use the bicinchoninic acid assay to determine the protein concentration of the lysate samples.

Adjust the protein samples to 0.5, or one microgram per microliter concentrations, in 4-X sample buffer, and boil the samples at 96 degrees Celsius for five minutes. For western blot analysis of the samples, assemble ethanol-cleaned glass plates and spacers, and load the mold with an 8%polyacrylamide gel with the appropriate experimental supplements. While the gel is polymerizing, add stacking gel solution to an appropriate mold, insert the comb, and allow the stacking gel to polymerize for 20 to 30 minutes at room temperature.

When both gels have polymerized, place them in the electrophoresis apparatus and fill the tank with running buffer. Then load one 20-to 30-microliter volume of each protein sample into each well, and run the gel at 180 volts. Stop the electrophoresis after approximately 60 minutes, once the dye front flows out of the gel, and wash the gel with tris-buffered saline, supplemented with Tween, for one to two minutes.

Transfer the proteins onto a polyvinylidene fluoride membrane by semi-dry blotting for one and 1/2 hours at a constant current of 300 milliamps. Block the membrane with 5%nonfat dry milk diluted with tris-buffered saline plus Tween solution for one hour at room temperature. Next, probe the membrane with the appropriate antibodies of interest at four degrees Celsius overnight.

The next morning, wash the membranes with three 10-minute washes of fresh tris-buffered saline per wash before exposing the membrane to the appropriate secondary antibody for one to one and 1/2 hours at room temperature. Then wash the membrane five times with fresh tris-buffered saline per wash and expose the membrane to enhanced chemiluminescence solution for signal visualization using films. Here, the decrease observed in the cell proliferation of parental PC-9 cells in response to increasing concentrations of afatinib is illustrated, confirming that PC-9 cells are sensitive to afatinib exposure.

However, none of the three afatinib-resistant cell lines demonstrate a suppression of cell proliferation under afatinib exposure. Afatinib-resistant cell lines exhibit significantly slower growth curves than parental PC-9 cells. Afatinib-resistant cells express significantly higher levels of EGFR genomic DNA and EGFR protein expression than do parental PC-9 cells.

EGFR sequencing demonstrates that PC-9 cells exhibit 15 base pair deletions in EGFR exon 19, and wild type EGFR in exon 20. Afatinib-resistant one and two cell line cells, however, exhibit an amplification of wild type EGFR exon 19. Afatinib-resistant cell line three cells contain the same 15 base pair deletions in EGFR exon 19 as in PC-9 cells, but a point mutation in T790M is observed in EGFR exon 20.

Cell growth can become quite slow when the inhibitor concentration is close to IC-50 value. Do not discuss culture, as the cell will continue to proliferate gradually. These strategies were developed to mimic the conditions under which cancer patients develop clinically-relevant resistance to assess acquired resistance mechanisms and to develop safe and effective therapeutic strategies.