Dose Escalation: A Method for Developing Drug Resistance in Cancer Cells

Protocolo

1. Establishment of Three Independent Afatinib-resistant PC-9 Cell Lines

1. Determination of the initial afatinib exposure concentration for PC-9 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay.
   1. Culture PC-9 cells in growth medium containing fetal bovine serum (10%), penicillin (100 U/mL), and streptomycin (100 µg/mL) in a cell-culture treated 10-cm dish in a 5% CO₂ incubator at 37 °C.
   2. Resuspend PC-9 cells at 4 x 10⁴ cells/mL in growth medium and then seed at 50 µL/well in a 96-well microplate. The final concentration of cells is 2.0 x 10³ cells/50 µL/well. Incubate overnight in a 5% CO₂ incubator at 37°C.
   3. Add 50 µL of afatinib solution at different concentrations: 0, 0.002, 0.006, 0.02, 0.06, 0.2, 0.6, 2, 6, and 20 µM to the wells containing the growth medium (50 µL). The final volume and concentrations of afatinib are 100 µL and 0, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 µM, respectively.
   4. Incubate the 96-well plate for 96 h in a 5% CO₂ incubator at 37 °C.
   5. Add 15 µL of the dye solution (see Table of Materials) to each well and incubate for 4 h in a 5% CO₂ incubator at 37 °C, and then add 100 µL of solubilization/stop-solution (see Table of Materials) to each well and incubate overnight in a 5% CO₂ incubator at 37 °C.
   6. Measure the optical density at 570 nm (OD₅₇₀) using a microplate reader (see Table of Materials). Prepare 6-12 replicates and repeat the experiments at least three times.
   7. Use statistical software (see Table of Materials) to graphically plot these data as a semi-log graph and calculate the IC₅₀ value, which is the drug concentration that reduces response to 50% of its maximum (see Table of Materials).
2. Continuous exposure of PC-9 cells to the irreversible EGFR-TKI, afatinib, by stepwise dose escalation in three independent 10 cm dishes
   1. Culture PC-9 cells in p100 dishes containing 10 mL of growth medium. When the PC-9 cells reach the sub-confluent stage, transfer 1 mL of cell suspension into three new p100 dishes, with 9 mL of growth medium. The 1:10 diluted PC-9 cells become sub-confluent in 3-4 days, with a cell number of approximately 4-5 x 10⁵ cells/mL.
   2. On the next day, add 1/10 of the IC₅₀ value of afatinib into each of the three p100 dishes.
      NOTE: Afatinib is reconstituted in DMSO at stock concentrations of 1 μM, 10 µM, 100 µM, 1 mM, and 5 mM. 1 to 10 µL of afatinib-solution is added into 10 mL of growth medium in the culture, as per the required final concentrations.
   3. When the cells in the afatinib-containing p100 dishes become sub-confluent, mix well by aspiration with a 1 mL pipette and add 1 mL of the cell suspension to 9 mL of fresh growth medium in a new p100 dish. Next, add 10-20% higher concentrations of afatinib to the new culture.
   4. Increase the afatinib concentration of 0.1 nM to 1 μM in the medium by the stepwise dose escalation with the afatinib concentration increased by 10-20% at each step over the period of 10-12 months.
      NOTE: When the afatinib concentration approaches the IC₅₀ value, cell growth becomes quite slow. If the cells are split 1:9, they may not grow, as these cells are killed by higher concentrations of afatinib. Therefore, at higher afanitib concentrations, the cells can be split at a ratio of 1:2. The most resistant cells were grown in afatinib-contained medium for 3-14 days, and the medium was not changed until the resistant cells needed to be passaged.
   5. Culture the afatinib-resistant cells for 2-3 months in 1 µM afatinib-containing growth medium. At an afatinib concentration of 1 µM, 10-12 months are required for developing resistance to afatinib in this model. Perform the MTT assay to confirm that the cells are resistant to afatinib. The three independently established afatinib resistance cell lines were named AFR1, AFR2, and AFR3.

Materiais

Name	Company	Catalog Number	Comments
FBS	gibco	26140-079
Pen Strep	gibco	15140-163
cell-culture treated 10 cm dish	Violamo	2-8590-03
afatinib	Selleck	S1011
96 well microplate	Thermo	130188
CellTiter 96	Promega	G4100	Non-Radioactive Cell Proliferation Assay; Dye solution and Solubilization/Stop solution
NanoDrop Lite spectrophotometer	Thermo	NA	spectrophotometer
GraphPad Prism v.7 software	GraphPad, Inc.	NA	a statistical software
DMSO	Wako	043-07216