Fig. 4. The combination of osimertinib and trametinib with different schedules delays the emergence of osimertinib resistance in vitro.

A, PC-9 cells seeded in 24-well plates were exposed to DMSO, 500 nM osimertinib (Osim), 20 trametinib (Tram) and osimertinib plus trametinib, respectively. The same treatments were repeated every 4 days. The cells were stained and pictured on the indicated days. After 39 days, the cells treated with osimertinib alone were switched to treatment with the combination of osimertinib and trametinib. B, PC-9 cells seeded in 96-well plates were exposed to the treatments as described in A. Cell densities were scored weekly for 10 weeks. At the end of the experiment, the cells were fixed and stained with SRB (right panel). The data (left panel) are the means ± SDs of 3 plates, each of which had 60 replicate wells. C, The experimental procedure was basically the same as described in A except for the combination treatments as follows: the combination of osimertinib and trametinib was included for 2 weeks after every 2-weeks or 4-weeks osimertinib treatment. After 98 days, resistant cells treated with osimertinib alone were switched to treatment with the combination of osimertinib and trametinib.