Fig. 2: MLD therapy minimises therapeutic resistance and is effective in EGFRi-resistant PC9 cells.

a MLD therapy abrogates cell proliferation and induces apoptosis in PC9 cells. PC9 cells were plated and incubated overnight to allow attachment to the plate. Cells were then treated with DMSO, with EGFR, RAF, MEK, ERK inhibitors at low dose, with 3D Combo (RAF + MEK + ERK inhibitors at LD) or with 4D Combo (EGFR + RAF + MEK + ERK inhibitors at LD) and placed in the IncuCyte®. Confluence (left) and caspase 3/7 activation (right) over time was measured by the IncuCyte®. Standard error of the mean (SEM) from 3 replicates is plotted. b MLD therapy prevents the acquisition of drug resistance in PC9 cells. PC9 cells were cultured with DMSO, with EGFR, RAF, MEK and ERK inhibitors at low dose (for 7 days) and with high dose (HD) of Osimertinib (200 nM), HD of Gefitinib (280 nM) and with 3D and 4D Combinations (for 1 month), after which plates were stained and scanned; A representative image from 3 biologically independent replicates is displayed. c EGFRi-resistant PC9 cells remain sensitive to MLD therapy. PC9, PC9-OR (Osimertinib-resistant) and PC9-GR (Gefitinib-resistant) cells (see methods) were cultured with DMSO, with low doses of EGFR, RAF, MEK or ERK inhibitors, with 3D or 4D combinations or with HD of Gefitinib or Osimertinib for 4 days, after which cell viability was measured using CellTiter-Blue®. Standard deviation (SD) from 3 biologically independent replicates is plotted. d MLD therapy blocks MAPK pathway in EGFRi-resistant PC9 cells. PC9, PC9-OR and PC9-GR cells were cultured with DMSO, HD of Osimertinib, HD of Gefitinib or with 3D or 4D combinations. Protein for western blotting was harvested after 24 h of treatment; The level of pathway inhibition was measured by examining pERK and pRSK protein levels and the level of EGFR inhibition was measured by examining pEGFR protein levels in the western blot. Tubulin and Vinculin were used as loading control.