Extended Data Fig. 2: DRPs have higher percentages of persisters than drug-naive population.
(a) The ratio of percent of persisters between PC9 DRPs that were generated by 9 days of gefitinib treatment and then been on drug holiday for 4–10 weeks and PC9 naïve cells is presented for two cell lines treated with the indicated drugs. Data are presented as mean values, bars represent the standard deviation of n = 4 independent cultures for PC9 or n = 3 independent cultures for G361. P-values were obtained by two tailed Student’s t-test. (b) The CTPgefitinib of the naïve PC9 population and selected 10 of its sub-clones as measured after 9 days of treatment. CTP of the sub-clones was normalized to the CTP of the naïve PC9 non-clonal population. Data are presented as mean values, error bars represent the standard deviation of n = 3 independent cultures. P-values were obtained by two tailed Student’s t-test. (c) PC9 clones were seeded on day 0 at 1*10^6 cells per 10 cm plate. Gefitinib or DMSO were added on day 1 at 500 nM. Floating and adherent cells were collected 48 h later, fixed and stained with anti cleaved-CASP3 (SCT #9664) followed by anti-rabbit alexa647 secondary antibody, according to the antibody manufacturer commended protocol. Flow cytometry data was acquired and analyzed by CytoFLEX LX. P-value was calculated by two tailed Student’s t-test. (d) The CTPosimertinib of PC9 clones with known high or low CTPgefitinib was measured after 7 days of treatment. CTP was normalized to the number of cells just before the initiation of treatment. Data are presented as mean values, error bars represent the standard deviation of n = 2 independent cultures. (e,f) Histograms of the CTP distribution of PC9 (e) and G361 (f) cell lines are presented for both the drug-naïve populations (blue) as well as for DRPs of these cell lines (red). CTP of clones was measured after 7 days of treatment with gefitinib. To generate DRP clones, single cells that survived 9 days of gefitinib treatment were let to proliferate into clones without drug. A right shift in the distribution is demonstrated for the DRPs. Modeling of the expected distribution by pure selection of clones according to their CTP is demonstrated in grey.
