Fig. 6: ALK inhibition hypersensitizes cancer cells to paracrine growth factors secreted from dying neighbor cells.

A The ErkKTR reporter indicates ERK activity through nuclear-cytoplasmic translocation of a fluorescent protein. B Sensitivity of single cells (STE-1) to 15 min EGF stimulation. Plot shows fold change of ERK activity in single cells upon stimulation with the indicated amount of EGF in the presence or absence of ALKi (crizotinib, 1 µM). Boxplot shows median and upper/lower quartiles, and whiskers extend to 1.5*IQR. Significance tests indicate increased response above 0 ng/mL EGF. ****p < 0.0001 by Hsu multiple comparison with the best (MCB) test. n = 316, 384, 421, 285, 307, 256, 294, 421 cells respectively for DMSO and 473, 727, 536, 595, 541, 425, 439, 415 cells respectively for ALKi. See Supplementary Movie 3. C Live-cell imaging of STE-1 cells expressing ErkKTR in the presence or absence of ALKi (1 µM crizotinib). See Supplementary Movie 4. D Representative single-cell traces of cytoplasmic/nuclear ErkKTR intensity ratio from conditions shown in (C). E Quantification of ErkKTR activity in the presence of ALKi or its combination with EGFRi (1 µM erlotinib) or MMPi (10 µM marimastat). F Quantification of ERK activity pulses. Boxplot shows median and upper/lower quartiles, whiskers show 1.5*IQR. Significance assessed by Hsu MCB test. n = 177 (control), 198 (ALKi), 170 (ALKi/EGFRi), and 182 (ALKi/MMPi) single cells. G Percent of cells that exhibited any pulses over 22 h of imaging. Error bars indicate 95% CI. Significance assessed by the Hsu MCB test. n = 200 cells per condition. H Apoptotic cells secrete paracrine EGFR ligands to their neighbors. Paracrine signaling can be blocked by inhibiting either EGFR or the MMPs that mediate shedding of EGFR ligands from the surface of the sender cell. I ErkKTR activity pulses are primarily observed surrounding a dying cell during ALK inhibition but not in the absence of drug or in the added presence of EGFR or MMP inhibitors. J Definition of neighbors and non-neighbors of a death event. K Quantification of pulses per cell for each death event in neighbors or in equal number of randomly chosen subset of cells not near a death event (see “Methods” for more details), n = 91, 103, 149, 20 events for ALKi, ALKi/EGFRi, ALKi/MMPi and DMSO, respectively. Boxplot shows median and upper/lower quartiles, whiskers show 1.5*IQR. L Fraction of total neighbor vs random non-neighbor cells that show any pulsing. Cell numbers as in (F). Error bars show 95% CI. Significance in (K, L) determined by independent T-tests (within treatment conditions) or by ANOVA followed by the Hsu MCB test (across treatment conditions).