Figure 2.

Erlotinib-resistant subclones underwent epithelial-to-mesenchymal transition and up-regulation of AXL and are characterized by resistance to apoptosis. (A) Erlotinib sensitivity of HCC827 parental cells and the erlotinib-resistant subclones (ER3 and ER10) was measured by resazurin assay after 5 days of erlotinib treatment. The mean IC50 value for HCC827 cells was 5.6 nM, and greater than 10 μM for the two erlotinib-resistant subclones ER3 and ER10 (n = 11–13, independent experiments). (B) Representative confocal images of HCC827, ER3, and ER10 cells immunofluorescently labeled for epithelial cell adhesion molecule E-cadherin (CDH1, green) and mesenchymal intermediate filament vimentin (VIM, red); DAPI nuclear stain (blue); scale bar: 50 μm. Single channel displays are shown in Supplementary Figure 1. (C) Western blot of lysates from HCC827 parental and ER cells using the indicated antibodies for epithelial marker E-cadherin (CDH1, 135 kDa), and mesenchymal markers N-cadherin (CDH2, 135 kDa), vimentin (VIM, 54 kDa), and AXL (140 kDa). β-Actin (42 kDa) is shown as a loading control. (D) Relative resistance to apoptosis was measured by induction of caspase activity of the HCC827 parental cells and ER10 cells upon 4 hours treatment with increasing doses of staurosporine by caspase 3/7 Glo luciferase assay. Y-axis represents bioluminescence as photon flux (photons/s). One-way analysis of variance was followed by Dunnett’s multiple comparisons test comparing every treatment against vehicle (DMSO) control (*** p < 0.001, **** p < 0.0001). (E) Time-lapse cytotoxicity assay to determine the cytotoxic effect of staurosporine treatment in HCC827, ER3, and ER10 cells. Green area (stained with IncuCyte CytoTox Green reagent 200 μM) representing dead cells is plotted relative to the percent confluence for each well ± SE. Three wells per condition and four images per well per time point were used for the calculations using the IncuCyte software. (F) Assessment of apoptosis induction in HCC827, ER3, and ER10 cells upon 24-hour treatment with 1 μM staurosporine by Western blot; cleaved PARP (89 kDa), procaspase 3 (32 kDa), cleaved caspase 3 (19, 17 kDa) and cleaved caspase 8 (CC8; 43, 41, and 18 kDa) expression is shown. HeLa cells were included as a positive control in this experiment. Actin (42 kDa) serves as a loading control. (G) High-dimensional analysis of the HCC827 parental cell line and the erlotinib-resistant clone ER3 using a 22-parameter time of flight mass cytometry (CyTOF) panel. Nonlinear dimensionality reduction, tSNE³³ created a 2D projection of the three cell lines on the basis of their E-cadherin (CDH1), vimentin (VIM), CD44, EGFR, AXL, and PD-L1 expression. Each point in the Cytobank visual representation of data (viSNE plot) represents an ion-cloud representing a single cell (equal downsampling at 10,000 cells per sample, 5000 iterations were applied). Distribution of cells within the viSNE plot is shown as viSNE contour plots colored by density of ion clouds representing single cells. (H) The color range represents the intensity of markers included in the CyTOF panel and the distribution is shown for each for the cell lines in separate viSNE plots. DAPI, 4′,6-diamidino-2-phenylindole PARP, poly (ADP-ribose) polymerase; PD-L1, programmed death-ligand 1; tSNE, t-distributed stochastic neighbor embedding.