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. 2017 Dec 6;10(2):294–308. doi: 10.15252/emmm.201708076

Figure 1. Both third‐generation TKIs and a triple mAb mixture inhibit erlotinib‐resistant tumors, but their mechanisms of action might differ.

Figure 1

  • A
    PC9ER cells (4 × 106 cells per animal) were subcutaneously implanted in CD1‐nu/nu mice. Thereafter, tumor‐bearing mice were randomized into groups of 9–10 animals that were later treated with the indicated antibodies (0.2 mg/mouse/injection) once every 3 days, for 30 days. Thereafter, tumor growth was followed without any further treatment. Data are means ± SEM from nine mice in each group. CTX, cetuximab; TRZ, trastuzumab; 33, a monoclonal anti‐human HER3 antibody; PBS, saline control.
  • B
    Metabolic activity of PC9ER and H1975 cells cultured for 4 days in the presence of increasing concentrations of erlotinib, osimertinib, CO‐1686 (rociletinib), or the triple antibody combination (CTX, TRZ, and mAb33). Data are means ± SD values from three experiments.
  • C
    PC9ER cells were treated overnight with the indicated TKIs, or with 3×mAbs, and whole‐cell extracts were prepared. Cleared extracts were electrophoresed, and resolved proteins were transferred onto filters. Filters were immunoblotted for the indicated proteins or for their phosphorylated forms. Blots are representative of two independent experiments.
  • D
    H1975 NSCLC cells (3 × 106 cells per animal) were subcutaneously grafted in the flanks of CD1‐nu/nu mice. Animals were randomized into groups of eight mice after tumors became palpable. Erlotinib (50 mg/kg/dose) and osimertinib (5 mg/kg/dose) were daily administered using oral gavage, whereas the triple antibody combination (3×mAbs; CTX, TRZ, and mAb33; 0.2 mg/mouse/injection) and saline (vehicle) were administered intraperitoneally once every 3 days. Data are means ± SEM values. The broken horizontal line marks the initial tumor volume.
  • E
    H1975 NSCLC cells (3 × 106 cells per animal) were subcutaneously grafted in the flanks of four groups of CD1‐nu/nu mice. Animals were subjected to the following treatments: erlotinib (50 mg/kg/a daily treatment), osimertinib (5 mg/kg/a daily treatment), or 3×mAbs (CTX, TRZ and mAb33; 0.2 mg/mouse/injection) administered twice a week. Immunohistochemical staining for KI67 in paraffin‐embedded sections was performed (see Fig EV1D), and the results are presented in box and whisker plots, where the ends of the box are the upper and lower quartiles, the median is marked by a line inside the box and the whiskers mark the highest and lowest values. Depicting quantifications of KI67 staining using 6–8 sections/tumor. Statistical calculations refer to the control group as reference. ***P < 0.001; ****P < 0.0001; n = 5; one‐way ANOVA with Tukey's test.
  • F, G
    H1975 NSCLC cells (3 × 106 cells per animal) were subcutaneously grafted in the flanks of three groups of CD1‐nu/nu mice. Animals were subjected to erlotinib treatment (50 mg/kg/dose), which continued until tumors reached 800 mm3. Thereafter, each group received one of the following treatments: erlotinib (50 mg/kg/dose), osimertinib (5 mg/kg/dose) or 3×mAbs (CTX, TRZ and mAb33; 0.2 mg/mouse/injection) administered as in (D). Data are means ± SEM from seven mice in each group. Also shown are tumors harvested from each group of animals. Scale bar, 1 cm.

Source data are available online for this figure.