Fig. 4
Afatinib inhibits EGFRvIII-cMET signaling crosstalk in SP/CSCs cells. a, b Afatinib reduces TMZ-resistant U87EGFRvIII SP/CSCs. U87MG and U87EGFRvIII cells treated with either TMZ (25 μM), afatinib (1 μM) alone or combination for 48 h were trypsinized, stained with Hoechst 33342 (5 μg/ml) and analyzed for CSCs and NSP cells using flow cytometry. The bar diagram shows the mean (± SD) percentage of SP/CSCs from U87MG and U87EGFRvIII (n = 3). c, d Afatinib decreases U87EGFRvIII-mediated self-renewal properties of CSCs. U87MG and U87EGFRvIII cells treated with TMZ (25 μM), afatinib (1 μM), or combination for 48 h were plated (2 × 103 cells/well in 100 μl of stem cell medium) on a 96-well ultra-low attachment plate. Neurosphere/tumor spheres formed after 10 days were quantified and photographed (X20 magnification). The graph shows the mean (± SD) number of tumor spheres formed by U87MG and U87EGFRvIII cells (n = 4). (*$ P ≤ 0.05); * significant compared to control; $ significant compared to TMZ. e U87, U87EGFR WT, U87EGFRvIII and U87EGFRvIII DK cell lysates were analyzed for pcMET (Tyr-1234/1235) by western blot analysis. f U87EGFRvIII cells were treated with either TMZ (25 μM), afatinib (1 μM), or combination for 48 h and analyzed for pcMET (Tyr-1234/1235). g Expression of Nanog, Oct3/4 (self-renewal marker) and pcMET (Tyr-1234/1235) were analyzed in SP/CSCs and NSP cells by western blot analysis. h U87EGFRvIII SP cells were treated with TMZ (25 μM), afatinib (1 μM), or combination for 48 h and analyzed for pcMET (Tyr-1234/1235) and stemness markers Nanog and Oct3/4 by western blot analysis. i U87EGFRvIII cells were treated with varying concentrations of the cMET specific inhibitor, SU-11274 (1 – 20 μM) or afatinib (1 μM) for 48 h and lysates were analyzed for pcMET (Tyr-1234/1235), Nanog and Oct3/4