Fig. 4.

EMT and acquired resistance. (A) H1975 cells were cultured in the presence of the irreversible EGFR inhibitor PF00299804 until drug resistance developed, as demonstrated by Syto60 viability assays. (B) Images of the parental and drug-resistant H1975 cells by bright-field microscopy demonstrate that the resistant cells have developed a spindle-like morphology. (C) Parental and resistant H1975 cells were lysed and probed with antibodies against E-cadherin, vimentin, and actin, revealing loss of E-cadherin expression and gain of vi-mentin expression among drug-resistant H1975 cells. For comparison, HCC827 cells and the derived HCC827 GR6 cell line (HCC827 cells that acquired resistance to gefitinib via MET amplification), which do not undergo an EMT, are shown. (D) Example of a case (patient 28) whose drug-resistant tumor shows evidence of an EMT (top, pretreatment specimens; bottom, drug-resistant posttreatment specimens). Left, H&E staining; middle, staining for vimentin; right, staining for E-cadherin. Notably, the pretreatment cancer had an adenocarcino-ma histology (panel 1), does not stain for vimentin (panel 2), and shows preserved membranous staining with E-cadherin (panel 3). The vimentin-positive areas in panel 2 include alveolar macrophages (red circles), inflammatory and stromal cells in fibro-vascular cores (black arrows), but not tumor cells lining papillary structures (yellow arrows). The drug-resistant posttreatment specimen has sarcomatoid histology (panel 4), is positive for vimentin (panel 5), and is negative for E-cadherin (panel 6), consistent with an EMT.