Figure 3.

EGFR expression is diminished following EMT-induced metastasis. (A) Following primary tumor removal, control (NS) and TGF-β1–pretreated (TGF-β) NME primary tumors were disassociated and subcultured in the presence of Puromycin (5 μg/ml). The resultant cultures were analyzed by flow-cytometry for cell surface EGFR. Isolation of the distinct EGFR-negative population within the post-EMT tumors by FACS analysis (denoted by the red box) resulted in a highly mesenchymal cell population. (B) Ex-vivo subcultured, post-EMT primary tumor cells were co-stained with antibodies against Ecad and EGFR (i), or co-stained with phalloidin and antibodies against EGFR (ii). Arrows denote cells with a highly mesenchymal phenotype (either Ecad negative or F-actin positive) that fail to express EGFR. (C) NME-LM cells and their parental NME counterparts were analyzed by flow cytometry for cell surface expression of EGFR. (D) NME and NME-LM cells were treated for 48 hours with EGF (50 ng/ml) or the EGFR inhibitor, AG1478 (1 μM). Afterward, whole cell lysates were analyzed by immunoblot for cleavage of PARP and expression levels of EGFR. (E) The outgrowth of NME and NME-LM cells grown under 3D-culture conditions in the presence of EGF or AG1478 was quantified by bioluminescence. Data are the mean (± SE) of 3 independent experiments completed in triplicate resulting in the indicated P values. (F) NME and NME-LM cells were serum starved for 6 hours and subsequently stimulated with the indicated concentrations of EGF for 30 minutes. Whole cell lysates were analyzed for phosphorylated EGFR (pEGFR). This blot was striped and reprobed for total EGFR (tEGFR). Erk1/2 served as a loading control. Data in panels A-D and F are representative of at least two independent experiments yielding similar results.