Impact of the 3D Microenvironment on Phenotype, Gene Expression, and EGFR Inhibition of Colorectal Cancer Cell Lines
A) Schematic representation of the EGFR signalling pathway. B) Immunoblot analysis of AKT, phospho-AKT (S473), p44/42 MAPK and phospho p44/42 MAPK (Thr202/Tyr204). Equal amounts of total protein isolated from cells cultivated as 2D or lrECM 3D cultures were analyzed by SDS/PAGE/immunoblotting as indicated. β−actin served as loading control. SKBR3 served as a positive control for the expression of EGFR signaling molecules. C) Expression of the EGFR protein in 2D or lrECM (3D) cultivated CACO-2, DLD-1, HT-29, SW-480, LOVO, COLO 205 and COLO-206F cells. Cell lysate (40 µg protein per lane) was analyzed by SDS/PAGE/immunoblotting using monoclonal anti EGFR antibody. β-actin served as loading control. D–G) Treatment of 2D or lrECM cultivated CRC cells with AG1478. Two-tailed P-values were calculated by the paired t-test (** indicates a P-value <0.001; *** indicates a P-value <0.0001; ns = not significant). CACO-2 cells were treated for 48 h (D) or 96 h (E) with different concentrations of AG1478 as indicated. F–G) DLD-1 and HT-29 cells were incubated with the EGFR inhibitor AG1478 for 48 h.