FIGURE 5 from Antibody–Drug Conjugate αEGFR-E-P125A Reduces Triple-negative Breast Cancer Vasculogenic Mimicry, Motility, and Metastasis through Inhibition of EGFR, Integrin, and FAK/STAT3 Signaling

Inhibition of EGFR signaling is not critical for inhibition of VM by αEGFR-E-P125A. A, αEGFR-E-P125A treatment inhibits VM tube formation in a dose-dependent manner compared with cetuximab and E-P125A controls. B, Quantification of meshes by Image J Angiogenesis Analyzer plugin demonstrates that compared with controls, αEGFR-E-P125A treatment significantly reduced mesh (tube) formation in MDA-MB-231-4175 cell plated on matrigel. C, Increasing doses of cetuximab treatment does not inhibit VM tube formation. D, Western blot analysis demonstrating reduction of EGFR Y1069 phosphorylation upon increasing doses of cetuximab treatment. E, Quantification of meshes by Image J Angiogenesis Analyzer plugin demonstrates that increasing doses of cetuximab do not inhibit VM tube formation. F, siRNA knockdown of EGFR was conducted on MDA-MB-231-4175 cells and a VM tube formation assay was conducted to determine whether knockdown of EGFR inhibited VM tube formation. G, Quantification of meshes demonstrates that number of meshes remained the same upon EGFR knockdown. H, Western blot analysis validating efficiency of EGFR siRNA knockdown. I, Increasing doses of E-P125A results in the formation of fewer VM tubes. J, Quantification of meshes demonstrates that increasing concentrations of E-P125A reduces number of complete meshes. K, Western blot analysis demonstrating reduction of FAK Y397 phosphorylation upon increasing concentrations of E-P125A treatment. VM tube formation assays were imaged at 5x magnification. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.