FIGURE 6 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

α5β1 integrin/FAK signaling regulates VM and tumor cell motility. A, siITGA5 knockdown in MDA-MB-231-4175 cells demonstrates total inhibition of VM tube formation after 16 hours at 4x magnification. B, Number of VM meshes significantly decreased upon ITGA5 knockdown. C, Western blot validation of siRNA knockdown of ITGA5. Knockdown of ITGA5 subsequently reduces phosphorylation of FAK Y397. D, Cell motility tracking demonstrated that ITGA5 knockdown cells had reduced motility and migratory capabilities. E, Quantification of motility analysis of ITGA5 knockdown cells demonstrated a reduction in the accumulated and Euclidean distances and decreased velocity. F, siFAK knockdown in MDA-MB-231-4175 cells demonstrates total inhibition of VM tube formation after 16 hours at 4x magnification. G, Number of VM meshes significantly decreased upon FAK knockdown. H, Western blot validation of siRNA knockdown of FAK. I, Cell motility tracking demonstrated that FAK knockdown cells had reduced motility and migratory capabilities. J, Quantification of motility analysis of FAK knockdown cells demonstrated a reduction in the accumulated and Euclidean distances and decreased velocity. K, Incucyte scratch wound migration assay of MDA-MB-231-4175 cells over 16 hours at 10x magnification. Wound closure was partially inhibited in cells treated with a combination of cetuximab of and E-P125A. Wound closure was maximally inhibited in cells treated with either the FAKi, PF-573228, or αE-E-P125A. L, Compared to the other controls, the remaining wound width after 16 hours of treatment remained the widest in the FAKi or αEGFR-E-P125A–treated cells. M, FAKi and αEGFR-E-P125A treatment had the least changed wound confluence over time. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.