FIGURE 7 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

αEGFR-EP125A reduces primary tumor growth and tumor cell infiltration in vivo. A, Schematic for in vivo workflow in NSG mice. MDA-MB-231-4175 TNBC cells were implanted into the mammary fat pad on day 0 and by day 2 tumors had engrafted and mice were randomized and split into four treatment groups: PBS, E-P125A, cetuximab or αEGFR-E-P125A (n = 7 mice per group). Mice were treated intravenously with respective treatments 2x a week. By day 26, mice were sacrificed, and tumors and lungs were resected for downstream analysis. B, Tumor growth curve demonstrates that mice treated with αEGFR-E-P125A had reduced primary tumor growth compared with mice treated with controls. C, Quantification of primary tumor growth at the final day 26 demonstrated that tumors treated with αEGFR-E-P125A were significantly smaller compared with the controls. D, Tumor sizes of five representative tumors from each treatment group (n = 5 for PBS due to death of 2 mice, and n = 7 for E-P125A, cetuximab, and αEGFR-E-P125A groups). E, Tumor weight of tumors demonstrated that tumor from mice treated with αEGFR-E-P125A were significantly smaller than the PBS-treated tumors, unlike the controls. F, Western blot run on protein extracted from three representative tumor samples demonstrated enhanced downregulation of EGFR Y1069, FAK Y397, and STAT3 Y705 phosphorylation in mice treated with αEGFR-E-P125A compared with the controls. G, NanoString validated gene expression changes associated with IL6 JAK STAT3 and Angiogenesis signaling in RNA isolated from in vivo tumors (n = 3 tumors per group). H, Cytokeratin staining of lungs depicts decreased tumor cell infiltration to the lungs at 20x magnification (n = 3 tumors per group). I, mCD31 staining and huLaminin costaining outlines angiogenic mouse vessels and human VM channels and their respective colocalization within tumors at 20x magnification (n = 3 tumors per group). ns, not significant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001.