β1-integrin signaling is targeted to suppress invasive recurrence post-IR in MCF10A-Akt cells in three-dimensional lrECM. (A) On Day 30 cultures, up-regulated α5β1-integrin and down-regulated E-cadherin expression observed on the post-IR cultures. Columns, mean intensity (n = 3; **, P < 0.01). (B) Up-regulation of FN and EDA+FN was observed in culture medium of cells post-IR. Columns, mean intensity (n = 5; *, P < 0.05; ***, P < 0.001). (C) Phase-contrast images. IR-induced invasive phenotype was abrogated by AIIB2 compared to IgG. The antibodies were added from day 0 of the second three-dimensional cultures. Bar = 50 μm. (D) Apoptosis was measured by TUNEL-positive cells in AIIB2-treated cultures -/+ IR (mean = 18.1% ± 3.9, P < 0.01). (E-F) Matrigel chemoinvasion was significantly increased in the surviving cells post-IR, and inhibited by β1-integrin (AIIB2, five-fold, P < 0.01) or α5-integrin (P1D6, two-fold, P < 0.05) inhibitory antibodies. DCIS, ductal carcinoma in situ; FN, fibronectin; IgG, immunoglobulin G; IR, ionizing radiation; lrECM, laminin-rich extracellular matrix; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.