An invasive phenotype emerged from a sub-population of cells surviving post-IR in three-dimensional lrECM. (A) Experimental schema of the recurrence model. At Day 12, cultures were exposed to Sham or 8 Gy IR. On Day 15, the colonies were taken out of three-dimensional lrECM, dissociated to make single cells, and expanded on two dimensional. Single cells were re-plated on three-dimensional lrECM and propagated until Day 30 (12 additional days). (B) Phase-contrast micrographs show that a distinct phenotype emerged by Day 30 of culture. Bar = 50 μm. IF images show α6-integrin or β1-integrin (green). Bar = 50 μm. (C) Invasive activity of MCF10A-Akt cells post-IR was quantified using invasion chambers. Graphical representation of the invasive cell numbers were normalized with control, non-irradiated cultures (n = 3; **, P < 0.01). (D) Gelatin zymography shows that MMP-9 secretion was increased in culture medium of IR-treated MCF10A-Akt. (E) Matrix degradation activity was confirmed by fluorescently labeled DQ-gelatin matrix. Degraded gelatin is shown in green (22% ± 7 invasive cells versus 3% ± 1; n = 3; **, P < 0.01). DCIS, ductal carcinoma in situ; IF, immunofluorescence; IR, ionizing radiation; lrECM, laminin-rich extracellular matrix; MMP-9, matrix metalloproteinase-9.