Figure 2. Coexpression of αvβ3 and Slug reveals unique stem-like cells in a broad-spectrum of clinical subtypes but does not impact EMT.

(A and B) Representative images of immunohistochemical staining for β3 (blue) and Slug (brown) in breast cancer samples from patient-derived xenografts (A and B, bottom) and tissue microarrays (B, top). (A) Shown is a tumor with heterogeneous staining for β3 and Slug. n = 5/30, β3⁺Slug⁺ tumors/total. Scale bars: 40 μm and 10 μm (enlarged insets). (B) β3⁺Slug⁺ cells (arrows) are shown for ER^– and ER⁺ tumors (top) as well as tumors representing different intrinsic molecular subtypes (bottom). (B, top) n = 19/125, β3⁺Slug⁺ tumors/total: n = 10, ER⁺; n = 4, HER2⁺; n = 5, triple-negative (TN). (B, bottom) n = 4/12, β3⁺Slug⁺ tumors/total: n = 2, luminal B; n = 2, basal-like. Scale bars: 20 μm. (C) Frequency of β3⁺Slug⁺ cells in immunohistochemically stained “baseline” breast cancer samples from recurrence-free patients and patients who later progressed to form distant recurrences. Numbers above each bar indicate the number of β3⁺Slug⁺ tumors per total number of tumors. P = 0.0068 (ER^–) and P = 0.0329 (ER⁺), for no recurrence versus distant recurrence. *P < 0.05 and **P < 0.01. Statistical analysis was performed by Fisher’s exact test. (D–G) Western blot analysis for the indicated proteins in a panel of breast cancer cell lines representing distinct subtypes (D), BT549 cells stably expressing β3 shRNA (shβ3) or a nonsilencing control shRNA (shCtrl) (E and G), or BT474 cells expressing ectopic β3 cDNA (β3) or a vector-only control (Ctrl) (F and G). For all immunoblots, data shown are representative of 3 independent experiments, and β-actin was used as a loading control. (H) Representative immunofluorescence images showing vimentin (red) and E-cadherin (green) in LM2-4 and MCF7 cells, with or without β3. Nuclei are stained blue in all panels. Scale bars: 20 μm. See also Supplemental Figures 2 and 3.