Figure 4

Treatment with the ErbB2-specific inhibitor trastuzumab (Herceptin) restores LOXL2-expressing acini phenotype to a more normal phenotype. Acini were cultured as described in Figure 2; 300 nM Herceptin (Her) dissolved in water was added to cells at day 6 (10A cont+her; 10A L2+her), and an equivalent amount of human IgG was added as a control (10A cont+hIgG; 10A L2+hIgG). Acini were fixed, stained, quantified, and presented as described in Figure 2. All data are based on at least three independent experimental repeats. Scale bar, 20 μm. (A) Immunofluorescence staining of acini with anti-activated caspase-3 antibody to detect apoptotic cells on day 8. Staining revealed increased activation of caspase-3 in 10A L2+her acini when compared with 10A L2+hIgG acini. (B)Immunofluorescence staining of acini with anti-Ki67 antibody to detect proliferating cells on day 13. Staining revealed a decrease in proliferating cells in 10A L2+her acini when compared with 10A L2+hIgG acini. (C) Immunofluorescence staining of acini with anti-GM130 antibody to assess cell polarity on day 10. Staining revealed GM-130 ring-like staining effect in 10A L2+her acini, compared with the scrambled and disorganized staining observed in 10A L2+hIgG acini. (D) Quantitative analysis of activated caspase-3 staining (left panel; P = 0.0364 for 10A cont+hIgG and 10A L2+hIgG; P = 0.0189 for 10A L2 ± her), Ki67 staining (middle panel; P = 0.0268 for 10A cont+hIgG and 10A L2+hIgG; P = 0.0073 for 10A L2 ± her), and GM130-ring structures (right panel; P = 0.0083 for 10A cont+IgG and 10A L2+IgG; P = 0.0007 for 10A L2 ± her) in acini. These results suggest that Herceptin treatment of 10A L2 acini significantly reverted the acinar morphology to a more-normal phenotype.