Comparison of the frequency of clonogenic progenitors in the isolated Aldehyde Dehydrogenase (ALDH)^high and ALDH^low fractions of bovine mammary cells (n = 3).

<p>Samples are: 1. 11 months old Piedmontese; 2. 7 months old Red Angus; 3. 12 months old Black Angus.</p

FACS profiles of bovine mammary cells stained for ALDH activity.

<p>Cells were stained with ALDEFLUOR with (a) or without (b) diethylamino-benzaldehyde (DEAB), an ALDH inhibitor. Panel c shows the proportional distribution of total clonogenic luminal and myoepithelial progenitors between the Aldehyde Dehydrogenase (ALDH)^high and ALDH^low fractions (% of all progenitors of a given type in the fraction shown ± SEM, n = 3).</p

Human <i>β-CASEIN</i> expression in regenerated bovine alveoli.

<p>Representative photomicrographs showing in bovine lactating mammary tissue the presence of milk proteins in the lumen of an alveolus (a) and the absence of crossreactivity of the antibody used to detect human <i>β-CASEIN</i> (b). In (c) the expression and localization in the lumen of recombinant human <i>β-CASEIN</i> is evident in outgrowths formed by cells transduced with the human β-cas-pWPI lentiviral vector. Scale bar is 25 µm.</p

Met Receptor Acts Uniquely for Survival and Morphogenesis of EGFR-Dependent Normal Mammary Epithelial and Cancer Cells

<div><p>Mammary gland development and breast cancer growth require multiple factors both of endocrine and paracrine origin. We analyzed the roles of Epidermal Growth Factor Receptor (EGFR) and Hepatocyte Growth Factor Receptor (Met) in mammary epithelial cells and mammary tumor cells derived from a mutated-ErbB2 transgenic mice. By using highly specific tyrosine kinase inhibitors we found that MCF-10A and NMuMG mammary epithelial cell lines are totally dependent on EGFR activation for their growth and survival. Proliferation and 3D-morphogenesis assays showed that HGF had no role in maintaining mammary cell viability, but was the only cytokine able to rescue EGFR-inhibited mammary cells. Insulin-Like Growth Factor-I (IGF-I), basic-Fibroblast Growth Factor (b-FGF) and Neuregulin, which are well known mammary morphogenic factors, did not rescue proliferation or morphogenesis in these cell lines, following EGFR inhibition. Similarly, ErbB2-driven tumor cells are EGFR-dependent and also display HGF-mediated rescue. Western-blot analysis of the signaling pathways involved in rescue after EGFR inhibition indicated that concomitant ERK1/2 and AKT activation was exclusively driven by Met, but not by IGF-I or b-FGF. These results describe a unique role for EGFR and Met in mammary epithelial cells by showing that similar pathways can be used by tumorigenic cells to sustain growth and resist to EGFR-directed anti-tumorigenic drugs.</p> </div

HGF act as a recovery agent in EGFR inhibited mammary epithelial cells. A.

<p>Viable cell count by trypan-blue exclusion staining at 48 h of MCF-10A (left panel) and NMuMG (right panel) cells cultivated in their respective growth medium and treated with the indicated factors (HGF 10 ng/ml (H), IGF-I 100 ng/ml (I), b-FGF 20 ng/ml (F), Neuregulin 20 ng/ml (N); AG1478 250 nM (A)). Untreated control (Ctrl) was set to 100%. Columns, mean (n = 6); bars, S.E.M. *P<0.05. <b>B.</b> MCF-10A cells (left panels; composite of 9 fields) and NMuMG cells (right panels) cultivated in their respective growth medium for 24 h with the indicated factors (HGF 10 ng/ml; AG1478 250 nM). Bar  = 1000 µm. <b>C.</b> Extended death of field composite image obtained from multiple Z-stacking pictures of MCF-10A cells (left panels) and NMuMG cells (right panels) grown in collagen for 4 days with the indicated factors (HGF 10 ng/ml; AG1478 250 nM). Bar  = 500 µm. <b>D.</b> Cell cycle distribution examined by propidium iodide staining and FACS analysis of MCF-10A cells (left panels; 8 h) and NMuMG cells (right panels; 16 h) cultivated in growth medium and treated with the indicated factors (HGF 10 ng/ml, AG1478 250 nM).</p

HGF is a survival factor of EGFR inhibited ErbB2 mammary tumor cells. A.

<p>Western-blot analysis of ErbB2, EGFR and Met expression in mammary tumors obtained from three separate transgenic ErbB2 mutated mice (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044982#s4" target="_blank">materials and methods</a>). Tubulin was used as an internal loading control. <b>B.</b> Phase contrast images of primary cells from ErbB2 mammary tumors (bar  = 500 µm). Cells were grown in proliferation medium and either left untreated (Ctrl) or treated with AG1478 (250 nM) or PHA-665752 (250 nM) for 96 h. <b>C.</b> Viable cell count by trypan-blue exclusion staining at 48 h, of immortalized ErbB2 tumor cells treated with the indicated factors (HGF 10 ng/ml (H), IGF-I 100 ng/ml (I), b-FGF 20 ng/ml (F), Neuregulin 20 ng/ml (N); AG1478 250 nM (A)). Untreated control (Ctrl) was set to 100%. Columns, mean (n = 6); bars, S.E.M. *P<0.05. <b>D.</b> Cell cycle distribution examined by propidium iodide staining and FACS analysis of ErbB2 mammary tumor cells cultivated in growth medium and treated with the indicated factors for 16 h (HGF 10 ng/ml, AG1478 250 nM). <b>E.</b> Immortalized ErbB2 tumor cells cultivated in their respective growth medium for 48 h with the indicated factors (HGF 10 ng/ml; AG1478 250 nM; IRESSA 1 µM; TARCEVA 1 µM). Bar  = 500 µm.</p

MCF-10A and NMuMG mammary epithelial cells show EGFR dependence. A:

<p>MTT assay of MCF-10A, NMuMG and BME-UV cells at 0 h, 24 h and 48 h. Cells were cultivated in their growth medium (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044982#s4" target="_blank">materials and methods</a>) and either left untreated (Ctrl) or treated with PHA-665752 (PHA) or AG1478 (both 250 nM). MTT value at 0 h was set to 100%. Columns, mean (n = 6); bars, S.E.M. *P<0.01. <b>B.</b> Western-blot analysis of ERK1/2 and AKT phosphorylation in mammary cells cultivated in their respective growth medium without (Ctrl) or with the RTK inhibitors (250 nM, 1 h) PHA-665752 (PHA) or AG1478 (AG). EGFR, Met, ERK1/2 and AKT were used to show comparable loadings between the lanes.</p

Expression and activation of EGFR and Met in mammary epithelial cells. A.

<p>Western-blot analysis of EGFR and Met expression in human MCF-10A, murine NMuMG and bovine BME-UV mammary epithelial cells. Mouse liver extract was used as a positive control for both Met and EGFR. <b>B.</b> Western-blot analysis of EGFR, Met, ERK1/2 and AKT phosphorylation in MCF-10A and NMuMG cells cultivated in serum-starving medium and treated with EGF or HGF (20 ng/ml) for 10, 30 and 60 min. EGFR, Met, ERK1/2 and AKT were used to show comparable loadings between the lanes.</p

HGF activates Met, ERK1/2 and AKT in EGFR inhibited mammary cells: roles for ERK1/2 and AKT pathways. A.

<p>Western-blot analysis of the EGFR, Met, ERK1/2 and AKT phosphorylation status in MCF-10A and NMuMG cells and ErbB2 tumor cells cultivated in starving medium for 16 h and then treated with the RTK inhibitors AG1478 (250 nM, 1 h) before adding the indicated cytokines (10 min; EGF 10 ng/ml, HGF 10 ng/ml, IGF-I 100 ng/ml). <b>B.</b> Viable cell count by trypan-blue exclusion staining at 48 h of NMuMG cells cultivated in growth medium and the indicated inhibitors (AG/A = AG1478 250 nM; UO/U = UO126 15 µM; WORT/W = wortmannin 100 nM) in presence or absence of HGF 10 ng/ml (H). Untreated control (Ctrl) was set to 100%. %. Columns, mean (n = 6); bars, S.E.M. *P<0.05. <b>C.</b> Representative pictures of NMuMG cells grown in collagen gels for 4 days in growth medium with the indicated factors (concentrations as in B; bar  = 250 µm).</p