Expression of MFGE8 in human breast carcinoma and effect of MFGE8 on MDA-MB-231 cells.

<p>(<b>a</b>–<b>b</b>) Scoring of MFGE8 expression, analyzed by immunohistochemistry as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072708#pone-0072708-g001" target="_blank">Figure 1</a>, in 59 breast tumor biopsies from Institut Curie’s patients, as a function of tumor grade (a) or phenotype in terms of Hormon Receptor (HR) or HER2 expression (<b>b</b>). ***: P = 0.0001 for distribution of tumors overexpressing (score 2-3) or not (score 0-1) MFGE8 between grade 2 versus grade 3 tumors (a), or between HR+/HER2- versus triple-negative tumors (b). (<b>c</b>) Analysis of mRNA expression levels (Log₂(Affymetrix U133plus2.0 signal)) of <i>MFGE8</i> in public results of Affymetrix microarray data of the Broad-Novartis Cancer Cell Line Encyclopedia as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072708#pone-0072708-g002" target="_blank">Figure 2</a>. Black arrow indicates the MDA-MB-231 cell line (HR-/HER2- triple negative phenotype) selected for subsequent experiments. (<b>d</b>) Quantification of MFGE8 by ELISA in MDA-MB-231 conditioned medium (24 hr). Secreted MFGE8 is expressed in ng/mL per 10⁶ cultured cells. Mean of two experiments + SD. (<b>e</b>) FACS analysis of αvβ3 (left panel) and αvβ5 (right panel) integrin expression at the surface of MDA-MB-231 cells. Specific antibody (blue line). Isotype control (red line). (<b>f</b>) MDA-MB-231 adhesion assay on 5µg/mL MFGE8, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072708#pone-0072708-g003" target="_blank">Figure 3-5</a>, and inhibition by 10µg/mL hMC3, represented as slope values between 0 and 1 hour. (<b>g</b>) MDA-MB-231 migration assay to 5µg/mL MFGE8 as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072708#pone-0072708-g003" target="_blank">Figure 3-5</a>, and inhibition by 10µg/mL 215A9 antibody. Data represented as slope values calculated between 0 and 18 hours. (<b>h</b>) MDA-MB-231 survival assay in the presence of 10 µg/mL MFGE8, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072708#pone-0072708-g003" target="_blank">Figure 3-5</a>, and inhibition by 10 µg/mL 399A12. 100% survival corresponds to 5µg/mL MFGE8 without antibodies. Box-and-whiskers graphs as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072708#pone-0072708-g003" target="_blank">Figure 3</a>. N=4 (except in (g): PBS N=6, MFGE8 + 215A9 N=5, and in (h): ctrl N=6, MFGE8: N=5). ***: P<0.001, **: P<0.01, compared to Ctrl = MFGE8.