Effect of EVs from MDA-MB-468 on stem cell and EMT markers in U2OS cells.

<p>U2OS cells were incubated with 0 or 50 µg/mL of MDA-MB-468-derived EVs and quantitative polymerase chain reaction analysis was conducted to examine the expression of the transcription factors <i>Nanog</i>, <i>Oct-4</i>, <i>Sox2</i>, <i>Zeb1</i> and <i>Snaill</i> involved in development and maintenance of stem cells [data are mean ±SD; <i>n</i> = 3, <i>p</i><0.05, 0.05, 0.01, 0.05 and 0.01, respectively, relative to the values in the control].</p

Human Mammospheres Secrete Hormone-Regulated Active Extracellular Vesicles

<div><p>Breast cancer is a leading cause of cancer-associated death worldwide. One of the most important prognostic factors for survival is the early detection of the disease. Recent studies indicate that extracellular vesicles may provide diagnostic information for cancer management. We demonstrate the secretion of extracellular vesicles by primary breast epithelial cells enriched for stem/progenitor cells cultured as mammospheres, in non-adherent conditions. Using a proteomic approach we identified proteins contained in these vesicles whose expression is affected by hormonal changes in the cellular environment. In addition, we showed that these vesicles are capable of promoting changes in expression levels of genes involved in epithelial-mesenchymal transition and stem cell markers. Our findings suggest that secreted extracellular vesicles could represent potential diagnostic and/or prognostic markers for breast cancer and support a role for extracellular vesicles in cancer progression.</p></div

Cancer-related networks detected by the data mining IPA software.

<p>A list of 37 proteins was recognized by ingenuity pathway analysis software to build a connectivity network. This network was highly associated to cancer (<i>p</i>-value = 5.9E-12) and integrates the data from the list of proteins identified in control (grey-filled circles), in estrogen (green-filled circles), in tamoxifen (red-filled circles) or in both estrogen and tamoxifen (yellow-filled circles) treatments. Some molecules (unfilled) were added by IPA software to complete the pathway. The proteins known to be regulated by estrogen (surrounded by an orange outline), by tamoxifen (surrounded by violet outline) or both (surrounded by blue outline) are also indicated.</p

Characterization and comparison of primary mammosphere-derived EVs cultured with or without hormone treatments.

<p>(A) Representative cryo-electron micrographs (Bar, 100 nm). (B) Normalized representation of size distribution by NTA analysis of EVs in one primary breast epithelial cell preparation. Mean, SD and particle concentration values are indicated in the table (EtOH in green, E2 in orange and TAM in light green). (C) Western blot analysis of cell extracts and EVs derived from mammospheres treated with ethanol (EtOH), estrogen (E2) or tamoxifen (TAM). Antibodies against exosomes (Flotillin-1, CD63, CD81 and MFGE8), early (EEA1) and recycling (Rab11) endosomes, or endoplasmic reticulum (Grp78) protein markers were assayed. The molecular mass (kDa) for each protein is indicated.</p

EV content is modulated by hormone treatment.

<p>Western blot analysis of total cell extracts and EVs of two independent human primary mammosphere preparations cultured in the presence of ethanol (EtOH), estrogen (E2) or tamoxifen (TAM). Protein expression was analyzed by immunoblotting with antibodies against CD13, Flotillin-1 and CD81 proteins. The molecular mass (kDa) for each protein is indicated. Representative micrographs of mammospheres formed under the different conditions are also shown. <i>Bar, 50</i> µ<i>m</i>.</p

Uptake of EVs by cell lines of different tissue origin as putative metastatic destinations.

<p>Left panels represent images of M1 (fibroblast), SK-Hep1 (endothelial, liver adenocarcinoma), U2SO (osteosarcoma), SH-SY5Y (neuroblastoma) and BXPC3 (pancreatic adenocarcinoma) human acceptor cells under control conditions. Right panels are individual frames showing the internalization of CD133-positive EVs from MDA-MB-468 into the acceptor cells (<i>Bar, 40 µm</i>).</p

Proteomic profiling of Evs secreted by breast primary human mammospheres.

<p>^a Number of peptides identified in the mass spectrometry analysis.</p><p>^b Functional categorization using Ingenuity Pathway Analysis software. The functional categories more represented in the proteomics analysis were Cancer (Can), Proliferation (Prol) (including cellular growth, cell cycle and cell death) and Development (Dev) (including cell signaling, cellular assembly and organization, tissue remodeling and maintenance).</p

Effect of EVs secreted by MDA-MB-468 mammospheres on MCF7 cells.

<p>(A) MCF-7 cells were incubated with 0, 25 and 50 µg/mL of MDA-MB-468 EVs and the number of mammospheres formed after 7-days was counted [data are mean ±SD; <i>n</i> = 3, *<i>p</i><0.05, **<i>p</i><0.01, relative to the values in the control]. (B) Quantitative polymerase chain reaction analysis was conducted to examine the expression of the factors <i>Zeb1</i> and <i>Snaill</i> [data are mean ±SD; <i>n</i> = 3, <i>p</i><0.05 and 0.05 respectively, relative to the values in the control].</p

Validation of proteomics profiling of EVs from primary human mammospheres by immunoblotting.

<p>Western blot analysis of protein extracts obtained from cells (left) or EVs (right) secreted by mammospheres cultured in the presence of ethanol (EtOH), estrogen (E2) or tamoxifen (TAM). In addition to EV protein markers (Flotillin-1, CD63, CD81 and MFGE8) used as positive controls, the presence of proteins detected in the proteomic analysis, including prominin1/CD133, annexin A2 and catalase, were evaluated. Three different tissue samples were examined and one representative example is shown. The molecular mass (kDa) for each protein is indicated.</p

Microglial phagocytic response during in vivo acute and chronic inflammatory challenge.

<p>(<b>A</b>) Experimental design and apoptosis in the DG of c57BL/6 fms-EGFP 1-mo mice injected systemically with LPS (1mg/kg; <i>n</i> = 5) or vehicle (saline; <i>n</i> = 4) 8 h prior to sacrifice. Apoptotic cells were identified by pyknosis/karryorhexis. <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.g002" target="_blank">Fig 2A</a></b> was generated from data that was originally published as part of [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.ref009" target="_blank">9</a>]. (<b>B</b>) Weighted Ph capacity of microglia (in parts per unit, ppu) in control and LPS mice. (<b>C</b>) Number of microglial cells in control and LPS mice. (<b>D</b>) Ph/A coupling in the 1-mo mouse hippocampus (in fold change) during acute inflammatory challenge. (<b>E</b>) Experimental design and representative confocal z-stacks of the DG of PND21 Swiss mice fed during gestation and lactation with a diet balanced (Ω3 bal; <i>n</i> = 7) or deficient (Ω3 def; <i>n</i> = 7) in the omega 3 polyunsaturated fatty acid, a diet that induces chronic inflammation in the hippocampus. Microglia were labeled with Iba1 (cyan) and apoptotic nuclei were detected by pyknosis/karyorrhexis (white, DAPI). Arrows point to apoptotic cells engulfed by microglia (M). Scale bars = 50 μm; z = 22.5μm. (<b>F</b>) Number of apoptotic (pyknotic/karyorrhectic) cells in mice fed with Ω3 balanced and deficient diets. (<b>G</b>) Ph index in the PND21 hippocampus (in % of apoptotic cells) in mice fed with Ω3 balanced and deficient diets. (<b>H</b>) Weighted Ph capacity of microglia (in ppu) in PND21 mice. (<b>I</b>) Histogram showing the Ph capacity distribution of microglia (in % of cells) in PND21 mice. (<b>J</b>) Total number of microglial cells (Iba1⁺) in PND21 mice. (<b>K</b>) Ph/A coupling in PND21 mice. Bars represent mean ± SEM. * indicates <i>p</i> < 0.05 and ** indicates <i>p</i> < 0.01 by one-tail Student´s <i>t</i> test. Underlying data is shown in <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s001" target="_blank">S1 Data</a></b>.</p