Expression profile of miR-200f and EMT-transcriptional inducers in breast tumors.

<p>The expression levels of miR-200 family members (<b>A</b>) and EMT-transcriptional inducers (<b>B</b>) were quantified by qRT-PCR in 70 breast cancer samples. Data are depicted as box-and-whisker plots. Adjusted <i>p</i>-values are shown where significant differences were found (Wilcoxon rank-sum test). ER+ (estrogen receptor positive tumors), HER2+ (HER2-positive tumors), TN (triple negative; ER−, PR−, HER2−), MBC (metaplastic breast carcinomas). (<b>C</b>) Upper heatmap represents Pearson coefficient (R) correlation values between the expression of EMT-transcriptional inducers and miR200f members. Bottom heatmap depicts the level of statistical significance (P) of the correlations.</p

miR-200f and EMT-transcriptional inducers are modulated during <i>in vitro</i> EMT in breast basal cell lines.

<p>Expression of (<b>A</b>) miR-200f and (<b>B</b>) EMT-transcriptional inducers was evaluated by qRT-PCR in the sorted epithelial (EpCAM+) and mesenchymal (Fibros) subpopulations within MCF12A and Myo1089 cell lines. Data are normalized to the expression of <i>RNU48</i> and <i>18S</i> respectively. Bars represent mean expression changes ±SE in Fibros subpopulation relative to EpCAM+ cells (baseline). Three biological replicates were measured. Moderated t-test was performed to evaluate statistical significance (<i>p</i><0.001 for all miRNAs analyzed in panel A. <i>NS</i>, non significant). (<b>C</b>) Methylation status of <i>miR-200f</i> promoter sequences in EpCAM+ and Fibros subpopulations within MCF12A and Myo1089 cell lines. Histogram bars represent averaged methylation levels (±SE) for the promoter sequences of <i>miR-200f loci</i>. Statistically significant differences are indicated (*<i>p</i><0.001, Student’s t-test).</p

Characterization of MSC.

<p>(A) <i>In vitro</i> differentiation potential of MSC. <i>In vitro</i> adipogenic differentiation. Representative images show the light microscopic view of lipid drop accumulation after 3 weeks of culture in control medium and in differentiation induction medium. <i>In vitro</i> chondrogenic potential of MSC. Representative images show the light microscopic view of alkaline phosphatase activity (detected by a blue-dark staining) and mineralization after culture in control medium and in differentiation induction medium (detected by red staining). (B) Growth rate evaluation in MSC-HD and MSC-P. Cells were counted with trypan blue at each passage. Trypan blue selectively colors dead cell in blue whereas live cells are observed shinning since this marker is unable to enter the cytoplasm when the cell membrane in intact. Both sets of samples presented similar growth rates and were in continuous growth during these experiments. (C) Immunophenotypic characterization of MSC: positive expression of CD105, CD90, CD73 and CD106 and negative expression of hematopoietic markers CD34, CD45, CD19 and HLA-DR (light gray represents control and darker gray represents sample with antibody).</p

Comparative study between MSC, EWS cell lines and EWS samples.

<p>(A) Hierarchical unsupervised cluster analysis showed two different clusters, one consisting of all EWS cell lines and the other of all MSC samples. Control was performed using Fluorescence minus one. The following markers: CD105, CD34, CD45 and CD90 were used in every tube in order to diminish the auto-fluorescence presented by MSC, therefore these were not contemplated in this analysis, and were validated in frozen tissue from EWS samples. (B) IHC studies in frozen tissue from EWs samples. Upper panel represents EWS typical morphology as well as CD99 strong expression. Middle panel shows that a strong expression of CD90 and positivity for CD105. Finally, lower panel shows that EWS present negative expression of both CD45 and CD34.</p

CD99 expression in MSC derived from healthy tissues.

<p>CD99 expression levels in MSC derived from normal tissue, including BM; dental pulp; adipose tissue; chorion; amnios and placenta. Again, intensity levels for CD99 expression were variable with 11/19 samples presenting moderate/high expression (scores +++ and ++); 8/19 presenting low levels of expression and 4/19 with negative CD99 expression. Samples were studied between passages 4 and 6.</p>1<p>MFI stands for Median Fluorescence Intensity,</p>2<p>PN stands for Passage Number.</p

Cytofluorimetric analysis of CD99 expression on RD-ES cell line and BM-MSC.

<p>A) Dotted line represents cells stained with secondary antibody alone; solid profile represents cells stained with anti-CD99 antibody. In each panel, the ordinate represents the number of cells. All the 5 MSC used for functional assays presented similar levels of CD99 expression. (B) Time-course analysis of apoptosis after exposure to anti-CD99 0662 MAb. RD-ES is used as positive control. Cell death was determined by examining annexin V-FLUOS binding with flow cytometry. Early-apoptotic cells were annexin V-positive and PI -negative (Lower-Right region). Late apoptotic/necrotic cells were annexin V- and PI-positive (Upper-Right region). Results shown here are representative of all the 5 CD99-highly positive MSC. (C) Percentage of living, apoptotic and necrotic cells observed in cells (two representative cultures of BM-MSC and RD-ES EWS cell line) treated with anti-CD99 MAb for 15 min or (* represents statistical significance in comparison to control p<0,05)(D) 4 hrs. The assay was performed on detached cells. The EWS cell line shows an increment in the percentage of apoptotic and necrotic cells after CD99 engagement, whereas the BM-MSC show similar percentages of alive and dying cells in treated or untreated conditions. (* represents statistical significance in comparison to control p<0,05).</p

MSC-P lack the presence of the EWSR1-FLI1 chromosomal translocation.

<p>(A) Study of the chromosomal translocation between genes EWSR1 and FLI1 using a EWSR1-FLI 1 homemade-fusion probe(1–3). MSC-P showed two normal copies of both EWSR1 and FLI1 (1 and 2) whereas in the TC71 cell line we observed not only two normal copies but also one EWSR1-FLI1 fusion, marked by a white arrow (3) Rearrangements of the EWSR1 gene were studied using a EWSR1 break-apart probe. (4–6) Representative images of MSC-P, EWS-P-01 and EWS-P-02, (4 and 5) failed to present a break in the EWSR1 gene, whereas the positive control here represented by the TC71 cell line (6), showed a distinctive rearrangement of this gene, marked by white arrows. (B) Results were validated by q-RT-PCR using EWS cell lines as a positive control. MSC-P, here represented by Patients 1;2;3;5;6 and 7 as well a healthy donor display a clear negative expression of all the transcripts.</p

Concordance between ALK IHC and <i>ALK</i> FISH.

<p><i>IHC, immunohistochemistry; FISH, fluorescence in situ hybridization; CI, confidence interval; PPV, positive predictive value; NPV, negative predictive value.</i></p><p>Concordance between ALK IHC and <i>ALK</i> FISH.</p

Box plots for number of <i>ALK</i> positive cells by FISH automatized technique versus intensity of the ALK IHC staining.

<p>With the Ventana anti-ALK antibody (A) and with Novocastra (5A4) antibody (B). Kruskal-Wallis test was performed. The comparisons between the categories in each antibody were statistically significant (p<0,001).</p

Study design and specimen selection.

<p>Study design and specimen selection.</p