Western blot of primary breast cancer-associated fibroblasts (CAFs).

<p>CAFs are derived from patients with ERα-positive breast cancer and have been cultured in serumfree media to allow detection of basal ERK phosphorylation levels (lower band: ERK2 42kDa, upper band: ERK1 44kDa).</p

Prognostic and molecular parameters.

1<p>Mann-Whitney <i>U</i>,</p>2<p>Pearson’s chi-square,</p>3<p>Spearman.</p><p>Distribution of CAF-pERK staining categorization according to clinico-pathological and molecular characteristics. (CAF: Cancer-associated fibroblasts, percentages in parenthesis).</p

Kaplan-Meier plots.

<p>Recurrence-free survival according to CAF-pERK level (A-C) and CAF-SMAα expression (D-F) of patients in cohort I (ERα-positive patients). Plots represent prognostic (A, D) or tamoxifen treatment-predictive information (B, C and E, F) (<i>P</i>-value: Univariate Cox regression, HR: Hazard Ratio, CI: Confidence Interval, RFS: Recurrence-Free Survival).</p

MiR-92a expression in normal breast and breast cancer samples.

<p><i>A)</i> and <i>B)</i> Two examples of miR-92a expression in normal breast tissue compared to tumour areas.</p

Analysis of mRNA expression in primary human breast cancer samples.

<p>Expression data was combined from multiple studies into a combined cohort of ERBB2 (n = 194) Basal (172) Luminal A (n = 336) and Luminal B (161) and normal-like (n = 244) subtypes of breast cancer. A) mRNA expression data was displayed in a heatmap: decreased (green) or increased (red) expression compared to the mean mRNA expression. % of samples with high gene expression are detailed and denoted as green when expression is decreased and red when expression is increased. B) Kaplan Meier plots Wnt gene expression show the years of recurrence free survival, where red indicates lower gene expression and blue indicates higher gene expression. * Red ≤upper quartile: Blue >upper quartile ** Red ≤lower quartile: Blue >lower quartile. P values were generated and significant results were highlighted in red.</p

Recurrence-free survival among breast cancer patients according to miR-92a expression.

<p><i>A)</i> Tumours were divided into four groups based on the staining intensity of miR-92a and recurrence-free survival for the patients was determined, <i>p</i> = 0.065. <i>B)</i> Recurrence-free survival analysis after merging of tumours in Q1–Q2 and Q3–Q4, <i>p</i> = 0.008.</p

Distribution of miR-92a expression in breast cancer tumours according to clinico-pathological parameters.

†<p>Correlations were calculated using Spearman's ρ(two-sided) unless otherwise specified. <i>P</i> values were not adjusted for multiple testing.</p>‡<p>Kruskal-Wallis test (two-sided).</p

Univariate Cox proportional hazard regression model of the risk of recurrence according to clinico-pathological factors and miR-92a expression.

<p>NHG = Nottingham Histological Grade; ER = estrogen receptor; HR = hazard ratio; CI = confidence interval.</p

Validation of miR-92a <i>in situ</i> hybridization probe.

<p><i>A)</i> Downregulated of miR-92a in MDA-231 cells was validated and confirmed by qRT-PCR. <i>B)</i> Detection of miR-92a using <i>in situ</i> hybridization in MDA-231 cells after downregulation of miR-92a. Lower panel shows the definition of positive pixels identified by the digital image analysis program. <i>C)</i> Quantification of positive pixels by the image analysis program.</p

Modulation of Wnt signalling in normal and primary breast cancer samples (Normal n = 3; ER+ve n = 3; ER-ve n = 3).

<p>Single cells were plated in non-adherent conditions and treated with increasing concentrations of either Wnt3a (0–50 ng/ml) or DKK1 (0–100 ng/ml) and cultured for 7 days and number of mammospheres counted. Wnt3a treatments are displayed in the left panel and DKK1 treatments in the right panel. Light grey bars represent untreated control A) primary normal breast cells (Wnt3a) B) ER+ve primary breast cancer cells (Wnt3a) C) ER-ve primary breast cancer cells (Wnt3a) D) primary normal breast cells (DKK1) E) ER+ve primary breast cancer cells (DKK1) F) ER−ve primary breast cancer cells (DKK1). Data is expressed as % mammosphere formation units. P values were generated by ANOVA. Asterisks mark individual comparisons which reached statistical significance * >0.01 ** >0.001 generated by a T-test. G) Image of a normal primary mammosphere H) Image of an ER positive primary tumour mammosphere I) Image of an ER negative primary tumour mammosphere. Scale bar represents 50 µM.</p