High magnification deconvolved images for WNT4, MMP7, CD1 and c-MYC co-expression in control and malignant penile tissue.

<p>Composite, representative, micrographs of immunofluorescently stained penile tissue cores used to quantify co-localization of four proteins WNT4 (Alexa 405-blue), MMP7 (FITC-green), CD1 (Cy3-orange) and c-MYC (Cy5-red) after deconvolution. Tissue cores from 8 patients (control), 25 malignant tissue cores (11 grade I and 14 grade II) were imaged using an Olympus FV1000 confocal microscope. High magnification imaging (at 1024x1024 resolution) was performed for deconvolution analysis using Huygens Profesional software. Areas of squamous cell carcinoma in malignant tissue cores were identified, visually (see box) from a serial H&E section of the tissue, and imaged at high magnification (40x with 4–6x zoom). Two representative immunofluorescently labelled cores are shown: A is control and B and C are grade I and grade II, squamous cell carcinoma samples, respectively; Inset is H&E serial sections of at low magnification. High magnification images were deconvolved to reduce noise and converted to 16bit tif files for each fluorophore (Alexa Fluor-405, FITC, Cy3 and Cy5) were imported into Huygens deconvolution software and pseudo colored (FITC, green; Alexa Fluor-405, blue; Cy3, orange; and Cy5, red). Z-projections for up to 53 images for each tissue core. Representative images of controls and malignant carcinoma samples are shown. Scale bar = 3μm. High magnification images were used for quantitative co-localization measurements.</p

Quantitative analysis of protein expression in PeCa.

<p>The expression of fluorophore labelled proteins (CD1, Cy3; MMP7, FITC; c-MYC, Cy5; Wnt4 Alexa Fluor-405, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124395#pone.0124395.g001" target="_blank">Fig 1</a>) was quantified in an unbiased manner, by using a reproducible, semi-automated particle analysis protocol (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124395#sec002" target="_blank">methods</a>) using grayscale images of the stained tissue from 141 tissue cores (102 malignant and 37 non-malignant control, normal or cancer adjacent penile tissue cores). Data is presented as means ± SD for control vs malignant samples (all grades). Fold increase is relative to control for each protein and significance of difference was calculated using Mann Whitney U test.</p><p>Quantitative analysis of protein expression in PeCa.</p

Representative images from Zeiss AxioScan Z1 slide scanner.

<p>Composite, overlay, of four fluorophores (FITC, Alexa Fluor-405, Cy3 and Cy5 for MMP7, Wnt4, CD1 and c-MYC, respectively) in (A) PeCa tissue cores (core B2 of PE241) and (B) control (core D5 of PE241, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124395#sec002" target="_blank">methods</a>). C-F are individual images used to construct the composite of PeCa tissue core, for MMP7 (C, green), Wnt4 (D, blue), CD1 (E, orange) and c-MYC (F, red). G-J are individual images used to construct the composite of control tissue core, for MMP7 (G, green), Wnt4 (H, blue), CD1 (I, orange) and c-MYC (J, red). Quantitative analysis was performed on the non-enhanced, original, gray images for which all settings were identical in all tissue core images analyzed. Scale bar = 250 μm.</p

Box plot of differential pattern for WNT4, MMP7, CD1 and c-MYC co-expression in control and malignant penile tissue.

<p>Quantitative co-localization was performed from high magnification images (e.g. as represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124395#pone.0124395.g003" target="_blank">Fig 3</a>) and image files used for quantitation as described in materials and methods in WCIF ImageJ software co-localization plugin to measure the Pearson coefficient of co-localization. 35 individual tissue cores that included control (gradient from centre ) and malignant grade I (gradient from left to right ) and grade II (gradient from right to left ) were used for each fluorophore to calculate the co-localization of expression of WNT4, MMP7, CD1 and c-MYC, generating 12 comparisons: CD1 and WNT4 (orange/blue), MMP7 and WNT4 (green/blue), c-MYC and WNT4 (pink/blue), CD1 and c-MYC (orange/pink), MMP7 and c-MYC (green/pink), MMP7 and CD1 (green/orange). Significance of difference in the Pearson coefficient of co-localization between control and grades I and II (*0.02–0.04) and between grade I and grade II (§ p<0.04, range 0.04–0.0001) samples was calculated using the Mann-Whitney U test; boxes showing comparisons without symbols are not significantly different.</p

Box plot of the quantitation of expression of WNT4, MMP7, CD1 and c-MYC in PeCa tissue classified according to pathological grade.

<p>97% of the samples used in this study were classified as grade I or II. The data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124395#pone.0124395.t001" target="_blank">Table 1</a> was segregated into expression according to pathological grades and plotted as box plots. Filled circles are 99% value and horizontal line within the box is the median value. Protein expression (mean gray value per core) in control samples (green, n = 37); malignant tumor sample classified as grade I by a pathologist (blue, n = 82) or grade II (purple, n = 18). All proteins showed a significant difference in expression between control and malignant tumor samples. Significance of difference was measured using Mann-Whitney U test: * = p<0.001 and ^§ = p<0.01 between control and malignant grade I or grade II samples or within grade I and grade II. There was no significant difference in protein expression for any marker between grade I and grade II samples.</p

Supplementary Figure 5 from Epigenetics Markers of Metastasis and HPV-Induced Tumorigenesis in Penile Cancer

Supplementary Figure 5. A) Receiver Operator Curve (ROC), for the accuracy of the epigenetic lymph node prediction signature in cross validation. B) Examples of FLI1 and IRX4 immunohistochemical staining of a PeCa TMA, in samples showing either methylation.</p

Supplementary Table 2 from Epigenetics Markers of Metastasis and HPV-Induced Tumorigenesis in Penile Cancer

Supplementary Table 2. Primers sequences for A) HPV primers, B)analysis of CDO1 and AR gene expression and C) MSP analysis of HMX3, PPP2R5C, IRF4, FLI1.</p

Supplementary Figure 3 from Epigenetics Markers of Metastasis and HPV-Induced Tumorigenesis in Penile Cancer

Supplementary Figure 3. Methylation profiles of PeCa (green) and normal penile tissue (red) across canonical gene structure</p

Supplementary Table 1 from Epigenetics Markers of Metastasis and HPV-Induced Tumorigenesis in Penile Cancer

Supplementary Table 1. Patient details for A) Fresh Frozen test cohort and B) archival validation cohort</p

Supplementary Figure 6 from Epigenetics Markers of Metastasis and HPV-Induced Tumorigenesis in Penile Cancer

Supplementary Figure 6. Heatmap of 6 HNSCC cell lines (in duplicate) showing the methylation of the 30 probe set HPV classifier</p