1. Epidermal Growth Factor Facilitates Melanoma Lymph Node Metastasis by Influencing Tumor Lymphangiogenesis 
Andreas Bracher, Ana Soler Cardona, Stefanie Tauber, Astrid M. Fink, Andreas Steiner, Hubert Pehamberger, Heide Niederleithner, Peter Petzelbauer, Marion Gröger, Robert Loewe 
Journal of Investigative Dermatology 
Volume 133, Issue 1, Pages (January 2013)
DOI: /jid
Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Effect of epidermal growth factor (EGF) knockdown on M24 and A375 melanoma cell lines in vitro. (a) Relative messenger RNA (mRNA) expression of EGF in M24 control (Ctrl) and A375 Ctrl cell lines represented as 2(-ΔΔCt) ±SD normalized to M24 Ctrl, *P<0.05. (b) Detection of pro-EGF protein in M24 and A375 cell lines in vitro by western blot analysis. β-Actin has been used as loading control. Images from one representative experiment out of three independent experiments are shown. (c) Migration of EGFkd and control cell lines over a period of 72hours after artificial wounding of a two-dimensional cell monolayer in vitro. Shown are representative images from one out of three independent experiments. Bar graph shows mean of three experiments ±SD, *P<0.05.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Epidermal growth factor (EGF) knockdown in EGF-high expressing melanoma cells reduces sentinel lymph node (SLN) metastasis. (a) Growth curve of intradermally inoculated M24 control (Ctrl) (n=7), M24 mirNeg (n=7), and M24 EGFkd (n=7) tumors (left) and of A375 Ctrl (n=5), A375 mirNeg (n=4), and A375 EGFkd (n=5) tumors (right). Data from one out of two independent experiments are represented as mean volumes ±SEM. (b) Weight of explanted SLNs from M24 Ctrl (n=11), M24 mirNeg (n=11), M24 EGFkd (n=12), A375 Ctrl (n=5), A375 mirNeg (n=4), and A375 EGFkd (n=5) tumor–bearing animals represented as mean±SEM, *P<0.05. (c) Classification of SLNs using immunohistochemical detection of human melanoma cells by using an antihuman vimentin antibody. SLNs within each group are given as percentages per group. Bar=400μm.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Epidermal growth factor (EGF) knockdown reduces tumor lymphangiogenesis in EGF-high expressing tumors. Immunohistochemistry of paraffin sections from (a) M24 control (Ctrl) (n=7), M24 mirNeg (n=7), and M24 EGFkd (n=7) tumors and (b) A375 EGFkd (n=5), A375 mirNeg (n=4), and from A375 Ctrl (n=5) tumors are shown. Blood vessel marker CD34, vessel marker CD31, and lymph vessel markers Lyve-1 and podoplanin were used. Representative areas were extracted from whole-sample scans with Scan Scope (Aperio) using the Image Scope software. Bar=200μm. Quantification of staining using positive (pos.) pixel evaluation is represented as mean ±SD, *P<0.05.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Epidermal growth factor (EGF) knockdown affects lymphangiogenesis by influencing vascular endothelial growth factor-C (VEGF-C) in vivo. (a) Induction of immortalized lymphatic endothelial cells (LECT) sprouting with tumor cell–derived conditioned media, conditioned media and EGF-neutralizing antibody (EGF neut. AB), recombinant EGF, and fetal calf serum (FCS) for 6hours. Representative images from one out of six independent experiments are shown. Quantification is displayed as mean sprout pixel area ±SD, *P<0.05. (b, Left) VEGF-C relative messenger RNA (mRNA) expression in M24 control (Ctrl), mirNeg, and EGFkd (n=7, each) tumors and in A375 Ctrl (n=5), mirNeg (n=4), and EGFkd (n=5) tumors, shown as mean 2(−ΔΔCt) ±SD and normalized to the respective Ctrl tumors. *P<0.05, as indicated. (Right) Correlation of EGF versus VEGF-C relative mRNA expression in xenotransplanted tumors; statistically significant linear regression line is shown (P<0.05).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Epidermal growth factor (EGF) expression correlates with vascular endothelial growth factor-C (VEGF-C), Prox-1, and sentinel lymph node (SLN) status in human primary melanoma. (a) Correlation of EGF/VEGF-C (left, n=42) and EGF/Prox-1 (right, n=27) relative messenger RNA (mRNA) expression levels in human primary melanoma. Results are shown as ΔCt values; individual tumors are represented as single dots; and statistically significant linear regression line is shown (P<0.05). (b) Correlation of EGF and VEGF-C protein expression in human melanoma by immunohistochemistry on tissue microarrays. Individual samples are represented as single dots; statistically significant linear regression line is shown (P<0.05). Two representative samples are shown. Bar=100μm. (c) Detection of EGF in sera of melanoma patients (n=60) undergoing Sentinel node biopsy and control patients (n=11) using ELISA. Tumor-positive SLNs were present in 18 patients, tumor-negative SLNs in 42 patients; *P<0.05. H&E, hematoxylin and eosin.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions