Epidermal Growth Factor Facilitates Melanoma Lymph Node Metastasis by Influencing Tumor Lymphangiogenesis

ORIGINAL ARTICLE

See related commentary on pg 14

Epidermal Growth Factor Facilitates Melanoma Lymph Node Metastasis by Influencing Tumor Lymphangiogenesis Andreas Bracher1,7, Ana Soler Cardona1,7, Stefanie Tauber1,2, Astrid M. Fink3, Andreas Steiner4, Hubert Pehamberger5, Heide Niederleithner1, Peter Petzelbauer1,5, Marion Gro¨ger1,6 and Robert Loewe1,5 Alterations in epidermal growth factor (EGF) expression are known to be of prognostic relevance in human melanoma, but EGF-mediated effects on melanoma have not been extensively studied. As lymph node metastasis usually represents the first major step in melanoma progression, we were trying to identify a potential role of primary tumor–derived EGF in the mediation of melanoma lymph node metastases. Stable EGF knockdown (EGFkd) in EGF-high (M24met) and EGF-low (A375) expressing melanoma cells was generated. Only in EGF-high melanoma cells, EGFkd led to a significant reduction of lymph node metastasis and primary tumor lymphangiogenesis in vivo, as well as impairment of tumor cell migration in vitro. Moreover, EGF-induced sprouting of lymphatic but not of blood endothelial cells was abolished using supernatants of M24met EGFkd cells. In addition, M24met EGFkd tumors showed reduced vascular endothelial growth factor-C (VEGF-C) expression levels. Similarly, in human primary melanomas, a direct correlation between EGF/VEGF-C and EGF/Prox-1 expression levels was found. Finally, melanoma patients with lymph node micrometastases undergoing sentinel node biopsy were found to have significantly elevated EGF serum levels as compared with sentinel lymph node–negative patients. Our data indicate that tumor-derived EGF is important in mediating melanoma lymph node metastasis. Journal of Investigative Dermatology (2013) 133, 230–238; doi:10.1038/jid.2012.272; published online 6 September 2012

INTRODUCTION Cutaneous melanoma is a highly malignant tumor and, once metastasized, a life-threatening disease. Metastases evolve (i) 1

Skin and Endothelium Research Division, Department of Dermatology, Medical University of Vienna, Vienna, Austria; 2Center for Integrative Bioinformatics Vienna, Max F. Perutz Laboratories, Vienna, Austria; 3 Department of Dermatology, Wilhelminenspital, Vienna, Austria; 4 Department of Dermatology, Hospital Hietzing, Vienna, Austria; 5 Department of Dermatology, Division of General Dermatology, Medical University of Vienna, Vienna, Austria and 6Core Facility Imaging, Clinical Institute for Medical and Chemical Laboratory Diagnostics, Medical University of Vienna, Vienna, Austria 7

These authors contributed equally to this work.

Correspondence: Robert Loewe or Andreas Bracher, Skin and Endothelium Research Division, Department of Dermatology, Medical University of Vienna, Waehringer Guertel 18–20, A-1090 Vienna, Austria. E-mail: [email protected] or [email protected] Abbreviations: A375 Ctrl, A375 (wild-type cell line); A375 EGFkd, A375 stable pLenti6/V5-GW/ þ EmGFP-Hmi405014; A375 mirNeg, A375 stable pLenti6/V5-GW/ þ EmGFPwith nontargeting miRNA; EGFkd, EGF knockdown; EGFR, EGF receptor; FFPE, formalin-fixed paraffin-embedded; HUVEC, human umbilical vein endothelial cells; LECT, immortalized (TERT) lymphatic endothelial cells; M24 Ctrl, M24met (wild-type cell line); M24 EGFkd, M24met stable pLenti6/V5-GW/ þ EmGFP-Hmi405014; M24 mirNeg, M24met stable pLenti6/V5-GW/ þ EmGFP with nontargeting miRNA; mRNA, messenger RNA; SCID, severe combined immunodeficient; SLN, sentinel lymph node; VEGF, vascular endothelial growth factor Received 28 November 2011; revised 5 May 2012; accepted 6 June 2012; published online 6 September 2012

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lymphogenic into regional and distant lymph nodes and (ii) hematogenic into distant organs, including lungs and the brain (Avraamides et al., 2008; Steeg et al., 2011). In cutaneous melanoma, metastases in locoregional lymph nodes usually evolve before metastases in distant organs. Although many general mechanisms of metastasis such as tumor cell proliferation, apoptosis inhibition, regulation of tumor cell motility, local invasiveness, and induction of neoangiogenesis are well understood, the question as to why melanoma first metastasizes into lymph nodes still remains unanswered (Hanahan and Weinberg, 2000; Steeg, 2006; Klein, 2009). Epidermal growth factor (EGF) belongs to a group of growth factors that are able to activate the EGF receptor (EGFR) signaling pathway (Yarden, 2001; Yarden and Shilo, 2007; Avraham and Yarden, 2011). EGF is expressed as a large pro-form (pro-EGF) on cellular membranes of many different cell types. After proteolytic processing of pro-EGF, the so-called ectodomain shedding, EGF may act in an autocrine or paracrine manner (Dempsey et al., 1997; Harris et al., 2003). After entering circulation, EGF may also bind EGFR at distant sites (Singh and Harris, 2005). In many human cancers, e.g., squamous cell carcinoma of the head and neck, non-small cell lung cancer, or breast cancer, constitutive activation of the EGFR pathway is associated with increased proliferation, migration, and survival of tumor cells (Maurer et al., 2011). Recently, EGF has been associated with other tumor-related effects, e.g., expression of matrix & 2013 The Society for Investigative Dermatology

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For identifying suitable cell lines, EGFR-ligand (EGFR-L) expression levels were analyzed in different melanoma cell lines using real-time PCR in vitro (Supplementary Figure S1 online, Supplementary Table S1 online). EGF mRNA expression was highest in M24met (designated M24 Ctrl throughout this paper) cells and lowest in A375 melanoma cells; therefore, these cell lines were chosen for EGFkd (Figure 1a, Supplementary Table S1 online). Successful EGFkd in both stable transduced cell lines (M24 EGFkd and A375 EGFkd) could be demonstrated at the mRNA (Supplementary Figure S2a online) and protein levels (Figure 1b). Effects of EGFkd on melanoma cells were first tested in vitro. Cell cycle distribution of M24 EGFkd/A375 EGFkd and of the control transduced cell lines M24 mirNeg/A375 mirNeg was not significantly altered when compared with M24 Ctrl and A375 Ctrl cells as examined by FACS analysis (Supplementary Figure S3a online). Of note, no significant increase of sub-G0 populations in any of the transduced cell lines could be identified when compared with control cell

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lymphangiogenesis in vivo. Reduction of EGF expression in vivo also decreased VEGF-C expression. Moreover, a direct correlation between EGF and VEGF-C, as well as between EGF and Prox-1 messenger RNA (mRNA) and protein expression levels, could be found in human primary melanomas. Analysis of sera from melanoma patients with tumor-positive sentinel lymph nodes (SLNs) also revealed significantly increased EGF levels compared with sera from patients with tumor-free SLNs. In summary, our data established an important role of EGF in the development of melanoma lymph node metastases.

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metalloproteinases (Kajanne et al., 2007; Zuo et al., 2011) or adhesion molecules (Lafky et al., 2008), initiation of epithelial to mesenchymal transition (Hardy et al., 2010), or induction of tumor neoangiogenesis (Kim et al., 2011). New vessel formation by angiogenesis and lymphangiogenesis is a central process in tumor progression and subsequent development of metastases. EGF is a potent inducer of various proangiogenic and prolymphangiogenic growth factors such as VEGF-A, VEGF-C, IL-8, basic fibroblast growth factor, angiopoetin-1, angiopoetin-2, and plasminogen-activator-inhibitor-1 (De Luca et al., 2008; Luangdilok et al., 2011). EGF may also induce revascularization of tumor vessels after antiangiogenic treatment with an anti-VEGF-A antibody (bevacizumab), as shown in a mouse xenotransplantation model of lung adenocarcinoma (Cascone et al., 2011). As EGFR could be detected on microvascular endothelial cells as well as on tumor-derived endothelial cells (Baker et al., 2002; Amin et al., 2006), EGF seems to be able to act directly on endothelial cells. Nevertheless, although the importance of EGF-mediated effects on angiogenesis has already been established, the effects of EGF on tumor lymphangiogenesis still remain to be elucidated (Alders et al., 2009; Hogan et al., 2009). To address the question whether differences in EGF expression levels could affect lymph node metastasis, a key step in melanoma progression, EGF knockdown (EGFkd) was carried out in two different melanoma cell lines (one EGFhigh expressing, the other EGF-low expressing). The generated cell lines together with their wild-type control cell lines were tested for their ability to develop lymph node metastases in a mouse melanoma xenotransplantation model. Knockdown of EGF in an EGF-high expressing melanoma cell line (M24met) led to reduced tumor cell migration in vitro, reduced lymph node metastasis, and impaired primary tumor

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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(DDCt) ±SD normalized to M24 Ctrl, *Po0.05. (b) Detection of pro-EGF protein in M24 and A375 cell lines in vitro by western blot analysis. b-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 72 hours 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, *Po0.05.

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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 ¼ 7), M24 mirNeg (n ¼ 7), and M24 EGFkd (n ¼ 7) tumors (left) and of A375 Ctrl (n ¼ 5), A375 mirNeg (n ¼ 4), and A375 EGFkd (n ¼ 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 ¼ 11), M24 mirNeg (n ¼ 11), M24 EGFkd (n ¼ 12), A375 Ctrl (n ¼ 5), A375 mirNeg (n ¼ 4), and A375 EGFkd (n ¼ 5) tumor–bearing animals represented as mean±SEM, *Po0.05. (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 ¼ 400 mm.

lines (data not shown). Examination of cell growth did not reveal any alterations in cell proliferation rates between M24 EGFkd or A375 EGFkd and their respective controls (Supplementary Figure S3b online). The migratory behavior of transduced melanoma cells was examined using an in vitro scratch (wounding) assay. In M24 EGFkd, wound closure was significantly delayed (Po0.05) when compared with M24 mirNeg or M24 Ctrl cells (Figure 1c). A375 EGFkd cells did not show any significant differences in wound closure kinetics compared with A375 mirNeg and A375 Ctrl (Figure 1c). EGFkd reduces lymph node metastasis in a spontaneously metastasizing xenotransplantation melanoma mouse model

To evaluate the impact of EGFkd on melanoma growth and metastasis in vivo, M24 EGFkd, A375 EGFkd, and their respective control cell lines were examined in a CB17 severe combined immunodeficient (SCID) xenotransplantation model (Supplementary Figures S4a and S5 online). Persistent EGFkd in tumor cells was confirmed by mRNA expression analysis in primary tumors after their removal (Supplementary Figure S4b online). Primary tumor growth did not differ among individual M24 or A375 tumors (EGFkd, mirNeg, and Ctrl tumors; Figure 2a). In mice inoculated with M24 tumor cells, SLNs were harvested on day 28 after tumor cell injection. Mean weight of M24 EGFkd SLNs (n ¼ 12) was 7.6 mg (SEM±2.2 mg), compared with 36.8 mg in M24 mirNeg SLNs (n ¼ 11, SEM±11.1 mg) and with 57.9 mg in M24 Ctrl SLNs (n ¼ 11, SEM±17.4 mg, Figure 2b). The weight of SLNs in A375 tumor–bearing animals did not differ 232

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among all three groups of A375 tumors (Figure 2b). Depending on the amount of tumor cells detected by immunohistochemistry, SLNs were further classified as tumor-negative, micrometastatic, or macrometastatic (see Supplementary Materials and Methods online, Figure 2c). In mice bearing M24 EGFkd tumors, 1/12 SLNs (8.3%) were classified as macrometastasis, 3/12 SLNs (25.0%) were classified as micrometastases, and 8/12 SLNs (66.7%) were classified as tumor free. In mice bearing M24 mirNeg tumors, 7/11 SLNs were classified as macrometastasis (63.6%), 3/11 SLNs as micrometastasis (27.3%), and 1/11 (9.1%) as tumorfree SLNs. In all, 10/11 (90.9%) macrometastatic and 1/11 (9.1%) tumor-free SLNs were found in animals with M24 Ctrl tumors. In mice bearing A375 tumors, no significant differences in the amount of SLN metastases could be found among different groups. In A375 EGFkd (n ¼ 10), four SLNs were classified as macrometastasis, two as micrometastasis, and four as tumor free. In A375 Ctrl animals (n ¼ 10), four SLNs were classified as macrometastasis, four as micrometastasis, and two as tumor free. In A375 mirNeg animals (n ¼ 8), three macrometastatic, three micrometastatic, and two tumor-free SLNs were found (Figure 2c). EGFkd influences tumor lymphangiogenesis

Lymphogenous metastasis depends not only on the migratory ability of individual tumor cells but also on the extent of the lymphatic network in primary tumors. To examine a possible effect of EGFkd on tumor vasculature in primary tumors, blood and lymph vessels were examined by

A Bracher et al. Role of EGF in Melanoma Lymphangiogenesis

immunohistochemistry. Tumor lymph vessels, as assessed by staining with antibodies against Lyve-1, Podoplanin (Figure 3a), and Prox-1 (data not shown), were significantly reduced in M24 EGFkd tumors when compared with M24 Ctrl and M24

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mirNeg tumors (Po0.05). Blood vessel density, assessed with CD34 staining, did not differ significantly between M24 EGFkd tumors and M24 Ctrl or M24 mirNeg tumors (Figure 3a). Staining with pan-vessel marker CD31 showed a slight but

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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 ¼ 7), M24 mirNeg (n ¼ 7), and M24 EGFkd (n ¼ 7) tumors and (b) A375 EGFkd (n ¼ 5), A375 mirNeg (n ¼ 4), and from A375 Ctrl (n ¼ 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 ¼ 200 mm. Quantification of staining using positive (pos.) pixel evaluation is represented as mean ±SD, *Po0.05.

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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 6 hours. Representative images from one out of six independent experiments are shown. Quantification is displayed as mean sprout pixel area ±SD, *Po0.05. (b, Left) VEGF-C relative messenger RNA (mRNA) expression in M24 control (Ctrl), mirNeg, and EGFkd (n ¼ 7, each) tumors and in A375 Ctrl (n ¼ 5), mirNeg (n ¼ 4), and EGFkd (n ¼ 5) tumors, shown as mean 2(DDCt) ±SD and normalized to the respective Ctrl tumors. *Po0.05, as indicated. (Right) Correlation of EGF versus VEGF-C relative mRNA expression in xenotransplanted tumors; statistically significant linear regression line is shown (Po0.05).

still not significant reduction of total vessels, most likely reflecting the highly reduced amount of lymphatic vessels (Figure 3a). In A375 tumors, no changes in CD34, CD31, Lyve-1, and Podoplanin staining could be observed upon EGFkd (Figure 3b). EGF induces sprout formation of lymphatic endothelial cells in vitro

To identify a direct effect of EGF on endothelial cells, the sprout formation ability of immortalized lymphatic endothelial cells (LECT) and human umbilical vein endothelial cells (HUVEC) was tested in vitro. Recombinant human EGF (peak efficacy at 10 ng ml1) was significantly more potent in inducing sprout formation in LECT spheres than control media containing 3% fetal calf serum (Po0.05, Figure 4a). Conditioned media derived from EGF-high producing cell lines M24 Ctrl and M24 mirNeg potently induced LECT sprouting—the extent of sprouting exceeded sprouting 234

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induced by recombinant EGF alone. The addition of EGFneutralizing antibody to supernatants from EGF high-producing cell lines M24 Ctrl and M24 mirNeg significantly reduced induced sprouting (Po0.05, Figure 4a). Supernatants of EGF-low expressing cells and even that of recombinant EGF exhibited significantly less sprout formation in LECT spheres as compared with M24 Ctrl and M24 mirNeg conditioned media (Po0.05, Figure 4a). With regard to blood endothelial cells, neither M24 EGFkd nor A375 EGFkd conditioned media altered the amount of sprout formation of HUVEC spheres compared with their respective controls (data not shown). In addition, HUVEC spheres remained largely refractory to stimulation with recombinant EGF (data not shown). These differences could not be explained with different EGFR expression on endothelial cells, as LECT and HUVEC cell lines showed similar EGFR mRNA (data not shown) and protein levels (Supplementary Figure S6a online).

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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 ¼ 42) and EGF/Prox-1 (right, n ¼ 27) relative messenger RNA (mRNA) expression levels in human primary melanoma. Results are shown as DCt values; individual tumors are represented as single dots; and statistically significant linear regression line is shown (Po0.05). (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 (Po0.05). Two representative samples are shown. Bar ¼ 100 mm. (c) Detection of EGF in sera of melanoma patients (n ¼ 60) undergoing Sentinel node biopsy and control patients (n ¼ 11) using ELISA. Tumorpositive SLNs were present in 18 patients, tumor-negative SLNs in 42 patients; *Po0.05. H&E, hematoxylin and eosin.

EGFkd reduces VEGF-C expression in vivo

As M24 Ctrl or M24 mirNeg conditioned media induced significantly more sprouting than recombinant EGF alone, the existence of additional lymphangiogenic-/sprout-inducing factors seemed likely. Therefore, the expression of the most potent lymphangiogenic and angiogenic factors, VEGF-C and VEGF-A, were examined in primary tumors using real-time PCR. VEGF-C mRNA expression was significantly reduced in M24 EGFkd tumors, when compared with M24 Ctrl and M24 mirNeg (Po0.05, Figure 4b). In A375 EGFkd tumors, VEGF-C expression was not altered, compared with respective control tumors (Figure 4b). Evaluation of EGF and VEGF-C mRNA expression levels among all different tumor groups (M24 and A375) demonstrated a direct correlation between EGF and VEGF-C expression levels (Figure 4b). In contrast, alterations in EGF expression levels did not lead to changes in VEGF-A expression levels (Supplementary Figure S6b online).

EGF correlates with VEGF-C and Prox-1 in human primary melanoma and is significantly elevated in serum of patients with tumor-positive SLNs

To test whether a correlation of EGF with VEGF-C and lymphangiogenesis could be found also in human melanoma, formalin-fixed paraffin-embedded (FFPE) samples of human primary melanoma were analyzed for EGF, VEGF-C, and Prox1 mRNA expression (Figure 5a). Real-time PCR–based analysis revealed a significant correlation between EGF and VEGF-C mRNA expression (Po0.05, R ¼ 0.441), as well as between EGF and Prox-1 mRNA expression (Po0.05, R ¼ 0.409). For detection of EGF and VEGF-C protein expression, immunohistochemistry on a tissue microarray generated from samples of primary human melanomas was performed (Figure 5b). Again, a significant correlation between EGF and VEGF-C protein levels as evaluated by immunohistochemistry staining could be demonstrated (P ¼ o0.05, R ¼ 0.693). In normal skin www.jidonline.org

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adjacent to melanocytic lesions, EGF or VEGF-C expression was detectable, but was not as strong as that found in some (EGF-high) of the tumor tissues (Supplementary Figure S7 online). To further support the prometastatic effect of EGF, serum samples of melanoma patients with primary melanomas 41 mm in thickness (pT2–pT4) were analyzed. Interestingly, patients with tumor-positive SLNs had significantly higher EGF serum levels than patients with tumor-negative SLNs (Po0.05, Figure 5c). No correlation could be found between EGF serum levels and tumor thickness (Supplementary Figure S8 online). DISCUSSION EGF is known to stimulate tumor cell proliferation and migration (Maurer et al., 2011). In our xenotransplantation model, EGFkd had no effect on proliferation but markedly reduced migration of EGF-high expressing M24 cells. This finding is in line with recent data demonstrating distinct downstream effects of different ligands binding to the EGFR (Glogowska et al., 2008; Mascia et al., 2009). Interestingly, we were not able to observe alterations after EGFkd in our EGF-low expressing melanoma cell line A375. Therefore, these observed effects in our EGF-high expressing tumor cell line seem to be specifically EGF mediated. Observed effects in our xenotransplantation model were induced by human EGF acting on mouse tissue; therefore, the extent of cross-species similarity is of relevance. In silico analysis displays a 92% similarity between shed 53 amino acids of mature mouse and human EGF, making EGF cross-species reactivity highly likely. Additional support for this assumption comes from data showing that the phenotype of EGFR-knockdown mice can be rescued by knock-in of human EGFR (Sibilia et al., 2003). Directed tumor cell migration and primary tumor lymphangiogenesis are important mechanisms for the development of lymph node metastases (Skobe et al., 2001). EGFkd reduced tumor cell migration in EGF-high expressing M24 cells in vitro; impaired migration may also be, at least partially, accountable for the impairment or delay of lymph node metastases seen in our in vivo xenotransplantation model. By demonstrating the ability of EGF to stimulate lymphatic endothelial cell sprouting, we found a direct effect of EGF on lymphatic endothelial cells. Interestingly, sprout induction induced by M24met Ctrl supernatants exceeded EGF-induced sprouting, suggesting the presence of an additional lymphangiogenic stimulus. VEGF-C could be identified as this additional factor by detecting a direct correlation between EGF and VEGF-C expression. EGFkd also reduced VEGF-C expression; therefore, it seems reasonable to speculate that EGF is able to induce VEGF-C in melanoma cells. A similar induction of VEGF-C expression by EGF has already been shown in endothelial cells, keratinocytes, and human fibroblasts but not in melanoma (Enholm et al., 1997; Trompezinski et al., 2004). We were able to elucidate a positive correlation between EGF and VEGF-C and between EGF and Prox-1 in human primary cutaneous melanoma. Correlation of EGF with Prox1, a marker for lymphangiogenesis, suggests that primary melanomas with higher levels of EGF also have increased 236

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lymph vessel density, which is a predictor for the development of metastases in primary melanoma (Dadras et al., 2003; Emmett et al., 2010). Moreover, peritumoral VEGF-C expression levels have been recently reported of being predictive for SLN status in human melanoma (Dadras et al., 2005; Gallego et al., 2011). Importantly, we also found significantly increased EGF serum levels in SLNpositive melanoma patients when compared with patients with tumor-negative SLNs, indicating that EGF might be a new predictive marker for metastasis and prognosis. In summary, we demonstrated an influence on melanoma lymph node metastasis by high expression levels of tumorderived EGF in vitro and in vivo. We could show that EGF mediates its effect on lymph node metastasis (i) by directly stimulating lymphatic endothelial cells, (ii) by inducing VEGF-C expression, and (iii) by increasing tumor cell motility. EGF and VEGF-C seem to act synergistically on lymph endothelial cell sprouting and lymphangiogenesis. Our data also indicate that EGF might serve as a new prognostic marker predicting lymph node metastasis. Such a marker would be of paramount importance for the development of new therapy and follow-up strategies. Nevertheless, to fully answer this question, further clinical studies will be required. MATERIALS AND METHODS RNA extraction and relative mRNA expression analysis RNA from tumor samples stored in RNA later (Ambion, Austin, TX) and from cell culture was extracted using RNeasy Mini RNA kits (Qiagen, Valencia, CA), according to the manufacturer’s instructions. RNA extraction from formalin-fixed paraffin-embedded tissue (Ethics committee of the Medical University of Vienna, approval number 901/2009) was performed using RNeasy FFPE Kits (Qiagen). RNA reverse transcription into complementary DNA (cDNA) was performed using RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas, Vienna, Austria). Relative mRNA expression analysis using real-time PCR was performed as described previously (Loewe et al., 2006). For specific target detection, primer-probe sets for EGF (Hs01099999_m1), TGFa (Hs00608187_m1), Amphiregulin (Hs00950669_m1), Epigen (Hs02385425_m1), Betacellulin (Hs01101204_m1), HB-EGF (Hs00181813_m1), Epiregulin (Hs00914312_m1), VEGF-C (Hs01099203_m1), VEGF-A (Hs00173626_m1), and Prox-1 (Hs00896293_m1) were purchased as Assay-on-demand (Applied Biosystems, Vienna, Austria). Normalization was performed with a glyceraldehyde 3-phosphate dehydrogenase or b-2 microglobulin internal control, respectively (Applied Biosystems). Reactions and data analysis were carried out on an Applied Biosystems Step One Plus cycler using the Step One Software v.2.1 (Applied Biosystems). Differences in mRNA expression were calculated according to the DDCt method (Livak and Schmittgen, 2001) and displayed as 2(DDCt).

Cell culture Human melanoma cell lines (Supplementary Table S2 online) A375 (ATTC cell line, LGC Promochem, Wesel, Germany), SK-MEL 28, 607B, 518A2, IGR37, and the stable transduced A375 cell lines were cultivated in DMEM supplemented with 10% fetal calf serum (Lifetechnologies, Vienna, Austria), 2 mM l1 glutamine (Life

A Bracher et al. Role of EGF in Melanoma Lymphangiogenesis

Technologies, Vienna, Austria), and 50 IU ml1 penicillin-streptomycin (Lifetechnologies). M24met melanoma cells (kindly provided by RA Reisfeld (Mueller et al., 1991)) and the stable M24 met met cell lines were grown in RPMI instead of DMEM with identical supplements. Blasticidin (8 mg ml1) (Invitrogen, Carlsbad, CA) was added for selection when necessary.

tumor volumes, tumors including the surrounding skin were explanted and skin defects sutured. Primary tumors were harvested and cut, and the pieces were stored in 4% paraformaldehyde and in RNA later (Ambion) for further experiments. As soon as lymph nodes were palpable in one group, animals were killed. Lymph nodes were removed, fixed in 4% paraformaldehyde, and embedded in paraffin.

Generation of stable cell lines

Immunohistochemistry

For stable EGFkd in M24met and A375 cells, microRNAs (EGF BLOCK-IT miR RNAi Select, Invitrogen) were purchased, tested for efficacy, and transferred into lenitviral vectors to perform stable knockdown according to the manufacturer’s instructions (see Supplementary Materials and Methods online). The generated M24met and A375 cells containing the stable pLenti6/V5-GW/ þ EmGFP-miR were cultivated under 8 mg ml1 Blasticidin (Invitrogen). In consecutive experiments, wild-type M24met was named M24 Ctrl and the wild-type A375 was named A375 Ctrl. M24met and A375 containing pLenti6/V5-GW/ þ EmGFPwith nontargeting miRNA were designated M24 mirNeg and A375 mirNeg, respectively; M24met or A375 stable pLenti6/V5-GW/ þ EmGFPHmi405014 cell lines were named M24 EGFkd and A375 EGFkd, respectively.

For immunohistochemistry, the following primary antibodies have been used: anti-vimentin Clone V9 (Dako, Glostrup, DK), mouse anti-Lyve-1 (DP3513, Acris, Herford, DE), mouse CD31 (Rb-10333, Neomarkers, Fremont, CA), mouse anti-podoplanin (DM3501, Acris), mouse CD34 (553731, BD Pharmingen, San Jose, CA), human EGF (ab9695, Abcam, Cambridge, UK), and human VEGF-C (AF752, R&D Systems Europe, Abingdon, UK). Detection was carried out using appropriate second-step antibodies: goat antirabbit biotinylated IgG(H þ L), goat anti-hamster biotinylated IgG(H þ L), rabbit anti-rat biotinylated IgG(H þ L), and rabbit antigoat biotinylated IgG(H þ L) (Vector Laboratories, Burlingame, CA). Staining was performed as previously described (Loewe et al., 2006; Valero et al., 2010) (see Supplementary Materials and Methods online).

Western blotting

Spheroid sprout formation assay

For western blotting of pro-EGF and EGFR, cell lines were lysed with Tris-Lysis-Buffer supplemented with Triton X-100 (10 mg ml1) (BioRad, Hercules, CA), IGEPAL CA-630 (10 mg ml1) (Sigma Aldrich, St. Louis, MO), and protease inhibitor cocktail (Sigma Aldrich) as previously described (Loewe et al., 2002) (see Supplementary Materials and Methods online).

Induction of sprout formation was tested on freshly isolated HUVECs (Gro¨ger et al., 2004) and LECT spheroids. HUVEC and LECT hanging drops were generated to form spheroids containing 450 cells per sphere. For stimulation purposes, conditioned media from M24met and A375 cell lines were generated. After 24 hours, spheres were transferred into collagen as described elsewhere (Korff et al., 2001) and incubated with conditioned medium, conditioned medium supplemented with EGF-neutralizing antibody (R&D Systems Europe), and recombinant human EGF (R&D Systems Europe) or fetal calf serum. In all, 15 spheres per condition were documented over a period of 24 hours and analyzed for sprout formation with the Image Scope software version 10.2.2.2319 (Aperio; see Supplementary Materials and Methods online).

In vitro wounding assay 8  105 M24met and 8  105 A375 cell lines were plated in triplicates. After reaching confluency, the cellular monolayer was disrupted (scratched) twice using a 200-ml pipette tip. Scratches were documented every 24 hours for 3 days using a Zeiss AxioCam ICc3 and Zeiss Axio Vision Rel.4.8 (Zeiss, Jena, Germany) camera for cell culture and analyzed using Image Scope software version 10.2.2.2319 (Aperio, Vista, CA; see Supplementary Materials and Methods online).

CB17 SCID xenotransplantation experiments All procedures were carried out in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care guidelines and Guide for the Care and Use of Laboratory Animals (Department of Health and Human Services, NIH, Publication no. 86-23). In addition, all experiments were approved by the ethics committee of the Medical University of Vienna and by the Austrian government committee on animal experimentation (ethics commission: BMBWK-66.009/0033BrGT/2006). Pathogen-free, 4- to 6-week-old female CB17 scid/scid (SCID) mice (Charles River, Sulzfeld, Germany) were housed and used as described previously (Valero et al., 2010). For xenotransplantation experiments, M24met and A375 wildtype and stable transduced cell lines were used. For M24met cell lines 1.5  106 cells and for A375 cell lines 2.0  106 A375 cells were suspended in 50 ml of phosphate-buffered saline and injected intradermally into the right flank of the animals. Tumor volume was assessed as previously described (Loewe et al., 2006). At indicated

Human serum ELISA This study was approved by the institutional review board of the Medical University of Vienna and has been carried out in accordance with the Declaration of Helsinki Principles. Written informed patient consent was obtained before blood was taken from patients with cutaneous melanoma. Serum was separated and stored at 80 1C. Serum samples from patients (n ¼ 60) with known SLN status (Supplementary Table S3.1 and S3.2 online) and control sera from healthy donors (n ¼ 11) were analyzed using Human EGF Quantikine ELISA (R&D Systems Europe) according to the manufacturer’s instructions.

Statistics Statistical testing of parametric data was done by performing unpaired two-tailed t-test or adjusted analysis of variance in case of multigroup testing. All graph preparations and statistical testing were performed using SigmaPlot Version 11.0 (Systat Software, Chicago, IL). Group differences were considered to be statistically significant when Pp0.05. Data are represented as mean±SD or mean±SEM as indicated. www.jidonline.org

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CONFLICT OF INTEREST The authors state no conflict of interest.

Harris RC, Chung E, Coffey RJ (2003) EGF receptor ligands. Exp Cell Res 284:2–13

ACKNOWLEDGMENTS

Hogan BM, Bos FL, Bussmann J et al. (2009) ccbe1 is required for embryonic lymphangiogenesis and venous sprouting. Nat Genet 41:396–8

This study was supported in part by grants from the Jubila¨umsfonds of the ¨ sterreichische Nationalbank (ONB-13046, MG; ONB-13672, RL), from the O Medical Scientific Fund of the Mayor of the City of Vienna (grant 11036, RL), and from the Austrian Science Fund (FWF P20940B11, PP). We thank Udo Losert and the staff of the Biomedical Sciences Center, Medical University of Vienna, Austria. We also thank Silvia Steele, Fahira Basota, Monika Weiss, Claudia Kokesch, and Alex Eteleng for their excellent technical assistance. All experiments were carried out at SERD, Vienna, Austria. SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http:// www.nature.com/jid

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Journal of Investigative Dermatology (2013), Volume 133

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