Activin A Is Anti-Lymphangiogenic in a Melanoma Mouse Model Magdalena Heinz, Heide Leb Niederleithner, Emmi Puujalka, Ana Soler-Cardona, Michael Grusch, Hubert Pehamberger, Robert Loewe, Peter Petzelbauer  Journal of Investigative Dermatology  Volume 135, Issue 1, Pages 212-221 (January 2015) DOI: 10.1038/jid.2014.328 Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Human primary melanomas have an increased INHBA to follistatin ratio compared with nevi and metastasis. (a) Quantified expression of immunohistochemistry staining for the inhibin βA subunit (INHBA) and follistatin (FST) in human nevi (n=19), primary melanoma (SSM; n=10), and metastasized melanoma (Mel met; n=81). *P<0.05. (b) Examples for immunohistochemistry stainings for FST and INHBA expression in a nevus and a primary melanoma. Antibody binding is visualized by red color; counterstaining with hematoxylin is blue. Isotype controls for the respective antibodies are shown at the bottom; corresponding H&E sections are shown at the right side. Bars = 100 μm. H&E, hematoxylin and eosin; INHBA, inhibin βA subunit; SSM, superficial spreading melanoma. Journal of Investigative Dermatology 2015 135, 212-221DOI: (10.1038/jid.2014.328) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Protein and gene expression profiles of melanomas transduced with the indicated transgenes. After intradermal injection of A375 cells overexpressing the indicated genes, arising tumors were excised at day 22 and tested for the expression of transgenes by immunohistochemistry (a) and real-time PCR (b). (a) Cell lines used are indicated in the top row; respective primary antibodies are shown at the left border; antibody binding is visualized by red color, and counterstaining with hematoxylin is blue. Images in the right row are isotype controls. Bar = 100 μm (for anti-vimentin stains 500 μm). (b) mRNA expression determined by real-time PCR. Of note, INHBA does not reduce VEGF-C expression; mean±SD; *P<0.05; n=5 per group. FST, follistatin; INHBA, inhibin βA subunit; VEGF, vascular endothelial growth factor. Journal of Investigative Dermatology 2015 135, 212-221DOI: (10.1038/jid.2014.328) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Activin does not interfere with melanoma metastasis. (a) Examples for anti-Lyve-1 stainings (red color) for lymphatic vessels in primary melanoma overexpressing indicated genes; arrows indicate lymphatic vessels filled with tumor cells in control and INHBA tumors, but narrow and empty vessels in Wnt1+ melanoma. Bar = 100 μm. (b) Lymph vessels surrounding primary tumors that contained tumor cells were counted and calculated as the percentage of the total lymph vessels in each section. Mean±SD; *P<0.05. (c) Summary of positive sentinel lymph nodes through an observation time of 70 days following tumor excision. An example for a negative and a positive lymph node metastasis, as determined by anti-vimentin stainings (red color), is exemplified in the inserted images. Bar = 200 μm. FST, follistatin; INHBA, inhibin βA subunit. Journal of Investigative Dermatology 2015 135, 212-221DOI: (10.1038/jid.2014.328) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Activin A reduces lymph vessel formation. (a) Peritumoral Lyve-1+ lymphatic vessels and (b) CD31+ vessels were counted in immunohistochemistry sections stained with the respective antibody. Positive vessels were counted in a 250-μm zone surrounding the tumor, as exemplified in images to the right (bar = 100 μm). Data summarize the results of two independent experiments with n>5 in each group. In addition, Lyve-1 mRNA expression was determined in melanoma samples by real-time PCR and is expressed as fold change compared with controls (insert in a), n=5 per group; mean±SD; *P<0.05. FST, follistatin; INHBA, inhibin βA subunit. Journal of Investigative Dermatology 2015 135, 212-221DOI: (10.1038/jid.2014.328) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Activin A increases melanoma migration in vitro. (a) A375 cells overexpressing the indicated genes were seeded in 96-well plates (2,000 cells per well), and proliferation was determined using the EZ4U kit, n=6 per group. (b) In vitro wound healing assay of indicated melanoma cells, representative example of three independent experiments. (c) Comparative analysis of human melanoma cells VM-1, -30, and -34, stimulated with medium as control, human recombinant Wnt3a (25 ng), ActA (10 ng), or TGF-β (10 ng). Top: western blot for SMAD2 (loading control) and phosphorylated SMAD2 (pSMAD2); the quantification of pSMAD2 is given as % increase compared with controls. Middle: proliferation assays (104 cells per well in 24-well plates; quantification by Picogreen kit). Bottom: migration is depicted as percent wound closure. ActA, activin A; FST, follistatin; INHBA, inhibin βA subunit; TGF-β, transforming growth factor-β. Journal of Investigative Dermatology 2015 135, 212-221DOI: (10.1038/jid.2014.328) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Activin A decreases lymphatic sprout formationin vitro. In vitro spheroid sprouting assay of lymphatic endothelial cells; summary of three independent experiments with n=10 per group. VEGF-C was used as a positive control. Fresh medium without the addition of growth factors served as a negative (Neg.) control; mean±SD; *P<0.05. Inserted images are examples of spheroids stimulated with supernatants of control or INHBA A375 cells. INHBA, inhibin βA subunit; VEGF, vascular endothelial growth factor. Journal of Investigative Dermatology 2015 135, 212-221DOI: (10.1038/jid.2014.328) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions