RANK Is Expressed in Metastatic Melanoma and Highly Upregulated on Melanoma- Initiating Cells  Verena Kupas, Carsten Weishaupt, Dorothee Siepmann, Maria-Laura Kaserer, Mareike Eickelmann, Dieter Metze, Thomas A. Luger, Stefan Beissert, Karin Loser  Journal of Investigative Dermatology  Volume 131, Issue 4, Pages 944-955 (April 2011) DOI: 10.1038/jid.2010.377 Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Increased RANK expression in melanoma cells from malignant melanoma (MM) stage IV compared with MM stage I patients. (a, b) Quantitative real-time PCR of receptor activator of NF-κB (RANK) (a) and receptor activator of NF-κB ligand (RANKL) (b) gene expression in peripheral nucleated blood cells from healthy donors (n=15), MM stage I patients (n=37), and MM stage IV patients (n=40). *P<0.05 versus MM stage I patients. (c) Quantitative real-time PCR of RANK and RANKL mRNA expression in sorted dendritic cells, monocytes, T cells, and melanoma cells from the peripheral blood of healthy donors, MM stage I patients, and MM stage IV patients. One representative out of three independent experiments is depicted. Histogram overlays from a MM stage IV patient are depicted to demonstrate the purity of sorted cell populations. (d) Quantitative real-time PCR of RANK (left) and RANKL (right) gene expression in peripheral nucleated blood cells from healthy donors (n=8), basal cell carcinoma patients (n=7), and squamous cell carcinoma patients (n=7). Journal of Investigative Dermatology 2011 131, 944-955DOI: (10.1038/jid.2010.377) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 RANK expression is upregulated in nonhematopoietic melanoma cells from MM stage IV patients compared with malignant melanoma (MM) stage I patients. (a, b) MART-1+ melanoma cells in peripheral blood from MM stage I (n=5) and stage IV patients (n=5) were analyzed for receptor activator of NF-κB (RANK) (a) and receptor activator of NF-κB ligand (RANKL) (b) expression by flow cytometry. Representative dot plots gated for CD45+ hematopoietic and CD45- nonhematopoietic cells, as well as statistical evaluation of the percentage of CD45-MART-1+ cells coexpressing either RANK (a) or RANKL (b) are shown. *P<0.05 versus MM stage I patients. MART-1 staining was performed after cell permeabilization. Journal of Investigative Dermatology 2011 131, 944-955DOI: (10.1038/jid.2010.377) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Receptor activator of NF-κB (RANK)-expressing melanoma cells are present in primary melanomas and lymph node (LN) metastases from malignant melanoma (MM) stage IV patients. (a) Immunofluorescence staining of melanoma tissue using antibodies directed against MART-1 and RANK. Cells expressing both markers are marked by arrows. Original magnification × 100 (hematoxylin and eosin (H&E) staining) or × 400 (immunofluorescence staining), bar=25μm. (b, c) Statistical evaluation of MART-1 and RANK expression in melanoma tissue. Total numbers of MART-1+ cells (b) and percentages of MART-1+ cells coexpressing RANK (c) were quantified in primary tumor tissues from MM stage I (n=7), or MM stage IV patients (n=7), and in lymph node metastases from MM stage IV patients (n=7). *P<0.05 versus primary tumors of the same stage IV patients. Journal of Investigative Dermatology 2011 131, 944-955DOI: (10.1038/jid.2010.377) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Receptor activator of NF-κB (RANK) is not increased in malignant melanoma (MM) stage IV patients presenting with bone metastases. (a) RANK gene expression levels in peripheral nucleated blood cells from MM stage IV patients with (n=7) and without (n=11) bone metastases were quantified by real-time PCR. (b) RANK expression in melanoma cells of primary tumors from MM stage IV patients with and without bone metastases (n=7 patients each group) was analyzed by immunofluorescence staining using antibodies against RANK and MART-1. One representative image is shown, original magnification × 100 (hematoxylin and eosin (H&E) staining) or × 400 (immunofluorescence staining), bar=25μm. (c) FACS analysis of MART-1+ melanoma cells coexpressing RANK in peripheral blood from MM stage IV patients with and without bone metastases (n=5 patients each group). MART-1 staining was performed after cell permeabilization. Journal of Investigative Dermatology 2011 131, 944-955DOI: (10.1038/jid.2010.377) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 MART-1+RANK+ melanoma cells coexpress the tumor stem cell markers ABCB5 and CD133. (a) Immunofluorescence staining of human skin, primary tumors, and lymph node (LN) metastases using antibodies against MART-1, receptor activator of NF-κB (RANK), and ATP-binding cassette (ABC) B5. Cells expressing all three markers are marked, original magnification × 400, bar=25μm. (b) Statistical evaluation of MART-1+RANK+ melanoma cells coexpressing ABCB5 and MART-1- cells coexpressing RANK+ABCB5 in primary tumors and lymph node (LN) metastases from MM stage IV patients (n=5 patients). (c) Immunofluorescence staining of MART-1+RANK- and MART-1+RANK+ cells from peripheral blood of malignant melanoma (MM) stage IV patients (n=3). Representative merged images are shown, MART-1+ melanoma cells coexpressing ABCB5 are marked and anti-MART-1 staining was performed after cell permeabilization. (d) FACS analysis of peripheral nucleated blood cells from MM stage I (n=4) and MM stage IV patients (n=4) using antibodies against CD45, MART-1, RANK, and CD133. One representative histogram overlay and the statistical evaluation is shown (*P<0.05 vs. MM stage I patients). MART-1 staining was performed after cell permeabilization. Journal of Investigative Dermatology 2011 131, 944-955DOI: (10.1038/jid.2010.377) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 MART-1+RANK+ melanoma cells coexpress the tumor stem cell markers ABCB5 and CD133. (a) Immunofluorescence staining of human skin, primary tumors, and lymph node (LN) metastases using antibodies against MART-1, receptor activator of NF-κB (RANK), and ATP-binding cassette (ABC) B5. Cells expressing all three markers are marked, original magnification × 400, bar=25μm. (b) Statistical evaluation of MART-1+RANK+ melanoma cells coexpressing ABCB5 and MART-1- cells coexpressing RANK+ABCB5 in primary tumors and lymph node (LN) metastases from MM stage IV patients (n=5 patients). (c) Immunofluorescence staining of MART-1+RANK- and MART-1+RANK+ cells from peripheral blood of malignant melanoma (MM) stage IV patients (n=3). Representative merged images are shown, MART-1+ melanoma cells coexpressing ABCB5 are marked and anti-MART-1 staining was performed after cell permeabilization. (d) FACS analysis of peripheral nucleated blood cells from MM stage I (n=4) and MM stage IV patients (n=4) using antibodies against CD45, MART-1, RANK, and CD133. One representative histogram overlay and the statistical evaluation is shown (*P<0.05 vs. MM stage I patients). MART-1 staining was performed after cell permeabilization. Journal of Investigative Dermatology 2011 131, 944-955DOI: (10.1038/jid.2010.377) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Reduced Ki67 proliferation index in MART-1+RANK+ melanoma cells. (a) Peripheral nucleated blood cells from MM stage IV patients (n=5) were stained for CD45, MART-1, RANK, and Ki67 and analyzed by flow cytometry. Cells are gated for CD45-MART-1+ and one representative dot plot is shown. (b) Statistical evaluation of Ki67 expression in CD45-MART-1+RANK+ cells (n=4 patients each group), *P<0.05 versus MM stage I patients. (c) FACS analysis of peripheral nucleated blood cells from MM stage I and stage IV patients (n=5 patients each group) using antibodies against MART-1, Ki67, RANK, and CD133. One representative dot plot and the statistical evaluation of RANK+Ki67- cells coexpressing MART-1 and CD133 are shown (*P<0.05 vs. MM stage I patients). MART-1 and Ki67 staining was performed after cell permeabilization. (d) MART-1+RANK+ melanoma cells initiate tumor growth in immunodeficient NSG mice. Mice (n=3) were inoculated with 2.5 × 104 MART-1+RANK+ or MART-1+RANK- cells from the peripheral blood of MM stage IV patients and one representative histological analysis of skin biopsies from recipients is depicted, original magnification × 200, bar=25μm. Journal of Investigative Dermatology 2011 131, 944-955DOI: (10.1038/jid.2010.377) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions