Mesenchymal Contribution to Recruitment, Infiltration, and Positioning of Leukocytes in Human Melanoma Tissues  Rafael Samaniego, Ana Estecha, Miguel Relloso, Natividad Longo, José L. Escat, Isabel Longo-Imedio, José A. Avilés, Miguel Á. del Pozo, Amaya Puig-Kröger, Paloma Sánchez-Mateos  Journal of Investigative Dermatology  Volume 133, Issue 9, Pages 2255-2264 (September 2013) DOI: 10.1038/jid.2013.88 Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Tumor microenvironment imaged by multicolor fluorescence microscopy. (a) Subcutaneous melanoma metastasis (no. 223) stained as follows: (A) NG2 (white), CD31 (red), and nuclei (blue); (B) CD31 (red) and Duffy antigen receptor for chemokine (DARC) (green); (C) von Willebrand factor (vWF) (red) and CD90 (green); (D) vWF (red) and fibroblast activation protein (FAP) (white). (E) Type I collagen (COL1) (red) and CD68 (white); and (F) CD45 (white). Panels are composites of three adjacent fields imaged at original magnification × 10. (b, c) Magnified peritumoral vessels stained for CD31 (red) and DARC (green); and vWF (white), FAP (red), and CD90 (green), respectively. Scale bars, as indicated. DAPI, 4′,6-diamidino-2-phenylindole. Journal of Investigative Dermatology 2013 133, 2255-2264DOI: (10.1038/jid.2013.88) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Ex vivo expression of CCL2/CCR2. (a) CD90, fibroblast activation protein (FAP), NG2, and Hmb45 expression levels in tumor cells (TCs)1 and mesenchymal cells (MCs)1, as assessed by flow cytometry. Images correspond to TCs19 and MCs19 stained for FN (red) and nuclei (white). (b) Peripheral blood monocyte/lymphocyte chemotaxis toward medium ±CCL2 (10ngml−1), or conditioned media from different tumor-associated MCs. Fold migrations are shown. (c) ELISA detection of CCL2 secreted by tumor-associated MCs, and control skin MCs. (d) Monocyte chemotaxis toward medium ±CCL2, or MC19-conditioned medium. Assays were performed in the presence of 50μgml−1 control mAb or anti-CCL2 mAb, or pre-incubating monocytes for 15 minutes with 50μgml−1 of either anti-CCR2-04 or anti-CCR2-05 antibodies. Fold migration ±SD is shown (*P<0.05, Student’s t-test). (e) CCL2, CCR2a, and CCR2b mRNA levels, as assayed by quantitative PCR from three “total tumors”; isolated TCs and MCs; control skin CD90+ MC; melanoma–tumor-associated macrophages (TAMs); and peripheral blood monocytes. Means ±SD are shown. (f) TC19 incubated in medium ±tumor necrosis factor-α (TNFα)/transforming growth factor-β1 (TGFβ1) (15ngml−1 each) for 24h. CCL2 mRNA and protein expressions are shown (two anti-CCL2 antibodies were used). Nuclei, in blue. DAPI, 4′,6-diamidino-2-phenylindole; PB, peripheral blood. Journal of Investigative Dermatology 2013 133, 2255-2264DOI: (10.1038/jid.2013.88) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 CCL2/CCR2 expression in melanoma tissues. (a) Metastasis no. 223 (see Figure 1a) stained as follows: (A) CCL2 (white) and CD31 (red); (B) CD68 (white); (C) CCL2 (white), CD31 (red), and CD90 (green); and (D) CCL2 (white), CCR2 (green), CD45 (red), and nuclei (blue); images are z-stack projections, and vessel contour is depicted. Arrows along panels point to the same peritumoral vessel. (b) Clark II (no. 158) lesion stained in consecutive sections for Hmb45, CD90, von Willebrand factor (vWF), CD31, Duffy antigen receptor for chemokine (DARC), CCL2, and nuclei, as indicated. Magnified peritumoral vessels surrounded by CCL2+CD90+ mesenchymal cells (MCs) are shown on the right. Cyan-dotted lines indicate the tumor border. Scale bars, as indicated. DAPI, 4′,6-diamidino-2-phenylindole. Journal of Investigative Dermatology 2013 133, 2255-2264DOI: (10.1038/jid.2013.88) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Stroma increases leukocyte infiltration of tumors. (a) Correlation analysis of CD45+ leukocyte density (mm2) and type I collagen (COL1) content (% stained area). Values/field are plotted (n=22 metastases, 66 intratumoral fields), and Pearson’s correlation (R) and statistical significance (P) are indicated. Dot colors differentiate two groups of metastases (see b). (b) Fibroblast activation protein (FAP) labeling (% stained area) allows the quantitative discrimination between metastases infiltrated by FAP+ mesenchymal cells (MCs) (↑FAP, n=11) and non/weakly infiltrated (↓FAP, n=11). The two groups were compared for leukocyte density, COL1 content, and percentage of leukocytes in contact with COL1 fibers (Mann–Whitney). (c) Distinct samples of the same metastasis, ↑FAP (no. 93.1) and ↓FAP (no. 93.3) stained for COL1, von Willebrand factor (vWF) and CD45, as indicated. (d) ↓FAP (no. 97) and ↑FAP (no. 223) metastases co-stained for CD45, COL1, and vWF, as indicated. Bars=100μm. DAPI, 4′,6-diamidino-2-phenylindole; NS, not significant. Journal of Investigative Dermatology 2013 133, 2255-2264DOI: (10.1038/jid.2013.88) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Mesenchymal cells (MCs) promote leukocyte infiltration of melanoma xenografts in vivo and T-lymphocyte migration in vitro. (a) A375±MCs19 were subcutaneously inoculated at opposite flanks of nude mice (n=5), as shown in the scheme. Contralateral tumors were compared for fibroblast activation protein (FAP+) MC and fibronectin (FN) content, host F4/80+, and Gr1+-cell infiltration, CD31+ vessel density, and infiltration of transferred CD8+ T cells. Three intratumoral fields by tumor were quantified and significant differences are shown (Wilcoxon). Representative images of the two tumor groups stained for CCL2, FN, FAP (host and human), F4/80, CD8a, and DAPI are shown, as indicated. (b) Human T lymphoblasts were allowed to migrate for 1h along A375±MC19 monolayers. Representative tracks (frame minute−1, left) and dot plots of net displacement, mean velocity, and migratory persistence of T cells pretreated ±pertussis toxin (PTX). Mean ±SEM (n=65 cells per condition) and significant differences (Mann–Whitney) are shown. Data correspond to one representative assay out of three. (c) Simultaneous chemotactic assay as control of PTX effect. DAPI, 4′,6-diamidino-2-phenylindole; NS, not significant; TC, tumor cell. Journal of Investigative Dermatology 2013 133, 2255-2264DOI: (10.1038/jid.2013.88) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Organization of poorly and highly infiltrated melanomas. (a) All melanoma tissues analyzed in this study can be classified in one of these two schematic groups. Tumors poorly infiltrated by leukocytes (<500cells per mm2) are also poorly infiltrated by mesenchymal cells (MCs), showing variable densities of both small and large vessels; on the contrary, highly leukocyte-infiltrated tumors are characterized by a distinctive fibro-cellular network of extracellular matrix (ECM) and MC (fibroblast activation protein (FAP+)CD90low/−), where most leukocytes (~90%) contact ECM fibrils while they face the neighbor tumor cells (TCs). Peritumoral stroma is quite similar in both scenarios: it is rich in elongated MCs (FAP+CD90+) and leukocytes (>2,000cells per mm2) and contains vessels surrounded by perivascular MCs (FAP−CD90hi), many of them with a DARC+ endothelium (∼40%). (b) Phenotype transition (CD90/FAP) from perivascular to peritumoral and intratumoral MCs. Journal of Investigative Dermatology 2013 133, 2255-2264DOI: (10.1038/jid.2013.88) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions