CXCL5 as Regulator of Neutrophil Function in Cutaneous Melanoma Agnes Forsthuber, Katharina Lipp, Liisa Andersen, Stefanie Ebersberger, 'Osvaldo Graña-Castro, Wilfried Ellmeier, Peter Petzelbauer, Beate M. Lichtenberger, Robert Loewe  Journal of Investigative Dermatology  Volume 139, Issue 1, Pages 186-194 (January 2019) DOI: 10.1016/j.jid.2018.07.006 Copyright © 2018 The Authors Terms and Conditions

Figure 1 Expression of neutrophil-associated genes is increased in CXCL5high melanoma samples of publicly available datasets. (a) Log2FC expression of neutrophil-associated genes in CXCL5high expressing tumors compared with CXCL5low expressing primary melanomas or metastases for five different GEO datasets. (b) Median z-scores of 40 neutrophil-associated genes positively correlate with increasing z-scores of CXCL5 (ranked values) for each individual sample of GEO and TCGA datasets combined (n = 690). Journal of Investigative Dermatology 2019 139, 186-194DOI: (10.1016/j.jid.2018.07.006) Copyright © 2018 The Authors Terms and Conditions

Figure 2 Neutrophil phenotypes in different tissues. (a) Representative dot plots of the gating scheme for CD11b+LY6G+ neutrophils isolated from bone marrow (BMNs), peripheral blood (PBNs), lung (LungNs), and primary tumor (TANs). (b) Percentages of neutrophils of B16F1-CXCL5 and HCmel12-CXCL5 tumor-bearing mice positive for indicated surface markers (n = 6–8). (c) Fold change expression of indicated genes analyzed from mRNA of sorted TANs, LungNs, and PBNs of tumor-bearing mice normalized to PBNs of tumor-free mice. (d) Percent proliferation of CD8+ T cells cocultured with TANs at different ratios. Proliferation in the absence of TANs was set as 100% (summary of all performed experiments, n = 4). Statistical analysis by one-way analysis of variance with the Tukey post-hoc test (b, c). Before statistical testing of (b), data were transformed to log10 and normal distribution was confirmed by QQ-Plots. Data are represented as individual data points as mean ± standard error of the mean (b–d); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Journal of Investigative Dermatology 2019 139, 186-194DOI: (10.1016/j.jid.2018.07.006) Copyright © 2018 The Authors Terms and Conditions

Figure 3 Neutrophils suppress antitumoral activity of T cells. (a) Representative anti-CD3 immunohistochemical staining (red) of HCmel12-CXCL5 tumor sections after Poly(I:C) or NaCl treatment; melanin (brown). (b) Representative immunofluorescent staining of Poly(I:C)-treated HCmel12-CXCL5 primary tumors. Anti-CD3 (red), anti-LY6G (green), DAPI (blue). The dashed white line represents the border between the tumor and the microenvironment. (c) Growth curves of Poly(I:C)- (n = 5) or NaCl-treated (n = 4) HCmel12-control tumors. (d) Representative anti-MAC-2 immunohistochemical staining and isotype control on consecutive histological sections from an HCmel12-control tumor. Brownish color in isotype staining is melanin taken up by macrophages. (e) Growth curves of Poly(I:C)- (n = 8) or NaCl-treated (n = 4) HCmel12-CXCL5 tumors. Data are shown as mean diameter (c, e). Scale bar 100 μm (a, b left, d) and 10 μm (b right). Journal of Investigative Dermatology 2019 139, 186-194DOI: (10.1016/j.jid.2018.07.006) Copyright © 2018 The Authors Terms and Conditions

Figure 4 CXCL5 expression in a syngeneic mouse melanoma model. (a) Representative growth kinetics of B16F1 (n = 10) and HCmel12 (n = 10) control and CXCL5 overexpressing tumors. (b) Image analysis of LY6G-stained sections of B16F1-control and CXCL5 overexpressing primary tumors (n = 16). (c) Significant differences in fold change expression of mRNA levels of chemokines and chemokine receptors of freshly dissected primary tumors: B16F1-CXCL5 (n = 10 per group) and HCmel12-CXCL5 (n = 5 per group) normalized to controls. (d) Fold change expression of Vegfa and Vegfc in freshly dissected primary tumors: B16F1-CXCL5 (n = 10 per group) and HCmel12-CXCL5 (n = 5 per group) normalized to controls. Scale bar 100 μm. Statistical analysis by the t-test (b–d), including P-value correction by B-H adjustment (c). Data are represented as mean ± standard error of the mean; *P ≤ 0.05, ***P ≤ 0.001. IHC, immunohistochemistry. Journal of Investigative Dermatology 2019 139, 186-194DOI: (10.1016/j.jid.2018.07.006) Copyright © 2018 The Authors Terms and Conditions

Figure 5 Neutrophils protect the lung against metastases. (a) Experimental protocol of the spontaneous melanoma metastasis model. Percentages of animals that developed metastasis from intracutaneously injected primary B16F1 melanoma cells (n = 31 per group). (b) Representative pictures of lymph node and lung metastases in mice and anti-LY6G immunohistochemical stainings for neutrophils in metastatic foci. (c) Top: experimental protocol of the experimental melanoma metastasis model and FACS blots showing neutrophil depletion. Bottom: percentages of animals with lung metastases and number of metastatic nodules per lung of intravenous-injected B16F1-CXCL5 and B16F1-control cells in anti-LY6G- or isotype-treated mice. (d) Median fluorescence intensities (FI) of DCFDA ROS detection reagent in lung neutrophils (LungNs) and primary tumor (TANs) (n = 4). Scale bar 100 μm. Statistical analysis by the χ2-test (a, c) and t-test (d); *P ≤ 0.05, **P ≤ 0.01. Journal of Investigative Dermatology 2019 139, 186-194DOI: (10.1016/j.jid.2018.07.006) Copyright © 2018 The Authors Terms and Conditions