Symptomatic Improvement in Human Papillomavirus-Induced Epithelial Neoplasia by Specific Targeting of the CXCR4 Chemokine Receptor  Floriane Meuris, Françoise Gaudin, Marie-Laure Aknin, Patrice Hémon, Dominique Berrebi, Françoise Bachelerie  Journal of Investigative Dermatology  Volume 136, Issue 2, Pages 473-480 (February 2016) DOI: 10.1016/j.jid.2015.11.004 Copyright © 2015 The Authors Terms and Conditions

Figure 1 AMD3100 treatment reduces ear thickening in K14-HPV16 mice. Progression of ear thickness in untreated K14-HPV16 mice (n = 14) and in AMD3100-treated K14-HPV16 mice (n = 14) from 5 to 9 weeks of age (mean ± SEM). Two-way analysis of variance; *P = 0.0482. Representative photographs of the left ear of wild-type FVB/n mice (n = 12) at 5 weeks of age (top left panel) and untreated K14-HPV16 FVB/n mice (n = 16) (top right and middle right panels) and AMD3100-treated K14-HPV16 mice (n = 20) (bottom right panel) at 5 and 9 weeks of age (start and end of the treatment, respectively). Journal of Investigative Dermatology 2016 136, 473-480DOI: (10.1016/j.jid.2015.11.004) Copyright © 2015 The Authors Terms and Conditions

Figure 2 Histologic analyses of untreated and AMD3100-treated K14-HPV16 mice. (a) Representative hematoxylin-eosin–saffron stained sections of ears from untreated (top, n = 16, dysplastic specimen) and AMD3100-treated K14-HPV16 mice (bottom, n = 20, hyperplastic specimen) at the end of the treatment period. Scanned slides are shown. DD, deep dermis; E, Epidermis; SD, superficial dermis. Bar = 100 μm. (b) Ear thicknesses (distances from a to d in panel a) were measured at four points along the scanned samples, in a blinded manner for each sample. (c) Epidermal thicknesses (distances from a to b and from c to d in panel a) were measured at eight points along the scanned samples, in a blinded manner for each sample. (a-c) Representative results from one of three independent experiments are shown. Unpaired t test; ***P < 0.0001. Journal of Investigative Dermatology 2016 136, 473-480DOI: (10.1016/j.jid.2015.11.004) Copyright © 2015 The Authors Terms and Conditions

Figure 3 AMD3100 treatment reduces the inflammatory infiltrate in the ear skin lesions of K14-HPV16 mice. Representative images of ear tissue from untreated and AMD3100-treated K14-HPV16 mice stained (a) for CD3 that labels T cells (red); (b) with toluidine blue, which specifically labels mast cells (purple); (c) for CD11c, which labels dendritic cells (DC); (d) for Foxp3, which labels regulatory T cells (Treg); and (e) for F4/80, which labels macrophages (macroφ). (a-e) Images were captured from scanned slides from ears harvested at the end of the treatment and are representative of three independent experiments. (a, d) Nuclei were counterstained with 4′,6-diamidino-2-phenylindole. Quantification of positive cells in the dermis is shown beside each image. Values are given as mean ± SEM. **P < .01; ***P < 0.001. Bar = 100 μm; inset bar = 10 μm. Journal of Investigative Dermatology 2016 136, 473-480DOI: (10.1016/j.jid.2015.11.004) Copyright © 2015 The Authors Terms and Conditions

Figure 4 Reduced proliferation and increased apoptosis in ear tissue keratinocytes of AMD3100-treated K14-HPV16 mice. (a) Representative Ki-67–stained ear tissue sections from untreated and AMD3100-treated K14-HPV16 mice and quantification of Ki-67–positive cells. (b) TUNEL assay for identifying apoptotic cells in ear tissue sections from untreated and AMD3100-treated K14-HPV16 mice and quantification of apoptotic cells. Positive cells are shown in green, and nuclei were counterstained with 4′,6-diamidino-2-phenylindole. (a, b) Images were captured from scanned slides of ear tissues harvested at the end of the treatment and are representative of three independent experiments. Values are given as mean ± SEM. ***P < 0.0001. Bar = 100 μm; inset bar = 10 μm. Journal of Investigative Dermatology 2016 136, 473-480DOI: (10.1016/j.jid.2015.11.004) Copyright © 2015 The Authors Terms and Conditions

Figure 5 AMD3100 treatment inhibits p16INK4a and Cxcl12 expression in keratinocytes of K14-HPV16 epidermis. (a) Representative p16INK4a staining in ear tissue from untreated and AMD3100-treated K14-HPV16 mice. (b) Representative Cxcl12 staining in ear tissue from untreated and AMD3100-treated K14-HPV16 mice. (a, b) Images were captured from scanned slides of ear harvested at the end of the treatment and are representative of three independent experiments. Bar = 100 μm; inset bar = 10 μm. Journal of Investigative Dermatology 2016 136, 473-480DOI: (10.1016/j.jid.2015.11.004) Copyright © 2015 The Authors Terms and Conditions