Simon R. Junankar, Alexandra Eichten, Annegret Kramer, Karin E

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Analysis of Immune Cell Infiltrates during Squamous Carcinoma Development Simon R. Junankar, Alexandra Eichten, Annegret Kramer, Karin E. de Visser, Lisa M. Coussens Journal of Investigative Dermatology Symposium Proceedings Volume 11, Issue 1, Pages 36-43 (September 2006) DOI: 10.1038/sj.jidsymp.5650024 Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Flow cytometric analysis of leukocytes during neoplastic progression in HPV16 mice. (a) Single-cell suspensions derived from ear-tissue from negative littermate (-)LM wt and ear-tissue from HPV16 mice were analyzed by flow cytometry to determine the percentage of positively stained cells as a percentage of total viable cells after gating on forward and side scatter and subsequent gating on live cells by exclusion of 7AAD-positive cells. (b, c) Representative histograms for CD45, CD11b, GR-1, and F4/80, and dot blots for CD4-, CD8-, and CD19/B220-positive cells are shown reflecting (-)LM versus dysplastic HPV16 skin. (d) Quantitative analysis reflecting presence of CD45-, CD11b-, GR-1-, F4/80-, CD4-, CD8-, and CD19/B220-positive cells in single-cell suspensions derived from the skin of (-)LMs and pre-malignant (hyperplastic, dysplastic) skin and carcinomas from HPV16 mice was determined as a percentage of total viable cells. Results shown are mean percentages (n=3–7 mice). Error bars represent SEM. Journal of Investigative Dermatology Symposium Proceedings 2006 11, 36-43DOI: (10.1038/sj.jidsymp.5650024) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 CD45 immunostaining during neoplastic progression. (a–f) Immunodetection of CD45-positive leukocytes (brown staining) in paraffin-embedded sections of staged neoplastic tissue reveals incremental increases in the number of CD45-positive leukocytes in the successive neoplastic stages. e, Epidermis; d, dermis; c, cartilage; f, hair follicle. Bar=50 μm. Journal of Investigative Dermatology Symposium Proceedings 2006 11, 36-43DOI: (10.1038/sj.jidsymp.5650024) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Infiltration of neoplastic skin by mast cells and granulocytes. (a) Mast cells (blue staining) in (-)LM and pre-malignant skin and carcinomas of HPV16-transgenic mice. (b) Immunostaining for GR-1+ granulocytes (neutrophils) in (-)LM and pre-malignant skin and carcinomas of HPV16-transgenic mice. Graphical representation of mast cell and GR-1+ neutrophil staining averaged from five high-power fields per mouse and five mice per category. Error bars represent SEM and asterisk (*) indicates statistically significant differences between HPV16 and wt (P<0.05 Mann–Whitney). e, epidermis; d, dermis; c, cartilage; f, hair follicle; SCC (C), central region of carcinoma; SCC(LE), leading edge of carcinoma. Bar=50 μm. Journal of Investigative Dermatology Symposium Proceedings 2006 11, 36-43DOI: (10.1038/sj.jidsymp.5650024) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Distinct characteristics of F4/80+ versus CSF-1R+ macrophages during neoplastic progression. Macrophages were visualized by immunoreactivity to either F4/80 (a–c, g) or CSF-1-R (d–f, h) in (-)LM and pre-malignant skin and carcinomas of HPV16-transgenic mice. Higher magnification images are shown in panels i–l representing F4/80, CSF-1-R, GR-1, or mast cell staining at the leading edge of the carcinomas shown in g and h. e, epidermis; d, dermis; c, cartilage; f, hair follicle. Bar=50 μm. Journal of Investigative Dermatology Symposium Proceedings 2006 11, 36-43DOI: (10.1038/sj.jidsymp.5650024) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions