In Vivo Detection of Small Subsurface Melanomas in Athymic Mice Using Noninvasive Fiber Optic Confocal Imaging  Peter Anikijenko, Liem T. Vo, Elise R. Murr, Jennifer Carrasco, Wendy J. McLaren, Roger G. King  Journal of Investigative Dermatology  Volume 117, Issue 6, Pages 1442-1448 (December 2001) DOI: 10.1046/j.0022-202x.2001.01592.x Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 FOCI of living melanoma cells in vitro. Plasma membrane labeling following incubation with (a) CD51/61-FITC 1:5 dilution; HY cells; (b) 14.G2a-FITC (1:5 dilution; NMD cells); (c) 9.2.27-FITC (1:5 dilution; NMD cells). In the absence of antibody, normal human keratinocytes (d) and MW melanoma cells (e) showed little or no fluorescence. Scale bars: (a, c–e) 60 µm; (b) 45 µm. Journal of Investigative Dermatology 2001 117, 1442-1448DOI: (10.1046/j.0022-202x.2001.01592.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Subsurface confocal images of implanted melanoma tumors in athymic mice in vivo, after administration of 0.2 ml of 1:5 diluted antimelanoma antibodies. (a) MW melanoma tumor site in an athymic mouse before administration of the antibodies. (b) Normal mouse skin image following CD51/61-FITC administration intravenously into an athymic mouse with no implanted tumor. (c) NMD melanoma after CD51/61-FITC administration. (d) NMD melanoma following administration of 9.2.27-FITC. (e) NMD melanoma tumor following administration of 14.G2a-FITC. (f) Implanted normal human skin cells in an athymic mouse after the administration of CD51/61-FITC showing little or no fluorescence. (g) NMD melanoma tumor following administration of FITC-conjugated mouse IgG1, κ monoclonal Ig isotype control showing little, scattered fluorescence. (h) NMD melanoma tumor after the administration of FITC-conjugated mouse IgG1, κ monoclonal Ig isotype control followed by the administration of CD52/61-FITC. (i) MW melanoma tumor following administration of FITC-conjugated mouse IgG1, κ monoclonal Ig isotype control showing little, scattered fluorescence. (j) MW melanoma tumor after the administration of FITC-conjugated mouse IgG1, κ monoclonal Ig isotype control followed by the administration of CD52/61-FITC. (k) HY melanoma tumor following administration of FITC-conjugated mouse IgG1, κ monoclonal Ig isotype control. (l) HY melanoma tumor after the administration of FITC-conjugated mouse IgG1, κ monoclonal Ig isotype control followed by the administration of CD52/61-FITC. Scale bars: (a, b, g, k) 30 µm; (c) 60 µm; (d, l) 50 µm; (e, f, h–j) 75 µm. Journal of Investigative Dermatology 2001 117, 1442-1448DOI: (10.1046/j.0022-202x.2001.01592.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 FOCI of excised melanoma tumor sites from the dermal surface. (a) NMD melanoma imaged from the dermal surface after epidermal in vivo imaging, intravenous administration of CD51/61-FITC (0.2 ml, 1:5 dilution). (b) Imaged from the dermal surface NMD melanoma tumor after in vivo intravenous administration of 14.G2a-FITC (0.2 ml, 1:5 dilution). (c) HY melanoma imaged from the dermal surface after in vivo intravenous administration of 9.2.27-FITC (0.2 ml, 1:5 dilution). (d) MW melanoma imaged from the dermal surface with no fluorescent antibody administered, showing no fluorescence. Scale bars: (a, c, d) 45 µm; (b) 75 µm. Journal of Investigative Dermatology 2001 117, 1442-1448DOI: (10.1046/j.0022-202x.2001.01592.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Conventional immunoperoxidase-stained cross-section of mouse skin. (a) S100-stained melanoma cells (→) below the epidermis (Epi). Hair follicles (HF), collagen fibers (C). Nerves cells (N) are also positively stained. (b) Negative control (no S100) of cross-section through athymic mouse skin showing no positively stained cells below the epidermis (Epi). Scale bars: (a, b) 30 µm. Journal of Investigative Dermatology 2001 117, 1442-1448DOI: (10.1046/j.0022-202x.2001.01592.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Changes in melanoma-associated vasculature as imaged by FOCI, in vivo. (a) Confocal image of normal murine dermal vasculature following intravenous administration of FITC-dextran (0.3 ml, 10 mg per ml) in an athymic mouse without a melanoma implant. (b) FOCI image of the dermal vasculature in and around an implanted melanoma tumor of an athymic mouse, following intravenous administration of (0.3 ml, 10 mg per ml) FITC-dextran. (c) Dual channel confocal image showing melanoma-associated dermal microvasculature labeled red (Texas Red-dextran; 0.2 ml, 10 mg per ml, intravenous) and the green labeled melanoma tumor (CD51/61-FITC; 0.2 ml, 1:5 dilution, intravenous). Scale bars: (a) 200 µm; (b) 250 µm; (c) 150 µm. Journal of Investigative Dermatology 2001 117, 1442-1448DOI: (10.1046/j.0022-202x.2001.01592.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions