Actinic keratosis modelling in mice: A translational study

<div><p>Background</p><p>Actinic keratoses (AK) are pre-malignant cutaneous lesions caused by prolonged exposure to ultraviolet radiation. As AKs lesions are generally accepted to be the initial lesions in a disease continuum that progresses to squamous cell carcinoma (SCC), AK lesions have to be treated. They are also the second most common reason for visits to the dermatologist. Several treatments are available but their efficacy still needs to be improved. The UV-B-induced KA lesion mouse model is used in preclinical studies to assess the efficacy of novel molecules, even though it is often more representative of advanced AK or SCC.</p><p>Objectives</p><p>Here we report on a translational study, comparing the various stages of AK development in humans and in the UV-B irradiated mouse model, as well as the optimization of photograph acquisition of AK lesions on mouse skin.</p><p>Methods</p><p>Human and mouse skin lesions were analysed by histology and immunohistochemistry. Mouse lesions were also assessed using a digital dermatoscope.</p><p>Results</p><p>An histological and phenotypic analysis, including p53, Ki67 and CD3 expression detection, performed on human and mouse AK lesions, shows that overall AK modelling in mice is relevant in the clinical situation. Some differences are observed, such as disorganization of keratinocytes of the basal layer and a number of atypical nuclei which are more numerous in human AK, whereas much more pronounced acanthosis is observed in skin lesion in mice. Thanks to this translational study, we are able to select appropriate experimental conditions for establishing either early or advanced stage AK or an SCC model. Furthermore, we optimized photograph acquisition of AK lesions on mouse skin by using a digital dermatoscope which is also used in clinics and allows reproducible photograph acquisition for further reliable assessment of mouse lesions. Use of this camera is illustrated through a pharmacological study assessing the activity of CARAC^®.</p><p>Conclusion</p><p>These data demonstrate that this mouse model of UV-B-induced skin lesions is predictive for the identification of novel therapeutic treatments for both early and advanced stages of the disease.</p></div

Comparison of macroscopic skin lesions.

<p>Photographs taken with either a standard camera (A1-A3) or a calibrated digital dermatoscope (B1-B3). Mice were exposed to UV-B irradiation for 108 days and photographs were taken on the indicated days after irradiation initiation. Scale bars represent 500 μm.</p

Patients and skin lesion characteristics.

<p>Patients and skin lesion characteristics.</p

Phenotypic analysis of human (A) and mouse UV-B-induced (B) AK lesions. P53 and Ki67 staining.

<p>Histological sections of normal human skin (pictures labelled “A”) and normal murine skin (pictures labelled “B”) as well as human and murine AK lesions (labelled “A” and “B” respectively) at various stages were stained with P53 (pictures with 1, 2 or 3 numbers) or Ki67 antibodies (pictures with 4, 5 or 6 numbers). Representative cases: <u>Human, normal skin, case 399</u>: A1) p53 staining, A4) Ki67 staining. <u>Human, early-stage AK, case P14-1532</u>: A2) p53 staining, A5) Ki67 staining. <u>Human, advanced-stage AK, case P14-1521</u>: A3) p53 staining, A6) Ki67 staining. <u>Mouse, normal skin case D181P14</u>: B1) p53 staining, B4) Ki67 staining. <u>Mouse, early-stage AK, case D195P16</u>: B2) p53 staining, B5) Ki67 staining. <u>Mouse, advanced-stage AK, case D-221-1</u>: B3) p53 staining, B6) Ki67 staining. Scale bars represent 100 μm.</p

0.5% 5-FU inhibits UV-B-induced AK lesions.

<p>Mice were exposed to UV-B irradiation for 74 days, then topically treated for 30h with either 0.05% 5-FU or control vehicle (5% DMSO, 70% Glycerol and 25% H₂O), starting on day 78 after irradiation initiation. The chamber systems containing the 0.05% 5-FU cream were then removed for 20h, and mice were again treated topically for 30 additional hours. Skin appearance after either control vehicle (A) or 5-FU (B) treatment, at indicated days after treatment initiation. Scale bars represent 500 μm. C) Assessment of the number of skin lesions in control vehicle and 5-FU treated mice, before and after treatment (on day 28 after treatment initiation). Experiments involved five mice per experimental group.</p

Ingenol mebutate inhibits UV-B-induced AK lesions.

<p>Mice were exposed to UV-B irradiation for 74 days, and then topically treated for 5h with either 0.015% ingenol mebutate or control vehicle (5% DMSO, 70% Glycerol and 25% H₂O) on day 78 after irradiation initiation. A) Skin appearance after ingenol mebutate treatment, at days indicated after irradiation initiation. B) Histology of UV-B-irradiated mouse skin topically treated with either control vehicle or 0.015% ingenol mebutate. B1-B2) Hematoxylin and eosin staining of control vehicle-treated skin. B3) Ki67 staining of control vehicle-treated skin. B4) p53 staining of control vehicle-treated skin. B5 and B6) Hematoxylin and eosin staining of 0.015% ingenol mebutate-treated skin. B7) Ki67 staining of 0.015% ingenol mebutate-treated skin. B8) p53 staining of 0.015% ingenol mebutate-treated skin. Experiments involved three mice per experimental group. Scale bars represent 100 μm.</p

Comparative histology of human and mouse UV-B-induced AK lesions.

<p>Hematoxylin and eosin staining. Representative cases: A1) Human normal skin, case 399. A2) Human early-stage AK, case P14-1532. A3) Human early-stage AK, case P14-1358. A4) Human intermediate-stage, case P14-1363. A5) Human advanced-stage AK, case P14-1521. A6) Human advanced-stage AK, case P14-1828. B1) Mouse normal skin, case D181P14. B2) Mouse early-stage AK, case 203P21. B3) Mouse early-stage AK, case D195P16. B4) Mouse intermediate-stage, case 198P39. B5) Mouse advanced-stage AK, case 488. B6) Mouse advanced-stage AK, case D221-1. Scale bars represent 100 μm.</p

Skin appearance after UV-B irradiation and procedure for topical treatment of skin lesions.

<p>(A) Photographs of UV-B-irradiated mouse skin at indicated days post irradiation initiation. (B) Example of tattooed area and of the use of the chamber system to perform topical application.</p