Comparative Study of Cutaneous Squamous Cell Carcinogenesis in Different Hairless Murine Models

Abstract

Background: In recent decades, a significant global increase in the incidence of non-melanoma skin cancer has been observed. To explore the pathogenesis of and potential therapeutic approaches for squamous cell carcinoma, various in vivo studies using mouse models have been conducted. However, investigations comparing different hairless mouse models, with or without melanin, as well as models with hypercholesterolemia and immunosuppression, in terms of their ability to induce squamous cell carcinoma have yet to be undertaken. Methods: Four mouse strains, namely SKH-hr1, SKH-hr2, SKH-hr2+ApoE, and immunodeficient Nude (Foxn1 knockout), were exposed to UVA and UVB radiation three times per week, initially to 1 Minimal Erythemal Dose (MED), incrementally increased weekly to a maximum dose of 3 MED. Clinical evaluation, photodocumentation, and biophysical parameters were monitored, along with proteasome protein activity and histopathological assessments. Results: The SKH-hr1 model primarily developed actinic keratosis without significant progression to invasive squamous cell carcinoma (SCC), while the SKH-hr2 and SKH-hr2+ApoE models exhibited a higher likelihood and intensity of papilloma and aggressive SCC formation, with the latter showing upregulated proteasome activity. Histopathological analysis confirmed the presence of poorly differentiated, invasive SCCs in the SKH-hr2 and SKH-hr2+ApoE models, contrasting with the less aggressive SCCs in the Nude mice and the mixed lesions observed in the SKH-hr1 mice. Conclusions: The SKH-hr2+ApoE and SKH-hr2 mice were identified as the most suitable for further exploration of squamous cell carcinogenesis. In contrast, the SKH-hr1 mice were found to be the least suitable, even though they are albino. Notably, proteasome analysis revealed a potential role of proteasome activity in squamous cell carcinogenesis.

Keywords: UV radiation; cutaneous squamous cell carcinoma; hairless mouse models; photo-carcinogenesis; skin tumors.

Figure 1

Evolution of carcinogenesis over time. Representative images of the four irradiated mouse models (SKH-hr1, SKH-hr2, SKH-hr2+ApoE, and Nude) at various time points. Differences are evident as early as the first month across all the groups, with increased melanin observed in the skin of the SKH-hr2 and SKH-hr2+ApoE models by the third month. Papilloma formation began after the third month, and tumor development was noted after the seventh month in the SKH-hr2, SKH-hr2+ApoE, and Nude models. In contrast, the SKH-hr1 mice primarily exhibited actinic keratosis toward the study’s end, with minimal papilloma and tumor formation.

Figure 2

Cumulative counts of papillomas and tumors over time. (A) Cumulative number of papillomas per mouse strain. (B) Cumulative number of tumors per mouse strain. (C) Cumulative number of animals exhibiting carcinogenesis per mouse strain (n = 10 SKH-hr1, 10 SKH-hr2, 10 SKH-hr2+ApoE, and 9 Nude mice).

Figure 3

Histopathological assessment of squamous cell carcinoma. (A) SCC exhibiting numerous keratin pearls (indicated by black arrows), extending from the epidermis into the dermis. (B) Aggressively, poorly differentiated SCC characterized by the presence of abundant keratin pearls (marked by black arrows). (C) Actinic keratosis is noted, alongside an evolving SCC in situ that remains non-invasive and has yet to breach the basement membrane. (D) Aggressive SCC displaying significant mitotic activity and the presence of enlarged, atypical nuclei. To the right of the image, a well-differentiated region of the SCC features numerous keratin pearls (black arrows), in contrast to the left side, which shows a poorly differentiated region of the SCC with cohesive cell clusters (blue arrows). Panels (A–D) correspond to Nude and SKH-hr2+ApoE, SKH-hr1, and SKH-hr2 mice, respectively. (Magnification: ×100).

Figure 4

Graphical representation of the alterations in the skin parameters per month. (A) Transepidermal water loss. (B) Hydration. (C) Melanin. (D) Skin thickness. Each mouse model is depicted with a different color (blue for SKH-hr1, red for SKH-hr2, green for SKH-hr2+ApoE, orange for Nude).

Figure 5

Proteasome status in the SKH-hr2 and SKH-hr2+ApoE mice: (A) chymotrypsin-like (CT-L), (B) caspase-like (C-L) and (C) trypsin-like (T-L) activities measured in normal (N) and tumorous (T) skin of hr2 (left) and hr2-ApoE (right) animals. Tissues from (A) 5 (N) -8 (T) hr2 animals and 9 (N) -11 (T) hr2-ApoE animals, (B) 6 (N) -8 (T) hr2 animals and 9 (N and T) hr2-ApoE animals, and (C) 3 (N) -4 (T) hr2 animals and 4 (N and T) hr2-ApoE animals were analyzed (paired t-test for (A,B), Wilcoxon test for (C), * p value < 0.05, ns, non-significant). (D) Representative immunoblots and (E) Band densitometry for the β1 and β2 proteasome subunits, depicting the mean band density ± SEM. The braces indicate that the normal (N) and tumorous (T) tissue came from the same animal. The β1 and β2 protein levels were normalized to gels stained with Coomassie Brilliant Blue. Tissues from 5–8 animals per group were analyzed (unpaired t-test, ns, non-significant). The original Western blot figures can be found in Supplemental Materials.

Figure 6

Sebum measurements across different murine models over time. While no statistically significant differences in the skin sebum levels were detected across the groups, the SKH-hr2+ApoE mice consistently exhibited higher average sebum concentrations compared to the other models.