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. Author manuscript; available in PMC: 2020 Aug 1.
Published in final edited form as: J Invest Dermatol. 2019 Feb 8;139(8):1837–1840.e3. doi: 10.1016/j.jid.2019.01.028

Differential hairless mouse strain-specific susceptibility to skin cancer and sunburn

Anita Y Voigt 1, Michael Michaud 1, Kenneth Y Tsai 2, Julia Oh 1,*,^, John P Sundberg 3,^
PMCID: PMC6650314  NIHMSID: NIHMS1526719  PMID: 30742806

LETTER TO THE EDITOR

While there is an ever growing variety of hairless allelic mutations on a diverse number of inbred and outbred strains (Benavides et al., 2009), albino hairless mice, particularly outbred (largely uncharacterized/non-pedigreed) Crl:SKH1-Hrhr/hr (hereafter SKH1) mice have been used extensively, almost exclusively, in UV light-induced skin cancer studies for many decades. These mice lack hair (but not hair follicles) and grow long curved nails as adults. They have essentially an intact immune system, and some experts claim that the skin resembles human skin. In fact, these mice develop the equivalent of the human genetic disease, papular atrichia (Sundberg et al., 1989) and develop skin cancers that resemble UV-induced squamous cell carcinoma (SCC) at both the molecular and morphological levels (Benavides et al., 2009). When these outbred mice are crossed with inbred or insipient congenic strains to study the effect of a mutated gene, investigators often disregard the fact that they have essentially no controls. As the vast majority of genetically engineered mice are created on the C57BL/6J or 6N inbred backgrounds (or made congenic on these and other closely related substrains e.g. (Aylor et al., 2011)), having the same Hrhr allele on one of these strains would potentially provide a better model to study the genetics underlying UV light-induced skin cancer. Such a congenic line was created, the C57BL/6(B6).Cg-Tyrc-2J Hrhr/hr/J strain (hereafter B6.Cg). Initial studies indicated that in acute UV light studies these B6.Cg mice exhibited a more pronounced sunburn response to UVB-irradiation than SKH1 mice (Konger et al., 2016).

To determine the comparative effects of prolonged exposure to UV light between these strains, congenic B6.Cg (Stock Number 017840, The Jackson Laboratory; 7 f /2 m) and outbred albino SKH1 (Strain Code 477, Charles River; 7 f /7 m), both homozygous for the hairless mutation, were chronically irradiated with 180 mJ/cm2 of UVB, 3× per week. Following euthanasia, representative lesions of each size were processed for histologic evaluation. Two or more skin sections of ≥1cm length containing two or more neoplasms or ulcers were evaluated. Lesions were scored using criteria previously described (Benavides et al., 2009). Briefly, tumors were graded as premalignant papillomas (grades 1–3), microinvasive SCC (grades 1–3), or fully invasive SCCs. Spindle cell neoplasms were labeled by immunohistochemistry with antibodies against S-100, keratin 5 (KRT5), keratin 14 (KRT14), pankeratin and vimentin. All work was approved by the Institutional Animal Care and Use Committees. Detailed materials and methods are in the Supplementary Material.

The B6.Cg strain developed wounds or ulcers that attempted to heal by pseudoepitheliomatous hyperplasia (Figure 1). These wounds often developed in the neck area and became progressively worse by scratching. Ulceration was observed as early as 4 weeks after UV light exposure and became pronounced at 13–19 weeks into the study requiring euthanasia (Table S1). All B6.Cg mice (9/9) developed cutaneous ulcers. Four out of 9 B6.Cg mice developed papillomas, 3 mice with grade 1 and one mouse with grade 2. Three out of 9 B6.Cg mice developed grade 2 malignant microinvasive SCC, all of which were very small. None of the B6.Cg mice developed fully invasive SCC (Table 1).

Figure 1: B6.Cg (upper) vs SKH1 (lower panel).

Figure 1:

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B6.Cg mice developed severe scab-covered ulcers (arrow, a) overlying granulation tissue, exophytic papillomas (b) and small, microinvasive carcinomas (c) with apoptosis (circles) and karyorrhexis (box/insert, d). SKH1 mice developed extensive pseudoepitheliomatous hyperplasia with focal hyperchromatic areas (box, e) with cellular atypia (hyperchromatic, multinucleated keratinocytes (boxes, f), high nuclear-to-cytoplasmic ratio, numerous mitotic figures (arrows, f), and keratin pearl formation (circle, f). SKH1 mice had numerous benign exophytic papillomas (g). Well-differentiated, cornified SCCs with keratin pearl formation (circle) (h, i) invaded to the panniculus carnosus muscle (arrows, i). Scale bars = 500 μm apply for panels a-c, e, g-h and 50 μm for panels d, f, i.

Table 1. Summary of histologic based diagnoses of representative lesions.

Lesion diagnosis and staging followed that described by Benavides et al., 2009. SCC: Squamous cell carcinoma.

Mouse strain Ulcer (%) Premalignant papilloma Grade (%) Malignant microinvasive Grade (%) Fully invasive SCC (%) Total malignant SCC lesions Summary (%)
1 2 3 1 2 3
B6.Cg 9/9 (100) 3/9 (33.3) 1/9 (11.1) 0/9 (0) 0/9 (0) 3/9 (33.3) 0/9 (0) 0/9 (0) 3/9 (33.3)
SKH1 6/14 (42.9) 3/14 (21.4) 2/14 (14.3) 1/14 (7.1) 6/14 (42.9) 6/14 (42.9) 4/14 (28.6) 6/14 (42.9) 13/14 (92.9)
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By contrast, fewer SKH1 mice developed cutaneous ulcers (one of which was histologically confirmed); however, none of the ulcers progressed to the same level of severity as the B6.Cg mice. Ulcers observed in SKH1 mice developed significantly later compared to B6.Cg (mean 28 vs. 14.8 weeks, p>0.05) (Table S1). Severe ulcer formation in B6.Cg was also observed at the Moffitt Cancer Center (KYT) in (3/4) UV-irradiated B6.Cg mice (12.50 kJ/m2 per week). In SKH1 mice, UV-related skin alterations (erythema and epidermal hyperplasia; the earliest signs of DMBA/TPA (Binder et al., 1997) and UV light-induced lesions (Konger et al., 2013)) were noted before palpable tumors were identified, with a mean of 18.4 weeks significantly earlier than for B6.Cg mice (p<0.05, Table S1). The frequency of SCCs (based on gross observation) in surviving SKH1 mice plateaued at 100% 23 weeks into the study. One mouse was euthanized in week 26 and the lesion was diagnosed as papilloma, hence, the frequency rate decreased to 92.9%. By contrast, tumor frequency in B6.Cg mice reached 55% of which 33.3% were histologically confirmed as malignant at the endpoint (Figure S1, Table 1). However, the lesion size in the B6.Cg mice was very small compared to those in the SKH1 mice (Figure 1, Figure S1). Measurement of malignant (micro- and fully invasive) lesions using the available histologic sections resulted in an average width of 3.9 ± 1.1 mm and height of 1.3 ± 0.5 mm for B6.Cg mice (n=3) and in a width of 4.4 ± 3 mm and height of 1.9 ± 1.1 mm (n=30) for SKH1 mice (p>0.05). However, caliper measurements at the study endpoint of all observed lesions (pre- and malignant) resulted in significant differences between the two strains for the average cumulative tumor area per mouse and the average tumor area. The average cumulative tumor area per mouse reached 21 ± 24 mm2 for B6.Cg (26 lesions from 6 mice) and 133 ± 115 mm2 for SKH1 mice (114 lesions from 14 mice) (p<0.05, Figure S1). Comparison of average total ulcer length resulted a significant difference with 32 ± 14 mm for B6.Cg (19 ulcers from 9 mice) and 10 ± 5 mm for SKH1 mice (6 ulcers from 6 mice) (p<0.05, Figure S1).

Other types of neoplasms observed included 1/14 SKH1 mice with a spindle cell tumor (S100 weakly positive, KRT5 and 14 negative and pankeratin negative, vimentin positive; Figure S2), and 2/9 B6.Cg mice had mammary ductal adenocarcinomas.

Since the B6.Cg is an inbred strain, Konger et al. (Konger et al., 2016) suggested the strain could be an improved model to assess the influence of the genetic background in cutaneous environmental or toxicological and photobiology studies. However, the current study shows limitations of this strain for chronic UV-irradiation studies. Although very small, premalignant papilloma and microinvasive SCC were diagnosed in a subset of B6.Cg mice. Larger groups of these mice might develop invasive SCCs over time in an adjusted experimental setup (e.g. reduced UVB dosage), since ulceration might be decreased through less UVB exposure; however, under the current experimental settings this strain developed large ulcers necessitating euthanasia.

These observations support the critical role of genetics on the ability of skin to respond to UVB light by sunburn with subsequent clinically severe ulceration or malignant transformation but not necessarily both together. Predilection to ulcer formation in the B6.Cg strain is a known spontaneous dermatologic condition in B6 substrains (Sargent et al., 2015, Sundberg et al., 1994, Sundberg et al., 2011). While chronic UV exposure could potentially exacerbate this strain predisposition to ulceration, the B6.Cg mice in this study lacked the characteristic follicular dystrophy of ulcerative dermatitis in B6 mice (Sundberg et al., 2011).

C57BL6/J mice are known to be resistant to UV-induced skin tumor formation (Kitajima et al., 1995, Naito and DiGiovanni, 1989). Dissecting the genetic differences between the outbred SKH1 and congenic B6.Cg albino hairless mice underlying the differential UV response is difficult due to the outbred and uncharacterized/non-pedigreed status of SKH1. However, extensive genomic and gene expression analysis could allow for identification of cross-strain genomic drivers explaining the susceptibility to tumor or ulcer development (Chitsazzadeh et al., 2016, Fujiwara et al., 2018, Nagase et al., 1996). An inbred SKHIN/Sprd strain was created and described; however, these mice are no longer available (Perez et al., 2012). Alternatively, if similar or more extreme response differences can be shown among inbred strains, such as the collaborative cross strains, or large populations of outbred strains, such as the diversity outcross, genetic susceptibility to sunburn versus squamous cell carcinomas can be easily defined and are attainable future goals.

ACKNOWLEDGEMENTS

We acknowledge the mouse work support by Tyler Gavitt, Jennifer Chung, Adrian Rodriquez, and Michelle Spoto. The authors thank Nicholas E. Gott for preparing the histology and immunohistochemistry in our Histopathology Shared Service. We also thank JAX Creative, especially Jane Cha, for support with figure composition. We are grateful for the funding of AYV through the Pyewacket PostDoc Fund. Research reported in this publication was partially supported by JO’s New Investigator funds from The Jackson Laboratory’s Cancer Center Support (CORE) Grant P30CA034196 which also supports The Jackson Laboratory Shared Services. The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This research received further funding through JO’s American Cancer Society (RSG-16–255-01 and IRG-82003–33 NCCC-01), and JO’s Mackenzie Foundation. KYT was supported by 5R01CA194617 and JPS by 5R01CA089713.

Abbreviations:

B6.Cg

C57BL/6(B6).Cg-Tyrc-2J Hrhr/hr/J

Hrhr

hairless gene mutation

SCC

squamous cell carcinoma

SKH1

Crl:SKH1-Hrhr/hr

Tyrc-J

tyrosinase mutation (albino)

Footnotes

CONFLICT OF INTEREST

JPS had or has sponsored research with Takeda, Theravance, and Curadim and is a consultant for Bioniz, all of which have no relevance to this project. AYV, MM, KYT, and JO state no conflicts of interest.

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