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. 2013 Oct;52(10):760-9.
doi: 10.1002/mc.21901. Epub 2012 Mar 16.

Imiquimod attenuates the growth of UVB-induced SCC in mice through Th1/Th17 cells

Maki Yokogawa  1 , Mikiro Takaishi, Kimiko Nakajima, Reiko Kamijima, John Digiovanni, Shigetoshi Sano
Affiliations

Imiquimod attenuates the growth of UVB-induced SCC in mice through Th1/Th17 cells

Maki Yokogawa et al. Mol Carcinog. 2013 Oct.

Abstract

Imiquimod (IMQ), a Toll-like receptor (TLR) 7/8 agonist, has been used to treat various skin neoplasms, including genital warts, actinic keratoses, and superficial basal cell carcinomas. Although IMQ has been recognized to activate both innate and adaptive immunity, the underlying mechanism(s) by which IMQ exerts its anti-tumor activity in vivo remains largely unknown. In this study, we took advantage of skin cancer-prone mice to characterize the effects of IMQ on ultraviolet irradiation (UV)-induced de novo carcinogenesis. Transgenic mice with keratinocytes expressing constitutively activated Stat3 (K5.Stat3C mice) developed squamous cell carcinomas (SCC in situ) as early as after 14 wk of UVB irradiation, while wild-type mice required much higher doses of UVB with more than 25 wk of UVB irradiation to produce SCC. Topical treatment of K5.Stat3C mice with IMQ attenuated UVB-induced epidermal dysplasia (SCC in situ). In addition, SCC growth due to increased total irradiation doses was significantly attenuated by IMQ treatment. Topical IMQ treatment induced T cell and plasmacytoid dendritic cell infiltrates at the tumor sites, where levels of IL-12/23p40, IL-12p35, IL-23p19, IL-17A, and IFN-γ mRNAs were up-regulated. Immunohistochemistry revealed T cell infiltrates consisting of T1, Th17, and CD8(+) T cells. We speculate that topical IMQ treatment attenuates the de novo growth of UVB-induced SCC through activation of Th17/Th1 cells and cytotoxic T lymphocytes.

Keywords: TLR agonist; Th1/Th17; UVB-induced skin cancer.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors state no conflict of interest.

Figures

Figure 1
Figure 1
K5.Stat3C transgenic mice are prone to UVB-induced skin cancers. (A) Development of skin cancers in K5.Stat3C mice at 18 weeks of UVB irradiation. At this time point, SCCs are present on the dorsal and ear skin of all K5.Stat3C mice tested, while no tumors are observed in non-transgenic control mice. (B, C) Percentage of mice without skin tumors (B) and the average number of skin tumors per mouse (C; mean ± SD) over time with UVB irradiation. black lines, non-transgenic mice (n = 12); red lines, K5.Stat3C mice (n = 7). *, p<0.05; **, p<0.01; Mann-Whitney U-test. (D, E) Representative images and corresponding histology of ear skins from control mice (D) and from K5.Stat3C mice (E) at 0, 12 and 16 weeks of UVB irradiation. K5.Stat3C mice show atypical cells harboring nuclear hyperchromatin (E, inset) at 12 weeks, and progressive SCC at 16 weeks. No cell atypicality or cancer in the epidermis is observed in control mice over this same time period, except for mild acanthosis. Scale bars in D, E, 50μm; except for that in the right bottom panel of E, indicating 200μm. Inset, an enlarged magnification of the epidermis showing atypical keratinocytes.
Figure 2
Figure 2
IMQ prevents the progression of UVB-induced SCCs in situ. Mice are UVB irradiated for 13 weeks followed by no treatment (A, C, E) on the left ears or by topical treatment with IMQ on the right ears (B, D, F) for 6 weeks. Representative results of hematoxylin-eosin staining (A, B), immunohistochemical staining with anti-CD3 (C, D) and with anti-pDC (E, F). There is downward growth of the epidermis, in which disorientated atypical keratinocytes were observed in the left ear (A, arrows) at 6 weeks after the discontinuation of the 13 weeks of UVB irradiation. Note that IMQ attenuated the cell atypicality in the epidermis with underlying cell infiltrates (B). Anti-CD3 staining reveals a number of T cells in the epidermis and in the dermis of the IMQ-treated right ear skin (D), in contrast to the left ear skin, where the growing tumor is infiltrated with very few T cells (C). (G) Number of T cells (mean numbers ± SD per 0.1 mm2, n=4). Black bar, untreated; red bar, IMQ-treated. p<0.05. Student’s t-test. Anti-pDC staining reveals an increased number of pDCs at the epidermal-dermal border and in the upper dermis of IMQ-treated ear skin (F), whereas very few pDCs are present in tumors on the left ear (E). (H) Number of pDCs (mean numbers ± SD per 0.1 mm2). Black bar, n = 4 mice, untreated; red bar, n = 4 mice, IMQ-treated. p<0.05. Student’s t-test. Scale bar, 50 μm in A to F.
Figure 3
Figure 3
IMQ induces apoptosis and attenuates the proliferation of epidermal cells in UVB-irradiated skin. TUNEL staining shows an increased number of apoptotic cells at the epidermal-dermal border in the right ear at 6 weeks of IMQ treatment after UVB irradiation for 13 weeks (B), whereas very few apoptotic cells are found in SCCs in situ in the untreated ear skin (A,). Staining with anti-Ki-67 reveals that cell proliferation is reduced in IMQ-treated skin (D), while a number of proliferating cells were observed in SCCs on the untreated right ear (C). Quantitative evaluation of apoptotic cells (E, mean numbers of TUNEL+ cells ± SD per 1mm) and cell proliferation (F, mean numbers of Ki67+ cells ± SD per 100μm of the epidermis). Black bar, n = 4 mice, untreated; red bar, n = 4 mice, IMQ-treated. **p<0.01, *p<0.05. Student’s t-test. Scale bars, 50μm in A to D.
Figure 4
Figure 4
IMQ attenuates the progression of established SCCs. Full-fledged SCCs induced by UVB-irradiation for 16 weeks are subsequently treated for 4 weeks with IMQ on the right ears or were untreated on the left ears. (A) Macroscopic views of mice #1 (top) and #7 (bottom), in which tumor masses are present on the right ears whereas only flattened, scaly lesions are observed on the left ears. (B) Histological views of untreated SCCs on the left ears (left panels) and the IMQ-treated right ears (right panels) from 7 mice as individually numbered. IMQ-treated SCCs appear to be decreased in size (right panels) compared with those on the left ears (left panels) except for mouse #6 (asterisk). Scale bar, 2 mm. (C) Tumor thickness of untreated SCCs (left) compared with IMQ-treated SCCs (right) from individual mice as numbered. All mice but #6 demonstrated decreased thickness of SCCs following IMQ treatment. **, p<0.01, Student’s t-test. (D, E) Close-up histologic views of representative SCCs on the ears (from mouse #2) treated (E) or untreated (D) with IMQ. Four weeks after the discontinuation of 16 weeks of UVB irradiation, SCCs became more progressive including cell atypia, hyperkeratosis, pseudo horncysts and downward invasion (D), whereas IMQ treatment greatly attenuated that malignant nature, but instead band-like inflammatory cell infiltrates were noted (E). Scale bars, 100 μm (E, same magnification as D).
Figure 5
Figure 5
IMQ-induced regression of SCCs is associated with Th1/Th17 cell infiltrates. Immunohistochemistry reveals dense T cell infiltrates in the regressing SCCs (B), whereas very few T cells are observed in untreated SCCs (A). Scale bar, 100μm in A, B. (C) Immunofluorescence staining of an IMQ-treated regressing tumor with anti-IFN-γ (red) and anti-CD3 (green); a merged image is shown. Arrows indicate IFN-γ+CD3+ cell infiltrates, namely Tc1/Th1 cells, in the dermis. (D) Staining with anti-IL-17A (red) and anti-CD4 (green); a merged image is shown. Arrows indicate Th17 cells. *, unspecific staining of the cornified layer. (E) Quantitative RT-PCR SCC lesions from IMQ-treated versus untreated controls. Expression of each gene was normalized to HPRT mRNA and relative mRNA levels from IMQ-treated lesions (mean ± SD) relative to those from untreated SCCs are shown. The horizontal dotted line is the basal level of each gene expression from untreated SCC samples being set to 1. IL-12, IL-23, IFN-γ and IL-17A mRNAs were up-regulated over 2-fold. n = 4 mice, p<0.05, Mann-Whitney U-test. (F) CD8+ cells infiltrating the epidermis and the dermis. Dotted lines, epidermal-dermal borders. Scale bars, 100μm in A to D; 50μm in F.
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