Introduction: The cells of the immune system are thought to contribute to the development of skin cancers, such as basal cell carcinoma (BCC). One possible mechanism may be the interaction between mast cells and regulatory T cells (Tregs), resulting in immunosuppression. Methods: Fresh-frozen biopsies from the lesional and nonlesional skin of 16 patients with BCC were processed for the enzymehistochemical staining of mast cell tryptase, immunohistochemical staining of FoxP3 (a marker of Tregs) as well as for the double-staining method to label tryptase+ cells and FoxP3+ cells on the same cryosection. The cell numbers and apparent morphological contacts (AMCs) between these cell types were counted. Results: There was a high increase in the number of tryptase+ cells, FoxP3+ cells, and AMCs between them in the lesional compared to corresponding nonlesional skin (p < 0.0001) in all cases. Conclusion: A morphological basis is theoretically present in BCC, suggesting an immune evasive microenvironment.

The ultraviolet radiation from the sun is the essential risk factor in cutaneous carcinogenesis through immunosuppression and DNA damage [1]. The primary events take place in the epidermis, but the cells of the immune system are thought to fail in the eradication of mutated cells [2]. Mast cells are increased in number in the most common skin cancer type, basal cell carcinoma (BCC) [3]. However, it is not clear whether mast cells play a protumorigenic or antitumorigenic role, as mast cells may have a dual role in the skin immune system [2, 4]. Regulatory T cells (Tregs) are characterized by their expression of the transcription factor forkhead box P3 (FoxP3), and these cells have generally been associated with immunosuppression or tolerance [5]. In a variety of experimental models, the interaction of Tregs with mast cells has been found to be one essential mechanism for immunosuppression [6‒8]. This cellular interaction may act in the isomorphic psoriatic lesion by preventing its development [9]. Therefore, the double-staining technique previously developed for demonstrating tryptase+ mast cells and FoxP3+ cells on the same cryosection [9] was utilized in this study to examine whether these cells interact in BCC.

Patients

The study included 16 adult patients with nodular (n = 10) or superficial spreading (n = 6) BCC (5 females, 11 males, age 55–97 years, mean 74), but without marked immunosuppressive state or medication. Four mm punch biopsies were taken after local anesthesia with lidocaine-adrenalin from the lesional and nonlesional (about 2 cm apart from the lesion) skin at varying body sites.

Histochemical Staining Methods

After fixation of cryosections in ice-cold acetone for 10 min, FoxP3 was stained immunohistochemically using 10 μg/mL mouse monoclonal anti-human FoxP3 antibody (clone 236A/E7) (Abcam, Cambridge, UK), and the avidin-biotin-peroxidase (ABC) technique (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA) [9]. Unrelated mouse IgG was used as the control. Mast cell tryptase was stained enzymehistochemically using 1 mm Z-Gly-Pro-Arg-4-methoxy-2-naphthylamide as the substrate (Bachem, Bubendorf, Switzerland) and Fast Black K as the chromogen (Sigma, St. Louis, MO) [3]. The numbers of FoxP3+ and tryptase+ cells were counted on separate cryosections using a 0.2 × 0.2-mm ocular grid in an area of 0.6 mm (depth) ×2.0 mm (width) immediately beneath the lesion or papillary dermis. The results are presented as cells/mm2.

In the double-staining method for tryptase+ and FoxP3+ cells [9], cryosections were treated with anti-FoxP3 mAb and then with biotin-conjugated secondary Ab. Thereafter, tryptase+ cells were identified enzymehistochemically, resulting in dark blue to violet mast cells. Finally, FoxP3+ cells were visualized with the ABC technique that produced black staining. The AMCs were counted in an area described above, and the results are presented as the percentage of tryptase+ mast cells in AMC with at least one FoxP3+ cell. In addition, the total number of AMCs was counted. Only clearly stained tryptase+, FoxP3+ cells, and AMCs were counted.

Statistical Analyses

The results were analyzed using Wilcoxon signed rank test, and p < 0.05 was considered significant.

A representative micrograph for illustrating AMCs is shown in Figure 1. The numbers of tryptase+ cells, FoxP3+ cells, and AMCs between these cells were highly and significantly increased in the lesional skin compared to the corresponding nonlesional skin in all 16 patients and in both BCC types (Table 1).

Fig. 1.

FoxP3+ immunoreactive cells (black) in AMCs with tryptase+ mast cells (violet) in a BCC lesion. The micrograph was taken using a ×40 objective.

Fig. 1.

FoxP3+ immunoreactive cells (black) in AMCs with tryptase+ mast cells (violet) in a BCC lesion. The micrograph was taken using a ×40 objective.

Close modal
Table 1.

Tryptase+ mast cells, FoxP3+ cells and apparent morphological contacts (AMCs) between these cells in the nonlesional and lesional skin of 16 patients with BCC

Tryptase+ cells, cells/mm2FoxP3+ cells, cells/mm2AMCs, %AMCs, contacts/mm2
Lesional skin 189 ± 61* 171 ± 123* 13.5 ±10.1* 25.6 ± 21.7* 
Nonlesional skin 86 ± 34* 24 ± 22* 1.2 ±1.2* 1.0 ± 1.0* 
Tryptase+ cells, cells/mm2FoxP3+ cells, cells/mm2AMCs, %AMCs, contacts/mm2
Lesional skin 189 ± 61* 171 ± 123* 13.5 ±10.1* 25.6 ± 21.7* 
Nonlesional skin 86 ± 34* 24 ± 22* 1.2 ±1.2* 1.0 ± 1.0* 

The results are expressed as the mean ± SD. “*” denotes p < 0.0001 between the lesional and nonlesional skin (Wilcoxon signed rank test).

“AMCs (%)” denotes the percentage of tryptase+ cells in AMC with at least one FoxP3+ cell.

The novel finding is that the numbers of tryptase+ cells, FoxP3+ cells, and especially AMCs between them are highly increased in BCC. Previously, high numbers of FoxP3+ cells and tryptase+ mast cells have been counted in BCC being in line with this study [3, 10]. However, the present results are descriptive because it is not known whether these cellular interactions are functional leading to an immunosuppressive state, even though previous studies suggest so [6‒8, 11]. Furthermore, considerable heterogeneity exists among FoxP3+, CD4+ Tregs, and the transcription factor can also be expressed to a varying extent and time by other cells, including activated CD4+ T cells, CD8+ Tregs, natural killer T cells, macrophages, B cells, and even some cancer cells [5]. In addition, inflammatory cytokines, including interleukin-1β and IL-6, can antagonize FoxP3 expression or function [5]. Interestingly, TNF-α, a pleiotropic cytokine, can regulate FoxP3 expression and Treg activity [12], and TNF-α immunoreactivity is increased in mast cells in BCC [13].

In summary, the results suggest that the morphological basis is present in BCC possibly permitting an immune evasive microenvironment [14]. Future research should be focused on experimental models of skin cancer.

Ms Anne Koivisto is acknowledged for expert technical assistance.

This study protocol was reviewed and approved by the Ethics Committee of Kuopio University Hospital, Kuopio, Finland (approval number 2/2007). A written informed consent was obtained from the patients.

The authors have no conflicts of interest to declare.

The Cancer Center of Eastern Finland of the University of Eastern Finland, Finnish Cancer Research Foundation, and the VTR-funding of Kuopio University Hospital.

Antti P. Kaukinen, Rauno J. Harvima, and Ilkka T. Harvima have made substantial contributions to the conception and design of the work; the acquisition, analysis, and interpretation of data; and the writing or reviewing of the manuscript.

A preprint version of this article is available on medRxiv [14].

Additional Information

Edited by: H.-U. Simon, Bern.

Data are not publicly available due to legal reasons. Further inquiries can be directed to the corresponding author.

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. modified on 26th October, 2023.