Journal of Cranio-Maxillo-Facial Surgery 42 (2014) 1827e1833
Contents lists available at ScienceDirect
Journal of Cranio-Maxillo-Facial Surgery journal homepage: www.jcmfs.com
Cutaneous and oral squamous cell carcinomaedual immunosuppression via recruitment of FOXP3þ regulatory T cells and endogenous tumour FOXP3 expression? Stephanie Schipmann a, *, 1, Kai Wermker b, 1, Hans-Joachim Schulze c, Johannes Kleinheinz d, Georg Brunner a a
Department of Cancer Research, Skin Cancer Centre Hornheide, Dormbaumstraße 300, 48157 Muenster, Germany Department of Oral and Maxillofacial Surgery, Skin Cancer Centre Hornheide, Muenster, Germany Department of Dermatology, Skin Cancer Centre Hornheide, Muenster, Germany d Department of Oral and Maxillofacial Surgery, University Hospital Muenster, Muenster, Germany b c
a r t i c l e i n f o
a b s t r a c t
Article history: Paper received 18 April 2014 Accepted 26 June 2014 Available online 8 July 2014
Regulatory T cells (Tregs) are an essential component of the immune system, but are also involved in the suppression of anti-tumour immune responses. The study examines their immunoregulatory role including their transcription factor, FOXP3, in oral and cutaneous SCC. Tregs were detected by double-immunohistochemistry. FOXP3-mRNA-expression was examined in tumour tissue, as well as in skin-derived primary cells and cell lines of different malignancy. Tregs were found in the tumour microenvironment, and FOXP3-mRNA-expression was significantly higher than in normal skin. Intriguingly, single FOXP3þ cells exhibited morphologic characteristics of SCC cells. Consistent with this, endogenous FOXP3-mRNA-expression was indeed detected in the epidermal cell lineage and dramatically increased with increasing malignancy of the cells. SCCs recruit Tregs into their microenvironment, presumably in order to suppress immunosurveillance, thus avoiding destruction by the immune system. Endogenous FOXP3-expression in malignant epidermoid cells might present a novel mechanism of immune escape. © 2014 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.
Keywords: FOXP3 Regulatory T cells Tregs Squamous cell carcinoma Head and neck cancer
1. Introduction In 1909, Paul Ehrlich was the first to suggest a relationship between the immune system and the elimination of cancer. In the 1950s, Burnet and Thomas developed the concept of cancer immunosurveillance, proposing that lymphocyte subpopulations of the immune system recognise and eliminate cancer cells, before they develop into clinically detectable tumours (Burnet, 1957, 1964; Ehrlich, 1909). However, in some cases tumour cells escape the surveillance of the immune system, becoming manifest in tumour cell progression and metastasis. While this mechanism is not fully understood yet, previous studies indicate that regulatory T cells
* Corresponding author. Tel.: þ49 1732874640; fax: þ49 251 32 87 655. E-mail addresses: [email protected] (S. Schipmann), kai. [email protected] (K. Wermker), schulze@fachklinik-hornheide. de (H.-J. Schulze), [email protected] (J. Kleinheinz), georg. [email protected] (G. Brunner). 1 These authors contributed equally to the study.
(Tregs) may play an important role in carcinogenesis and tumour progression (Knutson et al., 2007; Salama et al., 2009). Tregs serve as an essential component of the immune system, maintaining immunologic self tolerance and immune homeostasis by suppressing the expansion of effector cells directed against self antigens. High numbers of Tregs have been found in the tumour microenvironment and the peripheral blood of patients with various types of cancer, e.g., pancreas, breast, liver, gastrointestinal, and skin cancer (Liyanage et al., 2002; Ormandy et al., 2005; Salama et al., 2009; Viguier et al., 2004; Wolf et al., 2003), indicating that the negative regulatory activity of Tregs may be counterproductive, as Tregs might suppress adequate immune responses against the tumour (Coffer and Burgering, 2004). As a consequence, tumours might recruit Tregs via specific mechanisms into their microenvironment, in order to suppress immunosurveillance, thus avoiding destruction by the immune system and procuring advantages in tumour cell survival and proliferation (Knutson et al., 2007; Wang and Wang, 2007). The transcription factor, forkhead box P3 (FOXP3), has been identified as a specific marker of Tregs (Fontenot et al., 2003;
http://dx.doi.org/10.1016/j.jcms.2014.06.022 1010-5182/© 2014 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.
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Sakaguchi, 2005). FOXP3 activity is essential in the development and functionality of CD4þCD25þ Tregs, serving as a master control gene. Consequently, mutations causing loss of function in FOXP3 result in a lack of Tregs, leading to severe autoimmune disorders and the IPEX syndrome (immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome) in humans. These findings demonstrate, that FOXP3 plays an important role in the regulation of the immune system (Coffer and Burgering, 2004; Hori et al., 2003). Oral squamous cell carcinoma (oSCC) is the tenth most frequent cancer worldwide with dramatically increasing tumour incidence and constitutes, therefore, a significant public health problem (Jemal et al., 2011). In this study, we examined the expression patterns of Tregs and its marker FOXP3 in vivo in oSCC, cutaneous squamous cell carcinoma (cSCC) and metastases thereof as well as in vitro in the epidermoid cell lineage.
and 18S rRNA were: 10 min at 95 C, followed by 40 cycles of denaturation at 95 C for 15 s and annealing/extension at 60 C for 60 s mRNA copy numbers were estimated from CT values, assuming that a single copy yields a CT value of 40 and that, in each cycle, the amount of PCR product is doubled. 2.4. Immunohisto/cytochemistry
Following informed consent of the patients, fresh-frozen tissue samples from primary oSCCs and cSCCs, from metastases (lymph node and skin) thereof, as well as from normal skin were recruited for this study between 1986 and 2009, at the Skin Cancer Center Hornheide, Münster, Germany. Tissue samples were archived in our tumour tissue bank and stored frozen at 80 C. Cryosections of the tumour tissue samples were reviewed, following haematoxylineosin staining, in order to confirm sample quality and determine stroma content. Procedures for tissue collection and analysis were €lische Wilapproved by the local ethics committee of the Westfa helms University of Münster. For the determination of FOXP3 mRNA expression, 13 cSCC, 8 oSCC, and 14 SCC metastases were studied. Immunohistochemistry was performed on 10 cSCC, 8 oSCC, and 4 SCC metastases. Normal skin served as a control (n ¼ 10).
For immunohistochemistry, formalin-fixed paraffin-embedded sections of 5 mm thickness were dewaxed and dehydrated using xylene and graded alcohol washes. For antigen retrieval, slides were autoclaved and placed in 10 mM citrate buffer pH 6.0. For immunocytochemistry, cells were plated on cover slips and fixed with methanol and glacial acetic acid. In order to discriminate between expression of FOXP3 by Tregs and tumour cells, respectively, double-staining was employed using anti-FOXP3 and anti-HMW cytokeration antibodies. Slides were incubated with the primary antibodies (anti-FOXP3, clone 236A/E7, eBioscience, San Diego, CA, USA, 1:100 dilution, incubation overnight in the dark at 4 C; antihuman Cytokeratin High Molecular Weight, clone 34bE12, Dako, Glostrup, DK, 1:50 dilution, incubation for 1 h at RT). Biotinylated mouse IgG was used as a negative control (eBioscience, San Diego, CA, USA, 1:200 dilution). Antibody binding was visualized by using the EnVision™ Gj2 Doublestain System (Dako, Glostrup, DK) containing different detection systems and chromogens. Detection of the primary antibodies was carried out sequentially, visualising one with HRP/ DABþ and the other with alkaline phosphatase/permanent red. Sections were counterstained with Papanicolaou's hematoxylin (Merck, Darmstadt, D), dehydrated through ascending alcohol concentrations to xylene, and mounted. Staining was evaluated using a Leitz Diaplan microscope, by two investigators blinded to the patient's clinical information and diagnosis, and pictures were taken with a Leica DFC 320 digital camera.
2.2. Cell lines and culture
2.5. Statistical analysis
Primary human adult skin fibroblasts (isolated from normal human skin) were maintained in Quantum 333 medium (PAA Laboratories, Piscataway, NJ, USA). Primary human adult keratinocytes were cultured in EpiLife Medium, supplemented with HKGS (Cascade Biologics, Darmstadt, D). All cell lines were passaged using trypsin/EDTA (US Biologicals, Salem, MA, USA). The human squamous cell carcinoma cell line, A431, and the immortalized human HaCaT keratinocytes (Boukamp et al., 1988) (generous gift of Norbert Fusenig, German Cancer Research Centre Heidelberg, Germany) were cultured in DMEM supplemented with 4.5 g/l glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, and 10 mM Hepes buffer. All cells were cultured at 6% CO2 and 37 C in a humidified atmosphere.
Statistical significance of differences in FOXP3 expression between tumour tissue, metastases and skin were evaluated with the Students t-test. Significance was defined as the probability of type one error of
Contents lists available at ScienceDirect
Journal of Cranio-Maxillo-Facial Surgery journal homepage: www.jcmfs.com
Cutaneous and oral squamous cell carcinomaedual immunosuppression via recruitment of FOXP3þ regulatory T cells and endogenous tumour FOXP3 expression? Stephanie Schipmann a, *, 1, Kai Wermker b, 1, Hans-Joachim Schulze c, Johannes Kleinheinz d, Georg Brunner a a
Department of Cancer Research, Skin Cancer Centre Hornheide, Dormbaumstraße 300, 48157 Muenster, Germany Department of Oral and Maxillofacial Surgery, Skin Cancer Centre Hornheide, Muenster, Germany Department of Dermatology, Skin Cancer Centre Hornheide, Muenster, Germany d Department of Oral and Maxillofacial Surgery, University Hospital Muenster, Muenster, Germany b c
a r t i c l e i n f o
a b s t r a c t
Article history: Paper received 18 April 2014 Accepted 26 June 2014 Available online 8 July 2014
Regulatory T cells (Tregs) are an essential component of the immune system, but are also involved in the suppression of anti-tumour immune responses. The study examines their immunoregulatory role including their transcription factor, FOXP3, in oral and cutaneous SCC. Tregs were detected by double-immunohistochemistry. FOXP3-mRNA-expression was examined in tumour tissue, as well as in skin-derived primary cells and cell lines of different malignancy. Tregs were found in the tumour microenvironment, and FOXP3-mRNA-expression was significantly higher than in normal skin. Intriguingly, single FOXP3þ cells exhibited morphologic characteristics of SCC cells. Consistent with this, endogenous FOXP3-mRNA-expression was indeed detected in the epidermal cell lineage and dramatically increased with increasing malignancy of the cells. SCCs recruit Tregs into their microenvironment, presumably in order to suppress immunosurveillance, thus avoiding destruction by the immune system. Endogenous FOXP3-expression in malignant epidermoid cells might present a novel mechanism of immune escape. © 2014 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.
Keywords: FOXP3 Regulatory T cells Tregs Squamous cell carcinoma Head and neck cancer
1. Introduction In 1909, Paul Ehrlich was the first to suggest a relationship between the immune system and the elimination of cancer. In the 1950s, Burnet and Thomas developed the concept of cancer immunosurveillance, proposing that lymphocyte subpopulations of the immune system recognise and eliminate cancer cells, before they develop into clinically detectable tumours (Burnet, 1957, 1964; Ehrlich, 1909). However, in some cases tumour cells escape the surveillance of the immune system, becoming manifest in tumour cell progression and metastasis. While this mechanism is not fully understood yet, previous studies indicate that regulatory T cells
* Corresponding author. Tel.: þ49 1732874640; fax: þ49 251 32 87 655. E-mail addresses: [email protected] (S. Schipmann), kai. [email protected] (K. Wermker), schulze@fachklinik-hornheide. de (H.-J. Schulze), [email protected] (J. Kleinheinz), georg. [email protected] (G. Brunner). 1 These authors contributed equally to the study.
(Tregs) may play an important role in carcinogenesis and tumour progression (Knutson et al., 2007; Salama et al., 2009). Tregs serve as an essential component of the immune system, maintaining immunologic self tolerance and immune homeostasis by suppressing the expansion of effector cells directed against self antigens. High numbers of Tregs have been found in the tumour microenvironment and the peripheral blood of patients with various types of cancer, e.g., pancreas, breast, liver, gastrointestinal, and skin cancer (Liyanage et al., 2002; Ormandy et al., 2005; Salama et al., 2009; Viguier et al., 2004; Wolf et al., 2003), indicating that the negative regulatory activity of Tregs may be counterproductive, as Tregs might suppress adequate immune responses against the tumour (Coffer and Burgering, 2004). As a consequence, tumours might recruit Tregs via specific mechanisms into their microenvironment, in order to suppress immunosurveillance, thus avoiding destruction by the immune system and procuring advantages in tumour cell survival and proliferation (Knutson et al., 2007; Wang and Wang, 2007). The transcription factor, forkhead box P3 (FOXP3), has been identified as a specific marker of Tregs (Fontenot et al., 2003;
http://dx.doi.org/10.1016/j.jcms.2014.06.022 1010-5182/© 2014 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.
1828
S. Schipmann et al. / Journal of Cranio-Maxillo-Facial Surgery 42 (2014) 1827e1833
Sakaguchi, 2005). FOXP3 activity is essential in the development and functionality of CD4þCD25þ Tregs, serving as a master control gene. Consequently, mutations causing loss of function in FOXP3 result in a lack of Tregs, leading to severe autoimmune disorders and the IPEX syndrome (immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome) in humans. These findings demonstrate, that FOXP3 plays an important role in the regulation of the immune system (Coffer and Burgering, 2004; Hori et al., 2003). Oral squamous cell carcinoma (oSCC) is the tenth most frequent cancer worldwide with dramatically increasing tumour incidence and constitutes, therefore, a significant public health problem (Jemal et al., 2011). In this study, we examined the expression patterns of Tregs and its marker FOXP3 in vivo in oSCC, cutaneous squamous cell carcinoma (cSCC) and metastases thereof as well as in vitro in the epidermoid cell lineage.
and 18S rRNA were: 10 min at 95 C, followed by 40 cycles of denaturation at 95 C for 15 s and annealing/extension at 60 C for 60 s mRNA copy numbers were estimated from CT values, assuming that a single copy yields a CT value of 40 and that, in each cycle, the amount of PCR product is doubled. 2.4. Immunohisto/cytochemistry
Following informed consent of the patients, fresh-frozen tissue samples from primary oSCCs and cSCCs, from metastases (lymph node and skin) thereof, as well as from normal skin were recruited for this study between 1986 and 2009, at the Skin Cancer Center Hornheide, Münster, Germany. Tissue samples were archived in our tumour tissue bank and stored frozen at 80 C. Cryosections of the tumour tissue samples were reviewed, following haematoxylineosin staining, in order to confirm sample quality and determine stroma content. Procedures for tissue collection and analysis were €lische Wilapproved by the local ethics committee of the Westfa helms University of Münster. For the determination of FOXP3 mRNA expression, 13 cSCC, 8 oSCC, and 14 SCC metastases were studied. Immunohistochemistry was performed on 10 cSCC, 8 oSCC, and 4 SCC metastases. Normal skin served as a control (n ¼ 10).
For immunohistochemistry, formalin-fixed paraffin-embedded sections of 5 mm thickness were dewaxed and dehydrated using xylene and graded alcohol washes. For antigen retrieval, slides were autoclaved and placed in 10 mM citrate buffer pH 6.0. For immunocytochemistry, cells were plated on cover slips and fixed with methanol and glacial acetic acid. In order to discriminate between expression of FOXP3 by Tregs and tumour cells, respectively, double-staining was employed using anti-FOXP3 and anti-HMW cytokeration antibodies. Slides were incubated with the primary antibodies (anti-FOXP3, clone 236A/E7, eBioscience, San Diego, CA, USA, 1:100 dilution, incubation overnight in the dark at 4 C; antihuman Cytokeratin High Molecular Weight, clone 34bE12, Dako, Glostrup, DK, 1:50 dilution, incubation for 1 h at RT). Biotinylated mouse IgG was used as a negative control (eBioscience, San Diego, CA, USA, 1:200 dilution). Antibody binding was visualized by using the EnVision™ Gj2 Doublestain System (Dako, Glostrup, DK) containing different detection systems and chromogens. Detection of the primary antibodies was carried out sequentially, visualising one with HRP/ DABþ and the other with alkaline phosphatase/permanent red. Sections were counterstained with Papanicolaou's hematoxylin (Merck, Darmstadt, D), dehydrated through ascending alcohol concentrations to xylene, and mounted. Staining was evaluated using a Leitz Diaplan microscope, by two investigators blinded to the patient's clinical information and diagnosis, and pictures were taken with a Leica DFC 320 digital camera.
2.2. Cell lines and culture
2.5. Statistical analysis
Primary human adult skin fibroblasts (isolated from normal human skin) were maintained in Quantum 333 medium (PAA Laboratories, Piscataway, NJ, USA). Primary human adult keratinocytes were cultured in EpiLife Medium, supplemented with HKGS (Cascade Biologics, Darmstadt, D). All cell lines were passaged using trypsin/EDTA (US Biologicals, Salem, MA, USA). The human squamous cell carcinoma cell line, A431, and the immortalized human HaCaT keratinocytes (Boukamp et al., 1988) (generous gift of Norbert Fusenig, German Cancer Research Centre Heidelberg, Germany) were cultured in DMEM supplemented with 4.5 g/l glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, and 10 mM Hepes buffer. All cells were cultured at 6% CO2 and 37 C in a humidified atmosphere.
Statistical significance of differences in FOXP3 expression between tumour tissue, metastases and skin were evaluated with the Students t-test. Significance was defined as the probability of type one error of
