Biology and immunology of cancer stem(-like) cells in head and neck cancer.

ONCH-1961; No. of Pages 9

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Critical Reviews in Oncology/Hematology xxx (2015) xxx–xxx

Biology and immunology of cancer stem(-like) cells in head and neck cancer Xu Qian a,b , Chenming Ma b , Xiaobo Nie a , Jianxin Lu a , Minoo Lenarz b , Andreas M. Kaufmann c , Andreas E. Albers b,∗ a

Key Laboratory of Laboratory Medicine, Ministry of Education, Zhejiang Provincial Key Laboratory of Medical Genetics, Wenzhou Medical University, Zhejiang, PR China b Department of Otorhinolaryngology, Head and Neck Surgery, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany c Clinic for Gynecology, Charité-Universitätsmedizin Berlin, Campus Mitte and Benjamin Franklin, Berlin, Germany Accepted 30 March 2015

Contents 1. 2.

3.

4. 5.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The CSC hypothesis in HNSCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. CSCs in cancer progression and metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Interactions between human papillomavirus (HPV) and CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Resistance to current therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CSC-induced immune-responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Recognition of CSCs by the immune system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Immune escape of CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Immune suppression by CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CSC-based vaccination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biographies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract Immunological approaches against tumors including head and neck squamous cell carcinoma (HNSCC) have been investigated for about 50 years. Such immunotherapeutic treatments are still not sufficiently effective for therapy of HNSCC. Despite the existence of immunosurveillance tumor cells may escape from the host immune system by a variety of mechanisms. Recent findings have indicated that cancer stem(-like) cells (CSCs) in HNSCC have the ability to reconstitute the heterogeneity of the bulk tumor and contribute to immunosuppression and resistance to current therapies. With regard to the CSC model, future immunotherapy possibly in combination with other modes of treatment should target this subpopulation specifically to reduce local recurrence and metastasis. In this review, we will summarize recent research findings on immunological features of CSCs and the potential of immune targeting of CSCs. © 2015 Elsevier Ireland Ltd. All rights reserved.

Keywords: Cancer stem cells; ALDH1; Epithelial–mesenchymal transition; Human papillomavirus; Immunotherapy; Vaccination ∗ Corresponding author at: Department of Otorhinolaryngology, Head and Neck Surgery, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, D-12200 Berlin, Germany. Tel.: +49 30 84454586. E-mail address: [email protected] (A.E. Albers).

http://dx.doi.org/10.1016/j.critrevonc.2015.03.009 1040-8428/© 2015 Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: Qian X, et al. Biology and immunology of cancer stem(-like) cells in head and neck cancer. Crit Rev Oncol/Hematol (2015), http://dx.doi.org/10.1016/j.critrevonc.2015.03.009

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X. Qian et al. / Critical Reviews in Oncology/Hematology xxx (2015) xxx–xxx

1. Introduction The current treatments for head and neck squamous cell carcinoma (HNSCC) have been challenged by the cancer stem(-like) cell (CSC) hypothesis. These cells play a central role in initiation, progression, invasion, metastasis, recurrence of tumors and resistance to therapies [1]. In vitro and in vivo studies of HNSCC have shown that putative CSCs or CSC-enriched non-adherent spheroid cells present with stem cell-like self-renewal properties, invasion capacity and therapy resistance [2–5]. The CSC model is closely related to the phenomenon that HNSCC initially respond well to conventional treatments, but local and distant relapses occur frequently. It is interesting to note that phenotypic heterogeneity and plasticity of CSCs was observed to be associated with epithelial-mesenchymal transition (EMT), which collectively promotes metastasis [6]. Subsequently, CSCs require a special microenvironment to regulate their stemness, and to initiate and promote cancer development by recruiting and activating special cell types [7–10]. The development and the introduction of immunotherapy for HNSCC holds promise as an attractive supplement to traditional treatments such as surgery, chemotherapy, and radiation therapy. Since immunotherapies are designed to target directly the tumor cells the incidence of side effects is expected to be low. Many approaches based on bulk tumor cells have been developed and successfully monitored, but a correlation with good clinical responses has been sparse so far [11,12]. The main issues in developing cancer immunotherapy are the strengthening of cytotoxic T cell responses and prevention or reversal of tumor-induced immune-escape. Emerging evidence indicates that the host immune system is able to recognize CSCs and mount an effector response against them, but CSCs may also play a role in mediating immunosuppression within the tumor microenvironment [13,14]. Therefore, it is necessary to gain further insight into the immunological features of CSCs and explore potential immunotherapeutic approaches against CSCs. In this review, we discuss the biology of CSC in HNSCC with regard to their potential as targets for future immunotherapy.

2. The CSC hypothesis in HNSCC Accumulating evidence suggests that in a heterogeneic tumor, a subpopulation of tumor cells with stem cell-like selfrenewal capacity, known as CSCs or tumor-initiating cells (TICs) have the ability to give rise to a proliferative bulk tumor cell mass and to survive systemic treatments [1]. CSCs have been identified in many types of solid tumors including HNSCC [15,16]. One of the first studies of CSCs in HNSCC using an immunodeficient mouse as model demonstrated that a minor population of CD44+ cancer cells, which account for less than 10% of cells in a HNSCC primary tumor, could give rise to new tumors in vivo and displayed the ability of self-renewal and differentiation [2]. In consistency with this

finding, important advances have been achieved in the study of the role of HNSCC CSCs in the progression of malignancies in in vitro or in vivo mouse models and patient-derived clinical samples. 2.1. CSCs in cancer progression and metastasis Once initiated, CSCs may generate macroscopic tumors through the stem cell processes of self-renewal and differentiation into multiple cell variants. Furthermore, CSCs may undergo EMT, a process involved in embryogenesis and considered also to be involved in metastatic dissemination [17]. During EMT, cells of epithelial phenotype convert to migratory and invasive cells with mesenchymal phenotype. When the migrating mesenchymal cells have reached their destination, they may undergo a reverse process, a mesenchymal–epithelial transition (MET), to regain the epithelial phenotype. Recent studies highlight that tumor cells undergoing EMT acquire stem cell-like properties, and EMT can also induce non-CSC to acquire a CSC-like state (Fig. 1) [18–20]. We previously showed that aldehyde dehydrogenase 1 (ALDH1)+ -CSC enriched cell populations from 3 dimensional spheroid cultures generated from HNSCC cell lines displayed EMT characteristics with enhanced colony forming ability and invasiveness [4]. Further, the presence of HNSCCCSCs with the ability to undergo both EMT and MET by switching between their epithelial and mesenchymal phenotypes has been discovered by Biddle et al. [6]. Migratory CD44high epithelial-specific antigen (ESA)low EMT-CSC expressed EMT markers and a mesenchymal phenotype, while CD44high ESAhigh non-EMT-CSC had epithelial characteristics. Importantly, EMT-CSC thereby required an ALDH+ phenotype to switch to non-EMT-CSC and to develop metastasis successfully. More recently, a CD44regulated signaling pathway mediated by phosphorylation of glycogen synthase kinase 3␤ (GSK3␤) has been identified and has shown the potential to affect CSC phenotypes [21]. Inhibition of GSK3␤ could reduce the formation of CSCs-enriched tumor spheres and “holoclone” colonies. Reduction of the expression of stem cell markers and upregulation of the differentiation markers were also found in the CD44high ESAhigh cell fraction by GSK3␤ inhibition. GSK3␤ knockdown could induce CSCs reversing from EMT and back to the epithelial CSC phenotype. In another study, Yang et al. identified a mechanism in which the EMT inducer Twist1 elicits cancer cell movement through activation of RAC1 [22]. They found that Twist1 cooperates with BMI1 to suppress let-7i expression, which results in up-regulation of NEDD9 and DOCK3, leading to RAC1 activation and enabling mesenchymal-mode movement in three-dimensional environments. Moreover, the suppression of let-7i contributes to Twist1-induced stem-like properties. These tumor cells expressing a stem-like cancer cell phenotype could transit from non-motile, epithelial-like cells to motile mesenchymal cells. Reversing EMT in prostate cancer



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Fig. 1. Cancer-stem(-like)-cells-induced therapy resistance and its interaction with tumor microenvironments contribute to tumor recurrence and the development of distant metastasis. Abbreviations: cancer stem(-like) cell (CSC); circulating tumor cell (CTC); cytotoxic T lymphocyte (CTL); infiltrating T regulatory cell (Treg); tumor-associated macrophage (TAM).

cells by forced re-expression of miR-200, which also significantly inhibited the self-renewal capacity, was reported by Kong et al. [23]. Together, inhibition or reversal of the EMT process appears to be an attractive therapeutic strategy in HNSCC and other human cancers. Although there is accumulating experimental evidence supporting the role of CSCs in driving tumor growth and metastasis, clinical evidence for their driving role in the formation and progression of cancer is still sparse. In a recent study, we investigated the ALDH1+ CSCs frequency in primary oropharyngeal squamous cell carcinoma (OSCC) and its corresponding metastases [24]. OSCC with higher frequency of ALDH1+ cells present a more aggressive phenotype characterized by higher nodal classification and lower differentiation. A higher number of ALDH1-expressing cells were found more frequently present in lymph node metastases than its corresponding primary tumors indicating the ability of CSCs to complete the metastatic cascade and develop metastases. Studies from carcinomas of different origins also presented traits of high-grade malignancy that could be specifically traced back to the presence of CSCs [25–27]. 2.2. Interactions between human papillomavirus (HPV) and CSCs A subgroup of head and neck cancer is associated with persistent HPV infection thus rendering HPV infection a target

for therapy and prevention. The incidence of HPV+ cases in HNSCC is rising, in particular in OSCC, while the incidence of HPV− cases induced by alcohol- or tobacco-abuse has decreased in recent years with successfully established antismoking campaigns [28]. Besides the epidemiological role of HPV in HNSCC, studies also addressed the role of HPV in cancer progression and the differences of age, genetic background and prognosis between HPV+ and HPV− HNSCC patients [28]. With regard to these two different etiologies, investigations on the role of HPV with CSCs in initiating and spreading HNSCC are emerging but still have not been well documented. In a recent study, Jung et al. investigated the role of HPV16-E6 and E7 oncogenes during the process of EMT in vitro [29]. A stable expression of HPV16-E6 or E7 induced morphological conversion of epithelial cells to a mesenchyme-like phenotype. In addition to these morphological changes, both E6 and E7 induced expression of the EMT-activating transcription factors Slug, Twist, ZEB1 and ZEB2. This study suggests that HPV16 could induce an EMTlike process. Tang et al. investigated whether the behavior of CSCs is affected by the HPV-status or not in in vitro and in vivo studies [30]. In this study, the HPV+ cells and cells transduced with HPV E6/E7 demonstrated a greater clone forming capacity than HPV− cells. The HNSCC-CSCs were shown to be more resistant to cisplatin than the nonCSCs. However, there was no difference of the proportion


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of CSCs between HPV+ and HPV− HNSCC. Moreover, the HPV status did not affect the response of CSCs to cisplatin therapy. Our previous study on human primary OSCC demonstrated that fewer ALDH1-expressing CSCs were found in high-risk (HR)-HPV-DNA+ /p16+ primary OSCC compared to HR-HPV-DNA− /p16− primaries [24]. There were, however, no similar findings in the corresponding metastases. Similarly, Rietbergen et al. has shown that HPV+ OSCC primary tumors harbored a lower percentage of CSCs with the markers of CD44 and CD98 than HPV− tumors. A better survival was found in HPV+ patients with a lower percentage of CD98+ tumor cells compared to HPV+ patients with high fraction of CD98+ tumor cells [31]. Further studies are warranted to further elucidate the interactions between HPV and CSCs to identify a possible use of immunological targets specific to the HPV-related etiology of this subgroup of HNSCC.

3. CSC-induced immune-responses Current immunotherapy is mainly based on antigens presented to effector T cells by dendritic cells (DCs). Generally, these antigens are selected and derived from bulk tumor cells and target non-CSC tumor cells. They are not derived of CSCs that may not express immunogenic differentiation antigens [37]. CSCs also may be defective in antigen presentation due to the downregulation of human leukocyte antigen (HLA) surface expression [38]. Therefore, in a heterogeneous tumor entity, escaping from the attack of current immunotherapy, CSCs may lead to a treatment failure and disease progression. Thus, a better knowledge of the cross-talk between CSCs and the immune system (Table 1, Fig. 1) and a development of specific therapies targeted at CSCs are of interest for further improvement of cancer immunotherapies. 3.1. Recognition of CSCs by the immune system

2.3. Resistance to current therapy In patients with locally advanced HNSCC treated by surgery and/or chemo-radiotherapy, loco-regional control can be gained for some time but patients will frequently develop a recurrence and/or metastasis. Despite intensive investigation of resistance mechanisms to chemo- and radiation therapy, our understanding of the relationship between resistance mechanisms and therapeutic success is still limited and cannot be predicted. The CSC model offers explanations for these treatment failures [1,32]. The tumor cells in a stem cell-like state could escape from therapies acting against proliferating cells as “dormant” cells, a feature characteristic for CSC, and resume proliferation once the toxic pressure is removed. Radioresistance of CSCs has been attributed to their self-renewal capacity, DNA repair capacity and enhanced reactive oxygen species (ROS) defence [15,33]. In a study, CD44+ ALDH1+ cells isolated from HNSCC showed higher tumorigenicity, radioresistance, and high expression of stemness-related genes (Bmi-1/Oct-4/Nanog). When treated with Cucurbitacin I additionally, the induction of apoptosis and sensitivity to radiation of this CSC-population was enhanced [34]. Chen et al. reported that silencing of Bmi-1 significantly enhanced the sensitivity of HNSCCALDH1+ CSCs to chemo-radiation that may be explained be an increased degree of chemo-radiation mediated apoptosis. Moreover, the experiment showed that knockdown of Bmi-1 increased the effectiveness of radiotherapy and resulted in inhibition of tumor growth in nude mice transplanted with ALDH1+ CSCs [35]. Recently, in HNSCC cell lines, Bourguignon et al. [36] observed that CD44v3high ALDH1high cells were more resistant to cisplatin, the most commonly used chemotherapy drugs for treatment of HNSCC. They also discovered a new HA-CD44v3-mediated signaling pathway leading to the stimulation of apoptosis and enhanced chemosensitivity in CSCs.

One important issue for future immune therapy targeting CSCs is the recognition and distinction of CSCs from other cells by the host immune system. The immunogenicity of HNSCC-CSCs has been observed recently. Among reported CSC markers, ALDH1 is considered a more specific CSC marker than any of the other phenotypes used to identify the small population of highly tumorigenic cells present in HNSCC and other carcinomas, as well [5,39–41]. ALDH1 has already been recognized as an antigen-source eliciting a humoral immune response in HNSCC. Visus et al. [42] showed that ALDH1A188–96 peptide was an HLA-A2-restricted, naturally presented, CD8+ T cell-defined tumor-antigen. ALDH1 peptide-specific CD8+ T cells could only recognize HLA-A2+ HNSCC cell lines overexpressing ALDH1 but not a human MRC fibroblast cell line. Importantly, potential toxicity against CD34+ hematopoietic progenitor cells by ALDH1A1 peptide-specific CD8+ T cells was excluded. By ELISPOT interferon (IFN)-␥ assays, CD34+ hematopoietic progenitor cells isolated from HLAA2+ bone marrow cells were not recognized by ALDH1A1 peptide-specific CD8+ T cells. Most importantly, target recognition was blocked by anti-HLA class I and antiHLA-A2 antibodies. In addition to this defined CSC-tumor antigen, we recently compared the susceptibility of putative CSC-enriched spheroid culture-derived cells (SDC) generated from two HNSCC cell lines and one cervical cancer cell line to immunological recognition and killing by alloantigenspecific CD8+ cytotoxic T lymphocytes (CTL) [13]. This study was important to investigate immune suppression by CSC and its reversal by IFN-␥ treatment. In order to avoid antigen-specific bias we used alloantigen-specific responses as strong and generic T cell targets. We found that CSC populations were less sensitive to MHC class I-restricted alloantigen-specific CD8+ CTL lysis, as compared to their monolayer-derived cells (MDC). However, an additional pretreatment with IFN-␥ resulted in over-proportionally enhanced lysis of SDC. We also observed that the subset


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Table 1 The relationship between CSCs and the immune system. Immunological molecules expressed by CSCs

Model

Ref.

Similar expression of MHC class I between CSCs and bulk tumor cells Low expression of MHC class I No expression of MHC class I, low expression of MHC class II Cancer/testis antigens

HNSCC Cervical cancer Glioma Acute myeloid leukemia, renal cell carcinoma Lung adenocarcinoma, colon adenocarcinoma, breast adenocarcinoma Colon cancer, pancreatic cancer, breast cancer

[13] [13] [14] [43,44]

HNSCC Glioma

[42,67] [14]

Breast cancer Glioma Colon and lung cancer

[54] [14] [66]

Pancreatic ductal adenocarcinoma

[8]

Cancer/testis genes

Defect in antigen presentation due to downregulation of HLA surface expression Immune responses induced by CSCs ALDH1-specific CD8+ T cells recognize and eliminate CSCs Inhibition of the effector function of T lymphocytes including T cell proliferation and T cell apoptosis by CSCs EMT CSCs inhibit CTL-mediated tumor cell lysis Induction of FoxP3+ Tregs by CSCs TAMs increase the tumorigenicity and drug resistance of CSCs by activating STAT3 and the Sonic Hedgehog pathway TAMs induce CSCs by activating STAT3; In turn, CSCs induce immunosuppression in TAM and CD8+ T cell responses

of ALDHhigh expressing SDC was more sensitive than their counterpart of ALDHlow SDC toward cognate CD8+ CTL killing. Moreover, the potentially different immunogenicity of CSCs and the differentiated bulk tumor cells was observed in our study. We found SDC derived of the cell line CaSki appeared to be more resistant to the recognition and destruction by MHC class I-restricted alloantigen-specific CD8+ CTL than the matched MDC. Together, the data presented by the groups above have shown that the host immune system is able to recognize and distinguish CSCs with ALDH1 phenotype from non-CSC cells. In addition to ALDH1, some cancer/testis (CT) antigens were found to be preferentially expressed in CSCs. CyclinA1 was reported in leukemic stem cells of acute myeloid leukemia [43]. DNAJB8 was identified as novel CT antigen in renal CSCs [44]. Eighteen CT genes (MAGEA2, MAGEA3, MAGEA4, MAGEA6, MAGEA12, MAGEB2, GAGE1, GAGE8, SPANXA1, SPANXB1, SPANXC, XAGE2, SPA17, BORIS, PLU-1, SGY-1, TEX15 and CT45A1) exhibited higher expression levels in CSCs than in non-CSCs derived from LHK2 lung adenocarcinoma, SW480 colon adenocarcinoma and MCF-7 breast adenocarcinoma cells lines [45]. The specific expression of CT antigens may enable us to target CSCs specifically in this manner.

3.2. Immune escape of CSCs It has been well documented that tumor cells can evade the immune system by altering their phenotype or by suppressing immunity [46]. In HNSCC, defects in the MHC class I antigen processing machinery are observed and responsible for the escape of tumor cells from recognition by cytotoxic T lymphocytes (CTL) [47,48]. This correlates with poor

[45]

[38]

prognosis in patients with HNSCC [49]. Cytokines such as IFN-␥ can restore antigen processing [47]. In addition, tumor cells may downregulate MHC expression to escape immune surveillance [50] as has also been shown for CSC [12]. A recent study in melanoma and epithelial cancer cell lines has shown that SDC containing putative CSCs showed equal or higher mRNA expression levels of molecules involved in antigen processing and presentation (APM) such as LMP2, LMP7, and MECL-1, of APM molecules transportersassociated with antigen presentation (TAP) 1 and TAP2 and, also of TAA including differentiation antigens [38]. Downregulation or loss of MHC class I and MHC class II molecules in SDC was observed which was associated with decreased recognition of peptide-loaded disrupted SDC by CD8+ T cells. Interestingly, MHC expression on tumor SDC was not responsive to stimulation with IFN-␥. Previously, we investigated the expression of MHC class I, MHC class II, and of immune recognition associated molecules on SDC and MDC generated from two HNSCC cell lines and one cervical cancer cell line CaSki [13]. We found that MHC class I was expressed on SDC and MDC by all cell lines with different levels. Its expression on CaSki SDC was nearly two times lower than on corresponding MDC, but there were no difference in HNSCC cell lines. MHC class II was negative on SDC and MDC in all cell lines. IFN-␥ treatment upregulated the expression of MHC class I and induced MHC class II. In the above two studies, IFN-␥ treatment showed different effects on the upregulation of MHC class I and MHC class II. This may be due to different immune evasion mechanisms selected for in different tumor entities, individual patients and tumors or the isolated cell lines employed for the experimental research. Therefore theses data can demonstrate the diversity of potential mechanisms of immune evasion. These findings, nevertheless, revealed a


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potential defect of antigen-presenting functions of CSCs that might lead to the protection of CSCs from T cell rejection. Some studies suggest that immunoedited tumor cells may evade the immunosurveillance through EMT [51–53]. As we described above, CSCs can metamorphose between an EMT-state and non-EMT-state. The plasticity of the CSC phenotype enables migration and tumor formation at distant sites. Thus, one can hypothesize that CSCs undergoing EMT may survive the initial immune response and drive the regrowth of the tumor and development of metastasis. An important feature of CSCs with their contribution to tumor evasion of the immunosurveillance has been observed recently. Wei et al. reported that glioma CSCs could inhibit T cell proliferation and effector responses, trigger T cell apoptosis and induce FoxP3+ regulatory T cells [14]. Later, Akalay et al. found that the EMT phenotype acquired in various derivatives of MCF-7 human breast cancer cells was associated with dramatic morphologic changes and actin cytoskeleton remodeling, with CD24− /CD44+ /ALDH+ stem cell populations present exhibiting a higher degree of EMT relative to parental cells [54]. In the same study, acquisition of an EMT-CSC phenotype was found to be associated with an inhibition of CTL-mediated tumor cell lysis. In conclusion, these data indicate that the acquisition of an EMT phenotype by CSCs may be an additional mechanism of immune-escape by the tumor. 3.3. Immune suppression by CSCs Immunotherapeutic approaches for HNSCC are complicated due to the profound immune suppression induced by this disease. Mechanisms such as increased apoptosis of tumor-specific CD8+ T cells and increased tumor-infiltrating T regulatory cells (Tregs) in peripheral blood and at the tumor site have been demonstrated [55–57]. HPV-encoded oncogenic proteins have been reported to downregulate expression of MHC class I [58] and a higher level of tumor-infiltrating lymphocytes was observed in HPV+ OSCC patients than HPV− OSCC patients [59]. While the majority of these studies performed in regard to bulk tumor cells, immunosuppressive properties mediated by CSCs have been investigated recently. Work by Krishnamurthy et al. showed that CSCs are located in close proximity to blood vessels and that endothelial cell-initiated signaling could enhance survival and self-renewal of HNSCC-CSCs [7]. Clinically, patients with recurrent HNSCC showed an increased concentration of IL-6 in serum, in comparison with patients with primary HNSCC [60]. Elevated IL-6 levels could independently predict tumor recurrence, poor survival, and tumor metastasis [61,62]. In line with this finding, Yu et al. demonstrated that secretion levels of IL-6 and sIL-6R from CSCs were vital to maintain the self-renewal and tumorigenic properties of CSCs in HNSCC [60]. Tumor-associated macrophages (TAMs) may play a critical role in tumor progression by interacting with the tumor microenvironment [63]. Tregs are thought to promote tumor progression and link to a worse prognosis [64].

It was reported that glioma TICs could induce FoxP3+ Tregs that were mediated by the costimulatory inhibitory molecule B7-H1 and soluble Galectin-3 [14]. Further, the induction was diminished by altering the differentiation of the TICs. In a study of primary human gliomas and a orthotopically transplanted syngeneic glioma model, the distribution of TAM at the invasive tumor front was correlated with the presence of CD133+ glioma CSCs. TAM could significantly enhance the invasive capability of glioma stem cells through paracrine production of TGF-␤1 [65]. In another study of Jinushi et al. [66], the role of TAMs in the regulation of CSC-activity in relation to drug resistance was identified. They found a large amount of TAM-derived milk-fat globule EGF-8 (MFGE8) increased tumorigenicity and anticancer drug resistance in CD44+ ALDH+ colon tumor cells and CD133+ ALDH+ lung cancer cells. Furthermore, MFG-E8 was found mainly to activate signal transducer and activator of transcription-3 (STAT3) and Sonic Hedgehog pathways in CSCs and to further amplify their anticancer drug resistance in cooperation with IL-6. More recently, a similar finding was addressed by Mitchem et al. [8] in pancreatic ductal adenocarcinoma. They reported that TAMs directly induce TICs properties in pancreatic cancer cells by activating STAT3. In turn, TICs induce immunosuppression in TAMs, and block antitumor CD8+ T-lymphocyte responses during chemotherapeutic treatment. Targeting TAMs by inhibiting either the myeloid cell receptors colony-stimulating factor-1 receptor or chemokine (C-C motif) receptor 2 decreases the numbers of pancreatic TICs, improves chemotherapeutic efficacy, inhibits metastasis and increases antitumor T cell responses. Together, all these findings validated the interplay between CSCs and the tumor immune microenvironment. However, very little is known about the immune suppressive role of CSCs of HNSCC and other solid tumors so far.

4. CSC-based vaccination Previous investigations conducted on the immunity of CSC with ALDH1A1 phenotype presented an attractive potential immunotherapeutic approach by targeting these cells. Studies on vaccination against antigen ALDH1A1+ of CSCs have been performed and achieved significant progress. Visus et al. [67] have demonstrated the ability of in vivo generated ALDH1A1-specific CTLs to eliminate ALDHbright cells present in HLA-A2+ HNSCC, breast, and pancreas carcinoma cell lines, xenografts, and surgically removed lesions in vitro. They also found antitumor activity by adoptive immunotherapy with ALDH1A1-specific CTLs in vivo. ALDH1A1-specific CD8+ T cells could eliminate ALDHbright cells, inhibit tumor growth and metastases, or prolong survival of xenograft-bearing immunodeficient mice. Of note, normal hepatocytes expressing ALDH1A1 were shown to express little to no MHC class I Ag that made them unlikely to be recognized by MHC class I-restricted,




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Table 2 Studies of CSC-based vaccination. Approach CD8+

Recognition of ALDH1A1-CSCs by ALDH188–96 peptide-specific T cells Elimination of ALDHbright cancer cells by ALDH1A1-specific CD8+ T cells SOX6-peptide-specific-CTLs could lyse glioma stem cells CD8 defined prostate stem cell antigen specific T cell epitope could activate CD8+ effector T cells Dendritic cells (DC) loaded with neurospheres enriched with glioma CSCs could eliminate glioma tumors Vaccination by DC loaded with CSCs could induce higher IFN-␥ production and prolong survival Vaccination by DC loaded with CSCs could eliminate CSCs in vitro

Murine prostate stem cell antigen encoding cDNA vaccination can induce long-term protection against prostate cancer development Vaccine containing lysates of CSCs-enriched tumor cells could reduce tumor volume and occurrence Vaccination with defined human embryonic stem cells (hESCs) could effectively immunize against mouse and rat ovarian cancer

ALDH1A1-specific CD8+ T cells. Later, different to these previous studies, an in vivo study on the host immune system was used to selectively target CSCs. Ning et al. [68] investigated immunogenicity induced by murine ALDHhigh CSC used as source of antigen to prime DCs as a vaccine for malignant melanoma and squamous cell carcinoma in immunocompetent mice used as hosts. ALDHhigh CSCs were immunogenic and more effective as an antigen source than unselected tumor cells in inducing protective antitumor immunity. A high level of IgG produced by splenocytes subjected to CSC-tumor-lysate-pulsed DCs and the binding of the antibody from CSC-vaccinated murine hosts to the CSCs which resulted in the CSCs lysis via complementdependent cytotoxicity have been observed. CTLs generated from peripheral blood mononuclear cells or splenocytes harvested from CSC-vaccinated hosts were capable of killing CSCs in vitro. Consistent with the findings of the above groups, Duarte et al. [69] first demonstrated an ALDHhigh CSC-based vaccine could reduce both tumor volume and occurrence in a rat colon carcinoma syngeneic model. In this study, 50% of the CSC-based vaccinated animals became resistant to tumor development and CSC-based vaccination induced a 99.5% reduction in tumor volume compared to the control group. These studies provided a greater view of the immune biology of CSCs. Vaccination with CSCs (Table 2) has shown to be effective in killing CSCs specifically, reducing tumor volume and preventing tumor recurrence.

Model

Source

Ref.

HNSCC HNSCC, breast and pancreatic carcinoma Glioma Prostate cancer Murine glioma

Human Human

[42] [67]

Human Human Murine

[70] [71] [72]

Glioblastoma Murine melanoma and squamous cell carcinoma Murine prostate cancer Rat colon carcinoma Ovarian cancer

Rat Murine

[50] [68]

Murine

[73]

Rat Human

[69] [74]

other hand, CSCs can promote tumor progression either by immunoediting for CSCs that are more suitable to survive in an immunocompetent host or by establishing conditions that facilitate tumor outgrowth within the tumor immune-microenvironment. Therefore, specific targeting of CSCs by immunotherapeutic approaches may lead to more efficacious and lasting therapeutic results in the future. Up till now, the discovery of specific tumor-associated antigens expressed by CSCs as well as the progress in developing CSC-based vaccination, provides the potential applicability of targeting CSCs and may enable more rapid development of combinational therapies that act effectively on CSCs. Nonetheless, it seems necessary to address several points before immunotherapeutic approaches targeting CSCs can be brought into clinical trials. These include the effective isolation of CSCs from bulk tumor mass to measure potential immunotherapeutic effects on CSC, to determine the antigen-profile presented on CSCs specifically to identify specific CSC targets as well as the induction and enhancement of antigen processing and presentation of CSC epitopes. In addition, it is also necessary to combine other aspects of tumor-mediated interference with the host immune system, in particular in HPV+ and HPV− HNSCC (reviewed by Albers et al. [12]) with the effect of CSCs, and to develop novel therapy and improve therapeutic outcome.

Conﬂict of interest The authors have no conflict of interest to declare.

5. Conclusion Understanding how the immune system affects CSCs in cancer development and progression has been one of the most challenging questions in immunology. Recent research indicates a dual role of CSCs with the immune system. On one hand, CSCs can be recognized and destroyed or inhibited in their outgrowth by the immune system. On the

Reviewers Kazuaki Chikamatsu, M.D., Ph.D., Gunma University Graduate School of Medicine, Otolaryngology-Head and Neck Surgery, 3-39-22, Showa-machi, Maebashi 371-8511, Japan.



8


Grzegorz Dworacki, M.D., Ph.D., Dept. of Clinical Immunology, Rokietnicka 5D, Poznan, 60-806, Poland. [19]

Acknowledgments This study was supported by the Key Science and Technology Innovation Team of Zhejiang (2010R50048), Zhejiang Provincial Program for the Cultivation of High-level Innovative Health Talents and Key Laboratory of Laboratory Medicine, Ministry of Education, China.

[20]

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[22] [23]

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Biographies Andreas M. Kaufmann PhD is a biologist in gynecologic research. His interest is in HPV, cancer biology, and immunotherapy. Andreas E. Albers MD, PhD is a specialist in otorhinolaryngology, head and neck surgery. His basic scientific interests are HPV, the tumor- and immunobiology of head and neck cancer and its cancer stem cells.


Immunology and Immunotherapy of Head and Neck Cancer.

Cancer stem cells in head and neck cancer.

Head and neck cancer.

Head and neck cancer.

Responses of killer cells in head and neck cancer patients.

On the immunology of head and neck cancer--a prognostic index. Preliminary communication.

Cancer stem cells in head and neck squamous cell carcinoma.

Head and neck cancer surgery.

Advanced head and neck cancer.

Scavenger Receptors: Emerging Roles in Cancer Biology and Immunology.

Human papillomavirus in head and neck cancer.

Thermochemotherapy in inoperable head and neck cancer.

Adjuvant chemotherapy in head and neck cancer.

Histone modifications: Targeting head and neck cancer stem cells.

Targeting angiogenesis in head and neck cancer.

Human papillomavirus in head and neck cancer.

Immunity in head and neck cancer.

PPARγ in head and neck cancer prevention.

Decision analysis in head and neck cancer.

MicroRNAs in Head and Neck Cancer.

Adjuvant chemotherapy in head and neck cancer.

Surgical treatment in head and neck cancer.

Chemotherapy in head and neck cancer.

Ethical dilemmas in head and neck cancer.