Andreas E. Albers

Evidence for Epithelial-Mesenchymal Transition in Cancer Stem Cells of Head and Neck Squamous Cell Carcinoma

Initiation, growth, recurrence, and metastasis of head and neck squamous cell carcinomas (HNSCC) have been related to the behavior of cancer stem cells (CSC) that can be identified by their aldehyde-dehydrogenase-isoform-1 (ALDH1) activity. We quantified and enriched ALDH1+ cells within HNSCC cell lines and subsequently characterized their phenotypical and functional properties like invasion capacity and epithelial-mesenchymal transition (EMT). Spheroid culture enriched CSC from five HNSCC cell lines by up to 5-fold. In spheroid-derived cells (SDC) and the parental monolayer-derived cell line ALDH1, CD44, CD24, E-Cadherin, α-SMA, and Vimentin expression was compared by flow-cytometry and immunofluorescence together with proliferation and cell cycle analysis. Invasion activity was evaluated by Matrigel assay and expression of stemness-related transcription factors (TF) Nanog, Oct3/4, Sox2 and EMT-related genes Snail1 and 2, and Twist by real-time PCR. All cell lines formed spheroids that could self-renew and be serially re-passaged. ALDH1 expression was significantly higher in SDC. ALDH1+ cells showed increased colony-formation. The proportion of cells with a putative CSC marker constellation of CD44+/CD24− was highly variable (0.5% to 96%) in monolayer and spheroid cultures and overlapped in 0%–33% with the CD44+/CD24−/ALDH1+ cell subset. SDC had significantly higher invading activity. mRNA of the stemness-related genes Sox2, Nanog, and Oct3/4 was significantly increased in SDC of all cell lines. Twist was significantly increased in two while Snail2 showed a significant increase in one and a significant decrease in SDC of two cell lines. SDC had a higher G0 phase proportion, showed high-level expression of α-SMA and Vimentin, but significantly decreased E-Cadherin expression. HNSCC-lines harbor potential CSC, characterized by ALDH1 and stemness marker TF expression as well as properties like invasiveness, quiescence, and EMT. CSC can be enriched by anchorage-independent culture techniques, which may be important for the investigation of their contribution to therapy resistance, tumor recurrence and metastasis.

Tumor-derived microvesicles in sera of patients with head and neck cancer and their role in tumor progression.

Tumor‐derived membranous vesicles (MV) isolated from sera of the patients with squamous cell carcinomas of the head and neck (HNSCC) induce apoptosis of activated CD8+ T cells. We tested if MV molecular profile and activity correlate with disease progression.

Epithelial-to-mesenchymal transition and cancer stem(-like) cells in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the 6th commonest cancer worldwide. Relapse, thought to involve cancer stem(-like) cells (CSCs), and the development of metastases are common and survival rates remain low. Epithelial-to-mesenchymal transition (EMT) is a key event in metastasis and increasing evidence suggests a link between EMT and CSCs. MicroRNAs regulate multiple cellular processes including EMT and have been implicated in a CSC phenotype. This review aims to highlight key events that are involved in EMT, discusses their relevance in HNSCC progression and metastasis and explores the possibility of targeting EMT as a novel therapy in HNSCC.

Stem cells in squamous head and neck cancer

The initiation and metastasis of head and neck squamous cell carcinomas (HNSCC) and other cancers have recently been related to the presence of cancer stem cells (CSC). CSC are cancer initiating, sustaining and are mostly quiescent. Specific markers that vary considerably depending on tumor type or tissue of origin characterize putative CSC. Compared to the bulk tumor mass, CSC are less sensitive to chemo- and radiotherapy and may also have low immunogenicity. Therapeutic targeting of CSC may improve clinical outcome of HNSCC which has two distinct etiologies: infection of epithelial stem cells by high-risk types of the human papillomavirus, or long-term tobacco and alcohol abuse. Recent knowledge on the role of CSC in HNSCC is reviewed and where necessary parallels to CSC of other origin are drawn to give a more comprehensive picture.

Susceptibility to cytotoxic T cell lysis of cancer stem cells derived from cervical and head and neck tumor cell lines.

PurposeTo explore cancer stem cell susceptibility to a host’s cytotoxic T lymphocyte (CTL)-mediated immune response.MethodsWe compared the susceptibility of putative CSC generated from cancer cell lines to immunologic recognition and killing by alloantigen-specific CD8+ CTL. CSC-enriched spheroid culture-derived cells (SDC) exhibited higher expression of ALDH, ICAM1 and of stem/progenitor cell markers on all 3 tumor cell lines investigated and lower MHC class I on the cervical cancer cell line as compared to their monolayer-derived cells (MDC).ResultsThe expression of ICAM1 and MHCI was upregulated by IFN-γ treatment. CSC populations were less sensitive to MHC class I-restricted alloantigen-specific CD8+ CTL lysis as compared to matched MDC. IFN-γ pretreatment resulted in over-proportionally enhanced lysis of SDC. Finally, the subset of ALDHhigh expressing SDC presented more sensitivity toward CD8+ CTL killing than the ALDHlow SDC.ConclusionsTumor therapy resistance has been attributed to cancer stem cells (CSC). We show in vitro susceptibility of CSC to CTL-mediated lysis. Immunotherapy targeting of ALDH+ CSC may therefore be a promising approach. Our results and method may be helpful for the development and optimization of adjuvants, as here exemplified for INF-γ, for CSC-targeted vaccines, independent of the availability of CSC-specific antigens.

A comprehensively characterized large panel of head and neck cancer patient-derived xenografts identifies the mTOR inhibitor everolimus as potential new treatment option

Patient‐derived xenograft (PDX) models have shown to reflect original patient tumors better than any other preclinical model. We embarked in a study establishing a large panel of head and neck squamous cell carcinomas PDX for biomarker analysis and evaluation of established and novel compounds. Out of 115 transplanted specimens 52 models were established of which 29 were characterized for response to docetaxel, cetuximab, methotrexate, carboplatin, 5‐fluorouracil and everolimus. Further, tumors were subjected to sequencing analysis and gene expression profiling of selected mTOR pathway members. Most frequent response was observed for docetaxel and cetuximab. Responses to carboplatin, 5‐fluorouracil and methotrexate were moderate. Everolimus revealed activity in the majority of PDX. Mutational profiling and gene expression analysis did not reveal a predictive biomarker for everolimus even though by trend RPS6KB1 mRNA expression was associated with response. In conclusion we demonstrate a comprehensively characterized panel of head and neck cancer PDX models, which represent a valuable and renewable tissue resource for evaluation of novel compounds and associated biomarkers.

ALDH1-positive cancer stem-like cells are enriched in nodal metastases of oropharyngeal squamous cell carcinoma independent of HPV status

Oropharyngeal squamous cell carcinoma (OSCC) is caused by high-risk (HR) human papillomavirus (HPV) or alcohol and tobacco abuse. Aldehyde dehydrogenase 1 (ALDH1) is a confirmed marker for cancer stem-like cells (CSCs) of OSCC responsible for therapy resistance, recurrence and metastasis. Associations between HR-HPV/p16, CSC frequency and clinicopathological parameters in patients with metastatic OSCC were investigated. In the present study, HPV genotypes and expression of ALDH1 and p16 was analyzed in 40 paired OSCC and metastases. A significant correlation between ALDH1 positivity with lower primary tumor differentiation grade (P=0.009) and higher nodal status (P=0.015) was noted. Compared to primary tumors, the proportion of ALDH1-expressing cells was significantly increased in metastases (P=0.012), while significantly fewer ALDH1-expressing cells were found in HR-HPV-DNA⁺/p16⁺ primary tumors (P=0.038) compared to HR-HPV-DNA⁻/p16⁻ primary tumors. Metastases showed no difference. ALDH1⁺ CSCs are detectable in OSCC and metastases. ALDH1 high-grade OSCC exhibits a more aggressive phenotype characterized by higher nodal classification and lower differentiation. This suggests a subpopulation contained in the ALDH1-positive OSCC cell pool able to complete the metastatic cascade and subsequently enriching in metastasis independent of tumor etiology and ALDH1 content.

T cell-tumor interaction directs the development of immunotherapies in head and neck cancer.

The competent immune system controls disease effectively due to induction, function, and regulation of effector lymphocytes. Immunosurveillance is exerted mostly by cytotoxic T-lymphocytes (CTLs) while specific immune suppression is associated with tumor malignancy and progression. In squamous cell carcinoma of the head and neck, the presence, activity, but also suppression of tumor-specific CTL have been demonstrated. Functional CTL may exert a selection pressure on the tumor cells that consecutively escape by a combination of molecular and cellular evasion mechanisms. Certain of these mechanisms target antitumor effector cells directly or indirectly by affecting cells that regulate CTL function. This results in the dysfunction or apoptosis of lymphocytes and dysregulated lymphocyte homeostasis. Another important tumor-escape mechanism is to avoid recognition by dysregulation of antigen processing and presentation. Thus, both induction of functional CTL and susceptibility of the tumor and its microenvironment to become T cell targets should be considered in CTL-based immunotherapy.

Role of caveolin-1 in the pathogenesis of tissue fibrosis by keloid-derived fibroblasts in vitro.

Background  Recent studies have suggested that caveolin‐1 (cav‐1) plays an important role in the regulation of transforming growth factor (TGF)‐β1 signalling and participates in the pathogenesis of tissue fibrosis. However, its effects on dermal fibrosis keloids are unknown.

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

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.