Extracellular vesicles from Aggregatibacter actinomycetemcomitans exhibit potential antitumorigenic effects in oral cancer: a comparative in vitro study.

Aggregatibacter actinomycetemcomitans is an opportunistic Gram-negative periodontopathogen strongly associated with periodontitis and infective endocarditis. Recent evidence suggests that periodontopathogens can influence the initiation and progression of oral squamous cell carcinoma (OSCC). Herein we aimed to investigate the effect of A. actinomycetemcomitans-derived extracellular vesicles (EVs) on OSCC cell behavior compared with EVs from periodontopathogens known to associate with carcinogenesis. EVs were isolated from: A. actinomycetemcomitans and its mutant strains lacking the cytolethal distending toxin (CDT) or lipopolysaccharide (LPS) O-antigen; Porphyromonas gingivalis; Fusobacterium nucleatum; and Parvimonas micra. The effect of EVs on primary and metastatic OSCC cells was assessed using cell proliferation, apoptosis, migration, invasion, and tubulogenesis assays. A. actinomycetemcomitans-derived EVs reduced the metastatic cancer cell proliferation, invasion, tubulogenesis, and increased apoptosis, mostly in CDT- and LPS O-antigen-dependent manner. EVs from F. nucleatum impaired the metastatic cancer cell proliferation and induced the apoptosis rates in all OSCC cell lines. EVs enhanced cancer cell migration regardless of bacterial species. In sum, this is the first study demonstrating the influence of A. actinomycetemcomitans-derived EVs on oral cancer in comparison with other periodontopathogens. Our findings revealed a potential antitumorigenic effect of these EVs on metastatic OSCC cells, which warrants further in vivo investigations.

Identity matters: cancer stem cells and tumour plasticity in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) represents frequent yet aggressive tumours that encompass complex ecosystems of stromal and neoplastic components including a dynamic population of cancer stem cells (CSCs). Recently, research in the field of CSCs has gained increased momentum owing in part to their role in tumourigenicity, metastasis, therapy resistance and relapse. We provide herein a comprehensive assessment of the latest progress in comprehending CSC plasticity, including newly discovered influencing factors and their possible application in HNSCC. We further discuss the dynamic interplay of CSCs within tumour microenvironment considering our evolving appreciation of the contribution of oral microbiota and the pressing need for relevant models depicting their features. In sum, CSCs and tumour plasticity represent an exciting and expanding battleground with great implications for cancer therapy that are only beginning to be appreciated in head and neck oncology.

The prognostic influence of lymphatic endothelium-specific hyaluronan receptor 1 in cancer: A systematic review.

Lymphangiogenesis is a key process in cancer development and metastasis. Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) is a widely used marker for lymphatic endothelial cells (LEC), which also mediates immune and cancer cell migration. Recently, LYVE-1-positive tumor cells were shown to acquire LEC-like phenotype and exploit this receptor for lymphatic dissemination. Furthermore, selective targeting of LYVE-1 impaired the growth of cancer-related vasculature and reduced metastasis in vivo, signifying its role in therapeutic and prognostic applications. Although numerous studies have investigated the role of LYVE-1 in cancer, a unifying detailed review of its prognostic utility is lacking to date. Thus, we compiled and critically appraised evidence from clinical studies comprising a total of 2352 patients diagnosed with different types of cancer and using a variety of experimental approaches. Collectively, most studies revealed a significant association between LYVE-1 overexpression and dismal outcome of at least one survival estimate. Furthermore, the importance of vasculature location, intra- or peritumoral, and the influence of various lymphangiogenesis-related parameters, such as lymphatic vessel density and invasion, were discussed. However, the specificity of LYVE-1 staining is challenged by its expression in non-LEC cells, implying the need for double labelling to better estimate its prognostic significance. In conclusion, this is to our knowledge the first comprehensive systematic review on the prognostic value of LYVE-1 in cancer. More well-designed studies across different populations and the development of standardized protocols would be paramount for the consistency of LYVE-1 findings and for its potential transferability to clinical practice in future.

The effect of interleukin-17F on vasculogenic mimicry in oral tongue squamous cell carcinoma.

Oral tongue squamous cell carcinoma (OTSCC) is one of the most common cancers worldwide and is characterized by early metastasis and poor prognosis. Recently, we reported that extracellular interleukin-17F (IL-17F) correlates with better disease-specific survival in OTSCC patients and has promising anticancer effects in vitro. Vasculogenic mimicry (VM) is the formation of an alternative vasculogenic system by aggressive tumor cells, which is implicated in treatment failure and poor survival of cancer patients. We sought to confirm the formation of VM in OTSCC and to investigate the effect of IL-17F on VM formation. Here, we showed that highly invasive OTSCC cells (HSC-3 and SAS) form tube-like VM on Matrigel similar to those formed by human umbilical vein endothelial cells. Interestingly, the less invasive cells (SCC-25) did not form any VM structures. Droplet-digital PCR, FACS, and immunofluorescence staining revealed the presence of CD31 mRNA and protein in OTSCC cells. Additionally, in a mouse orthotopic model, HSC-3 cells expressed VE-cadherin (CD144) but lacked Von Willebrand Factor. We identified different patterns of VM structures in patient samples and in an orthotopic OTSCC mouse model. Similar to the effect produced by the antiangiogenic drug sorafenib, IL-17F inhibited the formation of VM structures in vitro by HSC-3 and reduced almost all VM-related parameters. In conclusion, our findings indicate the presence of VM in OTSCC and the antitumorigenic effect of IL-17F through its effect on the VM. Therefore, targeting IL-17F or its regulatory pathways may lead to promising therapeutic strategies in patients with OTSCC.

The effect of fascin 1 inhibition on head and neck squamous cell carcinoma cells.

Fascin 1 plays important pro-metastatic roles in head and neck carcinoma (HNSCC) migration, invasion, and metastasis. However, limited advancement in targeting metastasis remains a major obstacle in improving HNSCC patients' survival. Therefore, we assessed the therapeutic potential of fascin 1 targeted inhibition and its potential prognostic value in HNSCC patients. Using in vitro and in vivo approaches, we investigated the effect of compound G2, a novel fascin 1 inhibitor, on HNSCC cells migration, invasion, and metastasis. High-throughput screening (HTS) was used to assess cytotoxic activity of compound G2 alone or combined with irradiation. We also evaluated the prognostic potential of fascin 1 in HNSCC patients. Interestingly, compound G2 reduced carcinoma cells migration and invasion in vitro and inhibited metastasis in vivo. Moreover, HTS revealed a modest cytotoxic activity of the compound G2 on HNSCC cell lines. Irradiation did not synergistically enhance the compound G2-mediated cytotoxic activity. Survival analyses showed that high fascin 1 immunoexpression, at the tumor invasive front, was associated with cancer-specific mortality in the advanced stages of HNSCC. Collectively, our findings suggest that fascin 1 represents a promising anti-metastatic therapeutic target and a useful prognostic marker in patients with HNSCC. Novel anti-metastatic agents could provide a valuable addition to cancer therapy.

Nothing to sneeze at: Histamine and histamine receptors in oral carcinogenesis.

Oral squamous cell carcinoma (OSCC), the most common oral malignancy, shows an increasing rate of incidence worldwide. In spite of the recent advances in cancer research, OSCC therapy continues to have unfavourable outcomes, and thus, patient's prognosis remains relatively poor. Current research has been devoted to identifying novel therapeutic targets also in the tumour microenvironment (TME). Histamine and its G-protein-coupled receptors (H1R-H4R) play vital roles in multiple cancer-associated processes in TME, where histamine is mainly produced by mast cells. However, oral epithelial cells were recently shown to produce low concentrations of histamine in autocrine and paracrine modes. These findings, together with the discovery of the high-affinity histamine H4 receptor, have led to a massive increase in our understanding of histamine functions. In this review, we aim to summarize the most recent findings regarding histamine and its receptors and their involvement in oral carcinogenesis-from oral potentially malignant disorders (OPMDs) to invasive OSCC. Importantly, histamine receptors are differentially expressed in OPMDs and OSCC. Furthermore, H1R and H4R are associated with clinicopathological characteristics of OSCC patients, suggesting a role in prognosis. Due to the enormous success of histamine-based medications, histamine receptors may also represent promising and viable drug targets in oral cancer.

No detection of TSH or TSHR in oral lichen planus lesions in patients with or without hypothyroidism.

Objective: An association between hypothyroidism (HT) and oral lichen planus (OLP) has been reported. However, the mechanisms that could explain this association remain unresolved. This study aimed to evaluate the expression of thyroid-stimulating hormone (TSH) and thyroid-stimulating hormone receptor (TSHR) in healthy oral mucosa and in OLP lesions of individuals with and without HT.Material and methods: Immunohistochemical expression of TSH and TSHR was studied in oral mucosal biopsies obtained from 14 OLP patients with HT, 14 OLP patients without HT and 10 healthy controls without oral mucosal lesions. Gene expression of TSHR was investigated by using three different PCR techniques in oral mucosal samples from 7 OLP patients with HT, 3 OLP patients without HT, 9 healthy controls and in cultured human oral epithelial cells. Gene expression of TSH was examined by employing 2 PCR techniques in oral mucosal samples from 2 OLP patients with HT, 2 OLP patients without HT and 4 healthy controls.Results: TSH and TSHR stainings were negative in the studied oral mucosal specimens. Gene quantification assays demonstrated negative gene expression of TSH and TSHR in clinical and in vitro samples.Conclusions: These results suggest that TSH and TSHR may not be commonly involved in the pathogenetic mechanism that could explain the association between OLP and hypothyroidism.

Role of Cyclooxygenase-2 in Head and Neck Tumorigenesis.

The cyclooxygenase-2 (COX-2) is a potent enzyme that converts arachidonic acid to prostaglandins (PG), including PGE2, a key mediator of inflammation and angiogenesis. Importantly, COX-2 is activated in response to inflammatory stimuli, where it is also believed to promote the development and progression of head and neck cancers (HNC). COX-2 can mediate its protumorigenic effect through various mechanisms, such as inducing cell proliferation, inhibition of apoptosis, and suppressing the host's immune response. Furthermore, COX-2 can induce the production of vascular endothelial growth factors, hence, promoting angiogenesis. Indeed, the ability of COX-2 inhibitors to selectively restrict the proliferation of tumor cells and mediating apoptosis provides promising therapeutic targets for cancer patients. Thus, in this comprehensive review, we summarized the reported differential expression patterns of COX-2 in different stages of head and neck carcinogenesis-from potentially premalignant lesions to invasive carcinomas. Furthermore, we examined the available meta-analysis evidence for COX-2 role in the carcinogenesis of HNC. Finally, further understanding of the biological processes of COX-2 and its role in orchestrating cell proliferation, apoptosis, and angiogenesis may give therapeutically beneficial insight to develop the management plan of HNC patients and improve their clinical outcomes.

The Prognostic Value of Toll-Like Receptors in Head and Neck Squamous Cell Carcinoma: A Systematic Review and Meta-Analysis.

Head and neck squamous cell carcinoma (HNSCC) is a group of tumours which exhibit low 5 year survival rates. Thus, there is an urgent need to identify biomarkers that may improve the clinical utility of patients with HNSCC. Emerging studies support a role of toll-like receptors (TLRs) in carcinogenesis. Therefore, this systematic review and meta-analysis was performed to assess the prognostic value of TLR immunoexpression in HNSCC patients. We compiled the results of thirteen studies comprising 1825 patients, of which six studies were deemed qualified for quantitative synthesis. The higher immunoexpression of TLR-1 to 5 and 9 was associated with a worsening of the clinical parameters of patients with HNSCC. Furthermore, induced levels of TLR-3, 4, 5, 7 and 9 were found to predict the patients' survival time. The meta-analysis revealed that TLR-7 overexpression is associated with a decreased mortality risk in HNSCC patients (HR 0.51; 95%CI 0.13-0.89; I2 34.6%), while a higher expression of TLR-5 predicted shorter, but non-significant, survival outcome. In conclusion, this review suggests that TLRs may represent some prognostic value for patients with HNSCC. However, due to small sample sizes and other inherent methodological limitations, more well designed studies across different populations are still needed before TLRs can be recommended as a reliable clinical risk-stratification tool.

Prognostic value of blood and lymphatic vessel markers in tongue cancer: A systematic review.

Tongue squamous cell carcinoma (TSCC) has a poor prognosis due to its early metastasis through blood and lymphatic vessels. We undertook a systematic review to investigate the prognostic significance of blood microvessel density (MVD) and lymphatic vessel density (LVD) in TSCC patients. We carried out a systematic search in Ovid Medline, Scopus, and Cochrane libraries. All studies that evaluated the prognostic significance of MVD/LVD markers in TSCC were systematically retrieved. Our results showed that MVD/LVD markers, CD31, CD34, CD105, factor VIII, lymphatic vessel endothelial hyaluronan receptor-1, and D2-40 were evaluated in TSCC patients until 28 June 2018. Six out of 13 studies reported markers that were associated with poor prognosis in TSCC. Two out of three studies suggested that a high number of D2-40+ vessels predicated low overall survival (OS); the third study reported that the ratio of D2-40+ over factor VIII+ vessels is associated with low OS. Most of the other markers had controversial results for prognostication. We found higher expression of MVD/LVD markers were commonly, but not always, associated with shorter survival in TSCC patients. It is therefore not currently possible to recommend implementation of these markers as reliable prognosticators in clinical practice. More studies (especially for D2-40) with larger patient cohorts are needed.

PCR-based zebrafish model for personalised medicine in head and neck cancer.

BACKGROUND: Currently, in vivo model for personalised cancer drug testing is challenging. A zebrafish larvae xenograft model has been applied in recent years to cancer research, particularly for drug testing purposes, showing promising results in drug testing against patient-derived tumour xenografts. Currently, these xenograft models apply imaging techniques to measure drug efficacy. However, this method carries several limitations, including timely imaging, thereby reducing the available number of tested fish and drugs. Here, we propose a PCR-based fast assay to evaluate drug efficacy in a zebrafish larvae xenograft model. METHODS: We tested two primary and corresponding metastatic head and neck squamous cell carcinoma (HNSCC) cell lines and patient-derived tongue cancer sample applying zebrafish larvae xenograft model. Cisplatin efficacy was tested using imaging technique and compared the results with PCR-based methods. Drug screening of eight compounds was applied on both cell lines and patient sample using PCR. RESULTS: In a head-to-head comparison, all the three techniques (imaging, quantitative PCR, and droplet digital PCR) showed similar reduction of the cancer cells growth after cisplatin treatment. Using the quantitative PCR assay, we demonstrated a dose-dependent response of HNSCC cells to cisplatin. Drug screening results of four HNSCC cell lines and patient sample revealed different drug efficacy between tested cancer cells. CONCLUSION: We introduce a novel, easy, fast and cost-effective PCR-based in vivo zebrafish larvae assay to test the response of cell lines and clinical tumour samples to anti-cancer drugs. This method goes hand-by-hand with the commonly used imaging assay.

Human ß-Defensin 2 Expression in Oral Epithelium: Potential Therapeutic Targets in Oral Lichen Planus.

Human ß-defensin 2 (hBD-2) is a potent antimicrobial peptide that participates in defense against invading bacteria. We recently showed that bacterial components and histamine, through histamine H4 receptor (H4R), are involved in the pathogenesis of the potentially malignant lesion, oral lichen planus (OLP). However, the underlying mechanisms remain unknown. We, therefore, investigated the role of hBD2-histamine crosstalk signaling in promoting OLP pathology. Biopsies from OLP and oral tongue squamous cell carcinoma (OTSCC) patients, and healthy controls were used. Two OTSCC cell lines and normal human oral keratinocytes (HOKs) were used. HBD-2 and other targets were mapped by immunostaining and analyzed by ImageJ2 software. The highly sensitive droplet-digital PCR technology and qRT-PCR were utilized to study the clinically derived and in vitro samples, respectively. H4R was challenged with the specific agonist HST-10 and inverse agonist ST-1007. HBD-2 was highly induced in OLP lesions. In contrast, hBD2 expression was attenuated in OTSCC tissues, while very low levels of hBD-2 messenger RNA (mRNA) were observed in OTSCC cells. Together with tumor necrosis factor-α (TNF-α), histamine upregulated hBD-2 mRNA expression in HOKs. Activation of H4R seems to modulate the expression of epithelial hBD-2. These findings suggest the involvement of hBD-2 in the pathogenesis of OLP and may, thus, be harnessed for therapeutic interventions in OLP.

Role of the high mobility group box 1 signalling axes via the receptor for advanced glycation end-products and toll-like receptor-4 in the immunopathology of oral lichen planus: a potential drug target?

High mobility group box 1 (HMGB1) is an extremely conserved DNA-binding protein that stabilizes nucleosomes and facilitates gene transcription in mammalian cells. When released extracellularly, HMGB1 becomes an alarmin that can mediate systemic diseases. High mobility group box 1 signals via two main receptors: receptor for advanced glycation end-products (RAGE) and toll-like receptor-4 (TLR4). We hypothesized that HMGB1 expression is increased in patients with oral lichen planus (OLP) relative to healthy controls. Therefore, HMGB1 and its receptors were mapped in tissue biopsies from 25 patients with OLP and from 20 healthy controls by immunostaining and ImageJ analysis. High mobility group box 1 was induced in oral keratinocytes in all patients with OLP. The band-like cell infiltrate in patients with OLP revealed very strong staining for RAGE. Likewise, TLR4 was overexpressed throughout OLP mucosa which co-localized with HMGB1. In conclusion, we suggest that OLP could partly be an HMGB1-mediated condition by creating a proinflammatory loop cycle via RAGE- and TLR4-signalling axes, which may contribute to the chronicity of this disease.

Altered Expression of Toll-like Receptors in Human Oral Epithelium in Oral Lichenoid Reactions.

Oral lichenoid reactions (OLRs) are chronic inflammatory lesions induced by contact with allergens. Toll-like receptors (TLRs) are members of pattern-recognition receptor superfamily. Once activated, TLRs induce production of cytokines and chemokines, thus leading to inflammatory reaction in host tissue. In the present study, we aimed to investigate the potential role of TLRs in the initiation and perpetuation of OLRs, in which TLRs induce innate immune responses mounted against allergens. TLRs, 1 through 10, were mapped in tissue samples obtained from healthy donors and OLR patients using real-time quantitative reverse transcription polymerase chain reaction, immunostaining, and image analyses. We found that the immunoreactivity for all TLRs was increased in OLRs, except for TLR5, which was noticeably reduced. Gene analysis revealed that TLR1, TLR2, TLR4, TLR7, TLR8, and TLR9 transcripts were upregulated in OLRs compared with controls. In contrast, expression of TLR3, TLR5, and TLR6 genes were negatively regulated in OLRs. TLR10 remained unchanged in both groups. In conclusion, TLRs expression is deranged in OLRs in which TLRs could be sensitized by allergens and haptens derived from dental restorations. TLR reactivity is further enhanced by recruitment of T lymphocytes forming a diffuse lymphocytic infiltrate and thus creating a proinflammatory loop cycle. These findings suggest that TLRs are involved in OLRs and pave the way for alternative cost-effective therapeutic intervention.

Toll-like receptors and their soluble forms differ in the knee and thumb basal osteoarthritic joints.

Background and purpose - Although the pathogenesis of osteoarthritis (OA) is not well understood, chondrocyte-mediated inflammatory responses (triggered by the activation of innate immune receptors by damage-associated molecules) are thought to be involved. We examined the relationship between Toll-like receptors (TLRs) and OA in cartilage from 2 joints differing in size and mechanical loading: the first carpometacarpal (CMC-I) and the knee. Patients and methods - Samples of human cartilage obtained from OA CMC-I and knee joints were immunostained for TLRs (1-9) and analyzed using histomorphometry and principal component analysis (PCA). mRNA expression levels were analyzed with RT-PCR. Collected synovial fluid (SF) samples were screened for the presence of soluble forms of TLR2 and TLR4 by enzyme-linked immunosorbent assay (ELISA). Results - In contrast to knee OA, TLR expression in CMC-I OA did not show grade-dependent overall profile changes, but PCA revealed that TLR expression profiles clustered according to their cellular compartment organization. Protein levels of TLR4 were substantially higher in knee OA than in CMC-I OA, while the opposite was the case at the mRNA level. ELISA assays confirmed the presence of soluble forms of TLR2 and TLR4 in SF, with sTLR4 being considerably higher in CMC-I OA than in knee OA. Interpretation - We observed that TLRs are differentially expressed in OA cartilage, depending on the joint. Soluble forms of TLR2 and TLR4 were detected for the first time in SF of osteoarthritic joints, with soluble TLR4 being differentially expressed. Together, our results suggest that negative regulatory mechanisms of innate immunity may be involved in the pathomolecular mechanisms of osteoarthritis.

Distinctive expression pattern of interleukin-17 cytokine family members in colorectal cancer.

Colorectal cancer (CRC) is one of the most common cancers in both genders. Even though interleukin (IL)-17A was shown to play an important role in intestinal tumourigenesis and CRC, other IL-17 family members were not studied well. We therefore studied the expression of IL-17 cytokine family members in CRC. Ten healthy colons and ten CRC mucosa were immunostained for IL-17B, IL-17C, IL-17E, and IL-17F, and their receptors IL-17RA, IL-17RB, and IL-17RC. Double immunofluorescence staining of the CRC mucosa was done for IL-17B with markers of neutrophils, endothelial cells, macrophages, T cells, mast cells, or fibroblasts. While IL-17B was increased in CRC with a strong presence both in the epithelial and stromal compartments, IL-17C showed different expression depending on the grade of differentiation and IL-17E remained unchanged. In contrast, IL-17F was decreased in CRC compared to healthy control. Colon epithelial cells stained positive for IL-17RA, IL-17RB, and IL-17RC in both healthy control and CRC. Neutrophils were the main source of IL-17B in the stroma. IL-17 family members demonstrated distinct expression patterns in CRC, suggesting a differential role exerted by each member in colon carcinogenesis.

Activation of histamine H4 receptor inhibits TNFα/IMD-0354-induced apoptosis in human salivary NS-SV-AC cells.

Apoptosis is involved in the pathogenesis of Sjögren's syndrome (SS), an autoimmune disease affecting exocrine glands. Our recent studies revealed diminished histamine H4 receptor (H4R) expression and impaired histamine transport in the salivary gland epithelial cells in SS. The aim was now to test if nanomolar histamine and high-affinity H4R signaling affect apoptosis of human salivary gland epithelial cell. Simian virus 40-immortalized acinar NS-SV-AC cells were cultured in serum-free keratinocyte medium ± histamine H4R agonist HST-10. Expression and internalization of H4R were studied by immunofluorescence staining ± clathrin inhibitor methyl-ß-cyclodextrin (MßCD). Apoptosis induced using tumor necrosis factor-α with nuclear factor-κB inhibitor IMD-0354 was studied using phase contrast microscopy, Western blot, flow cytometry and polymerase chain reaction (qRT-PCR). HST-10-stimulated H4R internalization was inhibited by MßCD. Western blotting revealed diminished phosphorylated c-Jun N-terminal kinase JNK, but unchanged levels of phosphorylated extracellular signal regulated kinase pERK1/2 in H4R-stimulated samples compared to controls. qRT-PCR showed up-regulated expression of anti-apoptotic B cell lymphoma-extra large/Bcl-xL mRNAs and proteins, whereas pro-apoptotic Bcl-2-associated X protein/BAX remained unchanged in H4R-stimulated samples. H4R stimulation diminished cleavage of PARP and flow cytometry showed significant dose-dependent inhibitory effect of H4R stimulation on apoptosis. As far as we know this is the first study showing inhibitory effect of H4R activation on apoptosis of human salivary gland cells. Diminished H4R-mediated activation may contribute to loss of immune tolerance in autoimmune diseases and in SS in particular.

IL-17C and its receptor IL-17RA/IL-17RE identify human oral epithelial cell as an inflammatory cell in recurrent aphthous ulcer.

BACKGROUND: Recurrent aphthous ulcer (RAU) is an ulcerative disease of non-keratinized oral mucosa. Colon and bronchial epithelial cells produce interleukin-17C (IL-17C) upon stimulation of Toll-like receptor 2 (TLR2), TLR3 and TLR5, which are highly expressed in epithelial cells in RAU lesions. We therefore investigated the eventual presence and function of IL-17C in cultured human oral keratinocytes (HOK) and control biopsies compared to RAU lesions. METHODS: Expression of IL-17A, IL-17C, IL-17RA and IL-17RE was analysed in cultured HOK cells using quantitative real-time polymerase chain reaction (qRT-PCR). HOK cells were stimulated with IL-17C and analysed for IL-8 and tumour necrosis factor-α (TNF-α) using qRT-PCR. Control mucosa (n = 5) was immunostained for IL-17A, IL-17C, IL-8, TNF-α and mast cell tryptase and compared with RAU lesions (n = 5) using the mean grey scale value. RESULTS: IL-17C, but no IL-17A, mRNA was found in cultured HOK cells. Components of the heterodimeric IL-17RA/IL-17RE receptor for IL-17C were also highly expressed. Stimulation of HOK with IL-17C increased TNF-α mRNA (P = 0.03; IL-8 increase was not statistically significant). HOK in RAU lesions stained intensively for IL-17C compared to controls (P = 0.006). This was associated with increased epithelial immunostaining of TNF-α (P = 0.04) and IL-8 (P = 0.02). Most of the inflammatory cells which stained for IL-17A in control mucosa and RAU lesions were also mast cell tryptase positive. CONCLUSION: IL-17C is highly expressed in epithelial cells in RAU lesions, where it seems to stimulate oral keratinocytes via IL-17RA/IL-17RE to produce pro-inflammatory cytokines. Human oral epithelial cells are probably important inflammatory cells in RAU.

Ex Vivo Evaluation of Antiangiogenic Drugs in Oral Cancer: Potential Implications for Targeting Vasculogenic Mimicry.

BACKGROUND/AIM: Oral squamous cell carcinoma (OSCC) is characterized by early metastasis, clinical resistance and poor prognosis. Recently, we showed that aggressive OSCC cells co-express endothelial cell markers and can form tube-like structures, known as vasculogenic mimicry (VM), a process associated with poor prognosis in head and neck cancers. Given the limited success of current antiangiogenic therapy in treating OSCC, this study sought to explore the efficiency of these drugs in targeting an ex vivo model of VM. MATERIALS AND METHODS: OSCC cell lines from the tongue and floor of the mouth in addition to human endothelial cells were used. The treatments comprised a set of clinically relevant antiangiogenic drugs: sorafenib, sunitinib, and axitinib, which were administered in different doses. Multiple ex vivo approaches including cell tubulogenesis, proliferation, apoptosis, and migration assays were used. RESULTS: Although these drugs inhibited the formation of endothelial cell capillaries, they showed clear differential effects on OSCC cell-derived VM and cell morphology. Sorafenib inhibited the tubulogenesis of aggressive OSCC cells compared with the limited effect of sunitinib and axitinib. Furthermore, our data consistently demonstrated a preferential efficacy of certain drugs over others. Sorafenib and sunitinib exhibited anti-cancer effects on tumor cell proliferation, apoptosis, and cell migration, compared with the limited effect of axitinib. CONCLUSION: The antiangiogenic drugs, except sorafenib, had limited effect on VM formation in vitro and exhibited varying anti-cancer effects on OSCC cells. These data support the notion that VM formation may in part explain the development of drug resistance in OSCC cells.

Vasculogenic Mimicry in Head and Neck Squamous Cell Carcinoma-Time to Take Notice.

Head and neck squamous cell carcinoma (HNSCC) is a group of common cancers characterized by a swift growth pattern, early metastasis, and dismal 5-year survival rates. Despite the recent advances in cancer management, the multimodality approach is not effective in eradicating HNSCC. Moreover, the clinical response to the antiangiogenic therapy remains considerably limited in HNSCC patients, suggesting that tumor perfusion can take place through other non-angiogenic pathways. Tumor cell-induced angiogenesis is one of the main hallmarks of cancer. However, at the end of the previous millennium, a new paradigm of tumor cell-associated neovascularization has been reported in human melanoma cells. This new phenomenon, which was named "vasculogenic mimicry" or "vascular mimicry" (VM), describes the ability of aggressively growing tumor cells to form perfusable, matrix-rich, vessel-like networks in 3-dimensional matrices in vitro. Similar matrix-rich VM networks were also identified in tissue samples obtained from cancer patients. To date, myriad studies have reported intriguing features of VM in a wide variety of cancers including HNSCC. We aim in this mini-review to summarize the current evidence regarding the phenomenon of VM in HNSCC-from the available detection protocols and potentially involved mechanisms, to its prognostic value and the present limitations.