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.

[Application of superselective lingual artery embolization in treatment of severe hemorrhange in patients with carcinoma of tongue].

To explore the clinical value of superselective lingual artery embolization in treating the severe hemorrhage in patients with advanced carcinoma of tongue. Four patients with advanced tongue cancer hemorrhage from March 2014 to February 2016 were enrolled in this study. T3N2M0 (2 cases) and T4N1M0 (2 cases) were diagnosed preoperatively. Two cases of advanced tongue carcinoma tumors had severe bleeding and the other 2 cases of hemorrhage were after radiotherapy. All cases including 3 squamous cell carcinoma and 1 adenocarcinoma were firstly demonstrated by arterigraphy under seldinger technique with digtial subtraction angiogarphy to ensure the rupture site and then all cases were followed by superselective artery embolization. The efficacy and complications of interventional embolizationg were observed. There was no serious complication of central nervous system injury such as hemorrhage and hemiplegia during follow-up. Superselective lingual artery embolization can accurately locate the responsibility of blood vessels, and the injury is small, significant effect, fewer complications.

High relative density of lymphatic vessels predicts poor survival in tongue squamous cell carcinoma.

Tongue cancer has a poor prognosis due to its early metastasis via lymphatic vessels. The present study aimed at evaluating lymphatic vessel density, relative density of lymphatic vessel, and diameter of lymphatic vessels and its predictive role in tongue cancer. Paraffin-embedded tongue and lymph node specimens (n = 113) were stained immunohistochemically with a polyclonal antibody von Willebrand factor, recognizing blood and lymphatic endothelium and with a monoclonal antibody podoplanin, recognizing lymphatic endothelium. The relative density of lymphatic vessels was counted by dividing the mean number of lymphatic vessels per microscopic field (podoplanin) by the mean number of all vessels (vWf) per microscopic field. The high relative density of lymphatic vessels (≥80 %) was associated with poor prognosis in tongue cancer. The relative density of lymphatic vessels predicted poor prognosis in the group of primary tumor size T1-T2 and in the group of non-metastatic cancer. The lymphatic vessel density and diameter of lymphatic vessels were not associated with tongue cancer survival. The relative density of lymphatic vessels might have clinically relevant prognostic impact. Further studies with increased number of patients are needed.

Vascularity as assessed by Doppler intraoral ultrasound around the invasion front of tongue cancer is a predictor of pathological grade of malignancy and cervical lymph node metastasis.

OBJECTIVES: To quantitatively evaluate the relationship of vascularity of tongue cancer as demonstrated on intraoral ultrasonography images and tumour thickness with pathological grade of malignancy and the presence of cervical lymph node metastases. METHODS: 18 patients with tongue cancer were enrolled in this retrospective study. Using Doppler ultrasonography images of the invasion front of the cancers along the length of their tumour boundaries, three vascular indexes were analysed quantitatively, namely ratio of blood flow signal area within the cancer to whole tumour area (BAR), blood flow signal number ratio (BNR) and blood flow signal width ratio (BWR). The associations between these three indexes and occurrence of cervical lymph node metastasis and pathological grade of malignancy [Yamamoto-Kohama (YK) classification] were assessed. Furthermore, the relationship between tumour thickness and occurrence of cervical lymph node metastasis was evaluated on B-mode intraoral ultrasonography images. RESULTS: There was no significant association between BAR and tumour thickness or occurrence of cervical lymph node metastasis. The BNRs and BWRs of patients with cervical lymph node metastasis were significantly higher than those of patients without nodal involvement. The BWRs of patients with high-grade malignancy (YK-4C) were significantly higher than those of patients with low-grade malignancy (YK-2 or 3). CONCLUSIONS: BNR and BWR on the invasion front of the tongue cancer are predictors of pathological grade of malignancy and cervical lymph node metastasis.

Neurovascular Invasion and Histological Grade Serve as the Risk Factors of Cervical Lymph Node Metastases in Early Tongue Squamous Cell Carcinoma.

The objectives of this study were to analyze the regional characteristics of the cervical lymph node metastasis and to investigate the factors associated with the risk of lymph node involvement. One hundred seventy-one patients suffering from early primary squamous cell carcinoma (SCC) of the tongue (cT1-2N0) were enrolled. Gender, age, growth site, T stage, histological grade, and neurovascular invasion were statistically analyzed by K-M survival analysis and Cox multivariate analysis to evaluate the relationship between the factors and the neck lymph node metastasis. Of the 171 cases divided into the neck dissection group and observation group, 40 ended up with lymph node metastasis, of which 17 were metastasized to level I, 27 to level II, 10 to level III, 2 to level IV, and 1 to level V. Histological grade and neurovascular invasion were significantly associated with lymph node involvement in univariate and multivariate analyses. Age distribution was found to be significantly associated with the lymph node metastasis in multivariate analysis. The metastasis of early tongue SCC has a certain regularity at different sites. Age was not a critical risk factor for cervical lymph node metastasis after surgery. Tumor size was suspected to exert a negative effect on metastasis by influencing tumor invasion. Histological grade and neurovascular invasion were significantly associated with the risk of cervical lymph node metastasis of early tongue SCC.

Endostatin induces proliferation of oral carcinoma cells but its effect on invasion is modified by the tumor microenvironment.

The turnover of extracellular matrix liberates various cryptic molecules with novel biological activities. Endostatin is an endogenous angiogenesis inhibitor that is derived from the non-collagenous domain of collagen XVIII. Although there are a large number of studies on its anti-tumor effects, the molecular mechanisms are not yet completely understood, and the reasons why endostatin has not been successful in clinical trials are unclear. Research has mostly focused on its anti-angiogenic effect in tumors. Here, we aimed to elucidate how endostatin affects the behavior of aggressive tongue HSC-3 carcinoma cells that were transfected to overproduce endostatin. Endostatin inhibited the invasion of HSC-3 cells in a 3D collagen-fibroblast model. However, it had no effect on invasion in a human myoma organotypic model, which lacks vital fibroblasts. Recombinant endostatin was able to reduce the Transwell migration of normal fibroblasts, but had no effect on carcinoma associated fibroblasts. Surprisingly, endostatin increased the proliferation and decreased the apoptosis of cancer cells in organotypic models. Also subcutaneous tumors overproducing endostatin grew bigger, but showed less local invasion in nude mice xenografts. We conclude that endostatin affects directly to HSC-3 cells increasing their proliferation, but its net effect on cancer invasion seem to depend on the cellular composition and interactions of tumor microenvironment.

Expression of interleukin-33 is correlated with poor prognosis of patients with squamous cell carcinoma of the tongue.

OBJECTIVE: The aim of this study was to clarify the role of IL-33 in tumor progression. METHODS: Surgical specimens from 81 patients with squamous cell carcinoma of the tongue were studied using immunohistochemistry. Primary tumor sections were analyzed for IL-33 and ST2 expression. To examine the influence of IL-33 on the microenvironment of the tumor, we determined the mast cell density (MCD) and microvessel density of the stroma. RESULTS: Patients with high IL-33 expression had a significantly worse prognosis (p=0.004). IL-33 expression was significantly elevated in patients with local and nodal recurrence (p=0.014 and p=0.019). ST2 expression was also associated with a worse prognosis (p=0.024) and was significantly elevated in patients with nodal recurrence (p=0.004). MCD was associated with worse prognosis (p=0.038) and correlated significantly with IL-33 expression (r=0.626, p<0.001). Micovessels in the stroma were significantly increased in the high IL-33 group (p<0.001). CONCLUSION: These data suggest that the IL-33/ST2 axis contributes to tumor aggressiveness and affects the tumor microenvironment. Immunohistochemical evaluation of IL-33 and ST2 is useful for identifying patients at a high risk for poor prognosis.

Prognostic significance of amino-acid transporter expression (LAT1, ASCT2, and xCT) in surgically resected tongue cancer.

BACKGROUND: Amino-acid transporters are necessary for the tumour cell growth and survival, and have a crucial role in the development and invasiveness of cancer cells. But, it remains unclear about the prognostic significance of L-type amino-acid transporter 1 (LAT1), system ASC amino-acid transporter-2 (ASCT2), and xCT expression in patients with tongue cancer. We conducted the clinicopathological study to investigate the protein expression of these amino-acid transporters in tongue cancer. METHODS: Eighty-five patients with surgically resected tongue cancer were evaluated. Tumour sections were stained by immunohistochemistry for LAT1, ASCT2, xCT, 4F2hc/CD98hc (4F2hc), Ki-67, and microvessel density (MVD) determined by CD34, and p53. RESULTS: L-type amino-acid transporter 1 and 4F2hc were highly expressed in 61% (52 out of 85) and 45% (38 out of 47), respectively. ASC amino-acid transporter-2 and xCT were positively expressed in 59% (50 out of 85) and 21% (18 out of 85), respectively. The expression of both LAT1 and ASCT2 was significantly associated with disease staging, lymph-node metastasis, lymphatic permeation, 4F2hc expression and cell proliferation (Ki-67). xCT expression indicated a significant association with advanced stage and tumour factor. By univariate analysis, disease staging, lymphatic permeation, vascular invasion, LAT1, ASCT2, 4F2hc, and Ki-67 had a significant relationship with overall survival. Multivariate analysis confirmed that LAT1 was an independent prognostic factor for predicting poor prognosis. CONCLUSIONS: L-type amino-acid transporter 1 and ASCT2 can serve as a significant prognostic factor for predicting worse outcome after surgical treatment and may have an important role in the development and aggressiveness of tongue cancer.

Overexpression of chemerin was associated with tumor angiogenesis and poor clinical outcome in squamous cell carcinoma of the oral tongue.

OBJECTIVE: The present study aimed to explore expression and clinical significance of chemerin, a newly discovered adipokine, in squamous cell carcinoma of the oral tongue (SCCOT). METHODS: mRNA expression of chemerin in 19 pairs of fresh SCCOT samples matched with peritumoral mucosa tissues was quantified by real-time quantitative transcription polymerase chain reaction (qRT-PCR). Chemerin protein expression and microvessel density (MVD) were measured by immunohistochemistry on 147 cases of primary SCCOT specimen and their corresponding peritumoral noncancerous tissues. The relationship of chemerin expression with angiogenesis, clinicopathologic parameters, and cancer-related survival of patients was evaluated. RESULTS: Both qRT-PCR and immunohistochemistry results revealed that chemerin was overexpressed in SCCOT compared with peritumoral noncancerous tissues (P < 0.01). Overexpression of chemerin in SCCOT was significantly associated with poor differentiation, lymph node metastasis, and high clinical stage (P = 0.000, 0.012, and 0.015, respectively). In addition, overexpression of chemerin was positively related to MVD in SCCOT (r = 0.671, P = 0.002). SCCOT patients with overexpressed chemerin had a shorter cancer-related survival (P = 0.027). Moreover, multivariate survival analysis indicated that chemerin was an independent prognostic factor for SCCOT patients (P = 0.016). CONCLUSION: These results demonstrated that overexpression of chemerin in SCCOT was correlated with tumor angiogenesis and poor clinical outcomes of SCCOT patients. CLINICAL RELEVANCE: Our research implied that chemerin was a novel prognostic factor for SCCOT patients, and chemerin could be a new therapeutic target for regulating tumor angiogenesis and blocking tumor progression.

[Medial sural artery perforator fasciocutaneous flap for the reconstruction of tongue and mouth base]. / Arteria suralis medialis perforator fasciocutan lebeny alkalmazása a nyelv és a szájfenék rekonstrukciójában.

BACKGROUND: Despite the well-known high donor site morbidity of the radial forarm flap, it has still remained the first option for the reconstruction of the tongue and the floor of mouth. However, a desire for an alternative, thin fasciocutaneous flap has led to the use of the median sural artery perforator flap. METHODS: Three patients had reconstructive surgery used MSAP flaps, after radical tumor excision. RESULTS: The flap was based in all cases on a dominant perforator vessel. The size of the skin paddles ranged between 20-32 cm2, and the length of the vascular pedicle between 8.2-11 cm. The mean thickness of the flap was 6.5 mm. CONCLUSION: MSAP flap is an ideal solution for surgical reconstruction in the oral cavity.

Tumor-infiltrating macrophage and microvessel density in oral squamous cell carcinoma.

Tumor-associated macrophages (TAM) are the main cellular component in stroma of many tumors and participate in tumor angiogenesis. The aim of present study was to compare the microvascular density (MVD) and infiltrating macrophage density (IMD) in oral squamous cell carcinomas (OSCCs) with different histological grades. A histomorphometric analysis was performed after immunohistochemistry using antibodies such as von-Willebrand factor and CD68. A significant difference in MVD was found between well and moderately differentiated OSCCs (p<0.05). TAM were largely present in all studied tumors and the IMD was not different among OSCCs with different histological grades (p=0.381). Significant correlation between MVD and IMD was not observed (p=0.870). In conclusion, these results suggest that TAM and angiogenesis have an influence at different histological grades of OSCC. However, the lack of correlation between MVD and IMD could suggest that angiogenesis does not depend on the number of macrophages present in OSCC, but their predominant phenotype. Further studies involving distinct phenotypes of macrophages should be done to better understand the influence of TAM on the tumor angiogenesis.

Tumor-Infiltrating Macrophage and Microvessel Density in Oral Squamous Cell Carcinoma

Tumor-associated macrophages (TAM) are the main cellular component in stroma of many tumors and participate in tumor angiogenesis. The aim of present study was to compare the microvascular density (MVD) and infiltrating macrophage density (IMD) in oral squamous cell carcinomas (OSCCs) with different histological grades. A histomorphometric analysis was performed after immunohistochemistry using antibodies such as von-Willebrand factor and CD68. A significant difference in MVD was found between well and moderately differentiated OSCCs (p<0.05). TAM were largely present in all studied tumors and the IMD was not different among OSCCs with different histological grades (p=0.381). Significant correlation between MVD and IMD was not observed (p=0.870). In conclusion, these results suggest that TAM and angiogenesis have an influence at different histological grades of OSCC. However, the lack of correlation between MVD and IMD could suggest that angiogenesis does not depend on the number of macrophages present in OSCC, but their predominant phenotype. Further studies involving distinct phenotypes of macrophages should be done to better understand the influence of TAM on the tumor angiogenesis.

Macrófagos associados a tumores (MAT) representam o componente principal do estroma de muitos tumores, além de participar da angiogênese tumoral. Este estudo comparou a microdensidade vascular (MDV) e densidade de macrófagos infiltrando o tumor (DMIT) em carcinoma escamocelular da boca (CEC) com diferentes graus histológicos de malignidade. Análise histomorfométrica foi empregada após técnica imuno-histoquímica para os anticorpos fator von-Willebrand e CD68. Uma diferença significante entre MDV e carcinomas bem e moderadamente diferenciados foi observada (p<0,05). MAT estavam fortemente presentes em todos os tumores estudados e a DMIT não foi diferente entre os diferentes graus histológicos de malignidade do CEC (p=0,381). Correlação significante entre MDV e DMIT não foi observada (p=0,870). Em conclusão, os resultados desse estudo sugerem a influência de MAT e angiogênese nos diferentes graus histológicos de malignidade do CEC. Entretanto, a ausência de correlação entre MDV e DMIT sugere que a angiogênese não depende do número de macrófagos presentes neste tipo de câncer, mas do fenótipo predominante. Outros estudos devem ser realizados a fim de contribuir para melhor compreensão da participação de MAT na angiogênese tumoral.

[Vasculogenic mimicry in tongue squamous cell carcinoma].

OBJECTIVE: To investigate the presence of vasculogenic mimicry (VM) in tongue squamous cell carcinoma and explore its clinical significance. METHODS: Forty-two surgical specimens of tongue squamous cell carcinoma were examined for the presence of VM using HE staining and double staining of CD34 and PAS. RESULTS: Of the 42 specimens, 18 (42.86%) showed the presence of VM. VM was not correlated with the patients' age or gender, but with lymph node metastasis and the grade of tumor differentiation. Compared with tumors without VM, the tumors with VM had a significantly higher rate of lymph node metastasis (P<0.05) and a lower grade of differentiation (P<0.05). CONCLUSION: VM can be present in tongue squamous cell carcinoma, and the poorly differentiated tumors contain more VM, which is associated with a greater likeliness of lymph node metastasis and a poorer prognosis.

The role of angiogenin in pT1-T2 tongue carcinoma neo-angiogenesis and cell proliferation: an exploratory study.

BACKGROUND: Angiogenin (ANG) is a member of the ribonuclease superfamily and of medical interest largely because it supports the growth of primary and metastatic malignancies. This study is the first to investigate the potential role of ANG in tongue carcinoma neo-angiogenesis and cancer cell proliferation. METHODS: Angiogenin expression (in carcinoma cells and endothelial intratumor vessel cells), CD105-assessed micro-vessel density (MVD), and MIB-1 expression were correlated with prognostic parameters in 28 primarily consecutively operated pT1-T2 tongue carcinomas (squamous cell carcinoma [SCC]). Whenever feasible, a computer-based image analysis system was used for the immunohistochemical reaction analysis. RESULTS: No significant correlations emerged between ANG expression in the tongue carcinoma cells or endothelial intratumor vessel cells and tongue SCC recurrence rate or disease-free survival (DFS). ANG expression was also unrelated to CD105-assessed MVD or MIB-1 expression. Conversely, CD105-assessed MVD correlated directly with recurrence rate (P = 0.02) and DFS was significantly shorter in cases with CD105-assessed MVD >167 micro-vessels/mm(2) than in those with CD105-assessed MVD ≤167 micro-vessels/mm(2) (P = 0.042). CONCLUSIONS: Our results support the hypothesis that CD105-assessed MVD would be a valuable parameter for predicting which patients with tongue SCC are at greatest risk of disease recurrence. Despite our study results, the role of ANG in tongue carcinoma warrants further investigation in larger series.

A novel endovascular technique for temporary balloon occlusion and permanent vessel deconstruction with a single microcatheter.

OBJECTIVE: To report a novel technique for temporary and permanent vessel occlusion with a single microcatheter and investigate its efficacy in endovascular management of cerebral aneurysms and invasive head and neck tumors. METHODS: A 58-year-old woman with a history of T3N0 squamous cell carcinoma of the tongue with extensive surgical resections and radiotherapy presented to the emergency department with further wound dehiscence and exposure of the left cervical internal carotid artery (ICA). Digital subtraction angiography (DSA) was performed with the patient under conscious sedation. An Ascent balloon, 4 mm × 15 mm, was prepared and introduced into the proximal ICA. While the patient was fully awake, temporary balloon occlusion (TBO) was performed for 30 minutes. The Ascent balloon was kept inflated. Through the inflated Ascent balloon, coil embolization of the proximal ICA was initiated. The interstices of the coils were filled with Onyx 18 and Onyx 34 liquid embolic agents (ev3 Neurovascular, Inc, Irvine, California, USA). RESULTS: The Ascent balloon can be used as a temporary balloon device and as a coil or Onyx embolization device. As a result, fewer catheters, guidewires, and balloon inflations and deflations are required for the procedure, which is both cost-effective and time-efficient. The combination of initial coiling to create a framework for the deconstruction with a liquid embolic agent (Onyx) is a safe and efficient technique for vessel deconstruction. Flow arrest with proximal balloon occlusion minimizes the risks of distal thromboembolic events during embolization. CONCLUSIONS: This novel technique for temporary and permanent vessel occlusion with a single microcatheter is both cost-effective and time-efficient.

Arresten, a collagen-derived angiogenesis inhibitor, suppresses invasion of squamous cell carcinoma.

The turnover of extracellular matrix liberates various cryptic molecules with novel biological activity. Among these are the collagen-derived anti-angiogenic fragments, some of which are suggested to affect carcinoma cells also directly. Arresten is an endogenous angiogenesis inhibitor that is derived from the non-collagenous domain of the basement membrane collagen IV α1 chain. As the mere prevention of tumor angiogenesis leads to hypoxia that can result in selection of more aggressive cell types and reduces the efficacy of chemotherapy, we aimed here to elucidate how arresten influences the aggressive human carcinoma cells. Arresten efficiently inhibited migration and invasion of HSC-3 tongue carcinoma cells in culture and in an organotypic model. Subcutaneous Arr-HSC xenografts grew markedly more slowly in nude mice and showed reduced tumor cell proliferation, vessel density and local invasiveness. In the organotypic assay, HSC-3 cells overproducing arresten (Arr-HSC) showed induction of cell death. In monolayer culture the Arr-HSC cells grew in aggregated cobblestone-like clusters and, relative to the control cells, showed increased expression and localization of epithelial marker E-cadherin in cell-cell contacts. Application of electric cell-substrate impedance sensing (ECIS) further supported our observations on altered morphology and motility of the Arr-HSC cells. Administration of a function-blocking α1 integrin antibody abolished the impedance difference between the Arr-HSC and control cells suggesting that the effect of arresten on promotion of HSC-3 cell-cell contacts and cell spreading is at least partly mediated by α1ß1 integrin. Collectively, our data suggest novel roles for arresten in the regulation of oral squamous carcinoma cell proliferation, survival, motility and invasion through the modulation of cell differentiation state and integrin signaling.

A comparative study of microvessel density in squamous cell carcinoma of the oral cavity and lip.

OBJECTIVES: The objective of this study was to comparatively evaluate the density of lymphatic vessels (LVD) and neoformed microvessels (NMVD) in squamous cell carcinoma of the oral cavity (OCSCC) and lip (LSCC). Association between LVD/NMVD and vascular endothelial growth factor (VEGF)-A/-C was also assessed. STUDY DESIGN: OCSCC and LSCC were compared with regard to immunoexpression of LVD, NMVD, and vascular endothelial growth factor-A (VEGF)-A/-C. Association between VEGF-A/-C with vascularity was also assessed. Statistical analyses were performed using t test, Pearson χ(2), and Mann-Whitney tests. Statistical significance was accepted at P less than .05. RESULTS: The NMVD and VEGF-C expressions were significantly higher in OCSCC compared with LSCC. NMVD was associated with VEGF-C in OCSCC, but not in LSCC. CONCLUSIONS: Differences in NMVD and VEGF-C were found between OCSCC and LSCC. Positive association between VEGF-C and NMVD was observed in OCSCC, but not in LSCC, which may be one of the contributing factors that account for the distinctive clinical-biological behavior of these lesions.

Immunohistochemical expression of MMP-9 and VEGF in squamous cell carcinoma of the tongue.

Squamous cell carcinomas (SCCs) account for approximately 95% of all oral malignant neoplasms and for about 38% of all malignant head and neck tumors, especially affecting the tongue and lips. The aim of this study was to evaluate the immunohistochemical expression of MMP-9 and VEGF in oral SCC according to the occurrence of metastasis. Eighteen cases of tongue SCC without metastases and 17 cases of tongue SCC with metastases were subjected to immunohistochemical methods. High immunohistochemical expression of MMP-9 and VEGF by neoplastic cells and stroma was observed in tongue SCCs at the invasion front. Metastatic tumors tended to express higher levels of MMP-9 and VEGF than non-metastatic tumors, but the difference was not significant (P > 0.05). Spearman's correlation test showed no significant correlation between VEGF-immunopositive vessels and metastasis (P > 0.05). The present results demonstrate the importance of the expression of MMP-9 and VEGF for the development of SCC of the tongue. However, no significant association was observed between the overexpression of MMP-9 or VEGF and the presence of metastases.