Identification of Proteomic Markers in Head and Neck Cancer Using MALDI-MS Imaging, LC-MS/MS, and Immunohistochemistry.

PURPOSE: The heterogeneity of squamous cell carcinoma tissue greatly complicates diagnosis and individualized therapy. Therefore, characterizing the heterogeneity of tissue spatially and identifying appropriate biomarkers is crucial. MALDI-MS imaging (MSI) is capable of analyzing spatially resolved tissue biopsies on a molecular level. EXPERIMENTAL DESIGN: MALDI-MSI is used on snap frozen and formalin-fixed and paraffin-embedded (FFPE) tissue samples from patients with head and neck cancer (HNC) to analyze m/z values localized in tumor and nontumor regions. Peptide identification is performed using LC-MS/MS and immunohistochemistry (IHC). RESULTS: In both FFPE and frozen tissue specimens, eight characteristic masses of the tumor's epithelial region are found. Using LC-MS/MS, the peaks are identified as vimentin, keratin type II, nucleolin, heat shock protein 90, prelamin-A/C, junction plakoglobin, and PGAM1. Lastly, vimentin, nucleolin, and PGAM1 are verified with IHC. CONCLUSIONS AND CLINICAL RELEVANCE: The combination of MALDI-MSI, LC-MS/MS, and subsequent IHC furnishes a tool suitable for characterizing the molecular heterogeneity of tissue. It is also suited for use in identifying new representative biomarkers to enable a more individualized therapy.

Development of a new software and test setup for analyzing hVOR in very young children by vHIT.

INTRODUCTION: Earlier work revealed that vHIT examination is often difficult to perform on very young children. In particular, the calibration of the system can be difficult, as active cooperation of the patient is required. Additionally, the patient must be able to follow the examiner's instructions, which is challenging for very young children. Therefore, the aim of the present study was to develop and validate a new, software-based approach enabling vHIT testing of young children and infants. METHODS AND MATERIALS: Six patients (3 boys and 3 girls) aged 5-36 months were included in a prospective, monocentric study between January 2015 and August 2015.The newly developed intuitive software enabled calibration of the eye position signal with the subjects fixating on animated animal graphics which were projected on a screen. Testing ten healthy adults validated this new calibration and measurement method. After calibration, a vHIT goggle (EyeSeeCam ©) was used to perform head impulses in the horizontal plane while the patient was watching a movie sitting on their parent's lap or in a baby chair. At least 15 impulses to each side were obtained and the occurrence of refixation saccades was analyzed. All tests were performed by one of two experienced examiners. RESULTS: The new calibration method and modified test setup provided reproducible results for all patients tested. An increased incidence of artifacts was not observed. In 2 patients, more than one test was needed. None of the included children showed catch-up overt or catch-up covert saccades. There was no gain reduction of more than two standard deviations as compared to the normative results published in the literature on vHIT examinations of children. CONCLUSION: The proposed protocol allows vHIT testing in very young children and infants (aged 5 months to 3 years). The study emphasizes that vHIT is an easy and sensitive screening tool to evaluate vestibular function in children and should be used as the gold standard in pediatric vestibular assessment.

Communicate or Die - A Model for HPV+ and HPV- CSCs and Their Interactions with SDF-1α.

BACKGROUND/AIM: Cancer stem cells (CSCs) are suspected of being a reason for limited therapy in head and neck squamous cell carcinoma (HNSCC). Stromal cell-derived factor-1α (SDF-1α) plays a critical role in the communication between CSCs and their microenvironment. We investigated the influence of SDF-1α on HPV+/HPV- SCC cell lines to find an approach of explanation for the superior prognosis of HPV+ HNSCCs. MATERIALS AND METHODS: We evaluated the expression of CD44/CXCR4 on HPV+/HPV- SCC cell lines and monitored the influence of SDF-1α on proliferation, morphology and migration of HPV+/HPV- SCCs. RESULTS: HPV- SCCs showed a significant increase of podia formation and an intensified migration towards SDF-1α. HPV+ SCCs rested nearly unaffected by SDF-1α. CONCLUSION: Weakened reaction to SDF-1α in HPV+ SCC could lead to an impaired communication between CSCs and their niche, that would result in an increased exposure of CSCs to the harming influence of e.g. chemotherapeutic agents.

[Using a Standardized Questionnaire for Coagulation Assessment in Children Undergoing Tonsillectomy]. / Standardisierte Gerinnungsanamnese vor Tonsillektomie und Adenotomie im Kindesalter.

Introduction A 2006 position paper suggests assessing coagulation status via a standardized questionnaire instead of performing routine coagulation testing for children undergoing tonsillectomy/adenotomy. The aim of the presented study was to evaluate whether this paradigm change led to a change in the incidence of secondary bleeding. Methods Descriptive statistical analysis of existing clinical data was performed to evaluate the incidence and characteristics of secondary bleeding in children after tonsillectomy/adenotomy in 2003 vs. 2009. Result In 2003, 352 children underwent surgery. Secondary bleeding occurred in 25 cases (7.1%), 18, (6.1%) of which required surgical treatment. In 2009, 20 out of 293 children who had undergone tonsillectomy/adenotomy suffered from secondary bleeding, 14 required (4.7%) surgical treatment. There was no significant difference in the incidence of bleeding between those years. In 5 children who suffered from secondary bleeding in 2003, preoperative diagnostic blood coagulation testing was performed, none of them showed abnormal results. Furthermore, none of the diagnostic blood coagulation tests performed after secondary bleeding in both groups showed any abnormalities. Conclusion Using a standardized questionnaire instead of a diagnostic blood coagulation testing for preoperative coagulation assessment does not have an influence on the incidence of secondary bleeding after tonsillectomy/adenotomy. The results of this study suggest that secondary bleeding is not is not caused by abnormal hemostasis.

Lapatinib-induced mesenchymal-epithelial transition in squamous cell carcinoma cells correlates with unexpected alteration of ß-catenin expression.

The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer progression, and may promote resistance of cancer cells to therapy. Inhibiting EMT appears to be crucial to inhibit drug resistance. The mesenchymal-epithelial transition (MET), which is the reverse program of EMT in metastases, is characterized by the upregulation of epithelial adhesive proteins such as E-cadherin, and downregulation of mesenchymal proteins such as vimentin. The sensitivity of cancer cells to epithelial growth factor receptor (EGFR) inhibitor may be increased by inducing MET in these cells. Therefore, it is of clinical importance to specify the phenotype of cancer cells in order to overcome the phenomenon of drug resistance. The aim of the present study was to investigate the expression pattern of specific markers in squamous cell carcinoma (SCC) cells following stimulation with lapatinib and gefitinib. For this purpose, the head and neck (HN) SCC cell lines HNSCC22B and HNSCC11A were incubated with 0.5 and 2 µg/ml lapatinib and gefitinib, and the levels of E-cadherin, vimentin, matrix metalloproteinase-14, c-kit and ß-catenin were detected by immunocytochemistry and enzyme-linked immunosorbent assay at 5, 24 and 96 h post-incubation. The results indicated that, compared with HNSCC22B cells, the protein expression levels of vimentin increased, whereas those of E-cadherin reduced, in non-stimulated HNSCC11A cells. In addition, the protein expression levels of ß-catenin were altered in the epithelial- and mesenchymal-associated SCC cell lines following treatment with lapatinib and gefitinib. Furthermore, lapatinib induced the downregulation of vimentin and upregulation of E-cadherin in HNSCC11A cells in a time-dependent manner. This suggests that the sensitivity of cancer cells to lapatinib may be improved by inducing MET in these cells. In summary, the results of the present study demonstrated that lapatinib-induced MET led to an unexpected alteration of the protein expression levels of ß-catenin in SCC cells. Further studies on the mechanistic role of MET are required in order to increase the sensitivity of cancer cells to EGFR inhibitor and block the EMT process in these cells.

MMP9, Cyclin D1 and ß-Catenin Are Useful Markers of p16-positive Squamous Cell Carcinoma in Therapeutic EGFR Inhibition In Vitro.

BACKGROUND/AIM: In the United States 53,640 new cases of head and neck cancer were estimated in 2013. Over 95% of these cases were evaluated as squamous cell carcinoma (SCC). At present, smoking, drinking alcohol, chewing betel and infection with high-risk types of human papilloma virus (HPV) are classified as risk factors of oropharyngeal squamous cancer cell carcinoma (OPSCC). It could be suggested that patients with HPV-positive OPSCC have a better response to chemoradiotherapy than patients without. In many studies, there was observed an inverse correlation between epithelial growth factor receptor (EGFR) expression and HPV status in p16-positive SCC. Therefore, it is of great clinical interest to specify the phenotype of cancer cells in order to further individualize treatment modalities. The aim of the study was to investigate the expression pattern of specific markers in p16-positive SCC cells after stimulation with lapatinib and gefitinib. MATERIALS AND METHODS: We incubated p16-positive CERV196 cells with lapatinib and gefitinib (2 µg/ml) and after 5, 24 and 96 h determined E-cadherin, vimentin, matrix metalloproteinase-9 (MMP9), cyclin D1 and ß-catenin by immunocytochemistry, enzyme-linked immunosorbent assay and quantitative polymerase chain reaction (PCR). RESULTS: We found an increase of E-cadherin and a decrease of vimentin in unstimulated cells. We detected an alteration of expression of vimentin and E-cadherin level after treatment with lapatinib and gefitinib. We demonstrated a statistically significant lapatinib- and gefitinib-induced repression of cyclin D1, MMP9 and ß-catenin in CERV196 cells dependent on incubation time. CONCLUSION: Cyclin D1 and MMP9 expression profiles may represent an early measure of sensitivity and level of response to lapatinib and gefitinib. The presented cell culture model is, therefore, well-suited for further study of epigenetic regulation of molecular targeted-therapy by EGFR inhibition and prevention of mesenchymal transition in p16-positive SCC cells.

Targeting mTOR and AREG with everolimus, sunitinib and sorafenib in HPV-positive and -negative SCC.

BACKGROUND/AIM: Head and neck squamous cell carcinoma (HNSCC) is an aggressive epithelial malignancy. It is the most common neoplasm appearing in the upper aerodigestive tract and the sixth most common cancer worldwide. The five-year survival rate remains poor despite advances in surgery, radiation and chemotherapy. Furthermore, the incidence of human papillomavirus (HPV)-associated oropharyngeal cancer is rising. Thus, innovative therapy approaches are imperative in order to improve the situation. Everolimus, an inhibitor of the mammalian target of rapamycin (mTOR) and sorafenib and sunitinib, multityrosine kinase inhibitors, have been notably effective in the therapy of different tumor entities. The modest side-effects and oral application of the drugs might improve patient compliance. Expression levels of mTOR and Amphiregulin (AREG) in p16-positive and -negative SCC (squamous cell carcinoma) and the effect of everolimus, sorafenib or sunitinib on the expression levels of these target proteins were assessed. As far as we are aware of, this is one of the first in vitro studies to evaluate the effect of these small-molecule drugs with regard to the p16 status of SCC cells. MATERIALS AND METHODS: p16-negative HNSCC 11A and 14C cells and p16-positive CERV196 cells were exposed to different concentrations of everolimus, sorafenib and sunitinib for 2-8 days. Expression levels of mTOR and AREG were determined by enzyme-linked immunosorbent assay (ELISA) and compared against a chemonaïve control. RESULTS: AREG and mTOR were expressed in all tested cell lines. CERV196 displayed a remarkable increase of mTOR expression compared to p16-negative HNSCC. On the contrary, AREG levels were reduced by 50% in CERV196. Everolimus, sorafenib and sunitinib significantly reduced mTOR expression. Everolimus significantly decreased AREG expression independently of the HPV status. Sunitinib and sorafenib increased AREG expression in HNSCC 11A and 14C but not in CERV196. CONCLUSION: The applied drugs showed remarkable suppression of mTOR expression, which might delay tumor progression. Interestingly, sorafenib and sunitinib increased AREG in HNSCC 11A and 14C, which could be a possible evasive mechanism following incubation with these drugs. On the contrary, p16-positive CERV196 showed increased susceptibility to sorafenib and sunitinib concerning suppression of AREG expression. Further studies are required to evaluate the HPV-dependent differences of therapy response and the possible consequences for treatment options.

Small molecules alter VEGFR and PTEN expression in HPV-positive and -negative SCC: new hope for targeted-therapy.

BACKGROUND/AIM: Prognosis for patients with head and neck squamous cell carcinoma (HNSCC) is poor in most cases and has not improved despite advances in therapy. Novel therapeutic approaches are mandatory in order to improve the situation. Everolimus, an inhibitor of mammalian target of rapamycin, as well as the multi-tyrosine kinase inhibitors sorafenib and sunitinib, has demonstrated a substantial therapeutic effect in various types of human cancer with moderate side-effects. Expression of vascular endothelial growth factor receptor (VEGFR) 1 and 2, and of the tumor-suppressor protein phosphatase and tensin homolog deleted on chromosome 10 (PTEN) were evaluated in chemonaïve human papillomavirus (HPV)-positive and -negative squamous cell carcinoma (SCC) and after exposure to everolimus, sorafenib or sunitinib. MATERIALS AND METHODS: p16-positive CERV196 and p16-negative HNSCC 11A and 14C cells were incubated with different drug concentrations for 48-192 h. Expression of VEGFR1 and -2 as well as PTEN were determined by enzyme-linked immunosorbent assay and was compared to a chemonaïve control. RESULTS: VEGFR1 and -2, as well as PTEN, were expressed in all three cell lines. Sunitinib, sorafenib and everolimus significantly reduced the expression of VEGFR1 and -2, especially in p16-positive CERV196 cells. Sunitinib appeared to be more effective in reducing VEGFR1 and -2 expression than sorafenib and everolimus. PTEN levels were remarkably lower in HPV-positive CERV196 cells. PTEN expression increased significantly under sunitinib and sorafenib in HNSCC 11A and CERV196 cells. Everolimus, on the other hand, led to a significant decrease of PTEN expression in these cell lines. CONCLUSION: The tested drugs displayed a remarkable anti-angiogenic effect by inhibition of VEGFR1 and -2 expression. Sunitinib and sorafenib were able to increase PTEN expression, which might induce apoptosis of cancer cells. HPV-positive CERV196 cells were characterized by an increased susceptibility to these small-molecule drugs. Further studies are imperative to scrutinize HPV status-dependent differences in drug response and possible implications for future treatment options.

Incomplete epithelial-mesenchymal transition in p16-positive squamous cell carcinoma cells correlates with ß-catenin expression.

BACKGROUND: The epithelial-mesenchymal transition (EMT) is suggested to be a crucial factor for the development of an invasive and metastatic cell phenotype, which is characterized by down-regulation of epithelial adhesive proteins (e.g. E-cadherin) and induction of mesenchymal proteins (e.g. vimentin). Therefore, there is a great clinical interest to specify this phenotype. Different growth factors induce EMT, such as epithelial growth factor (EGF) and transforming growth factor beta 1 (TGFß1). The role of EMT in human papilloma virus (HPV)-positive squamous cell carcinoma (SCC) is still not understood. The aim of this study was to investigate the expression pattern in p16-positive and -negative SCC cells of vimentin, ß-catenin and E-cadherin after stimulation with growth factors. MATERIALS AND METHODS: We incubated the p16-positive CERV196 and p16-negative HNSCC22B SCC cell lines with EGF and EGF/TGFß1 (10 ng/ml) and detected E-cadherin, vimentin and ß-catenin by immunocytochemistry and enzyme-linked immunosorbent assay after 5, 24 and 96 h. RESULTS: We found a low expression of vimentin in all studied tumor cell lines. The negative control of HNSCC22B cells showed a higher intrinsic level of membranous E-cadherin and ß-catenin. We found statistically significant EGF/TGFß1-induced expression of vimentin dependent on incubation time in p16-negative HNSCC22B cells. Particularly in the presence of EGF, we detected an increase of ß-catenin and vimentin expression in p16-positive SCC tumor cell lines in addition to induced cell scattering and unexpected expression of E-cadherin. CONCLUSION: In conclusion, E-cadherin, ß-catenin and vimentin expression are important features to characterize EMT-like events. We were able to show incomplete EGF-induced EMT with ß-catenin expression in p16-positive SCC. Extended studies are required to investigate the mechanistic role of EMT markers, especially in p16-positive SCC, in order to develop new anti-SCC therapies to block EMT progression.

Matrix metalloproteinase-2 and -14 in p16-positive and -negative HNSCC after exposure To 5-FU and docetaxel In Vitro.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world. While the incidence of HNSCC associated with tobacco and alcohol abuse is falling, the incidence of HNSCC associated with human papilloma virus (HPV) is rising. Proliferation, cell migration and formation of metastases are dependent on interactions between the tumor cells, tumor stromal cells and the extracellular matrix (ECM). Degradation of the ECM is a crucial step in the process of local tumor infiltration and formation of locoregional and distant metastases. Matrix metalloproteinases (MMPs) are a family of enzymes that are able to degrade the ECM. Locally advanced HNSCC with cervical node metastases are treated with docetaxel in induction chemotherapy (ICT) combined with platinum-based chemotherapy and 5-fluorouracil (5-FU) as standard clinical anti-neoplastic regimens. This study evaluated the expression of MMP-14 and MMP-2 in HPV-positive (CERV196) and HPV-negative squamous cell carcinoma (HNSCC 11A and 14C) and the alteration of expression levels after exposure to either docetaxel or 5-FU. MATERIALS AND METHODS: Tumor cells were exposed to 5-FU or docetaxel in concentrations of 1.0 and 5.0 µmol/ml. MMP-protein expression was evaluated by enzyme-linked immunosorbent assay (ELISA) after 2, 3, 5, 8 and 10 days of incubation. RESULTS: Docetaxel exposure significantly decreased MMP-14 expression in HNSCC 11A and especially 14C but not in CERV196 apart from an apoptotic process. 5-FU had no significant effect on MMP-14 expression independent of the HPV-status. Significant alterations of MMP-2 could be detected in HNSCC 11A only. CONCLUSION: Although neither of the applied drugs were selective inhibitors of MMP-expression, surprisingly docetaxel significantly decreased MMP-14 in HNSCC 14C and 11A in this study. Interestingly, HPV-positive CERV196 was not sensitive to decreased MMP-14 or -2 expression following incubation with 5-FU or docetaxel.

Imatinib-associated matrix metalloproteinase suppression in p16-positive squamous cell carcinoma compared to HPV-negative HNSCC cells in vitro.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer worldwide. The growth and invasion of HNSCC are strongly influenced by the extracellular matrix (ECM), which is modified by matrix metalloproteinases (MMPs). The MMP family is still relevant to cancer research, as it promotes malignant transformation, cell proliferation and modulation of angiogenesis even in the early stages of cancer. The proteolytic processing of bioactive molecules by MMP-14 (MT1-MMP) causes severe abnormalities in connective tissue, defective angiogenesis and premature death. MMP-2 (gelatinase A) and MMP-14 also play a role in degradation of basement membrane and cell carcinoma invasion. Imatinib blocks the PTK receptor c-kit and forestalls its PTK activity. The aim of the present study was to investigate the expression pattern of MMP-14 and MMP-2 in human papilloma virus (HPV)-negative and p16-positive SCC and to evaluate the chemosensitivity of the tumour cells to the chemotherapeutic agents, imatinib and 5-fluorouracil (5-FU). We incubated the SCC cell lines with imatinib (18 and 30 µmol/ml) and 5-FU (1 and 5 µmol/ml) and detected MMP-14 and MMP-2 by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) after 48, 72, 120, 192 and 240 h. We detected expression of MMP-2 and MMP-14 in all incubated tumour cell lines. With imatinib in particular, we found a reliable trend towards decreased MMP-2 and MMP-14 expression levels in p16-positive and p16-negative SCC tumour cell lines in addition to an induced apoptotic effect. We found statistically significant imatinib-induced suppression of MMP-2- and MMP-14, dependent on the incubation time and the cell line. We detected a significant suppression of MMP-2 and MMP-14 especially in p16-negative HNSCC14C cells after prolonged treatment time with imatinib. Dose escalation of imatinib and 5-FU had no statistically significant effect on the expression of MMP-2 or MMP-14. The p16-positive SCC cells exhibited higher expression of total protein. We detected a significant suppression of MMP-2 and MMP-14 in all the incubated SCC cell lines, partially after treatment with imatinib. We found higher suppression of MMP-2 in the CERV196 cells after incubation with imatinib. We detected a reliable trend towards increased chemosensitivity of p16-positive tumour cells in vitro after treatment with imatinib. Extended studies and clinical trials are needed to further investigate these findings in HPV-associated HNSCC.

Interaction of a CD44+ head and neck squamous cell carcinoma cell line with a stromal cell-derived factor-1-expressing supportive niche: An in vitro model.

The cancer stem cell (CSC) theory implies that CSCs are surrounded by supportive stromal cells, which are known as the CSC niche. Stromal cell-derived factor-1 (SDF-1) shows a multitude of functional effects in head and neck squamous cell carcinoma (HNSCC) cells, including migration and polarization. Therefore, the SDF-1-CXCR4 axis may be involved in the pathophysiology of the progression, recurrence and metastasis of malignant diseases of the head and neck. In the present study, the CD44+ HNSCC UM-SCC-11A cell line was used as a model for CSCs. The interaction between the UM-SCC-11A cells and the supportive microenvironmental cells, including fibrocytes, human umbilical vein endothelial cells (HUVECs) and human microvascular vein endothelial cells (HMVECs) was evaluated. All the cell types that were tested were shown to secrete different concentrations of SDF-1 into the surrounding culture medium [mean (m)fibro, 1243.3±156.2 pg/ml; mHMVEC, 1061.4±23.2 pg/ml; mHUVEC, 849.6±110.9 pg/ml]. The migration of the UM-SCC-11A cells towards the supportive cells was increased by a higher supply of SDF-1 (contrfibro, 315.23±61.55 µm; mfibro, 477.73±143.7 µm; Pfibro=0.003; contrHMVEC, 123.41±66.68 µm; mHMVEC, 249.04±111.95 µm; PHMVEC=0.004; contrHUVEC, 189.7±93.26 µm; mHUVEC, 260.82±161.58 µm). The amount of the UM-SCC-11A cells that migrated towards the differentiated fibrocytes was significantly higher than that which migrated towards the HMVECs or HUVECs (Pfibro/HMVEC=2.12E-11; Pfibro/HUVEC=2.28E-5). Cell-cell interaction by podia formation of the UM-SCC-11A cells was observed in all the supportive cell types that were tested. Broadly based cell-cell contacts were observed. By contrast, digitiform podia formations presented by the UM-SCC-11A cells were determined using fluorescence microscopy. The SDF-1-CXCR4 axis is postulated to be a crucial pathway in the interaction between CSCs and their surrounding supportive cells. Understanding the cell-cell interactions in the CSC niche using in vitro models may aid in gaining further insight into these mechanisms and finding new strategies of therapy in this field.

Small molecule-based chemotherapeutic approach in p16-positive and -negative HNSCC in vitro.

BACKGROUND: Incidence of oropharyngeal head and neck squamous cell carcinoma (HNSCC) induced by the human papilloma virus (HPV) is rising. HNSCC is the sixth most common neoplasia worldwide. The survival rate remains poor, thus innovative therapy approaches are necessary. Everolimus, an inhibitor of the mammalian target of rapamycin, as well as the multi-tyrosine kinase inhibitors sorafenib (targeting vascular endothelial growth factor receptor, platelet-derived growth factor receptor and RAF) and sunitinib (targeting vascular endothelial growth factor receptor, platelet-derived growth factor receptor, stem cell factor receptor, RET proto-oncogene and colony-stimulating factor), have shown a remarkable antitumor effect against various tumor entities, with moderate side-effects. These drugs are administered orally, which should lead to higher patient compliance and less hospitalisation. AIM: This study sought to evaluate the expression of PDGFR α/ß and hypoxia-inducible factor-1α (HIF-1α) and their alterations induced by everolimus, sorafenib and sunitinib in chemonaïve HPV-positive and HPV-negative HNSCC. To our knowledge, this is the first in vitro study to investigate such cases. MATERIALS AND METHODS: We incubated HPV-positive CERV196 and HPV-negative HNSCC 11A and 14C cells for 2 to 8 days with increasing concentration of drugs. Expression of PDGFR α/ß and HIF-1α was measured by enzyme-linked immunosorbent assay and compared to a chemonaïve controls. RESULTS: Our study showed that PDGFR α/ß and HIF-1α were expressed in all three cell lines. Incubation with everolimus, sorafenib or sunitinib led to a decrease in PDGFR α/ß and HIF-1α expression, depending on the HPV status. A statistically significant alteration of PDGFR α/ß was detected in CERV196 only. Thus, HPV-positive HNSCC exhibited a higher sensitivity to the drugs used compared to HPV-negative HNSCC 11A and 14C tumor cells. A significant reduction of HIF-1α was measured for HNSCC 11A and 14C only. An escalation of drug concentration had no significant effect. CONCLUSION: We showed that these novel agents led to a significant reduction of PDGFR and HIF-1α, depending on the HPV status. HPV positivity is associated with increased chemosensitivity and may be associated with better locoregional control and overall patient survival compared to HPV negativity. Further studies are necessary to investigate the efficacy and safety of these agents in the treatment of HPV-positive and -negative HNSCC in vivo.

Unexpected alteration of ß-catenin and c-KIT expression by 5-FU and docetaxel in p16-positive squamous cell carcinoma compared to HPV-negative HNSCC cells in vitro.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer worldwide. In several tumour entities, the tyrosine kinase receptor c-KIT is associated with tumour transformation in the epithelial tissue in cases of aberrant expression. Furthermore, tumour development and dissemination are a result of dysregulated cellular pathways such as the WNT/ß-catenin pathway. ß-Catenin is a multifunctional protein within the canonical WNT signalling pathway and a pivotal factor for the stabilization of cell-cell interactions. In malignant tissues, ß-catenin triggers tumour proliferation and progression. The aim of this study is to investigate the expression patterns of c-KIT and ß-catenin in human papillomavirus-negative and p16-positive SCC and to evaluate the chemosensitivity of the tumour cells to the chemotherapeutical agents docetaxel and 5-fluorouracil (5-FU). MATERIALS AND METHODS: We incubated the tumour cell lines with docetaxel (5 µmol/ml) and 5-FU (1 µmol/ml) and detected ß-catenin and c-KIT by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) after 48, 72, 120, 192 and 240 h. RESULTS: We found a reliable trend towards decreased ß-catenin expression levels in p16-positive and p16-negative tumour cell lines when incubated with docetaxel, in addition to induced apoptotic effect. At best, 5-FU had a slight influence on the alteration of the expression of ß-catenin. Dose escalation of docetaxel and 5-FU had no statistically significant effect on the expression of ß-catenin or c-KIT. In HPV-negative HNSCC, a reduced expression level of ß-catenin and c-KIT was detected in an incubation period-dependent manner. p16-transformed SCC (CERV196) cells were characterized by a reduced susceptibility to docetaxel induced alteration of ß-catenin expression. CONCLUSION: We were unable to confirm the clinically-substantiated increased chemosensitivity of p16-positive tumour cells in vitro. Extended studies and clinical trials are needed to investigate these findings further in HPV-associated HNSCC.

Chemotherapeutic alteration of VEGF, PDGF and PDGFRα/ß expression under 5-FU vs. docetaxel in HPV-transformed squamous cell carcinoma compared to HPV-negative HNSCC in vitro.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the most common malignant epithelial tumor in the upper aerodigestive tract. The incidence of HNSCC induced by the oncogenic human papilloma virus (HPV) is rising, indicating a growing importance of the viral etiology. Cell proliferation, migration and tumor vascularization are regulated by a set of angiogenic peptides such as PDGF (platelet-derived growth factor), PDGFRα/ß (platelet-derived growth factor receptor α/ß) and VEGF (vascular endothelial growth factor). In locally advanced HNSCC docetaxel is used for induction chemotherapy (ICT) combined with platinum-based chemotherapy and 5-fluorouracil (5-FU). This study sought to evaluate the expression of angiogenic factors (VEGF, PDGF and PDGFRα/ß) in HPV-positive (CERV196) and HPV-negative squamous cell carcinoma (HNSCC 11A and 14C) and the efficacy of chemotherapy with docetaxel as a potential treatment modality, compared to 5-FU as a single-drug application. MATERIALS AND METHODS: Tumor cell lines were incubated with 5-FU or docetaxel at a concentration of 1.0 and 5.0 µmol/ml. Enzyme-linked immunosorbent assay (ELISA) and immunohistochemical analyses were carried out after 48, 72, 120, 192 and 240 hours, in order to identify changes in protein expression of VEGF, PDGF and PDGFRα/ß. RESULTS: We demonstrated a significant reduction of VEGF and PDGFRß expression after incubation with docetaxel by ELISA and of PDGF by immunohistochemistry, irrespective of the HPV status, whereas the application of 5-FU had a significantly weaker impact on the expression of angiogenic peptides. HPV-positive CERV196 cells were characterized by a reduced susceptibility to a docetaxel-altered expression. CONCLUSION: Although neither of the applied drugs are selective anti-angiogenic agents, docetaxel surprisingly was demonstrated to cause a significant decrease of angiogenic factors in this study.

SDF-1-CXCR4 axis: cell trafficking in the cancer stem cell niche of head and neck squamous cell carcinoma.

Stromal cell-derived factor-1α (SDF-1α), also known as CXCL12, has variable effects on a plurality of cells. CXCR4 has been identified as its corresponding receptor. The SDF-1-CXCR4 axis is postulated to be a crucial key pathway in the interaction between (cancer) stem cells and their surrounding supportive cells in the cancer stem cell niche. We evaluated the expression of CD44 as a cancer stem cell marker and of CXCR4 in human HNSCC tissue samples. Afterwards, we monitored the concentration of SDF-1 in peripheral blood samples of HNSCC patients and healthy donors. We showed that CD44 and CXCR4 are expressed in human HNSCC tissues. Markedly, CD44 showed a high expression in HNSCC cells bordering cancer stromal cells. CXCR4 was mainly expressed in HNSCC tumor nests, but not in the surrounding stromal cells. No significant difference was noted between the SDF-1 concentration in the peripheral blood of HNSCC patients compared to healthy donors. We showed that CD44, as a stem cell marker in HNSCC, is located mainly at the borderline of HNSCC tumor nests with the surrounding cells. In addition, we demonstrated that CXCR4 as the corresponding receptor to SDF-1 is highly expressed in HNSCC tumor nests, but not in the tumor stroma. We collected evidence that SDF-1-CXCR4 interaction may be a crucial pathway in cell trafficking in the cancer stem cell niche of HNSCC, while SDF-1 was not detected in the peripheral blood of HNSCC patients. The SDF-1-CXCR4 axis may play an important role in the cancer stem cell theory of HNSCC. As SDF-1α also exhibits a multitude of functional effects on HNSCC cells, such as migration and polarization, it may be possible that the SDF-1-CXCR4 axis is also involved in the pathophysiology of the progression, recurrence and metastasis of malignant disease. Understanding these interactions may help to gain further insight into these mechanisms and as such help to discover new strategies of therapy.

EGF/TGFß1 co-stimulation of oral squamous cell carcinoma cells causes an epithelial-mesenchymal transition cell phenotype expressing laminin 332.

Epithelial-mesenchymal transition (EMT) is suggested to be crucial for the development of an invasive and metastatic carcinoma cell phenotype. Therefore, the definition of this phenotype is of great clinical interest. We recently evidenced vimentin positive cells in oral squamous cell carcinoma (OSCC) invasive front expressing laminin γ2 chain mRNA implicating an EMT origin of these cells. To further elucidate the nature of these cells, we have investigated the relation between EMT criteria and laminin-332 expression in a cell culture model of transforming growth factor beta-1 (TGFß1)/epithelial growth factor (EGF) long time co-stimulation. We demonstrate that in contrast to TGFß1 or EGF alone, co-stimulation induces phenotype transition in OSCC cells which fulfils the criteria of EMT in terms of vimentin up-regulation and E-cadherin down-regulation on protein level as well as cell scattering. Furthermore, cells displayed a strongly enhanced invasiveness and adhesion to type I-IV collagens. Phenotype transition is accompanied by an enhanced expression of laminin-332, especially of its γ2 chain. We further analyse the expression of extracellular matrix related genes by RT-PCR profiling. With respect to strongly enhanced proteins, data confirm the EMT phenotype of co-stimulated OSCC cells and expression of laminin-332. Furthermore, alpha catenin, collagen type 16, the integrin α7 and ß1 chains, and MMP11 are suggested as candidates with potential role in EMT in OSCC. In summary we are able to show that EMT in OSCC is mediated by multiple growth factors and is accompanied by laminin γ2 chain up-regulation evidencing the existence of an intermediate Vim(+) /Ln332(+) EMT phenotype as seen in situ.

Stromal laminin chain distribution in normal, hyperplastic and malignant oral mucosa: relation to myofibroblast occurrence and vessel formation.

BACKGROUND: The contribution of stromal laminin chain expression to malignant potential, tumour stroma reorganization and vessel formation in oral squamous cell carcinoma (OSCC) is not fully understood. Therefore, the expression of the laminin chains alpha2, alpha3, alpha4, alpha5 and gamma2 in the stromal compartment/vascular structures in OSCC was analysed. METHODS: Frozen tissue of OSCC (9x G1, 24x G2, 8x G3) and normal (2x)/hyperplastic (11x) oral mucosa was subjected to laminin chain and alpha-smooth muscle actin (ASMA) immunohistochemistry. Results were correlated to tumour grade. The relation of laminin chain positive vessels to total vessel number was assessed by immunofluorescence double labelling with CD31. RESULTS: Stromal laminin alpha2 chain significantly decreases and alpha3, alpha4, alpha5 and gamma2 chains and also ASMA significantly increase with rising grade. The amount of stromal alpha3, alpha4 and gamma2 chains significantly increased with rising ASMA positivity. There is a significant decrease in alpha3 chain positive vessels with neoplastic transformation. CONCLUSIONS: Mediated by myofibroblasts, OSCC development is associated with a stromal up-regulation of laminin isoforms possibly contributing to a migration promoting microenvironment. A vascular basement membrane reorganization concerning alpha3 and gamma2 chain laminins during tumour angioneogenesis is suggested.

Expression of Snail is associated with myofibroblast phenotype development in oral squamous cell carcinoma.

Snail is a regulator of epithelial-mesenchymal transition (EMT) and considered crucial to carcinoma metastasis, myofibroblast transdifferentiation, and fibroblast activation. To investigate the role of Snail in oral squamous cell carcinoma (OSCC), its immunohistochemical expression was analysed in 129 OSCC samples and correlated to nodal metastasis, histological grade, E-cadherin, and alpha smooth-muscle-actin (alpha SMA). The results were compared to findings in 23 basal cell carcinomas (BCC). Additionally, the influence of TGF beta 1 and EGF on Snail, E-cadherin, vimentin, and alpha SMA expression was analysed in two OSCC cell lines. As a result, Snail-positive cells were mainly found in the stroma of the OSCC invasive front without statistically significant correlation to histological grade or nodal metastasis. Snail was co-localised to alpha SMA but not to E-cadherin or cytokeratin and showed a significant correlation to the loss of membranous E-cadherin. All BCCs were Snail negative. In OSCC culture, the growth-factor-mediated EMT-like phenomenon was accompanied by alpha SMA down-regulation. In summary, Snail expression in OSCC is a stromal phenomenon associated with the myofibroblast phenotype and not related to growth-factor-mediated transdifferentiation of the carcinoma cells themselves. Consequently, Snail immunohistochemistry cannot contribute to the prediction of the metastatic potential. Furthermore, stromal Snail expression is suggested to be the result of mutual paracrine interaction of fibro-/myofibroblasts and dedifferentiated carcinoma cells leading to the generation of a special type of carcinoma-associated fibroblasts.