A Tissue Engineered 3D Model of Cancer Cell Invasion for Human Head and Neck Squamous-Cell Carcinoma.

Head and neck squamous-cell carcinoma (HNSCC) is associated with aggressive local invasiveness, being a main reason for its poor prognosis. The exact mechanisms underlying the strong invasive abilities of HNSCC remain to be elucidated. Therefore, there is a need for in vitro models to study the interplay between cancer cells and normal adjacent tissue at the invasive tumor front. To generate oral mucosa tissue models (OMM), primary keratinocytes and fibroblasts from human oral mucosa were isolated and seeded onto a biological scaffold derived from porcine small intestinal submucosa with preserved mucosa. Thereafter, we tested different methods (single tumor cells, tumor cell spots, spheroids) to integrate the human cancer cell line FaDu to generate an invasive three-dimensional model of HNSCC. All models were subjected to morphological analysis by histology and immunohistochemistry. We successfully built OMM tissue models with high in vivo-in vitro correlation. The integration of FaDu cell spots and spheroids into the OMM failed. However, with the integration of single FaDu cells into the OMM, invasive tumor cell clusters developed. Between segments of regular epithelial differentiation of the OMM, these clusters showed a basal membrane penetration and lamina propria infiltration. Primary human fibroblasts and keratinocytes seeded onto a porcine carrier structure are suitable to build an OMM. The HNSCC model with integrated FaDu cells could enable subsequent investigations into cancer cell invasiveness.

Surgical management of tympanojugular paragangliomas using the flexible CO2 laser.

PURPOSE: Surgery is a standard therapy for tympanojugular paragangliomas (TJP). Maintaining the quality of life (QoL) requires functional preservation. The flexible CO2 laser allows contact-free tumor removal. This retrospective study compares the postoperative functional outcomes of TJP surgery with and without the flexible CO2 laser. METHODS: Between 2005 and 2019, 51 patients with TJP were surgically treated at a tertiary hospital. Until 2012, 17 patients received conventional surgery. Thereafter, the flexible laser was used in 34 patients. Tumor extend, pre- and postoperative cranial nerve function, and complications were compared between the groups. RESULTS: The cohort consisted of 33 class A and B tumors and 18 class C and D tumors. Preoperative embolization was performed in 17 cases. Class C/D TJP were usually removed via an infratemporal fossa type A approach. Gross total tumor removal was achieved in 14/18 class C/D tumors. 3/51 patients suffered from long-term partial or complete facial palsy. No differences in post-therapeutic cranial nerve function or complications were noted between the conventional and laser group. One recurrence was observed after complete tumor resection. CONCLUSION: The flexible CO2 laser was shown to be a safe and effective alternative to conventional bipolar cauterization, which is appreciated by the surgeon in these highly vascularized tumors. Both techniques allowed a high tumor control rate and good long-term results also from a functional point of view.

Cultivation of Head and Neck Squamous Cell Carcinoma Cells with Wound Fluid Leads to Cisplatin Resistance via Epithelial-Mesenchymal Transition Induction.

Locoregional recurrence is a major reason for therapy failure after surgical resection of head and neck squamous cell carcinoma (HNSCC). The physiological process of postoperative wound healing could potentially support the proliferation of remaining tumor cells. The aim of this study was to evaluate the influence of wound fluid (WF) on the cell cycle distribution and a potential induction of epithelial-mesenchymal transition (EMT). To verify this hypothesis, we incubated FaDu and HLaC78 cells with postoperative WF from patients after neck dissection. Cell viability in dependence of WF concentration and cisplatin was measured by flow cytometry. Cell cycle analysis was performed by flow cytometry and EMT-marker expression by rtPCR. WF showed high concentrations of interleukin (IL)-6, IL-8, IL-10, CCL2, MCP-1, EGF, angiogenin, and leptin. The cultivation of tumor cells with WF resulted in a significant increase in cell proliferation without affecting the cell cycle. In addition, there was a significant enhancement of the mesenchymal markers Snail 2 and vimentin, while the expression of the epithelial marker E-cadherin was significantly decreased. After cisplatin treatment, tumor cells incubated with WF showed a significantly higher resistance compared with the control group. The effect of cisplatin-resistance was dependent on the WF concentration. In summary, proinflammatory cytokines are predominantly found in WF. Furthermore, the results suggest that EMT can be induced by WF, which could be a possible mechanism for cisplatin resistance.

The Impact of Tissue Preparation on Salivary Gland Tumors Investigated by Fourier-Transform Infrared Microspectroscopy.

Due to the wide variety of benign and malignant salivary gland tumors, classification and malignant behavior determination based on histomorphological criteria can be difficult and sometimes impossible. Spectroscopical procedures can acquire molecular biological information without destroying the tissue within the measurement processes. Since several tissue preparation procedures exist, our study investigated the impact of these preparations on the chemical composition of healthy and tumorous salivary gland tissue by Fourier-transform infrared (FTIR) microspectroscopy. Sequential tissue cross-sections were prepared from native, formalin-fixed and formalin-fixed paraffin-embedded (FFPE) tissue and analyzed. The FFPE cross-sections were dewaxed and remeasured. By using principal component analysis (PCA) combined with a discriminant analysis (DA), robust models for the distinction of sample preparations were built individually for each parotid tissue type. As a result, the PCA-DA model evaluation showed a high similarity between native and formalin-fixed tissues based on their chemical composition. Thus, formalin-fixed tissues are highly representative of the native samples and facilitate a transfer from scientific laboratory analysis into the clinical routine due to their robust nature. Furthermore, the dewaxing of the cross-sections entails the loss of molecular information. Our study successfully demonstrated how FTIR microspectroscopy can be used as a powerful tool within existing clinical workflows.

Evaluation of the cytotoxic and genotoxic potential of printer toner particles in a 3D air-liquid interface, primary cell-based nasal tissue model.

Printer toner particles (TPs) are a common, potentially hazardous substance, with an unclear toxicological impact on the respiratory mucosa. Most of the airways surface is covered by a ciliated respiratory mucosa, therefore appropriate tissue models of the respiratory epithelium with a high in vivo correlation are necessary for in vitro evaluation of airborne pollutants toxicology and the impact on the functional integrity. The aim of this study is the evaluation of TPs toxicology in a human primary cell-based air-liquid-interface (ALI) model of respiratory mucosa. The TPs were analyzed and characterized by scanning electron microscopy, pyrolysis and X-ray fluorescence spectrometry. ALI models of 10 patients were created using the epithelial cells and fibroblasts derived from nasal mucosa samples. TPs were applied to the ALI models via a modified Vitrocell® cloud and submerged in the dosing 0.89 - 892.96 µg/ cm2. Particle exposure and intracellular distribution were evaluated by electron microscopy. The MTT assay and the comet assay were used to investigate cytotoxicity and genotoxicity, respectively. The used TPs showed an average particle size of 3 - 8 µm. Mainly carbon, hydrogen, silicon, nitrogen, tin, benzene and benzene derivates were detected as chemical ingredients. By histomorphology and electron microscopy we observed the development of a highly functional, pseudostratified epithelium with a continuous layer of cilia. Using electron microscopy, TPs could be detected on the cilia surface and also intracellularly. Cytotoxicity was detected from 9 µg/ cm2 and higher, but no genotoxicity after ALI and submerged exposure. The ALI with primary nasal cells represents a highly functional model of the respiratory epithelium in terms of histomorphology and mucociliary differentiation. The toxicological results indicate a weak TP-concentration-dependent cytotoxicity. AVAILABILITY OF DATA AND MATERIALS: The datasets used and analysed during the current study are available from the corresponding author on reasonable request.

Differentiation of Salivary Gland and Salivary Gland Tumor Tissue via Raman Imaging Combined with Multivariate Data Analysis.

Salivary gland tumors (SGTs) are a relevant, highly diverse subgroup of head and neck tumors whose entity determination can be difficult. Confocal Raman imaging in combination with multivariate data analysis may possibly support their correct classification. For the analysis of the translational potential of Raman imaging in SGT determination, a multi-stage evaluation process is necessary. By measuring a sample set of Warthin tumor, pleomorphic adenoma and non-tumor salivary gland tissue, Raman data were obtained and a thorough Raman band analysis was performed. This evaluation revealed highly overlapping Raman patterns with only minor spectral differences. Consequently, a principal component analysis (PCA) was calculated and further combined with a discriminant analysis (DA) to enable the best possible distinction. The PCA-DA model was characterized by accuracy, sensitivity, selectivity and precision values above 90% and validated by predicting model-unknown Raman spectra, of which 93% were classified correctly. Thus, we state our PCA-DA to be suitable for parotid tumor and non-salivary salivary gland tissue discrimination and prediction. For evaluation of the translational potential, further validation steps are necessary.

Head and neck squamous cancer cells enhance the differentiation of human mesenchymal stem cells to adipogenic and osteogenic linages in vitro.

Human mesenchymal stem cells (hMSC) are multipotent cells with the ability to differentiate into a range of different cell types, including fat, bone, cartilage or muscle. A pro-tumorigenic effect of hMSC has been previously reported as part of the tumor stroma. In addition, studies have previously revealed the influence of hematopoietic and lymphoid tumors on hMSC differentiation to support their own growth. However, this possible phenomenon has not been explored in solid malignancies. Therefore, the aim of the present study was to investigate the effects of head and neck squamous cell carcinoma (HNSCC) lines Cal27 and HLaC78 on the induction of osteogenic and adipogenic differentiation in hMSCs. Native hMSCs were co-cultured with Cal27 and HLaC78 cells for 3 weeks. Subsequently, hMSC differentiation was assessed using reverse transcription-PCR and using Oil Red O and von Kossa staining. Furthermore, the effects of differentiated hMSCs on Cal27 and HLaC78 were examined. For this purpose, hMSCs differentiated into the adipogenic (adipo-hMSC) and osteogenic (osteo-hMSC) lineages were co-cultured with Cal27 and HLaC78. Cell viability, cytokine secretion and activation of STAT3 signaling were measured by cell counting, dot blot assay (42 cytokines with focus on IL-6) and western blotting (STAT3, phosphorylated STAT3, ß-actin), respectively. Co-culturing hMSCs with Cal27 and HLaC78 cells resulted in both adipogenic and osteogenic differentiation. In addition, the viability of Cal27 and HLaC78 cells was found to be increased after co-cultivation with adipo-hMSCs, compared with that of cells co-cultured with osteo-hMSC. According to western blotting results, Cal27 cells incubated with adipo-hMSCs exhibited increased STAT3 activation, compared with that in cells co-cultured with native hMSCs and osteo-hMSCs. IL-6 concentration in the media of Cal27 and HLaC78 after co-cultivation with respectively incubation with conditioned media of hMSCs, adipo-hMSCs and osteo-hMSCs were also found to be increased compared with that in the media of Cal27 and HLaC78 cells incubated with DMEM. To conclude, HNSCC cell lines Cal27 and HLaC78 induced hMSC differentiation towards the adipogenic and osteogenic lineages in vitro. Furthermore, a proliferative effect of adipo-hMSCs on Cal27 and HLaC78 cells was revealed with STAT3 activation as a possible mechanism. These results warrant further investigation of the interaction between HNSCC cells and hMSCs, with focus on the mechanism underlying the differentiation of hMSCs.

Investigation of the Immune Modulatory Potential of Zinc Oxide Nanoparticles in Human Lymphocytes.

Zinc oxide nanoparticles (ZnO-NP) are commonly used for a variety of applications in everyday life. In addition, due to its versatility, nanotechnology supports promising approaches in the medical sector. NP can act as drug-carriers in the context of targeted chemo- or immunotherapy, and might also exhibit autonomous immune-modulatory characteristics. Knowledge of potential immunosuppressive or stimulating effects of NP is indispensable for the safety of consumers as well as patients. In this study, primary human peripheral blood lymphocytes of 9 donors were treated with different sub-cytotoxic concentrations of ZnO-NP for the duration of 1, 2, or 3 days. Flow cytometry was performed to investigate changes in the activation profile and the proportion of T cell subpopulations. ZnO-NP applied in this study did not induce any significant alterations in the examined markers, indicating their lack of impairment in terms of immune modulation. However, physicochemical characteristics exert a major influence on NP-associated bioactivity. To allow a precise simulation of the complex molecular processes of immune modulation, a physiological model including the different components of an immune response is needed.

Pilot study on the value of Raman spectroscopy in the entity assignment of salivary gland tumors.

BACKGROUND: The entity assignment of salivary gland tumors (SGT) based on histomorphology can be challenging. Raman spectroscopy has been applied to analyze differences in the molecular composition of tissues. The aim of this study was to evaluate the suitability of RS for entity assignment in SGT. METHODS: Raman data were collected in deparaffinized sections of pleomorphic adenomas (PA) and adenoid cystic carcinomas (ACC). Multivariate data and chemometric analysis were completed using the Unscrambler software. RESULTS: The Raman spectra detected in ACC samples were mostly assigned to nucleic acids, lipids, and amides. In a principal component-based linear discriminant analysis (LDA) 18 of 20 tumor samples were classified correctly. CONCLUSION: In this proof of concept study, we show that a reliable SGT diagnosis based on LDA algorithm appears possible, despite variations in the entity-specific mean spectra. However, a standardized workflow for tissue sample preparation, measurement setup, and chemometric algorithms is essential to get reliable results.

Systematic analysis of button batteries', euro coins', and disk magnets' radiographic characteristics and the implications for the differential diagnosis of round radiopaque foreign bodies in the esophagus.

OBJECTIVES: Button battery (BB) ingestion can cause life-threatening complications such as esophageal perforation, and mediastinitis. Chest radiography is the method of choice to diagnose BB ingestion. Therefore, an adequate interpretation of X-ray imaging by physicians is mandatory. The study aimed to analyze relevant diagnostic aspects of BB X-ray imaging, and retrospective analysis of the imaging of radiopaque foreign bodies. METHODS: All commercially available BBs and different foreign bodies such as euro coins (EC) and disk magnets (DM) were listed according to their sizes and compositions. Furthermore, an X-ray analysis of a test set of BBs, ECs and DMs was performed at angles of 0°, 45°, and 90°. Retrospective assessment of radiological characteristics was performed for patients with a round and radiopaque foreign body in the esophagus RESULTS: Only BBs with a diameter of at least 20 mm had a reliable positive double rim sign at scan angles of 90°, and 45°. The step-off effect was visible in all BBs regardless of the diameter. DMs and ECs presented a homogeneous X-ray weakening at all investigated scan angles. In the retrospective analysis all five cases of BB ingestion showed a double rim sign, all fifteen cases of coin ingestion a homogeneous X-ray weakening. A corrugated margin was detected for 10 cent, 20 cent and 50 cent coins. CONCLUSIONS: BBs with a diameter of at least 20 mm showed, depending on the battery model, a more or less pronounced double rim sign and step-off effect in conventional X-ray imaging. However the double rim sign can be simulated by for example a pot magnet.

The Radiosensitizing Effect of Zinc Oxide Nanoparticles in Sub-Cytotoxic Dosing Is Associated with Oxidative Stress In Vitro.

Radioresistance is an important cause of head and neck cancer therapy failure. Zinc oxide nanoparticles (ZnO-NP) mediate tumor-selective toxic effects. The aim of this study was to evaluate the potential for radiosensitization of ZnO-NP. The dose-dependent cytotoxicity of ZnO-NP20 nm and ZnO-NP100 nm was investigated in FaDu and primary fibroblasts (FB) by an MTT assay. The clonogenic survival assay was used to evaluate the effects of ZnO-NP alone and in combination with irradiation on FB and FaDu. A formamidopyrimidine-DNA glycosylase (FPG)-modified single-cell microgel electrophoresis (comet) assay was applied to detect oxidative DNA damage in FB as a function of ZnO-NP and irradiation exposure. A significantly increased cytotoxicity after FaDu exposure to ZnO-NP20 nm or ZnO-NP100 nm was observed in a concentration of 10 µg/mL or 1 µg/mL respectively in 30 µg/mL of ZnO-NP20 nm or 20 µg/mL of ZnO-NP100 nm in FB. The addition of 1, 5, or 10 µg/mL ZnO-NP20 nm or ZnO-NP100 nm significantly reduced the clonogenic survival of FaDu after irradiation. The sub-cytotoxic dosage of ZnO-NP100 nm increased the oxidative DNA damage compared to the irradiated control. This effect was not significant for ZnO-NP20 nm. ZnO-NP showed radiosensitizing properties in the sub-cytotoxic dosage. At least for the ZnO-NP100 nm, an increased level of oxidative stress is a possible mechanism of the radiosensitizing effect.