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.

Differentiation Behaviour of Adipose-Derived Stromal Cells (ASCs) Seeded on Polyurethane-Fibrin Scaffolds In Vitro and In Vivo.

Adipose-derived stromal cells (ASCs) are a promising cell source for tissue engineering and regenerative medicine approaches for cartilage replacement. For chondrogenic differentiation, human (h)ASCs were seeded on three-dimensional polyurethane (PU) fibrin composites and induced with a chondrogenic differentiation medium containing TGF-ß3, BMP-6, and IGF-1 in various combinations. In addition, in vitro predifferentiated cell-seeded constructs were implanted into auricular cartilage defects of New Zealand White Rabbits for 4 and 12 weeks. Histological, immunohistochemical, and RT-PCR analyses were performed on the constructs maintained in vitro to determine extracellular matrix (ECM) deposition and expression of specific cartilage markers. Chondrogenic differentiated constructs showed a uniform distribution of cells and ECM proteins. RT-PCR showed increased gene expression of collagen II, collagen X, and aggrecan and nearly stable expression of SOX-9 and collagen I. Rabbit (r)ASC-seeded PU-fibrin composites implanted in ear cartilage defects of New Zealand White Rabbits showed deposition of ECM with structures resembling cartilage lacunae by Alcian blue staining. However, extracellular calcium deposition became detectable over the course of 12 weeks. RT-PCR showed evidence of endochondral ossification during the time course with the expression of specific marker genes (collagen X and RUNX-2). In conclusion, hASCs show chondrogenic differentiation capacity in vitro with the expression of specific marker genes and deposition of cartilage-specific ECM proteins. After implantation of predifferentiated rASC-seeded PU-fibrin scaffolds into a cartilage defect, the constructs undergo the route of endochondral ossification.

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.

Effect of Hypoxia on Proliferation and the Expression of the Genes HIF-1α and JMJD1A in Head and Neck Squamous Cell Carcinoma Cell Lines.

BACKGROUND/AIM: The aim of the study was to investigate the effects of hypoxia on proliferation and the expression of HIF-1α (hypoxia-inducible factor 1 alpha) and JMJD1A (jumonji domain 1A) in head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: FaDu and HLaC78 cells were incubated for 1-24 h in hypoxia and normoxia. Cell proliferation, mRNA and protein levels of HIF-1α and JMJD1A were quantified by counting, PCR and western blot. RESULTS: Hypoxia led to a constant decrease in cell proliferation. Short hypoxia resulted in an increase in HIF-1α mRNA levels. This effect was reversed after longer incubation. The western blot for HIF-1α showed a maximum accumulation after 3-6 h of hypoxia. In FaDu cells, the concentration of JMJD1A reached a peak after 6 h and decreased thereafter, whereas in HLaC78 cells, it presented a second peak after 48 h. CONCLUSION: The transcription factors HIF-1α and JMJDA1 were confirmed as relevant hypoxia-dependent regulators of carcinogenesis in HNSCC.

Superparamagnetic Iron Oxide Particles (VSOPs) Show Genotoxic Effects but No Functional Impact on Human Adipose Tissue-Derived Stromal Cells (ASCs).

Adipose tissue-derived stromal cells (ASCs) represent a capable source for cell-based therapeutic approaches. For monitoring a cell-based application in vivo, magnetic resonance imaging (MRI) of cells labeled with iron oxide particles is a common method. It is the aim of the present study to analyze potential DNA damage, cytotoxicity and impairment of functional properties of human (h)ASCs after labeling with citrate-coated very small superparamagnetic iron oxide particles (VSOPs). Cytotoxic as well as genotoxic effects of the labeling procedure were measured in labeled and unlabeled hASCs using the MTT assay, comet assay and chromosomal aberration test. Trilineage differentiation was performed to evaluate an impairment of the differentiation potential due to the particles. Proliferation as well as migration capability were analyzed after the labeling procedure. Furthermore, the labeling of the hASCs was confirmed by Prussian blue staining, transmission electron microscopy (TEM) and high-resolution MRI. Below the concentration of 0.6 mM, which was used for the procedure, no evidence of genotoxic effects was found. At 0.6 mM, 1 mM as well as 1.5 mM, an increase in the number of chromosomal aberrations was determined. Cytotoxic effects were not observed at any concentration. Proliferation, migration capability and differentiation potential were also not affected by the procedure. Labeling with VSOPs is a useful labeling method for hASCs that does not affect their proliferation, migration and differentiation potential. Despite the absence of cytotoxicity, however, indications of genotoxic effects have been demonstrated.

Erratum: Scherzad, A., et al. Molecular Mechanisms of Zinc Oxide Nanoparticle-Induced Genotoxicity Short Running Title: Genotoxicity of ZnO NPs. Materials 2017, 10, 1427.

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Interaction of head and neck squamous cell carcinoma cells and mesenchymal stem cells under hypoxia and normoxia.

Mesenchymal stem cells (MSCs) exhibit strong tropism towards tumor tissue. While MSCs generally surround tumors, they can also infiltrate tumors and thereby influence their proliferation. Interactions between MSCs and tumor cells are usually tested under normoxia, but the majority of solid tumors, including head and neck squamous cell carcinoma (HNSCC), are also characterized by hypoxic areas. Hence, the present study aimed to assess the interaction between MSCs and tumor cells under hypoxic conditions. MSCs were cultivated under normoxia and hypoxia, and conditioned media were used to cultivate the HNSCC cell line FaDu. The cell cycle distribution and viability of MSCs and the proliferation of FaDu cells were analyzed under normoxia and hypoxia, and changes in cytokine levels in the conditioned media were evaluated. No cell cycle changes were observed for MSCs after 24 h of cultivation under hypoxia, but the cell viability had declined. Hypoxia also led to a decrease in the proliferation of FaDu cells; however, FaDu cells proliferated faster after 48 h under hypoxia compared with normoxic conditions. This effect was reversed after incubation under normoxia for 72 h and hypoxia for 72 h. While these changes constituted a trend, these differences were not statistically significant. A cytokine assay showed an increase in interleukin (IL)-6 in the hypoxic medium. Overall, the results indicated that there was an interaction between MSCs and tumor cells. The presence or absence of oxygen seemed to influence the functionality of MSCs and their protumorigenic properties, in which IL-6 was identified as a potential mediator. Since MSCs are a component of the tumor stroma, further in vitro and in vivo studies are needed to investigate this interaction in order to develop novel approaches for tumor therapy.

Effects of Zinc Oxide Nanoparticles in HUVEC: Cyto- and Genotoxicity and Functional Impairment After Long-Term and Repetitive Exposure in vitro.

PURPOSE: The present study focuses on threshold levels for cytotoxicity after long-term and repetitive exposure for HUVEC as a model for the specific microvascular endothelial system. Furthermore, possible genotoxic effects and functional impairment caused by ZnO NPs in HUVEC are elucidated. METHODS: Thresholds for cytotoxic effects are determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Annexin V assay. To demonstrate DNA damage, single-cell microgel electrophoresis (comet) assay is performed after exposure to sub-cytotoxic concentrations of ZnO NPs. The proliferation assay, dot blot assay and capillary tube formation assay are also carried out to analyze functional impairment. RESULTS: NPs showed to be spherical in shape with an average size of 45-55 nm. Long-term exposure as well as repetitive exposure with ZnO NPs exceeding 25 µg/mL lead to decreased viability in HUVEC. In addition, DNA damage was indicated by the comet assay after long-term and repetitive exposure. Twenty-four hours after long-term exposure, the proliferation assay does not show any difference between negative control and exposed cells. Forty-eight hours after exposure, HUVEC show an inverse concentration-related ability to proliferate. The dot blot assay provides evidence that ZnO NPs lead to a decreased release of VEGF, while capillary tube formation assay shows restriction in the ability of HUVEC to build tubes and meshes as a first step in angiogenesis. CONCLUSION: Sub-cytotoxic concentrations of ZnO NPs lead to DNA damage and functional impairment in HUVEC. Based on these data, ZnO NPs may affect neo-angiogenesis. Further investigation based on tissue cultures is required to elucidate the impact of ZnO NPs on human cell systems.

DNA Stability, Regeneration Capacity, and Mucociliary Differentiation of Human Nasal Mucosa Cells in Tissue Systems.

For culture models of primary cells of the human nasal mucosa, monocultures with epithelial cells (ECs) are used as well as cocultures with ECs and fibroblasts (FBs). Well-differentiated models of the respiratory nasal epithelium can be used for ecogenotoxicological assessments, for experiments on host/pathogen interactions, or tissue engineering. However, long-term cultivation and repeated passaging may induce a loss of DNA integrity or cell functionality. The aim of this study was to evaluate these parameters in test systems created from primary nasal mucosa cells. Enzymatic and sequential cell isolation from nasal tissue was performed. EC monocultures and compartment-separated EC-FB cocultures were cultivated over three passages under air/liquid interface conditions. DNA stability and regenerative capacity at the DNA and chromosomal level as well as proliferation and cell differentiation were examined. Both methods showed equivalent levels of DNA stability and regenerative capacity over all passages. Sequential growth of the coculture provided higher cell purity, while enzymatic cell harvest was associated with FB contamination in EC culture. Mucociliary differentiation was verified with electron microscopy in both methods. Functionality measured by lipopolysaccharide stimulation of interleukins was constant over long-term cultivation. Our data confirm DNA stability in long-term cell cultivation as well as functional integrity in both culture methods. Sequential cell isolation should be favored over enzymatic isolation due to higher culture purity. Impact statement Cell culture models are frequently used for ecogenotoxicological assessments, for experiments on host/pathogen interactions, or tissue engineering. However, DNA stability and functional integrity after long-term cultivation in such tissue models have not been investigated, yet. This study is the first showing systematic and evident data on DNA damage and functional aspects in primary human cell culture models of nasal epithelium.

Toxicity and Functional Impairment in Human Adipose Tissue-Derived Stromal Cells (hASCs) Following Long-Term Exposure to Very Small Iron Oxide Particles (VSOPs).

Magnetic nanoparticles (NPs), such as very small iron oxide NPs (VSOPs) can be used for targeted drug delivery, cancer treatment or tissue engineering. Another important field of application is the labelling of mesenchymal stem cells to allow in vivo tracking and visualization of transplanted cells using magnetic resonance imaging (MRI). For these NPs, however, various toxic effects, as well as functional impairment of the exposed cells, are described. The present study evaluates the influence of VSOPs on the multilineage differentiation ability and cytokine secretion of human adipose tissue derived stromal cells (hASCs) after long-term exposure. Human ASCs were labelled with VSOPs, and the efficacy of the labelling was documented over 4 weeks in vitro cultivation of the labelled cells. Unlabelled hASCs served as negative controls. Four weeks after labelling, adipogenic and osteogenic differentiation was histologically evaluated and quantified by polymerase chain reaction (PCR). Changes in gene expression of IL-6, IL-8, VEGF and caspase 3 were determined over 4 weeks. Four weeks after the labelling procedure, labelled and unlabelled hASCs did not differ in the gene expression of IL-6, IL-8, VEGF and caspase 3. Furthermore, the labelling procedure had no influence on the multidifferentiation ability of hASC. The percentage of labelled cells decreased during in vitro expansion over 4 weeks. Labelling with VSOPs and long-term intracellular disposition probably have no influence on the physiological functions of hASCs. This could be important for the future in vivo use of iron oxide NPs.

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.

Long-Term Impact of Zinc Oxide Nanoparticles on Differentiation and Cytokine Secretion of Human Adipose-Derived Stromal Cells.

Zinc oxide nanoparticles (ZnO-NPs) are widely utilized, for example in manufacturing paints and in the cosmetic industry. In addition, there is raising interest in the application of NPs in stem cell research. However, cytotoxic, genotoxic and pro-inflammatory effects were shown for NPs. The aim of this study was to evaluate the impact of ZnO-NPs on cytokine secretion and differentiation properties of human adipose tissue-derived stromal cells (ASCs). Human ASCs were exposed to the subtoxic concentration of 0.2 µg/mL ZnO-NPs for 24 h. After four weeks of cultivation, adipogenic and osteogenic differentiation procedures were performed. The multi-differentiation potential was confirmed histologically and using polymerase chain reaction (PCR). In addition, the gene expression of IL-6, IL-8, vascular endothelial growth factor (VEGF) and caspase 3 was analyzed. Over the course of four weeks after ZnO-NPs exposure, no significant differences were detected in the gene expression of IL-6, IL-8, VEGF and caspase 3 compared to non-exposed cells. The differentiation was also not affected by the ZnO-NPs. These findings underline the fact, that functionality of ASCs is likely to be unaffected by ZnO-NPs, despite a long-term disposition of NPs in the cells, supposing that the starting concentration was safely in the non-toxic range. This might provide important information for single-use nanomedical applications of ZnO-NPs.

[In vitro exposure of the shisha tobacco ingredient glycerol to human mucosa cells and lymphocytes]. / In vitro-Exposition des Shisha-Tabak-Bestandteils Glycerin an humanen Mukosazellen und Lymphozyten.

Shisha tobacco has a higher amount of glycerol than cigarette tobacco. Moreover, new legislation in Germany cancels the old limitation of humectants in shisha tobacco. Although higher amounts of glycerol in tobacco are expected, the knowledge of the toxicological profile of glycerol regarding human cells is incomplete. Aim of the study was to test glycerol for cytotoxic and genotoxic effects and to discuss the risk of humectants in shisha tobacco and the situation of German tobacco control.Lymphocytes and nasal mucosa cells of 10 patients were exposed to different glycerol levels (0.001 mol/l to 6.0 mol/l). Cytotoxic effects were examined by trypan blue exclusion test, genotoxic effects by comet assay and micronucleus test.The trypan blue exclusion test revealed significant cytotoxic effects on lymphocytes and nasal mucosa cells for glycerol concentrations of 1.0 mol/l and higher. In the comet assay a significant DNA damage could be shown for glycerol levels of 1.0 mol/l and higher. No significant micronucleus formation was monitored.While the geno- and cytotoxicity were seen in concentrations of glycerol clearly exceeding the concentrations in main stream smoke of shishas, genotoxicity is a stochastic risk occurring even at subtoxic levels. Furthermore, toxicity in lower levels could result from tobacco combustion or interactions with other smoke components. For an extensive evaluation of the risks of humectants in shisha tobacco further studies are needed. In addition, there is an enormous need for introducing further measures of tobacco control policy in Germany.

Wound fluid enhances cancer cell proliferation via activation of STAT3 signal pathway in vitro.

Wound healing begins immediately after surgery with a modification of the microenvironment via a well­orchestrated interaction between cells, cytokines and growth factors. Some of these growth factors and cytokines have mitogenic effects on cancer cells, which may lead to enhanced cancer cell proliferation and early metastatic events. The present study aimed to investigate the effects of wound fluid (WF) on the head and neck squamous carcinoma cell lines FaDu and HLaC78 in vitro. WF was harvested from 7 patients who had undergone a planned neck dissection. The presence of cytokines and growth factors was evaluated with the dot blot assay. Proliferation and cell viability were investigated via MTT assay and Ki-67 staining. Cell invasion was measured via tree­dimensional invasion assay. Western blotting was used to investigate STAT 3 activation. WF contained several cytokines and growth factors responsible for pro­ and anti­inflammation, chemotaxis, proliferation and angiogenesis. The proliferation effect of WF on FaDu and HLaC78 was concentration dependent. Media with 40% WF resulted in the highest proliferation effect. FaDu and HLaC78 exhibited enhanced motility after cultivation with 40% WF compared with cultivation with expansion medium. Cultivating cancer cells with WF had no advantageous effect on cell viability after the paclitaxel treatment. Western blot analysis revealed enhanced activation of the STAT3 signaling pathway by WF in both FaDu and HLaC78. In conclusion, surgery leads to excessive release of mitogenic factors. The contact of non­resected cancer cells and these factors may have a negative impact on patient outcome. Future investigations should specifically focus on the inhibition of mitogenic factors following cancer surgery in order to prevent early metastasis and cancer recurrence.

Influence of nasal polyp tissue on the differentiation and activation of T lymphocytes in a co-culture system.

T cell subpopulations in nasal polyps differ from peripheral lymphocytes in patients with chronic rhinosinusitis with nasal polyps (CRSwNP). However, little is known about the modulatory influence of the inflamed nasal polyp epithelial cells on the phenotype of the T cells. The aim of the present study was to assess this interaction. Tissue and blood samples were collected from 16 patients undergoing paranasal sinus surgery. Polypoid tissue was cultured under air-liquid interface conditions. Subsequently, cluster of differentiation (CD)3/CD28 activated peripheral lymphocytes from the same patients were added. After 3 days lymphocytes were separated from co-culture and analyzed by multicolor flow cytometry. Additionally, cytokine expression of the polyp tissue was measured using a human T helper cell (TH)1/TH2/TH17 antibody array. Viability staining of CD3+ lymphocytes detected fewer apoptotic cells under co-culture conditions compared with in mono-culture. There was a significantly higher frequency of CD4+ and CD8+ T cells in the co-culture system than in PBMC culture alone. Human leukocyte antigen (HLA)-DR isotype was significantly downregulated on co-cultured CD3+ lymphocytes and CD3+CD4+ T cells compared with the mono-cultured counterparts. Conventional Forkhead box P3- memory CD4+ T cells and activated regulatory T cells increased in frequency, and resting regulatory T cells decreased in the co-culture. Cytokine analysis identified expression of interleukin (IL)-6, IL-6 receptor, granulocyte-macrophage colony-stimulating factor, transforming growth factor-ß and macrophage inflammatory protein-3 in the polyp tissue. In summary, the present study performed a comparison between peripheral lymphocytes cultured with and without nasal polyp tissue cells was performed. The downregulation of HLA and the differentiation of Treg and Tconv by nasal polypoid tissue on PBMCs was demonstrated. Interestingly, the in vivo downregulation of HLA-DR on CD3+ lymphocytes, as reported previously, was confirmed in vitro. The inhibitory effect of polypoid tissue on the activation of lymphocytes is a possible pathogenic mechanism underlying CRSwNP.

Investigation of Cellular Function and DNA Integrity during 2D in vitro Culture of Human Salivary Gland Epithelial Cells.

In vitro culture of human salivary gland epithelial cells (SGEC) is still a challenge. A high quantity and quality of cells are needed for the cultivation of 3D matrices. Furthermore, it is known that DNA damage is supposed to be an important factor involved in carcinogenesis. This study investigates cellular function and DNA integrity of human SGEC during 3 passage steps in 2 groups (group 1: n = 10; group 2: n = 9). Cellular function was analyzed by immunofluorescence, transmission electron microscopy (TEM), and quantitative real-time polymerase chain reaction (qPCR). DNA integrity was tested via the comet assay. Immunohistochemistry and qPCR results showed stable α-amylase and pan-cytokeratin levels; TEM revealed functional cells; and no significant DNA damage could be detected in the comet assay during 3 culture steps. The study shows that not only at cellular but also at DNA level human SGEC can be safely quantified over 3 passages for preclinical tissue engineering without loss of differentiation and function.

[Mesenchymal stem cells: Cancer promoting effects or tumor suppression - A current overview]. / Mesenchymale Stammzellen: Tumorfördernde oder -hemmende Eigenschaften ­ Ein aktueller Überblick.

The multimodal treatment of cancer deals with cancer cells as well as with the cancer surrounding stroma. This stroma contains non-malignant cells like fibroblasts, immune cells as well as mesenchymal stem cells (MSC). MSC have the ability to migrate towards cancer tissue. In the current literature the impact of MSC on cancer cells is discussed divergently. The majority of the current publications reveal an induction of cancer progression by MSC. Four main processes namely the secretion of soluble factors and cell-cell contact, the transdifferentiation of MSC into carcinoma associated fibroblasts, the improvement of neoangiogenesis and the induction of immune suppression are responsible for cancer progression. This publication gives an overview on the current literature.

Effects of the novel polyphenol conjugate DPP-23 on head and neck squamous cell carcinoma cells in vitro.

Despite partial advances in therapy for patients suffering from head and neck squamous cell carcinomas (HNSCC), prognosis still remains poor with minimal improvement in survival for over the last several decades. Some agents found are known to cause cancer cell death in vitro by promoting cellular reactive oxygen species (ROS) accumulation. This is particularly of interest as some cancer cells are more sensitive to ROS than normal cells. It could be shown that the novel polyphenol conjugate (E)-3-(3',5'-Dimethoxyphenyl)-1-(2'-methoxyphenyl)prop-2-en-1-one (DPP-23) offers antitumor effects by the selective generation of ROS without an indication of toxicity in normal tissues in vitro and in vivo. In order to further evaluate the role of DPP-23 as a potential agent in head and neck oncology, the present study investigated its cytotoxic effects on well-established HNSCC cell lines such as HLaC 78 and FaDu, as well as primary human bone marrow stem cells (hBMSCs) and human peripheral blood lymphocytes in vitro. As DPP-23 is not commercially available, it was synthesized via a 'cold' procedure of the Claisen-Schmidt condensation. Following cell treatment with DPP-23 for 24 h, viability and apoptosis were measured via a MTT assay and the Annexin V-propidium iodide test. The results suggest a dose-dependent cytotoxicity in the tested HNSCC tumor cell lines, as well as in hBMSC and lymphocytes. In contrast to previous findings, these preliminary results indicate that the cytotoxic effects of DPP-23 in benign cells may be notably greater than previously suspected. This may indicate a limitation for in the feasibility, or at least of the systemic application, of DPP-23 for patients with HNSCC.

Accumulation of CD69+ tissue­resident memory T cells in the nasal polyps of patients with chronic rhinosinusitis.

In patients with chronic rhinosinusitis with nasal polyps (CRSwNP), a relative accumulation of cluster of differentiation (CD)8+ T cells over CD4+ T cells occurs in nasal polyps compared with the peripheral blood. Nasal CD8+ T cells and CD4+ T cells predominantly present an effector memory phenotype. Immunological studies have reported that memory T cells recirculate from the tissues to the peripheral blood and a high percentage of these T cells persist within the tissue. The aim of the present study was to characterize CD69+ sphingosine­1­phosphate receptor 1 (S1PR1)­ tissue resident memory T cells (Trm) in the polyps of patients with CRSwNP. Tissue and blood samples were collected from 10 patients undergoing nasal sinus surgery. Expression of specific extra­ and intracellular molecules were analyzed using multicolor flow cytometry. A significantly higher level of CD8+ T cells than CD4+ T cells was present in nasal polyps, while significantly more CD4+ T cells than CD8+ T cells were detected in the peripheral blood of patients with CRSwNP. The frequency of CD69+ T cells was significantly higher in CD8+ and CD4+ T cells in nasal polyps compared with the peripheral blood. The frequency of CD69+ S1PR1­ Trm was also significantly higher in CD4+ and CD8+ T cells from nasal polyps compared with the peripheral blood. Within polyps, the frequency of CD69+ S1PR1­ Trm was again significantly higher in CD8+ compared with CD4+ T cells. In summary, a significantly higher frequency of CD69+ S1PR1­ T cells was observed in the nasal polyps compared with the peripheral blood in patients with CRSwNP. The results of the present study suggest that local regulation of the immune response occurs within nasal polyps. As such, Trm should be considered a potential stimulus in the pathogenesis of nasal polyps. However, the role of Trm in nasal polyps as a pathogenic trigger of the local inflammatory reaction requires further investigation.

Combination of salinomycin and radiation effectively eliminates head and neck squamous cell carcinoma cells in vitro.

The antibiotic drug salinomycin has been reported to mediate cancer cell-specific cytotoxicity, especially regarding cancer stem cells. Since salinomycin has also been reported to arrest cancer cells in the G2 phase, it may have possible radiosensitizing effects. Radiotherapy is a common therapeutic strategy for head and neck squamous cell carcinoma (HNSCC). The aim of the present study was to evaluate a possible influence of salinomycin on the radiosensitivity of the HNSCC cell line HLaC-78 in vitro. HLaC-78 cells were incubated with 5 µM salinomycin or control medium for 24 h and then received 5-Gy irradiation. Subsequently, analysis of cell viability, apoptosis, necrosis and motility through an MTT and a colony formation assay, as well as an Annexin V/propidium-iodide test, a consecutive cell count for four days and a scratch assay were conducted. Additionally, interleukin-8 secretion was assessed using ELISA, due to its role in tumor progression and angiogenesis. Combined treatment with salinomycin and radiation revealed a significantly higher reduction of tumor cell viability, proliferation, motility and secretory capacity compared to cells receiving only one of the treatments alone. Therefore, it is postulated that radiation and salinomycin are an effective combination therapy against HNSCC, a hypothesis which warrants further investigation in cell lines, as well as in an animal model.