Identification of neutrophils and eosinophils in upper airway mucosa with immunofluorescence multiplex image cytometry.

Characterization of inflammation in chronic rhinosinusitis with (CRSwNP) and without nasal polyps (CRSsNP) is an ongoing research process. To overcome limitations of current cytologic techniques, we investigated whether immunofluorescence multiplex image cytometry could quantify intact neutrophils, eosinophils, and other immune cells in solid upper airway mucosa. We used a four-channel immunofluorescence-microscopy technique for the simultaneous detection of the leukocyte marker CD45, the neutrophil marker myeloperoxidase, two eosinophil markers, i.e., major basic protein and eosinophil peroxidase, and DAPI (4',6-diamidin-2-phenylindole), in formalin-fixed paraffin-embedded upper airway tissue samples of patients with CRSwNP and CRSsNP, as well as of patients free of CRS with inferior turbinate hypertrophy (controls). Image acquisition and analysis were performed with TissueFAXS and StrataQuest (TissueGnostics, Vienna, Austria), respectively. Positive and negative immunostaining were differentiated with a specific fluorescence signal/background signal ratio. Isotype controls were used as negative controls. In six controls, nine patients with CRSsNP, and 11 patients with CRSwNP, the median area scanned and median cell count per patient were 14.2 mm2 and 34,356, respectively. In CRSwNP, the number of eosinophils was three times higher (23%) than that of neutrophils (7%). Three times more immune cells were encountered in CRSwNP (33%) compared to CRSsNP (11%). In controls, inflammation was balanced between the epithelial layer and lamina propria, in contrast to CRS (three times more pronounced inflammation in the lamina propria). The quantification of intact neutrophils, eosinophils, and other immune cells in solid tissue with undisrupted architecture seems feasible with immunofluorescence multiplex image cytometry.

Is an oropharyngeal HPV infection more frequently detectable in women with a genital HPV infection?

PURPOSE: If not eliminated by the immune system and persisting over years, oropharyngeal high-risk HPV infection can lead to cancer development in the oropharynx. HPV infection is very commonly found in the genital region and can serve as an HPV reservoir. In this study, we investigate whether women with a genital HPV infection are at a higher risk of harboring an undetected oropharyngeal HPV infection via genital-oropharyngeal transmission. METHODS: Women presenting for routine gynecological checkups were included in this study. All participants received an HPV brush test from the genital region as well as from the oropharynx. Additionally, probable risk factors for an HPV infection were assessed in a structured questionnaire. RESULTS: 142 women were included in this study. The rate of oropharyngeal HPV infection was low with 2/142 (1,4%) women positive for a low-risk HPV genotype. In the genital brush test, 54/142 (38%) women were tested HPV positive of which 41/142 (29%) were positive for a high-risk HPV genotype. CONCLUSIONS: The rate of an oropharyngeal HPV detection in our population was low with 2/142 women harboring a low-risk HPV infection.

Validation of RNA Extraction Methods and Suitable Reference Genes for Gene Expression Studies in Developing Fetal Human Inner Ear Tissue.

A comprehensive gene expression investigation requires high-quality RNA extraction, in sufficient amounts for real-time quantitative polymerase chain reaction and next-generation sequencing. In this work, we compared different RNA extraction methods and evaluated different reference genes for gene expression studies in the fetal human inner ear. We compared the RNA extracted from formalin-fixed paraffin-embedded tissue with fresh tissue stored at -80 °C in RNAlater solution and validated the expression stability of 12 reference genes (from gestational week 11 to 19). The RNA from fresh tissue in RNAlater resulted in higher amounts and a better quality of RNA than that from the paraffin-embedded tissue. The reference gene evaluation exhibited four stably expressed reference genes (B2M, HPRT1, GAPDH and GUSB). The selected reference genes were then used to examine the effect on the expression outcome of target genes (OTOF and TECTA), which are known to be regulated during inner ear development. The selected reference genes displayed no differences in the expression profile of OTOF and TECTA, which was confirmed by immunostaining. The results underline the importance of the choice of the RNA extraction method and reference genes used in gene expression studies.

Analysis of cells of epithelial, connective tissue and immune differentiation in HPV-positive-, HPV-negative oropharyngeal carcinoma and normal oropharyngeal tissue by immunofluorescence multiplex image cytometry: a preliminary report.

BACKGROUND: Epithelial, connective tissue and immune cells contribute in various ways to the pathophysiology of HPV positive (HPV+) and HPV negative (HPV-) oropharyngeal squamous cell carcinoma (OPSCC). We aimed to investigate the abundance of these cell lineages and their coexpression patterns in patients with HPV + and HPV- OPSCC. METHODS: We used a 4-channel immunofluorescence-microscopy technique for the simultaneous detection of three direct-conjugated antibodies (pancytokeratin, vimentin and CD45/CD18) and DAPI (4',6-Diamidin-2-phenylindole) in formalin fixed paraffin-embedded tissue samples (FFPE) of patients with HPV + and HPV- OPSCC, and of control patients. Image acquisition and analysis were performed with TissueFAXS and StrataQuest (TissueGnostics, Vienna, Austria), respectively, in tumor cell clusters/stroma in OPSCC specimens and epithelial layer/lamina propria in control specimens. Cell populations were created based on antibodies' coexpression patterns. Isotype and positive controls were examined for plausibility. RESULTS: The proportion of cells of epithelial differentiation in tumor cell clusters was higher in HPV + OPSCC (55%) than in HPV- OPSCC samples (44%). The proportion of connective tissue cells in tumor cell cluster was lower in HPV + OPSCC patients (18%) than in HPV- OPSCC patients (26%). The proportion of immune cells in tumor cell clusters was higher in HPV + OPSCC patients (25%) than in HPV- OPSCC patients (18%). The percentage of anaplastic, potentially de-differentiated cells, was 2% in control patients, and it was higher in HPV- OPSCC (21%) than in HPV + OPSCC samples (6%). CONCLUSIONS: This study provided the first quantitative data for the abundance of cells of epithelial, connective tissue and immune differentiation, in patients with OPSCC and control patients. The abundance of these different crucial cell populations was consistently originating from the same tissue sample. De-differentiation of tumor cells was higher in HPV- OPSCC than in HPV + OPSCC. In tumor cells clusters, the antitumoral host immune response was higher in HPV + OPSCC than in HPV- OPSCC, whereas the fibroblast response was higher in HPV- OPSCC than in HPV + OPSCC. This study contributed to the understanding of histopathologic differences between HPV + OPSCC and HPV- OPSCC patients.

Somatostatin receptor subtype expression and radiomics from DWI-MRI represent SUV of [68Ga]Ga-DOTATOC PET in patients with meningioma.

OBJECTIVE: This retrospective study aimed to analyse the correlation between somatostatin receptor subtypes (SSTR 1-5) and maximum standardized uptake value (SUVmax) in meningioma patients using Gallium-68 DOTA-D-Phe1-Tyr3-octreotide Positron Emission Tomography ([68Ga]Ga-DOTATOC PET). Secondly, we developed a radiomic model based on apparent diffusion coefficient (ADC) maps derived from diffusion weighted magnetic resonance images (DWI MRI) to reproduce SUVmax. METHOD: The study included 51 patients who underwent MRI and [68Ga]Ga-DOTATOC PET before meningioma surgery. SUVmax values were quantified from PET images and tumour areas were segmented on post-contrast T1-weighted MRI and mapped to ADC maps. A total of 1940 radiomic features were extracted from the tumour area on each ADC map. A random forest regression model was trained to predict SUVmax and the model's performance was evaluated using repeated nested cross-validation. The expression of SSTR subtypes was quantified in 18 surgical specimens and compared to SUVmax values. RESULTS: The random forest regression model successfully predicted SUVmax values with a significant correlation observed in all 100 repeats (p < 0.05). The mean Pearson's r was 0.42 ± 0.07 SD, and the root mean square error (RMSE) was 28.46 ± 0.16. SSTR subtypes 2A, 2B, and 5 showed significant correlations with SUVmax values (p < 0.001, R2 = 0.669; p = 0.001, R2 = 0.393; and p = 0.012, R2 = 0.235, respectively). CONCLUSION: SSTR subtypes 2A, 2B, and 5 correlated significantly with SUVmax in meningioma patients. The developed radiomic model based on ADC maps effectively reproduces SUVmax using [68Ga]Ga-DOTATOC PET.

EMT-related transcription factors and protein stabilization mechanisms involvement in cadherin switch of head and neck squamous cell carcinoma.

Epithelial to mesenchymal transition (EMT) describes a process where epithelial tumor cells acquire mesenchymal characteristics. EMT often correlates with invasion and an increased cell migration potential by losing cellular polarity and cell-cell junctions. It is mainly induced by tumor-microenvironment factors, such as TGF-beta 1 and IL-6, which activate the increased expression of the EMT-transcription factor (TF) Slug. We previously reported the Slug/Krüppel-like factor 4 (KLF4) switch in EMT in HNSCC, and found, that in human papilloma virus (HPV)-negative HNSCC Slug gene expression was significant higher represented, than in HPV-positive HNSCC. The purpose of this study was to investigate the impact of KLF4 and Slug on the regulation of the cadherin switch and on the EMT phenotype. Gene expression of KLF4 positive correlated with E-cadherin in 71 head and neck squamous cell carcinoma (HNSCC) patient tissue samples, which we also confirmed by the investigation of the Cancer Genome Atlas database (TCGA). HPV-transcripts contributed to stabilization of KLF4 at protein level, and simultaneously upregulated E-cadherin. Furthermore, ectopic KLF4 overexpression was associated with epithelial gene expression by induction of E-cadherin, ß-catenin and 70-kDa heat shock protein (HSP-70). The presence of HSP-70 ensures the membranous localization of E-cadherin, therefore, the ability of cells to form cadherin/catenin complexes and cellular linkages. In conclusion, KLF4 is a major regulator of the epithelial cadherin-adhesion in HNSCC.

Cancer stem cells and their unique role in metastatic spread.

Cancer stem cells (CSC) possess abilities generally associated with embryonic or adult stem cells, especially self-renewal and differentiation, but also dormancy and cellular plasticity that allow adaption to new environmental circumstances. These abilities are ideal prerequisites for the successful establishment of metastasis. This review highlights the role of CSCs in every step of the metastatic cascade from cancer cell invasion into blood vessels, survival in the blood stream, attachment and extravasation as well as colonization of the host organ and subsequent establishment of distant macrometastasis.

HCN channels in the mammalian cochlea: Expression pattern, subcellular location, and age-dependent changes.

Neuronal diversity in the cochlea is largely determined by ion channels. Among voltage-gated channels, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels open with hyperpolarization and depolarize the cell until the resting membrane potential. The functions for hearing are not well elucidated and knowledge about localization is controversial. We created a detailed map of subcellular location and co-expression of all four HCN subunits across different mammalian species including CBA/J, C57Bl/6N, Ly5.1 mice, guinea pigs, cats, and human subjects. We correlated age-related hearing deterioration in CBA/J and C57Bl/6N with expression levels of HCN1, -2, and -4 in individual auditory neurons from the same cohort. Spatiotemporal expression during murine postnatal development exposed HCN2 and HCN4 involvement in a critical phase of hair cell innervation. The huge diversity of subunit composition, but lack of relevant heteromeric pairing along the perisomatic membrane and axon initial segments, highlighted an active role for auditory neurons. Neuron clusters were found to be the hot spots of HCN1, -2, and -4 immunostaining. HCN channels were also located in afferent and efferent fibers of the sensory epithelium. Age-related changes on HCN subtype expression were not uniform among mice and could not be directly correlated with audiometric data. The oldest mice groups revealed HCN channel up- or downregulation, depending on the mouse strain. The unexpected involvement of HCN channels in outer hair cell function where HCN3 overlaps prestin location emphasized the importance for auditory function. A better understanding may open up new possibilities to tune neuronal responses evoked through electrical stimulation by cochlear implants.

Characterization of epithelial cells, connective tissue cells and immune cells in human upper airway mucosa by immunofluorescence multichannel image cytometry: a pilot study.

Epithelial, connective tissue and immune cells contribute in various ways to the pathophysiology of chronic rhinosinusitis (CRS). However, data of their distribution in upper airway mucosa are sparse. We aimed to provide quantitative, purely informative data on the distribution of these cell lineages and their coexpression patterns, which might help identifying, e.g., cells in the epithelium undergoing through epithelial-mesenchymal transition (EMT). For this purpose, we used immunofluorescence multichannel image cytometry (IMIC). We examined fixed paraffin-embedded tissue samples (FFPE) of six patients with chronic rhinosinusitis (CRS) and of three patients without CRS (controls). The direct-conjugated antibodies pancytokeratin, vimentin and CD45/CD18 were used for coexpression analysis in epithelial layer and lamina propria. Image acquisition and analysis were performed with TissueFAXS and StrataQuest, respectively. To distinguish positive from negative expression, a ratio between cell-specific immunostaining intensity and background was developed. Isotype controls were used as negative controls. Per patient, a 4.5-mm2 tissue area was scanned and a median of 14,875 cells was recognized. The most common cell types were cytokeratin-single-positive (26%), vimentin-single-positive (13%) and CD45/CD18-single-positive with CD45/CD18-vimentin-double-positive cells (29%). In the patients with CRS, CD45/CD18-single-positive cells were 3-6 times higher compared to the control patients. In the epithelial layer, cytokeratin-vimentin-double-positive EMT cells were observed 3-5 times higher in the patients with CRS than in the control patients. This study provided quantitative data for the distribution of crucial cell types in CRS. Future studies may focus on the distribution and coexpression patterns of different immune cells in CRS or even cancer tissue.

Does low-level laser therapy affect the survival of patients with head and neck cancer?

Low-level laser therapy (LLLT) is used in patients with head and neck cancer (HNC) for treatment-related mucositis. There is conflicting evidence as to whether LLLT leads to the proliferation of tumor cells and whether it interferes with the tumoricidal effect of radiotherapy or chemoradiotherapy, if the tumor lies within the LLLT field. Using fuzzy matching, 126 HNC patients who had received LLLT including the tumor region and 126 matching HNC patients without LLLT (controls) treated at the Department of Otorhinolaryngology, Head & Neck Surgery, Medical University of Innsbruck, were identified. The overall survival was compared using the Kaplan-Meier analysis. Fuzzy matching yielded 2 patient samples well comparable in terms of risk of death. The survival did not significantly differ between patients with and without LLLT (p = 0.18). An increased risk of death in HNC patients who received LLLT covering the tumor region was not observed in our study.

Transcriptome-Wide Analysis Reveals a Role for Extracellular Matrix and Integrin Receptor Genes in Otic Neurosensory Differentiation from Human iPSCs.

We analyzed transcriptomic data from otic sensory cells differentiated from human induced pluripotent stem cells (hiPSCs) by a previously described method to gain new insights into the early human otic neurosensory lineage. We identified genes and biological networks not previously described to occur in the human otic sensory developmental cell lineage. These analyses identified and ranked genes known to be part of the otic sensory lineage program (SIX1, EYA1, GATA3, etc.), in addition to a number of novel genes encoding extracellular matrix (ECM) (COL3A1, COL5A2, DCN, etc.) and integrin (ITG) receptors (ITGAV, ITGA4, ITGA) for ECM molecules. The results were confirmed by quantitative PCR analysis of a comprehensive panel of genes differentially expressed during the time course of hiPSC differentiation in vitro. Immunocytochemistry validated results for select otic and ECM/ITG gene markers in the in vivo human fetal inner ear. Our screen shows ECM and ITG gene expression changes coincident with hiPSC differentiation towards human otic neurosensory cells. Our findings suggest a critical role of ECM-ITG interactions with otic neurosensory lineage genes in early neurosensory development and cell fate determination in the human fetal inner ear.

Early appearance of key transcription factors influence the spatiotemporal development of the human inner ear.

Expression patterns of transcription factors leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), transforming growth factor-ß-activated kinase-1 (TAK1), SRY (sex-determining region Y)-box 2 (SOX2), and GATA binding protein 3 (GATA3) in the developing human fetal inner ear were studied between the gestation weeks 9 and 12. Further development of cochlear apex between gestational weeks 11 and 16 (GW11 and GW16) was examined using transmission electron microscopy. LGR5 was evident in the apical poles of the sensory epithelium of the cochlear duct and the vestibular end organs at GW11. Immunostaining was limited to hair cells of the organ of Corti by GW12. TAK1 was immune positive in inner hair cells of the organ of Corti by GW12 and colocalized with p75 neurotrophic receptor expression. Expression for SOX2 was confined primarily to the supporting cells of utricle at the earliest stage examined at GW9. Intense expression for GATA3 was presented in the cochlear sensory epithelium and spiral ganglia at GW9. Expression of GATA3 was present along the midline of both the utricle and saccule in the zone corresponding to the striolar reversal zone where the hair cell phenotype switches from type I to type II. The spatiotemporal gradient of the development of the organ of Corti was also evident with the apex of the cochlea forming by GW16. It seems that highly specific staining patterns of several transcriptions factors are critical in guiding the genesis of the inner ear over development. Our findings suggest that the spatiotemporal gradient in cochlear development extends at least until gestational week 16.

Therapy resistance mediated by cancer stem cells.

Cancer stem cells (CSC) possess abilities generally associated with embryonic or adult stem cells, especially self-renewal and differentiation. The CSC model assumes that this subpopulation of cells sustains malignant growth, which suggests a hierarchical organization of tumors in which CSCs are on top and responsible for the generation of intratumoral heterogeneity. Effective tumor therapy requires the eradication of CSC as they can support regrowth of the tumor resulting in recurrence. However, eradication of CSC is difficult because they frequently are therapy resistant. Therapy resistance is mediated by the acquisition of dormancy, increased DNA repair and drug efflux capacity, decreased apoptosis as well as the interaction between CSC and their supporting microenvironment, the CSC niche. This review highlights the role of CSC in chemo- and radiotherapy resistance as well as possible ways to overcome CSC mediated therapy resistance.

Growth and cellular patterning during fetal human inner ear development studied by a correlative imaging approach.

BACKGROUND: Progressive transformation of the otic placode into the functional inner ear during gestational development in humans leads to the acquisition of hearing perception via the cochlea and balance and spatial orientation via the vestibular organ. RESULTS: Using a correlative approach involving micro-computerized tomography (micro-CT), transmission electron microscopy and histological techniques we were able to examine both the morphological and cellular changes associated with human inner ear development. Such an evaluation allowed for the examination of 3D geometry with high spatial and temporal resolution. In concert with gestational progression and growth of the cochlear duct, an increase in the distance between some of the Crista ampullaris is evident in all the specimens examined from GW12 to GW36. A parallel increase in the distances between the macular organs - fetal utricle and saccule - is also evident across the gestational stages examined. The distances between both the utricle and saccule to the three cristae ampullares also increased across the stages examined. A gradient in hair cell differentiation is apparent from apex to base of the fetal cochlea even at GW14. CONCLUSION: We present structural information on human inner ear development across multiple levels of biological organization, including gross-morphology of the inner ear, cellular and subcellular details of hearing and vestibular organs, as well as ultrastructural details in the developing sensory epithelia. This enabled the gathering of detailed information regarding morphometric changes as well in realizing the complex developmental patterns of the human inner ear. We were able to quantify the volumetric and linear aspects of selected gestational inner ear specimens enabling a better understanding of the cellular changes across the fetal gestational timeline. Moreover, these data could serve as a reference for better understanding disorders that arise during inner ear development.

Therapy resistance mediated by exosomes.

Therapy resistance can arise within tumor cells because of genetic or phenotypic changes (intrinsic resistance), or it can be the result of an interaction with the tumor microenvironment (extrinsic resistance). Exosomes are membranous vesicles 40 to 100 nm in diameter constitutively released by almost all cell types, and mediate cell-to-cell communication by transferring mRNAs, miRNAs, DNAs and proteins causing extrinsic therapy resistance. They transfer therapy resistance by anti-apoptotic signalling, increased DNA-repair or delivering ABC transporters to drug sensitive cells. As functional mediators of tumor-stroma interaction and of epithelial to mesenchymal transition, exosomes also promote environment-mediated therapy resistance.Exosomes may be used in anticancer therapy exploiting their delivery function. They may effectively transfer anticancer drugs or RNAs in the context of gene therapy reducing immune stimulatory effects of these drugs and hydrophilic qualities facilitating crossing of cell membranes.

Brain-Derived Neurotrophin and TrkB in Head and Neck Squamous Cell Carcinoma.

We hypothesized that in head and neck squamous cell carcinoma (HNSCC), the neurotrophin brain-derived neurotrophic factor (BDNF) and its high affinity receptor TrkB regulate tumor cell survival, invasion, and therapy resistance. We used in situ hybridization for BDNF and immunohistochemistry (IHC) for TrkB in 131 HNSCC samples. Brain-derived neurotrophic factor was highly expressed in normal mucosa in HNSCC tissue and in cell lines, whereas only 42.74% of HNSCC tissue was TrkB⁺. One fourth of HNSCC cases was human papilloma virus (HPV)- positive, but the TrkB IHC frequency was not different in HPV-positive (HPV⁺) and negative cases. The UPCI-SCC090 cells expressed constitutive levels of TrkB. Transforming-growth-factor-ß1 (1 ng/mL TGF-ß1) induced TrkB in a subpopulation of SCC-25 cells. A single 10-µg/mL mitomycin C treatment in UPCI-SCC090 cells induced apoptosis and BDNF did not rescue them. The SCC-25 cells were resistant to the MMC treatment, and their growth decreased after TGF-ß1 treatment, but was restored by BDNF if it followed TGF-ß1. Taken together, BDNF might be ineffective in HPV⁺ HNSCC patients. In HPV- HNSCC patients, tumor cells did not die after chemotherapeutic challenge and BDNF with TGF-ß1 could improve tumor cell survival and contribute to worse patient prognosis.

[Head & Neck Merkel Cell Carcinoma]. / Merkelzellkarzinome der Kopf-Hals-Region.

Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine tumor of the skin. Even if it is quite rare, the incidence has increased about two fold during the last twenty years. Mortality is higher than in malignant melanoma. Risk factors are chronic UV exposition and immunosuppression. MCC are most common in patients over 70 years and half of them manifest in the head and neck region. They early metastasize to regional lymph nodes. Surgical therapy should include wide resection of the primary tumor and diagnostic lymph node excision. In the head and neck region this means usually ipsilateral selective neck dissection. Adjuvant radiotherapy of the primary tumor bed and associated lymph nodes of the head and neck region decreases recurrence and should be performed in every patient regardless of the T- and N-stage. In the head and neck region adjuvant radiotherapy can only be spared in selected patients with low-risk profile (wide excisional margins > 2 cm, primary tumor size > 1 cm, absent lymphovascular infiltration, no immunosuppression and pathologic negative cervical lymph nodes). Isolated radiotherapy or systemic therapies are usually applied in patients with metastasized MCC. Disease recurrence is most common in the first two years after initial diagnosis. Patients should be examined at short intervals during this time.

The role of exosomes in cancer metastasis.

Exosomes are small membrane vesicles with a size ranging from 40 to 100nm. They can serve as functional mediators in cell interaction leading to cancer metastasis. Metastasis is a complex multistep process of cancer cell invasion, survival in blood vessels, attachment to and colonization of the host organ. Exosomes influence every step of this cascade and can be targeted by oncological treatment. This review highlights the role of exosomes in the various steps of the metastatic cascade and how exosome dependent pathways can be targeted as therapeutic approach or used for liquid biopsies.

Photodynamic Effect of Methylene Blue and Low Level Laser Radiation in Head and Neck Squamous Cell Carcinoma Cell Lines.

Photodynamic therapy (PDT) is suggested to have an impact on the treatment of early stage head and neck cancers (HNSCC). We investigated the effect of PDT with methylene blue (MB) and a diode laser (660 nm) as the laser source on HNSCC cell lines as an in vitro model of surface oral squamous cell carcinoma. Cell-cultures were exposed to 160 µM MB for 4 min and to laser light for 8 min. Viability was proven via cell viability assay and clonogenic survival via clone counting assay. The combination of MB and diode laser evidenced high efficient loss of cell viability by 5% of the control, while treatment with the same concentration of MB for 4 min alone showed a viability of 46% of the control. In both SCC-25 and Detroit 562 HNSCC cells, MB combined with the laser allowed a significant abrogation of clonogenic growth (p < 0.01), especially in the case of Detroit 562 cells less than 1% of the suspension plated cells were able to grow tumor cell nests. Multiresistant (Detroit 562) HNSCC cells expressing cancer stem cell markers are sensitive to MB/red laser combined PDT.