Method for Collecting Single Epithelial Cells from the Mouse Larynx.

OBJECTIVE: The larynx is lined by specialized epithelial cell populations. Studying molecular changes occurring in individual epithelial cell types requires a reliable method for removing these cells from the larynx. Our objective was to develop a method to harvest individual epithelial cells from the mouse larynx while minimizing contamination from non-laryngeal sites and non-epithelial laryngeal cells. METHODS: Mice were euthanized, and the larynx was carefully exposed and separated from non-laryngeal sites. A small dental brush was inserted into the laryngeal inlet and rotated to obtain epithelial cells. Cells were transferred to collection media, counted, and cytospin preparations stained for laryngeal epithelial (i.e., Pan-Keratin, EpCAM, NGFR, p63, K5, ß-tubulin, MUC5AC) and non-epithelial (i.e., vimentin) cell markers. Histopathology was completed on brushed laryngeal tissue sections to evaluate the depth of cell collection. Preliminary Single-cell RNA sequencing (scRNA-seq) was performed to confirm this method can capture diverse laryngeal cell types. RESULTS: We collected 6000-8000 cells from a single larynx and 35000-40000 cells from combining brushings from three tissues. Histopathology demonstrated brushing removed the epithelial layer of the larynx and some underlying tissue. Immunofluorescence staining demonstrated the phenotype of harvested cells was primarily epithelial. Preliminary scRNA-seq was successfully conducted and displayed nine unique cell clusters. CONCLUSION: We developed a reliable method of harvesting individual epithelial cells from the mouse larynx. This method will be useful for collection of laryngeal cells for a variety of downstream cellular and molecular assays, including scRNA-seq, protein analyses, and cell-culture-based experiments, following laryngeal injury. LEVEL OF EVIDENCE: NA Laryngoscope, 134:786-794, 2024.

Introduction and expression of PIK3CAE545K in a papillary thyroid cancer BRAFV600E cell line leads to a dedifferentiated aggressive phenotype.

Anaplastic thyroid cancer (ATC) is a rare, aggressive form of undifferentiated thyroid cancer, which exhibits rapid progression and is almost universally fatal. At least a subset of ATC is thought to arise from pre-existing well-differentiated thyroid cancer, most frequently papillary thyroid cancer (PTC). While PIK3CA mutations are rare in PTC, they are common in ATC and tend to co-occur with BRAF mutations. This provided the rationale for our study to identify the potential role of PIK3CA mutations in the progression from well-differentiated to undifferentiated thyroid cancer. We introduced PIK3CAE545K into the LAM1 PTC cell line, which carries a BRAFV600E mutation. In culture, the engineered cell line (LAM1:PIK3CAE545K) proliferated faster and demonstrated increased clonogenic potential relative to the parental line carrying an empty vector (LAM1EV). Both the LAM1EV and LAM1:PIK3CAE545K edited lines were implanted into hind flanks of athymic nude mice for in vivo determination of disease progression. While tumour weights and volumes were not significantly higher in LAM1:PIK3CAE545K mice, there was a decrease in expression of thyroid differentiation markers TTF-1, thyroglobulin, PAX8 and B-catenin, suggesting that introduction of PIK3CAE545K led to dedifferentiation in vivo. Collectively, this study provides evidence of a role for PIK3CAE545K in driving disease progression from a well-differentiated to an undifferentiated thyroid cancer; however, over-expression was not a determinant of an accelerated growth phenotype in ATC.

3p Arm Loss and Survival in Head and Neck Cancer: An Analysis of TCGA Dataset.

Loss of the 3p chromosome arm has previously been reported to be a biomarker of poorer outcome in both human papillomavirus (HPV)-positive and HPV-negative head and neck cancer. However, the precise operational measurement of 3p arm loss is unclear and the mutational profile associated with the event has not been thoroughly characterized. We downloaded the clinical, single nucleotide variation (SNV), copy number aberration (CNA), RNA sequencing, and reverse phase protein assay (RPPA) data from The Cancer Genome Atlas (TCGA) and The Cancer Proteome Atlas HNSCC cohorts. Survival data and hypoxia scores were downloaded from published studies. In addition, we report the inclusion of an independent Memorial Sloan Kettering cohort. We assessed the frequency of loci deletions across the 3p arm separately in HPV-positive and -negative disease. We found that deletions on chromosome 3p were almost exclusively an all or none event in the HPV-negative cohort; patients either had <1% or >97% of the arm deleted. 3p arm loss, defined as >97% deletion in HPV-positive patients and >50% in HPV-negative patients, had no impact on survival (p > 0.05). However, HPV-negative tumors with 3p arm loss presented at a higher N-category and overall stage and developed more distant metastases (p < 0.05). They were enriched for SNVs in TP53, and depleted for point mutations in CASP8, HRAS, HLA-A, HUWE1, HLA-B, and COL22A1 (false discovery rate, FDR < 0.05). 3p arm loss was associated with CNAs across the whole genome (FDR < 0.1), and pathway analysis revealed low lymphoid-non-lymphoid cell interactions and cytokine signaling (FDR < 0.1). In the tumor microenvironment, 3p arm lost tumors had low immune cell infiltration (FDR < 0.1) and elevated hypoxia (FDR < 0.1). 3p arm lost tumors had lower abundance of proteins phospho-HER3 and ANXA1, and higher abundance of miRNAs hsa-miR-548k and hsa-miR-421, which were all associated with survival. There were no molecular differences by 3p arm status in HPV-positive patients, at least at our statistical power level. 3p arm loss is largely an all or none phenomenon in HPV-negative disease and does not predict poorer survival from the time of diagnosis in TCGA cohort. However, it produces tumors with distinct molecular characteristics and may represent a clinically useful biomarker to guide treatment decisions for HPV-negative patients.

All HPV-negative head and neck cancers are not the same: Analysis of the TCGA dataset reveals that anatomical sites have distinct mutation, transcriptome, hypoxia, and tumor microenvironment profiles.

PURPOSE: Head and neck squamous cell carcinoma (HNSCC) affects various anatomical sites, which often dictates whether the cancer is managed with primary surgery or radiation. This study aimed to assess differences in single nucleotide variation (SNV), copy number, mRNA abundance, methylation, and tumor microenvironment (TME) between HPV-negative oral cavity (OC), oropharyngeal (OPC), hypopharyngeal (HPC), and laryngeal (LC) cancers within The Cancer Genome Atlas (TCGA). METHODS: We downloaded the clinical information and molecular data for the TCGA HNSCC cohort from the data portal and published literature. The TME was estimated using mRNA abundance data. We conducted our analyses within the Bioconductor statistical framework in the R environment. CNA and mRNA abundance results were correlated and grouped with SNV results for downstream pathway analysis. RESULTS: LC had a higher mutational burden than OC and OPC (p <10-4). LC tumors were enriched in CSMD3, NSD1, DCHS2 and ANK2 SNVs, while OC tumors were enriched in CASP8 SNVs (FDR < 0.1). LCs were enriched for neuronal and glycosylation pathways, while OCs were enriched for extracellular matrix pathways. B cells and endothelial cells were more abundant in LC while monocytes were more abundant in OC (FDR < 0.1). OPC was the most hypoxic, followed by OC then LC (FDR < 0.05). OC had greater methylation of Hox genes than LC. Subsite analysis revealed that oral tongue cancers had fewer CASP8 and FBN2 mutations and higher dendritic cell abundance than other oral cavity cancers. CONCLUSIONS: We identified significant genomic, transcriptional, and microenvironmental differences between HPV-negative HNSCC. Further study is warranted to determine if these findings portend differential response to specific treatment modalities.

Sex disparities in head & neck cancer driver genes: An analysis of the TCGA dataset.

OBJECTIVES: Survival in head and neck squamous cell carcinoma (HNSCC) has been associated with patient sex, typically with males experiencing poorer outcomes. It is unclear if this disparity is based in divergent tumor biology. We analyzed the TCGA HNSCC cohort to uncover disparities in the somatic single nucleotide variation (SNV), copy number alteration (CNA) and mRNA abundance profiles between males and females. Critically, we stratified our results by tumor HPV status to control for this significant confounder. METHODS: SNV, CNA and mRNA abundance differences between males and females were compared separately for the HPV-positive (n = 67) and negative (n = 431) TCGA HNSCC cohorts. Overall survival outcomes were compared in males and females in both HPV-positive and HPV-negative subsets of patients. RESULTS: Females were found to have poorer overall survival than males (p = 0.048), largely due to higher rates of HPV-positive disease among men. SNV analysis revealed that in HPV-positive disease, there were no differences by sex after accounting for the false discovery rate (FDR). In HPV-negative tumors, BRWD3 mutations occurred more frequently in the tumors of female patients compared to males after adjusting for the FDR (p = 0.02). Further, HPV-negative BRWD3 mutant tumors were found to have significantly worse 5-year overall survival compared to wildtype on multivariate analysis (p = 0.02). There were 88 heterozygous deletions and 14 amplifications that were differentially altered between male and female HPV-negative tumors and associated with expression changes. Pathway analysis of these genes revealed that tumors from males were enriched in five pathways including chemokine and phosphophatidylinositol signaling. CONCLUSIONS: Reanalysis of the TCGA HNSCC dataset stratified by sex revealed that males in this cohort had a significant survival advantage, due to a higher proportion of HPV-positive disease. Mutations in BRWD3 were more frequent in HPV-negative tumors of females and were associated with poorer overall survival. BRWD3 may represent a novel biomarker of patient outcomes, but will require additional validation.

Choosing The Right Animal Model for Renal Cancer Research.

The increase in the life expectancy of patients with renal cell carcinoma (RCC) in the last decade is due to changes that have occurred in the area of preclinical studies. Understanding cancer pathophysiology and the emergence of new therapeutic options, including immunotherapy, would not be possible without proper research. Before new approaches to disease treatment are developed and introduced into clinical practice they must be preceded by preclinical tests, in which animal studies play a significant role. This review describes the progress in animal model development in kidney cancer research starting from the oldest syngeneic or chemically-induced models, through genetically modified mice, finally to xenograft, especially patient-derived, avatar and humanized mouse models. As there are a number of subtypes of RCC, our aim is to help to choose the right animal model for a particular kidney cancer subtype. The data on genetic backgrounds, biochemical parameters, histology, different stages of carcinogenesis and metastasis in various animal models of RCC as well as their translational relevance are summarized. Moreover, we shed some light on imaging methods, which can help define tumor microstructure, assist in the analysis of its metabolic changes and track metastasis development.

Analysis of the TCGA Dataset Reveals that Subsites of Laryngeal Squamous Cell Carcinoma are Molecularly Distinct.

Laryngeal squamous cell carcinoma (LSCC) from different subsites have distinct presentations and prognosis. In this study, we carried out a multiomic comparison of LSCC subsites. The Cancer Genome Atlas (TCGA) LSCC cohort was analyzed in the R statistical environment for differences between supraglottic and glottic cancers in single nucleotide variations (SNVs), copy number alterations (CNAs), mRNA abundance, protein abundance, pathway overrepresentation, tumor microenvironment (TME), hypoxia status, and patient outcome. Supraglottic cancers had significantly higher overall and smoking-associated SNV mutational load. Pathway analysis revealed upregulation of muscle related pathways in glottic cancer and neural pathways in supraglottic cancer. Proteins involved in cancer relevant signaling pathways including PI3K/Akt/mTOR, the cell cycle, and PDL1 were differentially abundant between subsites. Glottic and supraglottic tumors have different molecular profiles, which may partially account for differences in presentation and response to therapy.

Genomic and human papillomavirus profiling of an oral cancer cohort identifies TP53 as a predictor of overall survival.

BACKGROUND: The genomic landscape of head and neck cancer has been reported through The Cancer Genome Atlas project. We attempt to determine if high-risk human papillomavirus (HPV) or frequently mutated genes are correlated with survival in an oral cancer cohort. METHODS: Patient demographic data along with data from final pathology was collected. Tumor DNA was analyzed using a custom Illumina targeted sequencing panel. Five high-risk HPV types were tested by qPCR. Statistical analyses were used to identify associations between patient outcome and mutational status. RESULTS: High-risk HPV types were identified in 7% of cases; HPV status was not associated with survival. Mutations were identified in TP53, TERT promoter, & PIK3CA. Mutations in TP53 were significantly associated with poorer overall survival on multi-variate analysis (p = 0.03). CONCLUSIONS: Mutations in TP53 were associated with poor patient survival. Expanding our sample size may identify further predictors of outcome to direct customized cancer care.

The dental arch dimensions in Vietnamese children at 7 years of age, and their variation by gender and ethnicity.

PURPOSE: Dental arch dimensions are important not only in dentistry (e.g. orthodontists and prosthodontists, and forensic odontology), but also other medical fields, biology, biometrics, painting or sculpture. This study aimed to determine these dimensions in Vietnamese children and compare these measurements across four ethnic groups and genders. METHODS: A cross-sectional study was conducted on 3204 Vietnamese children at 7 years of age from four major ethnic groups in Vietnam (Kinh, Tay, Thai and Muong). RESULTS: The means variables in study subjects were 33.72 ±â€¯2.16 mm for upper inter-canine width (UCW); 52.74 ±â€¯2.55 mm for upper inter-molar width (UMW); 8.69 ±â€¯1.79 mm for upper anterior length (UAL); 29.59 ±â€¯1.97 mm for upper posterior length (UPL); 26.94 ±â€¯2.49 mm for lower inter-canine width (LCW); 45.89 ±â€¯2.59 mm for lower inter-molar width (LMW); 5.04 ±â€¯1.53 mm for lower anterior length (LAL); and 26.22 ±â€¯2.07 mm for lower posterior length (LPL). The UCM, UMW, and LMW of Muong were significantly wider in males, but narrower in females compared with other ethnic groups. The Kinh, Tay and Thai groups had no significant differences between genders in all dimensions, but these sizes were significantly larger in males than females of Muong group. CONCLUSIONS: This study presents the means of dental arch dimensions in 7 year-old Vietnamese children, and there is no statistical differences in these dimensions between genders of almost studied groups, except Muong group. Ethnic differences are observed only in UCW, UMW and LMW of Muong vs other groups. Furthermore, Vietnamese children have dental arch width similar to the African and Caucasian.

Mutational analysis of head and neck squamous cell carcinoma stratified by smoking status.

Smoking has historically been recognized as a negative prognostic factor in head and neck squamous cell carcinoma (HNSCC). This study aimed to assess the mutational differences between heavy smokers (>20 pack years) and never smokers among the HNSCC patients within The Cancer Genome Atlas (TCGA). Single nucleotide variation and copy number aberration differences between heavy smokers and never smokers were compared within human papillomavirus-positive (HPV-positive) (n = 67) and HPV-negative (n = 431) TCGA cohorts with HNSCC, and the impact of these mutations on survival were assessed. No genes were differentially mutated between smoking and never-smoking patients with HPV-positive tumors. By contrast, in HPV-negative tumors, NSD1 and COL1A11 were found to be more frequently mutated in heavy smokers, while CASP8 was more frequently altered in never smokers. HPV-negative patients with NSD1 mutations experienced significantly improved overall survival compared with NSD1 WT patients. This improved prognosis was validated in an independent cohort of 77 oral cavity cancer patients and a meta-analysis that included 2 additional data sets (688 total patients, hazard ratio for death 0.44, 95% CI, 0.30-0.65). NSD1 mutations are more common in HPV-negative heavy smokers, define a cohort with favorable prognosis, and may represent a clinically useful biomarker to guide treatment deintensification for HPV-negative patients.

Effects of cell-cell crosstalk on gene expression patterns in a cell model of renal cell carcinoma lung metastasis.

The median survival rate of patients with metastatic renal carcinoma is approximately 10 to 12 months, with up to 50% of patients developing metastases in the lung parenchyma. The molecular basis for metastatic development remains unclear. In the present study, we used renal cell carcinoma (RCC) cells and bronchial epithelial cells, representing metastasis target organ cells, conditioned medium and co-culture models to identify specific gene expression changes responsible for cancer cell viability in a metastatic microenvironment. RCC cell proliferation and migration increased when the culture was supplemented with conditioned medium from lung fibroblasts or pleural epithelial cells. Healthy epithelial cells were, in turn, also stimulated with conditioned medium from RCC cell lines. The mitogen-activated protein kinase (MAPK), interleukin (IL)-6, and phosphatidylinositol 4,5-bisphosphate (PIP2) signaling pathways were identified as deregulated upon cell­cell interaction. Thus, cell-cell communication may contribute to the development of the metastatic niche. The identified deregulated signaling pathways may be considered as potential therapeutic targets in metastatic renal carcinoma.

MicroRNA-26a-5p regulates the expression of inducible nitric oxide synthase via activation of NF-κB pathway in human osteoarthritis chondrocytes.

Inducible nitric oxide synthase (iNOS) expression is associated with the pathogenesis of osteoarthritis (OA). This study was undertaken to investigate whether interleukin-1ß (IL-1ß)-mediated induction of iNOS can be regulated by microRNA-26a-5p (hsa-miR-26a-5p) in OA. Bioinformatics approaches show that 3'UTR of iNOS mRNA contained the 'seed-matched-sequence' for hsa-miR-26a-5p. IL-1ß-induced expression of iNOS correlated with the down-regulation of miR-26a-5p in human OA chondrocytes. hsa-miR-26a-5p directly suppressed the luciferase activity of 3'UTR-iNOS reporter clone. Transfection with pre-miR-26a-5p induced marked silencing of iNOS expression. Activation of NF-κB pathway down-regulated the expression of hsa-miR-26a-5p and induced iNOS expression. In short, this is the first report that shows hsa-miR-26a-5p is a direct regulator of iNOS expression in human chondrocytes. hsa-miR-26a-5p may be an important regulator of human cartilage homeostasis and a new target for OA therapy.

Immunological functions of oxidized human immunoglobulin G in type 1diabetes mellitus: its potential role in diabetic smokers as a biomarker of elevated oxidative stress.

The role of oxidized immunoglobulin G in type 1 diabetic smokers has been investigated in the present study. Human immunoglobulin G (IgG) was modified by reactive oxygen species (ROS). The binding characteristics of circulating autoantibodies in type 1 diabetes patients against native and modified IgG were assessed by direct binding ELISA. High degree of specific binding by 68.5% of patients sera towards ROS-modified IgG was observed in comparison to its native analogue (p< 0.05). In addition, diabetic smokers (n=28) were examined and the results were compared with diabetic non-smokers (n=26). Circulating antibodies of diabetic smokers showed substantially stronger binding to modified IgG as compared with the antibodies present in diabetic non-smokers (p< 0.05). Normal human sera (n=53) showed negligible binding with either antigen. Competitive inhibition ELISA reiterates the direct binding results. The increase in total serum protein carbonyl levels in the diabetic smokers was largely due to an increase in oxidized IgG. Diabetic smokers showed substantially higher carbonyl contents in sera as well as in purified IgG as compared with sera and IgG of diabetic non-smokers. Collectively, the oxidation of plasma proteins, especially IgG, might enhance oxidative stress in type 1 diabetes smokers.