Single-cell analysis reveals the multiple patterns of immune escape in the nasopharyngeal carcinoma microenvironment.

BACKGROUND: Single-cell transcriptomics has revolutionised our understanding of the cellular composition of the tumour microenvironment (TME) in nasopharyngeal carcinoma (NPC). Despite this progress, a key limitation of this technique has been its inability to capture epithelial/tumour cells, which has hindered further investigation of tumour heterogeneity and immune escape in NPC. METHODS: In this study, we aimed to address these limitations by analysing the transcriptomics and spatial characteristics of NPC tumour cells at single-cell resolution using scRNA/snRNA-seq and imaging mass cytometry techniques. RESULTS: Our findings demonstrate multiple patterns of immune escape mechanisms in NPC, including the loss of major histocompatibility complex (MHC) molecules in malignant cells, induction of epithelial-mesenchymal transition in fibroblast-like malignant cells and the use of hyperplastic cells in tumour nests to protect tumour cells from immune infiltration. Additionally, we identified, for the first time, a CD8+ natural killer (NK) cell cluster that is specific to the NPC TME. CONCLUSIONS: These findings provide new insights into the complexity of NPC immune landscape and may lead to novel therapeutic strategies for this disease.

Cordycepin inhibits the proliferation and progression of NPC by targeting the MAPK/ERK and ß-catenin pathways.

Cordycepin is an extract from the Cordyceps genus of ascomycete fungi. In the present study, the anticancer potential of cordycepin against nasopharyngeal carcinoma (NPC), and the potential underlying mechanisms, were investigated. Using Cell Counting Kit 8, wound-healing and Transwell assays, cordycepin was found to reduce the viability and inhibit the migration of C666-1 cells in a dose-dependent manner. In addition, in colony formation assays, co-treatment with cordycepin and cisplatin inhibited the proliferation of C666-1 cells. Furthermore, RNA sequencing analysis identified 72 significantly differentially expressed genes and different signaling pathways that may be regulated by cordycepin. After treatment with cordycepin, the expression levels of ERK1/2, phosphorylated ERK1/2 and ß-catenin were significantly downregulated. Therefore, cordycepin may be a novel candidate for NPC treatment or a co-treatment candidate with cisplatin in chemotherapy.

Next-Generation Sequencing Identifies Pathogenic Variants in HGF, POU3F4, TECTA, and MYO7A in Consanguineous Pakistani Deaf Families.

Background: Approximately 70% of congenital deafness is attributable to genetic causes. Incidence of congenital deafness is known to be higher in families with consanguineous marriage. In this study, we investigated the genetic causes in three consanguineous Pakistani families segregating with prelingual, severe-to-profound deafness. Results: Through targeted next-generation sequencing of 414 genes known to be associated with deafness, homozygous variants c.536del (p. Leu180Serfs∗20) in TECTA, c.3719 G>A (p. Arg1240Gln) in MYO7A, and c.482+1986_1988del in HGF were identified as the pathogenic causes of enrolled families. Interestingly, in one large consanguineous family, an additional c.706G>A (p. Glu236Lys) variant in the X-linked POU3F4 gene was also identified in multiple affected family members causing deafness. Genotype-phenotype cosegregation was confirmed in all participating family members by Sanger sequencing. Conclusions: Our results showed that the genetic causes of deafness are highly heterogeneous. Even within a single family, the affected members with apparently indistinguishable clinical phenotypes may have different pathogenic variants.

Transcriptomic Analyses Reveal Gene Expression Profiles and Networks in Nasopharyngeal Carcinoma.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a rare but highly aggressive tumor that is predominantly encountered in Southeast Asia and China in particular. Aside from radiotherapy, no effective therapy that specifically treats NPC is available, including targeted drugs. Finding more sensitive biomarkers is important for new drug discovery and for evaluating patient prognosis. METHODS: mRNA expression datasets from the Gene Expression Omnibus database (GSE53819, GSE64634, and GSE40290) were selected. After all samples in each dataset were subjected to quality control using principal component analyses, the qualified samples were used for additional analyses. The genes that were significantly expressed in each dataset were intersected to identify the most significant of these. Gene functional enrichment analyses were performed on these genes, using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes analyses. The protein-protein interaction network of selected genes was analyzed using the Search Tool for the Retrieval of Interacting Genes database. Significantly, differentially expressed genes were further verified with two RNA-seq datasets (GSE68799 and GSE12452), as well as in clinical samples. RESULTS: In all, 34 (8 upregulated genes and 26 downregulated) genes were identified as significantly differentially expressed. The immune response and the regulation of cell proliferation were the most enriched biological GO terms. Using reverse transcription quantitative real-time PCR (RT-qPCR), the genes MMP1, AQP9, and TNFAIP6 were detected to be upregulated, and FAM3D, CR2, and LTF were downregulated in NPC tissue samples. CONCLUSION: This study provides information on the genes that may be involved in the development of NPC and suggests possible druggable targets and biomarkers for diagnosing and evaluating the prognosis of NPC.

Risk factors and survival outcomes of laryngeal squamous cell carcinoma patients with lung metastasis: A population-based study.

OBJECTIVE: It remains elusive which factors may influence the morbidity and mortality of lung metastasis (LM) in Laryngeal Squamous Cell Carcinoma (LSCC) patients. The aim of the present study was to investigate factors influencing LM and the survival outcomes of LSCC patients with LM. METHODS: We identified 10,935 patients with LSCC from 2010 to 2014 using the Surveillance, Epidemiology and End Results database. Multivariate logistic regression analysis was used to determine the factors associated with the presence of LM. Multivariate cox regression analysis was used to identify covariates associated with increased all-cause mortality in patients with LM. RESULTS: Among 10,935 patients with LSCC, 232 (2.12%) patients had LM. The median survival time of patients with LM was 8 months, and 8.37% of patients survived after 3 years. Patients with age ≥ 60 years old, unmarried status, supraglottis, overlapping lesion of larynx, subglottis, pathological grade III, T4 stage, N1 stage, N2 stage, N3 stage and bone, brain or liver metastases were more likely to have LM. Survival analysis showed that chemotherapy and radiotherapy suggested better survival of LSCC patients with LM while pathological grade IV was associated with an increased all-cause mortality. CONCLUSION: The incidence of LSCC patients with LM varied by age, married status, and tumor subtypes. LSCC patients with LM had poor survival, and only 8.37% of patients survived after 3 years. However, chemotherapy and radiotherapy were found as independent favorable prognostic factors for survival.

Publisher Correction: Vascular Supply of the Human Spiral Ganglion: Novel Three-Dimensional Analysis Using Synchrotron Phase-Contrast Imaging and Histology.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Vascular Supply of the Human Spiral Ganglion: Novel Three-Dimensional Analysis Using Synchrotron Phase-Contrast Imaging and Histology.

Human spiral ganglion (HSG) cell bodies located in the bony cochlea depend on a rich vascular supply to maintain excitability. These neurons are targeted by cochlear implantation (CI) to treat deafness, and their viability is critical to ensure successful clinical outcomes. The blood supply of the HSG is difficult to study due to its helical structure and encasement in hard bone. The objective of this study was to present the first three-dimensional (3D) reconstruction and analysis of the HSG blood supply using synchrotron radiation phase-contrast imaging (SR-PCI) in combination with histological analyses of archival human cochlear sections. Twenty-six human temporal bones underwent SR-PCI. Data were processed using volume-rendering software, and a representative three-dimensional (3D) model was created to allow visualization of the vascular anatomy. Histologic analysis was used to verify the segmentations. Results revealed that the HSG is supplied by radial vascular twigs which are separate from the rest of the inner ear and encased in bone. Unlike with most organs, the arteries and veins in the human cochlea do not follow the same conduits. There is a dual venous outflow and a modiolar arterial supply. This organization may explain why the HSG may endure even in cases of advanced cochlear pathology.

The prevalence and clinical characteristics of vestibular schwannoma among patients treated as sudden sensorineural hearing loss: A 10-year retrospective study in southern China.

PURPOSE: To review the prevalence and clinical characteristics of vestibular schwannoma (VS) in patients with sudden sensorineural hearing loss (SSNHL) in southern China. MATERIALS AND METHODS: This study examined the medical records and MRI findings of all the 1249 patients diagnosed with SSNHL between May 2009 and April 2019 in the Division of Otolaryngology at Peking University Shenzhen Hospital. RESULTS: Among the 1249 patients with SSNHL, VS was found in 14 (1.12%). Among 14 patients, 11 (78.6%) complained of tinnitus and 3 patients (21.4%) complained of dizziness as accompanying symptoms. There was one patient with SSNHL in right ear who had an incidental finding of VS in the contralateral ear. 2 patients (14.3%) had normal auditory brainstem response (ABR) test and 3 patients (21.4%) had hearing recovery. The size of tumors ranged from 6.1 mm to 24.2 mm, with 7 grade 1 tumors, 4 grade 2 tumors, and 3 grade 3 tumors. The total MRI screening cost was $130,857 and the average MRI cost for identifying a VS patient was $9346. CONCLUSION: The prevalence of VS among patients treated as SSNHL was 1.12%. Predicting the risk of VS in SSNHL by the audiogram patterns, pure tone audiometry or hearing recovery is not relivable. Compared with ABR, MRI is more suitable for the assessment of VS in patients with SSNHL in China.

Intratympanic versus intravenous corticosteroid treatment for sudden sensorineural hearing loss in diabetic patients: proposed study protocol for a prospective, randomized superiority trial.

BACKGROUND: Diabetes mellitus is associated with risk of sudden sensorineural hearing loss (SSNHL). Systemic and intratympanic corticosteroids are the two primary treatments for SSNHL in patients with diabetes mellitus. The benefit of intratympanic compared to systemic treatment is the reduced systemic steroid exposure and associated systemic adverse effects. Intratympanic corticosteroid administration may have potential benefits over standard systemic therapies. METHODS/DESIGN: The proposed study is a prospective, randomized superiority trial. A total of 96 patients (48 in each group) will be randomized into the experimental or control group. Patients in the experimental group will receive four 1-mL doses of 40 mg/mL of methylprednisolone over a 1-week period, with a dose administered every 2 days via tympanic membrane injection into the middle ear. The control group will be administered intravenous methylprednisolone (1 mg/kg/day, maximal dose 60 mg/day) for 5 days. The primary outcome for this study is the change in hearing threshold from the first audiogram to the 30-day follow-up audiogram. Secondary outcome measures will include pure-tone average (PTA) at 90-day follow up, visual analog tinnitus scale, visual analog vertigo scale, visual analog aural fullness scale, fasting blood glucose and 2-h postprandial blood glucose during treatment, and the change in glycosylated hemoglobin (HbA1C) levels. Vital signs and otological physical examination will be performed at each follow-up visit. DISCUSSION: The efficacy and safety of intratympanic methylprednisolone compared to intravenous methylprednisolone will be investigated in patients with diabetes mellitus and SSNHL. This trial may provide strong evidence for the efficacy and safety of intratympanic corticosteroid treatment and important clinical information for the treatment of patients with diabetes mellitus and SSNHL. TRIAL REGISTRATION: ChiCTR, ChiCTR1800015954. Registered on 2 May 2018, Retrospectively registered, http://www.chictr.org.cn/showproj.aspx?proj=25326.

Whole-exome sequencing identifies rare pathogenic and candidate variants in sporadic Chinese Han deaf patients.

Genetic causes of hearing loss are highly heterogeneous and often ethnically specific. In recent years, a variety of next-generation sequencing (NGS) panels have been developed to target deafness-causative genes. Whole-exome sequencing (WES), on the other hand, was rarely used for genetic testing for deafness. In this study, we performed WES in 38 sporadic Chinese Han deaf patients who have been pre-excluded for mutations in common deafness genes GJB2, SLC26A4 and MT-RNR1. Non-synonymous variants have been filtered based on their minor allele frequencies in public databases and ethnically matched controls. Bi-allelic pathogenic mutations in eight deafness genes, OTOF, TRIOBP, ESPN, HARS2, CDH23, MYO7A, USH1C and TJP2, were identified in 10 patients, with 17 mutations identified in this study not being associated with deafness previously. For the rest 28 patients, possibly bi-allelic rare non-synonymous variants in an averaged 4.7 genes per patient were identified as candidate pathogenic causes for future analysis. Our study showed that WES may provide a unified platform for genetic testing of deafness and enables retro-analyzing when new causative genes are revealed.

Three-dimensional imaging of the human internal acoustic canal and arachnoid cistern: a synchrotron study with clinical implications.

A thorough knowledge of the gross and micro-anatomy of the human internal acoustic canal (IAC) is essential in vestibular schwannoma removal, cochlear implantation (CI) surgery, vestibular nerve section, and decompression procedures. Here, we analyzed the acoustic-facial cistern of the human IAC, including nerves and anastomoses using synchrotron phase contrast imaging (SR-PCI). A total of 26 fresh human temporal bones underwent SR-PCI. Data were processed using volume-rendering software to create three-dimensional (3D) reconstructions allowing soft tissue analyses, orthogonal sectioning, and cropping. A scalar opacity mapping tool was used to enhance tissue surface borders, and anatomical structures were color-labeled for improved 3D comprehension of the soft tissues. SR-PCI reproduced, for the first time, the variable 3D anatomy of the human IAC, including cranial nerve complexes, anastomoses, and arachnoid membrane invagination (acoustic-facial cistern; an extension of the cerebellopontine cistern) in unprocessed, un-decalcified specimens. An unrecognized system of arachnoid pillars and trabeculae was found to extend between the arachnoid and cranial nerves. We confirmed earlier findings that intra-meatal vestibular schwannoma may grow unseparated from adjacent nerves without duplication of the arachnoid layers. The arachnoid pillars may support and stabilize cranial nerves in the IAC and could also play a role in local fluid hydrodynamics.

Human inner ear blood supply revisited: the Uppsala collection of temporal bone-an international resource of education and collaboration.

BACKGROUND: The Uppsala collection of human temporal bones and molds is a unique resource for education and international research collaboration. Micro-computerized tomography (micro-CT) and synchrotron imaging are used to investigate the complex anatomy of the inner ear. Impaired microcirculation is etiologically linked to various inner ear disorders, and recent developments in inner ear surgery promote examination of the vascular system. Here, for the first time, we present three-dimensional (3D) data from investigations of the major vascular pathways and corresponding bone channels. METHODS: We used the archival Uppsala collection of temporal bones and molds consisting of 324 inner ear casts and 113 macerated temporal bones. Micro-CT was used to investigate vascular bone channels, and 26 fresh human temporal bones underwent synchrotron radiation phase contrast imaging (SR-PCI). Data were processed by volume-rendering software to create 3D reconstructions allowing orthogonal sectioning, cropping, and soft tissue analyses. RESULTS: Micro-CT with 3D rendering was superior in reproducing the anatomy of the vascular bone channels, while SR-PCI replicated soft tissues. Arterial bone channels were traced from scala vestibuli (SV) arterioles to the fundus, cochlea, and vestibular apparatus. Drainage routes along the aqueducts were examined. CONCLUSION: Human inner ear vessels are difficult to study due to the adjoining hard bone. Micro-CT and SR-PCI with 3D reconstructions revealed large portions of the micro-vascular system in un-decalcified specimens. The results increase our understanding of the organization of the vascular system in humans and how altered microcirculation may relate to inner ear disorders. The findings may also have surgical implications.

Functional elucidation of miR-494 in the tumorigenesis of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma has very high incidence and high mortality worldwide. MiRNA is related to the tumorigenesis and metastasis of a variety of tumors. In the present study, we verify that the expression of miR-494 in NPC tissues and NPC-derived cells was down-regulated, respectively. The proliferation, colony formation, migration, and invasion of NPC-derived cells were suppressed, while the cell apoptosis was promoted, when miR-494 was over-expressed in these cells. GALNT7 and CDK16 were confirmed to be the direct targets of miR-494. These results suggested that miR-494 play an inhibitory role in the tumorigenesis of NPC.

MicroRNA-188 suppresses G1/S transition by targeting multiple cyclin/CDK complexes.

BACKGROUND: Accelerated cell cycle progression is the common feature of most cancers. MiRNAs can act as oncogenes or tumor suppressors by directly modulating cell cycle machinery. It has been shown that miR-188 is upregulated in UVB-irradiated mouse skin and human nasopharyngeal carcinoma CNE cells under hypoxic stress. However, little is known about the function of miR-188 in cell proliferation and growth control. RESULTS: Overexpression of miR-188 inhibits cell proliferation, tumor colony formation and G1/S cell cycle transition in human nasopharyngeal carcinoma CNE cells. Using bioinformatics approach, we identify a series of genes regulating G1/S transition as putative miR-188 targets. MiR-188 inhibits both mRNA and protein expression of CCND1, CCND3, CCNE1, CCNA2, CDK4 and CDK2, suppresses Rb phosphorylation and downregulates E2F transcriptional activity. The expression level of miR-188 also inversely correlates with the expression of miR-188 targets in human nasopharyngeal carcinoma (NPC) tissues. Moreover, studies in xenograft mouse model reveal that miR-188 is capable of inhibiting tumor initiation and progression by suppressing target genes expression and Rb phosphorylation. CONCLUSIONS: This study demonstrates that miR-188 exerts anticancer effects, via downregulation of multiple G1/S related cyclin/CDKs and Rb/E2F signaling pathway.

[Idenfication of microRNAs profiles in nasopharyngeal carcinoma].

OBJECTIVE: To filtrate and prove the different microRNAs (miRs) profiles in nasopharyngeal carcinoma. METHOD: Screening the different expressions of miRs between nasopharyngeal carcinoma and the inflammatory tissues by the application of expression profiling of chip high-throughput and large-scale microarray analysis. Then we used RT-QPCR technology to prove the accuracy of screening results. RESULT: There were significant expression differences of miRs between nasopharyngeal carcinoma and the control tissues, 144 human miRs had 2 or more fold the difference ratio. Compared with the inflammatory tissues, we have found that miRs-34b, miRs-449b and miRs-7-1 significantly low expressed in nasopharyngeal carcinoma, yet miRs-125b, miRs-184, miRs-196b, miRs-205 and miRs-24-1 expressed high. The results were consistent with the microarray analysis. CONCLUSION: The difference expressed miRs might be closely related to the process of nasopharyngeal carcinoma, and the research on miRs profiles maybe provide a powerful target basis for early diagnosis and therapy of nasopharyngeal carcinoma.