Exosomal circPVT1 promotes angiogenesis in laryngeal cancer by activating the Rap1b-VEGFR2 signaling pathway.

Laryngeal cancer (LC) is the second most common head and neck cancer and has a decreasing 5-year survival rate worldwide. Circular RNAs regulate cancer development in diverse ways based on their distinct biogenesis mechanisms and expansive regulatory roles. However, currently, there is little research on how exosomal circular RNAs are involved in the development of laryngeal cancer. Here, we demonstrated that circPVT1, a circular RNA derived from the well-studied long noncoding RNA PVT1, is correlated with disease progression in LC and promotes angiogenesis both in vivo and in vitro. Mechanistically, circPVT1 is loaded into LC cell-secreted exosomes and taken up by vascular epithelium cells. By sponging miR-30c-5p, exosomal circPVT1 promotes Rap1b expression, which dramatically enhances VEGFR2 and PI3K/AKT pathway activation, ultimately resulting in the induction of angiogenesis. Furthermore, our xenograft models demonstrated that the combination of shRNA-circPVT1 and cetuximab showed high efficacy in inhibiting tumor growth and angiogenesis. Collectively, these findings uncover a novel mechanism of exosomal circular RNA-mediated angiogenesis modulation and provide a preclinical rationale for testing this analogous combination in patients with LC.

Exosomal PD-L1 derived from head and neck squamous cell carcinoma promotes immune evasion by activating the positive feedback loop of activated regulatory T cell-M2 macrophage.

The positive feedback loop of activated regulatory T cells (aTregs) and M2 macrophages (M2) play a vital role in promoting the tumor immunosuppressive microenvironment of head and neck squamous cell carcinoma (HNSCC). However, the key factors regulating the positive feedback loop remain unclear. Herein, we investigated the effect of PD-L1 carried on exosomes derived from tumor cells (TEXs) on the aTreg-M2 positive feedback loop, as well as their role in mediating immunosuppression. In our study, TEXs with or without PD-L1 (TEX-PD-L1 or TEX-PD-L1KO) were treated with CD4+CD25- T cells and M0 macrophages, and the effect on the differentiation of aTregs, M2 and the aTreg-M2 positive feedback loop was assessed. TEXs carried more PD-L1 than tumor cells and not only promoted the differentiation of aTregs and M2, but also, most importantly, enhanced the positive feedback loop of aTreg-M2, which inhibited the proliferation of CD4+CD25- T cells and in turn led to tumor immune escape. Moreover, in vivo study showed that TEX-PD-L1KO could inhibit tumor growth and significantly improve the antitumor efficacy in both the peripheral and tumor microenvironments. Collectively this study revealed the role and mechanism of TEX-PD-L1 in negative immune regulation, and targeting TEX-PD-L1 may be a new idea and strategy for immunotherapy of HNSCC.

Cancer stem cell antigen nanodisc cocktail elicits anti-tumor immune responses in melanoma.

One of the major reasons for poor cancer outcomes is the existence of cancer stem cells (CSCs). CSCs are a small subpopulation of tumor cells that can self-renew, differentiate into the majority of tumor cells, and maintain tumorigenicity. As CSCs are resistant to traditional chemotherapy and radiation, they contribute to metastasis and relapse. Thus, new approaches are needed to target and eliminate CSCs. Here, we sought to target and reduce the frequency of CSCs in melanoma by therapeutic vaccination against CSC-associated transcription factors, such as Sox2 and Nanog, and aldehyde dehydrogenase (ALDH). Toward this goal, we have identified novel immunogenic peptide epitopes derived from CSC-associated Sox2 and Nanog and synthesized synthetic high-density lipoprotein (sHDL) nanodisc vaccine formulated with Sox2, Nanog, and ALDH antigen peptides together with CpG, a Toll-like receptor 9 agonist. Vaccination with nanodiscs containing six CSC antigen peptides elicited robust T cell responses against CSC-associated antigens and promoted intratumoral infiltration of CD8+ T cells, while reducing the frequency of CSCs and CD4+ regulatory T cells within melanoma tumors. Nanodisc vaccination effectively reduced tumor growth and significantly extended animal survival without toxicity toward normal stem cells. Overall, our therapeutic strategy against CSCs represents a cost-effective, safe, and versatile approach that may be applied to melanoma and other cancer types, as well as serve as a critical component in combined therapies to target and eliminate CSCs.

Emerging concepts regarding pro- and anti tumor properties of B cells in tumor immunity.

Controversial views regarding the roles of B cells in tumor immunity have existed for several decades. However, more recent studies have focused on its positive properties in antitumor immunity. Many studies have demonstrated a close association of the higher density of intratumoral B cells with favorable outcomes in cancer patients. B cells can interact with T cells as well as follicular dendritic cells within tertiary lymphoid structures, where they undergo a series of biological events, including clonal expansion, somatic hypermutation, class switching, and tumor-specific antibody production, which may trigger antitumor humoral responses. After activation, B cells can function as effector cells via direct tumor-killing, antigen-presenting activity, and production of tumor-specific antibodies. At the other extreme, B cells can obtain inhibitory functions by relevant stimuli, converting to regulatory B cells, which serve as an immunosuppressive arm to tumor immunity. Here we summarize our current understanding of the bipolar properties of B cells within the tumor immune microenvironment and propose potential B cell-based immunotherapeutic strategies, which may help promote cancer immunotherapy.

Low expression of lncRNA SBF2-AS1 regulates the miR-302b-3p/TGFBR2 axis, promoting metastasis in laryngeal cancer.

The 5-year survival rate of laryngeal cancer continues to decline, and the laryngeal particularity of the anatomy adversely affects the patient's quality of life. Emerging evidence suggests that long noncoding RNAs (lncRNAs) are closely correlated to key steps in the malignant progression of cancer cells. In this study, we report the role of lncRNA SBF2-AS1/miR-302b-3p/TGFBR2 interactions in the metastasis of laryngeal squamous cell carcinoma (LSCC). We verified that SBF2-AS1 was significantly downregulated in LSCC tissues and cell lines using qRT-PCR analysis. Its low expression was correlated to lymph node metastasis and an advanced clinical stage. More importantly, LSCC patients with low expression of SBF2-AS1 tended to have a poor prognosis. Based on this, we performed gain-of-function and loss-of-function experiments in LSCC cell lines. The results confirmed that knocking down SBF2-AS1 can promote the metastasis of LSCC cells and enhance epithelial-mesenchymal transition phenotype, while the upregulation of SBF2-AS1 expression resulted in the opposite. Our in vivo model verified that SBF2-AS1 overexpression could inhibit LSCC cell metastasis. Subsequent mechanistic studies revealed that SBF2-AS1 acted as a competing endogenous RNA that upregulated the expression of TGFBR2 by endogenous sponging for miR-302b-3p in LSCC cell lines. Moreover, miR-302b-3p overexpression reversed the inhibitory effects on LSCC metastasis induced by upregulation of SBF2-AS1 expression, and inhibition of TGFBR2 expression reversed the effect of SBF2-AS1 on metastasis. Our study proposes SBF2-AS1 as a biomarker to predict the prognosis of LSCC patients and a novel potential therapeutic target.

A novel immune-related long non-coding RNA signature improves the prognosis prediction in the context of head and neck squamous cell carcinoma.

The tumor immune microenvironment plays an important role in head and neck squamous cell carcinoma (HNSCC). Reliable prognostic signatures able to accurately predict the immune landscape and survival rate of HNSCC patients are crucial to ensure an individualized/effective treatment. Here, we used HNSCC transcriptomic and clinical data retrieved from The Cancer Genome Atlas and identified differentially expressed immune-related long non-coding RNAs (DEirlncRNAs). DEirlncRNA pairs were recognized using univariate analysis. Cox and Lasso regression analyses were used to determine the association between DEirlncRNA pairs and the patients' overall survival and build the prediction model. Receiver operating characteristic curves and Kaplan-Meier survival curves were used to validate the prediction model. We then reevaluated the model based on the clinical factors, tumor-infiltrating immune cells, chemotherapeutic efficacy, and immunosuppression biomarkers. We built a risk score model based on 18 DEirlncRNA pairs, closely related to the overall survival of patients (hazard ratio: 1.376; 95% confidence interval: 1.302-1.453; P < 0.0001). Compared with two recently published lncRNA signatures, our DEirlncRNA pair signature had a higher area under the curve, indicating better prognostic performance. Additionally, the signature score positively correlated with aggressive HNSCC outcomes (low immunity score, significantly reduced CD8 + T cell infiltration, and low expression of immunosuppression biomarkers). However, high-risk patients might have high chemosensitivity. Overall, the lncRNAs signature established here shows promising clinical prediction and the effective disclosure of the tumor immune microenvironment in HNSCC patients; therefore, such signature might help distinguish patients that could benefit from immunotherapy.

IL-21 Is an Accomplice of PD-L1 in the Induction of PD-1-Dependent Treg Generation in Head and Neck Cancer.

Regulatory T cells (Tregs) are immunosuppressive cells involved in antitumor immunity. However, the regulation of Treg generation by inflammation in the tumor microenvironment has not been carefully investigated. Here, we demonstrated that IL-21-polarized inflammation was enriched in the tumor microenvironment in head and neck squamous cell carcinoma (HNSCC) and that IL-21 could promote PD-L1-induced Treg generation in a PD-1-dependent manner. Moreover, generated Tregs showed a greater ability to suppress the proliferation of tumor-associated antigen (TAA)-specific T cells than naturally occurring Tregs. Importantly, an anti-PD-1 antibody could inhibit only Treg expansion induced by clinical tumor explants with high expression of IL-21/PD-L1. In addition, neutralizing IL-21 could enhance the anti-PD-1 antibody-mediated inhibitory effect on Treg expansion. Furthermore, simultaneous high expression of IL-21 and PD-L1 was associated with more Treg infiltrates and predicted reduced overall and disease-free survival in patients with HNSCC. These findings indicate that IL-21 in the tumor microenvironment may promote PD-L1-induced, Treg-mediated immune escape in a PD-1-dependent manner and that an IL-21 neutralization strategy may enhance PD-1 blockade-based antitumor immunotherapy by targeting Treg-mediated immune evasion in patients with high expression of IL-21 and PD-L1.

Using RNA sequencing to identify a putative lncRNA-associated ceRNA network in laryngeal squamous cell carcinoma.

Accumulating evidence indicates that lncRNAs can interact with miRNAs to regulate target mRNAs through competitive interactions. However, this mechanism remains largely unexplored in laryngeal squamous cell carcinoma (LSCC). In this study, transcriptome-wide RNA sequencing was performed on 3 pairs of LSCC tissues and adjacent normal tissues to investigate the expression profiles of lncRNAs, miRNAs and mRNAs, with differential expression of 171 lncRNAs, 36 miRNAs and 1709 mRNAs detected. Seven lncRNAs, eight mRNAs and three miRNAs were identified to be dysregulated in patients' tissues by using qRT-PCR. GO and KEGG pathway enrichment analyses were performed to elucidate the potential functions of these differentially expressed genes in LSCC. Subsequently, a ceRNA (lncRNA-miRNA-mRNA) network including 4631 ceRNA pairs was constructed based on predicted miRNAs shared by lncRNAs and mRNAs. Cis- and transregulatory lncRNAs were analysed by bioinformatics-based methods. Importantly, mRNA-related ceRNA networks (mRCNs) were further obtained based on potential cancer-related coding genes. Coexpression between lncRNAs and downstream mRNAs was used as a criterion for the validation of mRCNs, with the ZNF561-AS1-miR217-WNT5A and SATB1-AS1-miR1299-SAV1/CCNG2/SH3 KBP1/JADE1/HIPK2 ceRNA regulatory interactions determined, followed by experimental validation after siRNA transfection. Moreover, ceRNA activity analysis revealed that different activities of ceRNA modules existing in specific pathological environments may contribute to the tumorigenesis of LSCC. Consistently, both downregulated SATB1-AS1 and ZNF561-AS1 significantly promoted laryngeal cancer cell migration and invasion, indicating their important roles in LSCC via a ceRNA regulatory mechanism. Taken together, the results of this investigation uncovered and systemically characterized a lncRNA-related ceRNA regulatory network that may be valuable for the diagnosis and treatment of LSCC.

Third or fourth branchial pouch sinus lesions: a case series and management algorithm.

BACKGROUND: The purpose of this study was to develop an effective management algorithm for lesions of third or fourth branchial sinuses. STUDY DESIGN: Case series with chart review. METHODS: Data from patients who were identified as having third or fourth branchial pouch sinus lesions in a single institution between January 2014 and December 2018 were retrospectively collected. RESULTS: All 67 patients underwent fistulectomy. First, we classified the patients into five types based on their anatomic features. Then, we considered four optimized surgical methods and adopted the appropriate method with full consideration of the patient's clinical characteristics. The great majority of cases occurred on the left side of the neck (68.7%) and most commonly presented as either a recurrent low-neck abscess or cutaneous discharging fistula with neck infection. Effective preoperative examination included administering contrast agent prior to a computed tomography (CT) scan and in-office laryngoscopy during the quiescent period of inflammation. Ultrasound was also very helpful in determining the presence of thyroiditis. The mean follow-up duration after excision of the lesion was 25.8 months. To date, only 1 (1.5%) recurrence and no obvious complications have been observed. CONCLUSION: Refining fistula subtypes and adopting corresponding treatment measures can reduce the recurrence rate and improve curative effects. We propose and advocate this treatment algorithm for all third and fourth branchial pouch lesions.

Long Noncoding RNA GAS5 Acts As A Tumor Suppressor In Laryngeal Squamous Cell Carcinoma Via miR-21.

PURPOSE: Long noncoding RNAs (lncRNAs) have been identified as an important class of noncoding RNAs that are deeply involved in multiple biological processes in tumorigenesis. This study is to investigate the critical roles and biological function of lncRNA growth arrest-specific 5 (GAS5) in tumorigenesis of laryngeal squamous cell carcinoma (LSCC). PATIENTS AND METHODS: A total of 59 samples of LSCC and paired adjacent tissue, as well as corresponding clinicopathological information were collected. GAS5 expression in both LSCC tissues and human SUN1076 and SNU899 cell lines were analyzed by Real-time quantitative RT-PCR method. Ectopic expression of GAS5 by vector transfection in LSCC cell lines and followed by in vitro experiments was to investigate the critical roles and function of GAS5 in LSCC. Cell Counting Kit 8 (CCK8) assay and PE/7AAD Annexin V Apoptosis analysis was to evaluate cell proliferation ability and cell apoptosis. Co-transfection of GAS5 and miR-21 was to explore the interaction between GAS5 and miR-21 in LSCC. BAX and CDK6 protein level were analyzed by western blot method. RESULTS: This study demonstrated that GAS5 was significantly downregulated in LSCC tissue and human LSCC cell lines. GAS5 levels were correlated with the clinicopathological features of LSCC patients. In addition, the ectopic expression of GAS5 significantly inhibited cell proliferation and promoted apoptosis. Co-expression analyses indicated that GAS5 is negatively correlated with miR-21 in LSCC tissues. Overexpression of miR-21 eliminated GAS5-mediated cell apoptosis and proliferation suppression. Furthermore, GAS5, which upregulated BAX mRNA expression and downregulated CDK6 mRNA expression, was reversed by ectopic expression of miR-21. CONCLUSION: GAS5 suppresses LSCC progression through the negative regulation of miR-21 and its targets involved in cell proliferation and apoptosis, indicating that GAS5 may serve as a biomarker and potential target for LSCC therapy.

Smartphone Application WeChat for Clinical Follow-up of Discharged Patients with Head and Neck Tumors: A Randomized Controlled Trial.

BACKGROUND: Nowadays, social media tools such as short message service, Twitter, video, and web-based systems are more and more used in clinical follow-up, making clinical follow-up much more time- and cost-effective than ever before. However, as the most popular social media in China, little is known about the utility of smartphone WeChat application in follow-up. In this study, we aimed to investigate the feasibility and superiority of WeChat application in clinical follow-up. METHODS: A total of 108 patients diagnosed with head and neck tumor were randomized to WeChat follow-up (WFU) group or telephone follow-up (TFU) group for 6-month follow-up. The follow-ups were delivered by WeChat or telephone at 2 weeks, 1, 2, 3, and 6 months to the patients after being discharged. The study measurements were time consumption for follow-up delivery, total economic cost, lost-to-follow-up rate, and overall satisfaction for the follow-up method. RESULTS: Time consumption in WFU group for each patient (23.36 ± 6.16 min) was significantly shorter than that in TFU group (42.89 ± 7.15 min) (P < 0.001); total economic cost in WFU group (RMB 90 Yuan) was much lower than that in TFU group (RMB 196 Yuan). Lost-to-follow-up rate in the WFU group was 7.02% (4/57) compared with TFU group, 9.80% (5/51), while no significance was observed (95% confidence interval [CI]: 0.176-2.740; P = 0.732). The overall satisfaction rate in WFU group was 94.34% (50/53) compared with 80.43% (37/46) in TFU group (95% CI: 0.057-0.067; P = 0.034). CONCLUSIONS: The smartphone WeChat application was found to be a viable option for follow-up in discharged patients with head and neck tumors. WFU was time-effective, cost-effective, and convenient in communication. This doctor-led follow-up model has the potential to establish a good physician-patient relationship by enhancing dynamic communications and providing individual health instructions. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR-IOR-15007498; http://www.chictr.org.cn/ showproj.aspx?proj=12613.