TGFß carrying exosomes in plasma: potential biomarkers of cancer progression in patients with head and neck squamous cell carcinoma.

OBJECTIVES: Contributions of TGFß to cancer progression are well documented. However, plasma TGFß levels often do not correlate with clinicopathological data. We examine the role of TGFß carried in exosomes isolated from murine and human plasma as a contributor to disease progression in head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: The 4-nitroquinoline-1-oxide (4-NQO) mouse model was used to study changes in TGFß expression levels during oral carcinogenesis. In human HNSCC, TGFß and Smad3 protein expression levels and TGFB1 gene expression were determined. Soluble TGFß levels were evaluated by ELISA and TGFß bioassays. Exosomes were isolated from plasma using size exclusion chromatography, and TGFß content was quantified using bioassays and bioprinted microarrays. RESULTS: During 4-NQO carcinogenesis, TGFß levels in tumour tissues and in serum increased as the tumour progressed. The TGFß content of circulating exosomes also increased. In HNSCC patients, TGFß, Smad3 and TGFB1 were overexpressed in tumour tissues and correlated with increased soluble TGFß levels. Neither TGFß expression in tumours nor levels of soluble TGFß correlated with clinicopathological data or survival. Only exosome-associated TGFß reflected tumour progression and correlated with tumour size. CONCLUSIONS: Circulating TGFß+ exosomes in the plasma of patients with HNSCC emerge as potential non-invasive biomarkers of disease progression in HNSCC.

Tumor gene signatures that correlate with release of extracellular vesicles shape the immune landscape in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinomas (HNSCCs) evade immune responses through multiple resistance mechanisms. Extracellular vesicles (EVs) released by the tumor and interacting with immune cells induce immune dysfunction and contribute to tumor progression. This study evaluates the clinical relevance and impact on anti-tumor immune responses of gene signatures expressed in HNSCC and associated with EV production/release. Expression levels of two recently described gene sets were determined in The Cancer Genome Atlas Head and Neck Cancer cohort (n = 522) and validated in the GSE65858 dataset (n = 250) as well as a recently published single-cell RNA sequencing dataset (n = 18). Clustering into HPV(+) and HPV(-) patients was performed in all cohorts for further analysis. Potential associations between gene expression levels, immune cell infiltration, and patient overall survival were analyzed using GEPIA2, TISIDB, TIMER, and the UCSC Xena browser. Compared to normal control tissues, vesiculation-related genes were upregulated in HNSCC cells. Elevated gene expression levels positively correlated (P < 0.01) with increased abundance of CD4(+) T cells, macrophages, neutrophils, and dendritic cells infiltrating tumor tissues but were negatively associated (P < 0.01) with the presence of B cells and CD8(+) T cells in the tumor. Expression levels of immunosuppressive factors NT5E and TGFB1 correlated with the vesiculation-related genes and might explain the alterations of the anti-tumor immune response. Enhanced expression levels of vesiculation-related genes in tumor tissues associates with the immunosuppressive tumor milieu and the reduced infiltration of B cells and CD8(+) T cells into the tumor.

Editorial: Special Issue on "The Role of Exosomes in Cancer Diagnosis and Therapy".

As a result of extensive research in recent years, small extracellular vesicles (sEVs), also known as exosomes, are now considered major contributors to intercellular communication in health and disease [...].

Expression and Regulatory Mechanisms of MicroRNA in Cholesteatoma: A Systematic Review.

Cholesteatoma is a temporal bone disease characterized by dysfunctions of keratinocytes. MicroRNAs (miRNAs) are evolutionary conserved noncoding RNAs that regulate mRNA expression. They can be packaged into exosomes and transported to target cells that can be used in the future therapy of cholesteatoma. This study aimed to collect knowledge on the role of miRNAs and exosomal miRNAs in cholesteatoma and was conducted according to the PRISMA guidelines for systematic reviews. Four databases were screened: Pubmed/MEDLINE, Web of Science, Scopus, and the Cochrane Library. The last search was run on the 6th of June 2023. We included full-text original studies written in English, which examined miRNAs in cholesteatoma. The risk of bias was assessed using the Office of Health Assessment and Translation (OHAT) Risk of Bias Rating Tool, modified for the needs of this review. We identified 118 records and included 18 articles. Analyses revealed the downregulation of exosomal miR-17 as well as miR-10a-5p, miR-125b, miR-142-5p, miR34a, miR-203a, and miR-152-5p and the overexpression of exosomal miR-106b-5p as well as miR-1297, miR-26a-5p, miR-199a, miR-508-3p, miR-21-3p, miR-584-5p, and miR-16-1-3p in cholesteatoma. The role of differentially expressed miRNAs in cholesteatoma, including cell proliferation, apoptosis, the cell cycle, differentiation, bone resorption, and the remodeling process, was confirmed, making them a potential therapeutic target in this disease.

Characteristics of Exosomes and the Vascular Landscape Regulate Exosome Sequestration by Peripheral Tissues and Brain.

Exosomes mediate intercellular communication, shuttling messages between cells and tissues. We explored whether exosome tissue sequestration is determined by the exosomes or the tissues using ten radiolabeled exosomes from human or murine, cancerous or noncancerous cell lines. We measured sequestration of these exosomes by the liver, kidney, spleen, and lung after intravenous injection into male CD-1 mice. Except for kidney sequestration of three exosomes, all exosomes were incorporated by all tissues, but sequestration levels varied greatly among exosomes and tissues. Species of origin (mouse vs. human) or source (cancerous vs. noncancerous cells) did not influence tissue sequestration. Sequestration of J774A.1 exosomes by liver involved the mannose-6 phosphate (M6P) receptor. Wheatgerm agglutinin (WGA) or lipopolysaccharide (LPS) treatments enhanced sequestration of exosomes by brain and lung but inhibited sequestration by liver and spleen. Response to LPS was not predictive of response to WGA. Path and heat map analyses included our published results for brain and found distinct clusters among the exosomes and the tissues. In conclusion, we found no evidence for a universal binding site controlling exosome-tissue interactions. Instead, sequestration of exosomes by tissues is differentially regulated by both exosomes and tissues and may be stimulated or inhibited by WGA and inflammation.

RAGE and HMGB1 Expression in Orbital Tissue Microenvironment in Graves' Ophthalmopathy.

Graves' ophthalmopathy (GO) is a chronic autoimmune inflammatory disorder involving orbital tissues. A receptor for advanced glycation end products (RAGE) and its ligand high mobility group box 1 (HMGB1) protein trigger inflammation and cell proliferation and are involved in the pathogenesis of various chronic inflammatory diseases. This study was aimed to evaluate RAGE and HMGB1 expression in GO to determine its potential clinical significance. To the best of our knowledge, this is the first study showing RAGE and HMGB1 expression in orbital tissue using immunohistochemistry. Sections of orbital adipose tissue obtained from patients diagnosed with GO (23 patients; 36 orbits) and normal controls (NC) (15 patients; 15 orbits) were analyzed by immunohistochemistry for RAGE and HMGB1 expression. Expression profiles were then correlated with clinical data of the study group. RAGE and HMGB1 expression were elevated in GO patients in comparison with NC (p = 0.001 and p = 0.02, respectively). We observed a correlation between RAGE expression and occurrence of dysthyroid optic neuropathy (DON) (p = 0.05) and levels of TSH Receptor Antibodies (TRAb) (p = 0.01). Overexpression of RAGE and HMGB1 might be associated with GO pathogenesis. In addition, RAGE and HMGB1 proteins may be considered as promising therapeutic targets, but this requires further research.

Tumor-derived exosomes promote carcinogenesis of murine oral squamous cell carcinoma.

Circulating tumor-derived exosomes (TEX) interact with a variety of cells in cancer-bearing hosts, leading to cellular reprogramming which promotes disease progression. To study TEX effects on the development of solid tumors, immunosuppressive exosomes carrying PD-L1 and FasL were isolated from supernatants of murine or human HNSCC cell lines. TEX were delivered (IV) to immunocompetent C57BL/6 mice bearing premalignant oral/esophageal lesions induced by the carcinogen, 4-nitroquinoline 1-oxide (4NQO). Progression of the premalignant oropharyngeal lesions to malignant tumors was monitored. A single TEX injection increased the number of developing tumors (6.2 versus 3.2 in control mice injected with phosphate-buffered saline; P < 0.0002) and overall tumor burden per mouse (P < 0.037). The numbers of CD4+ and CD8+ T lymphocytes infiltrating the developing tumors were coordinately reduced (P < 0.01) in mice injected with SCCVII-derived TEX relative to controls. Notably, TEX isolated from mouse or human tumors had similar effects on tumor development and immune cells. A single IV injection of TEX was sufficient to condition mice harboring premalignant OSCC lesions for accelerated tumor progression in concert with reduced immune cell migration to the tumor.

Tumor-derived exosomes promote angiogenesis via adenosine A2B receptor signaling.

RATIONALE: One hallmark of tumor-derived exosomes (TEX) is the promotion of cancer progression by stimulating angiogenesis. This study was performed to evaluate the role of adenosine receptors in TEX-induced angiogenesis. METHODS: TEX produced by UMSCC47 head and neck cancer cell line were isolated by mini size exclusion chromatography (mini-SEC). Enzymatic activity of ectonucleotidases CD39/CD73 carried by TEX was measured by HPLC. Adenosine content of TEX was measured by UPLC-MS/MS. Primary human macrophages were co-incubated with TEX or exosomes derived from the plasma of head and neck cancer patients and their marker expression profile was analyzed by flow cytometry. The macrophage secretome was analyzed by angiogenesis arrays. The in vitro angiogenic potential of TEX was evaluated in endothelial growth studies. Results were validated in vivo using basement membrane extract plug assays in A1R-/-, A2AR-/- and A2BR-/- rats. Vascularization was analyzed by hemoglobin quantification and immunohistology with vessel and macrophage markers. RESULTS: TEX carried enzymatically active CD39/CD73 and adenosine. TEX promoted A2BR-mediated polarization of macrophages toward an M2-like phenotype (p < 0.05) and enhanced their secretion of angiogenic factors. Growth of endothelial cells was stimulated directly by TEX and indirectly via macrophage-reprogramming dependent on A2BR signaling (p < 0.01). In vivo, TEX stimulated the formation of defined vascular structures and macrophage infiltration. This response was absent in A2BR-/- rats (p < 0.05). CONCLUSION: This report provides the first evidence for adenosine production by TEX to promote angiogenesis via A2BR. A2BR antagonism emerges as a potential strategy to block TEX-induced angiogenesis.

Adenosine receptors regulate exosome production.

Exosomes, small-sized extracellular vesicles, carry components of the purinergic pathway. The production by cells of exosomes carrying this pathway remains poorly understood. Here, we asked whether type 1, 2A, or 2B adenosine receptors (A1Rs, A2ARs, and A2BRs, respectively) expressed by producer cells are involved in regulating exosome production. Preglomerular vascular smooth muscle cells (PGVSMCs) were isolated from wildtype, A1R-/-, A2AR-/-, and A2BR-/- rats, and exosome production was quantified under normal or metabolic stress conditions. Exosome production was also measured in various cancer cells treated with pharmacologic agonists/antagonists of A1Rs, A2ARs, and A2BRs in the presence or absence of metabolic stress or cisplatin. Functional activity of exosomes was determined in Jurkat cell apoptosis assays. In PGVSMCs, A1R and A2AR constrained exosome production under normal conditions (p = 0.0297; p = 0.0409, respectively), and A1R, A2AR, and A2BR constrained exosome production under metabolic stress conditions. Exosome production from cancer cells was reduced (p = 0.0028) by the selective A2AR agonist CGS 21680. These exosomes induced higher levels of Jurkat apoptosis than exosomes from untreated cells or cells treated with A1R and A2BR agonists (p = 0.0474). The selective A2AR antagonist SCH 442416 stimulated exosome production under metabolic stress or cisplatin treatment, whereas the selective A2BR antagonist MRS 1754 reduced exosome production. Our findings indicate that A2ARs suppress exosome release in all cell types examined, whereas effects of A1Rs and A2BRs are dependent on cell type and conditions. Pharmacologic targeting of cancer with A2AR antagonists may inadvertently increase exosome production from tumor cells.

Optimization of cell culture conditions for exosome isolation using mini-size exclusion chromatography (mini-SEC).

Extracellular vesicles (EVs) are emerging as a major intercellular communication system engaged in a variety of physiological and pathophysiological processes. Tumor-derived exosomes (TEX) are a subset of EVs of special interest as potential cancer biomarkers. Supernatants of tumor cell lines are widely used as the source of pure TEX for molecular/genetic studies. To optimize TEX isolation and characterization for these studies, we evaluated culture conditions for different tumor cell lines and used mini size exclusion chromatography (mini-SEC) for TEX isolation. Each tumor cell line showed unique culture requirements that determined the recovery, purity and total yield of TEX. Culture conditions for optimal TEX purity and recovery by mini-SEC could be modified by altering the media composition and numbers of seeded cells. TEX recovered from mini-SEC fraction #4 under optimized conditions were biologically active, were sized from 30 to 150 nm in diameter, had a typical vesicular morphology and carried endocytic markers. The most critical requirement for reproducible exosome recovery was re-seeding of tumor cells in numbers adjusted to reflect the optimized culture conditions for each tumor cell line. This study provides insights into a cell culture technique, which can be optimized for exosome production by various human or mouse tumor cell lines for isolation by mini-SEC.

Transport of Extracellular Vesicles across the Blood-Brain Barrier: Brain Pharmacokinetics and Effects of Inflammation.

Extracellular vesicles can cross the blood-brain barrier (BBB), but little is known about passage. Here, we used multiple-time regression analysis to examine the ability of 10 exosome populations derived from mouse, human, cancerous, and non-cancerous cell lines to cross the BBB. All crossed the BBB, but rates varied over 10-fold. Lipopolysaccharide (LPS), an activator of the innate immune system, enhanced uptake independently of BBB disruption for six exosomes and decreased uptake for one. Wheatgerm agglutinin (WGA) modulated transport of five exosome populations, suggesting passage by adsorptive transcytosis. Mannose 6-phosphate inhibited uptake of J774A.1, demonstrating that its BBB transporter is the mannose 6-phosphate receptor. Uptake rates, patterns, and effects of LPS or WGA were not predicted by exosome source (mouse vs. human) or cancer status of the cell lines. The cell surface proteins CD46, AVß6, AVß3, and ICAM-1 were variably expressed but not predictive of transport rate nor responses to LPS or WGA. A brain-to-blood efflux mechanism variably affected CNS retention and explains how CNS-derived exosomes enter blood. In summary, all exosomes tested here readily crossed the BBB, but at varying rates and by a variety of vesicular-mediated mechanisms involving specific transporters, adsorptive transcytosis, and a brain-to-blood efflux system.

Arginase-1+ Exosomes from Reprogrammed Macrophages Promote Glioblastoma Progression.

Interactions between tumor cells and tumor-associated macrophages (TAMs) are critical for glioblastoma progression. The TAMs represent up to 30% of the glioblastoma mass. The role of TAMs in tumor progression and in the mechanisms underlying tumor growth remain unclear. Using an in vitro model resembling the crosstalk between macrophages and glioblastoma cells, we show that glioblastoma-derived exosomes (GBex) reprogram M1 (mediate pro-inflammatory function) and M2 (mediate anti-inflammatory function) macrophages, converting M1 into TAMs and augmenting pro-tumor functions of M2 macrophages. In turn, these GBex-reprogrammed TAMs, produce exosomes decorated by immunosuppressive and tumor-growth promoting proteins. TAM-derived exosomes disseminate these proteins in the tumor microenvironment (TME) promoting tumor cell migration and proliferation. Mechanisms underlying the promotion of glioblastoma growth involved Arginase-1+ exosomes produced by the reprogrammed TAMs. A selective Arginase-1 inhibitor, nor-NOHA reversed growth-promoting effects of Arginase-1 carried by TAM-derived exosomes. The data suggest that GBex-reprogrammed Arginase-1+ TAMs emerge as a major source of exosomes promoting tumor growth and as a potential therapeutic target in glioblastoma.

Inhibition of the Adenosinergic Pathway in Cancer Rejuvenates Innate and Adaptive Immunity.

The adenosine pathway plays a key role in modulating immune responses in physiological and pathological conditions. Physiologically, anti-inflammatory effects of adenosine balance pro-inflammatory adenosine 5'-triphosphate (ATP), protecting tissues from damage caused by activated immune cells. Pathologically, increased adenosine monophosphatase (AMPase) activity in tumors leads to increased adenosine production, generating a deeply immunosuppressed microenvironment and promoting cancer progression. Adenosine emerges as a promising target for cancer therapy. It mediates protumor activities by inducing tumor cell proliferation, angiogenesis, chemoresistance, and migration/invasion by tumor cells. It also inhibits the functions of immune cells, promoting the formation of a tumor-permissive immune microenvironment and favoriting tumor escape from the host immune system. Pharmacologic inhibitors, siRNA or antibodies specific for the components of the adenosine pathway, or antagonists of adenosine receptors have shown efficacy in pre-clinical studies in various in vitro and in vivo tumor models and are entering the clinical arena. Inhibition of the adenosine pathway alone or in combination with classic immunotherapies offers a potentially effective therapeutic strategy in cancer.

Challenges in Exosome Isolation and Analysis in Health and Disease.

A growing body of evidence emphasizes the important role exosomes in different physiological and pathological conditions. Exosomes, virus-size extracellular vesicles (EVs), carry a complex molecular cargo, which is actively processed in the endocytic compartment of parental cells. Exosomes carry and deliver this cargo to recipient cells, serving as an intercellular communication system. The methods for recovery of exosomes from supernatants of cell lines or body fluids are not uniformly established. Yet, studies of the quality and quantity of exosome cargos underlie the concept of "liquid biopsy." Exosomes are emerging as a potentially useful diagnostic tool and a predictor of disease progression, response to therapy and overall survival. Although many novel approaches to exosome isolation and analysis of their cargos have been introduced, the role of exosomes as diagnostic or prognostic biomarkers of disease remains unconfirmed. This review considers existing challenges to exosome validation as disease biomarkers. Focusing on advantages and limitations of methods for exosome isolation and characterization, approaches are proposed to facilitate further progress in the development of exosomes as biomarkers in human disease.

Microvessel density in head and neck squamous cell carcinoma.

PURPOSE: Microvessel density (MVD) corresponds to the intensity of neo-angiogenesis. MVD assessments are based on the expression levels of the vascular endothelium markers such as, e.g., CD34 or CD105. The goal of this study was to assess MVD among patients with head and neck squamous cell carcinoma (HNSCC), and to evaluate the predictive value of MVD in head and neck cancers. METHODS: The study included 49 patients treated for HNSCC and 11 patients with dysplasia of the upper respiratory tract epithelium. Control tissues consisted of 12 normal mucous membranes of the throat. Expression levels of MVD markers were assessed by immunohistochemistry (IHC) using tissue microarrays (TMA). Clinicopathological factors and patients' survival over the 5-year follow-up period were analyzed. RESULTS: The MVD/CD34 values were found to be significantly elevated in the HNSCCs compared to the non-malignant control tissues (p = 0.001) and to dysplastic tissues. (p = 0.02). Significantly higher MVD/CD105 values were also seen in the tumor compared to the control tissues (p = 0.001) or the dysplastic tissues (p = 0.001). Unexpectedly, significantly lower MVD/CD34 values were seen in the tumor tissues of patients with the T3-T4 tumors compared to those with T1-T2 tumors (p = 0.01). CONCLUSIONS: HNSCCs have statistically higher MVD values compared to dysplasia of the upper respiratory tract epithelium. However, the MVD/CD34 values did not correlate with local invasiveness (the T feature) of HNSCCs. This counterintuitive observation suggests that assessments of MVD as performed on TMA by IHC using anti-CD34 or anti-CD105 antibodies considered to be specific for endothelial cell markers might underestimate the extent of the tumor vascularity in HNSCC.

CD24+ tumor-initiating cells from oral squamous cell carcinoma induce initial angiogenesis in vivo.

BACKGROUND: In oral squamous cell carcinoma (OSCC), a minor subset of cancer stem cells has been identified using the surface marker CD24. The CD24+ cell population is involved in initiating, maintaining, and expanding tumor growth, but has not been reported to be involved in angiogenesis to date. METHODS: NOD/SCID mice were equipped with dorsal skinfold chambers and gelatin sponges seeded with CD24+, CD24-, and unsorted cancer cells suspended in Matrigel® were implanted. Following intravital fluorescence microscopy, specimens were examined by immunohistology. RESULTS: Sponges seeded with CD24+ cells showed a significantly higher functional capillary density than those seeded with CD24- cells. The presence of endothelial cells was confirmed by immunohistochemistry for CD31. CONCLUSION: For the first time, CD24+ tumorigenic cells with angiogenic potential, which were isolated from OSCC, were characterized. Our findings provide a promising in vivo model to facilitate the development of therapeutic agents against cancer stem cells and their angiogenic pathways.

Evaluation of CNPase and TGFß1/Smad Signalling Pathway Molecule Expression in Sinus Epithelial Tissues of Patients with Chronic Rhinosinusitis with (CRSwNP) and without Nasal Polyps (CRSsNP).

Chronic rhinosinusitis with and without nasal polyps (CRSwNP and CRSsNP, respectively) is a chronic inflammatory disease affecting almost 5 to 12% of the population and exhibiting high recurrence rates after functional endoscopic sinus surgery (FESS). TGFß1-related pathways contribute to tissue remodelling, which is one of the key aspects of CRS pathogenesis. Additionally, adenosine signalling participates in inflammatory processes, and CNPase was shown to elevate adenosine levels by metabolizing cyclic monophosphates. Thus, the aim of this study was to assess the expression levels of Smad2, pSmad3, TGFß1, and CNPase protein via immunohistochemistry in sinus epithelial tissues from patients with CRSwNP (n = 20), CRSsNP (n = 23), and non-CRS patients (n = 8). The expression of Smad2, pSmad3, TGFß1, and CNPase was observed in the sinus epithelium and subepithelial area of all three groups of patients, and their expression correlated with several clinical symptoms of CRS. Smad2 expression was increased in CRSsNP patients compared to CRSwNP patients and controls (p = 0.001 and p < 0.001, respectively), pSmad3 expression was elevated in CRSwNP patients compared to controls (p = 0.007), TGFß1 expression was elevated in CRSwNP patients compared to controls (p = 0.009), and CNPase was decreased in CRSsNP patients compared to controls (p = 0.03). To the best of our knowledge, we are the first to demonstrate CNPase expression in the upper airway epithelium of CRSwNP, CRSsNP, and non-CRS patients and point out a putative synergy between CNPase and TGFß1/Smad signalling in CRS pathogenesis that emerges as a novel still undiscovered aspect of CRS pathogenesis; further studies are needed to explore its function in the course of the chronic inflammation of the upper airways.

The Relationship between Fine Particle Matter (PM2.5) Exposure and Upper Respiratory Tract Diseases.

PM2.5 is one of the most harmful components of airborne pollution and includes particles with diameters of less than 2.5 µm. Almost 90% of the world's population lives in areas with poor air quality exceeding the norms established by the WHO. PM2.5 exposure affects various organs and systems of the human body including the upper respiratory tract which is one of the most prone to its adverse effects. PM2.5 can disrupt nasal epithelial cell metabolism, decrease the integrity of the epithelial barrier, affect mucociliary clearance, and alter the inflammatory process in the nasal mucosa. Those effects may increase the chance of developing upper respiratory tract diseases in areas with high PM2.5 pollution. PM2.5's contribution to allergic rhinitis (AR) and rhinosinusitis was recently thoroughly investigated. Numerous studies demonstrated various mechanisms that occur when subjects with AR or rhinosinusitis are exposed to PM2.5. Various immunological changes and alterations in the nasal and sinonasal epithelia were reported. These changes may contribute to the observations that exposure to higher PM2.5 concentrations may increase AR and rhinosinusitis symptoms in patients and the number of clinical visits. Thus, studying novel strategies against PM2.5 has recently become the focus of researchers' attention. In this review, we summarize the current knowledge on the effects of PM2.5 on healthy upper respiratory tract mucosa and PM2.5's contribution to AR and rhinosinusitis. Finally, we summarize the current advances in developing strategies against PM2.5 particles' effects on the upper respiratory tract.

Levels of small extracellular vesicles in patients treated with hyperbaric oxygenation.

Introduction: Hyperbaric oxygen (HBO2) therapy involves the inhalation of pure oxygen in a pressure chamber under increased ambient pressure. Recent research indicates that circulating small extracellular vesicles (sEVs) play important roles in human physiology and pathology. Therefore, the objective of this pilot study was to monitor the impact of HBO2 therapy on the levels of circulating sEVs in the serum of patients with necrotizing soft-tissue infections (NSTI), aseptic bone necrosis (ABN) or idiopathic sudden sensory neural hearing loss (ISSNHL). Material and methods: Serum-derived sEVs were isolated and quantified in 80 patients before and after HBO2 therapy applied for NSTI, ISSNHL and ABN patients as well as in normal controls who received neither HBO2 therapy nor steroids. Results: We observed a significant increase of circulating sEVs in patients with ISSNHL after HBO2 therapy (p < 0.05), as well as significantly elevated levels of sEVs after HBO2 therapy compared to patients with NSTI (p < 0.05) and ABN (p < 0.01). Conclusions: The increase in the levels of sEVs in ISSNHL may be evidence for both the intended reduction of inflammation as a result of steroid therapy and the inhibitory effect of oxidative stress induced by HBO2 therapy. Thus, sEVs released during HBO2 therapy might play an important biological role in mediating the response to therapy and might be a promising approach to gain further insights into the therapeutic efficacy of HBO2 therapy.

Metformin Treatment Is Not Associated with Altered PD-L1 Expression in Diabetic Patients with Oral Squamous Cell Carcinoma.

Background: The anti-neoplastic activity of metformin is a subject of current debate. Preclinical data have suggested that metformin enhances PD-L1 anti-tumor effects in various cancer entities by decreasing insulin levels and inducing energetic stress. However, its impact on PD-L1 expression remains unclear in a clinical setting. Therefore, we aim to investigate the impact of metformin treatment in type 2 diabetes mellitus (DM) patients on PD-L1 expression in patients with oral squamous cell carcinoma (OSCC). Methods: We performed a retrospective analysis of patients with DM and OSCC treated at our tertiary referral center over a period of 12 years. The tumor proportion score (TPS), immune cell score (IC), and combined positive score (CPS) were used to quantify PD-L1 expression. PD-L1 expression of patients receiving metformin was compared to a control group without metformin prescription. Results: A total of 68 patients diagnosed with OSCC and DM were analyzed, with 24 receiving and 44 not receiving metformin therapy. No statistically significant differences were identified between the metformin and non-metformin groups for any of the scores (TPS: p = 0.818; IC: p = 0.748; CPS: p = 0.387). Conclusions: In contrast to previous studies, we could not find significant differences in PD-L1 expression between patients with and without metformin intake. Further research needs to shed light on the exact mechanism of metformin in different tumor entities. A comprehensive understanding of metformin's role in cancer therapy could provide valuable insights for potential use of metformin as an adjuvant treatment to immune checkpoint therapy.