Proteome profiling of salivary small extracellular vesicles in glioblastoma patients.

BACKGROUND: Extracellular vesicles (EVs) play a critical role in intercellular communication under physiological and pathological conditions, including cancer. EVs cargo reflects their cell of origin, suggesting their utility as biomarkers. EVs are detected in several biofluids, and their ability to cross the blood-brain barrier has highlighted their potential as prognostic and diagnostic biomarkers in gliomas, including glioblastoma (GBM). Studies have demonstrated the potential clinical utility of plasma-derived EVs in glioma. However, little is known about the clinical utility of saliva-derived EVs in GBM. METHODS: Small EVs were isolated from whole mouth saliva of GBM patients pre- and postoperatively. Isolation was performed using differential centrifugation and/or ultracentrifugation. EVs were characterized by concentration, size, morphology, and EVs cell-surface protein markers. Protein cargo in EVs was profiled using mass spectrometry. RESULTS: There were no statistically significant differences in size and concentration of EVs derived from pre- and post GBM patients' saliva samples. A higher number of proteins were detected in preoperative samples compared to postoperative samples. The authors found four highly abundant proteins (aldolase A, 14-3-3 protein ε, enoyl CoA hydratase 1, and transmembrane protease serine 11B) in preoperative saliva samples from GBM patients with poor outcomes. Functional enrichment analysis of pre- and postoperative saliva samples showed significant enrichment of several pathways, including those related to the immune system, cell cycle and programmed cell death. CONCLUSIONS: This study, for the first time, demonstrates the feasibility of isolating and characterizing small EVs from pre- and postoperative saliva samples from GBM patients. Preliminary findings encourage further large cohort validation studies on salivary small EVs to evaluate prognosis in GBM.

A pilot study: Metabolic profiling of plasma and saliva samples from newly diagnosed glioblastoma patients.

BACKGROUND: Despite aggressive treatment, more than 90% of glioblastoma (GBM) patients experience recurrences. GBM response to therapy is currently assessed by imaging techniques and tissue biopsy. However, difficulties with these methods may cause misinterpretation of treatment outcomes. Currently, no validated therapy response biomarkers are available for monitoring GBM progression. Metabolomics holds potential as a complementary tool to improve the interpretation of therapy responses to help in clinical interventions for GBM patients. METHODS: Saliva and blood from GBM patients were collected pre and postoperatively. Patients were stratified conforming their progression-free survival (PFS) into favourable or unfavourable clinical outcomes (>9 months or PFS ≤ 9 months, respectively). Analysis of saliva (whole-mouth and oral rinse) and plasma samples was conducted utilising LC-QqQ-MS and LC-QTOF-MS to determine the metabolomic and lipidomic profiles. The data were investigated using univariate and multivariate statistical analyses and graphical LASSO-based graphic network analyses. RESULTS: Altogether, 151 metabolites and 197 lipids were detected within all saliva and plasma samples. Among the patients with unfavourable outcomes, metabolites such as cyclic-AMP, 3-hydroxy-kynurenine, dihydroorotate, UDP and cis-aconitate were elevated, compared to patients with favourable outcomes during pre-and post-surgery. These metabolites showed to impact the pentose phosphate and Warburg effect pathways. The lipid profile of patients who experienced unfavourable outcomes revealed a higher heterogeneity in the abundance of lipids and fewer associations between markers in contrast to the favourable outcome group. CONCLUSION: Our findings indicate that changes in salivary and plasma metabolites in GBM patients can potentially be employed as less invasive prognostic biomarkers/biomarker panel but validation with larger cohorts is required.

Development of a customisable 3D-printed intra-oral stent for head-and-neck radiotherapy.

Intra-oral stents (including mouth-pieces and bite blocks) can be used to displace adjacent non-involved oral tissue and reduce radiation side effects from radiotherapy treatments for head-and-neck cancer. In this study, a modular and customisable 3D printed intra-oral stent was designed, fabricated and evaluated, to utilise the advantages of the 3D printing process without the interruption of clinical workflow associated with printing time. The stent design used a central mouth-opening and tongue-depressing main piece, with optional cheek displacement pieces in three different sizes, plus an anchor point for moulding silicone to fit individual patients' teeth. A magnetic resonance imaging (MRI) study of one healthy participant demonstrated the tissue displacement effects of the stent, while providing a best-case indication of its comfort.

Helical intensity-modulated radiotherapy with concurrent chemotherapy for oropharyngeal squamous cell carcinoma: A prospective investigation of acute swallowing and toxicity patterns.

BACKGROUND: Conformal radiotherapy modalities may minimize treatment toxicities. The purpose of this study was to document the extent and timing of dysphagia and related toxicities during helical intensity-modulated radiotherapy (IMRT) with chemotherapy for oropharyngeal squamous cell carcinoma (SCC). METHODS: We conducted a prospective study of 76 patients with oropharyngeal SCC undergoing helical IMRT with chemotherapy. Dysphagia and acute toxicity data were collected weekly during treatment and at 2, 4, and 12 weeks posttreatment using the Functional Oral Intake Scale, diet descriptors, and Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. RESULTS: Patients experienced maximum incidence of grade 3 dysphagia (61%), mucositis (30%), and thick saliva (38%), with grade 2 xerostomia (87%) and dysgeusia (97%). Only 14.5% were nil-by-mouth. Symptoms peaked in week 7 and improved thereafter. Grade 3 dysphagia was twice as common for T3 to T4 tumors compared with T2. CONCLUSION: Results confirm that patients with oropharyngeal SCC undergoing helical IMRT with chemotherapy continue to experience incidences of acute toxicities comparable with other conformal techniques, and need supportive cares.

Polyurethane acrylates as effective substrates for sustained in vitro culture of human myotubes.

Muscular disease has debilitating effects with severe damage leading to death. Our knowledge of muscle biology, disease and treatment is largely derived from non-human cell models, even though non-human cells are known to differ from human cells in their biochemical responses. Attempts to develop highly sought after in vitro human cell models have been plagued by early cell delamination and difficulties in achieving human myotube culture in vitro. In this work, we developed polyurethane acrylate (PUA) materials to support long-term in vitro culture of human skeletal muscle tissue. Using a constant base with modulated crosslink density we were able to vary the material modulus while keeping surface chemistry and roughness constant. While previous studies have focused on materials that mimic soft muscle tissue with stiffness ca. 12kPa, we investigated materials with tendon-like surface moduli in the higher 150MPa to 2.4GPa range, which has remained unexplored. We found that PUA of an optimal modulus within this range can support human myoblast proliferation, terminal differentiation and sustenance beyond 35days, without use of any extracellular protein coating. Results show that PUA materials can serve as effective substrates for successful development of human skeletal muscle cell models and are suitable for long-term in vitro studies. STATEMENT OF SIGNIFICANCE: We developed polyurethane acrylates (PUA) to modulate the human skeletal muscle cell growth and maturation in vitro by controlling surface chemistry, morphology and tuning material's stiffness. PUA was able to maintain muscle cell viability for over a month without any detectable signs of material degradation. The best performing PUA prevented premature cell detachment from the substrate which often hampered long-term muscle cell studies. It also supported muscle cell maturation up to the late stages of differentiation. The significance of these findings lies in the possibility to advance studies on muscle cell biology, disease and therapy by using human muscle cells instead of relying on the widely used animal-based in vitro models.