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.

The deterioration of calcified cartilage integrity reflects the severity of osteoarthritis-A structural, molecular, and biochemical analysis.

The calcified cartilage zone (CCZ) is a thin interlayer between the hyaline articular cartilage and the subchondral bone and plays an important role in maintaining the joint homeostasis by providing biological and mechanical support from unmineralized cartilage to the underlying mineralized subchondral bone. The hallmark of CCZ characteristics in osteoarthritis (OA) is less well known. The aim of our study is to evaluate the structural, molecular, and biochemical composition of CCZ in tissues affected by primary knee OA and its relationship with disease severity. We collected osteochondral tissue samples stratified according to disease severity, from 16 knee OA patients who underwent knee replacement surgery. We also used meniscectomy-induced rat samples to confirm the pathophysiologic changes of human samples. We defined the characteristics of the calcified cartilage layer using a combination of morphological, biochemical, proteomic analyses on laser micro-dissected tissue. Our results demonstrated that the Calcium/Phosphate ratio is unchanged during the OA progression, but the calcium-binding protein and cadherin binding protein, as well as carbohydrate metabolism-related proteins, undergo significant changes. These changes were further accompanied by thinning of the CCZ, loss of collagen and proteoglycan content, the occurrence of the endochondral ossification, neovasculature, loss of the elastic module, loss of the collagen direction, and increase of the tortuosity indicating an altered structural and mechanical properties of the CCZ in OA. In conclusion, our results suggest that the calcified cartilage changes can reflect the disease progression.

A salivary biomarker panel to detect liver cirrhosis.

Limited access to diagnostic tests for liver fibrosis remains one of the main reasons for late diagnosis, especially in rural and remote communities. Saliva diagnostics is accessible with excellent patient compliance. The aim of this study was to develop a saliva-based diagnostic tool for liver fibrosis/cirrhosis. Salivary concentrations of hyaluronic acid (HA), tissue inhibitor of metalloproteinase-1 (TIMP-1), and α-2-macroglobulin (A2MG) were significantly increased (p < 0.05) in patients with liver fibrosis/cirrhosis. By combining these biomarkers, we developed the Saliva Liver Fibrosis (SALF) score, which identified patients with liver cirrhosis with an area under the receiver operating characteristic curve (AUROC) of 0.970 and 0.920 in a discovery and validation cohorts, respectively. The SALF score had a performance that was similar to that of the current Fibrosis-4 (AUROC:0.740) and Hepascore (AUROC:0.979). We demonstrated the clinical utility of saliva to diagnose liver fibrosis/cirrhosis with a potential to improve the screening for cirrhosis in asymptomatic populations.

Isolation and Characterization of Salivary Exosomes for Cancer Biomarker Discovery.

Exosomes are small extracellular vesicles secreted by cells and are known to play a key role in intercellular communication. Several studies have associated exosomes with various roles in tumorigenesis and explored their potential as a source of biomarkers for diagnosis and prognosis in cancer research. Exosomes can be isolated from several body fluids, including those that are noninvasively accessible, such as human saliva. This book chapter provides a step-by-step detailed description of techniques that are used for the isolation, quantification, and characterization of exosomes from saliva. These include ultracentrifugation for the isolation, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blot (WB) for characterization of exosomes. The NTA approach explores the Brownian motion and light scattering of particles to predict size and concentration. TEM enables visualization of the exosomes which often present a cup-shaped morphology. Western blot is used to detect commonly expressed exosome-associated proteins. Finally, salivary exosomes isolated using these protocols can further be characterized for downstream analysis according to their cargo (proteins, DNA, RNA, miRNA) and utilized for cancer biomarker discovery.

The use of minimally invasive biomarkers for the diagnosis and prognosis of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a common cause of cancer-related deaths worldwide. Despite advances in systemic therapies, patient survival remains low due to late diagnosis and frequent underlying liver diseases. HCC diagnosis generally relies on imaging and liver tissue biopsy. Liver biopsy presents limitations because it is invasive, potentially risky for patients and it frequently misrepresents tumour heterogeneity. Recently, liquid biopsy has emerged as a way to monitor cancer progression in a non-invasive manner. Tumours shed content into the bloodstream, such as circulating tumour cells (CTCs), circulating nucleic acids, extracellular vesicles and proteins, that can be isolated from biological fluids of patients with HCC. These biomarkers provide knowledge regarding the genetic landscape of tumours and might be used for diagnostic or prognostic purposes. In this review, we summarize recent literature on circulating biomarkers for HCC, namely CTCs, circulating tumour DNA (ctDNA), RNA, extracellular vesicles and proteins, and their clinical relevance in HCC.