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Nano-scale extracellular vesicles are lipid-bilayer delimited particles that are naturally secreted by all cells and have emerged as valuable biomarkers for a wide range of diseases. Efficient isolation of small extracellular vesicles while maintaining yield and purity is crucial to harvest their potential in diagnostic, prognostic, and therapeutic applications. Most conventional methods of isolation suffer from significant shortcomings, including low purity or yield, long duration, need for large sample volumes, specialized equipment, trained personnel, and high costs. To address some of these challenges, our group has reported a novel insulator-based dielectrophoretic device for rapid isolation of small extracellular vesicles from biofluids and cell culture media based on their size and dielectric properties. In this study, we report a comprehensive characterization of small extracellular vesicles isolated from cancer-patients' biofluids at a twofold enrichment using the device. The three-fold characterization that was performed using conventional flow cytometry, advanced imaging flow cytometry, and microRNA sequencing indicated high yield and purity of the isolated small extracellular vesicles. The device thus offers an efficient platform for rapid isolation while maintaining biomolecular integrity., (© 2023. Springer Nature Limited.)

MicroRNAs (miRNAs) are small non-coding RNA that are powerful regulators of gene expression and can affect the expression of hundreds of genes. miRNAs can be packed in small extracellular vesicles (SEV) and released into the extracellular space by neurons and microglia to act locally as well as pass through the blood-brain barrier and act systemically. We sought to understand the differences in neuronal SEV miRNA expression between frontotemporal dementia (FTD), Alzheimer's disease (AD), and healthy aging. Plasma was obtained from FTD, AD, and healthy aging participants that were matched based on age, sex, and race/ethnicity. Additionally, a subset of participants also provided paired cerebrospinal fluid samples to compare neuronal SEV miRNAs in plasma and cerebrospinal fluid. Neuronal SEV were isolated using differential ultracentrifugation and antibody conjugated Dynabeads® for the neuronal surface marker, L1CAM. RNA sequencing was performed. 12 FTD, 11 with AD, and 10 healthy aging participants were enrolled in the study. In FTD, SEV miRNA-181c was downregulated compared to healthy controls. In AD, miRNA-122 and miRNA-3591 were downregulated compared to those in healthy controls and FTD. Using an FDR <0.2, only miRNA-21-5p was found to have increased expression in the cerebrospinal fluid compared to plasma in a group of AD and FTD participants. SEV miRNA-181c is significantly downregulated in FTD compared to healthy controls and may mediate its effects through microglial-directed neuroinflammation and interaction with TAR DNA-binding protein 43 (TDP-43) based on pathway analysis. Additionally, the FOXO and Hippo pathways may be important mediators of FTD, based on pathway analysis. Lastly, because only one SEV miRNA was differentially expressed between the plasma and cerebrospinal fluid in paired samples, plasma represents an appropriate biofluid for studying neuronal SEV miRNA., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2022 the Author(s). Published by Wolters Kluwer Health, Inc.)

Small extracellular vesicles (sEV) are nano-sized (40-150 nm), membrane-encapsulated vesicles that are released by essentially all cells into the extracellular space and function as intercellular signaling vectors through the horizontal transfer of biologic molecules, including microRNA (miRNA) and other small non-coding RNA (ncRNA), that can alter the phenotype of recipient cells. sEV are present in essentially all extracellular biofluids, including serum, urine and saliva, and offer a new avenue for discovery and development of novel biomarkers of various disease states and exposures. The objective of this study was to systematically interrogate similarities and differences between sEV ncRNA derived from saliva, serum and urine, as well as cell-free small ncRNA (cf-ncRNA) from serum. Saliva, urine and serum were concomitantly collected from 4 healthy donors to mitigate potential bias that can stem from interpersonal and temporal variability. sEV were isolated from each respective biofluid, along with cf-RNA from serum. sEV were isolated from the respective biofluids via differential ultracentrifugation with a 30% sucrose cushion to minimize protein contamination. Small RNA-sequencing was performed on each sample, and cluster analysis was performed based on ncRNA profiles. While some similarities existed in terms of sEV ncRNA cargo across biofluids, there are also notable differences in ncRNA class and ncRNA secretion, with sEV in each biofluid bearing a unique ncRNA profile, including major differences in composition by ncRNA class. We conclude that sEV ncRNA cargo varies according to biofluid, so thus should be carefully selected and interpreted when designing or contrasting translational or epidemiological studies., Competing Interests: The authors have declared that no competing interests exist.

Aim: The goal of this study was to comprehensively interrogate and map DNA methylation across 16 CpG-dense regions previously associated with oral and pharyngeal squamous cell carcinoma (OPSCC). Materials & methods: Targeted multiplex bisulfite amplicon sequencing was performed on four OPSCC cell lines and primary non-neoplastic oral epithelial cells. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed for a subset of associated genes.  Results: There was clear differential methylation between one or more OPSCC cell lines and control cells for the majority of CpG-dense regions. Conclusion: Targeted multiplex bisulfite amplicon sequencing allowed us to efficiently map methylation across the entire region of interest with a high degree of sensitivity and helps shed light on novel differentially methylated regions that may have value as biomarkers of OPSCC.

Ultracentrifugation remains the gold standard for isolation of small extracellular vesicles (sEV), particularly for cancer applications. The objective of this study was to determine if a widely used ultracentrifugation protocol for isolation of serum sEV could be modified to reduce the number of ultracentrifugation cycles and increase efficiency, while maintaining equal or better sample purity and yield. Serum was obtained from two healthy subjects. sEVs were isolated from 1 mL aliquots using three different ultracentrifugation protocols. Co-isolation of RNA carrier protein was assessed by performing Western blots for ApoA-I, ApoB, and Ago2. Small RNA-sequencing was performed on the sEV isolates, and differential detection of small ncRNA was compared across isolation protocols. Reduction from three- to two-ultracentrifuge cycles with no sucrose cushion resulted in a much higher sEV yield but also had the highest levels of lipoprotein and Ago2 contamination. However, the two-ultracentrifugation cycle protocol that incorporated a 30% sucrose cushion into the first cycle resulted in slightly higher sEV yields with lower levels of protein contamination compared to the lengthier three-ultracentrifugation cycle approach, therefore presenting a more efficient alternative approach for isolation of serum sEVs. It was also notable that there were some differences in sEV ncRNA cargo according to protocol, although it was less than expected given the differences in co-isolated RNA carrier proteins. Our results suggest that use of the modified serum sEV isolation protocol with two ultracentrifugation cycles and incorporating a 30% sucrose cushion offers a more efficient approach in terms of efficiency and purity.

Exosomes are nano-scale, membrane encapsulated vesicles that are released by cells into the extracellular space and function as intercellular signaling vectors through horizontal transfer of biologic molecules, including microRNA (miRNA). There is evidence that cancer-derived exosomes enable the tumor to manipulate its microenvironment, thus contributing to the capacity of the tumor for immune evasion, growth, invasion, and metastatic spread. The objective of this study was to characterize differential secretion of exosomal miRNA by head and neck squamous cell carcinoma (HNSCC) and identify a set of candidate biomarkers that could be detected in non-invasive saliva samples. We isolated exosomes from conditioned media from 4 HNSCC cell lines and oral epithelial control cells and applied miRNA-sequencing to comprehensively characterize their miRNA cargo and compare transcript levels of each HNSCC cell line to that of oral epithelial control cells. A candidate set of miRNA differentially secreted by all 4 HNSCC cell lines was further evaluated in saliva collected from HNSCC patients and healthy controls. We observed extensive differences in exosomal miRNA content between HNSCC cells when compared to normal oral epithelial control cells, with a high degree of overlap in exosomal miRNA profiles between the 4 distinct HNSCC cell lines. Importantly, several of the exosomal miRNA secreted solely by cancer cells in culture were detected at substantially elevated levels in saliva from HNSCC patients relative to saliva from healthy controls. These findings provide important insight into tumor biology and yields a promising set of candidate HNSCC biomarkers for use with non-invasive saliva samples., Competing Interests: CONFLICTS OF INTEREST The authors have no conflicts of interest to declare.