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.
2019) Balancing yield, purity and practicality: a modified differential ultracentrifugation protocol for efficient isolation of small extracellular vesicles from human serum, RNA Biology, 16:1, 5-12, ABSTRACT 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.
ARTICLE HISTORY
We identified and validated differential expression of 5 lncRNA in HPV-negative HNSCC. Low MEG3 expression was associated with favorable 3-year RFS, although the significance of this finding remains unclear.
Background: The objective was to assess secretion of small extracellular vesicular microRNA (exo-miRNA) in head and neck squamous cell carcinoma (HNSCC) according to human papillomavirus (HPV) status, and determine the translational potential as a liquid biopsy for early detection. Methods: This study employed a combination of cell culture and case-control study design using archival pretreatment serum. Small extracellular vesicles (sEV) were isolated from conditioned culture media and human serum samples via differential ultracentrifugation. miRNA-sequencing was performed on each sEV isolate. Results: There were clear exo-miRNA profiles that distinguished HNSCC cell lines from nonpathologic oral epithelial control cells. While there was some overlap among profiles across all samples, there were apparent differences in exo-miRNA profiles according to HPV-status. Importantly, differential exo-miRNA profiles were also apparent in serum from early-stage HNSCC cases relative to cancer-free controls.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.