Increased levels of circulating cell-free double-stranded nucleic acids in the plasma of glioblastoma patients.

Circulating cell-free nucleic acids are considered a promising source of biomarkers for diseases and cancer. Liquid biopsy biomarkers for brain tumours represent a major, still unmet, clinical need. In plasma, nucleic acids can be free or be associated with extracellular vesicles (EVs). Here we report an easy and reproducible method to analyse cell-free nucleic acids in plasma and EVs by conventional flow cytometry easy to translate into the clinics. Nucleic acids associated with the EVs or present in plasma samples are stained by Pyronin Y, which is a fluorescent dye that is preferably binding double-stranded nucleic acids. Fluorescent staining of EVs isolated from cell-conditioned media is suitable for DNA and RNA detection by flow cytometry. The nucleic acids are partially protected from degradation by the EVs' membrane. Additionally, DNA and RNA can be stained in plasma samples and plasma-derived EVs. Remarkably, analysis of plasma from patients and healthy individuals reveals a difference in their nucleic acid profiles. Taken together, our results indicate that the proposed methodology, which is based on conventional direct flow cytometry, is a promising easy tool for plasma nucleic acid analysis.

Extracellular vesicles as source for the identification of minimally invasive molecular signatures in glioblastoma.

The analysis of extracellular vesicles (EVs) as a source of cancer biomarkers is an emerging field since low-invasive biomarkers are highly demanded. EVs constitute a heterogeneous population of small membrane-contained vesicles that are present in most of body fluids. They are released by all cell types, including cancer cells and their cargo consists of nucleic acids, proteins and metabolites and varies depending on the biological-pathological state of the secretory cell. Therefore, EVs are considered as a potential source of reliable biomarkers for cancer. EV biomarkers in liquid biopsy can be a valuable tool to complement current medical technologies for cancer diagnosis, as their sampling is minimally invasive and can be repeated over time to monitor disease progression. In this review, we highlight the advances in EV biomarker research for cancer diagnosis, prognosis, and therapy monitoring. We especially focus on EV derived biomarkers for glioblastoma. The diagnosis and monitoring of glioblastoma still relies on imaging techniques, which are not sufficient to reflect the highly heterogenous and invasive nature of glioblastoma. Therefore, we discuss how the use of EV biomarkers could overcome the challenges faced in diagnosis and monitoring of glioblastoma.