Glioblastoma (GBM) is a rare, aggressive cancer of the brain which is associated with poor prognosis and short life expectancy. This cancer remains one of the most difficult to cure in adults, despite intensive research efforts<super-script>(1)<super-script>. The group in which Dr André-Grégoire works carries out cellular biology and translational research involving extracellular vesicles (EVs),which aims to link fundamental research to the clinic. Their research focuses on intra- and extracellular signalling in GBM, with a particular interest in a subpopulation of cells—namely GBM stem-like cells (GSCs)—to identify factors that are responsible for the aggressiveness and resistance to treatment. Early recurrence is often seen after surgery, radiation, or chemotherapy<super-script>(2)<super-script>, and GSCs have previously been shown to exhibit resistance to treatment and are thus implicated in relapse<super-script>(3)<super-script>.If the cells and mechanisms responsible for recurrence can be conclusively determined, it may open up potential avenues for the development of diagnostic tests or targeted therapeutics, which would be greatly beneficial to the field. Dr André-Grégoire’s interests in the interactions between tumour cells led her to the field of EVs due to their importance in cell-to-cell communication. Indeed, EVs play key roles in dissemination of oncogenic material and tumour progression in GBM<super-script>(4)<super-script>.
In addition to the role of EVs in communication within the tumour environment, Dr André-Grégoire is interested in the potential of these vesicles as diagnostic tools and biomarkers of relapse and treatment-resistant tumours. With this in mind, she is analysing circulating EVs in the blood of patients with GBM as well as studying a mouse model of GBM and in vitro cell culture. The results have been promising so far; one particularly important finding has been that the concentration of EVs (which the group refers to as “vesiclemia”) is higher in the blood of patients with GBM than healthy individuals <super-script>(5)<super-script>. Although the sample size for this study was not large, Dr André-Grégoire’s further studies on a mouse model of GBM revealed that shrinking the tumour using a novel drug therapy reduces the number of circulating EVs in the bloodstream<super-script>(6)<super-script>. These findings strongly indicate that there is a link between vesiclemia and the presence and size of GBM tumours, which could represent a potential biomarker.
An important aspect of Dr André-Grégoire’s work is the isolation of EVs from plasma samples and cell culture. She notes that isolation from plasma or cell culture is complicated by the presence of lipoproteins and other protein aggregates. Dr André-Grégoire has found Izon’s qEV Isolation columns to be highly valuable for separating soluble contaminants from EVs, and always uses these columns to overcome the problem of contamination when working with samples where serum is present. The group have compared several isolation techniques and found that precipitation-based techniques are not suitable when analysis of vesiclemia or size is required. DrAndré-Grégoire has found ultracentrifugation(UC) to have some merits; indeed, when working with in-vitro cultures without added serum, classical UC isolation (without the use of columns) can be used, as the EVs are cleaner in these conditions and the technique can result in high yields. However, plasma samples from mice or patients are too complex, and EVs isolated from these samples using UC alone often remain contaminated; therefore, the group currently uses a combination of qEV Isolation columns and UC to ensure cleaner samples and high yields. In order to standardise the isolation of EVs from patient plasma samples, Dr André-Grégoire uses Izon’s Automated Fraction Collector (AFC) to reduce user error or intra-operator variability, and believes that this piece of equipment will bean important benchtop instrument in the clinic when the research reaches a point where a biomarker has been identified and a reliable protocol for the EV-based diagnosis of GBM has been established.
For EV characterisation, the group confirm the identity of the particles by analysing CD9, CD63, and CD81 surface markers as well as detecting internal EV proteins including Alix and HSP70, and excluding major contamination from intracellular material or plasmatic proteins. qNano analysis is used for size and concentration measurements as the group have found this technique to be one of the most accurate for quantification of EVs. Dr André-Grégoire does highlight the importance of being both well trained and careful in experimental set up in order to obtain reliable results and optimise the qNano procedure.
Dr André-Grégoire acknowledges that there are currently some limitations in the field of EV research, one being the sensitivity of current techniques. Although single-particle detection has improved in the last decade, EVs remain on the limit of detection for classical optical systems, and many techniques require high concentrations of vesicles to enable detection. Tracing the cells of origin of EVs is an area of interest, especially the detection of small subpopulations in biofluids such as blood; however, this is highly challenging. In order to support their potential as biomarkers, techniques dedicated to the detection of EVs with specific markers or from particular cell types are needed. The acquisition of funding, as well as the interest of clinician and private companies will be a powerful support to boost research in this area, as in many other areas, of health science.
The work ofDr André-Grégoire and her research group highlights the value of EV analysis for future diagnosis, follow-up, and treatments for GBM. The promising initial results open up avenues for further study, and larger-cohort studies are warranted. Importantly, the research group notes the importance of protocol standardisation for sampling, storage, and analysis in order to achieve reliable and comparable results. The work of DrAndré-Grégoire and colleagues demonstrates the potential clinical utility of longitudinal analysis of vesiclemia and plasmatic EV cargo for monitoring the progression of GBM, making a significant step in transferring the findings of fundamental research to the clinic.
