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EVs and Glioblastoma Research: Insights from the Field

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Sharing the story of France-based researcher Dr Gwennan André-Grégoire, who is working on unravelling the EV-related pathophysiology of glioblastoma multiforme.

Glioblastoma multiforme (GBM) is a highly aggressive and heterogenous primary tumour occurring in the central nervous system, and the most common brain cancer in adults.<super-script>1,2<super-script> It is very difficult to treat and is associated with a very short life expectancy.<super-script>3<super-script> Therapeutic efforts have been thwarted by many challenges, including GBM’s rapid growth rate, molecular heterogeneity, treatment resistance, and ability to infiltrate vital brain structures.<super-script>4<super-script> Understanding the cellular and molecular mechanisms underlying GBM disease progression – and identifying early biomarkers – are therefore top priorities for many scientists in the field of cancer research.

France-based Dr Gwennan André-Grégoire is one of those researchers committed to unravelling GBM pathophysiology. Through her mission with Dr Julie Gavard’s research group, Gwennan is exploring extracellular vesicles (EVs) as potential factors mediating GBM’s aggressiveness and resistance to treatment. Recently, Izon spoke to Gwennan about her career in EV-GBM research, challenges, and hopes for the EV cancer research field.  

ICO and CRCINA: A collaborative network

Following her PhD and postdoctoral position in Paris, Gwennan landed a postdoctoral opportunity at the Cancerology and Immunology Research Center of Nantes-Angers (CRCINA) in the team ‘Signaling in Oncogenesis, Angiogenesis and Permeability’ (SOAP) led by Dr Julie Gavard. The group is affiliated with Nantes University, the National Institute of Health and Medical Research (Inserm), and the French National Centre for Scientific Research (CNRS).  

Gavard’s group aims to link their fundamental research to the clinic, and is focused on EV-related cellular biology and translational research. While Gwennan’s current work is still centred on GBM pathophysiology at the SOAP laboratory, her new mission as permanent researcher of the Integrated Center for Cancerology (ICO) is to focus on EVs as new potential diagnostic and/or predictive companion tools for innovative therapeutics.

From left to right: Gwennan André-Grégoire Laetitia Guevel, Julie Gavard and Quentin Sabbagh.

EVs from GBM stem-like cells as key players in glioblastoma

As part of their research on GBM intra- and extracellular signalling, the group has developed a particular interest in a subpopulation of cells called glioblastoma stem-like cells (GSCs).  

GSCs are considered promotors of glioblastoma initiation and progression and are also associated with therapy resistance in GBM.<super-script>5<super-script> Understanding and harnessing aspects of GSCs, therefore, represents a potential way forward for developing GBM diagnostic tests and treatments. As such, there is great interest in uncovering how GSCs interact with each other and their microenvironment to promote GBM aggressiveness and treatment resistance.  

Although the precise mechanisms are still being explored, EVs have been established as critical components of cellular communication and pathophysiology – and GBM is no exception.<super-script>6-8<super-script> Several potential mechanisms have been identified; for example, GSCs secrete proangiogenic VEGF-A factor in EVs,<super-script>9<super-script> and EVs carrying specific microRNA reportedly target RhoB and reduce apoptosis in vivo and in vitro.<super-script>10<super-script>

To explore potential mechanisms of resistance, the team obtained stem-like cells from glioblastoma tumour resections and cultured them in the presence or absence of temozolomide, a chemotherapeutic agent.<super-script>11<super-script> In line with the observation that temozolomide can be met with resistance by some tumours, the chemotherapeutic was barely toxic to spheroid cultures of patient-derived glioblastoma stem-like cells. Interestingly, tunable resistive pulse sensing (TRPS) analysis of the cell culture medium demonstrated that EV concentrations were elevated when GSCs were treated with temozolomide, despite temozolomide having no effect on cell viability. Further analysis of EVs from patient-derived temozolomide-resistant GSCs unveiled an enrichment of EVs with cargoes dedicated to cell adhesion pathways, paving the way for new perspectives about the role of EVs in chemoresistance to temozolomide and GBM tumour recurrence.

Investigating circulating EVs as biomarkers of glioblastoma

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 information tools from liquid biopsies and possible biomarkers of relapse and treatment-resistant tumours. Some of their early work included measurements of what they call ‘vesiclemia’, i.e., the number of particles per volume, in the circulation of healthy donors and in people with GBM.<super-script>8<super-script>  

Blood from GBM patients contained a greater abundance of EVs, compared with blood from healthy donors. The reasons for this are unknown, says Gwennan: “We are not saying the EVs are coming from the tumour – it could be a marker of disease status.” Of note, other studies have also reported increased EV abundance with disease, including myocardial infarction.<super-script>12<super-script> In addition to EV quantity, EV content has also been explored in individual GBM patients; recently, von Willebrand Factor (VWF) was reported as a selective protein hallmark for GBM, highlighting the potential for EVs to reflect individual status and changes in homeostasis.<super-script>13<super-script>

Gwennan’s further studies on a mouse model of GBM revealed that shrinking the tumour with a novel drug therapy also reduces the number of circulating EVs in the bloodstream.<super-script>14<super-script> Together, these findings indicate a link between vesiclemia and the presence and size of GBM tumours.

Overcoming challenges in EV isolation to support glioblastoma research

With access to patient samples and GBM mouse models, the isolation of EVs is an important aspect of the group’s work. However, plasma contains lipoproteins and other protein aggregates that skew studies of EV quantification and content. To obtain high quality data, therefore, care must be taken to ensure samples are sufficiently purified prior to EV analysis. When working with plasma, Gwennan uses Izon’s qEV columns to separate soluble contaminants. The columns harness principles of size exclusion chromatography (SEC) and enable the rapid collection (<15 minutes) of purified collection volumes containing intact EVs.  

To standardise the collection of purified sample volumes, the group uses Izon’s Automatic Fraction Collector (AFC). The AFC can be programmed to collect a desired fluid volume into collection tubes which are held by an in-built carousel, allowing greater walk-away time and reduced intra-operator variability. Gwennan believes the AFC may be an important benchtop instrument in the clinic once a biomarker has been identified and a reliable protocol for the EV-based diagnosis of GBM has been established: “We use the qEV Automatic Fraction Collector to standardise everything. We find it super nice to use and we like to tell the doctors that one day if we have a biomarker, then we already have an instrument that could be put on the bench,” says Gwennan.  

The laboratory’s workbench, featuring Izon’s Automatic Fraction Collector (AFC), qEV Rack, and qNano.

Once the purified collection volumes have been obtained, particles are identified using surface markers CD9, CD63, and CD81, as well as internal EV proteins, such as Alix and HSP70.  Izon’s TRPS technology is used for size and concentration measurements, as the group have found this technique to be an accurate solution for EV characterisation. Dr André-Grégoire highlights the need for sufficient training as an important factor in obtaining quality results.  

Aspirations for the future of the glioblastoma field

Ultimately, Gwennan hopes to see EVs become a mainstream source of biomarkers. Therefore, having the ability to identify the cell of origin would create unlimited possibilities for diagnostics and basic research: “GSCs are thought to be responsible for the aggressive properties of GBM. The hope is that one day we will be able to use EVs to detect early relapse or treatment resistance, to help provide a better outcome for patients,” says Dr André-Grégoire.  

The field of EV research and other therapeutic areas will benefit not only from advances in techniques capable of detecting EVs with specific markers, but also standardised techniques for the evaluation of size, concentration, and other physical characteristics. Despite major advances in single-particle analysis, however, many still rely on classical optical systems that are unsuited to the quantitative analysis of polydisperse, biological samples. The work of Dr André-Grégoire and her colleagues demonstrates how accurate EV analyses can provide a window into life-changing physiological processes like GBM progression.  

References

1. Bahadur S, Sahu AK, Baghel P, Saha S. Current promising treatment strategy for glioblastoma multiform: A review. Oncology Reviews. 2019;13(2). doi:10.4081/oncol.2019.417

2. Lan X, Jörg DJ, Cavalli FMG, et al. Fate mapping of human glioblastoma reveals an invariant stem cell hierarchy. Nature. 2017;549(7671):227-232. doi:10.1038/nature23666

3. Stupp R, Mason WP, van den Bent MJ, et al. Radiotherapy plus Concomitant and Adjuvant Temozolomide for Glioblastoma. New England Journal of Medicine. 2005;352(10):987-996. doi:10.1056/nejmoa043330

4. Jackson CM, Choi J, Lim M. Mechanisms of immunotherapy resistance: lessons from glioblastoma. Nature Immunology. 2019;20(9):1100-1109. doi:10.1038/s41590-019-0433-y

5. Chen J, Li Y, Yu T-S, et al. A restricted cell population propagates glioblastoma growth after chemotherapy. Nature. 2012;488(7412):522-526. doi:10.1038/nature11287

6. Yekula A, Yekula A, Muralidharan K, Kang K, Carter BS, Balaj L. Extracellular Vesicles in Glioblastoma Tumor Microenvironment. Frontiers in Immunology. 2020;10. doi:10.3389/fimmu.2019.03137

7. André-Grégoire G, Gavard J. Spitting out the demons: Extracellular vesicles in glioblastoma. Cell Adhesion & Migration. 2016;11(2):164-172. doi:10.1080/19336918.2016.1247145

8. Sabbagh Q, Andre-Gregoire G, Guevel L, Gavard J. Vesiclemia: counting on extracellular vesicles for glioblastoma patients. Vol. 39, Oncogene. Springer Nature; 2020. p. 6043–52.

9. Treps L, Perret R, Edmond S, Ricard D, Gavard J. Glioblastoma stem-like cells secrete the pro-angiogenic VEGF-A factor in extracellular vesicles. Journal of Extracellular Vesicles. 2017;6(1):1359479. doi:10.1080/20013078.2017.1359479

10. Yin J, Ge X, Shi Z, et al. Extracellular vesicles derived from hypoxic glioma stem-like cells confer temozolomide resistance on glioblastoma by delivering miR-30b-3p. Theranostics. 2021;11(4):1763-1779. doi:10.7150/thno.47057

11. André-Grégoire G, Bidère N, Gavard J. Temozolomide affects Extracellular Vesicles Released by Glioblastoma Cells. Biochimie. 2018;155:11-15. doi:10.1016/j.biochi.2018.02.007

12. Boulanger CM, Loyer X, Rautou P-E, Amabile N. Extracellular vesicles in coronary artery disease. Nature Reviews Cardiology. 2017;14(5):259-272. doi:10.1038/nrcardio.2017.7

13. Sabbagh Q, André-Grégoire G, Alves-Nicolau C, et al. The von Willebrand Factor stamps Plasmatic Extracellular Vesicles from Glioblastoma Patients. Biorxiv. 2021. doi: https://doi.org/10.1101/2021.03.25.437073

14. André-Grégoire G, Douanne T, Thys A, et al. The Necroptosis Effector MLKL drives Small Extracellular Vesicle Release and Tumour Growth in Glioblastoma. Biorxiv. 2021. doi: https://doi.org/10.1101/2021.01.12.426398

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