## 4 Discussion  
Brain tumors have as a rule a poor prognosis and treatments are mostly of limited efficaciousness. Therefore, there is a desperate need for disease biomarkers and relevant methods that can offer insight into pathogenesis and be useful in theranostics [37; 48]. To address this issue, in this work we focused on three elements pertinent to brain tumor biology, i.e., Hsp60, microRNAs that regulate Hsp60 expression [19; 20; 21; 22; 23; 24], and EVs, and we standardized a battery of techniques for measuring these elements. The standardized methods provided quantitative and qualitative information on the three elements. For example, the methods were useful for determining morphology, size distribution, and the presence of specific EVs markers (Alix, Hsc70, and CD81) in the EVs isolated from plasma. The purified vesicles showed typical characteristics of small (<100 nm) and medium (<200 nm) size EVs [49]. The EVs isolated from the plasma of GBM patients were more homogeneous in size distribution during follow-up compared to EVs from atypical meningioma. This variation in size of EVs from plasma of patients with atypical meningioma could be due to disease progression, and is in line with the high variability in biochemical and biophysical properties of tumor cells-derived EVs [50; 51; 52; 53]. The standardized methodology revealed quantitative variations of Hsp60 during time, for example, before and after ablative surgery, allowing surveyance of response to treatment. A major limitation in brain tumor diagnosis stems from the impossibility of molecular profiling using tissue biopsies. A viable alternative is liquid biopsy, which can be obtained via a minimally invasive method such as drawing venous blood. This provides enough material for isolating EVs from plasma to measure their contents in Hsp60 and miRNAs. However, purification and characterization of EVs must be carefully done for the information to be of use to clinicians and surgeons, and to scientists willing to penetrate the secrets of the malignancy of some brain tumors. There are pitfalls that must be avoided that pertain to blood collection; plasma extraction; and specimen manipulation, storage, and testing, and these include type of anticoagulant, sample processing time and temperature, and number of freeze-thaw cycles, just to name a few. Various strict criteria should be met while assessing the physical and biochemical characteristics of EVs for the information obtained to be reliable and reproducible. Currently, the method we recommend for isolating EVs from plasma is ultracentrifugation, including density gradient-based ultracentrifugation. This approach, based on the density of the EVs, ensures a high level of purity, higher than the size-based methods, such as size-exclusion chromatography. Moreover, differential ultracentrifugation can be complemented with ultrafiltration steps to increase the yield, but this imposes a pre-established cutoff. These procedures allow the isolation of defined subpopulations of EVs, such as exosomes, excluding the EVs of larger size. However, the latter larger EVs can also provide useful biomarkers. The EVs population may be highly heterogeneous, depending on the type and/or the state of the cell from which the vesicles derive. Therefore, validation and standardization of the EVs isolation and characterization methods must be carefully done, before applying them to the management of brain tumors patients [49]. Furthermore, the EVs cargo includes a wide range of biomarkers that can be different between EVs subtypes, a diversity not yet fully characterized for all tumors and conditions [49]. In the present work, we propose a set of standardized methods that produce a plethora of complementary results, providing a quantitative picture of three key disease players, Hsp60 and related miRNAs and EVs that can easily be sampled by liquid biopsy (Figure 4). The methods are now available to study more samples from more patients to reveal the