## 4 The Release of miRNAs and Chaperones through EVs as Molecular Signaling and Source of Biomarkers
Several studies suggest the oncogenic effect of miR-21, whose overexpression occurring in many tumor types favors the establishment of tumor-permissive pathways in glioblastoma [170; 171]. It has been observed that malignant brain tumor-derived EVs support neoangiogenesis through miR-21/VEGF signaling [172]. MicroRNAs can cross the blood-brain barrier (BBB) and reach far target sites through the circulation. For instance, the vesicular miR-21 has been identified in the blood [250] and CSF [173; 174], suggesting that these EVs carrying miRNAs could have diagnostic and prognostic usefulness. Both miR-105 [253] and miR-181c [254] have the ability to disrupt the blood-brain barrier and function as pro-oncogenes by downregulating tumor suppressor genes, and thereby favor invasiveness and metastasization. It has also been reported that miR-148a delivered by EVs promotes glioma-cell proliferation and metastasis via targeting CADM1 to activate the STAT3 pathway [175]. The content in miRNAs within EVs released by a tumor can vary depending on the stage of tumor progression, thus providing key diagnostic indicators, which is particularly convenient when it is possible to isolate the EVs from biological fluids such as plasma [163]. For instance, miR-222, miR-124-3p, miR-221, miR-320, miR-574-3p, and miR-301a were found to be increased in EVs derived from serum of high-grade glioma patients [176; 177] and their distinctive quantitative patterns allowed for distinguishing between tumoral and control samples [177]. The pathogenic role of some miRNAs depends on their tissue localization. For instance, miR-454 may act as an oncogene in gastric cancer [255], but is a tumor suppressor in gliomas [181]. The microRNA miR-454 is elevated in EVs from preoperative sera when compared with its levels after surgery, indicating that cancer cells can differentially secrete specific miRNAs into the circulation in EVs, which support the idea that these vesicles and their contents have potential as useful biomarkers accessible with minimally invasive procedures [182]. Despite their fundamental role in cell homeostasis and the knowledge that molecular chaperones are differentially expressed in cancer, little is known about the secretion of these proteins by brain tumors via EVs. The stress response of tumor cells, included those of brain tumors, consists of the overexpression of Hsps and their release in the extracellular environment also by EVs. However, little is known on the role of molecular chaperones in the brain tumor microenvironment; one working hypothesis postulates that the release of chaperones via EVs could be a defense mechanism from injury [242; 256]. CCT6A, a subunit of the molecular chaperone CCT (Chaperonin-containing Tcp-1) is present in EVs from glioma cells derived from surgical specimens [241]. Secretion of CCT6A via EVs could be linked to GBM cell invasion and has a negative association with patient survival [241] (Figure 1). After their release in the extracellular space, EVs can either perform a local autocrine/paracrine signalling, targeting other nearby tumor cells and nervous-system cells (neurons and astrocytes), or cross the blood-brain barrier and reach distant cells through the systemic circulation. In both cases, EVs release their cargo into the cytoplasm of recipient cells by fusing with the target-cell membrane. At this point, the delivered molecules can mediate many physiological and pathological processes. EVs cargo reflects the characteristics of the cells from which they originate. Therefore, it is likely that both miRNAs and Hsps normally produced by tumor cells may be selected for the sorting into the released EVs and affect the tumor microenvironment and other distant tissues. As indicated in the bracket at the top right half of Figure 1, miRNAs may act either as onomiR, favoring tumor cells proliferation and migration, or as tumor suppressor, by inhibiting cancer cells survival and proliferation.