**5. Microenvironment in glioblastoma**

the highest degree of vascular proliferation and endothelial cell hyperplasia [62]. Patients with high tumor microvascular densities exhibit shorter postoperative survival rates than

Angiogenesis and tumor cell invasion play a critical role in GBM development and growth,

Angiogenesis requires three distinct steps: (1) blood vessel breakdown, (2) degradation of the vessel basement membrane and the surrounding extracellular matrix (ECM), and (3) migration of endothelial cells for the formation of new blood vessels. The first step (1) in forming new blood vessels from existing vessels is the dissolution of aspects of native vessels [67].

Tumor angiogenesis results from a balance between pro-angiogenic factors and anti-angiogenic factors with a shift toward angiogenic factors that stimulate uncontrolled and disorganized vascular growth. These molecular factors can be secreted by cancer, endothelial,

Pro-angiogenic factors include vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), acidic fibroblast growth factor, basic fibroblast growth factor (bFGF), placental growth factor, angiopoietin-2 (Ang-2), and interleukins, whereas anti-angiogenic factors include angiostatin, endostatin, thrombospondin 1, and endothelial monocyte-activat-

The VEGF play a major role in GBM angiogenesis by stimulating capillary sprouting from pre-existing vessels toward VEGF-expressing tumor cells. Tumor VEGF expression and angiogenesis are mainly hypoxia-driven, but can also be promoted by other vascular cyto-

Another mechanism of neo-vascularization includes the recruitment of endothelial progenitor cells (EPCs) that have been proposed to originate from different sources, including bone

Upon BBB injury, endothelial progenitor cells (EPC) contribute (directly or as a source and carrier of pro-angiogenic factors) to BBB re-endothelialization. EPC are involved to the cerebral angiogenesis associated with physiological conditions (i.e., activity-induced neurogenesis) or pathological conditions (i.e., tumor progression). In adults, there are three origins of EPC:

**i.** Bone marrow origin (EPC derived from bone marrow multipotent hemangioblasts

**iii.** Tissue origin (EPC found at the sites of extensive angiogenesis but demonstrating no signs of hematopoietic origin, being, probably, derived from tissue multipotent cells) [73–75] In pathological conditions, angiogenesis and vascular remodeling are usually considered as significant components of brain tissue repair program after injury (hypoxic, ischemic, traumatic, inflammatory, toxic, etc.) and the mobilization of EPC from bone marrow correlates to

**ii.** Mesenchymal origin SC) (CD73(+)CD90(+)CD105(+)CD34(−)CD45(−)];

patients with low microvascular densities [63, 64].

stromal, and blood cells and by the extracellular matrix [68].

kines and constitutively expressed by genetic tumor mutations [71].

marrow, the existing vasculature, or adipose tissue [72, 73].

[VEGFR2(+)VEcadherin(+)CD45(−)

the severity of cerebral alterations [76].

even during the earliest phases [65, 66].

136 Liquid Biopsy

ing polypeptide 2 [69, 70].

Cytokines play a critical role in contributing to the complexity and lethality of glioblastoma characterized by diffuse invasiveness, immunosuppression, aggressive proliferation, vascularization, and resistance to conventional radiotherapy and chemotherapy. Cytokines consist in small molecules with diverse effects depending on the microenvironment. Cytokines comprise glycoproteins and polypeptides that exert pro-inflammatory, anti-inflammatory or immunosuppressive action. In addition to the tumor cells, immune cells, extracellular matrix, blood vessels, the cytokines are an integral part of the network GBM tumor microenvironment-associated. Glioblastoma arises from astrocytes and their precursors, neural stem cells. The resulting tumor is a heterogeneous cellular population composed of both undifferentiated and differentiated cells and containing subpopulations of tumor cell with different grade of tumorigenic property. Recently, study on GBM progression reveals a feed-forward mechanism with the epidermal growth factor receptor variant III (EGFRvIII) and STAT3 defined by the presence of cytokine receptor OSMR on the surface of non-tumorigenic and tumorigenic brain tumor cells. In particular, EGFRvIII-OSMR complex signals activates STAT3, and STAT3 signals upregulates OSMR expression. The result is a feed-forward signaling mechanism that drives oncogenesis in GBM. One of principal activator of the JAK-STAT signaling pathway is IL-6. Interleukin-6 (IL-6) is a pleiotropic cytokine that regulates the immune response, but also plays a role in promoting tumor growth and survival [88, 89]. In gliomas, the level of IL-6 gene expression increases with the grade of malignancy. In GBM, the, amplification/overexpression of the IL-6 gene appears to be a common feature [90]. Array studies have reported that the number of IL-6 gene copies was increased in 40–50% of GBM [91, 92]. In ependymoma, with inflammatory phenotype, was described a constitutive activation of the IL6/STAT3 pathway and crosstalk between tumor and immune cells with significant increase in STAT3 and IL8 secretion in tumor microenvironment. Moreover, IL-6 and IL-8, EGF, and other cytokines are involved on the regulation of endothelial function at BBB level, with several potential outcomes: increased permeability, generation of relaying signaling including another cytokine(s) and soluble mediators potentiating the effects of another cytokine(s), modulation of efflux transporters). Even though the BBB within the tumor is considered "permeable," in large parts of gliomas the BBB more closely resembles the intact BBB and prevents efficient passage of cancer therapeutics, such as small molecules and antibodies. Recently, was reported a tumor screening study that combined the detection and analysis of circulating tumor DNA with protein markers comprising some interleukins and the increase of chemokines (IL-6, IL-8) with encouraging results [93]. The big limit in the use of circulating tumor DNA in the screening phase of tumor disease is due to the poor specificity of this biomarker. In fact, mutated circulating DNA may be found in the peripheral blood of healthy subjects, it represents the genetic segments discarded during the normal proliferation of the cells in the body, for example during the hematopoiesis.

The detection of CTCs in the field of CNS represents a promising noninvasive technique to facilitate early diagnosis and monitoring tumor evolution [95, 96]. The significance of the CTCs detection in intracranial tumors, commonly related to metastatic phase of the cancer disease, was hardly applied for the very few case with extracranial metastases observed although the high malignancy and invasiveness of glioma [97]. More recently, it was reported that disseminating cells of medulloblastoma were able to infiltrate the leptomeninges through hematological way [98]. Garzia et al. described an in vivo experiment consisting in *via surgical* union of two mice by their flanks. The cancer cells of medulloblastoma implanted in one mouse model were able via hematological to give leptomeningeal metastatic disease in the other one mouse. The surgical union shared not only the cells but also the biochemical environment. The biochemical composition of cancer microenvironment consists by a mixture of permeabilizing and inflammatory cytokines. Many types of cytokines like Histamine, TNFa, IL1b and in particular the axis, chemokine CCL2-CCR2 receptor, sustain BBB alterations. Their action plays on BBB by activating cytoskeleton redistribution, downregulating structural proteins and favoring the tight-junction opening [99]. Garzia et al. to obtain leptomeningeal relapsing lesion used an experiment that shared the microenvironment rather than to inject only cancer cells. The tumor cells, although expressing molecular equipment involved in cell movement, were not able, alone, to determine leptomeningeal metastasis. The tumor cytokines mediated

Blood-Brain Barrier Breakdown by Combined Detection of Circulating Tumor and Endothelial…

http://dx.doi.org/10.5772/intechopen.80594

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Moreover, due to the contribution of other studies demonstrating that CTCs can be detected in all pathological subtypes of glioma, irrespective of different malignant degree, now, the reductive misconception of *metastatic CTCs* has been challenged suggesting that CTCs should be considered a common property of glioma lesions [100]. The behavior of the CTCs suggest that the pathogenesis of their release in the blood stream is independent from the grade of malignancy of intracranial tumor, and such as happened in other tumor diseases, this phe-

The BBB confers to intracranial tumor diseases a different nosology. The presence of CTCs in the bloodstream of patients with intracranial tumors acquires a diverse significance respect to cancers located in extra-cranial sites. Regarding the intracranial cancers could be more appropriate to use CTCs as biomarkers of BBB-damage rather than biomarkers of a preferential/ alternative dissemination way. The understanding of this scenario is important to focus the better targeting strategy. The interference on the interaction between cancer-endothelial cells or the reduction of the cytokines levels could be the key targets for pharmacological interven-

Another emerging role in the study of glioblastoma and liquid biopsy are represented by the detection in the peripheral blood of extracellular vesicles (EVs) of tumor origin. EVs are secreted by tumor cells, are mediators of intercellular communication that transfer nucleic acids, proteins and lipids. The liquid biopsy of EVs in patients with GBM has the role to clarify their implications in tumor progression, as a tumor biomarker for tracking GBM progression and as a potential therapeutic target/delivery system [102]. Therapeutic experimental protocols involving EVs delivery of miRNA were performed for GBM. The deletions on chromosome 10 are a common chromosomal alteration found within GBM with leads to a loss of miR-146b normally located at 10q24 [103]. In GBM the loss of miR-146b facilitates migration and invasion. Moreover, in a study using an in vivo model, the exosomes derived

BBB-opening and successively favored cellular BBB-crossing.

nomenon happened in each phase of cancer development [101].

tions for the inhibition of cell spread by an intracranial tumor.
