**3. Blood-brain barrier damage**

Morphology and function of BBB are linked as shown by microscopic observation. Early ultrastructural studies where performed by administration of silver nitrate in the drinking water of rodents [33]. They showed the presence of very scarce quantity of silver around the capillaries in the cerebral cortex, medulla, and cerebellum. In such brain regions, capillaries are continuous. In other areas, such as neurohypofisis, area postrema, pineal body and intercolumnar tubercle, heavy accumulation of silver was present around fenestrated capillaries [34]. Using lanthanum and horseradish peroxidase as tracers, via intravenous injection, it has been demonstrated that these substances are unable to penetrate between the endothelial cells because of the tight junctions presence (zonulae occludentes). Regarding the contribution of astrocytes to the BBB, it is well known that its end-feet form a relatively complete layer, but the junctions between are gap junctions and not of the occluding kind [35]. Perivascular endfeet of astrocytes do not provide an effective barrier even if substances should pass through the endothelial cells into the brain. Indeed, after intravenous infusion of peroxidase, endothelial cells show micropinocytotic vesicles containing the tracer [36]. Such a transendothelial cell barrier is very selective and based on carrier-mediated transports, but is not furtherly

Caveolae are small, bulb-shaped, plasma membrane invaginations. They have been described to have a function in endocytosis and transcytosis and in in maintaining the lipid composition

While endothelial cells in peripheral organs, such as the lung and heart, are enriched in caveolae, in BBB only a small number of caveolae are detectable [24]. Mfsd2a (transporter of the major facilitator superfamily domain-containing (Mfsd) family) contributes to the regulation of vesicular traffic in BBB endothelial cells [37] through the transport of the essential omega-3 fatty acid docosahexaenoic acid (DHA). The expression of Mfsd2a becomes upregulated in ECs with the maturation of BBB. Gene ablation of Mfsd2a in mice results in BBB leakiness and

Caveolins (Cavs) are thought to play a role in the regulation of BBB function. Cavs, are a family of integral membrane proteins which represent both positive and negative regulators of intracellular signaling as scaffolding proteins that regulate the intracellular distribution of the signaling molecules. Cav-1 overexpression protects the integrity of the BBB mainly by

Cav-1 is a marker of caveolae in endothelial cells and is important in the regulation of various functions like endocytosis, transcytosis, signal transduction, and molecular transport. Recent studies on mice indicate that the suppression of the caveolae pathway requires the transport of lipids, notably DHA-containing phospholipids, by Mfsd2a to regulate CNS endothelial cell

Moreover, Cav-1 regulate the angiogenic response by influencing VEGF receptor 2 (VEGFR2)

In BBB the basement membrane (BM) represents the noncellular component. Astrocytes, PCs and ECs synthetize and secrete molecules which constitute the BM surrounding the external surface of the endothelial cell, composed by type IV collagen, fibronectin, heparan sulfate,

plasma membrane composition and to inhibit caveolae vesicle formation [39].

of the membrane, as well as acting as signaling background.

preventing the degradation of TJ proteins in rats [38].

phosphorylation and internalization [40, 41].

mechanically regulated.

132 Liquid Biopsy

increased vesicular traffic in ECs.

The endothelial cells (ECs) compose the wall of vessels and capillaries and represent the primary blood-tissue barrier. The ECs acting as a protective filter are able to regulate the passage of molecules and immune cells and the level of specialization of each blood-barrier is determined by the functions of endothelial-wall. In the brain, there is the higher level of specialization of the endothelial wall. The filter function of BBB is carried out by ECs strictly interconnect with numerous tight junctions.

Pathological conditions within the central nervous system like ischemia, inflammation or tumor growth lead to blood-brain barrier (BBB) dysfunction, emphasizing that the permeability barrier regulation is principally provided by the local microenvironment and its maintenance is a necessary condition in any circumstance. In many brain tumors morphological irregularities of the perivascular space correlate with a breakdown of the BBB [45–47].

Wolburg et al. [48] found that claudin-3, a key component of BBB tight junctions, is lost in glioblastoma. Further evidence on claudin-1 loss in tumor microvessels, as well as downregulation of claudin-5 and occludin in hyperplastic vasculature, result in a phenotypic change in BBB function due to leaky tight junctions and hyperpermeable endothelial cells.

A recent study by Watkins et al. [49] using a mouse model demonstrates that astrocytic endfeet displace from their position alongside endothelial cells with disruption of the communication between the astrocytes and vasculature and that single glioma cells were sufficient to produce local BBB opening.

The structural perturbations of the vascular barrier during tumor progression, in other organs was demonstrated, are successive to the release in the tumor microenvironment of specific cytokines that downregulate the transcription of these structural proteins [50].

The crucial point is the vascular damage that the tumor direct and indirect operas through the release on the next microenvironment of chemical factors able to increase the permeability of the tissue vessels reducing their protective barrier function. This could explain the pathogenesis of the BBB damage occurring during the development of glioblastoma. The BBB damage induced by the glioblastoma represents a strategy employed to control the BBB opening to allow the passage of drugs [51].

The detection of endogenous circulating molecules normally restricted by the BBB, namely albumin, immunoglobulin G, or fibrinogen, in the brain parenchyma, using immunohistochemistry or immunofluorescence is one of the most straightforward in situ techniques used to assess BBB impairment [52].

Measurement of exogenous tracer extravasation remains a technique of choice in preclinical studies testing for BBB permeability. A variety of detectable exogenous tracers has become available, and methods have been developed to evaluate the kinetics of their extravasation. Due to variations in tightness of the BBB, the extravasation of different BBB permeability tracers across an impaired BBB critically depends on their physicochemical properties and molecular weight.

In recent years, different in vivo imaging techniques have contributed to the understanding of BBB dysfunction in disease [52]. Among these, noteworthy:

• In vivo confocal microscopy, an important tool for the high-resolution dynamic fluorescence imaging in BBB research. This technique has the advantage of time-lapse imaging of the same subject. The apparent disadvantages of this technique are the technical complexity and invasive nature, because it requires opening of a cranial window in experimental animals.

**4. Glioblastoma's angiogenesis**

et al. IJC Heart & Vasculature [106]).

active, and predisposed to cellular necrosis [56].

Gliomas are a heterogeneous group of neoplasms derived from glial cells that account for 40–45% of all intracranial tumors. The most malignant type of glioma, Glioblastoma multiforme (GBM), account for approximately 12–15% of all intracranial neoplasms and 50–60% of all astrocytic tumors. In most European and North American countries, incidence is approximately 2–3 new cases per 100,000 people per year. Nearly 260,000 patients worldwide are diagnosed annually with primary malignant brain cancer [54, 55]. The World Health Organization defines GBM as a cancer of grade IV characterized as malignant, mitotically

**Figure 2.** Vessel's arrangement of endothelial cells acting as protective filter (A) mimics an array of warriors (B) placed to protect the tissues. In (A), primary human endothelial culture. Scale bar 100 μm (figure partially modified from Malara

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

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

135

Untreated patients with glioblastoma multiform uniformly die within 3 months from the diagnosis. Treated patients with a protocol including surgical resection, radiation therapy, and chemotherapy, have a median survival of 14.6 months, with fewer than 25% of patients surviving up to 2 years and fewer than 10% up to 5 years [52, 57, 58]. Moreover, males had a slight preponderance over females, with a male-to-female ratio of 1.6:1. GBM may manifest in persons of any age, but it affects adults preferentially, with a peak incidence at 45–70 years [55]. Glioblastomas are tumors that display extensive morphological and molecular heterogeneity, and thus may reflect their origin from different population of astrocytes, and possibly from oligodendrocytes and ependymal cell lineages. GBM rarely metastasize outside the brain [59]. GBM can be classified into primary type and secondary type, according to whether they are generated de novo or by progression of lower-grade tumors. Histologically, primary and secondary glioblastomas are largely indistinguishable, but they differ in their genetic and epigenetic profiles [60]. GBM is a highly aggressive tumor with distinct histopathological features, including high proliferation, necrosis and considerable neovascularization (i.e., angiogenesis), leading to vessels that exhibit morphological abnormalities and "leakiness" [60]. It is generally accepted that the degree of angiogenesis is correlated to the malignancy of the tumor [58, 61]. GBMs are the most lethal cancer and the most vascularized brain cancer, with


With the exception of brain tumors, where angiogenic biomarkers have been targeted for molecular imaging [53], cerebrovascular biomarkers have not yet been fully exploited for molecular imaging of brain diseases.

Despite this advance the problem to track BBB opening remains for insufficient selectivity, specificity of the tools provided and for the no sufficiently abundancy [52] of the biologicsbased molecular imaging probes today available. In this prospective the liquid biopsy could represent an opportunity to track in real time the BBB opening. The concept is based on the causal release of tumor cells in the systemic circulation through the BBB damage. The structural perturbation of the BBB and the consequent modification of its permeability could induce cell spread independently by the molecular equipment expressed by cancer cell. In this direction, the detection of the circulating cancer cells in glioblastoma cases could acquire a specific significance related to the permeability of the BBB more than a prognostic value related to the dissemination phase of the disease (**Figure 2**)**.**

Blood-Brain Barrier Breakdown by Combined Detection of Circulating Tumor and Endothelial… http://dx.doi.org/10.5772/intechopen.80594 135

**Figure 2.** Vessel's arrangement of endothelial cells acting as protective filter (A) mimics an array of warriors (B) placed to protect the tissues. In (A), primary human endothelial culture. Scale bar 100 μm (figure partially modified from Malara et al. IJC Heart & Vasculature [106]).
