**4. Glioblastoma's angiogenesis**

or immunofluorescence is one of the most straightforward in situ techniques used to assess

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

In recent years, different in vivo imaging techniques have contributed to the understanding of

• 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

• Noninvasive fluorescence imaging (NFI), image acquisition is fast (in the range of seconds), and the imaging equipment is comparably inexpensive, simple, and easy to use. However, NFI has a limited resolution (1–2 mm) and quantitation of the imaging signal has several

• Nuclear imaging, rarely used for clinical imaging of BBB impairment. Examples of nuclear imaging techniques are single photon emission computed tomography (SPECT) and posi-

• Magnetic resonance imaging, MRI upon intravenous injection of a contrast agent, usually gadolinium-diethylene triamine pentaacetic acid (Gd-DTPA; MW 550 Da), is the most commonly used noninvasive imaging technique for detection of BBB impairment in both clini-

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

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

BBB dysfunction in disease [52]. Among these, noteworthy:

BBB impairment [52].

134 Liquid Biopsy

molecular weight.

animals.

pitfalls.

tron emission tomography (PET).

cal and preclinical studies.

molecular imaging of brain diseases.

related to the dissemination phase of the disease (**Figure 2**)**.**

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 active, and predisposed to cellular necrosis [56].

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 the highest degree of vascular proliferation and endothelial cell hyperplasia [62]. Patients with high tumor microvascular densities exhibit shorter postoperative survival rates than patients with low microvascular densities [63, 64].

A key challenge in the field of BBB permeability is the current shortage of the knowledge on the most efficient way to cross BBB used by tumor cells. It has been suggested that the brain endothelial cells can actively participate in metastatic progression and stimulate increased

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

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

137

During vasculogenesis, EPCs are mobilized from the bone marrow by increased concentrations of chemokines, growth factors and other soluble factors in serum, including stromal cell-derived factor-1 (SDF-1), VEGF, and granulocyte-monocyte colony stimulating factor

Given the high rate of angiogenesis in glioblastomas and the lack of prognostic markers for anti-VEGF treatments in general, the kinetics and prognostic relevance of circulating endothelial cells was assessed relatively this their potential role. It is clear that molecules with high pro-angiogenic potential provide the recruitment of EPC from bone marrow (VEGF, IL-8, IGF, etc.) [81]. Moreover, EPC provide paracrine signaling to facilitate angiogenesis [79, 80]. It has been suggested that quantification of CECs is useful to identify patients who might benefit from anti-angiogenic treatments [81]. Batchelor et al., in a series of patients with glioblastoma treated with AZD2171, a pan-VEGF receptor tyrosine kinase inhibitor, found that viable CEC number increased when tumors escaped treatment [82]. Indeed, CECs, rare in healthy individuals, increase in vascular disorders and tumors due to vascular damage such as the GBM, in which, the VEGF is highly expressed and may mobilize endothelial precursors from the bone marrow [83–85]. During GBM progression, the extracellular matrix (ECM) changes its flexibility and structure (i.e., content of fibrillary proteins as collagen, fibronectin, etc.) and malignant infiltration of glioma may stimulate the development of distinct provisional ECM patterns. Indeed, tumor cells induce changes in ECM by secretion of growth factors and angiogenic factors such as VEGF, bFGF, PDGF, and TNF-α [86]. The combined release of these factors have a synergic effect on the mobilization of EPC [87]. Further studies are needed to

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.

assess the role of EPC in liquid biopsy as marker of BBB dysfunction.

**5. Microenvironment in glioblastoma**

BBB permeability.

(GM-CSF) [77, 78].

Angiogenesis and tumor cell invasion play a critical role in GBM development and growth, even during the earliest phases [65, 66].

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

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-activating polypeptide 2 [69, 70].

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

Another mechanism of neo-vascularization includes the recruitment of endothelial progenitor cells (EPCs) that have been proposed to originate from different sources, including bone marrow, the existing vasculature, or adipose tissue [72, 73].

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:


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 the severity of cerebral alterations [76].

A key challenge in the field of BBB permeability is the current shortage of the knowledge on the most efficient way to cross BBB used by tumor cells. It has been suggested that the brain endothelial cells can actively participate in metastatic progression and stimulate increased BBB permeability.

During vasculogenesis, EPCs are mobilized from the bone marrow by increased concentrations of chemokines, growth factors and other soluble factors in serum, including stromal cell-derived factor-1 (SDF-1), VEGF, and granulocyte-monocyte colony stimulating factor (GM-CSF) [77, 78].

Given the high rate of angiogenesis in glioblastomas and the lack of prognostic markers for anti-VEGF treatments in general, the kinetics and prognostic relevance of circulating endothelial cells was assessed relatively this their potential role. It is clear that molecules with high pro-angiogenic potential provide the recruitment of EPC from bone marrow (VEGF, IL-8, IGF, etc.) [81]. Moreover, EPC provide paracrine signaling to facilitate angiogenesis [79, 80]. It has been suggested that quantification of CECs is useful to identify patients who might benefit from anti-angiogenic treatments [81]. Batchelor et al., in a series of patients with glioblastoma treated with AZD2171, a pan-VEGF receptor tyrosine kinase inhibitor, found that viable CEC number increased when tumors escaped treatment [82]. Indeed, CECs, rare in healthy individuals, increase in vascular disorders and tumors due to vascular damage such as the GBM, in which, the VEGF is highly expressed and may mobilize endothelial precursors from the bone marrow [83–85]. During GBM progression, the extracellular matrix (ECM) changes its flexibility and structure (i.e., content of fibrillary proteins as collagen, fibronectin, etc.) and malignant infiltration of glioma may stimulate the development of distinct provisional ECM patterns. Indeed, tumor cells induce changes in ECM by secretion of growth factors and angiogenic factors such as VEGF, bFGF, PDGF, and TNF-α [86]. The combined release of these factors have a synergic effect on the mobilization of EPC [87]. Further studies are needed to assess the role of EPC in liquid biopsy as marker of BBB dysfunction.
