**6. Circulating biomarkers of glioblastoma**

The dissemination of cancer cells in the bloodstream transforms a limited disease in a systemic pathology with remarkable prognostic and therapeutic complications for the patients. The phase of dissemination is a complex plan of action, in which, independently by type of tumor and its location, there are two main actors, the endothelial and the tumor cells. The endothelial cells compose the wall of vessels and capillaries and represent the primary bloodtissue barrier. The adhesion molecules expressed on ECs surface condition the permeability and trans-endothelial resistance to cell crossing [6].

In the brain, the filter function of BBB is carried out by ECs strictly interconnect with numerous tight junctions. The tight junctions give continuity to the endothelial wall eliminating the intercellular space. Astrocyte end-feet, pericytes and microglia interacting with ECs draining fluids in the glymphatic system. Glymphatic system controls the levels of concentration of solutes in the neuronal interstitium and it is connected with the blood circulation [6].

Animal models combined with observations on humans, are still promising tools in order to clarify mechanisms of passage of malignant cells trough the BBB. Indeed, similarly to what is observed in epithelial tumors have been demonstrated by patient-derived GBM xenografts that acquisition of a mesenchymal phenotype is mandatory for malignant cells in order to enter the blood stream and behavior as CTCs [94].

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 BBB-opening and successively favored cellular BBB-crossing.

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 prolifera-

tion of the cells in the body, for example during the hematopoiesis.

The dissemination of cancer cells in the bloodstream transforms a limited disease in a systemic pathology with remarkable prognostic and therapeutic complications for the patients. The phase of dissemination is a complex plan of action, in which, independently by type of tumor and its location, there are two main actors, the endothelial and the tumor cells. The endothelial cells compose the wall of vessels and capillaries and represent the primary bloodtissue barrier. The adhesion molecules expressed on ECs surface condition the permeability

In the brain, the filter function of BBB is carried out by ECs strictly interconnect with numerous tight junctions. The tight junctions give continuity to the endothelial wall eliminating the intercellular space. Astrocyte end-feet, pericytes and microglia interacting with ECs draining fluids in the glymphatic system. Glymphatic system controls the levels of concentration of

Animal models combined with observations on humans, are still promising tools in order to clarify mechanisms of passage of malignant cells trough the BBB. Indeed, similarly to what is observed in epithelial tumors have been demonstrated by patient-derived GBM xenografts that acquisition of a mesenchymal phenotype is mandatory for malignant cells in order to

solutes in the neuronal interstitium and it is connected with the blood circulation [6].

**6. Circulating biomarkers of glioblastoma**

138 Liquid Biopsy

and trans-endothelial resistance to cell crossing [6].

enter the blood stream and behavior as CTCs [94].

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

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

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

in which therapeutic drugs may be favorite to cross BBB. Moreover, those patients in which the BBB opening is not detectable through liquid biopsy could be considered a different

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therapeutic approach using EVs.

Natalia Malara\* and Giuseppe Donato

\*Address all correspondence to: nataliamalara@unicz.it

School of Medicine, University Magna Graecia of Catanzaro, Italy

**Author details**

**References**

2017. pp. 298-299

nm.3407

**Figure 3.** Schematic representations of endothelial-cancer cells interaction. (A) Interstitium space at brain (blood-brain barrier, BBB) and at blood barrier (BB) of other organs. Vessels (red), pericyte (green-cell), astrocyte end-feet (yellow-cell) and cancer cells (blue cells). (B) Cancer cells cross BB through pre-existing inter-endothelial spaces, (C) while at BBB overcome the absence of intercellular spaces realizing permeabilizing cytokines (green balls).

from marrow cells were administered in intracranial rat tumors with consequent reduction of glioma cell invasion, migration, viability and expression of EGFR normally amplified in approximately 40% of all GBM [104]. The possibility of miRNA-based approaches using exosomes have a strong therapeutic potential, which could be achieved through the delivery and transfer agents in those cases of GBM in which the BBB opening is a rare event. In these cases, the liquid biopsy negative for the detection of EPC and CTCs could be useful to select the patients eligible to this type of approach (**Figure 3**).
