It is a well-known fact that effectiveness of oncotherapy in brain tumors remains under the expectations in comparison to anaplastic tumors of other organs. Knowing the very modest survival rates enormous efforts of neuro-oncological researches has been made, but only partial success is produced. Beside the extremely high proliferation rate of high grade glioma cells researches established the highly intensive invasiveness and angiogenesis as the main reasons of treatment failure. In this chapter the main molecular mechanisms of brain tumor invasion and angiogenesis will be discussed followed by the hopeful treatment possibilities that are already in studies and will be achievable in the near-future.
2. General a spects of glioma invasion
Malignant gliomas are the most common primary brain tumors. They are associated with the shortest survival time explained by their early recurrence due to their deep invasion of the normal brain, which makes them practically impossible to remove completely. Invasive anaplastic gliomas are almost invariably fatal, recurring close to the resection margin in almost all cases. Interestingly, primary brain tumors have a strong tendency to invade their environment, but with rare exceptions, do not metastasize outside the brain. [1-3].
To understand the invasion behaviour of gliomas, the cellular and molecular events of peritumoral infiltration have to be discussed. The most important medium for this process is the extracellular matrix (ECM). The ECM comprises a considerable proportion of the normal brain volume. The extracellular space (ECS) of the healthy brain tissue volume is approximatley 20%. The extracellular volume fraction in the majority of primary brain tumors is significantly increased, representing about 48% of the total tumor tissue volume especially in high grade gliomas. The structure and compounds of the ECM of the brain tissue have many specific differences from other human organs. The ECM of the brain contains mainly macromolecules like glycosaminoglycans (GAGs) and proteoglycans (PGs), and only moderately express fibrillary glycoproteins (e.g. collagens, fibronectin, elastin or reticulin). The compounds of ECM glycoproteins play a crucial role in peritumoral invasion forming structural elements for cellular attachment and migration. There is much evidence that ECM components can modulate brain tumor growth, proliferation, and invasion by many different mechanisms. Thus extracellular matrix plays a pivotal role in the tumorous infiltration of the surrounding tissue. The presence and functions of hyaluronic acid (hyaluronan, HA), PGs and various types of GAGs have already been intensively investigated to clarify the molecular mechanisms of invasion, and a positive correlation has been established many times. To allow cell adhesion and migration, the ECM components interact with specific receptors on the cell membrane, such as integrins, CD44, or CD168. Some proteases and synthases also strongly influence invasiveness because of their capacity to alter the actual levels of the ECM molecules or to degrade the pericellular network. [4-16]
Using the ECM macromolecules to their active movement, glioma cells infiltrate the enviroment and form it similar to the tumor tissue. The process of the peritumoral invasion depends on the confrontation zone of the tumor cells and the non-neoplastic cells and ECM. Glioma cells express mainly adhesion receptors and proteases, while host cells produce macromolecules to maintain original structure and to inhibit invading cell movement. Since brain ECM has no strong fibrillar, collagen-rich network, the brain parenchyma remains soft, that can not hinder significantly the migration of tumor cells.
In case of glial cell tumors there are two main factors that significantly promote peritumoral infiltration. First is the normal structure of the brain parenchyma composed mainly by tracts in the white matter and basement membranes, which are suitable for guiding cell migration. Second is the increased ability of glia cells to migration. Both factors are special for the brain and they can be easily understood knowing the connection of development, structure and function. [17, 18]
From neuro-oncological point of view the increased glioma cell mobility and extensive peritumoral infiltration leads to the following problems:
A. Total extirpation of a low grade tumor is not an easy and evident technical tool of therapy. This is one main reason why these tumors are “semi-benign” tumors. Thus in spite of the macroscopically radical surgical removal, the recurrence rate of these tumors is very high, and full recovery is not a general event.
B. In case of high grade tumors, neither open surgery, nor stereotactic radiosurgery can achieve radical tumor removal. This experience can explain the local recurrence that appears in almost every case.
C. Local chemotherapeutical treatment (intraparenchymal or post operatively administered intracavital drug) has low effectiveness.
3. Molecular aspects of glioma invasion
Molecules that are responsible for the cell migration are divided in three groups:
Cell-membrane associated molecules (receptors and adhesion molecules).
Extracellular matrix (ECM) components (targets for the receptors).
Enzymes that are synthesizing or lysating the ECM components.
3.1. Cell-membrane associated molecules (receptors and adhesion molecules)
Molecules with evident role in peritumoral invasion are located either on the cell surface, or form transmembrane structure. The main representatives of this group are the receptors and adhesion molecules as detailed below.
Integrins mediate also activation of
3.2. Extracellular Matrix (ECM) components (targets for the receptors)
Various components of brain ECM, like GAGs and PGs are overexpressed in gliomas. These molecules are binding sites for tumor cell receptors or they can inhibit cell migration, so they take an important part in peritumoral glioma invasion, and consequently could also serve as targets for anti-tumor therapy.
Depending on the GAG side chains the main types of PGs are chondroitin-sulphate (glycuronacid and N-acethylgalactosamine polymer and protein core), dermatan-sulphates (former name chondroitin-sulphate-B, composed of iduronacid and N-acethylgalactosamine polymer and protein core), heparansulphate (glycuronacid and N-sulphoglucosamine polymer and protein core) and keratansulphate (galactose and N-acethylgalactosamine polymer and protein core). Hyaluronic acid (hyaluronan, HA) is consisted of only GAGs (glycuronacid and N-acethylglucosamin polymer) that has no covalent bind to a protein, so it is not a PG by definition, but due to its tight relation to the PGs in general it is discussed together with them.
One of the most frequent adhesion glycoprotein in the ECM is
Another common component of the ECM is the molecular family of
The ECM glycoprotein
A number of proteinase families are capable of generating the proteolytic fragments of versican. Matrix metalloproteinase (MMP)-1, -2, -3, -7, and -9, ADAMTS-1, -4, -5 and -9 cleave versican and generates proteolytic fragments.The accumulation of proteolytic fragments of versican play an important role in cancer progression. The regulation of G1 and G3 versican levels by proteases is known to be important in regulating cancer cell motility and metastasis. Through the EGF-like motifs in the G3 domain versican can stimulate cell proliferation and its G1 domain destabilizes cell adhesion and promotes cell growth. Versican expression is associated with a high rate of proliferation and it is localized in HA-rich tissues and also accumulated in perivascular elastic tissues involved in peritumoral invasion. These features of versican make it a proliferative, anti-adhesive and pro-migratory molecule that facilitates tumor cell motility. In clinical samples the association of versican to invasiveness of astrocytoma could be evidently demonstrated. On the other side, the decreased expression of versican V0 and V1 isoforms in glioma ECM can be related to the marked local invasivity and rarity of extracranial metastasis of gliomas. [105-111]
3.3. Enzymes that are synthesizing or lysating the ECM components
4. Invasion process of tumor cells
Knowing the invasion potential of primary brain tumors, many of the molecular mechanisms of peritumoral infiltration have been already studied and some of the invasion processes have been defined. During malignant transformation, invasiveness is determined by the complex functions of tumor cells of distinct histological types. A four-step model of invasion has been applied, that is also valid for brain tumors. This model contains the following steps: 1) the tumor cells at the invasive site detach from the growing primary tumor mass; 2) they adhere to the extracellular matrix (ECM) via specific recebptors; 3) proteases secreted by the glioma cells locally degrade the ECM components, forming a pathway migration into the surrounding tissue, and 4) tumor cell movement due to cytosceletal processes. Each step of the peritumoral invasion requires a harmonized cooperation of numerous molecules resulted in active cellular movement into the normal brain parenchyma. [119, 120]
The detachment of invading glioma cells from the primary tumor mass is a complex process comprising the following steps: 1) Destabilization and disorganization of the cadherin mediated junctions that hold the primary mass together. 2) Decrease expression of cell adhesion molecule which provides adhesion to the primary tumor mass. This leads to a reduction in gap junction formation. Cell–cell communication is necessary for growth control and differentiation, and it is mainly achieved through gap junctions. Increased malignancy of gliomas is accociated with reduced in situ gap junction formation, and invasion of gliomas. 3) Cleavage of CD44, which anchors the primary mass to the ECM. This process is mediated by metalloproteinase ADAM. [119-123]
Tumor cell adherence to the ECM components is mediated by specific cell surface or transmembrane receptors like integrins binding to laminins, fibronectins and collagens or CD44 to hyaluronan.
Degradation of ECM components occurs due to the local enhancement and activation of protease suc as MMPs, hyaluronidase, cathepsins and chondroitin suphatase.
Due to migration the glioma cell must interact with the surrounding ECM, which forms a mechanical barrier to the cells, and serves as a substratum for traction for the moving cells. For cell movement changes in cell morphology occur: the cell becomes polarized and membrane protrusions develop, including the extensions at the leading edge of pseudopodia, lamellipodia, filopodia, and invadopodia. These extensions contain filamentous actin and various structural and signaling molecules. The formation of membrane anchors needs cytoskeletal contraction, which finally results a cell forward displacement. Glioma cell motility and contractility also require A and B isoforms of myosin II. Myosin II is the major source of cytoplasmic contractile force. Myosin II allows glioma cells to squeeze through pores smaller than their nuclear diameter, which is especially important for gliomas because the human brain tissue has particularly narrow extracellular spaces. The connection of ECM macromolecules and cytosceleton is mediated by dystroglycans. [69, 124]
5. The possible agents for antiinvasive therapy
Tumor cell invasion into the surrounding brain tissue is mainly responsible for the failure of radical surgical resection and successful treatment, with tumor recurrence as microdisseminated disease. ECM related molecules and their receptors predominantly participate in the invasion process, including the cell adhesion to the surrounding microenvironment and cell migration. Determination of the key molecules of invasion process can help toprovide possible targets for antiinvasive therapy. Regarding peritumoral infiltration activity of glioma cells, the following molecules are supposed to serve as antitumor agents.
Knowing the evident role of versican proteolytic fragments in cancer progression, its possible role as target for anti-cancer therapy has been arised. Although there are only a few results regarding anti-versican therapy in glioma patients, some possible agents are notable to mention for their potential future role.The tyrosine kinase inhibitor
Tumor formation of the pericellular matrix with HA and versican can be inhibited by treatment with
Since increased MMP levels are associated with glioma invasion and angiogenesis,
6. General aspects of angiogenesis
Rapidly growing tumors need to develop their own vasculature. The hypervascularisation of high grade gliomas can be visualized well on radioimaging and it can be a preoperative characteristic of glioblastoma. Furthermore, glioma angiogenesis is necessary for tumor expansion and survival, so its inhibition could be a potential tool in anti-tumor therapy.
There are two main angiogenic and invasive glioma phenotypes. Clusters of glioma cells perform single cell infiltrations into normal parenchyma independent of vasculature. Another group of glioma cells can be found around newly developed vessels in the normal brain parenchyma near to the tumor margin. These two different angiogenic and invasive phenotypes are called angiogenesis-dependent and angiogenesis independent invasions. High grade astrocytomas content both invasion phenotypes in a mixture of subclones present in different intratumoral regions. Molecular mechanisms of single cell migration were detailed above, but the role of neo-angiogenesis forms also a very important way to glioma expansion. 
In expanding, highly proliferate gliomas angiogenesis is activated when the pro-angiogenic stimuli dominates over the anti-angiogenic stimuli. These stimuli are mediated by factors secreted from glioma, endothelial or microglia cells, or arise from the extracellular matrix or other environmental sources like hypoxia induced cell productions. The pro- and anti-angiogenic forces are influenced strongly by tissue hypoxia and genetic alterations. The summation of these stimulileads to the so-called “angiogenic switch” in glioma angiogenesis.The most effective activator of angiogenesis in brain tumors is hypoxia that downregulates anti-angiogenic pathways and induces many pro-angiogenic ones. A well-known pathway is the HIF-1/VEGF-A pathway, which play a significant role in endothelial cell proliferation and migration. Another pathway mediator is interleukin-8, which is produced by microglia cells as a reaction to hypoxia. It is important to mention, that genetic instability of high grade gliomas provides the way of angiogenesis independently of hypoxia (such as chronic HIF activation via phosphoinositide 3-kinase (PI3K) or mitogenactivated protein kinase (MAPK) pathways. [146-152]
After activating the “angiogenic switch”, the tumor produces new vessels. The modes of new blood vessel formation in glioma occur by one of three different methods: 1) angiogenesis; 2) vasculogenesis; or 3) arteriogenesis. Angiogenesis is the formation of new blood vessels by rerouting or remodeling existing tumor vessels, and is supposed to be the main stream of neo-angiogenesis. Vasculogenesis means de novo production of blood vessels from circulating marrow-derived endothelial progenitor cells originally as the method of vasculature development in embryonic process. Since these progenitor cells have been also identified in tumors, they role in tumor angiogenesis cannot be denied. Vasculogenesis is probably regulated by tumor-secreted stromal-derived factor 1 under the control of the hypoxia-induced transcription factor hypoxia-inducible factor 1α (HIF1α). Arteriogenesis is the third mode of arteriolar networks formation representing a moderate proportion of tumor angiogenesis. [153-156]
The most significant way to form new blood vessels in gliomas is neoangiogenesis. Formation of new vessels from native vessels begins with breaking down the original vessel wall. The process of blood vessel breakdown is composed of three main phases. The first event in forming new vessels from existing ones is the disintegration of the vessel wall. Angiopoietin-1 (Ang-1) and its receptor Tie-2 play a pivotal role in this phase. Normally, Ang-1 binds to Tie-2 achieving a close association between pericytes and endothelial cells that is necessary for vasculature stability. In rapidly proliferating tumors like glioblastoma, tissue hypoxia increases and it induces Ang-2 upregulation in endothelial cells whereas Ang-1 is accumulated tumor cells. Increased Ang-2 expression, which is an antagonist of Tie-2, leads to the initial regression of blood vessels. Beside these, matrix-metalloproteinase (MMP)-2 expression is induced via Tie-2 signaling, and in conjunction with VEGF promotes angiogenesis. The second phase is the breakdown of ECM to provide place for the migration of endothel cells to form new blood vessels. Following dissolution of native vessel wall, degradation of the vessel basement membrane and relating ECM is the necessary condition for endothelial cells for invasion the surrounding microenvironment. MMPs play an integral role in this phase. In case of glioma angiogenesis, the collagenases MMP-2 and MMP-9 are involved in this process and their expression correlates with a poor prognosis in gliomas. Expression of MMP-2 and MMP-9 is also induced by hypoxia and through their proteolytic activity interaction of endothelial cells and tumor-ECM contents like VEGF and fibroblast growth factor (FGF) occurs. [157-166]
The third phase to form new blood vessels is the migration of endothelial cells. After dissolution of the basement membrane of the blood vessels and decomponent ECM, endothelial cells begins to proliferate and migrate toward tumor cells that expresses pro-angiogenic factors. Due to this process cell surface adhesion and migration molecules, such as integrins and CD44 upregulates.The activated endothelial cells secrets platelet-derived growth factor (PDGF) that induces pericytes to participate in creating a new basement membrane. For this reason beside migration of endothelial cells, pericyte migration also occurs as a necessary event of vasculogenesis. [167-169]
At the end of tumor blood vessel formation a significant change occurs in the extracellular environment, caused by increased expression of embryonic ECM molecules, such as tenascin-C. Elevation of VEGF and Ang-2 levels can be also detected, that probably explains the leakiness and pathologic structure of the new vessels. The result of glioma angiogenesis are highly tortuous dilated vessels and lots of small diameter vessels with alterations in endothelial cell adhesion molecule expression and disrupted basement membrane. [170-174]
7. Molecular aspects of glioma angiogenesis
Angiogenesis is mainly induced through growth hormone receptors, especially through the
Regarding glioma angiogenesis not only VEGFR but the hormone ligand
Other growth factors have also influence on angiogenesis. Epidermal growth factor (EGF),
In rapidly proliferating anaplastic gliomas oxigene supply remains constantly under the necessity, thus hypoxia remains a permanent stimuli for angiogenic factors. It seems to induce not only the secretion of growth factors, but also
Interestingly, there are some molecules involved in neuronal pattering during embryogenesis that have similar functions in vascular pattern during tumor angiogenesis. One of these molecules is the
Beside growth factors and their receptors, there are some ECM components that are overexpressed in glioma vessels in comparison to normal brain tissue, and have some stimulating effect on angiogenesis. One of the most important ECM proteins with an evident role in angiogenesis is
8. The possible agents for anti-angiogenic therapy
Since VEGFR play the most significant role in tumor angiogenesis, its inhibition bears the most effective possibility for decrease tumor growth. The VEGFR is a transmembrane tumor cell receptor, so blocking antibodies could close down its effect. On the other side blocking the intracellular tyrosine-kinase domain could also inhibit the activation of the signaling pathways. The latest way came into the front in past few years, when small-molecular tyrosine kinase inhibitors proved to be effective in vitro against glioma cell lines. Beside these, blocking the VEG-factor itself can also definitely decrease the stimulating effect of the receptor.
The most known VEGF neutralizing antibody is the bevacizumab that is already a possible tool of the oncotherapy for glioblastoma. In recurrent glioma patients treated with bevacizumab combined with the chemotherapy agent irinotecan the median survival can be prolonged. As the result of a significant antitumor effect 63% radiographic response, 6-month progression-free survival in 32% of GBM patients could be achieved. Based on these favorable observations further clinical trials have been initiated to combine bevacizumab with temozolomide, the current standard of care for newly diagnosed glioblastoma patients. Another clinical trial suggests that the presence of tumor hypoxia markers predicts probable radiographic response and better survival of patients treated with combinant chemotherapy of bevacizumab and irinotecan. Gliomas treated with bevacizumab often appear as nonenhancing infiltrating laesions on MRI proving the reduced vascularity beside the ongoing invasion, so induction of anti-angiogenic therapy combined with anti-invasive therapy seems to be a possible treatment method in the future. [198-203]
8.2. VEGF-receptor blocking
Anti-angiogenic therapy with VEGF receptor inhibitor
8.3. Other target molecules for anti-angiogenic therapy
Another ECM protein that has anti-angiogenic effect in glioma is secreted protein acidic and rich in cysteine (SPARC), also known as
8.4. Endogenous anti-angiogenic factors
A number of endogenous anti-angiogenic factors have been described that play pivotal role in tumor angiogenesis. Identifying these factors could offer some anti-cancer agent for neuro-oncological therapies. One of the best known endogenous anti-angiogenic proteins is
There are no simple and evidently succesful protocols for therapy of primary brain tumors. The intensive proliferation activity, the significant peritumoral infiltration and increased angiogenesis altogether are responsible for the extremely high recurrence rate of gliomas. The failure of recently administered chemotherapy arises the requirement of combination therapy. Thus besides searching a highly specific tumor marker, establishing the molecular spectrum of these tumors can be suggested. Supporting this theory, the mRNA expression pattern of the invasion-related molecules was found to be highly specific for various different histological tumor groups. So determination of the genetic signature of invasion of a glioma is thought to help in screening exact molecules as targets for individual chemotherapy.  Furthermore, complexity of oncotherapy with combination of antiproliferation, antiinvasive and antiangiogenic drugs could bring benefits in treatment effectiveness against brain tumors in the future.