**2. The Extracellular Matrix**

Gliomas differ from metastatic tumors in their ability to migrate into the surrounding brain parenchyma. While most recurrence occurs within 1–2 cm of the original tumor bed [5], seemingly multifocal disease or so-called "butterfly glioma" can develop at distant sites as a consequence of migration of cells along blood vessels and white matter tracts [6] (**Figure 1**). Although they are highly infiltrative, less than 2% of gliomas spread outside the CNS, suggesting that tumor cells are either not able to cross the basement membrane and enter the vasculature or that they require a specific neural environment containing specific molecules through which they can proliferate and migrate. Tumor cells switch from a proliferative to a migratory or mesenchymal phenotype, resorting to this more primitive state, which mimics the behavior of their migratory progenitors such as the radial glia that traversed white matter pathways and other structures during embryonic development [7]. The molecular mechanisms

**Figure 1.** Butterfly glioma in a patient with bihemispheric spread due to involvement of the corpus callosum.

In this chapter, we discuss the composition of the brain's extracellular matrix, as well as the mechanisms by which tumor cells transition to a migratory phenotype and remodel the ECM through degradation by novel proteases and their inhibitors. We discuss the search for ECM molecules expressed by the tumor cells, which then respond to chemoattractants in the environment in order to direct growth. Finally, we discuss potential targets of anti-infiltrative therapy and the obstacles that must yet be overcome to address this important neuro-oncologic

of this transition are currently poorly understood.

110 Neurooncology - Newer Developments

problem.

The ECM consists of three components: (1) perineural nets (PN) which surround neural cells and their processes and provide support and regulate plasticity, (2) a complex interstitial matrix between cells, and (3) basement membranes that surround blood vessels made up primarily of laminin, fibronectin, and collagens. ECM components are secreted by resident cells and serve to provide structural cell support, regulate cell-cell connectivity and commu‐ nication, and sequester growth factors and chemoattractants to regulate cell motility. The composition of the ECM varies from tissue to tissue and is comprised of proteoglycans such as chondroitin sulfate, heparin sulfate, keratin sulfate, and a lattice of interconnected fibrous proteins [8].

The neural extracellular matrix comprises 10–20% by volume of the brain and spinal cord and is structurally and functionally distinct from the ECM in other tissues [9] (**Figure 2**). The most abundant component of the neural ECM is hyaluronan (HA) and its associated glycoproteins. HA is a large hygroscopic glycosaminoglycan composed of alternating D-glucuronic acid and N-acetylglucosamine which is synthesized by hyaluronan synthase anchored to cell mem‐ branes [10]. In the developing brain, HA is organized into fiber-like structures along which neural precursors migrate. HA is anchored to astrocytes through its receptor, CD44, a transmembrane glycoprotein that couples the ECM to the actin cytoskeleton [11] and to hyaluronan synthase on neurons. Overexpression of CD44 was shown to increase the length of filopodia of neuroblastoma cells *in vitro* and promote invasion into a HA-rich matrix, demonstrating how overexpression of this single gene can affect the complex sequence of events for an invading cell to detach from its substrate, adhere to and degrade the surround‐ ing matrix, and migrate through it [12].

**Figure 2. Structure of neural extracellular matrix (ECM)**. Reproduced with permission from Miyata et al. [14].

HA is associated with a number of proteins that are organized into a scaffold within the ECM. The major group is chondroitin sulfate proteoglycans (CSPGs) whose structure consists of a core protein covalently linked to chondroitin sulfate glycosaminoglycan (CS-GAG) through serine residues [13, 14]. Chondroitin sulfate is a disaccharide chain consisting of glucuronic acid and N-acetylgalactosamine linked via a β-glycosidic bond and is polymerized into chains through the activity of chondroitin synthase and polymerizing factor [15]. Variability within CSPG derives from variation within the core protein of CSPGs as well as the number and

**Figure 3. Chondroitin sulfate proteoglycan structure**. (A) Binding of glycosaminoglycan side chains to core protein through serine residues (Xyl, xylose; Gal, galactose). Sulfate groups added at R. (B) CSPG core protein with N-terminal hyaluronan binding domain and a C-terminal tenascin-binding domain. Central domain binds GAG side chains. (C) Structure of Lecticans (Reproduced with permission from Miyata et al. [14]).

positions of sulfate groups, which are added via chondroitin sulfotransferases [16, 17]. This variability determines the CSPG binding properties and their function. Link proteins such as Bral-1 and 2, [18], Crtl1 [19], and HAPLN1 [20] stabilize the interaction between HA and CSPG within the PN. Mice lacking Crtl1 have attenuated PN and persistent plasticity in the visual cortex [21].

The classes of CSPG include (1) lecticans such as aggrecan, brevican, neurocan, and versican [22], (2) phosphacan (a tyrosine phosphatase) [23], and (3) small leucine-rich proteoglycans [24]. CSPG can be associated with the plasma membrane through a membrane-spanning domain [25] or a GPI-anchor [26], or can be secreted into the ECM (e.g., lecticans and phos‐ phacan). In the CNS, the chondroitin sulfate side chains act as chemorepellents, and CPSGs are known to inhibit axon projection and cell motility and limit neural plasticity [27]. The lecticans are the principal CSPGs in the CNS, whose core proteins consist of an N-terminal HA-binding domain and a C-terminal domain that binds tenascin-R [28] and tenascin-C [29] (**Figure 3**).

The tenascin family of glycoproteins has four members, tenascin-R, -C, -X, and -W, which are encoded by four genes with a number of splice variants [30] and are believed to modulate cell adhesion and migration. Tenascin-R (formerly called restrictin) is found exclusively in the adult CNS and forms trimers which crosslink CSPGs. Tenascin-R inhibits adhesion of neural cells to fibronectin [31]. Tenascin-C is expressed during embryonic development by migrat‐ ing neural crest cells, is re-expressed during wound healing and in gliomas, and is thought to be involved with increasing glioma cell proliferation and migration [32]. In tenascin-C knockout mice, CSPGs aggregate and fewer PN form [33]. Tenasacin-X is not found in the nervous system. Tenascin-Wis expressed in blood vessels within gliomas and may be involved in angiogenesis [34].

The interstitial ECM forms a highly compressible network of HA and CSPG filaments that is resistant to cell migration by virtue of the inhibitory actions of CPSG, especially their CS components, paucity of anchorage points in the water-rich environment, and the presence of sequestered inhibitory molecules such as slits [35], semaphorins [36], and netrins [37].

In contrast to the hydrated PN that surrounds neurons and the loose interstitial matrix of the brain parenchyma, the ECM around the brain's vasculature and subpial surfaces forms a more rigidbasal lamina that contains laminin, fibronectin, andtype IV and VI collagens andis similar to the ECM in other tissues [35]. This substrate is more likely to allow adhesion of migrating cells and for this reason, gliomas tend to follow blood vessels and subpial surfaces as they invade into the surrounding tissue [36]. They do not, however, degrade the basal lamina and thus do not generally intravasate and spread hematogenously to distant sites.
