Glioma Invasion: Mechanisms and Therapeutic Challenges: Mariano S. Viapiano Sean E. Lawler

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Glioma Invasion: Mechanisms and Therapeutic Challenges
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DOI: 10.1007/978-1-60327-553-8_49
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Chapter 49
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Glioma Invasion: Mechanisms and Therapeutic
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Challenges
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Mariano S. Viapiano and Sean E. Lawler
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13 Abstract A hallmark of gliomas is the ability of individual tumor cells to
14 infiltrate the neural tissue and extend beyond the visible borders of the tumor.
15 Current treatments fail to remove these invasive cells, which almost invariably
16 lead to tumor dissemination and therapy failure in the long term. The composi-
17 tion and properties of the extracellular matrix (ECM) in the adult central
nervous system are notoriously inhibitory to cell motility and axonal extension.
18
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19 However, glioma cells are uniquely able to remodel this microenvironment by
20 degradation of the neural ECM and production of a novel matrix that contains
21 neural-specific and mesenchymal components. Structural signals from the
22 ECM and soluble factors from the surrounding non-neoplastic cells regulate
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23 the molecular and cellular mechanisms of invasion, which include matrix
24 remodeling, cytoskeletal reorganization, and phenotype transition from highly
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proliferative to migratory. Specific strategies against invading glioma cells are
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in their infancy due to the paucity of appropriate targets and the difficulty of
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predicting the effects of targeting this highly plastic cell population in vivo.
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Identification of the key molecular mechanisms necessary for cell invasion and
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the major switches that regulate the inter-conversion of migratory and prolif-
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erative phenotypes will provide a wealth of novel targets to direct therapies
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against brain tumor progression and improve long-term patient survival.
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Contents
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49.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1220
36 49.2 Overview of Glioma Cell Invasion in the CNS . . . . . . . . . . . . . . . . . . . . . . . . 1220
37 49.3 Glioma Cell Microenvironment: Extracellular Matrix . . . . . . . . . . . . . . . . . . 1225
38 49.3.1 Neural ECM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1225
49.3.2 Basal Lamina. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1229
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49.4 Extracellular Remodeling and Glioma Invasion . . . . . . . . . . . . . . . . . . . . . . . 1230
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41
M.S. Viapiano (*)
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Center for Molecular Neurobiology, The Ohio State University,
43 226B Rightmire Hall, 1060 Carmack Rd, Columbus, Ohio 43210, USA
44 e-mail: viapiano.1@osu.edu
E.G. Van Meir (ed.), CNS Cancer, Cancer Drug Discovery 1219
and Development, DOI 10.1007/978-1-60327-553-8_49,
Ó Humana Press, a part of Springer ScienceþBusiness Media, LLC 2009
1220 M.S. Viapiano and S.E. Lawler
45 49.4.1 ECM Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1230

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49.4.2 ECM Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1234
49.5 Soluble Signals and Transduction Mechanisms in Glioma Invasion . . . . . . . . 1237
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49.5.1 Chemoattractants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1237
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49.5.2 Chemorepellents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1239
49 49.6 Targeting Strategies Against Glioma Cell Invasion . . . . . . . . . . . . . . . . . . . . . 1240
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50 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1244
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49.1 Introduction
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Malignant gliomas are the most common primary tumors of the central nervous
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system (CNS). A hallmark of these neoplasms is the ability of individual glioma
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cells to detach from the tumor mass and invade the neural tissue (Louis, 2006). This
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diffuse infiltration occurs with little distortion of the neural architecture and does
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not seem to trigger inflammatory or other immune responses, placing the migrating
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cells beyond the limits of current clinical detection (Claes et al., 2007). In addition,
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significant evidence has shown that migrating glioma cells divide more slowly than
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the cells at the core of the tumor (Demuth and Berens, 2004; Giese et al., 1996) and
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are consequently more resistant to cytotoxic therapies. Therefore, these cells
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remain in the CNS even after aggressive resection and treatment of the residual
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tumor, becoming a major factor for local recurrence and tumor dissemination
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throughout the CNS (Berens and Giese, 1999; Giese and Westphal, 2001).
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Current therapeutic strategies after surgical resection, or directly applied to
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inoperable tumors, target proliferating cells through a combination of cytotoxic
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and anti-angiogenic drugs (Anderson et al., 2008; Salgaller and Liau, 2006).
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Few attempts have been made to specifically target the migratory malignant
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cells located far from the tumor center, as an additional strategy to prevent
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recurrence and improve long-term management of the disease. This approach is
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of major relevance because cell infiltration is a cause of rapid disease progres-

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sion after initial response and therefore a major limitation against lasting
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success of current therapies (Lamszus et al., 2003). Understanding the cellular
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and molecular mechanisms underlying glioma invasion is thus a priority for
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developing effective therapeutic strategies against the spread and recurrence of
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these tumors (Claes et al., 2007; Louis, 2006). In this chapter we will focus on
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the composition and remodeling of the microenvironment that surrounds the
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motile glioma cells, briefly review the major signals and mechanisms involved in
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glioma cell invasion, and discuss potential anti-invasive strategies.
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85 49.2 Overview of Glioma Cell Invasion in the CNS

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87 In solid carcinomas, increased malignancy is characterized by the ’epithelial-to-

88 mesenchymal’ transition of the tumor cells (Guarino et al., 2007), a set of
89 genotypic and phenotypic changes that enables individual cells to detach from
49 Glioma Invasion 1221
90 the primary tumor, disrupt the basal lamina of the epithelial tissue, invade the
91 surrounding stroma, and eventually reach lymph and blood vessels that will
92 allow dissemination of the tumor to other organs (Stetler-Stevenson et al.,
93 1993). The dispersion of individual tumor cells from the primary lesion requires
94 a number of sequential steps that include detachment from the tumor core,
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95 receptor-mediated adhesion to the immediate extracellular matrix (ECM), local
96 degradation of the ECM to allow cell passage, and active motility mechanisms
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97 that drive the cells away from the tumor and/or toward attractants (Bellail et al.,
98 2004; Giese and Westphal, 1996; Nakada et al., 2007). The dispersion of glioma
99 cells within the CNS has been studied within this paradigm and in many ways
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reflects it. Accordingly, most assays to study glioma cell motility and invasion
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follow models of invasion and metastasis from other cancers (see Table 49.1 for
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the most common assays currently in use). However, there are important
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differences between the local dispersion of gliomas and the stromal infiltration
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and metastasis observed in non-neural carcinomas.
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Compared to peripheral tissues, central nervous tissue is highly refractory to
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tumor infiltration and tumors that metastasize to the CNS almost never invade
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it but rather push the neural tissue aside, even when those tumors may aggres-
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sively infiltrate their tissues of origin (Subramanian et al., 2002). Conversely,
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glioma cells invade the brain parenchyma well, despite the presence of an
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inhibitory ECM and the absence of most basal-lamina elements and supporting
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stromal cells (Giese et al., 2003). At the same time, glioma cells do not intrava-
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sate into blood vessels (Bernstein and Woodard, 1995) nor metastasize to close
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peripheral tissues. Moreover, when implanted peripherally, gliomas grow as
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compact, encapsulated masses (Bolteus et al., 2001; Pilkington, 1997). The
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ability of gliomas to colonize neural tissue as an ’intra-parenchymal metastasis’
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(Bernstein, 1996) together with their metastatic failure outside the CNS indi-
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cates a high degree of specialization of these tumors to the neural environment
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and suggests that glioma cells may have unique mechanisms of invasion
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adapted for the particular composition and structure of the CNS (Bellail
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et al., 2004). Experimental models should therefore strive to reproduce the
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distinguishing properties of glioma cells, and their differences with other tumors
should be kept in mind when analyzing glioma invasion in conditions that do
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not mimic the neural microenvironment (Claes et al., 2007) (see Table 49.1).
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Histological evidence demonstrates that glioma cells follow typical disper-
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sion routes and have preferential tropisms independently of the original loca-
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lization of the tumor (Giese and Westphal, 1996; Giese, 2003) (Fig. 49.1). Most
commonly, glioma cells disperse along white matter tracts, leading in many
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cases to the invasion of the tumor into the opposite hemisphere in the brain
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(’butterfly lesions’ across the corpus callosum, see Fig. 49.1A). Glioma cells also
131 migrate along the basal lamina of brain blood vessels or spread in the space
132 between the glia limitans and the pia mater, forming perivascular and subpial
133 foci of proliferation. Finally, glioma cells can also move through the network of
134 unmyelinated cell processes that form the grey matter neuropil and proliferate
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Table 49.1 Common models used to study glioma cell motility and invasion
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Good
Assay Timescale to analyze Advantages Disadvantages
ADH Adhesion assay: Cells plated on a Minutes Adhesive Fast, simple, easy to reproduce Static assay does not inform
substrate-coated surface to hour properties and quantify. about cell motility.
long of the cells
Motility ’Scratch’ assay: Cell monolayer Overnight Overall Relatively fast, simple. Gap-width is difficult to
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scratched with a tip and left to to 2 days population quantify. Not all cells form
reform. motility dense monolayers for this
assay. Other: (1),(2)
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Radial migration assay: Cells are Overnight Random Simple, easy to reproduce. Other: (1), (2), (3). Proper image
plated on the center of a pre- to a few dispersion analysis is required (see (Stein
coated surface and migrate days. from a et al., 2007)).
radially. focal point
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Boyden chamber assay: Cells Few hours Motility Relatively fast, easy to Pores are larger than inter-
move through a porous to under reproduce and quantify. Can cellular spaces in the CNS.
membrane toward chemo- or overnight directional be used to test repellent Cells that remain on the upper
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haptoattractants stimuli molecules. surface may skew
quantification if not
T thoroughly removed.
Other: (1)
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Scaffold-based migration: Cells Usually Topographic Scaffolds can be precisely Nanofiber composition and
plated on polymer scaffolds 24–36 influences engineered and functionalized engineering varies among
and allowed to move in 2D/ hours. on motility with neural-specific laboratories. Time-lapse
3D biomolecules. confocal cell tracking and
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complex image analysis
required.
Invasion Collagen invasion assay: Cell Several Random 3D version of the Radial Changes in medium composition
aggregates seeded inside a days invasion migration assay. Cell or hypoxia in the gel may
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collagen matrix and allowed through a morphologies approximate F develop over time.
to disperse radially. matrix those observed in intracranial Degradation of basal-lamina
tumors matrix is uncommon to
M.S. Viapiano and S.E. Lawler
gliomas in the brain. Other:

(2),(3),(4)
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Table 49.1 (continued)

Good
Assay Timescale to analyze Advantages Disadvantages
Glioma Invasion
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Matrigel invasion assay: Usually Directed A more complex version of the Degradation of basal-lamina
Cells seeded onto a matrix inside 2–4 days invasion Boyden Chamber assay. matrix is uncommon to
a Boyden Chamber and through a Allows quantitative gliomas in the brain. Other:
stimulated to migrate toward matrix. evaluation of pro-invasive (2),(4).
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an attractant factors, e.g., matrix proteases.
Slice invasion assay: Cell 3 days to a Invasion
Long and time-consuming. Substrate accurately mimics the
aggregates seeded on top of week through a neural architecture. Cell
Dying neural cells in the slice
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live brain slices realistic
may affect glioma cells. morphologies similar to those
matrix
Migrating cells may not show observed in intracranial
preferential attraction to tumors.
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white matter. Other: (2)
In vivo Tumor implantation: Cells Several True Tumor growth and invasion All subcutaneous gliomas and
injected subcutaneously or weeks invasion in
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under influence of neural, many intracranial xenografts
intracranially and allowed to vivo vascular, and immune factors. do not show invasion. Brain
form a tumor It is the most reliable assay to invasion in syngeneic models
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verify anti-tumoral strategies. is difficult to quantify and
requires morphometric
analysis.
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Common disadvantages in several models: (1) Motility on a hard surface may not be representative of natural movement.
(ADH: Adhesion) (2) Long-term assays must consider cell proliferation/viability as additional variables. (3) Radius and area of dispersion can be
biased by outlier cells and image analysis algorithms. (4) Matrix proteins used in these assays are usually absent in the CNS.
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245 Fig. 49.1 Anatomical pathways for glioma invasion. (A) Dramatic infiltration of a ‘‘butterfly’’
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glioblastoma multiforme across the corpus callosum (unfixed, gross specimen). (B) Micro-
photography of elongated, hyperchromatic tumor nuclei oriented along myelinated axons
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(Luxol fast blue and H&E stain, 400X, myelinated fibers indicated with arrows). (C) Glioma
248 cells show preferential dispersion along myelinated tracts, blood vessels, and the basal lamina
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249 of the subpial surface. Tumor cells can also move throughout the neuropil of the brain
250 parenchyma. Periaxonal and perineuronal migration often results in perineuronal satellitosis.
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Image in A reproduced from the collection of Dr. John J. Kepes, with permission from the
University of Kansas, Department of Pathology and Laboratory Medicine; images in B and C
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reproduced from Louis, D., Annual Review of Pathology: Mechanisms of Disease, Volume 1
253 # 2006, with permission from Annual Reviews (www.annualreviews.org)
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256 around individual neurons. Detailed illustrations of these routes of dispersion

257 can be found in reviews by Claes et al. (2007) and Louis (2006).
The patterns of periaxonal, perivascular,̀ and perineuronal accumulations,
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259 or satellitosis, known as secondary structures of Scherer, are typical of clinical

260 specimens and can be replicated experimentally (Guillamo et al., 2001),
261 suggesting that glioma cells may have a stereotyped set of substrate-dependent
262 migratory behaviors. The routes of migration likely follow both appropriate
biochemical cues as well as favorable anatomical structures that provide path-
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264 ways of least resistance to cell dispersion (Giese, 2003).

265 Regardless of the biochemical or structural influences on the migrating cells,
266 this invasive behavior is common to the overwhelming majority of gliomas. It
267 has been suggested that this reflects the acquisition of motility, a ’mesenchymal’
268 property (Wolf et al., 2003), early after transformation (Louis, 2006). Alterna-
269 tively, given the mounting evidence (see Chapter 44) suggesting that gliomas
270 may arise from transformed neural stem cells (Barami, 2007; Gilbertson and
271 Rich, 2007), the migratory phenotype could be a reflection of the motile nature
272 of the committed neural progenitors that derive from those stem cells (Claes
273 et al., 2007). Evidence from experimental models indicates that glioma cells
274 move through the neural architecture in a manner that largely resembles the
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275 migration of neural progenitors and that is quite different from glioma cell
276 motility in vitro (Beadle et al., 2008). The possibility that glioma-initiating cells
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277 derive from adult neural stem cells has raised interesting questions about the
278 possibility of predicting pathways of invasion radiating from neural stem cell-
279 rich zones. For example, recent results have shown that the depth of the tumor
280 in the brain and its association with the subventricular zone may correlate with
281 invasive and multifocal properties of glioblastomas (Lim et al., 2007).
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49.3 Glioma Cell Microenvironment: Extracellular Matrix
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Both grey and white matter in the adult neural parenchyma form an inhibitory
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environment for cellular motility and axonal extension (Busch and Silver,
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2007). To disseminate in this tissue, migrating glioma cells must interact with
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a variety of substrates with different topography and molecular composition,
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such as the amorphous ECM of the grey matter neuropil, the surface of white
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matter fibers, and the basal lamina of the neural vasculature and the subpial
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surface (Giese, 2003). The ability of glioma cells to interact with a wide variety
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of substrates suggests the existence of multiple migratory mechanisms that
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overcome the inhibitory elements and respond to ’motogenic’ signals to pro-
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mote cell dispersion (Bellail et al., 2004).
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299 49.3.1 Neural ECM

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301 The major barrier opposing glioma cell movement through intercellular spaces
302 is the ubiquitous and distinct neural ECM that comprises as much as 20% of the
adult brain and spinal cord volume (Novak and Kaye, 2000). This matrix lacks
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304 the typical proteins found in the fibrillar ECM of other tissues, such as col-
305 lagens, fibronectin and type-I laminin (Novak and Kaye, 2000), and is instead
306 composed of the polysaccharide hyaluronic acid (HA), with associated glyco-
307 proteins and proteoglycans. The major structural constituents of this matrix,
which in most cases are also expressed in gliomas, are listed in Table 49.2 and
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309 illustrated in Fig. 49.2. Of these, HA and its HA-binding proteins are the key
310 molecules that form the neural ECM scaffold (Yamaguchi, 2000); other illus-
311 trations of these ECM components can be found in reviews by Viapiano and
312 Matthews (2006) and Galtrey and Fawcett (2007).
313 HA is a very large (500–1000 kDa) hygroscopic glycosaminoglycan that
314 can retain large amounts of water, thus creating hydrated spaces used by cells to
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Table 49.2 Major structural components of the neural extracellular matrixa
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Neural tissue Gliomas

Molecules
(Mw) Properties Expressionb Functions Expressionc Functions References
Hyaluronic Occupies large " in early Compression "" (fourfold Cell proliferation Delpech
acid (HA) hydrated spaces development resistance. higher than and migration; (1993),
(>106 Da) and forms a # and less soluble in Facilitates cell normal adult HA fragments Laurent
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compressible the adult migration and CNS) induce synthesis (1996) and
mesh at high proliferation of ECM Novak
concentrations. during molecules and (2000)
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development MMPs
Lecticans Bind HA and cell- Variable profiles for Restrict cell Variable Cell proliferation Crespo (2007),
(150–600 kDa) surface receptors. each lectican. " migration and profiles. and resistance to Novak
Aggrecan Many isoforms neurocan during axonal extension Versican and apoptosis (2000),
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Neurocan produced by development. " in adult CNS. brevican are (versican); Viapiano
Versican alternative brevican and May guide highly enhanced (2006) and
Brevican splicing, versican in adult axonal extension expressed in adhesion and Yamaguchi
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proteolysis, and CNS. Total levels and neuroblast gliomas migration (2000)
differential of CSPGs are migration in the (brevican);
glycosylation higher in adult
T developing CNS binding to
than developing fibronectin;
CNS EGFR activation
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Link proteins Link HA to the # during early Formation of ## in glioma Unknown Oohashi (2005)
(HAPLN1 to lecticans; development. perineuronal tissue
4) condense the "" HAPLN2 and nets; might " HAPLN4 in
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(50–55 kDa) adult ECM HAPLN4 in adult modulate GSCs (MSV,
CNS synaptic unpublished)
plasticity.
RPTP-b/z Membrane-bound " in early Matrix remodeling, " RPTP-b/z in Pleiotropin Margolis
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Phosphacan K/CSPG; does development. neurite gliomas grade F signaling to (1996),
(250–450 kDa) not bind HA. outgrowth and II–III stimulate cell Rauch
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Neural tissue Gliomas

Molecules
(Mw) Properties Expressionb Functions Expressionc Functions References
Phosphacan is the ## in the adult synaptic " Phosphacan in migration and (2004) and
soluble form of (restricted to plasticity (both gliomas grade proliferation Ulbricht
RPTP-b/z. Major neurons) positive and IV (2003)
Glioma Invasion
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ligand: negative effects
pleiotropin reported)
Tenascins (Tn) Multimeric proteins; " in early Proliferation and
Tn-C combined Garwood "" Tenascin-C
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(180–250 kDa) bind to the development. migration of
with PDGF/LPA (2001), (expressed in the
Tn-C / Tn-R lecticans, to # in the adult neural precursors
signaling triggers Joester tumor
Tn-X / Tn-Y RPTP-b/z, and to (mostly expressed from the SVZ;
cell migration. (2001), vasculature)
several cell- in the white axonal guidance;
Tenascins also Lange (2008)
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surface receptors matter) adult ECM
increase glioma and Rauch
condensation
cell proliferation (2004)
SPARC family Small glycoproteins; " in early SPARC May be involved in
HSP27 activation; Au (2007),
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(40–50 kDa) bind to growth development. member of MMP neurite extension
Brekken
factors and basal Expressed by the family is and synapse
upregulation; cell (2000) and
lamina ECM astrocytes in adult
T
highly formation
migration on Vincent
proteins CNS expressed by basal lamina (2008)
ED
glioma cells matrix
a
This table includes secreted matrix molecules known to have a structural role in the neural ECM and to be also expressed in the glioma matrix.
Therefore, we have omitted the cleaved extracellular domains of membrane-bound proteins (heparan sulfate proteoglycans, NG2, and myelin-
associated inhibitors), small soluble factors, and cell/axon guidance factors that are not structurally associated to the ECM (semaphorins, Wnts, slits).
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b
("), high expression; (#) low expression.
c
Expression levels compared to normal adult CNS.
GSCs: Glioma stem cells; HAPLN: hyaluronan and proteoglycan link protein; HSP27: heat shock protein-27; K/CSPG: proteoglycan with keratan
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and chondroitin sulfate; MMP: matrix metalloprotease; PDGDF/LPA: plateled-derived growth factor and lysophosphatidic acid; RPTP-b/z:
receptor-type protein tyrosine-phosphatase b/z; SPARC: secreted protein acidic and rich in cystein. F
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Fig. 49.2 Simplified model of the neural ECM. The cartoon depicts the predominant mole-
434 cules that compose the ECM close to the surface of the neuronal and glial cells. The
435 chondroitin sulfate proteoglycans of the lectican family can bind hyaluronic acid, secreted
436 ECM proteins, and cell-surface receptors, thus acting as extracellular ‘anchors’. ERM pro-
437
teins: proteins of the Ezrin/Radixin/Moesin family; SGLs: sulfoglucuronyl-glycolipids. Fig-
ure and text reproduced from Viapiano M.S. and Matthews R. T., Trends in Molecular
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Medicine, Volume 12 # 2006, with permission from Elsevier
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441 proliferate and migrate during development (Laurent et al., 1996). HA associ-
442 ates with secreted and membrane-bound HA-binding proteins, which act as orga-
nizers of the matrix scaffold around neural cells (Yamaguchi, 2000). Accumulation
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443
444 of these HA-binding proteins in the adult CNS reduces the interstitial spaces and
445 renders the neural ECM largely insoluble, forming a restrictive environment for
446 axonal navigation and cell motility (Rauch, 2004; Viapiano and Matthews, 2006).
447 The major group of HA-binding proteins in the adult CNS is formed by the
448 secreted chondroitin sulfate proteoglycans (CSPGs) of the lectican family:
449 aggrecan, versican, neurocan, and brevican (Yamaguchi, 2000). These large,
450 heavily glycosylated proteins exhibit a remarkable heterogeneity of isoforms,

451 expression patterns and molecular partners, but in general predominate in the
452 adult CNS and connect the matrix scaffold to receptors on the surface of
453 neurons and glial cells (Viapiano and Matthews, 2006; Yamaguchi, 2000).
454 The lecticans have been recognized as a major group of inhibitory molecules
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455 for axonal extension, cellular adhesion, and motility (Rhodes and Fawcett,
456 2004), a property attributed in part to a chemorepellent effect of the side chains
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457 of chondroitin sulfate (Crespo et al., 2007). Surprisingly, some members of this
458 family are highly expressed in gliomas and promote glioma cell migration (see
459 below) (Viapiano and Matthews, 2006).
460 The composition of the neural ECM is very similar in the grey and white
461 matter, with some differences in the local expression of ECM molecules in
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462 subsets of neurons and grey matter neuropil (e.g., aggrecan, neurocan) or
463 around myelinated axons (e.g., versican and the tenascins). In the white matter,
464 the inhibitory effect of the secreted CSPGs on cell and neurite motility is
465 potentiated by the effect of ECM-associated molecules normally involved in
466 neuronal and axonal repulsion (most notably the netrins and slits (Barallobre
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467 et al., 2005; Wong et al., 2002)), as well as the well-known myelin-associated
468 inhibitors (Nogo, the myelin-associated glycoprotein MAG, and the myelin
469 oligodendrocyte glycoprotein (Xie and Zheng, 2008)). The latter are not matri-
470 cellular proteins but membrane-bound ’collapse signals’ that prevent extension
471 of glioma cell processes and motility in vitro (Hensel et al., 1998; Liao et al.,
T
472
2004), although at least one of these myelin inhibitors (MAG) exhibits ECM-
473
associated, highly inhibitory soluble forms in vivo (Tang et al., 2001).
EC
474
In addition to having strong inhibitory signals for cell migration and neurite
475
extension, the scaffold of the neural ECM also presents structural constraints
476
for cell motility. This scaffold is essentially a highly compressible mesh of HA
477
and proteoglycan filaments that does not favor cell traction because it provides
478
sparse, randomly organized anchorage points for the formation of intracellular
RR
479
stress fibers (Georges et al., 2006; Peyton et al., 2007). This structural limitation
480
may be partially attenuated in the white matter due to the high density of
481
parallel axons that may form guiding rails for motile cells. In vitro scaffolds
482
composed of aligned fibers have been shown to provide topographic guidance
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483
to neural cells and neurites and promote cell adhesion and motility better than
484
randomly oriented fibers (Yang et al., 2005) (also SEL and MSV, manuscript in
485
preparation), independently of other cues in the surrounding environment.
486
487
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488
489 49.3.2 Basal Lamina

490
491 In stark contrast with the HA- and proteoglycan-based matrix that fills the
492 extracellular space around neurons and glial cells, the ECM that covers the
493 abluminal surface of the brain–blood vessels and the subpial surface resembles
494 the typical basal lamina that separates epithelial cells from their underlying
495 stroma in peripheral tissues. This matrix contains some of the proteoglycans
496 and glycoproteins described in the neural ECM but is predominantly abundant
497 in matricellular proteins that form networks of fibrillar aggregates, such as
498 laminins, fibronectin, and vitronectin, and thus promote the adhesion of motile
499 cells (Bellail et al., 2004; Gladson, 1999). The basal lamina ECM also contains
F
500 several types of collagens, particularly collagen IV and VI, with lower amounts
501 of the interstitial collagens I and III (Gladson, 1999; Paulus et al., 1988). This
OO
502 dense, organized matrix provides higher resistance to compression than the
503 neural ECM and favors the formation of focal adhesions. Therefore, the basal
504 lamina serves as a preferred substrate for integrin-mediated cell adhesion and
505 motility along vascular and subpial surfaces (Goldbrunner et al., 1999). Inter-
506 estingly, glioma cells accumulate along this basal lamina but do not degrade it
PR
507 in vivo (Paulus et al., 1988), although they can traverse the basal lamina of
508 peripheral blood vessels when they are injected intravenously (Mandybur et al.,
509 1984). The mechanisms that prevent degradation of the basal lamina of CNS
510 blood vessels by glioma cells are unknown, and this phenomenon has been
511 postulated as the underlying reason for lack of intravasation and extra-axial
metastasis of these tumors (Bernstein and Woodard, 1995)
512
ED
513
514
515
516
49.4 Extracellular Remodeling and Glioma Invasion
T
517
As described above, tumor cells attempting to disseminate in the CNS are chal-
518
lenged by a variety of molecular and structural inhibitory factors in the neural
EC
519
parenchyma that limit their adhesion and motility. Peripheral tumors facing these
520
obstacles continue growing but individual cells rarely detach from the tumor mass
521
and almost never infiltrate CNS structures. Conversely, glioma cells actively
522
remodel the surrounding matrix to reach the anatomical structures along which
523
RR
they disseminate. This remodeling involves degradation of the pre-existing ECM,

524
overproduction of neural ECM molecules, secretion of novel ECM molecules that
525
are absent in the neural parenchyma, and expression of novel cell-surface receptors
526
for ECM signals. These processes are likely potentiated by additional tissue
527
remodeling produced by infiltrating immune cells and the proliferative endothe-
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528
lium of hyperplastic tumor blood vessels (Bellail et al., 2004; Gladson, 1999; Kaur
529
et al., 2005). For the scope of this review we will focus only on the major molecular
530
changes exerted by isolated glioma cells invading normal neural tissue.
531
532
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533
534 49.4.1 ECM Degradation

535
536 Compared to adult normal neural tissue, glioma cells exhibit increased expres-
537 sion of lysosomal hyaluronidases and secreted proteases. These enzymes can
538 degrade the pericellular matrix, opening spaces for cell motility and releasing
539 protein and glycosaminoglycan fragments that act as mitogenic and motogenic
540 signals (Junker et al., 2003; Rao, 2003). A wealth of evidence has demonstrated
541 that several families of proteases actively contribute to matrix remodeling in
542 gliomas, including the plasminogen activators, the members of the matrix
543 metalloprotease (MMP) family, and the lysosomal/secreted cathepsins (Levicar
544 et al., 2003; Rao, 2003). Recent investigations have expanded the set of glioma-
F
545 active proteases to include the disintegrin-and-metalloproteases (ADAMs) and
546 the ADAMs with thrombospondin motifs (ADAMTSs) (Rocks et al., 2008).
OO
547 The major members of these families that are expressed in gliomas, their
548 functional relevance, and their relationship with glioma progression are sum-
549 marized in Table 49.3.
550 Secreted metalloproteases, particularly members of the MMP family such as
551 MMP-2 and MMP-9, have been clearly demonstrated to promote brain tumor
PR
552 growth and progression, as well as cell proliferation and invasion in vitro
553 (Levicar et al., 2003; Rao, 2003). Accordingly, upregulation of those MMPs
554 in gliomas correlates with tumor grade and lower survival rates (Levicar et al.,
555 2003; Rao, 2003). Secreted MMPs and membrane-bound ADAMs have been
556 involved in several functions that promote tumor progression, including matrix
degradation, release of trophic factors, regulation of cell proliferation, stimula-
557
ED
558 tion of angiogenesis, and control of the immune response in the tumor (Egeblad
559 and Werb, 2002; Rocks et al., 2008).
560 Pericellular matrix degradation by infiltrating glioma cells in vivo, however,
561 may be far more restricted than what is thought from in vitro observations or
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562 results from metastatic tumors. For example, the predominant CSPGs in the CNS,
563 versican and brevican, are degraded by MMPs in vitro (Nakamura et al., 2000);
EC
564 however, MMP-dependent proteolysis of these proteins in intracranial gliomas is a

565 very minor event (Viapiano et al., 2008), suggesting that the scaffold of the neural
566 ECM could be less affected by degradation than by production of novel matrix
567 components, as described below. Similarly, despite of their high MMP expression,
568 glioma cells do not disrupt the basal lamina of the brain blood vessels in vivo, even
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569 though these enzymes degrade basal lamina components during in vitro invasion
570 assays (Bernstein and Woodard, 1995; Paulus et al., 1988).
571 It is thus possible that, in addition to regulated pericellular proteolysis,
572 proteases in glioma may promote cell infiltration through a variety of non-
degradative mechanisms. For example, ADAMTS enzymes cleave the neural
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573
574 lecticans preferentially at a single site, producing fragments that remain asso-
575 ciated to the ECM scaffold and act as pro-migratory signals (Ang et al., 1999;
576 Hu et al., 2008). In another example, MT1-MMP can induce EGFR transacti-
577 vation independently of its catalytic activity (Langlois et al., 2007). Moreover, it
has been shown that inhibition of metalloproteases may drive metastatic tumor
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578
579 cells to adopt an amoeboid phenotype and infiltrate by ’squeezing’ through the
580 intercellular spaces (Wolf et al., 2003). This causes tumor cells to acquire
581 elongated morphologies that strongly resemble those usually seen in migratory
582 glioma cells in the brain (Beadle et al., 2008), suggesting that these cells may
583 naturally adapt to the surrounding neuropil rather than degrade their way
584 through it.
629
628
627
626
625
624
623
622
621
620
619
618
617
616
615
614
613
612
611
610
609
608
607
606
605
604
603
602
601
600
599
598
597
596
595
594
593
592
591
590
589
588
587
586
585
Table 49.3 Proteases involved in glioma cell invasion
1232
Activity and
Protease Members studied in substrates in the Relationship with Endogenous
family gliomas CNS Functional relevance tumor progression inhibitors References
Plasminogen – Urokinase-type PA Serine proteases. Plasmin degrades High levels of uPA Plasminogen Gondi (2003,
activators (expressed by Convert pericellular matrix and uPAR activator 2007), Rao
(PAs) glioma cells) plasminogen to and activates correlate with a inhibitors (2003) and
UN
– Tissue-type PA activated plasmin MMPs. uPA also more invasive (PAI): PAI-1 Tsatas
(expressed in the signals for phenotype and to PAI-3 (2003)
endothelium of integrin- and poor prognosis.
glioma blood vitronectin- Inhibition of uPA/
CO
vessels) mediated cell uPAR reduces
adhesion through invasiveness and
its receptor, uPA- promotes
RR
R. tPA is apoptosis
presumed to play a
role in tumor
EC
angiogenesis
T rather than
invasion
Matrix gelatinases (MMP2, Zinc-dependent MMPs are secreted MMP2, MMP9 and Tissue Binder (2002),
Metallo- MMP9), endopeptidases. by glioma cells, MT1-MMP inhibitors of Chintala
ED
proteases stromelysins Degrade most endothelial cells strongly correlate metallo- (1999),
(MMPs) (MMP3, MMP7, proteins of the and infiltrating with glioma proteases Lakka
(28 members) MMP10, basal lamina and immune cells; progression. (TIMP): (2005) and
MMP11), proteoglycans of involved in Inhibition of these TIMP-1 to Nakada
PR
membrane-type the neural ECM perivascular MMPs sharply TIMP-4 (2003)
MMPs (MT1- invasion and reduces invasion in
MMP) tumor vitro and tumor
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angiogenesis. formation in vivo
Membrane-bound
MT-MMPs
F
implicated in white
matter invasion.
674
673
672
671
670
669
668
667
666
665
664
663
662
661
660
659
658
657
656
655
654
653
652
651
650
649
648
647
646
645
644
643
642
641
640
639
638
637
636
635
634
633
632
631
630
49
Activity and
Protease Members studied in substrates in the Relationship with Endogenous
family gliomas CNS Functional relevance tumor progression inhibitors References
ADAMS ADAM8, Similar to MMPs. Membrane-bound ADAMS TIMPs Held-Feindt
(40 members) ADAM17 (TACE), proteases upregulation (2006) and
ADAM19 upregulated in correlates with Rocks
Glioma Invasion
UN
brain tumors. glioma (2008)
Specific targets in invasiveness
CNS and
functional
CO
relevance yet to be
determined.
ADAMTS Aggrecanases Similar to MMPs. Lectican cleavage ADAMTS TIMP-1 and -3 Held-Feindt
RR
(20 members) (ADAMTS-4 and Degrade CSPGs releases N- upregulation (2006),
ADAMTS-5) of the neural ECM terminal correlates with Rocks
fragments that glioma (2008) and
EC
promote cell invasiveness Viapiano
proliferation, (2008)
T adhesion, and
migration
Cathepsins Cathepsin B and D Cystein proteases. Lysosomal Cathepsin Cystatins Gondi (2006),
ED
Degrade degradation of upregulation (extracellular Levicar
collagens, ECM molecules. correlates with inhibitors) (2002) and
fibronectin, Cathepsins are tumor grade and Rao (2003)
PR
laminin also secreted and more invasive
may remain active profile. Inhibition
in the ECM. reduces tumor
Cathepsin B growth, invasion,
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indirectly activates and angiogenesis
MMPs via uPA/
plasmin
F
1233
675 49.4.2 ECM Synthesis

676
677 Matrix remodeling depends not only on controlled pericellular ECM degrada-
678 tion but also on the formation of a new pericellular scaffold that contains novel
679 ECM molecules produced by glioma cells. These structural molecules can be
F
680 roughly grouped into three categories: molecules that are highly expressed
681 during early neural development (HA, SPARC, phosphacan), molecules that
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682
predominate in the adult neural ECM (lecticans), and molecules that are not
683
present in the normal neural ECM (basal lamina proteins).
684
Most gliomas contain high levels of HA comparable to those in the early
685
developing brain (Delpech et al., 1993). The overproduction of HA and sub-
686
sequent turnover by upregulated hyaluronidases in gliomas creates a regionally
PR
687
disorganized scaffold that becomes very soluble and permissive for cell prolif-
688
eration, detachment, and movement (Novak et al., 1999). In addition, soluble
689
HA acts as a pro-invasive signal by increasing the expression of metallopro-
690
teases and ECM proteins (MMP9 and SPARC, Kim et al., 2005a; Kim et al.,
691
2005b); it may also promote apoptosis of immune cells (Yang et al., 2002) and
692
ED
could activate tyrosine kinase signaling as demonstrated in other carcinomas
693
(Misra et al., 2006). Moreover, glioma cells express HA receptors that are
694
absent or expressed at very low levels in the normal CNS, such as CD168/
695
RHAMM and CD44, the latter being one of the hallmark cell-surface markers
696
in high-grade gliomas (Akiyama et al., 2001; Baltuch et al., 1995; Ranuncolo
T
697
et al., 2002). These receptors have a demonstrated role in promoting tumor cell
698
proliferation and migration through several signaling pathways in a variety of
EC
699
malignancies (Hall and Turley, 1995; Knudson, 1998) (Fig. 49.3), and their
700
blockade or downregulation in gliomas inhibits tumor proliferation and inva-
701
702
sion in vitro and in vivo (Akiyama et al., 2001; Ward et al., 2003).
703
Glioma cells secrete SPARC and phosphacan, proteins that abound in the
RR
704
permissive matrix of the developing CNS, but that are highly reduced in the
705
adult ECM. SPARC, which is overexpressed in many solid tumors (Brekken
706
and Sage, 2000), interacts with basal lamina proteins and promotes the prolif-
707
eration and migration of glioma cells in vitro, as well as tumor dispersion, but
not mass growth, in vivo (Schultz et al., 2002). At the molecular level, it has been
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708
709
shown that SPARC can modulate integrin-linked kinase activity (Barker et al.,
710
2005), upregulate MMP expression (McClung et al., 2007), and induce cytos-
711
keletal changes through the chaperone HSP-27 (Golembieski et al., 2008).
712
These activities result in the reorganization of cell morphology, matrix remo-
deling, and a net increase in cell motility.
UN
713
714 The membrane-bound receptor-type protein tyrosine-phosphatase RPTP-b/

715 z is highly upregulated in grades I–III gliomas and its soluble isoform, phos-
716 phacan, predominates in grade IV glioblastomas (Norman et al., 1998). This
717 complex proteoglycan activates NF-kappaB-dependent transcription,
718 promotes the association of glioma cells to the ECM and increases cell migra-
719 tion (Lorente et al., 2005), although the underlying molecular mechanisms for
720
721
722
723
724
F
725
726
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727
728
729
730
731
PR
732
733
734
735
736
737
ED
738
739
740
741 Fig. 49.3 Soluble factors and signaling pathways involved in glioma cell migration. The figure
T
742 depicts some of the major intracellular transduction pathways that respond to the well-
characterized extracellular signals that promote cell motility. The interactions in the figure
743
were summarized from several bibliographical sources and verified individually using the
EC
744
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, stored at the
745 Bioinformatics Center of Kyoto University and the Human Genome Center of the University
746 of Tokyo (http://www.genome.ad.jp/kegg/pathway.html). Small-sized receptors indicated on
747 both sides of the figure (p75NTR and Plexins/Neuropilins) represent novel, mostly unex-
plored signaling mechanisms recently implicated in glioma invasion
748
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749
750
751
these processes are mostly unknown. Recent evidence suggests that the major
752 ligand of phosphacan/RPTP-b/z, the soluble factor pleiotropin, is also upregu-
lated in gliomas and may be part of an autocrine loop that promotes glioma cell
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753
754 motility (Ulbricht et al., 2003). Accordingly, targeting strategies against RPTP-
755 b/z disrupt glioma cell motility in vitro and tumor growth in vivo (Foehr et al.,
756 2006; Ulbricht et al., 2006).
757 Somewhat surprisingly, gliomas also overexpress two CSPGs of the lectican
family that are well characterized as inhibitory molecules against cell motility in
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758
759 the adult CNS: versican and brevican (Viapiano and Matthews, 2006). These
760 CSPGs have a clear role in promoting tumor growth and dispersion in neural
761 tissue (Viapiano and Matthews, 2006), although their mechanisms of action are
762 poorly understood. Results from in vitro assays suggest that both versican and
763 brevican bind to fibronectin, which is present in the glioma ECM but not the
764 neural ECM (Hu et al., 2008; Wu et al., 2004). Moreover, both lecticans promote
765 fibronectin-dependent cell adhesion and may activate EGFR signaling (Hu et al.,
766 2008; Wu et al., 2004), suggesting that they could act through pathways well
767 characterized in brain tumor invasion.
768 In addition, recent evidence suggests that the role of these lecticans as moto-
769 genic signals in gliomas could also be consequence of the expression of isoforms
F
770 that are minor or absent in the normal adult CNS (Viapiano and Matthews,
771 2006). It is possible that the functions of those isoforms may differ from the
OO
772 predominant role of the lecticans as inhibitors of cell motility and axonal exten-
773 sion (Rauch, 2004; Viapiano and Matthews, 2006). For example, versican has
774 four known splice variants, V0–V3, of which V0/V1 predominates in the develop-
775 ing CNS while V2 is the major adult isoform. Coincidentally, the isoforms V0/V1
776 are upregulated in glioma cells and have proliferative and anti-apoptotic func-
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777 tions, while V2 versican is highly reduced in gliomas and does not protect glioma
778 cells from apoptosis (Paulus et al., 1996; Rauch, 2004; Sheng et al., 2005).
779 Similarly, brevican exhibits novel glycoforms in gliomas that are absent in normal
780 adult human brain but appear during early neural development (Viapiano et al.,
781 2005). Moreover, full-length brevican does not have effects on glioma cell adhe-
sion or migration, but a fragment produced by ADAMTS-4/5 cleavage is suffi-
782
ED
783 cient to act as a signal for cell dispersion in vitro and in vivo (Hu et al., 2008;
784 Viapiano et al., 2008). Consistent with this observation, cleavage of brevican is
785 increased in gliomas compared to normal CNS (Viapiano et al., 2005).
786 Finally, in stark contrast with normal neural cells, glioma cells secrete a group
T
787 of basal lamina components that are not expressed in the ECM of the neural
788 parenchyma, such as type-I laminin, fibronectin, and variable amounts of col-
EC
789 lagens type I, III, IV, and VI (Gladson, 1999; Paulus et al., 1988). The expression
790 of these proteins in culture has been attributed to the loss of distinctive glial
791 features in cell lines and phenotype adaptation to culture conditions, a phenom-
792 enon called mesenchymal drift (Paulus et al., 1994). However, basal lamina
793 proteins can be detected in culture conditions that attempt to avoid this drift,
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794 such as short-term primary cultures of gliomas and culture of glioma-derived

795 neurospheres in serum-free conditions (Paulus et al., 1994; Tso et al., 2006) (also
796 MSV and SEL, unpublished results). Similarly, these matrix proteins can be
797 detected in glioblastoma cells in vivo (Gladson, 1999; Paulus et al., 1994; Tso
et. al., 2006). Taken together, these observations suggest that subsets of glioma
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798
799 cells naturally have the ability to secrete mesenchymal ECM molecules into the
800 surrounding matrix in the brain. These molecules are key elements in the engage-
801 ment of integrin receptors to promote cell adhesion and migration in all solid
802 tumors and have a demonstrated role in glioma invasion (D’Abaco and Kaye,
2007; Giese and Westphal, 1996). In addition, type-VI collagen and fibronectin
UN
803
804 can interact directly with HA (Kielty et al., 1992; Yamada et al., 1980), which
805 could affect the structure of the HA-based matrix that surrounds infiltrating
806 glioma cells, thus favoring cell migration.
807 In sum, glioma cells are able to detach from the original surrounding matrix
808 and produce a novel ECM that contains a variety of neural-specific proteins
809 absent in other tissues (e.g., phosphacan, brevican) as well as mesenchymal
810 proteins absent from the brain parenchyma (fibronectin, collagens) (Bellail
811 et al., 2004). Thus, this ECM differs from the original neural matrix and at
812 the same time is distinct from the matrix secreted by peripheral tumors that
813 metastasize to the CNS. The glioma-specific ECM may be a unique source of
814 haptotactic cues and soluble signals that regulate and direct the migration of
F
815 glioma cells and could thus underlie the distinct ability of these tumors to invade
816 and disperse within the nervous tissue.
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817
818
819
49.5 Soluble Signals and Transduction Mechanisms
820
821
in Glioma Invasion
PR
822
823
In addition to the haptotactic and mechanical effects of the ECM on tumor
824
invasion, glioma cells have a complex paracrine interplay with neurons, glial,
825
endothelial, and immune cells in the microenvironment of the tumor
826
(Hoelzinger et al., 2007; Oliveira et al., 2005). These non-neoplastic cells secrete
soluble factors and provide substrate molecules that may promote glioma
827
ED
proliferation, enhance cell adhesion/motility, and regulate the overall process
828
829
of invasion (Giese and Westphal, 1996; Hoelzinger et al., 2007). There is a
830
multitude of potential signals and transduction mechanisms implicated or
831
proposed to be involved in glioma invasion (Nakada et al., 2007)(see Chapters
37–40); here we will consider only the major soluble factors that have been
T
832
833
shown to consistently influence glioma cell migration and the key signaling
pathways that respond to those factors by promoting cytoskeletal reorganiza-
EC
834
835
tion or transcription of pro-migratory genes.
836
837
838 49.5.1 Chemoattractants

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839
840 The best characterized chemoattractants in gliomas are the ligands of the ErbB
841 receptors (EGF, HB-EGF, TGF-a, heregulin, etc.), the scatter factor/hepato-
842 cyte growth factor (SF/HGF), transforming growth factor beta (TGF-b), and
more recently the stromal-cell-derived factor (SDF)-1 (Mueller et al., 2003).
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843
844 Most of these factors are produced by the neural and endothelial cells surround-
845 ing the tumor (paracrine stimulation) or the glioma cells themselves (autocrine/
846 paracrine loop) (Hoelzinger et al., 2007; Mueller et al., 2003). The major
847 signaling pathways that respond to these factors and activate a pro-migratory
phenotype are summarized in Fig. 49.3. Other soluble molecules shown to be
UN
848
849 involved in regulating glioma cell migration on ECM substrates include addi-
850 tional growth factors (basic fibroblast growth factor, platelet-derived growth
851 factor, and nerve growth factor), cytokines (IL12), molecules involved in
852 neuronal guidance, and some bioactive lipids (lysophosphatidic acid and
853 sphingosine-1-phosphate) (Hoelzinger et al., 2007; Lange et al., 2008; Young
854 and Van Brocklyn, 2007).
855 EGF and related factors are prototypical pro-migratory signals for glioma
856 cells in vitro (Pedersen et al., 1994). They act by activation of the receptor
857 tyrosine kinases (RTKs) of the ErbB family, of which the EGF receptor
858 (EGFR/ErbB1) is the most commonly amplified gene in malignant gliomas
859 (Ohgaki and Kleihues, 2007). Interestingly, amplification of this receptor could
F
860 have a direct relation to the process of invasion in high-grade gliomas; FISH
861 analysis has shown a gradient of EGFR amplification in glioblastoma samples,
OO
862 with the highest amplification levels found in the cells at the invasive border of
863 the tumor (Okada et al., 2003). Activation of a different RTK, c-Met, initiates
864 the effect of another pro-migratory signal, the soluble factor SF/HGF
865 (Moriyama et al., 1999). This receptor is also upregulated in gliomas, and
866 more importantly, localizes predominantly to the invasive pseudopodiae of
PR
867 motile glioma cells (Abounader and Laterra, 2005; Beckner et al., 2005).
868 The key role of RTK signaling on glioma cell proliferation in vitro and tumor
869 growth in vivo has been well established and makes these receptors one of the
870 major current therapeutic targets (Furnari et al., 2007; Nakada et al., 2007) (see
871 also Chapters 20 and 39) . Accordingly, pharmacological inhibition of EGFR
and c-Met, as well as antibody-mediated receptor blocking, has been shown to
872
ED
873 powerfully inhibit both cell proliferation and migration in vitro, as well as
874 tumor invasion in animal models (Abounader and Laterra, 2005; Furnari
875 et al., 2007). The molecular mechanisms by which RTKs promote glioma cell
876 motility are complex and involve multiple parallel and redundant pathways,
T
877 indicated in Fig. 49.3. RTK signaling results, among other effects, in the
878 upregulation of matrix metalloproteases, synthesis of ECM proteins, and
EC
879 cytoskeletal reorganization initiated by activation of actin-binding proteins

880 (Lal et al., 2002; Van Meter et al., 2004).
881 The growth factors TGF-b and SDF-1 act through their cognate receptors,
882 the serine/threonine-kinase TGF-b receptors I/II, and the G-protein-coupled
883 receptor CXCR4, respectively. These factors have been implicated in a variety
RR
884 of trophic effects for glioma progression, including stimulation of cell prolif-
885 eration, angiogenesis, resistance to apoptosis, and cell motility (Leivonen and
886 Kahari, 2007; Savarin-Vuaillat and Ransohoff, 2007; Wick et al., 2006).
887 Although these factors can be produced by glioma cells, another major source
is found in the microglial cells that invade the tumor mass in the brain, which
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888
889 has led to the hypothesis that microglia may actually promote glioma cell
890 invasion by paracrine stimulation (Wesolowska et al., 2008). The signaling
891 pathways initiated by these growth factors in gliomas are not fully determined,
892 but in other cancer models they have been shown to exhibit significant cross talk
with RTK-mediated signaling (Bhola and Grandis, 2008; Uchiyama-Tanaka
UN
893
894 et al., 2002), resulting in MAPK activation, upregulation of extracellular

895 MMPs and ECM proteins, and enhanced cell adhesion and motility (Leivonen
896 and Kahari, 2007; Savarin-Vuaillat and Ransohoff, 2007). Accordingly, inhibi-
897 tion of TGF-b receptors and CXCR4 reduces glioma cell invasion in vitro and
898 tumor growth in vivo (Ehtesham et al., 2006; Uhl et al., 2004) (see Chapter 34
899 for CXCR4 targeting). Interestingly, the expression patterns of SDF-1 in brain
900 tissue show good correlation with the dispersion pathways favored by glioma
901 cells in vivo (Zagzag et al., 2008). Consistent with this observation, the receptor
902 CXCR4 is highly expressed at the leading edge of the tumors (Ehtesham et al.,
903 2006; Zagzag et al., 2008).
904
F
905
906
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907
49.5.2 Chemorepellents
908
909
Most growth factors and cytokines that induce glioma cell motility have been
910
identified as attractants of the cells in vitro. However, glioma cells could also
911
respond to repellent factors that would stimulate dispersion outward from the
PR
912
tumor core. The existence of tumor-produced chemorepellents has been postu-
913
lated from the observation of cell-avoidance patterns during migration in vitro
914
(Mueller et al., 2003; Werbowetski et al., 2004).
915
Members of the Slit and semaphorin families are potential candidates for a
916
role as chemorepellents in gliomas. The members of the Slit family act in the
normal CNS as axon guidance molecules during development and promote the
917
ED
918
migration of neural progenitors away from the subventricular zone in the adult
919 brain (Wong et al., 2002). Recent results have demonstrated that Slit-2 can
920 effectively repel glioma and medulloblastoma cells in vitro through its receptor
921 Robo-1 (Mertsch et al., 2008; Werbowetski-Ogilvie et al., 2006). However, the
T
922 role of this protein as a repellent from the tumor core in vivo is uncertain,
923 because the Slit2 gene has been found frequently inactivated epigenetically in
EC
924 gliomas (Dallol et al., 2003).

925 The soluble class-III semaphorins constitute another group of well-known
926 chemorepellents for extending axons and neural progenitors, and, in addition,
927 have been implicated in tumor progression and angiogenesis (Neufeld and
928 Kessler, 2008). Glioma cells express the soluble semaphorins sema-3A
RR
929 and -3C as well as their cognate receptors, neuropilins and plexins, and can
930 retain sema-3A on the cell surface (Rieger et al., 2003). However, sema-3A does
931 not seem to affect glioma cell morphology or motility in vitro (Rieger et al.,
932 2003), making it an unlikely repellent from the tumor core.
It has been proposed that the gradually increasing hypoxic status of the
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933
934 growing tumor could act as a major repellent condition through exhaustion of
935 metabolic substrates and local decrease of pH (Werbowetski et al., 2004).
936 Indeed, glioma cells under hypoxia migrate at a faster rate in vitro, which has
937 been postulated as a possible explanation for the formation of hypercellular
zones (pseudopalisades) around necrotic foci (Brat et al., 2004) (see Chapter
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938
939 22). Reduced available oxygen is known to cause the stabilization and
940 transcriptional activation of the hypoxia-inducible factor HIF-1a, a master
941 transcriptional regulator with a well-established role in promoting glioma
942 angiogenesis and invasion (Kaur et al., 2005).
943 HIF-1a is upregulated in the invasive borders of the tumor and its activity is
944 increased by integrin- and RTK-initiated signaling (Zagzag et al., 2003),
945 suggesting that this transcription factor may act as a downstream regulator of the
946 pathways that promote glioma cell motility. Furthermore, HIF-1-dependent
947 transcription results in the upregulation of MMPs and the membrane receptors
948 c-Met and CXCR4 (Eckerich et al., 2007; Zagzag et al., 2006), likely resulting in
949 positive feedback mechanisms that mobilize glioma cells in response to both
F
950 hypoxia-induced and growth factor-induced signaling. This complex signaling
951 produces local tissue remodeling that favors the formation of new blood vessels
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952 (Kaur et al., 2005) and at the same time promotes glioma cell dispersion (Zagzag
953 et al., 2006).
954 The use of RNA interference against HIF-1a reduces hypoxia-induced
955 migration and invasion of several glioma cell lines in vitro (Fujiwara et al.,
956 2007). However, direct targeting of HIF-1a has shown some contradictory
PR
957 results in different gflioma models. For example, immortalized astrocytes
958 where HIF-1a has been deleted by recombination form small tumors when
959 injected subcutaneously in nude mice (Blouw et al., 2003); however, the same
960 cells show enhanced growth and invasion when implanted in the well-vascular-
961 ized brain parenchyma. These results suggest that the HIF-1-dependent
ED
962 response of these tumors is strongly dependent on their microenvironment
963 (Blouw et al., 2003). Further work is thus needed to define conditions where
964 HIF-1a could be an effective target of strategies against migratory tumor cells.
965
966
T
967
968 49.6 Targeting Strategies Against Glioma Cell Invasion

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969
970 The current standard of care for malignant gliomas after surgery is based on
971 the combination of irradiation and novel alkylating agents (temozolomide) to
972 promote apoptosis in the residual tumor. Novel stand-alone and adjuvant
973 approaches under clinical investigation are being directed against specific
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974 molecular targets in the tumor cells or blood vessels, to prevent cell
975 proliferation, angiogenesis, and local re-growth. These approaches have
976 been extensively reviewed elsewhere in this book and include, among others,
977 small-molecule inhibitors against RTK signaling (EGFR, c-Met), signal trans-
duction pathways (PKC, RAS, mTOR), integrin-mediated adhesion, and
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978
979 matrix degrading enzymes (MMPs), as well as antibody-based therapies

980 against major molecular targets in the tumor cells (EGFR and EGFRvIII)
981 and blood vessels (VEGF, VEGF receptors) (Salgaller and Liau, 2006; Stupp
982 et al., 2007). The points of contact between these anti-tumoral strategies and
the mechanisms underlying the migratory phenotype of glioma cells are
UN
983
984 summarized in Fig. 49.4.

985 Several of these strategies could have a direct impact on glioma cell disper-
986 sion, such as protease inhibitors and integrin blockers that could inhibit late
987 steps in the process of cell adhesion and migration. However, current therapies
988 essentially target the local re-growth of residual tumor and their effects on the
989 slowly proliferating, far-reaching migratory cells are at present difficult to
990
991
992
993
994
F
995
996
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997
998
999
1000
1001
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1002
1003
1004
1005
1006
1007
ED
1008
1009
1010
1011
T
1012
1013
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1014
1015
1016
1017
Fig. 49.4 Targeting strategies against invasive glioma cells. The figure summarizes the micro-
1018
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environmental factors that stimulate migration, the cellular processes that underlie the
1019
migratory phenotype (white boxes), and the autocrine/paracrine feedback during glioma cell
1020 dispersion (dashed lines). Glioma cell migration could be disrupted at several levels, including
1021 blocking of extracellular signals, inhibition of motogenic signal transduction, and interrup-
1022
tion of cell adhesion and matrix remodeling. The figure indicates pharmacological strategies
currently used against glioma cells and the steps where they could disrupt pro-migratory
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1023
processes
1024
1025
1026 gauge. Indeed, because migratory glioma cells have reduced expression of
1027 proliferative and pro-apoptotic genes (Mariani et al., 2001) they could be
expected to resist most cytotoxic insults better than the residual proliferating
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1028
1029 cells (Lefranc et al., 2005) (see Chapter 28). In addition, there is evidence that
1030 both radiation (Zhai et al., 2006) and angiostatic treatments (Lamszus et al.,
1031 2003) can stimulate glioma cell migration. Thus, there is a risk that current
1032 cytotoxic treatments not only may spare actively dispersing tumor cells but
1033 could also exert a selective pressure toward an enhanced migratory phenotype
1034 that would cause tumor recurrence in the long term (Lamszus et al., 2003).
1035 Therefore, there is a clear need for understanding the effect of current therapies
1036 on migrating glioma cells and designing novel therapeutic approaches with an
1037 explicitly formulated anti-invasive adjuvant component (Claes et al., 2007).
1038 A first obstacle for the design of an effective anti-invasive approach resides in
1039 the ’invisibility’ of infiltrating tumor cells to current detection methods and the
F
1040 anatomical difficulty of reaching them in the neural parenchyma, where they lie
1041 behind a functional blood–brain barrier (see Chapter 33) and too far from the
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1042 tumor core to be affected by drugs delivered locally into the post-resection
1043 cavity (Bolteus et al., 2001). These difficulties argue for the need of better
1044 delivery systems, such as the currently employed convection enhanced delivery
1045 (Ferguson et al., 2007), as well as improved ECM dispersion of the anti-invasive
1046 agents, in the hope of reaching the tumor cells located farthest from the original
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1047 lesion.
1048 The second, and likely largest, difficulty for an anti-invasive strategy is the
1049 identification of effective pro-migratory targets. Glioma invasion is, at the
1050 molecular level, a highly redundant process that responds to multiple signals
1051 and depends on diverse, overlapping, signaling pathways. This molecular
redundancy has made it difficult to envision and implement targeting strategies
1052
ED
1053 relying on individual ECM components, cell-surface receptors, or signaling
1054 molecules (Giese, 2003; Giese and Westphal, 1996). For example, inhibition
1055 of the catalytic activity of MMPs, which effectively reduces glioma cell invasion
1056 in animal models, has shown no efficacy in clinical trials (Salgaller and Liau,
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1057 2006), underscoring the limitations of current glioma models to identify or
1058 predict alternative mechanisms of glioma dispersion (Beadle et al., 2008; Wolf
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1059 et al., 2003).

1060 It is worth noting that extracellular targets, however, have proved useful to
1061 enhance the specificity and efficacy of adjuvant therapies (see Chapters 34–36).
1062 This has been observed, for example, in the modest but significant effect of
1063 radio-immunotherapy directed against the ECM protein tenascin-C that is
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1064 highly upregulated in gliomas (Goetz et al., 2003). In addition, pericellular

1065 and cell-surface molecules represent the most accessible targets in the tumor
1066 cells and a significant proportion of these potential targets in gliomas are
1067 restricted to the CNS (tenascin-R, phosphacan, Margolis et al., 1996) or are
tumor-specific (certain brevican isoforms, Viapiano et al., 2005), which could
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1068
1069 facilitate directed therapies with reduced non-specific and systemic responses
1070 (Viapiano and Matthews, 2006).
1071 To avoid the potentially limited efficacy of targeting individual signals and
1072 receptors, a significant bulk of research on pro-invasive molecular targets is
currently focused on downstream transduction pathways that integrate extra-
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1073
1074 cellular signals and intracellular mechanisms and may contain molecular
1075 ‘bottlenecks’ appropriate for intervention (Giese, 2003; Giese and Westphal,
1076 1996). The non-receptor tyrosine kinases of the Src family and the small
1077 GTPases of the Rho family have been highlighted as major examples of such
1078 molecular integrators. These molecules are key cross-signaling factors in path-
1079 ways initiated by a variety of extracellular signals and mediated by RTKs,
1080 integrins, CD44, and G-protein-coupled receptors (Fig. 49.3). Src kinases and
1081 the RhoA/ROCK pathway are key transduction mechanisms of glioma cell
1082 motility (Angers-Loustau et al., 2004; Goldberg and Kloog, 2006) and are
1083 required for the convergence of multiple signals on cytoskeletal reorganization
1084 and upregulation of pro-invasive proteins. The RhoA/ROCK pathway has
F
1085 been shown as a major target for radiosensitization and adjuvant therapy in
1086 solid cancers (Rattan et al., 2006), which makes it worthy of attention for future
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1087 anti-invasive approaches in gliomas. On the other hand, recent evidence has
1088 shown that the kinase Lyn constitutes more than 90% of the total Src kinase
1089 activity in glioblastomas (Stettner et al., 2005), making this single enzyme a
1090 potential major target of anti-Src strategies.
1091 Another interesting integrative target is the enzyme glycogen synthase
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1092 kinase-3 (isozymes GSK-3a and b), which can be regulated by both Rho and
1093 Src members in glioma cells (Kleber et al., 2008; Skuli et al., 2006). This multi-
1094 tasking enzyme is capable of interacting directly with more than 50 different
1095 substrates (Jope et al., 2007) and is a key point of convergence of many
1096 pathways that regulate expression of pro-invasive genes, cellular structure,
apoptosis, and motility (Jope et al., 2007; Meijer et al., 2004). Indeed, specific
1097
ED
1098 inhibition of GSK-3 can potently and specifically block glioma cell migration
1099 without causing other significant changes in the cell phenotype (Nowicki et al.,
1100 2008). Many small-molecule inhibitors, including, for example, lithium salts
1101 that are used in current psychiatric practice, are already available against this
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1102 enzyme (Meijer et al., 2004).
1103 Interestingly, strategies that were designed to prevent neo-vascularization,
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1104 such as VEGF targeting and integrin competition, may also impact the migra-
1105 tory ability of glioma cells. Anti-VEGF antibodies inhibit the association of this
1106 growth factor not only to VEGF receptors in endothelial cells but also to
1107 receptors in glioma cells, which are thought to regulate cell migration (Her-
1108 old-Mende et al., 1999). In a similar manner, soluble RGD-based peptides that
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1109 compete with integrin binding are well known to affect glioma cell adhesion and
1110 motility in vitro (D’Abaco and Kaye, 2007). Accordingly, the integrin compe-
1111 titor peptide cilengitide is currently being tested as adjuvant therapy of chemor-
1112 adiotherapy for gliomas, with positive preliminary results (Stupp et al., 2007).
A last major concern for the design of anti-invasive strategies is the difficulty
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1113
1114 of predicting the effects of anti-migratory compounds on the residual popula-

1115 tion(s) of tumor cells in vivo. Evidence from cultured glioblastoma cells sug-
1116 gests that proliferation and migration are highly plastic processes under control
1117 of poorly understood molecular switches that respond to microenvironmental
signals (Gao et al., 2005). Indeed, actively migrating glioma cells cultured
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1118
1119 within brain slices may revert to a phenotype of local proliferation if they
1120 become stalled on blood vessel branches (Farin et al., 2006), which has been
1121 suggested as a mechanism for the formation of the distant perivascular tumor
1122 foci observed in clinical specimens. Thus, sub-lethal doses of anti-invasive drugs
1123 unable to cause apoptosis in motile cells could instead have the risk of promot-
1124 ing the growth of secondary tumors (Giese and Westphal, 1996).
1125 Therefore, in addition to their inherent genetic instability, the ability of

1126 glioma cells to convert phenotype (proliferative <=> migratory) under micro-
1127 environmental influences (Giese et al., 2003) could significantly contribute to
1128 the appearance of resistant populations against therapies that act through a
1129 single type of selection pressure. Interestingly, seminal work by Goldie and
F
1130 Coldman (1979) suggested the use of rapidly alternating cytotoxic treatments to
1131 avoid single-pressure bias and deal more effectively with resistant clones during
OO
1132 adjuvant chemotherapy. It is tempting to speculate whether a similar strategy,
1133 alternating anti-proliferative and anti-migratory compounds acting through
1134 highly different mechanisms, could prove efficient against invasive glioma
1135
cells. Future research on the characterization of the major switches that regulate
1136
the conversion of migratory and proliferative phenotypes in vivo (Gao et al.,
PR
1137
2005) will be critical to better understand the influence of the microenvironment
1138
on residual tumor cells, and the evolution and weak points of these cell
1139
populations.
1140
In sum, the cellular mechanisms that make glioma cells invade efficiently and
1141
selectively within central nervous tissue, as well as the specific molecular med-
1142
ED
iators underlying these mechanisms, are still largely unexplored. Novel in vitro
1143
and in vivo models are required to reproduce more accurately the multiple
1144
influences that stimulate glioma cell migration in the CNS and to identify the
1145
molecules that control this process. This information must be combined with
1146
the wealth of data from molecular profiling of patients and seminal bioinfor-
T
1147
matic analysis (Phillips et al., 2006) (see Chapters 23–24), to identify glioma-
1148
specific, pro-invasive targets amenable to pharmacological intervention.
EC
1149
Together with current cytostatic and angiostatic strategies, inclusion of a
1150
’motostatic’ strategy is likely to maximize the efficacy of adjuvant therapies
1151
and improve progression-free survival and long-term management of these
1152
devastating tumors.
1153
RR
1154
1155 Acknowledgments The authors would like to thank the critical reading of the manuscript by
1156
Drs. E. Antonio Chiocca and Balveen Kaur from the Department of Neurosurgery at Ohio
State University (Columbus, OH), and Sarah Cork from the Department of Neurosurgery at
1157
Emory University (Atlanta, GA).
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1158
1159
1160
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Glioma Invasion: Mechanisms and Therapeutic Challenges: Mariano S. Viapiano Sean E. Lawler

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Glioma Invasion: Mechanisms and Therapeutic Challenges: Mariano S. Viapiano Sean E. Lawler

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Glioma Invasion: Mechanisms and Therapeutic Challenges

Chapter · May 2009

Mariano S. Viapiano Sean E. Lawler

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45 49.4.1 ECM Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1230

of major relevance because cell infiltration is a cause of rapid disease progres-

85 49.2 Overview of Glioma Cell Invasion in the CNS

87 In solid carcinomas, increased malignancy is characterized by the ’epithelial-to-

gliomas in the brain. Other:

Table 49.1 (continued)

256 around individual neurons. Detailed illustrations of these routes of dispersion

259 or satellitosis, known as secondary structures of Scherer, are typical of clinical

264 ways of least resistance to cell dispersion (Giese, 2003).

299 49.3.1 Neural ECM

Neural tissue Gliomas

Neural tissue Gliomas

450 heavily glycosylated proteins exhibit a remarkable heterogeneity of isoforms,

489 49.3.2 Basal Lamina

they disseminate. This remodeling involves degradation of the pre-existing ECM,

534 49.4.1 ECM Degradation

564 however, MMP-dependent proteolysis of these proteins in intracranial gliomas is a

675 49.4.2 ECM Synthesis

714 The membrane-bound receptor-type protein tyrosine-phosphatase RPTP-b/

794 such as short-term primary cultures of gliomas and culture of glioma-derived

838 49.5.1 Chemoattractants

879 cytoskeletal reorganization initiated by activation of actin-binding proteins

894 et al., 2002), resulting in MAPK activation, upregulation of extracellular

924 gliomas (Dallol et al., 2003).

968 49.6 Targeting Strategies Against Glioma Cell Invasion

979 matrix degrading enzymes (MMPs), as well as antibody-based therapies

984 summarized in Fig. 49.4.

1059 et al., 2003).

1064 highly upregulated in gliomas (Goetz et al., 2003). In addition, pericellular

1114 of predicting the effects of anti-migratory compounds on the residual popula-

1125 Therefore, in addition to their inherent genetic instability, the ability of

1208 Opin. Neurobiol., 17: 120–127

1388 Biochem. Biophys., 47: 300–320

1474 migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis.

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