**4.1. Degradation**

In 1946, Fisher [76] proposed that metastatic spread of tumors may be mediated by proteolytic degradation of the ECM. Since then, several classes of intracellular and extracellular proteases, both secreted and membrane-bound, have been identified which play roles in tumorigenesis including cell proliferation, adhesion, migration, angiogenesis, and apoptosis. The coordination between ECM degradation and subsequent cell adhesion to ECM components through integrin and other receptors followed by migration is the basis of glioma infiltration into brain parenchyma.

Lysosomal cathepsins are proteases, which are critical in removing other proteases which are in turn critical in removing unwanted cellular and extracellular components such as colla‐ gens, fibronectin, and laminin [77], and secreted cathepsins also mediate the activity of matrix metalloproteases (MMPs) by degrading their inhibitors TIMP-1 and TIMP-2 [78]. Cathepsin B promoter activity and protein levels are higher in high grade gliomas than in low-grade gliomas, and the protein is maximally expressed at the leading infiltrating edge of enlarging tumors [79]. Additionally, upregulation of cathepsins B and D has been shown to correlate with glioma tumor grade and invasiveness [80, 81], while inhibition inhibits glioma cell invasion *in vitro* [82]. Another class of intracellular proteases is the caspases, a family of intracellular cysteine proteases essential for apoptosis that are synthesized as inactive pro-

caspases and activated by pro-apoptotic signals [83]. Malignant cells have the ability to escape apoptosis and, in neuroblastoma cells, the caspase 8 gene (CASP8) has been shown to be either deleted or silenced [84]. Loss of CASP8 correlates with increased risk of metastasis in pa‐ tients with neuroblastoma. Other caspases, including CASP10, 3, 5, 6, and 7, have also found to be mutated in various tumor types [85–89].

HIF-1 is essential in the epithelial-mesenchymal transition by directly regulating the expres‐ sion of Twist, which is essential for cancer metastasis [64]. Twist is a basic helix-loop-helix transcription factor whose expression is regulated through a number of signal transduction pathways including Akt, Ras, and Wnt and whose expression correlates with higher tumor grade [65]. It inhibits the E-cadherin-mediated adhesion between cells, which enables tumor cells to adopt a more motile phenotype [66]. Twist also serves as a survival factor by inhibit‐ ing p53-induced apoptosis by counteracting the effects of c-MYC in neuroblastoma [67]. HIF-1 regulates expression of a number of adhesion molecules, such as alpha- and beta-integrins and E-cadherin [68–70], matrix metalloproteinase-2 and -9 [71, 72] as well as a number of chemo‐ kines and their receptors including c-Met and CXCR4 [73–75], suggesting how hypoxia may

Much remains to be elucidated regarding the molecular cascades through which cells transition to a migratory phenotype. Rapid proliferation creates a toxic microenvironmentthat, when sensed by the cell, sends a signal through the AMPK-mTORC axis or by HIF-1 and others to effect the changes in transcription needed to bring about the transformation to a migrato‐ ry phenotype so the cell may escape to a more favorable environment. As they leave the main tumor mass and move into the brain parenchyma which limits and inhibits their migration, glioma cells remodel their environment by secreting degradative enzymes and novel ECM components which attempt to recapitulate the more permissive, primitive structure of the

In 1946, Fisher [76] proposed that metastatic spread of tumors may be mediated by proteolytic degradation of the ECM. Since then, several classes of intracellular and extracellular proteases, both secreted and membrane-bound, have been identified which play roles in tumorigenesis including cell proliferation, adhesion, migration, angiogenesis, and apoptosis. The coordination between ECM degradation and subsequent cell adhesion to ECM components through integrin and other receptors followed by migration is the basis of glioma infiltration

Lysosomal cathepsins are proteases, which are critical in removing other proteases which are in turn critical in removing unwanted cellular and extracellular components such as colla‐ gens, fibronectin, and laminin [77], and secreted cathepsins also mediate the activity of matrix metalloproteases (MMPs) by degrading their inhibitors TIMP-1 and TIMP-2 [78]. Cathepsin B promoter activity and protein levels are higher in high grade gliomas than in low-grade gliomas, and the protein is maximally expressed at the leading infiltrating edge of enlarging tumors [79]. Additionally, upregulation of cathepsins B and D has been shown to correlate with glioma tumor grade and invasiveness [80, 81], while inhibition inhibits glioma cell invasion *in vitro* [82]. Another class of intracellular proteases is the caspases, a family of intracellular cysteine proteases essential for apoptosis that are synthesized as inactive pro-

play a role in triggering cell migration and digestion of the ECM.

developing brain.

**4.1. Degradation**

**4. ECM Remodeling**

116 Neurooncology - Newer Developments

into brain parenchyma.

Extracellular proteases are secreted by migrating tumor cells in order to degrade ECM components and to release chemoattractant and chemorepellent molecules to direct further tumor cell migration [90]. MMPs are a class of 28 zinc-dependent endopeptidases whose expression by tumor cells has been linked to increased invasion, proliferation, angiogenesis, and morbidity [91]. There are both membrane-bound MMPs and secreted forms which are released as inactive zymogens in response to growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and transforming growth factor beta (TGF-β) [92], as well as in response to cell–cell and cell–ECM interactions and signaling [93]. These inactive proenzymes contain a pro-peptide residue at the N-terminus, which masks the zinc ion within the catalytic region. Proteolytic cleavage of this region exposes the catalytic domain and activates the enzyme [94]. MMP2 and MMP9 are both expressed in human glioma cells *in vitro* [95, 96] and overexpression in gliomas correlates with higher tumor grade and poorer prognosis [97]. The MMPs localize to the leading edges of migrating cells, and many compo‐ nents of the neural ECM have been identified as substrates for MMP2 and MMP9 including the lecticans-aggrecan [98], versican [99], brevican [100], and neurocan, as well as link protein and tenascin [101] and components of the vascular basement membrane such as laminin, fibronectin, and collagen [102]. Although these substrates have been demonstrated *in vitro*, the exact role of MMP *in vivo* is unclear. Versican, for example, appears not be a major MMP substrate *in vivo*, and glioma cells do not degrade the basal laminae of blood vessels to enter the bloodstream. MMP activity is tightly regulated at the level of transcription, activation of the zymogen, and by activity of tissue inhibitors of metalloproteases (TIMS) [103]; the degree of ECMdigestion during migration is clearly a complex and carefully regulated process. MMPs are also involved in the "pro-angiogenic switch" that stimulates the production of new blood vessels into the growing tumor mass [104], a process mediated, in large part, by the VEGF signaling pathway [105]. The transmembrane metalloprotease, MT1-MMP, can directly degrade the ECM and also activate pro-MMP2 and upregulate VEGF expression [106], suggesting that this MMP may play a major role in tumor invasiveness and angiogenesis and may serve as a potential target for therapy.

Another class of zinc-dependent metalloproteases is the ADAMS (a disintegrin and metallo‐ proteinase) and the related ADAMTS (a disintegrin and metalloproteinase with thrombo‐ spondin motifs). ADAMS are membrane-bound, zinc-dependent metalloproteases that contain a pro-domain which is cleaved to activate the metalloprotease, a disintegrin domain which can bind to integrin receptors, a cysteine-rich domain, an EGF-like domain, and a transmembrane domain at the C terminus [107] (**Figure 5**). ADAMTS are extracellular proteases similar in structure but lack the EGF and transmembrane domains and contain an additional thrombospondin type I motif at the C terminus which may bind them to the ECM [108]. ADAM-8 and ADAM-19 are expressed at high levels in gliomas, and its expression correlates with invasiveness [109]. ADAM-17 activates the EGF/phosphoinositide-3 kinase/ serine/threonine kinase signal transduction pathway under hypoxic conditions and leads to increased tumor cell invasion [110]. ADAM-22 normally inhibits astrocyte proliferation in normal brain via interactions between its disintegrin domain and cell surface integrins. It is downregulated in high-grade gliomas leading to elimination of this growth inhibition [111]. ADAMTS-4 and ADAMTS-5 degrade the lectican and small leucine-rich repeat families of proteoglycans, and their expression also correlates with glioma invasiveness [112].

**Figure 5. ADAM/ADAMT structure**. ADAM and ADAMT members contain a propeptide domain (Pro) followed by the enzymatic metalloprotease domain containing the Zn-binding site followed by the disintegrin- and cystein-rich domains. ADAM members contain an EGF-like domain and ADAMTS contain additional thrombospondin type 1 mo‐ tifs (TSP1) transmembrane (TM).

Finally, plasmin degrades ECM components and activates several MMPs, and the plasmino‐ gen activators, urokinase-type PA (uPA), expressed by glioma cells, and tissue-type PA (tPA) transmembrane (TM) expressed in the endothelium of blood vessels, both play an important role in tumor cell invasion and angiogenesis [113, 114]. There is a higher level of expression of uPA in higher-grade gliomas than in lower-grade gliomas [115] and its binding to its recep‐ tor (uPAR) causes it to form a heteromeric complex with integrin receptors, which are also highly expressed on glioma cells. These then initiate signal transduction cascades that result in the upregulation of uPA, MMP, and other molecules that promote cell migration through the ECM [116].

#### **4.2. Synthesis**

It is evident that intracellular and extracellular proteases play important roles in the com‐ plex process of glioma cell-cell adhesion and attachment and detachment to ECM compo‐ nents during migration. This process is mediated by a well-regulated cascade of signal transduction pathways that also lead to the production of novel ECM components to create a new scaffold on which tumor cells can migrate. As migrating cells degrade the ECM, they

further change their microenvironment by secreting novel proteins and liberating peptides on which to migrate and generating chemoattractants and chemorepellents for guidance. In many ways, this new environment is reminiscent of that of the primitive CNS in which cell migra‐ tion was abundant in setting up the organization of the brain.

correlates with invasiveness [109]. ADAM-17 activates the EGF/phosphoinositide-3 kinase/ serine/threonine kinase signal transduction pathway under hypoxic conditions and leads to increased tumor cell invasion [110]. ADAM-22 normally inhibits astrocyte proliferation in normal brain via interactions between its disintegrin domain and cell surface integrins. It is downregulated in high-grade gliomas leading to elimination of this growth inhibition [111]. ADAMTS-4 and ADAMTS-5 degrade the lectican and small leucine-rich repeat families of

**Figure 5. ADAM/ADAMT structure**. ADAM and ADAMT members contain a propeptide domain (Pro) followed by the enzymatic metalloprotease domain containing the Zn-binding site followed by the disintegrin- and cystein-rich domains. ADAM members contain an EGF-like domain and ADAMTS contain additional thrombospondin type 1 mo‐

Finally, plasmin degrades ECM components and activates several MMPs, and the plasmino‐ gen activators, urokinase-type PA (uPA), expressed by glioma cells, and tissue-type PA (tPA) transmembrane (TM) expressed in the endothelium of blood vessels, both play an important role in tumor cell invasion and angiogenesis [113, 114]. There is a higher level of expression of uPA in higher-grade gliomas than in lower-grade gliomas [115] and its binding to its recep‐ tor (uPAR) causes it to form a heteromeric complex with integrin receptors, which are also highly expressed on glioma cells. These then initiate signal transduction cascades that result in the upregulation of uPA, MMP, and other molecules that promote cell migration through

It is evident that intracellular and extracellular proteases play important roles in the com‐ plex process of glioma cell-cell adhesion and attachment and detachment to ECM compo‐ nents during migration. This process is mediated by a well-regulated cascade of signal transduction pathways that also lead to the production of novel ECM components to create a new scaffold on which tumor cells can migrate. As migrating cells degrade the ECM, they

tifs (TSP1) transmembrane (TM).

118 Neurooncology - Newer Developments

the ECM [116].

**4.2. Synthesis**

proteoglycans, and their expression also correlates with glioma invasiveness [112].

One of the earliest events in glioma formation is the loss of the p53 tumor suppressor gene and upregulation of secreted protein acidic and rich in cysteine (SPARC), an ECM-associated glycoprotein that has an anti-adhesive role and leads to cell rounding and detachment from the ECM [117]. This is thought to be accomplished through the activation of P38 mitogenactivated protein kinase (MAPK)-heat shock protein (HSP)-27, Akt, and Shc-Raf-extracellu‐ lar signal-regulated kinase (ERK) signaling pathways [118]. SPARC is secreted at the leading edge of the invading cells [119] and has been shown to increase invasion *in vitro* [120] and *in vivo* [121]. Additionally, the combination of SPARC overexpression and loss of p53 may play a role in promoting cell survival by escaping immune surveillance [122]. SPARC is highly expressed in gliomas, and increased levels are associated with poorer prognosis [123]. Interestingly, SPARC levels are higher in developing brain, where cell migration is necessa‐ ry for setting up the architecture of the developing brain, so it is not surprising that invading glioma cells try to recapitulate this more permissive environment.

The Receptor-type protein tyrosine phosphatase mu (PTPμ) is a cell adhesion molecule normally found in neurons and glia but is absent in higher-grade, infiltrative gliomas. It is hypothesized to be involved with cell-cell adhesion and contact inhibition and that its loss allows for cell migration [124]. PTPμ is cleaved in human GBM tumor tissue and cell lines by a number of proteases including ADAMS, calpain, and serine proteases to generate protein fragments with unique biologic functions affecting cell adhesion and migration [125]. In addition to the degradation of the protein component of the ECM, glioma cells secrete hyaluronidases, which break down HA in the ECM, generating soluble HA, which activates MMP and promotes invasion [126]. Increased levels of HA and hyaluronectin are found in peripherally invasive regions of certain tumors [127] creating a more disorganized matrix through which cells can migrate. Receptor-type protein tyrosine phosphatase zeta (RPTP-ζ) is a membrane-bound proteoglycan expressed in developing and adult brains as well as in migrating glioma cells [128]. The soluble factor pleiotrophin is overexpressed in gliomas and, through binding to RPTP-ζ, promotes cytoskeletal changes through modification of betacatenin, beta-adducin, and Fyn [129]. RPTP-ζ undergoes differential splicing and one splice variant, phosphacan, is a soluble factor lacking the cytoplasmic phosphatase domain. Phosphacan is also highly expressed during embryogenesis and in migrating glioma cells and may regulate glioma migration through interactions with tenascin in the ECM [130] and axonin [131].

Two members of the lectican family, which are normally inhibitory to cell migration, versi‐ can and brevican, have unique isoforms that are present at different times of development, and these tumor-specific isoforms have been shown to promote invasion. Versican under‐ goes differential splicing to generate four different isoforms (V0, V1, V2, and V3), which vary in their GAG-binding domains. The V2 isoform is predominantin the adult CNS and is a potent inhibitor of axonal growth into the ECM [132]. The V0/V1 isoform, however, is found in the

primitive developing brain and is upregulated by TGF-β in malignant glioma [133], where it acts as a pro-migratory factor by upregulating membrane type 1 matrix metalloprotease (MT1- MMP) through the activation of microglial Toll-like receptor 2 (TLR2) [134]. Brevican, on the other hand, undergoes differential glycosylation and there are novel glycoforms in gliomas in developing and mature brain [135]. It is overexpressed in malignant gliomas, and its brevi‐ can knockdown inhibits proliferation, invasion, and spread of brevican-expressing glioma cells *in vitro* [136]. Not only is brevican overexpressed in gliomas, it is also proteolytically cleaved by metalloproteases of the ADAMTS family including ADAMTS-4 and -5, which are also overexpressed in gliomas [137]. If this posttranslational cleavage is blocked, brevican does not enhance glioma cell invasion *in vitro* or tumor progression *in vivo* [138].

Because the basal lamina of blood vessels presents a more favorable substrate for migration, it is not surprising that migrating glioma cells secrete basal lamina components to travel on. For example, certain laminin isoforms are secreted by glioma cells, and these cells interact with these isoforms and others on the tumor vasculature through the alpha3beta1 integrin during migration [139]. As tumor cells proliferate, the tumor mass becomes denser, and this mechan‐ ical stress induces secretion of collagens, their crosslinker LOX, and the angiogenic factor VEGF [140]. Collagens bind to integrins via integrin-binding domains at the cell surface and can thus activate signal transduction pathways that control proliferation, angiogenesis, and migration [141]. Integrins are associated with the actin cytoskeleton through the interaction with talin and with the microtubule network via paxillin and binding to components of the cytoskeleton modulates the affinity of integrins for the ECM [142]. Glioma cells can migrate along fibronectin in the vascular ECM, and both versican and brevican can increase synthe‐ sis of fibronectin through an EGFR-dependent mechanism by binding to β-1 integrin and β-3 integrin, respectively [143–145]. These newly synthesized fibronectin fibrils accumulate at the migrating cell surface and serve to reorganize the ECM and promote cell attachment [146, 147].

#### **4.3. Transcriptional control**

The regulation of the expression of novel proteins in gliomas is poorly understood but is surely a complex process involving many signal transduction pathways and transcription factors, and some candidates have emerged that may regulate cell migration. The Oct-3/4 transcrip‐ tion factor is involved in regulating self-renewal in stem cells and was recently found to be overexpressed in malignant gliomas. Oct-3/4 expressing-glioblastoma cells exhibited in‐ creased migration and invasion *in vitro* and resulted in upregulated FAK and c-Src expres‐ sion, which mediate integrin signals as well as increased MMP-13 expression [148]. ATF2, another transcription factor expressed in malignant glioma, is thought to be involved in the regulation of cell invasion as its level of expression is correlated with cell invasion *in vitro* [149]. Finally, suppressor of fused (Sufu) is a tumor suppressor which downregulates hedgehog, WNT, and other signaling pathways to prevent tumorigenesis [150, 151]. Overexpression suppresses glioma cell proliferation and invasiveness, angiogenesis, and *in vivo* tumor growth, while knockdown of Sufu promoted these effects, possibly by directly affecting Gli1, a transcription factor in the hedgehog signaling pathway [152].

## **4.4. Chemotaxis**

primitive developing brain and is upregulated by TGF-β in malignant glioma [133], where it acts as a pro-migratory factor by upregulating membrane type 1 matrix metalloprotease (MT1- MMP) through the activation of microglial Toll-like receptor 2 (TLR2) [134]. Brevican, on the other hand, undergoes differential glycosylation and there are novel glycoforms in gliomas in developing and mature brain [135]. It is overexpressed in malignant gliomas, and its brevi‐ can knockdown inhibits proliferation, invasion, and spread of brevican-expressing glioma cells *in vitro* [136]. Not only is brevican overexpressed in gliomas, it is also proteolytically cleaved by metalloproteases of the ADAMTS family including ADAMTS-4 and -5, which are also overexpressed in gliomas [137]. If this posttranslational cleavage is blocked, brevican does

Because the basal lamina of blood vessels presents a more favorable substrate for migration, it is not surprising that migrating glioma cells secrete basal lamina components to travel on. For example, certain laminin isoforms are secreted by glioma cells, and these cells interact with these isoforms and others on the tumor vasculature through the alpha3beta1 integrin during migration [139]. As tumor cells proliferate, the tumor mass becomes denser, and this mechan‐ ical stress induces secretion of collagens, their crosslinker LOX, and the angiogenic factor VEGF [140]. Collagens bind to integrins via integrin-binding domains at the cell surface and can thus activate signal transduction pathways that control proliferation, angiogenesis, and migration [141]. Integrins are associated with the actin cytoskeleton through the interaction with talin and with the microtubule network via paxillin and binding to components of the cytoskeleton modulates the affinity of integrins for the ECM [142]. Glioma cells can migrate along fibronectin in the vascular ECM, and both versican and brevican can increase synthe‐ sis of fibronectin through an EGFR-dependent mechanism by binding to β-1 integrin and β-3 integrin, respectively [143–145]. These newly synthesized fibronectin fibrils accumulate at the migrating cell surface and serve to reorganize the ECM and promote cell attachment [146, 147].

The regulation of the expression of novel proteins in gliomas is poorly understood but is surely a complex process involving many signal transduction pathways and transcription factors, and some candidates have emerged that may regulate cell migration. The Oct-3/4 transcrip‐ tion factor is involved in regulating self-renewal in stem cells and was recently found to be overexpressed in malignant gliomas. Oct-3/4 expressing-glioblastoma cells exhibited in‐ creased migration and invasion *in vitro* and resulted in upregulated FAK and c-Src expres‐ sion, which mediate integrin signals as well as increased MMP-13 expression [148]. ATF2, another transcription factor expressed in malignant glioma, is thought to be involved in the regulation of cell invasion as its level of expression is correlated with cell invasion *in vitro* [149]. Finally, suppressor of fused (Sufu) is a tumor suppressor which downregulates hedgehog, WNT, and other signaling pathways to prevent tumorigenesis [150, 151]. Overexpression suppresses glioma cell proliferation and invasiveness, angiogenesis, and *in vivo* tumor growth, while knockdown of Sufu promoted these effects, possibly by directly affecting Gli1, a

transcription factor in the hedgehog signaling pathway [152].

not enhance glioma cell invasion *in vitro* or tumor progression *in vivo* [138].

**4.3. Transcriptional control**

120 Neurooncology - Newer Developments

Once glial cells switch to a migratory phenotype, they degrade the ECM surrounding them, detach from the matrix and extend "invadopodia", actin-rich protrusions with ECM proteo‐ lytic activity that bind to and digest ECM components as a result of complex signal transduc‐ tion pathways linking the extracellular microenvironment to the actin cytoskeleton [153]. Cells respond to soluble molecules in this environment and use these cues to direct migration through various signal transduction pathways. These include growth factors, soluble pepti‐ des generated by proteolysis of cell surface adhesion molecules [118], and small chemotactic cytokines.

The EGF family of growth factors is known to stimulate cell proliferation and migration [154], and overexpression of EGF receptor (EGFR) is an important feature distinguishing high-grade from low-grade gliomas [155], and the highest level of expression is found at the invasive border of the expanding tumor [156]. EGFR is amplified in 40% of GBM and of these, half have a mutant form of the receptor (EGFRvIII) lacking the ligand-binding domain leading to constitutive activation [157]. Ligand binding induces dimerization and activation of EGFR, a receptor tyrosine kinase (RTK), whose signaling results in cell proliferation, angiogenesis as well as metastatic spread through the activation of PI3K-AKT-GSK3b-Rac1 and Ras-Raf-MEK-ERK signal transduction pathways [158]. However, the activation of wild-type EGFR pro‐ motes invasion independent of angiogenesis, whereas loss of its activity results in angiogenic tumor growth. EGFRvIII might only be involved in stimulating angiogenic tumor growth when wild-type EGFR expression is lost [159,160]. Formylpeptide receptor (FPR) is a Gprotein-coupled receptor that has been shown to be expressed in highly malignant gliomas [161]. Necrotic GBM cells release a number of potential ligands for FPR, and the activation of this pathway promotes chemotaxis as well as the production of VEGF [162, 163]. In addition, FPR has been shown to transactivate EGFR leading to increased chemotaxis and prolifera‐ tion [164].

Scatter factor/hepatocyte growth factor (SF/HGF) as well as its receptor which is encoded by the c-MET proto-oncogene are both are upregulated in malignant gliomas. MET is a trans‐ membrane RTK whose signal transduction cascade leads to increased gliomas motility *in vitro* as well as survival and angiogenesis [165]. Fibroblast growth factor (FGF) may act synergisti‐ cally with upregulated VEGF and SF/HGF in GBM cells to enhance malignancy [166].

Serine-threonine kinases also play a role in tumorigenesis. TGF-β is an important growth factor whose signaling is involved in invasion as well as proliferation and survival of glioma cells [167]. Its receptor is a serine-threonine kinase that, on ligand binding, oligomerizes and activates a signal transduction cascade that results in the translocation of activated Smads to the nucleus where they interact with othertranscription factors to regulate expression of genes involved in cell motility and proliferation [168]. TGF-β signaling upregulates MMP expres‐ sion and suppresses tissue inhibitors of metalloproteinase (TIMP), thus promoting invasion [169] and inhibition of TGF-β1 or knockdown of its receptorreduces invasiveness *in vitro* [170].

Chemotactic cytokines are a group of small molecules that have been found to regulate the migration of leukocytes in the immune system and have been found to be involved in

metastatic behavior of certain cancers [171]. Chemokine receptors are G-protein-coupled transmembrane receptors whose signaling pathways regulate many cellular activities including motility. Chemokines and their receptors are expressed throughout the CNS by neurons and glia and are overexpressed in high-grade gliomas [172]. Stromal-derived factor 1 (SDF-1) also called CXCL2 is a chemotactic cytokine, which, along with its receptor CXCR4, is overexpressed in gliomas as well as within the vascular endothelium along the hypoxic rim of the tumor [173, 174]. SDF-1 has been shown to promote the migration of glioma cells *in vitro* [175] by upregulating the expression of membrane type-2 matrix metalloproteinase (MT2- MMP) [176]. CXCL1 is another small chemokine known to be involved in the metastatic spread in melanoma [177] and has been shown to be highly expressed in glioma samples and promotes migration *in vitro* by upregulating MMP-2 and β1-integrin [178]. TGF-β signaling promotes invasion by reducing expression of neurotactin, a chemokine also known as CX3CL1 whose pro-adhesive properties must be overcome to allow cells to detach and migrate. The treat‐ ment of glioma cells with recombinant TGF-β1 reduced CX3CL1, expression and facilitated glioma cell detachment and dispersion [179].

Both positive and negative signals exist within the microenvironment of glioma cells, and hypoxia is an important chemorepellent as described previously which induces cell migra‐ tion away from the tumor mass. HIF-1α is stabilized at the leading tumor edge and mediates cell invasion and angiogenesis through integrin and RTK signaling pathways [180]. Slit glycoproteins are secreted into the ECM and normally serve as chemorepulsive factors but whose expression is diminished in invasive gliomas through promoter methylation. They normally bind to members of the Roundabout (Robo) family of transmembrane receptors and lead to depolymerization of the actin cytoskeleton within the invadopodia to promote cell adhesion [181, 182]. This may be accomplished through the inactivation of Cdc42, a Rho GTPase known to be involved in cell motility [183].

Semaphorins and their receptors (plexins and neuropilins) have been found to be involved in cell migration and metastasis as well as proliferation and angiogenesis in several types of cancers [184–188], and different members of the family have different functions depending on the type of tumor involved. For example, Sema 3A inhibits migration in GBM and has antiangiogenic properties in meningioma [189, 190]. Secreted semaphorins contain an N-termi‐ nal sema domain followed by variable numbers of PSI (plexins, semaphorins, and integrins) and immunoglobulin-like domains in their extracellular regions [191]. Sema 3A binds to the Neuropilin-1 receptorthatrecruits thePlexinA1 receptorto transduce a chemorepulsive signal. Sema3A also binds Neuropilin-2 but at a lower affinity than Neuropilin-1, and the binding of Neuropilin-2 acts to modulate cell signaling and converts the repulsive signal into an attractive one. Blocking Neuropilin-1 or Plexin A1 switches the Sema3A response from repulsion to attraction, while blockade of Neuropilin-2 suppresses Sema3A's typical chemorepulsive effect [192]. Similarly, Sema 4D which binds to PlexinB1 and acts through Rho [193], and Sema5A which binds to PlexinB3 and acts through Rac1, both act as chemorepellents by ultimately affecting the actin cytoskeleton and altering cell morphology [194].

Finally, Netrins are secreted laminin-associated chemotactic molecules that regulate embry‐ onic axon migration [195] which have also been shown to be involved in glioma cell migra‐ tion, mediated by binding to theirreceptors, deleted in colorectal cancer (DCC), neogenin, and uncoordinated-5 (UNC5) [196–198]. Netrins have been shown to localize to cell surfaces and interact with laminins in the basement membrane of blood vessels. Netrin-1 binding to DCC receptors on migrating glia promotes the formation of focal adhesions, limiting migration. GBMs have been shown to downregulate Netrins, thus releasing the inhibition and promot‐ ing loss of cell-cell interaction, promoting migration along basement membranes [199]. Though Netrin-1 binding to the DCC receptor tends to promote adhesion, limiting migration, UNC5 binding transforms this to repulsion [200]. This switch from attachment to motility is remi‐ niscent of that described earlier with the semaphorins.
