**5. Future Directions**

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

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

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‐

glioma cell detachment and dispersion [179].

122 Neurooncology - Newer Developments

GTPase known to be involved in cell motility [183].

Current therapy for malignant gliomas is aimed at reducing tumor burden and targeting dividing cells with cytotoxic chemotherapy, anti-angiogenic agents, or tumor-treating fields. New agents targeting new pathways are desperately needed as survival stillremains extreme‐ ly poor and glioma cells become resistant over time to current therapies. Delivering therapy to normal brain parenchyma containing infiltrating tumor cells is also difficult as the bloodbrain barrier remains largely intact, though several strategies have been attempted to overcome this [201]. Limiting the invasiveness of these aggressive tumors is desirable; however, the molecular pathways involved in this complex process remain incompletely understood and new targets and therapies are lacking. Additionally, because tumor cells seem to adopt either a proliferative or migratory phenotype, preventing migration may impose a selective advantage for cells to proliferate rather than migrate leading to more rapid local recurrences. There is also no way to visualize invading cells until they stop migrating and proliferate to create a radiographically evident tumor mass. Two-dimensional *in vitro* assays have been used to study this process using glass or plastic substrates and more recently, assays using hydrogels or nanofiber scaffolds have been developed to better simulate the threedimensional microenvironment encountered by migrating tumor cells [202–204].

Identifying molecular targets has been a priority in developing new therapies for GBM, and the complex process of cell migration offers many potential targets including ECM compo‐ nents, proteases, and members of signal transduction pathways. The neural ECM has many unique components that are potential targets for therapy. Because tenascin is upregulated in malignant gliomas and may be a stem cell marker [205], it is an attractive target and phase I trials are underway exploring the use of radiolabeled monoclonal antibodies to tenascin-C or tenascin-R to deliver a radiation boost to the resection cavity. Since many signal transduc‐ tion pathways involved in glioma invasion often involve RTKs, and since various RTKs are mutated or overexpressed in GBM, RTK inhibitors are an obvious choice of targeted therapy. The results, however, have been disappointing, and no clear RTK inhibitor or combination has demonstrated a significant survival benefit [206]. Similarly, several inhibitors of MMPs have been investigated and, though some have shown efficacy *in vitro*, no clear clinical benefit has yet been demonstrated [207–209]. Cilengitide is an arginine-glycine-aspartic acid containing peptide that targets integrins and, though it was promising in preclinical studies, it failed to

show improvement in progression-free survival or overall survival in both the CENTRIC [210] and CORE [211] trials investigating the addition of cilengitide to standard therapy in pa‐ tients with newly diagnosed GBM with or without MGMT promoter methylation.

Many molecules involved in regulating cell migration in malignant gliomas are also in‐ volved in angiogenesis, cell proliferation, and avoidance of apoptosis; so agents targeting these molecules would be expected to have multiple antitumor effects. However, results have been mixed and, though *in vitro* data are encouraging for many agents, none has proven success‐ ful in showing clinical improvement in survival. There is apparently much redundancy in these signaling pathways, requiring a more complete understanding of these molecular events as well as more accurate modeling with which to study the complex processes involved in tumor spread. The earliest events leading to a migratory phenotype would be ideal candi‐ dates for therapy, though inhibiting migration could select for a proliferating phenotype leading to faster local recurrence. Hopefully, as we achieve a better understanding of the genetics and molecular alterations leading to glioma invasion, new therapies will arise to limit this aggressive, deadly disease.
