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The ongoing characterization of the genetic and epigenetic alterations in the gliomas has already improved the classification of these heterogeneous tumors and enabled the development of rodent models for analysis of the molecular pathways underlying their proliferative and invasive behavior. Effective application of the targeted therapies that are now in development will depend on pathologists' ability to provide accurate information regarding the genetic alterations and the expression of key receptors and ligands in the tumors. Here we review the mechanisms that have been implicated in the pathogenesis of the gliomas and provide examples of the cooperative nature of the pathways involved, which may influence the initial therapeutic response and the potential for development of resistance.

Anti-angiogenic therapies would be particularly beneficial in the treatment of malignant gliomas. Peptides derived from the second type 1 repeat (TSR) of thrombospondin-1 (TSP-1) have been shown to inhibit angiogenesis in non-glioma tumor models and a modified TSR peptide, ABT-510, has now entered into Phase II clinical trials of its efficacy in non-glioma tumors. As microvascular endothelial cells (MvEC) exhibit heterogeneity, we evaluated the ability of the modified TSR peptide (NAcSarGlyValDallolleThrNvalleArgProNHE, ABT-510) to inhibit malignant glioma growth in vivo and to induce apoptosis of brain microvessel endothelial cells (MvEC) propagated in vitro. We found that daily administration of ABT-510 until euthanasia (days 7 to 19), significantly inhibited the growth of human malignant astrocytoma tumors established in the brain of athymic nude mice. The microvessel density was significantly lower and the number of apoptotic MvEC was significantly higher (3-fold) in the tumors of the ABT-510-treated animals. Similar results were found using a model in which the established tumor is an intracerebral malignant glioma propagated in a syngeneic mouse model. ABT-510 treatment of primary human brain MvEC propagated as a monolayer resulted in induction of apoptosis in a dose- and time-dependent manner through a caspase-8-dependent mechanism. It also inhibited tubular morphogenesis of MvEC propagated in collagen gels in a dose- and caspase-8 dependent manner through a mechanism that requires the TSP-1 receptor (CD36) on the MvEC. These findings indicate that ABT-510 should be evaluated as a therapeutic option for patients with malignant glioma.

Here we present a new technique to quantitatively characterize malignant glioma invasion in a syngeneic mouse model. The GL261 mouse malignant glioma cell line was injected intracerebrally into the C57B1/6 black mouse and allowed to propagate for 10 or 17 days, followed by euthanasia of the animal, harvesting of the brain, fixation, and serial sectioning. Histologic examination was performed and the primary tumor mass and discontinuous sites of tumor invasion were traced on digital images of serial microscopy sections, followed by analysis of the invasion characteristics using a custom-written MATLAB program. We found a significant increase in the number of discontinuous tumor invasion sites and in the distance of these sites from the tumor centroid in mice that were euthanized at 17 days post-tumor cell injection, as compared to mice euthanized at 10 days. Furthermore, a scatter plot analyses indicated that the invasion site data could be grouped based on the characteristics of area and distance from the tumor centroid to reveal significant differences between the two experimental groups of mice. This quantitative method will allow a future in vivo analysis of invasion characteristics in glioma cells expressing altered levels or function of invasion genes, and of new therapy targeting invading glioma cells.

Angiogenesis is the formation of new blood vessels from the existing vasculature and is necessary for tumor growth. Syndecan-2 (S2) is highly expressed in the microvasculature of mouse gliomas. When S2 expression was down-regulated in mouse brain microvascular endothelial cells (MvEC), this inhibited cell motility and reduced the formation of capillary tube-like structures in vitro. Pro-angiogenic growth factors and enzymes up-regulated during glioma tumorigenesis stimulated shedding of the S2 ectodomain from endothelial cells in vitro. The effect of shed S2 on angiogenic processes was investigated by incorporating recombinant S2 ectodomain (S2ED) into in vitro angiogenesis assays. S2ED promoted membrane protrusion, migration, capillary tube formation, and cell-cell interactions. We therefore propose that S2 is necessary for angiogenesis of MvEC, proangiogenic factors expressed during glioma progression regulate S2 shedding, and shed S2 ectodomain may increase endothelial cell angiogenic processes.

Host antiangiogenesis factors defend against tumor growth. The matricellular protein, thrombospondin-2 (TSP-2), has been shown to act as an antiangiogenesis factor in a carcinogen-induced model of skin cancer. Here, using an in vivo malignant glioma model in which the characteristics of the tumors formed after intracerebral implantation of GL261 mouse glioma cells are assessed, we found that tumor growth and microvessel density were significantly enhanced in tumors propagated in TSP-2(-/-) mice. Mechanistically, matrix metalloproteinase (MMP)-2 has been associated with neoangiogenesis and it has been proposed that the levels of available MMP-2 may be down-regulated by formation of a complex with TSP-2 that is internalized by low-density lipoprotein receptor-related protein 1 (LRP1). We found elevated expression of MMP-2 and MMP-9 in tumors propagated in TSP-2(-/-) mice, with a preferential localization in the microvasculature. In wild-type mice, MMP-2 was coexpressed with TSP-2 in the tumor microvasculature. In vitro, addition of recombinant (rec) TSP-2 to mouse brain microvessel endothelial cells reduced MMP-2 levels and invasion through mechanisms that could be inhibited by a competitive inhibitor of ligand binding to LRP1 or by siLRP1. Thus, the antiangiogenic activity of TSP-2 is capable of inhibiting the growth of gliomas in part by reducing the levels of MMP-2 in the tumor microvasculature. This mechanism is mediated by LRP1.

Angiogenesis is a promising target for the development of effective strategies for the treatment of malignant brain tumors in that it has the potential to starve large tumors and prevent the regrowth of residual margins. Two critical steps in angiogenesis, the proliferation of activated endothelial cells and their migration into the perivascular space (sprouting), require adherence of the endothelial cells to the extracellular matrix (ECM). Thus, the availability of the appropriate ligands within the ECM contributes to the regulation of angiogenesis. In addition, several components of the ECM can act through other mechanisms to further promote angiogenesis or inhibit it. Current evidence suggests that the regulation of angiogenesis is a dynamic process in which the endothelial cells can promote angiogenesis by secreting proteases that remodel the ECM, tumor cells can further promote angiogenesis by secreting ECM components and actively remodeling their environment, and stromal cells may respond to angiogenesis associated with tumors and inflammatory reactions by secreting inhibitory molecules. Here, we provide a critical review of the protein and proteoglycan components of the ECM that have been implicated in angiogenesis with an emphasis on their role in promoting or inhibiting angiogenesis in brain tumors.

Angiogenesis is necessary for tumor growth beyond a volume of approximately 2 mm(3). This observation, along with the accessibility of tumor vessels to therapeutic targeting, has resulted in a research focus on inhibitors of angiogenesis. A number of endogenous inhibitors of angiogenesis are found in the body. Some of these are synthesized by specific cells in different organs, and others are created by extracellular proteolytic cleavage of plasma-derived or extracellular matrix-localized proteins. In this review, we focus on angiostatin, endostatin, PEX, pigment epithelial-derived factor, and thrombospondin (TSP)-1 and -2, either because these molecules are expressed in malignant glioma biopsies or because animal studies in malignant glioma models have suggested that their therapeutic administration could be efficacious. We review the known mechanisms of action, potential receptors, expression in glioma biopsy samples, and studies testing their potential therapeutic efficacy in animal models of malignant glioma. Two conclusions can be made regarding the mechanisms of action of these inhibitors: (1) Several of these inhibitors appear to mediate their antiangiogenic effect through multiple protein-protein interactions that inhibit the function of proangiogenic molecules rather than through a specific receptor-mediated signaling event, and (2) TSP-1 and TSP-2 appear to mediate their antiangiogenic effect, at least in part, through a specific receptor, CD36, which initiates the antiangiogenic signal. Although not proven in gliomas, evidence suggests that expression of specific endogenous inhibitors of angiogenesis in certain organs may be part of a host antitumor response. The studies reviewed here suggest that new antiangiogenic therapies for malignant gliomas offer exciting promise as nontoxic, growth-inhibitory agents.

Glioblastoma (GBM) contains a self-renewing, tumorigenic cancer stem cell (CSC) population which contributes to tumor propagation and therapeutic resistance. While the tumor microenvironment is essential to CSC self-renewal, the mechanisms by which CSCs sense and respond to microenvironmental conditions are poorly understood. Scavenger receptors are a broad class of membrane receptors well characterized on immune cells and instrumental in sensing apoptotic cellular debris and modified lipids. Here, we provide evidence that CSCs selectively use the scavenger receptor CD36 to promote their maintenance using patient-derived CSCs and in vivo xenograft models. CD36 expression was observed in GBM cells in addition to previously described cell types including endothelial cells, macrophages, and microglia. CD36 was enriched in CSCs and was able to functionally distinguish self-renewing cells. CD36 was coexpressed with integrin alpha 6 and CD133, previously described CSC markers, and CD36 reduction resulted in concomitant loss of integrin alpha 6 expression, self-renewal, and tumor initiation capacity. We confirmed oxidized phospholipids, ligands of CD36, were present in GBM and found that the proliferation of CSCs, but not non-CSCs, increased with exposure to oxidized low-density lipoprotein. CD36 was an informative biomarker of malignancy and negatively correlated to patient prognosis. These results provide a paradigm for CSCs to thrive by the selective enhanced expression of scavenger receptors, providing survival, and metabolic advantages. S tem C ells 2014;32:1746-1758 [ABSTRACT FROM AUTHOR]

Abstract: Glioblastoma (GBM) is an extremely aggressive, infiltrative tumor with a poor prognosis. The regulatory approval of bevacizumab for recurrent GBM has confirmed that molecularly targeted agents have potential for GBM treatment. Preclinical data showing that SRC and SRC-family kinases (SFKs) mediate intracellular signaling pathways controlling key biologic/oncogenic processes provide a strong rationale for investigating SRC/SFK inhibitors, e.g., dasatinib, in GBM and clinical studies are underway. The activity of these agents against solid tumors suggests that they may also be useful in treating brain metastases. This article reviews the potential for using SRC/SFK inhibitors to treat GBM and brain metastases. [ABSTRACT FROM AUTHOR]

Abstract: Here we present a new technique to quantitatively characterize malignant glioma invasion in a syngeneic mouse model. The GL261 mouse malignant glioma cell line was injected intracerebrally into the C57B1/6 black mouse and allowed to propagate for 10 or 17 days, followed by euthanasia of the animal, harvesting of the brain, fixation, and serial sectioning. Histologic examination was performed and the primary tumor mass and discontinuous sites of tumor invasion were traced on digital images of serial microscopy sections, followed by analysis of the invasion characteristics using a custom-written MATLAB program. We found a significant increase in the number of discontinuous tumor invasion sites and in the distance of these sites from the tumor centroid in mice that were euthanized at 17 days post-tumor cell injection, as compared to mice euthanized at 10 days. Furthermore, a scatter plot analyses indicated that the invasion site data could be grouped based on the characteristics of area and distance from the tumor centroid to reveal significant differences between the two experimental groups of mice. This quantitative method will allow a future in vivo analysis of invasion characteristics in glioma cells expressing altered levels or function of invasion genes, and of new therapy targeting invading glioma cells. [Copyright &y& Elsevier]