**3.1. RTK, hypoxia and angiogenesis**

HB-EGF, which stimulate in turn wild-type EGFR forming an autocrine loop [17]. It was previously reported that EGFR and EphA2 are both expressed in GBM cells and co-localize to the cell surface. EphA2 phosphorylation is dependent on EGFR activity, and EphA2 downregulation inhibits EGFR phosphorylation, downstream signaling, and EGF-induced cell viability [17]. HGF indirectly activates alternative RTKs such as EGFR by upregulating expression of EGFR ligands such as TGF-α and HB-EGF [2]. Previous studies report that EphA4, whose expression is correlated with increasing glioma grade, forms a heteroreceptor complex with fibroblast growth factor receptor 1(FGFR1) in glioma cells and that the EphA4-FGFR1 complex potentiated FGFR-mediated downstream signaling such as Akt/MAPK, Rac1, and Cdc42 pathways, resulting in the promotion of invasion [18]. A few other reports suggest that Tie2 activation regulates angiogenesis in a highly context and tissue-dependent manner and

Human GBM is characterized with high degrees of intertumoral and intratumoral heterogeneity. For example, individual GBM tumors display striking histological variations. As a hallmark of GBM development, oncogenic RTK activation is highly responsible for malignant behaviors of multiple cells in the TME other than GBM cells, that is, endothelial cells, epithelial cells, astrocytes, infiltrated immune cells, glioma stem cells (GSC), etc. [2]. The malignant grade in human astrocytoma was associated with an upregulation of the PDGFRβ on vessel endothelial cells indicating the role of paracrine activation in tumor angiogenesis [21, 22]. Besides EGF, five other respective ligands activate EGFR including transforming growth factor alpha (TGF-α), amphiregulin, betacellulin, heparin-binding EGF-like growth factor (HB-EGF), and epiregulin, respectively. These ligands are secreted by glioma cells and received by tumor microenvironmental cells such as microglia and reactive astrocytes [2]. Axl/Gas6 signaling has multiple functions to regulate survival, proliferation, and migration in a variety of cells in vitro including tumor-derived cell lines of epithelial, mesenchymal, and hematopoietic origin [23]. Moreover, the Eph/ephrin system plays a role in many biological processes such as cell adhesion and migration during development, especially in the central nervous system [24]. In glioma, different Eph receptors are overexpressed not only in tumor cells but also in the surrounding tumor-infiltrating cells like tumor-associated

closely collaborates with VEGF and other angiogenesis regulators [19, 20].

macrophages (TAMs) [25], endothelial cells, stromal cells [26], as well as GSCs [27].

Activation of RTK pathways can lead to cellular transformation and result in genetic alteration in GSCs. Fully differentiated neural cells were able to generate malignant glioma upon PDGFA overexpression and showed high expression of stem and progenitor cell markers [28]. Growth factors such as PDGF, bFGF, and EGF were usually added to the serum-free media to maintain properties of cancer stem cells derived from patient tumor biopsies [29]. HGF/c-Met pathway was involved in brain tumorigenesis and malignant progression, and thus, HGF/c-Met signaling may maintain GSC properties [30]. Moreover, RTKs show various regional expression pattern within tumor in situ during tumor progression, for example, histopathological analysis on in vivo human glioma biopsies showed that Ang-2, MMP-2, MT1-MMP, and laminin5γ2 are co-overexpressed in the invasive areas but not in the central regions of the glioma tissues [31]. GBM is characterized with the unique pattern showing that necrotic areas are typically surrounded by "pseudopalisading" glioma cells, which are highly

**2.3. Heterogeneity of RTK expression within the TME**

34 Glioma - Contemporary Diagnostic and Therapeutic Approaches

As one of the most prominent features in human GBM, pseudopalisading necrosis, the area of hypercellularity surrounding necrotic regions, and associated active vascular proliferation and tumor invasion are driven by hypoxia [32, 33]. Tumor cells reside in these regions have a high expression of HIF-1α and release VEGF, which is one of the most important regulators of angiogenesis and neovascularization (**Figure 1**). VEGF family members signal predominantly through the cognate RTKs, VEGFR-1, VEGFR-2, and VEGFR-3, in association with the co-receptors [34] via both hypoxia-dependent and hypoxia-independent mechanisms. Moreover, pseudopalisading necrosis regions protect glioma stem cells (GSC) in the region from therapeutic agents, and this facilitates the GSC niche to expand and contribute to tumor growth [35]. HIF-1α is a transcription factor that regulates the expression of a variety of genes involved in glycolysis, angiogenesis, invasion and epithelial-mesenchymal transition (EMT), which are critical for tumor growth and progression, and likely cooperate and activate other aberrant RTK signaling pathways [36, 37]. We and other reports showed that, in response to hypoxia condition, significantly increased activity of EGFR, as well as its mutant protein EGFRvIII, which further promoted activation of convergent downstream signaling pathways including Ras/MAPK, PI3K/Akt, JAK2/STAT3, and NF-κB signaling, and enhanced malignant behaviors in GBM cells in vitro, and most likely to act in the same way in vivo [1, 38].

invasiveness and aggressiveness [53]. We demonstrated that hypoxia tumor microenvironment and ECM vitronectin could enhance tumor cell invasion and EGFRvIII activity via EGFRvIII and integrin β3 complex, emphasizing key roles of TME in tumor progression and metastasis [54]. Furthermore, as ECM may act as a reservoir for multiple growth factors such as VEGF, EGF, PDGF and TGF-β, release of these factors and their binding to their cognate receptors may also converge and further strengthen the activation of these signaling cascades, leading to uncontrolled cell behaviors in tumor growth and survival, angiogenesis, and invasion [55].

Receptor Tyrosine Kinase Interaction with the Tumor Microenvironment in Malignant…

http://dx.doi.org/10.5772/intechopen.76873

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Knockout studies show the role of integrins in overactive GBM angiogenesis, which highly depends on VEGF and bFGF [56], αvβ3, αvβ5, β1, and αvβ8 notably play an important role during the process. For example, endothelial cells-expressed αvβ3/αvβ5 can provide survival signals and traction for invading cells, which are necessary to angiogenesis [57, 58]. αvβ3/αvβ5 associated neovascularization is respectively dependent on tumor cell-secreted bFGF/TNFα and VEGF and involved in a process leading to active interaction between tumor cells and endothelial cells [59]. Overexpression of αvβ3/αvβ5 in endothelial cells facilitates adhesive interactions with ECM proteins such as vitronectin, fibronectin, fibrinogen, osteopontin, etc. In cooperation with bFGF/VEGF, αvβ3/avβ5 also activates signaling pathways including FAK/ ILK, PI3K/Akt, and SDF1-CXCR4 [60] that promote EC proliferation, survival, and migration [61], and initiation of tumor angiogenesis. Collectively, several key integrins such as αvβ3, αvβ5, β1, and αvβ8 appear to be potential targets in GBM to reduce tumor angiogenesis [62].

GBM patients show marked intratumoral and systemic immunosuppression. The tumor microenvironment contains multiple immunosuppressive factors including TGFβ2, prostaglandin-E2, IL-10; and receptor molecules B7-H1, Fas-ligand, etc. [63]. The tumor is heavily infiltrated by microglia/macrophages, which can represent up to 30% of viable cells in the tumor mass, but lymphocytes infiltration is not common [64]. These monocytes/macrophages in the tumor environment interact with GBM cells and develop immunosuppressive myeloidderived suppressor cells (MDSCs). Systemic immunosuppression in GBM patients shows that total T-cell counts are greatly decreased, especially CD4+ T cell counts [65]. Furthermore, T-cell function is markedly abnormal [66]. Besides other factors that may underlie T-cell dysfunction, increases in circulating cell populations such as regulatory T cells (Tregs) and

Because of immunosuppressive features of GBM, in recent years, new therapies such as tumor vaccines and peptides are tested in preclinical and clinical studies [63]. The mutant protein EGFRvIII is a cancer specific antigen bearing a targetable epitope that is almost exclusively present in GBM [69]. Rindopepimut is composed of an EGFRvIII-specific peptide conjugated with an adjuvant protein KLH (keyhole limpet hemocyanin). The vaccination produced active anti-tumor response with significant survival benefit in GBM patients. Furthermore, the underlying immune response is not only effective regarding specific removal of EGFRvIII-positive GBM cells, but also the increase in the titer of anti-EGFRvIII sera in beneficial patients [70]. More importantly, phase II clinical trial with the vaccine confirmed these results [71], and the randomized phase III clinical study is ongoing [63]. EGFRvIII lacks the ligand binding domain

**3.3. RTK-mediated immune suppression**

MDSCs may be more important [67, 68].

In addition to VEGF, supplementary proangiogenic factors including FGF, PDGF, placentalike growth factor (PLGF), integrins, HGF/scatter factor, ephrins, angiopoietins (ANGPT), and interleukin-8 (IL-8), matrix-metalloproteinase (MMP)-2, MMP-9, collagen type I α1 (COLIA1), endothelial markers CD34, Tenascin-C, neuron-glial antigen 2 (NG-2) on pericytes, insulin-like growth factor (IGF), and EGF present in GBM [39–42]. Interestingly, many of these factors are RTK ligands and may bind to respective RTK on vascular endothelial cells or GBM cells, act in autocrine or paracrine manner to stimulate the events of neo-angiogenesis. Activation of these proangiogenic factors interacts with a number of signaling pathways include activation of Ras/ Raf/MAPK [41, 42], PTEN/PI3K/AKT [40], PLC-γ/protein kinase C (PKC) [40], nitric oxide (NO) [43], PDGFB [44], and Notch1 [45]. GBMs are diagnosed at the advanced stages when they show hypoxia and leaky vasculatures [35]. The critical role that VEGF and these pro-angiogenic factors play in angiogenesis has rendered them appealing targets to exploit in cancer therapeutics [43].
