*4.1.1. EGFR family and EGFRvIII*

and is persistently activated, promoting tumor formation by activating aberrant signaling pathways, epigenetic mechanisms, and metabolic networks, and thus, is a promising cancer target [72]. Further with rindopepimut efficacy, chimeric antigen receptor (CAR) T cells transduced with humanized scFv against EGFRvIII were produced, and the studies are ongoing [73]. Other immunotherapy approaches that are tested or ongoing in GBM clinical studies include the administration of dendritic cells-based therapies; application of check-point inhibitor drugs,

EGFR also plays a protruding role in GBM cell invasion and aggressiveness [74]. It was previously showed that microglia cells stimulate GBM invasion via the EGFR signaling [75]. Coniglio et al. [76] demonstrated in vitro that microglia secreted EGF, which may activate EGFR and signaling pathways in GBM cells [77]. For example, EGFR or EGFRvIII may activate the STAT3 pathway [38], which is induced in various immune populations, and mediate immunosuppression potentiated by the GSCs [78]. Moreover, recent data implicated VEGF as a potent mediator of immunosuppression, again via GSC-associated mechanisms [79, 80]. A VEGF inhibitor, aflibercept, was applied in combination with an antitumor vaccine. Delayed tumor progression and survival extension were observed, which confirmed the efficacy of combining antiangiogenic and immunotherapy approaches, as well as the value of delineating tumor microenvironment [81]. Antiangiogenic therapy added to immunotherapeutic approaches toward glioma may show clinical benefits, among which the endogenous microenvironment or vaccine-

induced inflammatory responses is importantly subsidiary to its effectiveness [82].

promotes angiogenesis and supports the GSC tumor-initiating capacity [86, 87].

GSCs or glioma initiating cells (GICs) are preferentially located in perivascular and around necro/hypoxic zones where they closely react with the microenvironment and, thus, escape from apoptotic stimuli and preserve the capacity of self-renewal [83]. These interactions with microenvironment components, such as stromal cells or extracellular matrix (ECM) etc., seem important for GSC maintenance, possibly via metabolic and/or epigenetic modifications [83, 84]. Besides, GSCs may be protected from external factors via specific survival signals that they receive from the niche [85]. For instance, hypoxia induces VEGF expression, which

Besides VEGF/VERGFR signaling, HGF/Met signaling involves in regulating cell growth, motility and has a role in embryogenesis, degenerative disease and wound healing [88]. This RTK-mediated signaling also promotes the acquisition of stem-cell like properties in glioma cells and the formation and malignant progression of GBM [89, 90]; overexpressing of Met in vitro in glioma cells was highly clonogenic [88]. Met expression seems to be associated with genetic features with EGFR and the tumor suppressor PTEN inactivation, indicating cooperation among these RTK-mediated signaling in keeping GSC phenotype in glioma [91, 92]. A recent mouse study showed that EGFR inhibition induces increased c-Met expression and associated proliferation of GSCs expressing pluripotency TFs and displaying multi-lineage potential [89]. There is now the debate as to the long-term safety of anti-EGFR treatments, which may possibly induce MET-driven GSC populations [88, 89]. On the other hand, however, it implies the combination of targeting EGFRvIII and GSC as a new therapeutic approach.

and adoptive cell therapy (ACT), etc.

38 Glioma - Contemporary Diagnostic and Therapeutic Approaches

**3.4. RTK, GSC and tumor metabolism**

Small molecular weight kinase inhibitors include gefitinib (EGFR) and erlotinib (EGFR and EGFRvIII), and these two irreversible inhibitors, unfortunately, have achieved limited success either as a single agent or as combination therapies in numerous Phase I and II trials in patients with newly diagnosed or recurrent GBM [104–107]. The resistance may be driven by a subset of EGFR mutations, activation of alternate signaling pathways and suppression of EGFRvIII on extrachromosomal DNA, etc. [72]. Besides, irreversible inhibitors currently in clinical trials including lapatinib (EGFR, ErbB-2), AEE788 (EGFR, VEGFR), and dacomitinib (EGFR, HER2, HER4), alone or in combination with other agents, still attained minimal to moderate anti-tumor response in newly diagnosed GBM or recurrent patients [108, 109].

*4.1.3. PDGFR family*

or in combination with other therapies [82].

**4.2. Mechanisms of resistance to RTK-targeted therapy**

A number of PDGFR inhibitors with multiple targets are developed and tested, including imatinib mesylate (PDGFR, c-KIT, BCR-ABL), sunitinib (PDGFR, VEGFR, c-KIT), sorafenib (PDGFR, VEGFR, RAF), tandutinib (PDGFR, FLT3, c-KIT), vatalanib (PDGFR, VEGFR, c-KIT), pazopanib (PDGFR, c-KIT, EGFR) or dasatinib (PDGFRB, Src, BCR/Abl, c-KIT, ephrin A2) [125]. Among these, Imatinib (Gleevec®) is already used for the first-line treatment of myeloid malignancies and gastrointestinal stromal tumors; however, as a single agent, it shows minimal efficacy in GBMs. Previously, a combination of imatinib and hydroxyurea, a cytotoxic agent that inhibits DNA synthesis, showed a 20% response rate in progressive chemo- and radio-refractory GBM patients [125]; however, similar combination treatment achieved minimal response in recent studies [126]. Other combinations of imatinib with cytotoxic agents, or other kinase inhibitors, have been tested at the preclinical levels and clinical studies [127, 128]. Thus far, this class of targeted agents only achieved minimal anti-tumor activity either alone

Receptor Tyrosine Kinase Interaction with the Tumor Microenvironment in Malignant…

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

41

RTK targeted therapeutic strategies in cancer came into cancer practice since 2001, when FDA promptly approved imatinib (Gleevec®) as a first-line targeted agent for the treatment of patients with chronic myeloid leukemia (CML). In 2004, FDA approved bevacizumab (Avastin®) as a combination agent with standard chemotherapy to treat progressive disease such as metastatic lung cancer [129]. Thus far, however, RTK-targeted treatment strategies have achieved only moderate anti-tumor activity in patients with GBM [1, 35, 82]. Two RTK family as major targets including EGFR family and EGFRvIII, and anti-angiogenesis therapy against VEGF/VEGFR family are applied in newly diagnosed or recurrent GBM [82]. The experience with EGFR RTK inhibitors in GBM proved that, even if EGFR itself gets efficiently dephosphorylated in tumors, the treatment-induced EGFR-independent regulatory circuits may promote alternate activation of downstream signaling and render clinically ineffective [1]. Similarly, in the case of VEGFR-targeted therapy, alternative activation of other proangiogenic factors, such as FGF, PDGF, HGF, ANGPT2 and IL-8 et al. may still activate downstream effectors on converged signaling pathways [117–122]. Moreover, RTK heterogeneity and cooperation between RTKs, as well as secondary activation of downstream signaling pathways, may compensate for the loss of the targeted RTK [2]. For example, EGFRvIII transcriptionally inhibits PDGFRβ in tumor cells. EGFR TKIs reduces such inhibition, enabling

tumor cells to switch their dependence to PDGFRβ for growth and survival [1].

The inherent link between RTK and TME greatly contribute to the resistance or even failure with the RTK-targeted therapy and combinational therapies [31, 35, 82]. Treatment with the VEGFR2 inhibitor vatalanib only achieved transient benefits on reduction of tumor vascular volume but induced hypoxia and was related to the increased expression of several pro-angiogenic cytokines and chemokines such as VEGF, SDF-1, HIF-1α, FGF, Ephrin, and their receptors including VEGFR2, VEGFR3, and EGFR, which promoted aggressive tumor invasion [130] and alternative pathway of neovascularization [131, 132]. Other RTK inhibitors such as cediranib and sunitinib have been associated with higher toxicities in clinical trials [133–137]. Recent data suggest that tumors have several distinct mechanisms of

Monoclonal antibodies (mAbs) targeted against both wild-type EGFR and EGFRvIII have also been developed including cetuximab, which showed only minimal anti-tumor effect as a single agent in Phase I/II trials [110], but the drug showed chemosensitizing and radiosensitizing effect and may achieve better effect when combined with TMZ and radiotherapy [111]. Other anti-EGFR antibodies include panitumumab and nimotuzumab. Nimotuzumab, a humanized mAb against EGFR, has shown promising efficacy with significantly higher mean and median survival time in GBM patients in Phase I/II trials via its inhibition on tumor growth and angiogenesis; the antibody drug also shows least cutaneous toxicity [82]. Furthermore, targeting at EGFRvIII which acts as a GBM-specific antigen, rindopepimut is a promising peptide vaccine and has shown its effectiveness and induced strong and specific immune response in Phase II clinical trials [70, 112]. The vaccine is currently investigated in Phase II/III trials in newly diagnosed GBM patients alone, in combination treatment with other agents or standard treatment protocol in recurrent patients [82].

### *4.1.2. VEGFR family*

VEGFR is the most potent stimulator in angiogenesis, mainly including VEGFR1, VEGFR2, and VEGFR3. Several VEGFR inhibitors have been developed and applied in preclinical and clinical studies in GBM.

Cediranib (AZD2171) is a pan-VEGFR RTK inhibitor; in addition, it inhibits activity of RTKs including c-Kit, PDGFRA and PDGFRB. In Phase II trials, cediranib treatment quickly induces tumor vessel normalization and edema reduction which were related with the progressionfree survival (PFS) in GBM patients [113]. The treatment with cediranib is associated with improved overall survival (OS) only in newly diagnosed GBM patients [114]. Various other VEGFR inhibitors including aflibercept, BIBF 1120, pazopanib, AMG 386 (trebananib), Vandetanib are tested in combination with other drugs in their Phase I/II trials [115]. Among these, aflibercept inhibits both VEGF and placental growth factor (PGF), and acts as a decoy receptor dubbed VEGF trap, yet shows limited success in Phase II trials for recurrent GBM patients [116]. The mAb against VEGF, bevacizumab (Avastin®) is currently used in patients with GBM, mostly in combination with other treatment or drugs. When used in combination with irinotecan, a cytotoxic topoisomerase I inhibitor, the treatment resulted in objective radiographic responses and improvement in PFS [117]. Since bevacizumab was approved by FDA in 2004, over 60 countries apply it for the treatment of progressive disease including the USA and Japan [118–120]. Two completed Phase III trials indicated an improved PFS but not OS of newly diagnosed GBM patients with combination of bevacizumab with the standard protocol (TMZ and RT), yet showed inconsistent results on patient performance status during the treatment [121, 122].

Research data indicated that resistance to VEGF/VEGFR targeted inhibition in GBM may activate other angiogenic factors, such as FGF and PDGF, and thus, promote alternate signaling for neovascularization [123]. Moreover, the treatment-induced HGF/c-Met activation may contribute to robust invasion in the resistant GBMs [124]. Combinational targeting strategies in a good timing with VEGFR-targeted agents warrant further investigations.

### *4.1.3. PDGFR family*

(EGFR, HER2, HER4), alone or in combination with other agents, still attained minimal to moderate anti-tumor response in newly diagnosed GBM or recurrent patients [108, 109].

Monoclonal antibodies (mAbs) targeted against both wild-type EGFR and EGFRvIII have also been developed including cetuximab, which showed only minimal anti-tumor effect as a single agent in Phase I/II trials [110], but the drug showed chemosensitizing and radiosensitizing effect and may achieve better effect when combined with TMZ and radiotherapy [111]. Other anti-EGFR antibodies include panitumumab and nimotuzumab. Nimotuzumab, a humanized mAb against EGFR, has shown promising efficacy with significantly higher mean and median survival time in GBM patients in Phase I/II trials via its inhibition on tumor growth and angiogenesis; the antibody drug also shows least cutaneous toxicity [82]. Furthermore, targeting at EGFRvIII which acts as a GBM-specific antigen, rindopepimut is a promising peptide vaccine and has shown its effectiveness and induced strong and specific immune response in Phase II clinical trials [70, 112]. The vaccine is currently investigated in Phase II/III trials in newly diagnosed GBM patients alone, in combination treatment with other agents or

VEGFR is the most potent stimulator in angiogenesis, mainly including VEGFR1, VEGFR2, and VEGFR3. Several VEGFR inhibitors have been developed and applied in preclinical and

Cediranib (AZD2171) is a pan-VEGFR RTK inhibitor; in addition, it inhibits activity of RTKs including c-Kit, PDGFRA and PDGFRB. In Phase II trials, cediranib treatment quickly induces tumor vessel normalization and edema reduction which were related with the progressionfree survival (PFS) in GBM patients [113]. The treatment with cediranib is associated with improved overall survival (OS) only in newly diagnosed GBM patients [114]. Various other VEGFR inhibitors including aflibercept, BIBF 1120, pazopanib, AMG 386 (trebananib), Vandetanib are tested in combination with other drugs in their Phase I/II trials [115]. Among these, aflibercept inhibits both VEGF and placental growth factor (PGF), and acts as a decoy receptor dubbed VEGF trap, yet shows limited success in Phase II trials for recurrent GBM patients [116]. The mAb against VEGF, bevacizumab (Avastin®) is currently used in patients with GBM, mostly in combination with other treatment or drugs. When used in combination with irinotecan, a cytotoxic topoisomerase I inhibitor, the treatment resulted in objective radiographic responses and improvement in PFS [117]. Since bevacizumab was approved by FDA in 2004, over 60 countries apply it for the treatment of progressive disease including the USA and Japan [118–120]. Two completed Phase III trials indicated an improved PFS but not OS of newly diagnosed GBM patients with combination of bevacizumab with the standard protocol (TMZ and RT), yet showed inconsistent results on patient performance status during

Research data indicated that resistance to VEGF/VEGFR targeted inhibition in GBM may activate other angiogenic factors, such as FGF and PDGF, and thus, promote alternate signaling for neovascularization [123]. Moreover, the treatment-induced HGF/c-Met activation may contribute to robust invasion in the resistant GBMs [124]. Combinational targeting strategies

in a good timing with VEGFR-targeted agents warrant further investigations.

standard treatment protocol in recurrent patients [82].

40 Glioma - Contemporary Diagnostic and Therapeutic Approaches

*4.1.2. VEGFR family*

clinical studies in GBM.

the treatment [121, 122].

A number of PDGFR inhibitors with multiple targets are developed and tested, including imatinib mesylate (PDGFR, c-KIT, BCR-ABL), sunitinib (PDGFR, VEGFR, c-KIT), sorafenib (PDGFR, VEGFR, RAF), tandutinib (PDGFR, FLT3, c-KIT), vatalanib (PDGFR, VEGFR, c-KIT), pazopanib (PDGFR, c-KIT, EGFR) or dasatinib (PDGFRB, Src, BCR/Abl, c-KIT, ephrin A2) [125]. Among these, Imatinib (Gleevec®) is already used for the first-line treatment of myeloid malignancies and gastrointestinal stromal tumors; however, as a single agent, it shows minimal efficacy in GBMs. Previously, a combination of imatinib and hydroxyurea, a cytotoxic agent that inhibits DNA synthesis, showed a 20% response rate in progressive chemo- and radio-refractory GBM patients [125]; however, similar combination treatment achieved minimal response in recent studies [126]. Other combinations of imatinib with cytotoxic agents, or other kinase inhibitors, have been tested at the preclinical levels and clinical studies [127, 128]. Thus far, this class of targeted agents only achieved minimal anti-tumor activity either alone or in combination with other therapies [82].

### **4.2. Mechanisms of resistance to RTK-targeted therapy**

RTK targeted therapeutic strategies in cancer came into cancer practice since 2001, when FDA promptly approved imatinib (Gleevec®) as a first-line targeted agent for the treatment of patients with chronic myeloid leukemia (CML). In 2004, FDA approved bevacizumab (Avastin®) as a combination agent with standard chemotherapy to treat progressive disease such as metastatic lung cancer [129]. Thus far, however, RTK-targeted treatment strategies have achieved only moderate anti-tumor activity in patients with GBM [1, 35, 82]. Two RTK family as major targets including EGFR family and EGFRvIII, and anti-angiogenesis therapy against VEGF/VEGFR family are applied in newly diagnosed or recurrent GBM [82]. The experience with EGFR RTK inhibitors in GBM proved that, even if EGFR itself gets efficiently dephosphorylated in tumors, the treatment-induced EGFR-independent regulatory circuits may promote alternate activation of downstream signaling and render clinically ineffective [1]. Similarly, in the case of VEGFR-targeted therapy, alternative activation of other proangiogenic factors, such as FGF, PDGF, HGF, ANGPT2 and IL-8 et al. may still activate downstream effectors on converged signaling pathways [117–122]. Moreover, RTK heterogeneity and cooperation between RTKs, as well as secondary activation of downstream signaling pathways, may compensate for the loss of the targeted RTK [2]. For example, EGFRvIII transcriptionally inhibits PDGFRβ in tumor cells. EGFR TKIs reduces such inhibition, enabling tumor cells to switch their dependence to PDGFRβ for growth and survival [1].

The inherent link between RTK and TME greatly contribute to the resistance or even failure with the RTK-targeted therapy and combinational therapies [31, 35, 82]. Treatment with the VEGFR2 inhibitor vatalanib only achieved transient benefits on reduction of tumor vascular volume but induced hypoxia and was related to the increased expression of several pro-angiogenic cytokines and chemokines such as VEGF, SDF-1, HIF-1α, FGF, Ephrin, and their receptors including VEGFR2, VEGFR3, and EGFR, which promoted aggressive tumor invasion [130] and alternative pathway of neovascularization [131, 132]. Other RTK inhibitors such as cediranib and sunitinib have been associated with higher toxicities in clinical trials [133–137]. Recent data suggest that tumors have several distinct mechanisms of neovascularization including vascular mimicry (VM) [138]. VM is identified as tumor cells, most likely GSCs, transdifferentiate into endothelial cells and form neovascular structures to irrigate the hypoxic tumors for both nutrients and active metabolism [139, 140]. GSCs also transdifferentiate into pericytes to maintain VM [141]. Thus, VM is one of the key tumorinherent mechanisms to drive the resistance to anti-angiogenesis therapy in GBM [142–144]. Indeed, resistance to RTK-targeted and combination therapies is associated with accumulation of GSC as well as immune suppression. Achyut et al. reported that vatalanib treatment increased the number of CD68+ myeloid cells and the CD133+, CD34+, and Tie2+ endothelial cell signatures in a mouse model of GBM [145]. The enhanced myeloid cell infiltration in the TME following therapeutic resistance was associated with the activation of the CSF1–CSF1R pathway, which results in increased number of tumor-associated macrophages (TAM) within dynamic TME [146, 147].

in modulating cellular, molecular and epigenetic changes in a tumor cell, we propose that immunotherapy, especially vaccine-based treatment, targeting hypoxic cancer cells or HIF, and GSC-based therapies may be among the most promising strategies in GBM, in which reasonable and well-designed RTK-targeted therapy may at least partially contribute to the treatment success [31]. Dynamic treatment data measurement and personalized medicine with new imaging modalities (PET) using hypoxia radiotracers are key to delineating the hypoxic tumor regions, clinical tissue biopsy profile monitoring, and well-adjusted drug delivery sys-

, Quanye Sun<sup>1</sup>

2 Department of Neurosurgery, The 3rd Hospital Affiliated to Harbin Medical University,

[1] Furnari FB, Cloughesy TF, Cavenee WK, Mischel PS. Heterogeneity of epidermal growth factor receptor signalling networks in glioblastoma. Nature Reviews Cancer.

[2] Nakada M, Kita D, Teng L, Pyko IV, Watanabe T, Hayashi Y, Hamada J. Receptor tyrosine kinases: Principles and functions in glioma invasion. Advances in Experimental

[3] Li GZ, Qin Z, Chen L, Xie R, Wang HZ. Tumor Microenvironment in Treatment of Glioma. Open Medicine (Wars). 2017;**12**:247-251. DOI: 10.1515/med-2017-0035

[4] Wu M, Swartz MA. Modeling tumor microenvironments in vitro. Journal of Biome-

[5] Charles NA, Holland EC, Gilbertson R, Glass R, Kettenmann H. The brain tumor micro-

[6] Turner SG, Ahmad M, Toms SA. Mechanisms of glioma cell invasion.In: Agrawal A, editor. Neurooncology – Newer Developments. London: InTech; 2016. pp. 109-141. DOI:

Medicine and Biology. 2013;**986**:143-170. DOI: 10.1007/978-94-007-4719-7\_8

chanical Engineering. 2014;**136**:021011. DOI: 10.1115/1.4026447

environment. Glia. 2012;**60**:502-514. DOI: 10.1002/glia.21264

, Ziqi Zhen<sup>1</sup>

Receptor Tyrosine Kinase Interaction with the Tumor Microenvironment in Malignant…

, Yifan Gao<sup>1</sup>

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

, Jun Su<sup>2</sup>

and

43

tems may be rigorously applied to ensure therapeutic efficacy in GBM.

, Jingru Cui<sup>1</sup>

1 Department of Immunology, Harbin Medical University, Harbin, China

**Author details**

\*

, Yucui Dong1†

These authors contributed equally.

\*Address all correspondence to: ren\_huan99@qq.com

2015;**15**:302-310. DOI: 10.1038/nrc3918

Yujiao Gong1†

Harbin, China

**References**

10.5772/63126

†

Huan Ren<sup>1</sup>

Collectively, the mechanisms of resistance to RTK-targeted therapy include (1) intratumoral heterogenicity of RTKs, that is, cooperation of various RTKs and their downstream signaling pathways; (2) intertumoral heterogenicity of RTK expression and activity within TME; (3) the treatment-induced shaping and adaption of TME including secondary hypoxia, accumulation of GSC and immune suppression [77]. These mechanisms may cause from ineffectiveness to treatment failure, or even clinical toxicity, leading to GBM recurrence. Moreover, during RTKtargeted treatment, most clinical studies actually lack sufficient information regarding the measurement on intratumoral drug levels, target engagement and the degree of inhibition on the targeted RTK in real time [82]. Nonetheless, design of further combination therapies should consider such information, in addition to monitoring the tumor dynamic profiles, and treat the patients according to the corresponsive patterns in disease progression. Therefore, understanding the biology of CNS tumors and influence of TME on tumor progression is becoming increasingly important for developing new therapeutic strategies for this deadly disease.
