Table of Contents Author Guidelines Submit a Manuscript
Stem Cells International
Volume 2015, Article ID 427518, 11 pages
http://dx.doi.org/10.1155/2015/427518
Review Article

EGFR Amplification and Glioblastoma Stem-Like Cells

1Laboratory for Brain Tumor Biology, Department of Neurosurgery, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany
2Laboratory for Tumor Genetics, Department of Neurology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany

Received 15 February 2015; Revised 21 May 2015; Accepted 25 May 2015

Academic Editor: Giorgio Stassi

Copyright © 2015 Katrin Liffers et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Glioblastoma (GBM), the most common malignant brain tumor in adults, contains a subpopulation of cells with a stem-like phenotype (GS-cells). GS-cells can be maintained in vitro using serum-free medium supplemented with epidermal growth factor, basic fibroblast growth factor-2, and heparin. However, this method does not conserve amplification of the Epidermal Growth Factor Receptor (EGFR) gene, which is present in over 50% of all newly diagnosed GBM cases. GS-cells with retained EGFR amplification could overcome the limitations of current in vitro model systems and contribute significantly to preclinical research on EGFR-targeted therapy. This review recapitulates recent methodological approaches to expand stem-like cells from GBM with different EGFR status in order to maintain EGFR-dependent intratumoral heterogeneity in vitro. Further, it will summarize the current knowledge about the impact of EGFR amplification and overexpression on the stem-like phenotype of GBM-derived GS-cells and different approaches to target the EGFR-dependent GS-cell compartment of GBM.

1. Introduction

Glioblastoma (GBM) is the most common malignant brain tumor in adults. Despite advances in surgical procedures and therapeutic options, the live expectancy of GBM patients has remained poor with a median survival of only 12–15 months [1]. Although GBMs are characterized by extensive intra- and intertumoral heterogeneity at the histological and molecular level, they can be divided into four major subtypes based on their global expression profiles associated with distinct prognosis [2, 3]. According to Verhaak et al., these are the mesenchymal, neural, proneural, and classical subtypes, each defined by specific genetic aberrations or expression of marker genes (mesenchymal: NF1; neural: SYT1; proneuronal: PDGFRα/IDH1, classical: EGFR).

Pediatric and adult GBMs contain a subpopulation of cells with a stem-like phenotype (GBM stem-like cells; GS-cells), identified by the cell surface marker CD133 (also termed Prominin-1) [48]. Similar to adult neural stem cells (NSCs), GS-cells contain the ability to self-renew and to differentiate along neural lineages, that is, astrocytes, neurons, and oligodendrocytes, when cultured in differentiation medium (fetal calf serum, retinoic acid, and cyclic adenosine monophosphate) [5, 9]. For GS-cells, the ability to initiate tumors that recapitulate the heterogeneous phenotype of their parent tumor when implanted into the brain of immunodeficient mice is considered the central criterion to distinguish GS-cells from nonstem-like tumor cells.

At the molecular level, GS-cells in neurosphere cell culture have been described to closely mirror the genotype and the transcriptional phenotype of primary GBM tissue as opposed to conventional adherent monolayers which were established in the presence of serum [1015]. This is also reflected in the conservation of the molecular subtypes of GBM in GS-cells, while these are lost in conventional cell lines [2, 14, 16, 17].

GS-cells were found to be highly resistant to radio- and chemotherapy in vitro and in vivo [4, 8, 18] and to adapt rapidly to changes in the tumor microenvironment, that is, acidic stress [19] or hypoxia [20, 21]. Data from our lab could further demonstrate that GS-cells undergo a metabolic switch from glycolysis to the pentose phosphate pathway in response to hypoxia, resulting in decreased proliferation and increased migration [22]. This indicates an inherent metabolic plasticity, translated into phenotypic properties such as migration or proliferation, in order to adapt to microenvironmental oxygen changes. Finally, these mechanisms might also contribute to treatment resistance.

Clinically, a stem-cell related gene expression signature in patient-derived tumors (self-renewal signature [23]) was found to be associated with resistance to radio/chemotherapy in GBM patients [24]. Additionally, a high proportion of cells positive for putative GS-cell markers such as CD133, nestin, or PDPN was a negative prognostic factor for progression-free survival (PFS) and overall survival (OS) in GBM patients [11, 2528]. This has led to an investigation of GS-cell targeted therapies (reviewed in [2931]), including differentiation therapies [9, 32], oncolytic therapies with CD133-targeted measles virus [33], or indirect targeting of the perivascular GS-cell niche [20, 34, 35].

The most frequent genetic alteration in GBMs is an amplification of the Epidermal Growth Factor Receptor (EGFR) gene and/or its overexpression at the protein level, which is present in 40–60% of all GBM cases [36, 37]. Half of the amplified cases additionally express a constitutively active, oncogenic EGFR deletion variant lacking the ligand-binding domain (exons 2–7) termed EGFRvIII [38, 39]. EGFR/EGFRvIII expression is associated with increased proliferation and migration of GBMs, contributing to the malignant phenotype of these tumors in an angiogenesis-independent manner [4042]. Additionally, expression of EGFRvIII has been found to promote and accelerate angiogenesis in preclinical GBM models in vivo [43, 44]. However, therapeutic targeting of the EGFR by inhibiting tyrosine kinase activity or by interfering with ligand-induced activation has not improved overall life expectancy for GBM patients when compared to standard treatment [4548].

One of the major drawbacks for the analysis of the impact of EGFR amplification on targeted therapy is that it is rapidly lost when cells from EGFR-amplified GBM are taken into culture [49]. As a result of this limitation, preclinical models for studying EGFR biology in GBM largely relied on ectopic overexpression of EGFR and/or EGFRvIII in nonamplified GBM cell lines and a subsequent blockade of the overexpressed proteins [5052]. Over the years, different methods have been developed to overcome these limitations and to maintain EGFR amplification in addition to a stem-like phenotype in vitro, which allowed for the investigation of the contribution of the EGF/EGFR axis to a glioma stem cell phenotype in an EGFR-amplified background.

2. Glioblastoma Cells with a Stem-Like Phenotype In Vitro

Different approaches have been described to isolate and to expand GS-cells from GBM tissue in vitro based on phenotypic criteria or marker expression. Using cell culture conditions originally developed to promote in vitro growth of neural precursor cells from the neurogenic subependymal zone (serum-free medium supplemented with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF)), Ignatova et al. described cells with stem-like features isolated from cortical glial tumors (anaplastic astrocytoma, WHO grade III and GBM, and WHO grade IV) [7]. Phenotypically, cells selected under these conditions grew as neurospheres with a heterogeneous cellular morphology, were clonogenic, and expressed neural lineage markers such as nestin and glial fibrillary acidic protein (GFAP).

Using a similar approach, Galli et al. isolated stem-like cells from glioblastoma tissue which, in addition to their phenotypic analogy to neural precursor cells, established tumors upon orthotopic xenotransplantation in nude mice [4].

Pollard et al. described glioma stem cells propagated as adherent cultures on a laminin matrix using growth factor-supplemented neurosphere medium in the absence of serum, thereby preventing differentiation [53]. These cells exhibited stem-like features in vitro and also initiated tumors that recapitulated the cellular heterogeneity of primary GBM.

An alternative approach to isolate tumor stem-like cells is based on biological properties of these cells and enriches the “side population” of dissociated tumor tissue or established tumor cell lines, including glioblastoma [54, 55]. Here, GS-cells are identified by their high efflux capacity for chemical dyes like Hoechst 33342 due to the high expression of drug resistance-related ABC-transporters like ABCG2 [5658]. The side population of GBM cell lines has been shown to contain cells with stem-like properties [54, 59, 60]. However, this approach is presently challenged since a side population could not be detected in neurospheres derived from primary GBM tissue [61]. Furthermore, Golebiewska et al. could demonstrate that the side population derived directly from primary GBM tissue mostly contains brain endothelial cells and is nontumorigenic in vivo upon xenotransplantation [62].

Singh et al. isolated stem-like cells from GBM by enrichment of CD133-positive cells from primary tumor material. They could show that as little as 100 CD133-positive cells initiated a tumor upon orthotopic injection, while 100.000 CD133-negative cells did not, delivering key evidence that CD133+ cells are glioma stem-like cells. Based on these findings, CD133 has since been the most widely used marker for identifying GS-cells and is so far the most reliable molecule for isolation and/or identification of GS-cells. However, the idea of a restrictive model where CD133 expression defines GS-cells is currently under debate [63]. For example, expression of CD133 is subject to changes in the tumor microenvironment such as hypoxia, indicating that CD133 might be a marker for bioenergetic stress [14, 22, 64]. Additionally, different reports suggest that CD133-negative cells can also exhibit stem-like characteristics, most importantly the capacities for self-renewal and tumor initiation in vivo [53, 65]. A comprehensive overview of the complex regulation of CD133 in GBM is provided by Campos and Herold-Mende in [66].

At present, the most reliable method to propagate GS-cell lines from primary GBM is the selection for cells that grow as neurospheres in the absence of serum and in the presence of EGF and bFGF [67]. These cells then have to be extensively characterized for their capacities for serial self-renewal, differentiation, and in vivo tumorigenicity [68]. Although CD133 expression identifies a possible stem-like lineage within GBM, it is not the single universal marker identifying GS-cells [65]. In order to fully recapitulate the cellular heterogeneity of the stem-like compartment of GBM in vitro, different isolation approaches and cell culture protocols have to be combined and refined, for example, the modeling of the hypoxic stem-cell niche in vitro, which might increase the frequency of isolated GS-cells [20, 22].

3. EGFR-Amplified Glioblastoma Cells with a Stem-Like Phenotype In Vitro

One major shortcoming of the above-mentioned methods is that although GS-cells resemble the genetic and transcriptional phenotype of the original tumor closely [13, 14], EGFR amplification as the most frequent molecular alteration is usually not preserved in vitro [49]. EGFR amplification can only be maintained for a limited number of passages in vitro () either using conventional or GS cell culture conditions at normoxia (21% O2) or at hypoxia (1% O2) [14, 69]. Experimental systems to retain EGFR amplification present in the original tumor have thus largely relied on immediate orthotopic implantation of freshly resected tissue from GBM with EGFR amplification into nude mice [49, 70, 71] and by subsequent serial passaging in vivo of these xenograft tumors [42, 72, 73]. Apparently, the in vivo conditions provide a favorable microenvironment for EGFR-amplified cells, whereas standard in vitro conditions exert a negative selection pressure for those cells [38, 49]. However, after several passages in vivo (4-5), the serially transplanted tumors also lose their EGFR overexpression and histologically change from an invasive to a solid, vascularized morphology (“angiogenic switch”). These xenotransplantation approaches, although delivering valuable information, are laborious, time consuming (the time to development of symptoms ranges from 70 to 150 days), difficult to standardize, and limited to analyses in vivo. To study EGFR amplification in vitro, permanent cell lines with endogenous EGFR amplification and with stem-like features, such as self-renewal, clonogenicity, and the potential for in vivo tumorigenicity, would be the ideal model system.

In this regard, short-term culturing of GBM-derived primary cells as three-dimensional tumor spheres under stem cell conditions rather than as adherent monolayers has indicated that EGFR amplification can be maintained in vitro [7476]. When propagated in the absence of serum, tumor spheroids retained EGFR amplification and an associated polysomy of chromosome 7 as determined by FISH analysis. Additionally, heterogeneous EGFRvIII-expression, when present in the original tumor, was preserved in vitro as well [77]. Furthermore, tumor-derived spheroids from short-term cultures have been shown to initiate xenograft tumors that phenocopy the EGFR status of the original tumor in vivo, even when cultured in the presence of serum on agar-coated cell culture plates in order to avoid attachment [70, 78]. Culturing cells derived from EGFR-amplified GBMs as spheroids can conserve EGFR aberrations for a limited number of passages, thereby allowing for analyses of EGFR-related processes in a naturally EGFR-amplified background in vitro and in vivo, for example, response to EGFR-targeted therapy with tyrosine kinase inhibitors (TKIs) or monoclonal antibodies (mAbs) [70, 74, 76, 77].

One possible reason for the loss of EGFR amplification in vitro in addition to the growth pattern in vitro (adherent versus spheroid) is the propagation of tumor-derived cells in the presence of exogenous mitogens, especially EGF. EGF has been shown to inhibit the growth of EGFR-amplified MDA-468 breast cancer and A431 epidermoid carcinoma cells, which both strongly overexpress EGFR at the protein level [7981]. In addition, EGF can induce apoptosis by activating the EGFR in A431 cells, which can be abrogated by tyrosine kinase inhibition [82]. Abundance of EGFR signaling due to increased receptor expression and subsequent ligand-induced overstimulation of the EGFR pathway therefore seems to be a major negative selector for EGFR-amplified GBM cells in vitro.

Following this line of evidence, we recently demonstrated that the modulation of exogenous EGF concentrations and otherwise unaltered neurosphere conditions preserves genetic EGFR aberrations in an EGF-dependent manner. Omitting EGF from the cell culture medium when primary GBM cells are taken into culture preserved EGFR amplification and EGFRvIII expression with high success rates (approximately 40% of all tumors with EGFR amplification taken into culture) [69]. By applying different EGF concentrations (0 to 20 ng/mL), we were able to generate isogenic permanent cell lines from the same tumor with stable EGFR amplification and EGFRvIII expression (>15 passages) in the absence of EGF and nonamplified, EGFRvIII-negative cell lines in the presence of 20 ng/mL EGF. This method therefore allows for the conservation of EGFR-dependent intratumoral heterogeneity in vitro. The cell lines exhibited a stem-like phenotype; that is, they expressed CD133, showed the capacity for self-renewal, could be differentiated along astrocytic, oligodendrocytic, and neuronal lineages, and recapitulated the heterogeneous EGFR expression of the original tumor when implanted into immunocompromised mice. Importantly, tumorigenicity was enhanced for EGFR-amplified cells (median survival 102 versus 117 days, , log-rank test), emphasizing the relevance of EGFR expression for the progression of GBM in vivo.

Spontaneous conservation of EGFR amplification in permanent cell lines with a stem-like phenotype was however reported occasionally even in the presence of EGF. Mazzoleni et al. described two neurosphere cell lines with stem-like features and heterogeneous EGFR amplification maintained under standard neurosphere conditions, termed L0306 and L0627 with low and high EGFR amplification, respectively [83]. Importantly, the authors could show that reduction of exogenous EGF led to a reexpression of EGFR protein in cells formerly negative for EGFR. Furthermore, our own data recently indicated that the highly EGFR-amplified L0627 could be propagated permanently in the absence of EGF without any changes in proliferation, EGFR amplification, or stem-like features, while EGF withdrawal from L0306 led to significantly reduced proliferation [84, 85]. This finding indicates that high level EGFR amplification might be a prerequisite for the generation of permanent cell lines from EGFR-amplified GBM in the absence of EGF.

4. EGFR and a Stem-Like Phenotype in GBM

The apex cell of the cellular hierarchy of GBM still remains elusive [1, 86, 87]. However, experimental evidence from a genetically engineered mouse brain tumor model that allows for lineage tracking of neural stem/progenitor cells indicates that NSCs have to be considered the prime suspects for the cell of origin in GBM [88]. Furthermore, adult NSCs share key characteristics with GS-cells, such as the capacity for self-renewal and differentiation as well as spherical growth in vitro under the same cell culture conditions and a highly migratory phenotype, albeit with different dynamics [1, 86, 89]. Additionally, the reliance on stimulation with specific exogenous growth factors in order to maintain a stem-like phenotype is striking. For NSCs, EGF-induced activation of EGFR increases proliferation, survival, and migration, while inhibiting differentiation, whereas withdrawal of EGF from NSC cultures leads to differentiation and cell death (Figure 1(a)) [90]. For GS-cells, however, we and others could show that proliferation and maintenance of a stem-like phenotype were solely dependent on bFGF and not on EGF, even though GS-cells were still sensitive to stimulation with exogenous EGF and responded with enhanced proliferation and neurosphere size (Figure 1(b)) [69, 74, 9193]. Furthermore, withdrawal of EGF led to preservation or even a regain of molecular EGFR aberrations and/or EGFR protein overexpression, which is usually lost in the presence of EGF [69, 83, 84]. One possible explanation is that strong overexpression of EGFR renders cells autonomous of exogenous ligand stimulation through ligand-independent mechanisms or spontaneous receptor activation [9497]. Moreover, GS-cells with conserved EGFR amplification and protein overexpression secrete EGF in amounts that are sufficient to stimulate EGFR phosphorylation in an autocrine activation loop [69]. Even relatively small amounts of secreted EGF can activate EGFR signal transduction, since only a single EGF molecule is necessary to activate one EGFR dimer [98].

Figure 1: EGF/EGFR-dependent plasticity of neural stem/progenitor cells (NSC) and glioma stem-like cells (GS-cells). (a) In NSCs, EGF promotes self-renewal and proliferation, while withdrawal of EGF leads to terminal differentiation along astrocytic, neuronal, and oligodendrocytic lineages. (b) In GS-cells, EGF modulates the expression of EGFR at the protein level and EGFR amplification present in the original tumor. Withdrawing EGF from cell culture can in some cases lead to an upregulation of EGFR expression, while repeated stimulation with exogenous EGF reduces the amount of EGFR in the cells.

Within the stem-like compartment of EGFR-amplified GBM, EGFR seems to define a distinct cellular hierarchy [83, 99]. By dividing EGFR-amplified GS-cells into EGFRhigh and EGFRlow cells by fluorescence activated cell sorting, Mazzoleni et al. could determine an EGFR-dependent cellular hierarchy with distinct molecular and functional phenotypes. The authors described high EGFR expression to confer the highest malignancy to GS-cells. Similarly, we could demonstrate that GS-cells with retained EGFR amplification proliferated much faster in vivo than GS-cells from the same primary tumor without EGFR amplification. These results indicate a higher degree of “stemness” associated with EGFR amplification and EGFR overexpression [69, 83].

EGFR amplification and the EGFR gene rearrangement events leading to the loss of exons 2–7 resulting in EGFRvIII expression are considered to be early events in GBM development [99]. In analogy to the unaltered full-length EGFR, EGFRvIII is associated with a cellular hierarchy in EGFRvIII-positive GBM (Figure 2). Interestingly, EGFRvIII-positive cells can give rise to both EGFRvIII-positive and -negative cells. However, reexpression of EGFRvIII can only occur in a cell that has just recently lost EGFRvIII and has not persisted in an EGFRvIII-negative state for increased time duration [83, 99]. It has also been demonstrated that GBM contain a CD133+/EGFRvIIIhigh subpopulation of stem-like cells [100]. Additionally, EGFRvIII can keep glioma cells in an undifferentiated, stem-like state whereas differentiation of EGFRhigh/EGFRvIII+ GS-cells leads to downregulation of both receptors and a loss of stem-like potential [101, 102]. Vice versa, upregulation of EGFR in a telomerase reverse transcriptase- (TERT-) dependent manner allows differentiated glioma cells to acquire stem-like features [103]. Furthermore, EGFRvIII has been shown to enhance in vivo tumorigenicity of GBM cells in cooperation with EGFR, indicating an enhanced stem-like potential in the presence of EGFRvIII [50, 69, 102].

Figure 2: EGFRvIII-dependent hierarchy of glioma stem-like cells (GS-cells). An EGFRvIII-expressing GS-cell at the apex of the cellular hierarchy in GBM can divide into EGFRvIII-positive and negative cells, thereby maintaining the EGFRvIII-positive GS-cell reservoir. Within a limited time frame, EGFRvIII-negative cells can regain EGFRvIII expression, an ability which is lost further downstream in the EGFRvIII-dependent hierarchy [69, 83, 98].

5. Targeting Glioma Stem-Like Cells via EGFR/EGFRvIII

EGFR amplification and protein overexpression are considered potential therapeutic targets in neurooncology. In particular, the expression of EGFRvIII, which comprises a unique tumor-specific target in approximately 30% of all newly diagnosed GBM, offers many possibilities [38]. However, clinical trials targeting EGFR function have been so far disappointing since the heterogeneous distribution of EGFR throughout the tumor might render cells differentially sensitive towards EGFR inhibition, ultimately leading to therapy failure [51, 69, 70, 83]. Strikingly, EGFRvIII seems to be closely associated with an acquired resistance against targeted therapy with TKIs [104, 105]. Nathanson et al. described an EGFRvIII-positive subpopulation of tumor cells which they isolated from EGFRvIII-expressing GBM patients who developed resistance to TKI-therapy after an initial response [75]. This subpopulation persisted during TKI-treatment and expanded again after drug withdrawal. The authors described this subpopulation of cells to grow as neurospheres in vitro and to give rise to highly heterogeneous xenograft tumors, indicating that they possessed stem-like features.

The difficulties with TKI or mAbs targeting EGFR have sparked the development of alternative treatment strategies to exploit EGFR or EGFRvIII as a molecular target in GBM. Current approaches are utilizing EGFR/EGFRvIII as a unique tumor antigen to specifically identify GBM cells rather than targeting the EGFR’s biological function and have emphasized the significance of EGFR/EGFRvIII as a target for GBM therapy [106]. Arming the patients’ immune system against GBM with EGFR amplification and EGFRvIII expression appears to be especially promising. Currently, the most exciting systemic approach to exploit the exclusive expression of EGFRvIII by the tumor is a vaccination strategy with a peptide termed rindopepimut covering the neoepitope of EGFRvIII (i.e., a novel glycine at the exon 1-exon 8 junction) [107]. In a recent Phase II study, this approach could prolong the OS of patients with newly diagnosed EGFRvIII-positive GBM to 21.8 months with a 36-month OS of 26% [108]. Strikingly, nearly all patients had lost expression of EGFRvIII at recurrence [109]. In line with this data, it was found that treatment-naïve GBM patients already exhibit a strong endogenous immune response against EGFR as indicated by a high level of anti-EGFR serum autoantibodies, pointing towards a high immunogenic potential of EGFR [110].

In a different immunotherapeutic approach, T-cells are equipped with chimeric antigen receptors (CARs) recognizing EGFRvIII, which then effectively target EGFRvIII expressing GS-cells in vitro and exhibit significant cytotoxicity. CAR-expressing T-cells also infiltrate and kill established EGFRvIII-positive xenograft tumors in mice [111114]. In a similar approach, Muller et al. recently demonstrated that engineering NK-cells modified with an EGFRvIII-specific CAR to overexpress CXCR4 improves immunotherapy of CXCL12/SDF-1α-secreting glioblastoma in mice [115]. These strategies, although not specifically aiming at GS-cells, might also eradicate the stem-like compartment defined by EGFRvIII.

Emlet et al. developed a bispecific CD133/EGFRvIII antibody to specifically target the CD133+/EGFRvIIIhigh subpopulation of GBM [100]. In an in vitro cellular cytotoxicity assay, this antibody displayed superior toxicity for CD133+/EGFRvIIIhigh glioma cells than for CD133+ or EGFRvIIIhigh cells alone and also decreased stem-like properties such as self-renewal. Most importantly, the antibody significantly reduced tumorigenicity in vivo, most likely via antibody-dependent cellular cytotoxicity similar to cetuximab [70].

As mentioned, high EGFR/EGFRvIII expression designates an aggressive subtype of GS-cells [69, 100]. Therefore, downregulation of these molecules could represent a potential therapeutic strategy for EGFR-positive tumors [99]. Histone deacetylase inhibitors (HDACi) are an exciting class of anticancer agents. They inhibit the removal of acetyl residues from histones by histone deacetylases (HDAC), resulting in an open chromatin structure and increased transcription, including genetic loci that have been silenced during oncogenesis [116]. This leads to reexpression of proapoptotic and differentiation programs, which partially account for the anticancer effects of HDACi [117, 118]. In GS-cells, the HDACi valproic acid (VPA) induced differentiation and as a result decreased the expression of stem cell markers, rendering them more vulnerable to conventional therapy [119]. Importantly, nonneoplastic cells are relatively resistant to cell death induced by HDAC inhibition [120, 121]. Conversely, HDACi have been described to selectively induce transcriptional repression of high copy number genes such as amplified EGFR through blockade of RNA-polymerase II-dependent elongation [122]. As a consequence, the expression of EGFR and of EGFRvIII, which is controlled by epigenetic mechanisms in EGFR-amplified cells, can be reduced by HDACi such as Trichostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA) in conventional and GS-cells [75, 84, 99]. Furthermore, treatment of cancer cells with either acquired resistance or an inherent tolerance to EGFR TKIs with HDACi could resensitize these cells to the action of the inhibitor [123]. The combined effects of the TKI erlotinib and different HDACi (SAHA, TSA) were independent of cell culture conditions (neurosphere or containing serum), EGFR status (EGFR/EGFRvIII; EGFR+/EGFRvIII; EGFR+/EGFRvIII+) or acquired TKI resistance [69, 84]. This effect of HDACi might affect also the EGFR-dependent stem-like compartment of GBM and sensitize it to conventional, EGFR-targeted therapy.

6. Conclusions and Future Prospects

Endogenous amplification of the EGFR gene and overexpression of EGFR/EGFRvIII protein have been difficult to study in vitro in the past. Optimization of cell culture conditions for stem-like cells from GBM has enabled researchers to maintain EGFR-amplified GS-cells with high EGFR expression in combination with or without EGFRvIII expression at the protein level. These cell culture systems facilitated the analyses of the contribution of EGFR/EGFRvIII to a stem-like phenotype, the discovery of an EGFR/EGFRvIII-dependent cellular hierarchy within the stem-like compartment of GBM, and the development of targeted therapy approaches for EGFR/EGFRvIII-positive GS-cells.

The importance of representative model systems of EGFR-amplified GBM for research is highlighted by recent reports which described the occurrence of circulating tumor cells (CTCs) in the blood of more than 20% of GBM patients [124126]. The study by Müller et al. could demonstrate a significant association between an amplification of the EGFR gene in the primary tumor and the occurrence of CTCs in the blood. Importantly, these cells displayed preserved EGFR amplification. However, the occurrence of CTCs was not significantly associated with OS of the patient cohort. In other cancers than glioma, the ability of tumor cells to disseminate from the primary tumor mass, to remain dormant for many years, and to survive systemic chemotherapy unharmed has been attributed to cancer stem cell properties. Therefore, in EGFR-amplified GBM, cells of the EGFRhigh GS-cell pool might have the ability to extravasate into the blood stream and to potentially give rise to GBM metastases. Support for this notion comes from reports in the literature describing GBM metastases occurring with a relatively high frequency of 10–20% in transplant patients who received organs from GBM patients [127]. As GBM therapy continues to improve, especially for EGFR-amplified, EGFRvIII-positive tumors [108], the likeliness of extracranial metastases might increase from sporadic events to a veritable complication for these patients. Therefore, targeting the EGFRhigh GS-cell compartment could have prospective benefit for GBM patients with EGFR-amplified GBM.

Abbreviations

GBM:Glioblastoma
GS-cell:Glioma stem-like cell
NSC:Neural stem/progenitor cell
EGFR:Epidermal growth factor receptor
HDAC:Histone deacetylase
HDACi:Histone deacetylase inhibitor
TKI:Tyrosine kinase inhibitor
mAb:Monoclonal antibody.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgment

This work was supported by the Forschungs- und Wissenschaftsstiftung Hamburg (Katrin Liffers, Katrin Lamszus).

References

  1. M. Westphal and K. Lamszus, “The neurobiology of gliomas: from cell biology to the development of therapeutic approaches,” Nature Reviews Neuroscience, vol. 12, no. 9, pp. 495–508, 2011. View at Publisher · View at Google Scholar · View at Scopus
  2. H. S. Phillips, S. Kharbanda, R. Chen et al., “Molecular subclasses of high-grade glioma predict prognosis, delineate a pattern of disease progression, and resemble stages in neurogenesis,” Cancer Cell, vol. 9, no. 3, pp. 157–173, 2006. View at Publisher · View at Google Scholar · View at Scopus
  3. R. G. W. Verhaak, K. A. Hoadley, E. Purdom et al., “Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1,” Cancer Cell, vol. 17, no. 1, pp. 98–110, 2010. View at Publisher · View at Google Scholar · View at Scopus
  4. R. Galli, E. Binda, U. Orfanelli et al., “Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma,” Cancer Research, vol. 64, no. 19, pp. 7011–7021, 2004. View at Publisher · View at Google Scholar · View at Scopus
  5. H. S. Günther, N. O. Schmidt, H. S. Phillips et al., “Glioblastoma-derived stem cell-enriched cultures form distinct subgroups according to molecular and phenotypic criteria,” Oncogene, vol. 27, no. 20, pp. 2897–2909, 2008. View at Publisher · View at Google Scholar · View at Scopus
  6. H. D. Hemmati, I. Nakano, J. A. Lazareff et al., “Cancerous stem cells can arise from pediatric brain tumors,” Proceedings of the National Academy of Sciences of the United States of America, vol. 100, no. 25, pp. 15178–15183, 2003. View at Publisher · View at Google Scholar · View at Scopus
  7. T. N. Ignatova, V. G. Kukekov, E. D. Laywell, O. N. Suslov, F. D. Vrionis, and D. A. Steindler, “Human cortical glial tumors contain neural stem-like cells expressing astroglial and neuronal markers in vitro,” Glia, vol. 39, no. 3, pp. 193–206, 2002. View at Publisher · View at Google Scholar · View at Scopus
  8. S. K. Singh, C. Hawkins, I. D. Clarke et al., “Identification of human brain tumour initiating cells,” Nature, vol. 432, no. 7015, pp. 396–401, 2004. View at Publisher · View at Google Scholar · View at Scopus
  9. B. Campos, F. Wan, M. Farhadi et al., “Differentiation therapy exerts antitumor effects on stem-like glioma cells,” Clinical Cancer Research, vol. 16, no. 10, pp. 2715–2728, 2010. View at Publisher · View at Google Scholar · View at Scopus
  10. P. C. de Witt Hamer, A. A. G. van Tilborg, P. P. Eijk et al., “The genomic profile of human malignant glioma is altered early in primary cell culture and preserved in spheroids,” Oncogene, vol. 27, no. 14, pp. 2091–2096, 2008. View at Publisher · View at Google Scholar · View at Scopus
  11. A. Ernst, S. Hofmann, R. Ahmadi et al., “Genomic and expression profiling of glioblastoma stem cell-like spheroid cultures identifies novel tumor-relevant genes associated with survival,” Clinical Cancer Research, vol. 15, no. 21, pp. 6541–6550, 2009. View at Publisher · View at Google Scholar · View at Scopus
  12. T. M. Fael Al-Mayhani, S. L. R. Ball, J.-W. Zhao et al., “An efficient method for derivation and propagation of glioblastoma cell lines that conserves the molecular profile of their original tumours,” Journal of Neuroscience Methods, vol. 176, no. 2, pp. 192–199, 2009. View at Publisher · View at Google Scholar · View at Scopus
  13. J. Lee, S. Kotliarova, Y. Kotliarov et al., “Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines,” Cancer Cell, vol. 9, no. 5, pp. 391–403, 2006. View at Publisher · View at Google Scholar · View at Scopus
  14. A. Schulte, H. S. Günther, H. S. Phillips et al., “A distinct subset of glioma cell lines with stem cell-like properties reflects the transcriptional phenotype of glioblastomas and overexpresses CXCR4 as therapeutic target,” Glia, vol. 59, no. 4, pp. 590–602, 2011. View at Publisher · View at Google Scholar · View at Scopus
  15. M. Westphal and H. Meissner, “Establishing human glioma-derived cell lines,” Methods in Cell Biology, vol. 57, pp. 147–165, 1998. View at Google Scholar · View at Scopus
  16. A. Balbous, U. Cortes, K. Guilloteau et al., “A mesenchymal glioma stem cell profile is related to clinical outcome,” Oncogenesis, vol. 3, article e91, 2014. View at Publisher · View at Google Scholar
  17. P. Mao, K. Joshi, J. Li et al., “Mesenchymal glioma stem cells are maintained by activated glycolytic metabolism involving aldehyde dehydrogenase 1A3,” Proceedings of the National Academy of Sciences of the United States of America, vol. 110, no. 21, pp. 8644–8649, 2013. View at Publisher · View at Google Scholar · View at Scopus
  18. S. Bao, Q. Wu, R. E. McLendon et al., “Glioma stem cells promote radioresistance by preferential activation of the DNA damage response,” Nature, vol. 444, no. 7120, pp. 756–760, 2006. View at Publisher · View at Google Scholar · View at Scopus
  19. A. B. Hjelmeland, Q. Wu, J. M. Heddleston et al., “Acidic stress promotes a glioma stem cell phenotype,” Cell Death and Differentiation, vol. 18, no. 5, pp. 829–840, 2011. View at Publisher · View at Google Scholar · View at Scopus
  20. E. E. Bar, A. Lin, V. Mahairaki, W. Matsui, and C. G. Eberhart, “Hypoxia increases the expression of stem-cell markers and promotes clonogenicity in glioblastoma neurospheres,” The American Journal of Pathology, vol. 177, no. 3, pp. 1491–1502, 2010. View at Publisher · View at Google Scholar · View at Scopus
  21. J. M. Heddleston, Z. Li, R. E. McLendon, A. B. Hjelmeland, and J. N. Rich, “The hypoxic microenvironment maintains glioblastoma stem cells and promotes reprogramming towards a cancer stem cell phenotype,” Cell Cycle, vol. 8, no. 20, pp. 3274–3284, 2009. View at Publisher · View at Google Scholar · View at Scopus
  22. A. Kathagen, A. Schulte, G. Balcke et al., “Hypoxia and oxygenation induce a metabolic switch between pentose phosphate pathway and glycolysis in glioma stem-like cells,” Acta Neuropathologica, vol. 126, no. 5, pp. 763–780, 2013. View at Publisher · View at Google Scholar · View at Scopus
  23. A. V. Krivtsov, D. Twomey, Z. Feng et al., “Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9,” Nature, vol. 442, no. 7104, pp. 818–822, 2006. View at Publisher · View at Google Scholar · View at Scopus
  24. A. Murat, E. Migliavacca, T. Gorlia et al., “Stem cell-related ‘self-renewal’ signature and high epidermal growth factor receptor expression associated with resistance to concomitant chemoradiotherapy in glioblastoma,” Journal of Clinical Oncology, vol. 26, no. 18, pp. 3015–3024, 2008. View at Publisher · View at Google Scholar · View at Scopus
  25. B. H. Kong, J. H. Moon, Y. Huh et al., “Prognostic value of glioma cancer stem cell isolation in survival of primary glioblastoma patients,” Stem Cells International, vol. 2014, Article ID 838950, 6 pages, 2014. View at Publisher · View at Google Scholar
  26. D. R. Laks, M. Masterman-Smith, K. Visnyei et al., “Neurosphere formation is an independent predictor of clinical outcome in malignant glioma,” Stem Cells, vol. 27, no. 4, pp. 980–987, 2009. View at Publisher · View at Google Scholar · View at Scopus
  27. K. Mishima, Y. Kato, M. K. Kaneko, R. Nishikawa, T. Hirose, and M. Matsutani, “Increased expression of podoplanin in malignant astrocytic tumors as a novel molecular marker of malignant progression,” Acta Neuropathologica, vol. 111, no. 5, pp. 483–488, 2006. View at Publisher · View at Google Scholar · View at Scopus
  28. F. Zeppernick, R. Ahmadi, B. Campos et al., “Stem cell marker CD133 affects clinical outcome in glioma patients,” Clinical Cancer Research, vol. 14, no. 1, pp. 123–129, 2008. View at Publisher · View at Google Scholar · View at Scopus
  29. D.-Y. Cho, S.-Z. Lin, W.-K. Yang et al., “Targeting cancer stem cells for treatment of glioblastoma multiforme,” Cell Transplantation, vol. 22, no. 4, pp. 731–739, 2013. View at Publisher · View at Google Scholar · View at Scopus
  30. K. Lamszus and H. S. Günther, “Glioma stem cells as a target for treatment,” Targeted Oncology, vol. 5, no. 3, pp. 211–215, 2010. View at Publisher · View at Google Scholar · View at Scopus
  31. S. G. M. Piccirillo and A. L. Vescovi, “Brain tumour stem cells: possibilities of new therapeutic strategies,” Expert Opinion on Biological Therapy, vol. 7, no. 8, pp. 1129–1135, 2007. View at Publisher · View at Google Scholar · View at Scopus
  32. S. G. M. Piccirillo, B. A. Reynolds, N. Zanetti et al., “Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells,” Nature, vol. 444, no. 7120, pp. 761–765, 2006. View at Publisher · View at Google Scholar · View at Scopus
  33. P. Bach, T. Abel, C. Hoffmann et al., “Specific elimination of CD133+ tumor cells with targeted oncolytic measles virus,” Cancer Research, vol. 73, no. 2, pp. 865–874, 2013. View at Publisher · View at Google Scholar · View at Scopus
  34. C. Calabrese, H. Poppleton, M. Kocak et al., “A perivascular niche for brain tumor stem cells,” Cancer Cell, vol. 11, no. 1, pp. 69–82, 2007. View at Publisher · View at Google Scholar · View at Scopus
  35. C. Folkins, S. Man, P. Xu, Y. Shaked, D. J. Hicklin, and R. S. Kerbel, “Anticancer therapies combining antiangiogenic and tumor cell cytotoxic effects reduce the tumor stem-like cell fraction in glioma xenograft tumors,” Cancer Research, vol. 67, no. 8, pp. 3560–3564, 2007. View at Publisher · View at Google Scholar · View at Scopus
  36. T. A. Libermann, H. R. Nusbaum, N. Razon et al., “Amplification, enhanced expression and possible rearrangement of EGF receptor gene in primary human brain tumours of glial origin,” Nature, vol. 313, no. 5998, pp. 144–147, 1985. View at Publisher · View at Google Scholar · View at Scopus
  37. B. R. Voldborg, L. Damstrup, M. Spang-Thomsen, and H. Skovgaard Poulsen, “Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials,” Annals of Oncology, vol. 8, no. 12, pp. 1197–1206, 1997. View at Publisher · View at Google Scholar · View at Scopus
  38. P. A. Humphrey, A. J. Wong, B. Vogelstein et al., “Anti-synthetic peptide antibody reacting at the fusion junction of deletion-mutant epidermal growth factor receptors in human glioblastoma,” Proceedings of the National Academy of Sciences of the United States of America, vol. 87, no. 11, pp. 4207–4211, 1990. View at Publisher · View at Google Scholar · View at Scopus
  39. A. J. Wong, J. M. Ruppert, S. H. Bigner et al., “Structural alterations of the epidermal growth factor receptor gene in human gliomas,” Proceedings of the National Academy of Sciences of the United States of America, vol. 89, no. 7, pp. 2965–2969, 1992. View at Publisher · View at Google Scholar · View at Scopus
  40. A. Lal, C. A. Glazer, H. M. Martinson et al., “Mutant epidermal growth factor receptor up-regulates molecular effectors of tumor invasion,” Cancer Research, vol. 62, no. 12, pp. 3335–3339, 2002. View at Google Scholar · View at Scopus
  41. M. Lund-Johansen, R. Bjerkvig, P. A. Humphrey, S. H. Bigner, D. D. Bigner, and O.-D. Laerum, “Effect of epidermal growth factor on glioma cell growth, migration, and invasion in vitro,” Cancer Research, vol. 50, no. 18, pp. 6039–6044, 1990. View at Google Scholar · View at Scopus
  42. K. M. Talasila, A. Soentgerath, P. Euskirchen et al., “EGFR wild-type amplification and activation promote invasion and development of glioblastoma independent of angiogenesis,” Acta Neuropathologica, vol. 125, no. 5, pp. 683–698, 2013. View at Publisher · View at Google Scholar · View at Scopus
  43. R. Bonavia, M. M. Inda, S. Vandenberg et al., “EGFRvIII promotes glioma angiogenesis and growth through the NF-B, interleukin-8 pathway,” Oncogene, vol. 31, no. 36, pp. 4054–4066, 2012. View at Publisher · View at Google Scholar · View at Scopus
  44. Y. Katanasaka, Y. Kodera, Y. Kitamura, T. Morimoto, T. Tamura, and F. Koizumi, “Epidermal growth factor receptor variant type III markedly accelerates angiogenesis and tumor growth via inducing c-myc mediated angiopoietin-like 4 expression in malignant glioma,” Molecular Cancer, vol. 12, no. 1, article 31, 2013. View at Publisher · View at Google Scholar · View at Scopus
  45. B. Hasselbalch, U. Lassen, H. S. Poulsen, and M.-T. Stockhausen, “Cetuximab insufficiently inhibits glioma cell growth due to persistent EGFR downstream signaling,” Cancer Investigation, vol. 28, no. 8, pp. 775–787, 2010. View at Publisher · View at Google Scholar · View at Scopus
  46. J. N. Rich, D. A. Reardon, T. Peery et al., “Phase II trial of gefitinib in recurrent glioblastoma,” Journal of Clinical Oncology, vol. 22, no. 1, pp. 133–142, 2004. View at Publisher · View at Google Scholar · View at Scopus
  47. R. Stupp, W. P. Mason, M. J. van den Bent et al., “Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma,” The New England Journal of Medicine, vol. 352, no. 10, pp. 987–996, 2005. View at Publisher · View at Google Scholar · View at Scopus
  48. M. J. van den Bent, A. A. Brandes, R. Rampling et al., “Randomized phase II trial of erlotinib versus temozolomide or carmustine in recurrent glioblastoma: EORTC brain tumor group study 26034,” Journal of Clinical Oncology, vol. 27, no. 8, pp. 1268–1274, 2009. View at Publisher · View at Google Scholar · View at Scopus
  49. A. Pandita, K. D. Aldape, G. Zadeh, A. Guha, and C. D. James, “Contrasting in vivo and in vitro fates of glioblastoma cell subpopulations with amplified EGFR,” Genes Chromosomes and Cancer, vol. 39, no. 1, pp. 29–36, 2004. View at Publisher · View at Google Scholar · View at Scopus
  50. M.-D. Inda, R. Bonavia, A. Mukasa et al., “Tumor heterogeneity is an active process maintained by a mutant EGFR-induced cytokine circuit in glioblastoma,” Genes & Development, vol. 24, no. 16, pp. 1731–1745, 2010. View at Publisher · View at Google Scholar · View at Scopus
  51. I. K. Mellinghoff, M. Y. Wang, I. Vivanco et al., “Molecular determinants of the response of glioblastomas to EGFR kinase inhibitors,” The New England Journal of Medicine, vol. 353, no. 19, pp. 2012–2024, 2005. View at Publisher · View at Google Scholar · View at Scopus
  52. W. Yang, R. F. Barth, G. Wu et al., “Development of a syngeneic rat brain tumor model expressing EGFRvIII and its use for molecular targeting studies with monoclonal antibody L8A4,” Clinical Cancer Research, vol. 11, no. 1, pp. 341–350, 2005. View at Google Scholar · View at Scopus
  53. S. M. Pollard, K. Yoshikawa, I. D. Clarke et al., “Glioma stem cell lines expanded in adherent culture have tumor-specific phenotypes and are suitable for chemical and genetic screens,” Cell Stem Cell, vol. 4, no. 6, pp. 568–580, 2009. View at Publisher · View at Google Scholar · View at Scopus
  54. M. A. Harris, H. Yang, B. E. Low et al., “Cancer stem cells are enriched in the side population cells in a mouse model of glioma,” Cancer Research, vol. 68, no. 24, pp. 10051–10059, 2008. View at Publisher · View at Google Scholar · View at Scopus
  55. K. Weber, W. Paulus, and V. Senner, “The side population of gliomas exhibits decreased cell migration,” Journal of Neuropathology and Experimental Neurology, vol. 69, no. 6, pp. 623–631, 2010. View at Publisher · View at Google Scholar · View at Scopus
  56. A.-M. Bleau, D. Hambardzumyan, T. Ozawa et al., “PTEN/PI3K/Akt pathway regulates the side population phenotype and ABCG2 activity in glioma tumor stem-like cells,” Cell Stem Cell, vol. 4, no. 3, pp. 226–235, 2009. View at Publisher · View at Google Scholar · View at Scopus
  57. A.-M. Bleau, J. T. Huse, and E. C. Holland, “The ABCG2 resistance network of glioblastoma,” Cell Cycle, vol. 8, no. 18, pp. 2936–2944, 2009. View at Google Scholar · View at Scopus
  58. A. R. Rama, P. J. Alvarez, R. Madeddu, and A. Aranega, “ABC transporters as differentiation markers in glioblastoma cells,” Molecular Biology Reports, vol. 41, no. 8, pp. 4847–4851, 2014. View at Publisher · View at Google Scholar · View at Scopus
  59. R. Fukaya, S. Ohta, M. Yamaguchi et al., “Isolation of cancer stem-like cells from a side population of a human glioblastoma cell line, SK-MG-1,” Cancer Letters, vol. 291, no. 2, pp. 150–157, 2010. View at Publisher · View at Google Scholar · View at Scopus
  60. C. Hirschmann-Jax, A. E. Foster, G. G. Wulf et al., “A distinct ‘side population’ of cells with high drug efflux capacity in human tumor cells,” Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 39, pp. 14228–14233, 2004. View at Publisher · View at Google Scholar · View at Scopus
  61. K. W. R. Broadley, M. K. Hunn, K. J. Farrand et al., “Side population is not necessary or sufficient for a cancer stem cell phenotype in glioblastoma multiforme,” Stem Cells, vol. 29, no. 3, pp. 452–461, 2011. View at Publisher · View at Google Scholar · View at Scopus
  62. A. Golebiewska, S. Bougnaud, D. Stieber et al., “Side population in human glioblastoma is non-tumorigenic and characterizes brain endothelial cells,” Brain, vol. 136, no. 5, pp. 1462–1475, 2013. View at Publisher · View at Google Scholar · View at Scopus
  63. E. Binda, B. A. Reynolds, and A. L. Vescovi, “Glioma stem cells: turpis omen in nomen? (The evil in the name?),” Journal of Internal Medicine, vol. 276, no. 1, pp. 25–40, 2014. View at Publisher · View at Google Scholar
  64. C. E. Griguer, C. R. Oliva, E. Gobin et al., “CD133 is a marker of bioenergetic stress in human glioma,” PLoS ONE, vol. 3, no. 11, Article ID e3655, 2008. View at Publisher · View at Google Scholar · View at Scopus
  65. R. Chen, M. C. Nishimura, S. M. Bumbaca et al., “A hierarchy of self-renewing tumor-initiating cell types in glioblastoma,” Cancer Cell, vol. 17, no. 4, pp. 362–375, 2010. View at Publisher · View at Google Scholar · View at Scopus
  66. B. Campos and C. C. Herold-Mende, “Insight into the complex regulation of CD133 in glioma,” International Journal of Cancer, vol. 128, no. 3, pp. 501–510, 2011. View at Publisher · View at Google Scholar · View at Scopus
  67. E. S. Molina, M. M. Pillat, V. Moura-Neto, T. T. Lah, and H. Ulrich, “Glioblastoma stem-like cells: approaches for isolation and characterization,” Journal of Cancer Stem Cell Research, vol. 2, Article ID e1007, 2014. View at Publisher · View at Google Scholar
  68. A. L. Vescovi, R. Galli, and B. A. Reynolds, “Brain tumour stem cells,” Nature Reviews Cancer, vol. 6, no. 6, pp. 425–436, 2006. View at Publisher · View at Google Scholar · View at Scopus
  69. A. Schulte, H. S. Günther, T. Martens et al., “Glioblastoma stem-like cell lines with either maintenance or loss of high-level EGFR amplification, generated via modulation of ligand concentration,” Clinical Cancer Research, vol. 18, no. 7, pp. 1901–1913, 2012. View at Publisher · View at Google Scholar · View at Scopus
  70. T. Martens, Y. Laabs, H. S. Günther et al., “Inhibition of glioblastoma growth in a highly invasive nude mouse model can be achieved by targeting epidermal growth factor receptor but not vascular endothelial growth factor receptor-2,” Clinical Cancer Research, vol. 14, no. 17, pp. 5447–5458, 2008. View at Publisher · View at Google Scholar · View at Scopus
  71. J. N. Sarkaria, B. L. Carlson, M. A. Schroeder et al., “Use of an orthotopic xenograft model for assessing the effect of epidermal growth factor receptor amplification on glioblastoma radiation response,” Clinical Cancer Research, vol. 12, no. 7, pp. 2264–2271, 2006. View at Publisher · View at Google Scholar · View at Scopus
  72. P. Ø. Sakariassen, L. Prestegarden, J. Wang et al., “Angiogenesis-independent tumor growth mediated by stem-like cancer cells,” Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 44, pp. 16466–16471, 2006. View at Publisher · View at Google Scholar · View at Scopus
  73. J. Wang, H. Miletic, P. T. Sakariassen et al., “A reproducible brain tumour model established from human glioblastoma biopsies,” BMC Cancer, vol. 9, article 465, 2009. View at Publisher · View at Google Scholar · View at Scopus
  74. S. Eimer, F. Dugay, K. Airiau et al., “Cyclopamine cooperates with EGFR inhibition to deplete stem-like cancer cells in glioblastoma-derived spheroid cultures,” Neuro-Oncology, vol. 14, no. 12, pp. 1441–1451, 2012. View at Publisher · View at Google Scholar · View at Scopus
  75. D. A. Nathanson, B. Gini, J. Mottahedeh et al., “Targeted therapy resistance mediated by dynamic regulation of extrachromosomal mutant EGFR DNA,” Science, vol. 343, no. 6166, pp. 72–76, 2014. View at Publisher · View at Google Scholar · View at Scopus
  76. M. Witusik-Perkowska, P. Rieske, K. Hułas-Bigoszewska et al., “Glioblastoma-derived spheroid cultures as an experimental model for analysis of EGFR anomalies,” Journal of Neuro-Oncology, vol. 102, no. 3, pp. 395–407, 2011. View at Publisher · View at Google Scholar · View at Scopus
  77. M. T. Stockhausen, H. Broholm, M. Villingshøj et al., “Maintenance of EGFR and EGFRvIII expressions in an in vivo and in vitro model of human glioblastoma multiforme,” Experimental Cell Research, vol. 317, no. 11, pp. 1513–1526, 2011. View at Publisher · View at Google Scholar · View at Scopus
  78. R. Bjerkvig, A. Tonnesen, O. D. Laerum, and E.-O. Backlund, “Multicenter tumor spheroids from human gliomas maintained in organ culture,” Journal of Neurosurgery, vol. 72, no. 3, pp. 463–475, 1990. View at Publisher · View at Google Scholar · View at Scopus
  79. J. Filmus, M. N. Pollak, J. G. Cairncross, and R. N. Buick, “Amplified, overexpressed and rearranged epidermal growth factor receptor gene in a human astrocytoma cell line,” Biochemical and Biophysical Research Communications, vol. 131, no. 1, pp. 207–215, 1985. View at Publisher · View at Google Scholar · View at Scopus
  80. G. N. Gill and C. S. Lazar, “Increased phosphotyrosine content and inhibition of proliferation in EGF-treated A431 cells,” Nature, vol. 293, no. 5830, pp. 305–307, 1981. View at Publisher · View at Google Scholar · View at Scopus
  81. T. Kawamoto, J. Mendelsohn, A. Le, G. H. Sato, C. S. Lazar, and G. N. Gill, “Relation of epidermal growth factor receptor concentration to growth of human epidermoid carcinoma A431 cells,” The Journal of Biological Chemistry, vol. 259, no. 12, pp. 7761–7766, 1984. View at Google Scholar · View at Scopus
  82. L. F. Gulti, K. C. Palmer, Y. Q. Chen, and K. B. Reddy, “Epidermal growth factor-induced apoptosis in A431 cells can be reversed by reducing the tyrosine kinase activity,” Cell Growth and Differentiation, vol. 7, no. 2, pp. 173–178, 1996. View at Google Scholar · View at Scopus
  83. S. Mazzoleni, L. S. Politi, M. Pala et al., “Epidermal growth factor receptor expression identifies functionally and molecularly distinct tumor-initiating cells in human glioblastoma multiforme and is required for gliomagenesis,” Cancer Research, vol. 70, no. 19, pp. 7500–7513, 2010. View at Publisher · View at Google Scholar · View at Scopus
  84. K. Liffers, K. Kolbe, M. Westphal et al., “Histone deacetylase inhibitors resensitize EGFR/EGFRvIII-overexpressing, erlotinib-resistant glioblastoma cells to tyrosine kinase inhibition,” Targeted Oncology. In press.
  85. K. Liffers, S. Riethdorf, R. Galli, M. Westphal, K. Lamszus, and A. Schulte, “Histone deacetylase inhibitors sensitize glioblastoma cells to EGFR-directed therapy with tyrosine kinase inhibitors,” in Proceedings of the 65th Annual Meeting of the German Society of Neurosurgery (DGNC '14), Dresden, Germany, May 2014. View at Publisher · View at Google Scholar
  86. F. A. Siebzehnrubl, B. A. Reynolds, A. Vescovi, D. A. Steindler, and L. P. Deleyrolle, “The origins of glioma: E Pluribus Unum?” Glia, vol. 59, no. 8, pp. 1135–1147, 2011. View at Publisher · View at Google Scholar · View at Scopus
  87. M. Venere, H. A. Fine, P. B. Dirks, and J. N. Rich, “Cancer stem cells in gliomas: identifying and understanding the apex cell in cancer's hierarchy,” GLIA, vol. 59, no. 8, pp. 1148–1154, 2011. View at Publisher · View at Google Scholar · View at Scopus
  88. J. Chen, Y. Li, T.-S. Yu et al., “A restricted cell population propagates glioblastoma growth after chemotherapy,” Nature, vol. 488, no. 7412, pp. 522–526, 2012. View at Publisher · View at Google Scholar · View at Scopus
  89. M. Varghese, H. Olstorn, C. Sandberg et al., “A comparison between stem cells from the adult human brain and from brain tumors,” Neurosurgery, vol. 63, no. 6, pp. 1022–1033, 2008. View at Publisher · View at Google Scholar · View at Scopus
  90. A. Ayuso-Sacido, J. A. Moliterno, S. Kratovac et al., “Activated EGFR signaling increases proliferation, survival,and migration and blocks neuronal differentiation in post-natal neural stem cells,” Journal of Neuro-Oncology, vol. 97, no. 3, pp. 323–337, 2010. View at Publisher · View at Google Scholar · View at Scopus
  91. P. A. Clark, M. Iida, D. M. Treisman et al., “Activation of multiple ERBB family receptors mediates glioblastoma cancer stem-like cell resistance to EGFR-targeted inhibition,” Neoplasia, vol. 14, no. 5, pp. 420–428, 2012. View at Publisher · View at Google Scholar · View at Scopus
  92. J. J. P. Kelly, O. Stechishin, A. Chojnacki et al., “Proliferation of human glioblastoma stem cells occurs independently of exogenous mitogens,” Stem Cells, vol. 27, no. 8, pp. 1722–1733, 2009. View at Publisher · View at Google Scholar · View at Scopus
  93. N. Podergajs, N. Brekka, B. Radlwimmer et al., “Expansive growth of two glioblastoma stem-like cell lines is mediated by bFGF and not by EGF,” Radiology and Oncology, vol. 47, no. 4, pp. 330–337, 2013. View at Publisher · View at Google Scholar · View at Scopus
  94. O. M. Fischer, S. Hart, A. Gschwind, and A. Ullrich, “EGFR signal transactivation in cancer cells,” Biochemical Society Transactions, vol. 31, part 6, pp. 1203–1208, 2003. View at Publisher · View at Google Scholar · View at Scopus
  95. D. Liu, J. A. Aguirre Ghiso, Y. Estrada, and L. Ossowski, “EGFR is a transducer of the urokinase receptor initiated signal that is required for in vivo growth of a human carcinoma,” Cancer Cell, vol. 1, no. 5, pp. 445–457, 2002. View at Publisher · View at Google Scholar · View at Scopus
  96. C. Y. Thomas, M. Chouinard, M. Cox et al., “Spontaneous activation and signaling by overexpressed epidermal growth factor receptors in glioblastoma cells,” International Journal of Cancer, vol. 104, no. 1, pp. 19–27, 2003. View at Publisher · View at Google Scholar · View at Scopus
  97. Y. Wang, O. Roche, C. Xu et al., “Hypoxia promotes ligand-independent EGF receptor signaling via hypoxia-inducible factor-mediated upregulation of caveolin-1,” Proceedings of the National Academy of Sciences of the United States of America, vol. 109, no. 13, pp. 4892–4897, 2012. View at Publisher · View at Google Scholar · View at Scopus
  98. P. Liu, T. E. Cleveland IV, S. Bouyain, P. O. Byrne, P. A. Longo, and D. J. Leahy, “A single ligand is sufficient to activate EGFR dimers,” Proceedings of the National Academy of Sciences of the United States of America, vol. 109, no. 27, pp. 10861–10866, 2012. View at Publisher · View at Google Scholar
  99. C. A. Del Vecchio, C. P. Giacomini, H. Vogel et al., “EGFRvIII gene rearrangement is an early event in glioblastoma tumorigenesis and expression defines a hierarchy modulated by epigenetic mechanisms,” Oncogene, vol. 32, no. 21, pp. 2670–2681, 2013. View at Publisher · View at Google Scholar · View at Scopus
  100. D. R. Emlet, P. Gupta, M. Holgado-Madruga et al., “Targeting a glioblastoma cancer stem-cell population defined by EGF receptor variant III,” Cancer Research, vol. 74, no. 4, pp. 1238–1249, 2014. View at Publisher · View at Google Scholar · View at Scopus
  101. E. J. Kim, S. O. Kim, X. Jin et al., “Epidermal growth factor receptor variant III renders glioma cancer cells less differentiated by JAGGED1,” Tumor Biology, vol. 36, no. 4, pp. 2921–2928, 2015. View at Publisher · View at Google Scholar
  102. M.-T. Stockhausen, K. Kristoffersen, L. Stobbe, and H. S. Poulsen, “Differentiation of glioblastoma multiforme stem-like cells leads to downregulation of EGFR and EGFRvIII and decreased tumorigenic and stem-like cell potential,” Cancer Biology and Therapy, vol. 15, no. 2, pp. 216–224, 2014. View at Publisher · View at Google Scholar · View at Scopus
  103. S. Beck, X. Jin, Y.-W. Sohn et al., “Telomerase activity-independent function of TERT allows glioma cells to attain cancer stem cell characteristics by inducing EGFR expression,” Molecules and Cells, vol. 31, no. 1, pp. 9–15, 2011. View at Publisher · View at Google Scholar · View at Scopus
  104. C. A. Learn, T. L. Hartzell, C. J. Wikstrand et al., “Resistance to tyrosine kinase inhibition by mutant epidermal growth factor receptor variant III contributes to the neoplastic phenotype of glioblastoma multiforme,” Clinical Cancer Research, vol. 10, no. 9, pp. 3216–3224, 2004. View at Publisher · View at Google Scholar · View at Scopus
  105. A. Schulte, K. Liffers, A. Kathagen et al., “Erlotinib resistance in EGFR-amplified glioblastoma cells is associated with upregulation of EGFRvIII and PI3Kp110delta,” Neuro-Oncology, vol. 15, no. 10, pp. 1289–1301, 2013. View at Publisher · View at Google Scholar · View at Scopus
  106. M. E. Hegi, P. Rajakannu, and M. Weller, “Epidermal growth factor receptor: a re-emerging target in glioblastoma,” Current Opinion in Neurology, vol. 25, no. 6, pp. 774–779, 2012. View at Publisher · View at Google Scholar · View at Scopus
  107. A. B. Heimberger, L. E. Crotty, G. E. Archer et al., “Epidermal growth factor receptor VIII peptide vaccination is efficacious against established intracerebral tumors,” Clinical Cancer Research, vol. 9, no. 11, pp. 4247–4254, 2003. View at Google Scholar · View at Scopus
  108. J. Schuster, R. K. Lai, L. D. Recht et al., “A phase II, multicenter trial of rindopepimut (CDX-110) in newly diagnosed glioblastoma: the ACT III study,” Neuro-Oncology, 2015. View at Publisher · View at Google Scholar
  109. J. H. Sampson, A. B. Heimberger, G. E. Archer et al., “Immunologic escape after prolonged progression-free survival with epidermal growth factor receptor variant III peptide vaccination in patients with newly diagnosed glioblastoma,” Journal of Clinical Oncology, vol. 28, no. 31, pp. 4722–4729, 2010. View at Publisher · View at Google Scholar · View at Scopus
  110. A. Mock, R. Warta, C. Geisenberger et al., “Printed peptide arrays identify prognostic TNC serumantibodies in glioblastoma patients,” Oncotarget. In press, http://www.impactjournals.com/oncotarget/index.php?journal=oncotarget&page=article&op=view&path%5B%5D=3791.
  111. B. D. Choi, C. M. Suryadevara, P. C. Gedeon et al., “Intracerebral delivery of a third generation EGFRvIII-specific chimeric antigen receptor is efficacious against human glioma,” Journal of Clinical Neuroscience, vol. 21, no. 1, pp. 189–190, 2014. View at Publisher · View at Google Scholar · View at Scopus
  112. H. Miao, B. D. Choi, C. M. Suryadevara et al., “EGFRvIII-specific chimeric antigen receptor T cells migrate to and kill tumor deposits infiltrating the brain parenchyma in an invasive xenograft model of glioblastoma,” PLoS ONE, vol. 9, no. 4, Article ID e94281, 2014. View at Publisher · View at Google Scholar · View at Scopus
  113. M. Ohno, A. Natsume, K. Ichiro Iwami et al., “Retrovirally engineered T-cell-based immunotherapy targeting type III variant epidermal growth factor receptor, a glioma-associated antigen,” Cancer Science, vol. 101, no. 12, pp. 2518–2524, 2010. View at Publisher · View at Google Scholar · View at Scopus
  114. J. H. Sampson, B. D. Choi, L. Sanchez-Perez et al., “EGFRvIII mCAR-modified T-cell therapy cures mice with established intracerebral glioma and generates host immunity against tumor-antigen loss,” Clinical Cancer Research, vol. 20, no. 4, pp. 972–984, 2014. View at Publisher · View at Google Scholar · View at Scopus
  115. N. Müller, S. Michen, S. Tietze et al., “Engineering NK cells modified with an EGFRvIII-specific chimeric antigen receptor to overexpress CXCR4 improves immunotherapy of CXCL12/SDF-1α-secreting glioblastoma,” Journal of Immunotherapy, vol. 38, no. 5, pp. 197–210, 2015. View at Publisher · View at Google Scholar
  116. J. E. Bolden, M. J. Peart, and R. W. Johnstone, “Anticancer activities of histone deacetylase inhibitors,” Nature Reviews Drug Discovery, vol. 5, no. 9, pp. 769–784, 2006. View at Publisher · View at Google Scholar · View at Scopus
  117. G. Reid, R. Métivier, C.-Y. Lin et al., “Multiple mechanisms induce transcriptional silencing of a subset of genes, including oestrogen receptor α, in response to deacetylase inhibition by valproic acid and trichostatin A,” Oncogene, vol. 24, no. 31, pp. 4894–4907, 2005. View at Publisher · View at Google Scholar · View at Scopus
  118. W. S. Xu, R. B. Parmigiani, and P. A. Marks, “Histone deacetylase inhibitors: molecular mechanisms of action,” Oncogene, vol. 26, no. 37, pp. 5541–5552, 2007. View at Publisher · View at Google Scholar · View at Scopus
  119. A. A. Alvarez, M. Field, S. Bushnev, M. S. Longo, and K. Sugaya, “The effects of histone deacetylase inhibitors on glioblastoma-derived stem cells,” Journal of Molecular Neuroscience, vol. 55, no. 1, pp. 7–20, 2014. View at Publisher · View at Google Scholar · View at Scopus
  120. A. Burgess, A. Ruefli, H. Beamish et al., “Histone deacetylase inhibitors specifically kill nonproliferating tumour cells,” Oncogene, vol. 23, no. 40, pp. 6693–6701, 2004. View at Publisher · View at Google Scholar · View at Scopus
  121. J. S. Ungerstedt, Y. Sowa, W.-S. Xu et al., “Role of thioredoxin in the response of normal and transformed cells to histone deacetylase inhibitors,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 3, pp. 673–678, 2005. View at Publisher · View at Google Scholar · View at Scopus
  122. Y. J. Kim, C. B. Greer, K. R. Cecchini, L. N. Harris, D. P. Tuck, and T. H. Kim, “HDAC inhibitors induce transcriptional repression of high copy number genes in breast cancer through elongation blockade,” Oncogene, vol. 32, no. 23, pp. 2828–2835, 2013. View at Publisher · View at Google Scholar · View at Scopus
  123. S. V. Sharma, D. Y. Lee, B. Li et al., “A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations,” Cell, vol. 141, no. 1, pp. 69–80, 2010. View at Publisher · View at Google Scholar · View at Scopus
  124. K. M. MacArthur, G. D. Kao, S. Chandrasekaran et al., “Detection of brain tumor cells in the peripheral blood by a telomerase promoter-based assay,” Cancer Research, vol. 74, no. 8, pp. 2152–2159, 2014. View at Publisher · View at Google Scholar · View at Scopus
  125. C. Muller, J. Holtschmidt, M. Auer et al., “Hematogenous dissemination of glioblastoma multiforme,” Science Translational Medicine, vol. 6, no. 247, Article ID 247ra101, 2014. View at Publisher · View at Google Scholar
  126. J. P. Sullivan, B. V. Nahed, M. W. Madden et al., “Brain tumor cells in circulation are enriched for mesenchymal gene expression,” Cancer Discovery, vol. 4, no. 11, pp. 1299–1309, 2014. View at Publisher · View at Google Scholar
  127. G. Kalokhe, S. A. Grimm, J. P. Chandler, I. Helenowski, A. Rademaker, and J. J. Raizer, “Metastatic glioblastoma: case presentations and a review of the literature,” Journal of Neuro-Oncology, vol. 107, no. 1, pp. 21–27, 2012. View at Publisher · View at Google Scholar · View at Scopus