Glioblastoma quo vadis: Will migration and invasiveness reemerge as therapeutic targets?

Open AccessPublished:June 26, 2018DOI:https://doi.org/10.1016/j.ctrv.2018.06.017

      Abstract

      Purpose

      The purpose of the current review is to highlight, on one hand, the fact that the migratory pattern of glioma cells is the major obstacle to combat them with chemotherapy, and on the other one, the new treatment strategies to overcome this obstacle.

      Methods

      This review surveys several membrane and extracellular molecules involved in glioma cell migration, invasiveness and resistance to apoptosis.

      Results

      This review focuses on signaling pathways implicated in the positive regulation of glioblastoma cell migration, including glutamate and ion channel networks, microtubes and membrane-derived extracellular vesicles (EV) containing microRNAs. Glioma cells release glutamate to the extracellular matrix, inducing neuronal cell death, which may facilitate glioma growth and invasion. Glioma cell migration and invasion are further facilitated through ion channels and transporters that modify cellular volume. Microtubes and EV promote connections and communication among glioma cells and with the microenvironment and are associated with progression and resistance to therapy. Potential therapies linked to these pathways for glioblastoma are being developed.

      Conclusion

      Our view is evolving from an intracellular view of the complex intracellular signaling pathways to one of orchestral machinery, including connections between heterogeneous tumoral and nontumoral cells and with the microenvironment through channels, microtubes, and extracellular miRNA, generating different signals at different times. All of these elements give rise to a new perspective for the treatment of glioblastoma.

      Introduction

      Gliomas, including the most common variant, glioblastoma, are the most common primary malignant brain tumors, with an annual incidence of 5.26 cases per 100,000 population or 17,000 new diagnoses per year [
      • Ostrom Q.T.
      • Gittleman H.
      • Liao P.
      • et al.
      CBTRUS Statistical Report: Primary brain and other central nervous system tumors diagnosed in the United States in 2010–2014.
      ]. Glioblastoma remains one of the most lethal forms of cancer. The high spatial and temporal glioblastoma heterogeneity due to functionally diverse cell types, chaotic leaky vasculature and tumor cell infiltration contributes to poor prognosis in patients [
      • Reardon D.A.
      • Wen P.Y.
      Glioma in 2014: unravelling tumour heterogeneity-implications for therapy.
      ]. The current understanding of the biology and genetics of glioblastoma has greatly improved in the past two decades. Two major multi-institutional programs, the Glioma Molecular Diagnostic Initiative and The Cancer Genome Atlas (TCGA), have pursued comprehensive genomic characterization of a large number of clinical glioma samples using a variety of technologies to measure gene expression, chromosomal copy number alterations, somatic and germline mutations, DNA methylation, microRNA, and proteomic changes. These data will facilitate the discovery of therapeutic and diagnostic targets and pathway candidates, validation of research and clinical observations and generation of unanticipated hypotheses that can advance the molecular understanding of this lethal cancer [
      • Riddick G.
      • Fine H.A.
      Integration and analysis of genome-scale data from gliomas.
      ]. Sturm et al. identified six epigenetic glioblastoma subgroups displaying characteristic global DNA methylation patterns, harboring distinct hotspot mutations, DNA copy-number alterations, and transcriptomic patterns [
      • Sturm D.
      • Witt H.
      • Hovestadt V.
      • et al.
      Hotspot mutations in H3F3A and IDH1 define distinct epigenetic and biological subgroups of glioblastoma.
      ]. Brenann et al. provided a comprehensive and in-depth overview of the genetic, epigenetic and transcriptional profiles associated with glioblastoma [
      • Brennan C.W.
      • Verhaak R.G.
      • McKenna A.
      • et al.
      TCGA Research Network. The somatic genomic landscape of glioblastoma.
      ]. The new WHO 2016 classification, therefore, is not only based on the histological features of the tumor but also includes isocitrate dehydrogenase (IDH) mutation and chromosomal arms 1p and 19q codeletion status to classify diffuse gliomas of adulthood [
      • Louis D.N.
      • Perry A.
      • Reifenberger G.
      • et al.
      The 2016 world health organization classification of tumors of the central nervous system: a summary.
      ]. However, each individual glioblastoma should be considered as an ensemble of molecularly distinct subclones that reflect a spectrum of dynamic cell states. It is becoming clear that glioblastoma is a composite of multiple cell populations in the same tumor, and this heterogeneity makes the design of effective treatment modalities challenging [
      • Patel A.P.
      • Tirosh I.
      • Trombetta J.J.
      • et al.
      Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma.
      ,
      • Sottoriva A.
      • Spiteri I.
      • Piccirillo S.G.
      • et al.
      Intratumor heterogeneity in human glioblastoma reflects cancer evolutionary dynamics.
      ].

       Diffuse glioma genesis

      In the context of gliomagenesis, glial cells were once considered the only dividing cells in the postnatal brain, making these cells the only brain cells susceptible to transformation [
      • Sanai N.
      • Alvarez-Buylla A.
      • Berger M.S.
      Neural stem cells and the origin of gliomas.
      ,
      • Holland E.C.
      Progenitor cells and glioma formation.
      ]. It is now known that both multipotent and self-renewing neural stem cells and self-renewing glial progenitor cells produce astrocytes and oligodendrocytes in limited regions of the adult brain including the subventricular zone, the subgranular zone of the dentate gyrus and possibly other minor stem-cell niches throughout the brain [
      • Gould E.
      How widespread is adult neurogenesis in mammals?.
      ,
      • Nunes M.C.
      • Roy N.S.
      • Keyoung H.M.
      • et al.
      Identification and isolation of multipotential neural progenitor cells from the subcortical white matter of the adult human brain.
      ,
      • Canoll P.
      • Goldman J.E.
      The interface between glial progenitors and gliomas.
      ]. These stem cell and progenitor elements, in addition to differentiating into adult glia, are now thought to constitute a substrate for neoplastic transformation, termed glioma initiating cells or glioma stem cells (GSCs), a small population of self-renewal, therapy-resistant and slow-dividing malignant cells inside the main tumor bulk. Calabrese et al. found that a significant proportion of vessel-associated Nestin + tumor cells are proliferating and that these cells are distributed throughout tumors, from all regions of the cerebrum and cerebellum [
      • Calabrese C.
      • Poppleton H.
      • Kocak M.
      • et al.
      A perivascular niche for brain tumor stem cells.
      ]. In the early 70 s, Pierce proposed that tumors arise from carcinogenic events occurring in stem cells of a particular tissue that cause a defect in the control of normal stem cell function, including self-renewal and differentiation [
      • Pierce G.B.
      Neoplasms, differentiations and mutations.
      ]. In the adult brain, neural stem cells commonly possess features associated with gliomas, including a robust proliferative potential, high motility, diverse progeny, associations with blood vessels and white-matter tracts and an immature gene expression profile characterized by expression of nestin, epidermal growth factor receptor (EGFR), Notch and PTEN, telomerase activity, and Hedgehog and Wnt pathway activities [
      • Jackson E.L.
      • Garcia-Verdugo J.M.
      • Gil-Perotin S.
      • et al.
      PDGFR alpha-positive B cells are neural stem cells in the adult SVZ that form glioma-like growths in response to increased PDGF signaling.
      ,
      • Sanai N.
      • Tramontin A.D.
      • Quiñones-Hinojosa A.
      • et al.
      Unique astrocyte ribbon in adult human brain contains neural stem cells but lacks chain migration.
      ,
      • Merkle F.T.
      • Tramontin A.D.
      • García-Verdugo J.M.
      • et al.
      Radial glia give rise to adult neural stem cells in the subventricular zone.
      ,
      • Westphal M.
      • Lamszus K.
      The neurobiology of gliomas: from cell biology to the development of therapeutic approaches.
      ].
      GSCs expresse nestin (an intermediate filament protein) and CD133 (also known as prominin 1, a transmembrane glycoprotein), factors associated with neural stem cells, although some GSCs are CD133-negative [
      • Chen R.
      • Nishimura M.C.
      • Bumbaca S.M.
      • et al.
      A hierarchy of self-renewing tumor-initiating cell types in glioblastoma.
      ]. Although CD133 expression seems to be related to stemness, it might only indicate an intermediate, adaptive state of a cell rather than a stable phenotype [
      • Griguer C.E.
      • Oliva C.R.
      • Gobin E.
      • et al.
      CD133 is a marker of bioenergetic stress in human glioma.
      ]. A tumor is therefore a caricature of normal tissue and appears to be undifferentiated because of the preponderance of undifferentiated proliferating stem cells in relationship to the number of cells that have differentiated and are post-mitotic.
      Stem and progenitor cells in the central nervous system may be particularly at risk for malignant transformation since they constitutively activate the cellular machinery necessary for tumor initiation, progression or both (including growth factor receptor EGFR, PI3K, Akt, mTOR, Ras, mitogen-activated protein kinase (MAPK) members). Thus, neural stem cells may represent the path of least resistance in tumorigenesis since these cells already have the ability to bypass apoptosis and senescence. As a result, neural stem cells may require less than the estimated four to seven mutations needed to induce malignant change in a differentiated cell [
      • Scadden D.T.
      The stem-cell niche as an entity of action.
      ]. In gliomas, as in several solid tumors outside the central nervous system, only a small fraction of the total population of cancer cells are clonogenic cancerous neural stem cells. However, this resistant population is considered the primary cause of glioblastoma invasion and responsible for the maintenance of a minimal residual disease, which commonly leads to post-therapeutic recurrence [
      • Singh S.K.
      • Hawkins C.
      • Clarke I.D.
      • et al.
      Identification of human brain tumour initiating cells.
      ]. Evidence from human gliomas and rodent glioma models indicate that the glioma vascular niche supports glioma stem cell maintenance and nurtures tumor growth by offering nutrients necessary for unlimited glioma stem cell proliferation [
      • Calabrese C.
      • Poppleton H.
      • Kocak M.
      • et al.
      A perivascular niche for brain tumor stem cells.
      ,
      • Sharma A.
      • Shiras A.
      Cancer stem cell-vascular endothelial cell interactions in glioblastoma.
      ]. Several embryonic signaling pathways, such as Notch, Hedgehog and Wnt/b-catenin, have been reported to help maintain the GSC pool and thus provide potentials target for treating glioblastoma [
      • Thomas A.A.
      • Brennan C.W.
      • DeAngelis L.M.
      • et al.
      Emerging therapies for glioblastoma.
      ]. Paw et al. [
      • Paw I.
      • Carpenter R.C.
      • Watabe K.
      • et al.
      Mechanisms regulating glioma invasion.
      ] and Lee et al. [
      • Lee Y.
      • Lee J.K.
      • Ahn S.H.
      • et al.
      WNT signaling in glioblastoma and therapeutic opportunities.
      ] recently provided an overview of the specific signaling pathways that regulate glioblastoma stem cell maintenance and migration.
      On the basis of their histological appearance, gliomas have been traditionally classified as astrocytic, oligodendroglial or ependymal tumors and assigned WHO grades I-IV, which indicate different degrees of malignancy. Diffusely infiltrating gliomas in adults are now separated into three overarching tumor groups with distinct natural histories, responses to treatment and outcomes: IDH-mutant, 1p/19q co-deleted tumors with mostly oligodendroglial morphology that are associated with the best prognosis; IDH-mutant, 1p/19q non-co-deleted tumors with mostly astrocytic histology that are associated with intermediate outcome; and IDH wild-type, mostly higher WHO grade (III or IV) tumors that are associated with poor prognosis [
      • Weller M.
      • Wick W.
      • Aldape K.
      • et al.
      Glioma.
      ].

       Treatment

      The current standard treatment for glioblastoma includes surgical resection, followed by radiotherapy plus concomitant temozolomide (TMZ) followed by six cycles of maintenance TMZ [
      • Weller M.
      • van den Bent M.
      • Tonn J.C.
      • et al.
      European Association for Neuro-Oncology (EANO) Task Force on Gliomas. European Association for Neuro-Oncology (EANO) guideline on the diagnosis and treatment of adult astrocytic and oligodendroglial gliomas.
      ,

      Stupp R, Hegi ME, Mason WP, et al; European Organisation for Research and Treatment of Cancer Brain Tumour and Radiation Oncology Groups; National Cancer Institute of Canada Clinical Trials Group. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol 2009; 10: p. 459–66.

      ]. These treatments allow a median overall survival in the range of 16 months in clinical trial patient populations, but are never curative. Indeed, less than 10% of the 77,000 patients diagnosed each year with glioblastoma in the USA and Europe will survive more than 5 years. This dismal prognosis reflects the high rate of tumor recurrence approaching 100% which is linked to the profoundly infiltrative nature of this tumor, rendering curative surgical resection elusive, and its relative resistance not only to the conventional treatments of genotoxic radiotherapy and chemotherapy, but also more recently to targeted therapies. Antiangiogenic therapies have dominated the clinical trial landscape of glioblastoma. However, it has now become clear that this approach is hopeless, at least with a view to prolonging overall survival. Indeed, while anti-angiogenic treatment targeting VEGF exerts anti-edema effects in glioblastoma which may translate into clinical benefit in subgroups of patients, no clinical trial has demonstrated a survival benefit afforded by anti-angiogenic agents.

      Glioblastoma migration: What is new?

      In a previous review we highlighted the complex dynamic processes governing glioma cell migration in general and in glioblastoma in particular [
      • Lefranc F.
      • Brotchi J.
      • Kiss R.
      Possible future issues in the treatment of glioblastomas: special emphasis on cell migration and the resistance of migrating glioblastoma cells to apoptosis.
      ]. This dynamic process includes, among others, (i) cell adhesion to numerous components of a modified (when compared to normal parenchyma) extracellular matrix (ECM); (ii) cell motility, which involves the reorganization of the actin cytoskeleton, primarily through modifications in the field of integrin network–ECM component interactions; and (iii) invasion, which involves the degradation of matrix proteins by tumor-secreted proteolytic enzymes, primarily serine proteases, cathepsins and metalloproteinases (MMPs). Admittedly, no effective treatments for human glioma patients have evolved from these avenues of research.
      In the present review, we analyzed the recent literature on new promising therapies to combat glioblastoma with a focus on two pathognomonic features of glioblastoma biology, i.e., glioblastoma cell migration and invasion through the brain parenchyma and, associated with that, the apparent intrinsic resistance of migrating glioblastoma cells to pro-apoptotic stimuli, including cytotoxic chemotherapy and radiotherapy.
      We focused in the current review on glutamate, ion channels, tumor microtubules and several signaling pathways. While there is a myriad of signaling pathways that influence the migratory behavior of malignant astrocytes [
      • Lefranc F.
      • Brotchi J.
      • Kiss R.
      Possible future issues in the treatment of glioblastomas: special emphasis on cell migration and the resistance of migrating glioblastoma cells to apoptosis.
      ], we decided to restrain ourselves on the “glutamate pathway” because we are convinced that impairing this malignant astrocyte-migration avenue of invasion within the brain parenchyma would actually contribute therapeutic benefits to the patients. We detail below why we believe in this avenue to combat malignant gliomas.

       Interaction of glutamate, glutamate transporters and glutamate receptors

      The release of the neurotransmitter glutamate from glioma cells has been well studied in vitro and in vivo as well as in glioblastoma patient populations [
      • Ye Z.C.
      • Sontheimer H.
      Glioma cells release excitotoxic concentrations of glutamate.
      ,
      • Lyons S.A.
      • Chung W.J.
      • Weaver A.K.
      • et al.
      Autocrine glutamate signaling promotes glioma cell invasion.
      ,
      • Robert S.M.
      • Buckingham S.C.
      • Campbell S.L.
      • et al.
      SLC7A11 expression is associated with seizures and predicts poor survival in patients with malignant glioma.
      ]. The Na+-independent, Cl--dependent, cystine/glutamate exchanger system (SXC) is the only transporter identified in glioma cells that has the ability to release large amounts of glutamate [
      • Ye Z.C.
      • Sontheimer H.
      Glioma cells release excitotoxic concentrations of glutamate.
      ]. Glutamate released from malignant glioma cells into the extracellular space has been linked to seizure induction, and at higher concentration, glutamate is thought to induce neuronal cell death, mediated mainly by prolonged activation of N-methyl-D-aspartate (NMDA) receptors, but also via α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate receptors resulting in Ca2+ influx and Ca2+-mediated cell death (Fig. 1) [
      • Takano T.
      • Lin J.H.
      • Arcuino G.
      • et al.
      Glutamate release promotes growth of malignant gliomas.
      ,
      • Ye Z.C.
      • Rothstein J.D.
      • Sontheimer H.
      Compromised glutamate transport in human glioma cells: reduction-mislocalization of sodium-dependent glutamate transporters and enhanced activity of cystine-glutamate exchange.
      ,
      • Chung W.J.
      • Lyons S.A.
      • Nelson G.M.
      • et al.
      Inhibition of cystine uptake disrupts the growth of primary brain tumors.
      ]. In the healthy brain, excitatory amino acid transporters (EAAT), located on astrocytes (EAAT1 and-2) and neurons (EAAT2), are responsible for the majority of glutamate uptake, and astrocytes can rapidly reduce toxic concentrations of extracellular glutamate. Glioma cells lack EAAT-mediated glutamate transport which contributes to the elevated peritumoral levels observed in glioma patients and in animal models of glioma [
      • Robert S.M.
      • Sontheimer H.
      Glutamate transporters in the biology of malignant gliomas.
      ]. Moreover, Robert and Sontheimer hypothesized that glioma cells alter peritumoral astrocyte EAAT function, preventing the re-establishment of glutamate homeostasis, as typically occurs in the healthy brain [
      • Robert S.M.
      • Sontheimer H.
      Glutamate transporters in the biology of malignant gliomas.
      ]. Investigations effectively discovered that glioma cells lack EAAT2-mediated glutamate transport [
      • De Groot J.F.
      • Liu T.J.
      • Fuller G.
      • et al.
      The excitatory amino acid transporter-2 induces apoptosis and decreases glioma growth in vitro and in vivo.
      ]. These findings were corroborated by microarray analyses of human glioma biopsies demonstrating the absence of EAAT2 expression in patient glioma tissue [
      • De Groot J.F.
      • Liu T.J.
      • Fuller G.
      • et al.
      The excitatory amino acid transporter-2 induces apoptosis and decreases glioma growth in vitro and in vivo.
      ].
      Figure thumbnail gr1
      Fig. 1The glutamatergic system in glioblastoma. Glial tumor cells release glutamate by means of the Na+-independent, Cl--dependent, cystine/glutamate exchanger system (SXC) transporter. Glutamate released from malignant glioma cells in the extracellular matrix is responsible for seizure induction and, at a higher concentration, neuronal cell death, which is mediated by prolonged activation of neuronal N-methyl-D-aspartate (NMDA) receptors, resulting in Ca2+ influx and Ca2+-mediated cell death. This neuronal cell death may facilitate tumor growth. Glutamate also stimulates growth, migration and invasion of glial tumor cells through activation of Ca2+-permeable glutamate AMPA receptors on glioma cells in a paracrine and autocrine manner.
      By contrast, glioma cells express NMDA, a-amino-3-hydroxy-5-methyl-4-isoxaole propionic acid (AMPA) and metabotropic (mGlu) receptors [
      • Ishiuchi S.
      • Tsuzuki K.
      • Yoshida Y.
      • et al.
      Blockage of Ca(2+)-permeable AMPA receptors suppresses migration and induces apoptosis in human glioblastoma cells.
      ]. However, van Vuurden et al. showed that on protein level, AMPA-type glutamate receptor subunits are variably expressed in glioblastoma and are overall down-regulated as compared to the normal brain tissue [
      • van Vuurden D.G.
      • Yazdani M.
      • Bosma I.
      • et al.
      Attenuated AMPA receptor expression allows glioblastoma cell survival in glutamate-rich environment.
      ]. These authors further reported that down-regulation of AMPA receptor expression and mislocalization of these receptors enables the cells to survive in a high glutamate environment without undergoing excitotoxic cell death themselves [
      • van Vuurden D.G.
      • Yazdani M.
      • Bosma I.
      • et al.
      Attenuated AMPA receptor expression allows glioblastoma cell survival in glutamate-rich environment.
      ].
      Glutamate stimulates the growth, migration and invasion of neuroglial tumor cells through activation of Ca2+-permeable glutamate AMPA receptors on glioma cells in a paracrine and autocrine manner (Fig. 1). AMPA receptor expression correlates with an increase in surface expression of β1 integrin, a molecule implicated in glioma invasion, and AMPA receptor stimulation increases glioma cell de-adhesion from surrounding cells. Glial AMPA receptors activate focal adhesion kinase (FAK) which influences cytoskeletal rearrangement and focal adhesion contact formation [
      • Piao Y.
      • Lu L.
      • de Groot J.
      AMPA receptors promote perivascular glioma invasion via beta1 integrin-dependent adhesion to the extracellular matrix.
      ].
      Additionally, in response to reactive oxygen species (ROS), glioma cells upregulate SXC activity to provide intracellular cysteine for enhanced glutathione (GSH) synthesis, an important intracellular antioxidant that promotes cell survival [
      • Robert S.M.
      • Ogunrinu-Babarinde T.
      • Holt K.T.
      • et al.
      Role of glutamate transporters in redox homeostasis of the brain.
      ]. Increased GSH neutralizes ROS, preventing cell destruction triggered by hypoxia; inhibits apoptosis signaling, protecting cells from degradation; and via ROS antagonism, may play an important role in the development of resistance to radiotherapy and chemotherapy [
      • Oliva C.R.
      • Moellering D.R.
      • Gillespie G.Y.
      • et al.
      Acquisition of chemoresistance in gliomas is associated with increased mitochondrial coupling and decreased ROS production.
      ].

       Roles of ion channels, transporters and exchangers

      Increasing evidence suggests that cellular migration and invasion in cancer cells in general and glioma cells in particular results from shape and volume changes facilitated by ion channels and transporters [
      • Schwab A.
      • Fabian A.
      • Hanley P.J.
      • et al.
      Role of ion channels and transporters in cell migration.
      ,
      • Cuddapah V.A.
      • Sontheimer H.
      Ion channels and transporters [corrected] in cancer. 2. Ion channels and the control of cancer cell migration.
      ,
      • Cuddapah V.A.
      • Robel S.
      • Watkins S.
      • et al.
      A neurocentric perspective on glioma invasion.
      ,
      • Pardo L.A.
      • Stühmer W.
      The roles of K(+) channels in cancer.
      ]. Glioma cells principally use voltage-gated chloride and potassium channels as well as the KCa family of Ca2+-activated potassium channels ([
      • Cuddapah V.A.
      • Robel S.
      • Watkins S.
      • et al.
      A neurocentric perspective on glioma invasion.
      ] for a review of this topic) (Fig. 2).
      Figure thumbnail gr2
      Fig. 2Glioma cell volume and shape regulation: involvement of ion channels. Cell migration and glioblastoma cell migration in particular is characterized by the polarized cellular and molecular processes that promote the protrusion of the leading edge (local volume increase) and retraction of the trailing edge (local volume reduction). A net outflow of ions and water at the cell trailing edge and a net inflow of ions and water at the cell leading edge underlie these localized hydrodynamic changes. Ion channels and transporters display polarized subcellular distribution. At the trailing edge, calcium entry through calcium-permeable channels can activate calcium-activated potassium channels (KCa) to promote potassium efflux. The opening of chloride (KCl) and potassium channels (Kv) further enhances the outflow of ions Cl- and K+, followed by obligatory water efflux through aquaporins. At the leading edge, an assortment of inward ion channels, such as the Na+/K+ ATPase and the Na+-K+-Cl- cotransporters (NKCC1) together with aquaporins, regulate ion and water influx. This ion and water influx drives local cell volume increase in the protruding lamellipodium for leading edge extension.

       Potassium channels

      Among ion transporters, potassium channels show the highest variability and the most frequently altered pattern of expression in many tumor types [
      • Hervé J.C.
      Membrane channels and transporters in cancers.
      ]. Cell cycle, proliferation, migration, invasion, angiogenesis and apoptosis are all processes that can be modified by the expression of potassium channels [
      • Comes N.
      • Serrano-Albarrás A.
      • Capera J.
      • et al.
      Involvement of potassium channels in the progression of cancer to a more malignant phenotype.
      ]. Potassium channels have been implicated in every step of adhesion and migration [
      • Schwab A.
      • Fabian A.
      • Hanley P.J.
      • et al.
      Role of ion channels and transporters in cell migration.
      ]. Interactions of potassium channels with crucial contributors to cell adhesion, such as FAK and cortactin, an actin-interacting protein implicated in the cytoskeletal architecture and often amplified in several types of cancer, have been reported (Fig. 2) [
      • Herrmann S.
      • Ninkovic M.
      • Kohl T.
      • et al.
      Cortactin controls surface expression of the voltage-gated potassium channel K(V)10.1.
      ]. Potassium channels also associate with the main contributors to cell adhesion signaling, integrins. The most important understood mechanisms by which particular potassium channels contribute to cancer initiation and progression have recently been reviewed [
      • Pardo L.A.
      • Stühmer W.
      The roles of K(+) channels in cancer.
      ]. Here we focus on the literature on potassium channels involved in glioma progression and migration, e.g. Kv1.3 and Kv1.5 [
      • Preussat K.
      • Beetz C.
      • Schrey M.
      • et al.
      Expression of voltage-gated potassium channels Kv1.3 and Kv1.5 in human gliomas.
      ,
      • Lefranc F.
      • Pouleau H.B.
      • Rynkowski M.
      • et al.
      Voltage-dependent K+ channels as oncotargets in malignant gliomas.
      ,
      • Arvind S.
      • Arivazhagan A.
      • Santosh V.
      • et al.
      Differential expression of a novel voltage gated potassium channel–Kv 1.5 in astrocytomas and its impact on prognosis in glioblastoma.
      ], Kv10.1 [
      • Bai Y.
      • Liao H.
      • Liu T.
      • et al.
      MiR-296-3p regulates cell growth and multi-drug resistance of human glioblastoma by targeting ether-à-go-go (EAG1).
      ,
      • Pardo L.A.
      • Stühmer W.
      Eag1: an emerging oncological target.
      ,
      • Martínez R.
      • Stühmer W.
      • Martin S.
      • et al.
      Analysis of the expression of Kv10.1 potassium channel in patients with brain metastases and glioblastoma multiforme: impact on survival.
      ], Kv11.1 [
      • Masi A.
      • Becchetti A.
      • Restano-Cassulini R.
      • et al.
      hERG1 channels are overexpressed in glioblastoma multiforme and modulate VEGF secretion in glioblastoma cell lines.
      ], KCa1.1 [
      • Ransom C.B.
      • Liu X.
      • Sontheimer H.
      BK channels in human glioma cells have enhanced calcium sensitivity.
      ,
      • Weaver A.K.
      • Bomben V.C.
      • Sontheimer H.
      Expression and function of calcium-activated potassium channels in human glioma cells.
      ] and KCa3.1 [
      • Ruggieri P.
      • Mangino G.
      • Fioretti B.
      • et al.
      The inhibition of KCa3.1 channels activity reduces cell motility in glioblastoma derived cancer stem cells.
      ] (Fig. 2).
      The Kv1.3, Kv10.1, and Kv11.1 channels are the best studied voltage-gated K channels in the context of cell migration. They are of particular importance for cells of the immune system and tumor cells, including glioblastoma cells, constituting potential diagnostic or prognostic markers or even therapeutic targets [
      • Pardo L.A.
      • Stühmer W.
      The roles of K(+) channels in cancer.
      ,
      • Comes N.
      • Serrano-Albarrás A.
      • Capera J.
      • et al.
      Involvement of potassium channels in the progression of cancer to a more malignant phenotype.
      ].
      Kv10.1 (also known as EAG1, ether-à-go-go and KCNH1), a voltage-gated potassium channel that is virtually absent from normal cells outside the central nervous system, is expressed in limited areas of the brain (hypothalamus, hippocampus, cerebral cortex, cerebellum and olfactory nerve) but highly expressed in different cancers, including brain metastases and gliomas [
      • Martínez R.
      • Stühmer W.
      • Martin S.
      • et al.
      Analysis of the expression of Kv10.1 potassium channel in patients with brain metastases and glioblastoma multiforme: impact on survival.
      ]. Bai et al. also observed Kv10.1 overexpression in both glioma cell lines and clinical glioma samples [
      • Bai Y.
      • Liao H.
      • Liu T.
      • et al.
      MiR-296-3p regulates cell growth and multi-drug resistance of human glioblastoma by targeting ether-à-go-go (EAG1).
      ]. In several different cancers, Kv10.1 enhances proliferation. It has also been shown to be required for the maintenance of growth associated with the p53/miRNA34/E2F1 regulatory pathway in SHSY5Y human dopaminergic neuroblastoma cells [
      • Lin H.
      • Li Z.
      • Chen C.
      • et al.
      Transcriptional and post-transcriptional mechanisms for oncogenic overexpression of ether à go-go K+ channel.
      ]; this channel up-regulates HIF-1 and VEGF in the tumor environment [
      • Downie B.R.
      • Sánchez A.
      • Knötgen H.
      • et al.
      Eag1 expression interferes with hypoxia homeostasis and induces angiogenesis in tumors.
      ] and increases migration [
      • Toral C.
      • Mendoza-Garrido M.E.
      • Azorín E.
      • et al.
      Effect of extracellular matrix on adhesion, viability, actin cytoskeleton and K+ currents of cells expressing human ether à go-go channels.
      ]. Kv10.1 expression in Chinese Hamster Ovary (CHO) cells affects cytoskeletal organization, which might influence cancer cell proliferation, migration and metastasis [
      • Toral C.
      • Mendoza-Garrido M.E.
      • Azorín E.
      • et al.
      Effect of extracellular matrix on adhesion, viability, actin cytoskeleton and K+ currents of cells expressing human ether à go-go channels.
      ].
      A correlation between dismal prognosis and Kv10.1 expression in patients with brain metastasis from different carcinomas has been observed, and metastatic cancer patients receiving tricyclic antidepressants (such as imipramine, chlorimipramine, citalopram and amitriptyline) showed a significantly longer median overall survival than patients not treated with these agents [
      • Martínez R.
      • Stühmer W.
      • Martin S.
      • et al.
      Analysis of the expression of Kv10.1 potassium channel in patients with brain metastases and glioblastoma multiforme: impact on survival.
      ].
      Kv11.1 (also known as HERG or KCNH2) was among the first voltage-gated channels directly associated with cancer [
      • Bianchi L.
      • Wible B.
      • Arcangeli A.
      • et al.
      herg encodes a K+ current highly conserved in tumors of different histogenesis: a selective advantage for cancer cells?.
      ]. Further, Kv11.1 blockers impair the proliferation of tumor cells [
      • Crociani O.
      • Guasti L.
      • Balzi M.
      • et al.
      Cell cycle-dependent expression of HERG1 and HERG1B isoforms in tumor cells.
      ]. Kv11.1 physically interacts with β1 integrin at the plasma membrane of human cancer cells to regulate survival and migration [
      • Becchetti A.
      • Crescioli S.
      • Zanieri F.
      • et al.
      The conformational state of hERG1 channels determines integrin association, downstream signaling, and cancer progression.
      ] (Fig. 2), and this channel has also been implicated in the regulation of apoptosis of human glioblastoma cells (see below) [
      • Staudacher I.
      • Jehle J.
      • Staudacher K.
      • et al.
      HERG K+ channel-dependent apoptosis and cell cycle arrest in human glioblastoma cells.
      ].
      The Kv1.3 and Kv1.5 channels modulate Ca2+ signaling and cell volume and have been analyzed in a number of cancer cell types [
      • Felipe A.
      • Bielanska J.
      • Comes N.
      • et al.
      Targeting the voltage-dependent K(+) channels Kv1.3 and Kv1.5 as tumor biomarkers for cancer detection and prevention.
      ]. The levels of expression of the Kv1.5 and Kv1.3 channel subtypes vary between different glioma groups, and differential expression of Kv1.5 according to malignancy grade has been noted [
      • Preussat K.
      • Beetz C.
      • Schrey M.
      • et al.
      Expression of voltage-gated potassium channels Kv1.3 and Kv1.5 in human gliomas.
      ]. Kv1.5 also contributes to the resistance to pro-apoptotic stimuli of cancer cells, including gliomas (see below).
      The calcium-activated potassium channels KCa1.1 and KCa3.1 are important for the migration of glioma and glioma stem cells [
      • Ruggieri P.
      • Mangino G.
      • Fioretti B.
      • et al.
      The inhibition of KCa3.1 channels activity reduces cell motility in glioblastoma derived cancer stem cells.
      ,
      • Turner K.L.
      • Honasoge A.
      • Robert S.M.
      • et al.
      A Proinvasive Role for the Ca2+-Activated K+ Channel KCa3.1 in Malignant Glioma.
      ,
      • D’Alessandro G.
      • Catalano M.
      • Sciaccaluga M.
      • et al.
      KCa3.1 channels are involved in the infiltrative behavior of glioblastoma in vivo.
      ]. The REepository of Molecular BRAin Neoplasia DaTa (REMBRANDT) database allowed to determine that the gene KCNN4, which encodes KCa3.1, is overexpressed in 32% of gliomas, and that its expression correlated with short survival [
      • Turner K.L.
      • Honasoge A.
      • Robert S.M.
      • et al.
      A Proinvasive Role for the Ca2+-Activated K+ Channel KCa3.1 in Malignant Glioma.
      ]. Turner et al. generated U251 glioma cells that stably express an inducible knockdown shRNA to experimentally eliminate KCa3.1 expression [
      • Turner K.L.
      • Honasoge A.
      • Robert S.M.
      • et al.
      A Proinvasive Role for the Ca2+-Activated K+ Channel KCa3.1 in Malignant Glioma.
      ]. Subjecting these cells to a combination of in vitro and in situ invasion assays revealed that KCa3.1 expression may enhance glioma invasion and either specific pharmacological inhibition with TRAM-34 or elimination of this channel by gene silencing impairs invasion. Importantly, after intracranial implantation into SCID mice, ablation of KCa3.1 with inducible shRNA from U251 cells resulted in a significant reduction in tumor invasion into the surrounding brain [
      • Turner K.L.
      • Honasoge A.
      • Robert S.M.
      • et al.
      A Proinvasive Role for the Ca2+-Activated K+ Channel KCa3.1 in Malignant Glioma.
      ].
      Migration of glioblastoma cells can be stimulated by ionizing radiation (IR) [
      • Wild-Bode C.
      • Weller M.
      • Rimner A.
      • et al.
      Sublethal irradiation promotes migration and invasiveness of glioma cells: implications for radiotherapy of human glioblastoma.
      ]. Steinle et al. accordingly analyzed the IR-induced increase in Ca2+-activated potassium channel activity and its effect on the migration of T98G and U87MG cells [
      • Steinle M.
      • Palme D.
      • Misovic M.
      • et al.
      Ionizing radiation induces migration of glioblastoma cells by activating BK K channels.
      ]. IR dose-dependently stimulated migration of glioblastoma cells, which in turn was sensitive to the channel inhibitor paxilline. These authors concluded that IR stimulates potassium channel activity, resulting in the activation of Ca2+/calmodulin-dependent protein kinase II and enhanced glioblastoma cell migration [
      • Steinle M.
      • Palme D.
      • Misovic M.
      • et al.
      Ionizing radiation induces migration of glioblastoma cells by activating BK K channels.
      ].

       Chloride channels

      Approximately 20 years ago, Sontheimer proposed that the invasive migration of human glioma cells into the normal brain parenchyma requires shape and/or volume changes [
      • Soroceanu L.
      • Manning Jr, T.J.
      • Sontheimer H.
      Modulation of glioma cell migration and invasion using Cl(-) and K(+) ion channel blockers.
      ]. Chloride currents may contribute to such shape-volume changes by affecting net salt fluxes across glioma cell membranes, with accompanying water efflux, resulting in cell shrinkage that is conducive to glioma cell migration through minute extracellular spaces of the brain [
      • Ransom C.B.
      • O'Neal J.T.
      • Sontheimer H.
      Volume-activated chloride currents contribute to the resting conductance and invasive migration of human glioma cells.
      ]. Sontheimer and collaborators subsequently performed a direct examination of Cl- dynamics in human glioma cells and its functional relationship to cell biology [
      • Habela C.W.
      • Ernest N.J.
      • Swindall A.F.
      • et al.
      Chloride accumulation drives volume dynamics underlying cell proliferation and migration.
      ]. First, these authors observed that invasive tumor cells become morphologically polarized and develop membrane protrusions (Fig. 2). During this process, invasive glioma cells alter their cell shape and volume to fit into the narrow extracellular spaces available and invade the brain parenchyma. These features include secretion of Cl- and K+ through ion channels localized to lipid raft domains on invadopodia and water passively flowing through water channels or aquaporins [
      • Cuddapah V.A.
      • Sontheimer H.
      Ion channels and transporters [corrected] in cancer. 2. Ion channels and the control of cancer cell migration.
      ]. In addition, the interplay between Na+-K+-Cl- cotransporters and Na+/K+ ATPase leads to active accumulation of K+ and Cl-, establishing a gradient for KCl efflux (Fig. 2) [
      • Cuddapah V.A.
      • Sontheimer H.
      Ion channels and transporters [corrected] in cancer. 2. Ion channels and the control of cancer cell migration.
      ].
      Here, we do not review the roles of other channels involved in glioma migration, such as transient receptor potential (TRP) calcium channels, which also contribute to changes in Ca2+ by modulating the driving force for Ca2+ entry as Morrone et al. recently summarized this subject [
      • Morrone F.B.
      • Gehring M.P.
      • Nicoletti N.F.
      Calcium channels and associated receptors in malignant brain tumor therapy.
      ].

       Microtubes

      Osswald et al. reported that astrocytomas but not oligodendrogliomas develop functional multicellular network structures through ultra-long membrane protrusions and that astrocytoma cells use these distinct tumor microtubes as routes for brain invasion, proliferation, and interconnections over long distances [
      • Osswald M.
      • Jung E.
      • Sahm F.
      • et al.
      Brain tumour cells interconnect to a functional and resistant network.
      ]. The resulting network facilitates multicellular communication through microtube-associated gap junctions. Confocal microscopy revealed punctate connexin 43 (Cx43) gap junction immunoreactivity, particularly at the tumor microtubes of astrocytoma cells, which was not observed for other connexins. When damage to the network occurs, tumor microtube is used for repair. Moreover, microtube-connected astrocytoma cells, but not those remaining unconnected throughout tumor progression, were protected from cell death inflicted by radiotherapy. Osswald et al. hypothesized that intercellular tumor microtubes can serve as an individual tumor cell to distribute critical elevations of small molecules, such as calcium, within the large network (Fig. 3), achieving nonlethal levels for radiotherapy-induced cytotoxicity and for intrinsic apoptotic cell death in glioma cells [
      • Osswald M.
      • Jung E.
      • Sahm F.
      • et al.
      Brain tumour cells interconnect to a functional and resistant network.
      ]. Neuronal growth-associated protein 43 (GAP-43) is important for microtube formation and function and drives microtube dependent tumor cell invasion, proliferation, interconnection, and radioresistance. The disconnection of astrocytoma networks by disrupting their tumor microtubes may emerge as a new principle to reduce the treatment resistance of this disease, but clinical applications have not been introduced [
      • Osswald M.
      • Jung E.
      • Sahm F.
      • et al.
      Brain tumour cells interconnect to a functional and resistant network.
      ].
      Figure thumbnail gr3
      Fig. 3Pathways of intercellular communication in glioblastoma. Glioblastoma demonstrates intratumoral heterogeneity and contains a population of glioma stem cells (GSC) that contributes to tumor propagation, maintenance and therapeutic resistance. After secretion, glioblastoma EV microRNAs are taken up by other tumor cells or normal cells in the microenvironment to change their phenotype and support tumor progression. Structures called microtubes connect tumor cells, enabling these cells to act as a single, organism-like unit. Tumor microtubes facilitate invasion into healthy brain tissue, permitting the spread of toxic molecules, such as Ca2+, which build up during radiotherapy enabling the whole unit to share the burden of toxicity. Furthermore, if tumor tissue is surgically removed, then newly synthesized nuclei donated by tumor cells can travel down the microtube to the cell-free site to form new tumor cells.

       Extracellular MicroRNAs

      Over the past decade, there has been increased awareness of the possible role of microRNAs (miRNAs) in the development of cancer, including glioblastoma [
      • Lin S.
      • Gregory R.I.
      MicroRNA biogenesis pathways in cancer.
      ]. miRNAs are small non-coding RNAs of approximately 20–23 nucleotides that act as post-transcriptional regulators of gene expression. miRNAs repress gene expression by binding to complementary sequences in the 3′ untranslated region (3′ UTR) of mRNAs and targeting these molecules for degradation, thereby preventing their translation, resulting in a decreased level of encoded proteins that affect an array of cellular processes, e.g., differentiation, proliferation, migration, metabolism, apoptosis and stem cell maintenance [
      • Fu J.
      • Rodova M.
      • Nanta R.
      • et al.
      NPV-LDE-225 (Erismodegib) inhibits epithelial mesenchymal transition and self-renewal of glioblastoma initiating cells by regulating miR-21, miR-128, and miR-200.
      ]. Aberrant miRNA biogenesis in cancer occurs at different steps during miRNA maturation [
      • Lin S.
      • Gregory R.I.
      MicroRNA biogenesis pathways in cancer.
      ], and a single miRNA can regulate the expression of many genes. Depending on its target genes, miRNA can act as a tumor suppressor or tumor promoter. The accumulation of specific miRNAs in glioblastoma samples from patients and in glioblastoma cell lines was initially demonstrated a decade ago [
      • Ciafrè S.A.
      • Galardi S.
      • Mangiola A.
      • et al.
      Extensive modulation of a set of microRNAs in primary glioblastoma.
      ,
      • Chan J.A.
      • Krichevsky A.M.
      • Kosik K.S.
      MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells.
      ]. Since then, numerous reviews have highlighted the involvement of miRNAs in glioblastoma progression, migration and radio-chemotherapy resistance and suggested their potential as therapeutic targets [
      • Sana J.
      • Hajduch M.
      • Michalek J.
      • et al.
      MicroRNAs and glioblastoma: roles in core signalling pathways and potential clinical implications.
      ,
      • Karsy M.
      • Arslan E.
      • Moy F.
      Current progress on understanding MicroRNAs in glioblastoma multiforme.
      ,
      • Møller H.G.
      • Rasmussen A.P.
      • Andersen H.H.
      • et al.
      A systematic review of microRNA in glioblastoma multiforme: micro-modulators in the mesenchymal mode of migration and invasion.
      ,
      • Costa P.M.
      • Cardoso A.L.
      • Mano M.
      • et al.
      MicroRNAs in glioblastoma: role in pathogenesis and opportunities for targeted therapies.
      ,
      • Pal D.
      • Mukhopadhyay D.
      • Ramaiah M.J.
      • et al.
      Regulation of cell proliferation and migration by miR-203 via GAS41/miR-10b axis in human glioblastoma cells.
      ,
      • Godlewski J.
      • Krichevsky A.M.
      • Johnson M.D.
      • et al.
      Belonging to a network-microRNAs, extracellular vesicles, and the glioblastoma microenvironment.
      ,
      • Stojcheva N.
      • Schechtmann G.
      • Sass S.
      • et al.
      MicroRNA-138 promotes acquired alkylator resistance in glioblastoma by targeting the Bcl-2-interacting mediator BIM.
      ].
      Recently, microRNAs have also been localized to the extracellular space, often encapsulated in secreted extracellular vesicles (EV), nanometer size membrane-enclosed particles formed either from the fusion of an endosome with the plasma membrane (exosomes) or directly from the cell membrane (microvesicles) and released from numerous cell types, including glioblastoma cells, to mold the tumor microenvironment to their advantage (Fig. 3) [
      • Fanini F.
      • Fabbri M.
      Cancer-derived exosomic microRNAs shape the immune system within the tumor microenvironment: state of the art.
      ,
      • Naito Y.
      • Yoshioka Y.
      • Yamamoto Y.
      • et al.
      How cancer cells dictate their microenvironment: present roles of extracellular vesicles.
      ,
      • Rooj A.K.
      • Mineo M.
      • Godlewski J.
      MicroRNA and extracellular vesicles in glioblastoma – Small but powerful.
      ]. Neighboring or distant recipient normal and tumor cells can take up EVs containing tissue-specific expressed/secreted microRNAs [
      • Bronisz A.
      • Godlewski J.
      • Chiocca E.A.
      Extracellular vesicles and MicroRNAs: their role in tumorigenicity and therapy for brain tumors.
      ]. In a recent review, Rooj et al. discussed the current knowledge regarding the multifaceted roles of both cellular and EV microRNAs in shaping the glioblastoma microenvironment [
      • Rooj A.K.
      • Mineo M.
      • Godlewski J.
      MicroRNA and extracellular vesicles in glioblastoma – Small but powerful.
      ]. Glioblastoma EVs are enriched in several oncogenic microRNAs, including miR-21, miR-23a, miR-30a, miR-221 and miR-451 [
      • Li C.C.
      • Eaton S.A.
      • Young P.E.
      • et al.
      Glioma microvesicles carry selectively packaged coding and non-coding RNAs which alter gene expression in recipient cells.
      ]. However, the functions of most of these microRNAs remain unknown. Hoshino and colleagues elegantly demonstrated that tumor-derived EVs are internalized in organ-specific cells, and this process plays an important role in the preparation of the pre-metastatic niche [
      • Hoshino A.
      • Costa-Silva B.
      • Shen T.L.
      • et al.
      Tumour exosome integrins determine organotropic metastasis.
      ]. EVs containing miRNA were also detected in the blood or the cerebrospinal fluid of glioblastoma patients [
      • Akers J.C.
      • Hua W.
      • Li H.
      • et al.
      A cerebrospinal fluid microRNA signature as biomarker for glioblastoma.
      ]. Thus, microRNAs circulating in complexes with tumor-originated EVs represent a potential source of biomarkers for the early detection and monitoring of tumor responses to treatment and could be utilized for glioblastoma therapy using therapeutic mi-RNA [
      • Bronisz A.
      • Godlewski J.
      • Chiocca E.A.
      Extracellular vesicles and MicroRNAs: their role in tumorigenicity and therapy for brain tumors.
      ]. Arscott et al. showed that irradiation increases the abundance of exosomes released by glioblastoma cell lines and normal astrocytes [
      • Arscott W.T.
      • Tandle A.T.
      • Zhao S.
      • et al.
      Ionizing radiation and glioblastoma exosomes: implications in tumor biology and cell migration.
      ]. These authors showed that exosomes derived from irradiated cells enhanced the migration of recipient cells, and their molecular profiling revealed an abundance of molecules related to signaling pathways important for cell migration, including activation of neurotrophic tyrosine kinase receptor type 1 (TrkA) and FAK signaling [
      • Arscott W.T.
      • Tandle A.T.
      • Zhao S.
      • et al.
      Ionizing radiation and glioblastoma exosomes: implications in tumor biology and cell migration.
      ].

       Relationship between cell migration and resistance to pro-apoptotic stimuli

      Intracellular signaling pathways involved in the acquisition of resistance to pro-apoptotic stimuli by migrating glioma cells, including the PTEN/PI3K/Akt/mTOR and NF-kB pathways, were already an important topic more than 10 years ago [
      • Lefranc F.
      • Brotchi J.
      • Kiss R.
      Possible future issues in the treatment of glioblastomas: special emphasis on cell migration and the resistance of migrating glioblastoma cells to apoptosis.
      ]. These pathways are not all constitutively activated at the same time in all cells in any one glioma. Many inhibitors of these pathways have been developed, and numerous clinical trials have investigated these agents alone or in conjunction with conventional radiotherapy or chemotherapy. Several recent publications have provided an update on the state of EFGR and PI3K/Akt/mTOR inhibitors in clinical trials for glioblastoma [
      • Li X.
      • Wu C.
      • Chen N.
      • et al.
      PI3K/Akt/mTOR signaling pathway and targeted therapy for glioblastoma.
      ,
      • Zhao H.F.
      • Wang J.
      • Shao W.
      • et al.
      Recent advances in the use of PI3K inhibitors for glioblastoma multiforme: current preclinical and clinical development.
      ]. So far, targeted therapy directed to central intracellular signal pathways has not yet achieved satisfactory results in glioblastoma. A future perspective for glioblastoma therapy may be the combined inhibition of multiple targets and more tumor-tailored strategies.
      As indicated above, potassium channels may be involved both in the migratory processes and resistance to pro-apoptotic stimuli of glioblastoma cells. A volume change and decrease in intracellular K+ concomitantly occur with the initiation of apoptosis in glioma cells [
      • McFerrin M.B.
      • Turner K.L.
      • Cuddapah V.A.
      • et al.
      Differential role of IK and BK potassium channels as mediators of intrinsic and extrinsic apoptotic cell death.
      ]. Kv1.3, expressed at the plasma membrane, is also expressed at the inner membrane of mitochondria, where it is directly inhibited by the proapoptotic protein BAX, resulting in cytochrome c release and initiation of apoptosis [
      • Leanza L.
      • Henry B.
      • Sassi N.
      • et al.
      Inhibitors of mitochondrial Kv1.3 channels induce Bax/Bak-independent death of cancer cells.
      ]. Similar mechanisms are shared by other K+ channels implicated in apoptosis [
      • Checchetto V.
      • Azzolini M.
      • Peruzzo R.
      • et al.
      Mitochondrial potassium channels in cell death.
      ]. Staudacher et al. showed that the small molecule Kv11.1 ligand doxazosin induced concentration-dependent apoptosis of human LNT-229 and U87MG glioma cells, accompanied by cell cycle arrest in G0/G1 [
      • Staudacher I.
      • Jehle J.
      • Staudacher K.
      • et al.
      HERG K+ channel-dependent apoptosis and cell cycle arrest in human glioblastoma cells.
      ].
      In addition, compared with normal cells, several human cancers exhibit a high mitochondrial membrane potential and low expression of the Kv1.5 channel, which both contribute to the apoptosis resistance of cancer cells, including glioma cells [
      • Bonnet S.
      • Archer S.L.
      • Allalunis-Turner J.
      • et al.
      A mitochondria-K+ channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth.
      ]. Closing K+ channels or decreasing their expression in cancer cells increases the intracellular K+ concentration, which in turn increases the tonic inhibition that cytosolic K+ exerts on caspases [
      • Checchetto V.
      • Azzolini M.
      • Peruzzo R.
      • et al.
      Mitochondrial potassium channels in cell death.
      ,
      • Burg E.D.
      • Remillard C.V.
      • Yuan J.X.
      K+ channels in apoptosis.
      ].
      As indicated above, glioma cells can extend tumor microtubes for brain invasion, proliferation, and interconnection of single cells to a syncytium that is resistant to radiotherapy [
      • Osswald M.
      • Jung E.
      • Sahm F.
      • et al.
      Brain tumour cells interconnect to a functional and resistant network.
      ]. Recently Weil et al. evidenced using patient-derived glioblastoma stemlike cell lines (GBMSCs) implanted into the brain of mice that after a surgical lesion GBMSCs increasingly extended tumor microtubes toward the lesion area [
      • Weil S.
      • Osswald M.
      • Solecki G.
      • et al.
      Tumor microtubes convey resistance to surgical lesions and chemotherapy in gliomas.
      ]. In fact, an excessive “healing response” was observed in which tumor cell densities significantly exceeded those of unlesioned brain regions. Moreover, inhibition of tumor microtube formation and function by genetic targeting of growth associated protein-43 robustly suppressed this surgery-induced tumor growth reaction, in contrast to standard postsurgical anti-inflammatory treatment with dexamethasone. After one cycle of TMZ chemotherapy, intra- and intertumoral heterogeneity of tumor microtube formation and interconnection was strongly associated with therapy response: when tumor cells were integrated in tumor microtube networks, they were more likely to resist chemotherapy [
      • Weil S.
      • Osswald M.
      • Solecki G.
      • et al.
      Tumor microtubes convey resistance to surgical lesions and chemotherapy in gliomas.
      ].

      New anti-glioblastoma drugs related to migration, invasion and resistance to apoptosis

       Targeting glutamate

      The multitude of effects of glutamate in glioma biology has supported the rationale for the pharmacological targeting of glutamate receptors and transporters in the treatment of glioblastoma ([
      • de Groot J.
      • Sontheimer H.
      Glutamate and the biology of gliomas.
      ] for review). Using the web site http://www.clinicaltrials.gov/ as a reference, we analyzed the few clinical trials completed with therapies targeting glutamate (see also [
      • Blecic S.
      • Rynkowski M.
      • De Witte O.
      • et al.
      Glutamate and malignant gliomas, from epilepsia to biological aggressiveness: therapeutic implications.
      ]).
      Glutamate release can be inhibited by sulfasalazine, an FDA-approved drug for the treatment of inflammatory bowel disease, through inhibition of the CXC network. This drug may also facilitate death receptor-mediated apoptosis of glioma cells [
      • Hermisson M.
      • Weller M.
      NF-kB-independent actions of sulfasalazine dissociate the CD95L- and Apo2L/TRAIL-dependent death signalling pathways in human malignant glioma cells.
      ]. Sulfasalazine could be considered a therapeutic agent for glioma patients to reduce seizure activity and control invasive growth in patients, although a preliminary study of ten patients with recurrent glioblastoma indicated lack of clinical efficacy [
      • Robe P.A.
      • Martin D.H.
      • Nguyen-Khac M.T.
      • et al.
      Early termination of ISRCTN45828668, a phase 1/2 prospective, randomized study of sulfasalazine for the treatment of progressing malignant gliomas in adults.
      ] (Table 1). Yet, small series of 10 patients are insufficient to draw definitive conclusions about the potential therapeutic benefit of any given drug. Accordingly, Huberfeld and Vecht have discussed the potential for single therapeutic agents, such as the CXC system blocker sulfasalazine or histone deacetylase inhibitor valproic acid, to treat both gliomas and associated epilepsy [
      • Huberfeld G.
      • Vecht C.J.
      Seizures and gliomas–towards a single therapeutic approach.
      ]. However, the same year valproic acid was definitively excluded as a useful therapeutic approach after the analysis of survival of a pooled patient cohort (n = 1869) of four contemporary randomized clinical trials in newly diagnosed glioblastoma patients that received the antiepileptic drug at the start of chemoradiotherapy with temozolomide [
      • Happold C.
      • Gorlia T.
      • Chinot O.
      • et al.
      Does valproic acid improve survival in glioblastoma? A meta-analysis of randomized trials in newly diagnosed glioblastoma.
      ].
      Table 1Potential treatment approaches in clinical study.
      TargetsCompoundsIndicationsStudyRef.
      Glutamate release by CXC networkSulfasalazineRecurrent glioblastomaPhase I/II
      • Robe P.A.
      • Martin D.H.
      • Nguyen-Khac M.T.
      • et al.
      Early termination of ISRCTN45828668, a phase 1/2 prospective, randomized study of sulfasalazine for the treatment of progressing malignant gliomas in adults.
      AMPA receptorsTalampanelNewly diagnosed glioblastomaPhase II in association with radio- and temozolomide
      • Iwamoto F.M.
      • Kreisl T.N.
      • Kim L.
      • et al.
      Phase 2 trial of talampanel, a glutamate receptor inhibitor, for adults with recurrent malignant gliomas.
      ,
      • Grossman S.A.
      • Ye X.
      • Chamberlain M.
      • et al.
      Talampanel with standard radiation and temozolomide in patients with newly diagnosed glioblastoma: a multicenter phase II trial.
      ,
      • Grossman S.A.
      • Ye X.
      • Piantadosi S.
      • et al.
      Survival of patients with newly diagnosed glioblastoma treated with radiation and temozolomide in research studies in the United States.
      NMDA receptorsMemantineGlioblastomaPhase I in association with temozolomideOn going
      Cl-channelTM-601Anaplastic astrocytoma and glioblastomaPhases I/II and III intracavitary
      • Cuddapah V.A.
      • Sontheimer H.
      Ion channels and transporters [corrected] in cancer. 2. Ion channels and the control of cancer cell migration.
      ,
      • Mamelak A.N.
      • Rosenfeld S.
      • Bucholz R.
      • et al.
      Phase I single-dose study of intracavitary-administered iodine-131-TM-601 in adults with recurrent high-grade glioma.
      Talampanel, an allosteric inhibitor of AMPA receptors, entered phase II clinical trials, and it has been speculated that this drug may provide a survival advantage when used in combination with standard chemoradiotherapy [
      • Iwamoto F.M.
      • Kreisl T.N.
      • Kim L.
      • et al.
      Phase 2 trial of talampanel, a glutamate receptor inhibitor, for adults with recurrent malignant gliomas.
      ,
      • Grossman S.A.
      • Ye X.
      • Chamberlain M.
      • et al.
      Talampanel with standard radiation and temozolomide in patients with newly diagnosed glioblastoma: a multicenter phase II trial.
      ] (Table 1). However, Grossman and colleagues challenged their own conclusions one year later [
      • Grossman S.A.
      • Ye X.
      • Piantadosi S.
      • et al.
      Survival of patients with newly diagnosed glioblastoma treated with radiation and temozolomide in research studies in the United States.
      ]. Overall survival of 244 patients with newly diagnosed glioblastoma accrued to three consecutive therapeutic trials conducted by the New Approaches to Brain Tumor Therapy (NABTT) CNS Consortium was significantly higher than is reported with radiation and temozolomide alone [

      Stupp R, Hegi ME, Mason WP, et al; European Organisation for Research and Treatment of Cancer Brain Tumour and Radiation Oncology Groups; National Cancer Institute of Canada Clinical Trials Group. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol 2009; 10: p. 459–66.

      ]. The improvement in survival was evident as early as 6 months after study initiation (94% vs 86% survival, p < 0.003) and continued over two years (37% vs 27%, p < 0.02). In each of these trials a novel agent including talampanel for 72 patients was combined with standard radiation and temozolomide [
      • Grossman S.A.
      • Ye X.
      • Piantadosi S.
      • et al.
      Survival of patients with newly diagnosed glioblastoma treated with radiation and temozolomide in research studies in the United States.
      ]. This report documents significant improvements in the survival of patients with newly diagnosed glioblastoma. However, Grossman et al. emphasized that it remains uncertain if these results are attributable to the novel therapies themselves or reflect evolving patterns of care in this patient population [
      • Grossman S.A.
      • Ye X.
      • Piantadosi S.
      • et al.
      Survival of patients with newly diagnosed glioblastoma treated with radiation and temozolomide in research studies in the United States.
      ]. This argues strongly against the overinterpretation of survival data from uncontrolled phase II studies in newly diagnosed glioblastoma, as has recently been confirmed with the negative outcome of the phase III ACT IV trial [
      • Weller M.
      • Butowski N.
      • Tran D.D.
      • et al.
      ACT IV trial investigators: Rindopepimut with temozolomide for patients with newly diagnosed, EGFRvIII-expressing glioblastoma (ACT IV): a randomised, double-blind, international phase 3 trial.
      ].
      Memantine, a noncompetitive inhibitor of NMDA receptors approved for dementia treatment, is a candidate for modulating the effects of glutamate at the infiltrative rim of gliomas [
      • Blecic S.
      • Rynkowski M.
      • De Witte O.
      • et al.
      Glutamate and malignant gliomas, from epilepsia to biological aggressiveness: therapeutic implications.
      ,
      • Takano T.
      • Lin J.H.
      • Arcuino G.
      • et al.
      Glutamate release promotes growth of malignant gliomas.
      ]. Indeed, a phase I factorial trial of temozolomide, memantine, mefloquine, and metformin for post-radiation therapy glioblastoma is ongoing (https://clinicaltrials.gov/ct2/show/NCT01430351?term=memantine&cond=glioma&rank=3) (Table 1).
      The goal of this study is to identify the highest tolerable dose of TMZ in combination with a mixture of memantine, mefloquine, and/or metformin that can be administered to glioblastoma patients who have previously undergone radiation and chemotherapy in combination. The safety of these drug combinations will also be studied (https://clinicaltrials.gov/ct2/show/NCT01430351?term=memantine&cond=glioma&rank=3). However, whether such complex trials advance standards of care in glioblastoma remains doubtful, and exploring the addition of high doses of memantine alone to standard chemoradiotherapy might have been preferable. Yohay et al. demonstrated that the local polymer-based delivery of riluzole, a glutamate uptake blocker approved for the treatment of amyotrophic lateral sclerosis, or memantine improved survival in a rat intracranial glioma model when the polymer wafers were placed at the time of tumor engraftment [
      • Yohay K.
      • Tyler B.
      • Weaver K.D.
      • et al.
      Efficacy of local polymer-based and systemic delivery of the anti-glutamatergic agents riluzole and memantine in rat glioma models.
      ].
      We speculate that normal neurons might be protected against the high glutamate microenvironment by specific inhibitors of ionotropic receptors but further research is warranted [
      • Chen H.S.
      • Lipton S.A.
      The chemical biology of clinically tolerated NMDA receptor antagonists.
      ,
      • Catarzi D.
      • Colotta V.
      • Varano F.
      Competitive AMPA receptor antagonists.
      ].

       Targeting ion channels

      One Cl- channel inhibitor, TM-601, a synthetic version of the peptide chlorotoxin, a small 36 amino acid neurotoxin isolated from the venom of the giant yellow Israeli scorpion Leiurus quinquestriatus [
      • DeBin J.A.
      • Maggio J.E.
      • Strichartz G.R.
      Purification and characterization of chlorotoxin, a chloride channel ligand from the venom of the scorpion.
      ] that is covalently linked to iodine 131, has completed phase I and II clinical trials for the treatment of anaplastic astrocytomas and glioblastomas through repeated local intracavitary administrations (NCT00591058) [
      • Cuddapah V.A.
      • Sontheimer H.
      Ion channels and transporters [corrected] in cancer. 2. Ion channels and the control of cancer cell migration.
      ,
      • Mamelak A.N.
      • Rosenfeld S.
      • Bucholz R.
      • et al.
      Phase I single-dose study of intracavitary-administered iodine-131-TM-601 in adults with recurrent high-grade glioma.
      ] (Table 1). Intracavitary-administered 131I-TM-601 is well tolerated, remains highly localized to the treatment site, and preliminarily appears safe for repeated injections. As no major toxicity and no death due to 131I-TM-601 have been reported, the FDA allowed the trial to go to Phase III [
      • Mamelak A.N.
      • Jacoby D.B.
      Targeted delivery of antitumoral therapy to glioma and other malignancies with synthetic chlorotoxin (TM-601).
      ]. An FDA approval has also been obtained to investigate the effect of TM601 in newly diagnosed gliomas. TM601 appears to be stable, is not immunogenic and lacks toxicity in humans upon local administration. Parallel to this trial, the same team has worked on an intravenous injection protocol of this product but this time with an imaging application in perspective [
      • Hockaday D.C.
      • Shen S.
      • Fiveash J.
      • et al.
      Imaging glioma extent with 131I-TM-601.
      ]. However, to our knowledge no study has been published recently. Interestingly, intracranial injection of 131I-CTX detects brain tumors by gamma-ray scintigram scans in vivo, but also labels the stomach, indicating that the molecular target of chlorotoxin is also expressed in this organ. So far, TM-601 is the only derivative of chlorotoxin for which human clinical studies have been partially published. Besides the clinical trial of 131I-TM-601, many other chlorotoxin applications have been described that may be potentially useful to treat glioma. A large proportion of them rely on the administration of therapeutic agents, cDNA or siRNA to block oncogene expression, thanks to the use of nanoparticles [
      • Veiseh O.
      • Kievit F.M.
      • Fang C.
      • et al.
      Chlorotoxin bound magnetic nanovector tailored for cancer cell targeting, imaging, and siRNA delivery.
      ,
      • Huang R.
      • Ke W.
      • Han L.
      • et al.
      Targeted delivery of chlorotoxin-modified DNA-loaded nanoparticles to glioma via intravenous administration.
      ].
      Molecular targets for chlorotoxin include voltage gated chloride channels, calcium-dependent phospholipid-binding protein annexin-2, and matrix metalloproteinase-2 (MMP-2) [
      • Deshane J.
      • Garner C.C.
      • Sontheimer H.
      Chlorotoxin inhibits glioma cell invasion via matrix metalloproteinase-2.
      ]. Among these targets, MMP-2 has most anti-neoplastic potential [
      • Deshane J.
      • Garner C.C.
      • Sontheimer H.
      Chlorotoxin inhibits glioma cell invasion via matrix metalloproteinase-2.
      ].
      According to Pardo and Stühmer [
      • Pardo L.A.
      • Stühmer W.
      The roles of K(+) channels in cancer.
      ] and our previous study [
      • Lefranc F.
      • Pouleau H.B.
      • Rynkowski M.
      • et al.
      Voltage-dependent K+ channels as oncotargets in malignant gliomas.
      ], inhibition of potassium channels also has therapeutic potential for glioblastoma patients. Wulf et al. have previously discussed the pharmacological strategies for targeting potassium channels with venom peptides, antibodies or small molecules [
      • Wulff H.
      • Castle N.A.
      • Pardo L.A.
      Voltage-gated potassium channels as therapeutic targets.
      ]. The Kv10.1 and Kv11.1 inhibitors have been proposed for the treatment of various types of cancers. Two potent blockers of Kv10.1, the antihistamine, astemizole, and the tricyclic antidepressant, imipramine, have been shown to decrease tumor cell proliferation in vitro and, in the case of astemizole, also in vivo [
      • García-Quiroz J.
      • Camacho J.
      Astemizole: an old anti-histamine as a new promising anti-cancer drug.
      ]. Imipramine binds to the intracellular pocket and consequently inhibits the current of Kv10.1 [
      • Pardo L.A.
      • Gómez-Varela D.
      • Major F.
      • et al.
      Approaches targeting K(V)10.1 open a novel window for cancer diagnosis and therapy.
      ]. Desipramine and imipramine have been demonstrated to inhibit PI3K/Akt/mTOR signaling and induce autophagic cell death in vitro in murine C6 and human U87MG glioma cells [
      • Ma J.
      • Hou L.N.
      • Rong Z.X.
      • et al.
      Antidepressant desipramine leads to C6 glioma cell autophagy: implication for the adjuvant therapy of cancer.
      ,
      • Jeon S.H.
      • Kim S.H.
      • Kim Y.
      • et al.
      The tricyclic antidepressant imipramine induces autophagic cell death in U-87MG glioma cells.
      ].
      Recently, Sales et al. showed that in vitro suppression of the Kv10.1 channel using astemizole or silencing using a short-hairpin RNA expression vector sensitized U87MG glioma cells to temozolomide [
      • Sales T.T.
      • Resende F.F.
      • Chaves N.L.
      • et al.
      Suppression of the Eag1 potassium channel sensitizes glioblastoma cells to injury caused by temozolomide.
      ].
      Kv11.1 is affected by drugs that cause acquired long QT syndrome [
      • Kalyaanamoorthy S.
      • Barakat K.H.
      Development of Safe Drugs: The hERG Challenge.
      ]. Even if the intracellular channel pore openings of Kv10.1 and Kv11.1 are similar, although not identical, all of the known Kv10.1 blockers are also effective blockers of Kv11.1 and therefore share cardiac safety problems. For these reasons, the antihistamine astemizole was withdrawn from the market in 2000 [
      • Gottlieb S.
      Antihistamine drug withdrawn by manufacturer.
      ]. Because the risk benefit profile of an anticancer drug is radically different from that of compounds developed for the treatment of benign conditions, the potential for these compounds should be reconsidered for repositioning. Moreover, there are at least three alternative transcripts of Kv11.1, which are differentially expressed in heart versus tumor cells, suggesting the option of selective inhibition of this type of ion channel in tumors while preserving heart function. A specific peptide toxin that can achieve this result has yet to be reported. However, a monoclonal antibody (mAb56) has been developed that specifically blocks Kv10.1 without affecting Kv11.1 or its close relative Kv10.2 [
      • Napp J.
      • Pardo L.A.
      • Hartung F.
      • et al.
      In vivo imaging of tumour xenografts with an antibody targeting the potassium channel Kv10.1.
      ]. This antibody showed efficacy in vitro against human melanoma MDA-MB-435S and human pancreatic carcinoma AsPC-1 cell lines as well as in vivo in nude mice subcutaneously grafted with these tumor models, but the required doses were high, whereas the reduction of tumor growth was modest [
      • Napp J.
      • Pardo L.A.
      • Hartung F.
      • et al.
      In vivo imaging of tumour xenografts with an antibody targeting the potassium channel Kv10.1.
      ]. The experiments were performed in immunodeficient mice, so that the antibody could, in principle, act exclusively as a channel blocker. Finally, the anticancer compound roscovitine, a cyclin-dependent kinase inhibitor that has terminated phase II clinical trials for non-small cell lung cancer [
      • Ganapathi S.B.
      • Kester M.
      • Elmslie K.S.
      State-dependent block of HERG potassium channels by R-roscovitine: implications for cancer therapy.
      ], is an efficient blocker of Kv11.1, but does not induce arrhythmia, likely due to its low affinity for closed and inactivated states of the channel.
      Staudacher et al. recently identified the Kv11.1 ligand doxazosin as a small molecule compound that promotes apoptosis and exerts anti-proliferative effects in human glioblastoma cells in vitro [
      • Staudacher I.
      • Jehle J.
      • Staudacher K.
      • et al.
      HERG K+ channel-dependent apoptosis and cell cycle arrest in human glioblastoma cells.
      ]. Doxazosin and future derivatives have therefore been proposed as novel options for more effective glioblastoma treatment [
      • Staudacher I.
      • Jehle J.
      • Staudacher K.
      • et al.
      HERG K+ channel-dependent apoptosis and cell cycle arrest in human glioblastoma cells.
      ].
      Citalopram (Cipramil®), a selective serotonin reuptake inhibitor used for its antidepressive activity, acts on Kv1.5 currents as an open-channel blocker [
      • Lefranc F.
      • Pouleau H.B.
      • Rynkowski M.
      • et al.
      Voltage-dependent K+ channels as oncotargets in malignant gliomas.
      ]. Because both imipramine and citalopram have been commonly used to treat depression, which commonly occurs in glioma patients, the European Organization for Research and Treatment of Cancer (EORTC) conducted a large epidemiological study to investigate the actual benefit that these two drugs may provide for glioblastoma patients. D’Alessandro et al. demonstrated that (i) blockade of the calcium-activated potassium channel KCa3.1 with TRAM-34, a high affinity blocker of KCa3.1, exhibits good selectivity for these channels and increases the TMZ antitumor effects, reducing GL-261 glioma cell migration, invasion and colony forming activity, increasing apoptosis and forcing cells to pass the G2/M cell cycle phase, likely through cdc2 de-phosphorylation; (ii) KCa3.1 silencing potentiates the inhibitory effect of TMZ on glioma cell viability; (iii) the combination of TMZ and TRAM-34 attenuates the toxic effects of glioma conditioned medium on neuronal cultures through a microglia-dependent mechanism since the effect is abolished by clodronate-induced microglia killing; (iv) TMZ/TRAM-34 co-treatment increases the number of apoptotic tumor cells and the mean survival time in a syngeneic mouse glioma model (C57BL6 mice implanted with GL261 cells); and (v) TMZ/TRAM-34 co-treatment reduces the cell viability of glioblastoma cells and cancer stem cells freshly isolated from patients [
      • D'Alessandro G.
      • Grimaldi A.
      • Chece G.
      • et al.
      KCa3.1 channel inhibition sensitizes malignant gliomas to temozolomide treatment.
      ]. Taken together, these data demonstrate that TMZ/TRAM-34 co-treatment affects both glioma cells and infiltrating microglial cells, resulting in an overall reduction of tumor cell progression. Furthermore, calcium activated potassium channel blockade by paxilline, a toxin of the fungus Penicillium paxilli, inhibited ionizing radiation-induced migration in vivo in an orthotopic model of the human glioblastoma model [
      • Edalat L.
      • Stegen B.
      • Klumpp L.
      • et al.
      BK K+ channel blockade inhibits radiation-induced migration/brain infiltration of glioblastoma cells.
      ]. Additionally, ophiobolin-A, a sesterterpenoid phytotoxin produced by pathogenic fungi of the genus Bipolaris, induced marked changes in the dynamic organization of the F-actin cytoskeleton and inhibited proliferation and migration of glioblastoma cells, likely by inhibiting conductance Ca(2+)-activated potassium channel activity [
      • Bury M.
      • Girault A.
      • Mégalizzi V.
      • et al.
      Ophiobolin A induces paraptosis-like cell death in human glioblastoma cells by decreasing BKCa channel activity.
      ].

       Targeting extracellular miRNAs

      MicroRNAs are secreted into the extracellular space, at least in part by EV, and are subsequently taken up by normal and cancer cells to sustain and promote tumor growth (Fig. 3). Glioblastoma EVs transport several tumor-specific microRNAs; however, the functions of most of these microRNAs remain unknown. A better understanding of the role of secreted microRNAs may unveil a novel mechanism of glioblastoma progression and resistance to current treatments and may provide novel diagnostic and prognostic biomarkers. Rooj et al. suggested that these biomarkers can be used for the development of targeted therapies aimed at improving glioblastoma patient outcomes [
      • Rooj A.K.
      • Mineo M.
      • Godlewski J.
      MicroRNA and extracellular vesicles in glioblastoma – Small but powerful.
      ].

      Conclusion and perspectives

      Glioblastoma is an incurable neoplasm characterized by diffusely infiltrative growth. Given the highly invasive nature of glioblastoma, it remains unlikely that any single therapeutic approach will be able to specifically target and eliminate residual heterogeneous tumor cells. New therapeutic strategies to fight both migrating apoptotic resistant and proliferating glioma cells are necessary to effectively counteract glioblastoma progression. Novel treatment approaches have to integrate the intracellular perspective of the complex intracellular signaling pathways with the microenvironmental orchestral machinery, including connections between heterogeneous tumoral and nontumoral cells, potentially also through channels, microtubes, and extracellular miRNA, generating different messages at different times. New avenues for glioblastoma treatment must not ignore the very specific and peculiar interactions between tumor cells and their host tissue, and the prime targets currently appear to be glutamate- and ion channel-controlled processes.

      Conflict of interest

      The authors declared that there is no conflict of interest.

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