Autonomous rhythmic activity in glioma networks drives brain tumour growth.

Diffuse gliomas, particularly glioblastomas, are incurable brain tumours1. They are characterized by networks of interconnected brain tumour cells that communicate via Ca2+ transients2-6. However, the networks' architecture and communication strategy and how these influence tumour biology remain unknown. Here we describe how glioblastoma cell networks include a small, plastic population of highly active glioblastoma cells that display rhythmic Ca2+ oscillations and are particularly connected to others. Their autonomous periodic Ca2+ transients preceded Ca2+ transients of other network-connected cells, activating the frequency-dependent MAPK and NF-κB pathways. Mathematical network analysis revealed that glioblastoma network topology follows scale-free and small-world properties, with periodic tumour cells frequently located in network hubs. This network design enabled resistance against random damage but was vulnerable to losing its key hubs. Targeting of autonomous rhythmic activity by selective physical ablation of periodic tumour cells or by genetic or pharmacological interference with the potassium channel KCa3.1 (also known as IK1, SK4 or KCNN4) strongly compromised global network communication. This led to a marked reduction of tumour cell viability within the entire network, reduced tumour growth in mice and extended animal survival. The dependency of glioblastoma networks on periodic Ca2+ activity generates a vulnerability7 that can be exploited for the development of novel therapies, such as with KCa3.1-inhibiting drugs.

Brain tumour cells interconnect to a functional and resistant network.

Astrocytic brain tumours, including glioblastomas, are incurable neoplasms characterized by diffusely infiltrative growth. Here we show that many tumour cells in astrocytomas extend ultra-long membrane protrusions, and use these distinct tumour microtubes as routes for brain invasion, proliferation, and to interconnect over long distances. The resulting network allows multicellular communication through microtube-associated gap junctions. When damage to the network occurred, tumour microtubes were used for repair. Moreover, the microtube-connected astrocytoma cells, but not those remaining unconnected throughout tumour progression, were protected from cell death inflicted by radiotherapy. The neuronal growth-associated protein 43 was important for microtube formation and function, and drove microtube-dependent tumour cell invasion, proliferation, interconnection, and radioresistance. Oligodendroglial brain tumours were deficient in this mechanism. In summary, astrocytomas can develop functional multicellular network structures. Disconnection of astrocytoma cells by targeting their tumour microtubes emerges as a new principle to reduce the treatment resistance of this disease.

A vaccine targeting mutant IDH1 induces antitumour immunity.

Monoallelic point mutations of isocitrate dehydrogenase type 1 (IDH1) are an early and defining event in the development of a subgroup of gliomas and other types of tumour. They almost uniformly occur in the critical arginine residue (Arg 132) in the catalytic pocket, resulting in a neomorphic enzymatic function, production of the oncometabolite 2-hydroxyglutarate (2-HG), genomic hypermethylation, genetic instability and malignant transformation. More than 70% of diffuse grade II and grade III gliomas carry the most frequent mutation, IDH1(R132H) (ref. 3). From an immunological perspective, IDH1(R132H) represents a potential target for immunotherapy as it is a tumour-specific potential neoantigen with high uniformity and penetrance expressed in all tumour cells. Here we demonstrate that IDH1(R132H) contains an immunogenic epitope suitable for mutation-specific vaccination. Peptides encompassing the mutated region are presented on major histocompatibility complexes (MHC) class II and induce mutation-specific CD4(+) T-helper-1 (TH1) responses. CD4(+) TH1 cells and antibodies spontaneously occurring in patients with IDH1(R132H)-mutated gliomas specifically recognize IDH1(R132H). Peptide vaccination of mice devoid of mouse MHC and transgenic for human MHC class I and II with IDH1(R132H) p123-142 results in an effective MHC class II-restricted mutation-specific antitumour immune response and control of pre-established syngeneic IDH1(R132H)-expressing tumours in a CD4(+) T-cell-dependent manner. As IDH1(R132H) is present in all tumour cells of these slow-growing gliomas, a mutation-specific anti-IDH1(R132H) vaccine may represent a viable novel therapeutic strategy for IDH1(R132H)-mutated tumours.

Tweety-Homolog 1 Drives Brain Colonization of Gliomas.

Early and progressive colonization of the healthy brain is one hallmark of diffuse gliomas, including glioblastomas. We recently discovered ultralong (>10 to hundreds of microns) membrane protrusions [tumor microtubes (TMs)] extended by glioma cells. TMs have been associated with the capacity of glioma cells to effectively invade the brain and proliferate. Moreover, TMs are also used by some tumor cells to interconnect to one large, resistant multicellular network. Here, we performed a correlative gene-expression microarray and in vivo imaging analysis, and identified novel molecular candidates for TM formation and function. Interestingly, these genes were previously linked to normal CNS development. One of the genes scoring highest in tests related to the outgrowth of TMs was tweety-homolog 1 (TTYH1), which was highly expressed in a fraction of TMs in mice and patients. Ttyh1 was confirmed to be a potent regulator of normal TM morphology and of TM-mediated tumor-cell invasion and proliferation. Glioma cells with one or two TMs were mainly responsible for effective brain colonization, and Ttyh1 downregulation particularly affected this cellular subtype, resulting in reduced tumor progression and prolonged survival of mice. The remaining Ttyh1-deficient tumor cells, however, had more interconnecting TMs, which were associated with increased radioresistance in those small tumors. These findings imply a cellular and molecular heterogeneity in gliomas regarding formation and function of distinct TM subtypes, with multiple parallels to neuronal development, and suggest that Ttyh1 might be a promising target to specifically reduce TM-associated brain colonization by glioma cells in patients.SIGNIFICANCE STATEMENT In this report, we identify tweety-homolog 1 (Ttyh1), a membrane protein linked to neuronal development, as a potent driver of tumor microtube (TM)-mediated brain colonization by glioma cells. Targeting of Ttyh1 effectively inhibited the formation of invasive TMs and glioma growth, but increased network formation by intercellular TMs, suggesting a functional and molecular heterogeneity of the recently discovered TMs with potential implications for future TM-targeting strategies.

Meningiomas: Overview and New Directions in Therapy.

The majority of meningiomas, the most common primary brain tumor, are considered to be benign, and characteristic magnetic resonance imaging features allow a preliminary diagnosis. Meningiomas can be classified in the World Health Organization system as grade I, II, or III, depending on various histological features. In many cases, observation is the preferred management option, although this means the absence of a histological diagnosis. If necessary, standard therapy consists of surgery with or without adjuvant radiation, depending on the tumor grade and the degree of resection. To date, systemic therapies are not included in the standard of care. The level of evidence for treatment recommendations is low, and effective treatment regimens, especially for surgery-refractory and radiation-refractory meningiomas, are still very limited. Recent studies have broadened our knowledge of the genetics and pathogenesis of meningiomas and will lead to new therapeutic options. This review summarizes the epidemiology, pathogenesis and genetics, classification, and diagnosis of meningiomas, as well as management principles, including promising new avenues of therapy.

mTOR target NDRG1 confers MGMT-dependent resistance to alkylating chemotherapy.

A hypoxic microenvironment induces resistance to alkylating agents by activating targets in the mammalian target of rapamycin (mTOR) pathway. The molecular mechanisms involved in this mTOR-mediated hypoxia-induced chemoresistance, however, are unclear. Here we identify the mTOR target N-myc downstream regulated gene 1 (NDRG1) as a key determinant of resistance toward alkylating chemotherapy, driven by hypoxia but also by therapeutic measures such as irradiation, corticosteroids, and chronic exposure to alkylating agents via distinct molecular routes involving hypoxia-inducible factor (HIF)-1alpha, p53, and the mTOR complex 2 (mTORC2)/serum glucocorticoid-induced protein kinase 1 (SGK1) pathway. Resistance toward alkylating chemotherapy but not radiotherapy was dependent on NDRG1 expression and activity. In posttreatment tumor tissue of patients with malignant gliomas, NDRG1 was induced and predictive of poor response to alkylating chemotherapy. On a molecular level, NDRG1 bound and stabilized methyltransferases, chiefly O(6)-methylguanine-DNA methyltransferase (MGMT), a key enzyme for resistance to alkylating agents in glioblastoma patients. In patients with glioblastoma, MGMT promoter methylation in tumor tissue was not more predictive for response to alkylating chemotherapy in patients who received concomitant corticosteroids.

Protein kinase Cß as a therapeutic target stabilizing blood-brain barrier disruption in experimental autoimmune encephalomyelitis.

Disruption of the blood-brain barrier (BBB) is a hallmark of acute inflammatory lesions in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis. This disruption may precede and facilitate the infiltration of encephalitogenic T cells. The signaling events that lead to this BBB disruption are incompletely understood but appear to involve dysregulation of tight-junction proteins such as claudins. Pharmacological interventions aiming at stabilizing the BBB in MS might have therapeutic potential. Here, we show that the orally available small molecule LY-317615, a synthetic bisindolylmaleimide and inhibitor of protein kinase Cß, which is clinically under investigation for the treatment of cancer, suppresses the transmigration of activated T cells through an inflamed endothelial cell barrier, where it leads to the induction of the tight-junction molecules zona occludens-1, claudin 3, and claudin 5 and other pathways critically involved in transendothelial leukocyte migration. Treatment of mice with ongoing experimental autoimmune encephalomyelitis with LY-317615 ameliorates inflammation, demyelination, axonal damage, and clinical symptoms. Although LY-317615 dose-dependently suppresses T-cell proliferation and cytokine production independent of antigen specificity, its therapeutic effect is abrogated in a mouse model requiring pertussis toxin. This abrogation indicates that the anti-inflammatory and clinical efficacy is mainly mediated by stabilization of the BBB, thus suppressing the transmigration of encephalitogenic T cells. Collectively, our data suggest the involvement of endothelial protein kinase Cß in stabilizing the BBB in autoimmune neuroinflammation and imply a therapeutic potential of BBB-targeting agents such as LY-317615 as therapeutic approaches for MS.

Antigen dependently activated cluster of differentiation 8-positive T cells cause perforin-mediated neurotoxicity in experimental stroke.

Neuroinflammation plays a key role in secondary brain damage after stroke. Although deleterious effects of proinflammatory cytokines are well characterized, direct cytotoxic effects of invading immune cells on the ischemic brain and the importance of their antigen-dependent activation are essentially unknown. Here we examined the effects of adaptive and innate immune cells-cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells-that share the direct perforin-mediated cytotoxic pathway on outcome after cerebral ischemia in mice. Although CTLs and NK cells both invaded the ischemic brain, only brain-infiltrating CTLs but not NK cells were more activated than their splenic counterparts. Depletion of CTLs decreased infarct volumes and behavioral deficit in two ischemia models, whereas NK cell depletion had no effect. Correspondingly, adoptive CTL transfer from wild-type into Rag1 knock-out mice increased infarct size. Adoptive CTL transfer from perforin knock-out or interferon-γ knock-out mice into Rag1 knock-out mice revealed that CTL neurotoxicity was mediated by perforin. Accordingly, CTLs isolated from wild-type or interferon-γ knock-out but not from perforin knock-out mice induced neuronal cell death in vitro. CTLs derived from ovalbumin-specific T-cell receptor transgenic mice were not activated and infiltrated less into the ischemic brain compared with wild-type CTLs. Their transfer did not increase the infarct size of Rag1 knock-out mice, indicating antigen-dependent activation as an essential component of CTL neurotoxicity. Our findings underscore the importance of antigen-dependent, direct cytotoxic immune responses in stroke and suggest modulation of CTLs and their effector pathways as a potential new strategy for stroke therapy.

Towards optimizing the sequence of bevacizumab and nitrosoureas in recurrent malignant glioma.

Studies on the monoclonal VEGF-A antibody bevacizumab gave raise to questions regarding the lack of an overall survival benefit, the optimal timing in the disease course and potential combination and salvage therapies. We retrospectively assessed survival, radiological progression type on bevacizumab and efficacy of salvage therapies in 42 patients with recurrent malignant gliomas who received bevacizumab and nitrosourea sequentially. 15 patients received bevacizumab followed by nitrosourea at progression and 27 patients vice versa. Time to treatment failure, defined as time from initiation of one to failure of the other treatment, was similar in both groups (9.6 vs. 9.2 months, log rank p = 0.19). Progression-free survival on nitrosoureas was comparable in both groups, while progression-free survival on bevacizumab was longer in the group receiving bevacizumab first (5.3 vs. 4.1 months, log rank p = 0.03). Survival times were similar for patients with grade III (n = 9) and grade IV (n = 33) tumors. Progression-free survival on bevacizumab for patients developing contrast-enhancing T1 progression was longer than for patients who displayed a non-enhancing T2 progression. However, post-progression survival times after bevacizumab failure were not different. Earlier treatment with bevacizumab was not associated with better outcome in this series. The fact that earlier as compared to later bevacizumab treatment does not result in a different time to treatment failure highlights the challenge for first-line or recurrence trials with bevacizumab to demonstrate an overall survival benefit if crossover of bevacizumab-naïve patients after progression occurs.

Insights into cell-to-cell and cell-to-blood-vessel communications in the brain: in vivo multiphoton microscopy.

A complex and reciprocal communication of cells with each other and with relevant parts of the tissue stroma governs many biological processes in both health and disease. However, in the past, the study of these anatomical and molecular interactions has suffered from a lack of appropriate experimental models. An imaging methodology aimed at changing this should allow intravital display and quantification in an intact non-traumatized organ, imaging over a wide range of time spans including extended periods (i.e., months), many repetitive measurements of the same cell or area to permit the study of the cause and consequence of biological processes, the display of various cell types and their reciprocal interaction with each other in three dimensions, the co-registration of relevant physiological parameters and reporters for selected molecular pathways and as high as possible resolution to visualize sub-cellular structures such as organelles. Remarkably, intravital multiphoton microscopy (in vivo MPLSM) through a chronic cranial window allows us to do all these things, making the brain the inner organ of choice for this technology. Here, we give an overview of the application of in vivo MPLSM to study the choreography of cellular, vascular and molecular interactions in the healthy brain and in neurological diseases. We focus on brain tumor formation, progression and response to therapies. This review further aims at demonstrating that we stand at the beginning of full exploitation of the opportunities provided by this technology and gives clues to future directions that appear most promising.

Individual glioblastoma cells harbor both proliferative and invasive capabilities during tumor progression.

BACKGROUND: Glioblastomas are characterized by aggressive and infiltrative growth, and by striking heterogeneity. The aim of this study was to investigate whether tumor cell proliferation and invasion are interrelated, or rather distinct features of different cell populations. METHODS: Tumor cell invasion and proliferation were longitudinally determined in real-time using 3D in vivo 2-photon laser scanning microscopy over weeks. Glioblastoma cells expressed fluorescent markers that permitted the identification of their mitotic history or their cycling versus non-cycling cell state. RESULTS: Live reporter systems were established that allowed us to dynamically determine the invasive behavior, and previous or actual proliferation of distinct glioblastoma cells, in different tumor regions and disease stages over time. Particularly invasive tumor cells that migrated far away from the main tumor mass, when followed over weeks, had a history of marked proliferation and maintained their proliferative capacity during brain colonization. Infiltrating cells showed fewer connections to the multicellular tumor cell network, a typical feature of gliomas. Once tumor cells colonized a new brain region, their phenotype progressively transitioned into tumor microtube-rich, interconnected, slower-cycling glioblastoma cells. Analysis of resected human glioblastomas confirmed a higher proliferative potential of tumor cells from the invasion zone. CONCLUSIONS: The detection of glioblastoma cells that harbor both particularly high proliferative and invasive capabilities during brain tumor progression provides valuable insights into the interrelatedness of proliferation and migration-2 central traits of malignancy in glioma. This contributes to our understanding of how the brain is efficiently colonized in this disease.

Does age matter? - A MRI study on peritumoral edema in newly diagnosed primary glioblastoma.

BACKGROUND: Peritumoral edema is a characteristic feature of malignant glioma related to the extent of neovascularisation and to vascular endothelial growth factor (VEGF) expression.The extent of peritumoral edema and VEGF expression may be prognostic for patients with glioblastoma. As older age is a negative prognostic marker and as VEGF expression is reported to be increased in primary glioblastoma of older patients, age-related differences in the extent of peritumoral edema have been assessed. METHODS: In a retrospective, single-center study, preoperative magnetic resonance imaging (MRI) scans of steroid-naïve patients (n = 122) of all age groups were analysed. Patients with clinically suspected, radiologically likely or known evidence of secondary glioblastoma were not included.Extent of brain edema was determined in a metric quantitative fashion and in a categorical fashion in relation to tumor size. Analysis was done group-wise related to age. Additionally, tumor size, degree of necrosis, superficial or deep location of tumor and anatomic localization in the brain were recorded. RESULTS: The extent of peritumoral edema in patients >65 years (ys) was not different from the edema extent in patients ≤ 65 ys (p = 0.261). The same was true if age groups ≤ 55 ys and ≥ 70 ys were compared (p = 0.308). However, extent of necrosis (p = 0.023), deep tumor localization (p = 0.02) and frontal localisation (p = 0.016) of the tumor were associated with the extent of edema. Tumor size was not linearly correlated to edema extent (Pearson F = 0.094, p = 0.303) but correlated to degree of necrosis (F = 0.355, p < 0.001, Spearman-Rho) and depth of tumor (p < 0.001). In a multifactorial analysis of maximum edema with the uncorrelated factors age, regional location of tumor and degree of necrosis, only the extent of necrosis (p = 0.022) had a significant effect. CONCLUSION: Age at diagnosis does not determine degree of peritumoral edema, and tumor localization in the white matter is associated with greater extent of edema. The area of necrosis is reflective of volume of edema. In summary, the radiographic appearance of a glioblastoma at diagnosis does not reflect biology in the elderly patient.

Tumor cell plasticity, heterogeneity, and resistance in crucial microenvironmental niches in glioma.

Both the perivascular niche (PVN) and the integration into multicellular networks by tumor microtubes (TMs) have been associated with progression and resistance to therapies in glioblastoma, but their specific contribution remained unknown. By long-term tracking of tumor cell fate and dynamics in the live mouse brain, differential therapeutic responses in both niches are determined. Both the PVN, a preferential location of long-term quiescent glioma cells, and network integration facilitate resistance against cytotoxic effects of radiotherapy and chemotherapy-independently of each other, but with additive effects. Perivascular glioblastoma cells are particularly able to actively repair damage to tumor regions. Population of the PVN and resistance in it depend on proficient NOTCH1 expression. In turn, NOTCH1 downregulation induces resistant multicellular networks by TM extension. Our findings identify NOTCH1 as a central switch between the PVN and network niche in glioma, and demonstrate robust cross-compensation when only one niche is targeted.

Identification and Characterization of Cancer Cells That Initiate Metastases to the Brain and Other Organs.

Specific biological properties of those circulating cancer cells that are the origin of brain metastases (BM) are not well understood. Here, single circulating breast cancer cells were fate-tracked during all steps of the brain metastatic cascade in mice after intracardial injection over weeks. A novel in vivo two-photon microscopy methodology was developed that allowed to determine the specific cellular and molecular features of breast cancer cells that homed in the brain, extravasated, and successfully established a brain macrometastasis. Those BM-initiating breast cancer cells (BMIC) were mainly originating from a slow-cycling subpopulation that included only 16% to 20% of all circulating cancer cells. BMICs showed enrichment of various markers of cellular stemness. As a proof of principle for the principal usefulness of this approach, expression profiling of BMICs versus non-BMICs was performed, which revealed upregulation of NDRG1 in the slow-cycling BMIC subpopulation in one BM model. Here, BM development was completely suppressed when NDRG1 expression was downregulated. In accordance, in primary human breast cancer, NDRG1 expression was heterogeneous, and high NDRG1 expression was associated with shorter metastasis-free survival. In conclusion, our data identify temporary slow-cycling breast cancer cells as the dominant source of brain and other metastases and demonstrates that this can lead to better understanding of BMIC-relevant pathways, including potential new approaches to prevent BM in patients. IMPLICATIONS: Cancer cells responsible for successful brain metastasis outgrowth are slow cycling and harbor stemness features. The molecular characteristics of these metastasis-initiating cells can be studied using intravital microscopy technology.

Emerging intersections between neuroscience and glioma biology.

The establishment of neuronal and glial networks in the brain depends on the activities of neural progenitors, which are influenced by cell-intrinsic mechanisms, interactions with the local microenvironment and long-range signaling. Progress in neuroscience has helped identify key factors in CNS development. In parallel, studies in recent years have increased our understanding of molecular and cellular factors in the development and growth of primary brain tumors. To thrive, glioma cells exploit pathways that are active in normal CNS progenitor cells, as well as in normal neurotransmitter signaling. Furthermore, tumor cells of incurable gliomas integrate into communicating multicellular networks, where they are interconnected through neurite-like cellular protrusions. In this Review, we discuss evidence that CNS development, organization and function share a number of common features with glioma progression and malignancy. These include mechanisms used by cells to proliferate and migrate, interact with their microenvironment and integrate into multicellular networks. The emerging intersections between the fields of neuroscience and neuro-oncology considered in this review point to new research directions and novel therapeutic opportunities.

Differential Effects of Ang-2/VEGF-A Inhibiting Antibodies in Combination with Radio- or Chemotherapy in Glioma.

Antiangiogenic strategies have not shown striking antitumor activities in the majority of glioma patients so far. It is unclear which antiangiogenic combination regimen with standard therapy is most effective. Therefore, we compared anti-VEGF-A, anti-Ang2, and bispecific anti-Ang-2/VEGF-A antibody treatments, alone and in combination with radio- or temozolomide (TMZ) chemotherapy, in a malignant glioma model using multiparameter two-photon in vivo microscopy in mice. We demonstrate that anti-Ang-2/VEGF-A lead to the strongest vascular changes, including vascular normalization, both as monotherapy and when combined with chemotherapy. The latter was accompanied by the most effective chemotherapy-induced death of cancer cells and diminished tumor growth. This was most probably due to a better tumor distribution of the drug, decreased tumor cell motility, and decreased formation of resistance-associated tumor microtubes. Remarkably, all these parameters where reverted when radiotherapy was chosen as combination partner for anti-Ang-2/VEGF-A. In contrast, the best combination partner for radiotherapy was anti-VEGF-A. In conclusion, while TMZ chemotherapy benefits most from combination with anti-Ang-2/VEGF-A, radiotherapy does from anti-VEGF-A. The findings imply that uninformed combination regimens of antiangiogenic and cytotoxic therapies should be avoided.

Anti-Angiogenics: Their Role in the Treatment of Glioblastoma.

Angiogenesis is a hallmark of glioblastomas, but anti-angiogenic therapies have fallen short of the initial expectations to relevantly change the clinical course of the disease. Only one agent, the anti-vascular endothelial growth factor (VEGF)-A antibody bevacizumab, has shown meaningful efficacy in controlled clinical trials in glioblastoma, so far. In primary and recurrent glioblastoma, this efficacy is, however, limited to prolonging progression-free survival and to generating some additional palliative benefits, without affecting overall survival in the total population of glioblastoma patients. Here, we give an overview of the current status of anti-angiogenic therapy in glioblastoma, including how it is currently used in the clinic. Furthermore, we discuss avenues of biomarker research aiming to identify those glioblastoma patients with a higher likelihood of profiting from anti-VEGF-A therapies (and to identify those who will not). Together with novel anti-angiogenic treatment targets and combination regimens under development today, those might improve the current clinical benefits from this class of drugs in glioblastoma.

Treatment of glioblastoma in adults.

The diagnosis of a glioblastoma is mainly made on the basis of their microscopic appearance with the additional determination of epigenetic as well as mutational analyses as deemed appropriate and taken into account in different centers. How far the recent discovery of tumor networks will stimulate novel treatments is a subject of intensive research. A tissue diagnosis is the mainstay. Regardless of age, patients should undergo a maximal safe resection. Magnetic resonance imaging is the surrogate parameter of choice for follow up. Patients should receive chemoradiotherapy with temozolomide with the radiation schedule adapted to performance status, age and tumor location. The use of temozolomide may be reconsidered according to methylguanine DNA methyltransferase (MGMT) promoter methylation status; patients with an active promoter may be subjected to a trial or further molecular work-up in order to potentially replace temozolomide; patients with an inactive (hypermethylated) MGMT promoter may be counseled for the co-treatment with the methylating and alkylating compound lomustine in addition to temozolomide. Tumor-treating fields are an additive option independent of the MGMT status. Determination of recurrence is still challenging. Patients with clinical or radiographic confirmed progression should be counseled for a second surgical intervention, that is, to reach another macroscopic removal of the tumor bulk or to obtain tissue for an updated molecular analysis. Immune therapeutic approaches may be dependent on tumor types and molecular signatures. In newly diagnosed and recurrent glioblastoma, bevacizumab prolongs progression-free survival without affecting overall survival in an unselected population of glioblastoma patients. Whether or not selection can be made on the basis of molecular or imaging parameters remains to be determined. Some patients may benefit from a second radiotherapy. In our view, the near future will provide support for translating the amazing progress in understanding the molecular background of glioblastoma in to more complex, but promising therapy concepts.

Tumor microtubes convey resistance to surgical lesions and chemotherapy in gliomas.

BACKGROUND: Primary and adaptive resistance against chemo- and radiotherapy and local recurrence after surgery limit the benefits from these standard treatments in glioma patients. Recently we found that glioma cells can extend ultra-long membrane protrusions, "tumor microtubes" (TMs), for brain invasion, proliferation, and interconnection of single cells to a syncytium that is resistant to radiotherapy. We wondered whether TMs also convey resistance to the other 2 standard treatment modalities. METHODS: Patient-derived glioblastoma stemlike cell (GBMSC) lines were implanted under a cranial window in mice. Longitudinal in vivo two-photon laser scanning microscopy was used to follow tumor growth, including the fate of single glioma cells over months. RESULTS: After a cylindrical surgical lesion, GBMSCs increasingly extended TMs toward the lesion area, which contributed to the repopulation of this area over many weeks. In fact, an excessive "healing response" was observed in which tumor cell densities significantly exceeded those of unlesioned brain regions over time. Inhibition of TM 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 temozolomide chemotherapy, intra- and intertumoral heterogeneity of TM formation and interconnection was strongly associated with therapy response: when tumor cells were integrated in TM networks, they were more likely to resist chemotherapy. CONCLUSION: TMs can contribute to the resistance against standard treatment modalities in gliomas. Specific inhibition of TMs is a promising approach to reduce local recurrence after surgery and lower resistance to chemotherapy.

Tunneling Nanotubes and Gap Junctions-Their Role in Long-Range Intercellular Communication during Development, Health, and Disease Conditions.

Cell-to-cell communication is essential for the organization, coordination, and development of cellular networks and multi-cellular systems. Intercellular communication is mediated by soluble factors (including growth factors, neurotransmitters, and cytokines/chemokines), gap junctions, exosomes and recently described tunneling nanotubes (TNTs). It is unknown whether a combination of these communication mechanisms such as TNTs and gap junctions may be important, but further research is required. TNTs are long cytoplasmic bridges that enable long-range, directed communication between connected cells. The proposed functions of TNTs are diverse and not well understood but have been shown to include the cell-to-cell transfer of vesicles, organelles, electrical stimuli and small molecules. However, the exact role of TNTs and gap junctions for intercellular communication and their impact on disease is still uncertain and thus, the subject of much debate. The combined data from numerous laboratories indicate that some TNT mediate a long-range gap junctional communication to coordinate metabolism and signaling, in relation to infectious, genetic, metabolic, cancer, and age-related diseases. This review aims to describe the current knowledge, challenges and future perspectives to characterize and explore this new intercellular communication system and to design TNT-based therapeutic strategies.