Microphysiological systems to study tumor-stroma interactions in brain cancer.

Brain tumors still lack effective treatments, and the mechanisms of tumor progression and therapeutic resistance are unclear. Multiple parameters affect cancer prognosis (e.g., type and grade, age, location, size, and genetic mutations) and election of suitable treatments is based on preclinical models and clinical data. However, most candidate drugs fail in human trials due to inefficacy. Cell lines and tissue culture plates do not provide physiologically relevant environments, and animal models are not able to adequately mimic characteristics of disease in humans. Therefore, increasing technological advances are focusing on in vitro and computational modeling to increase the throughput and predicting capabilities of preclinical systems. The extensive use of these therapeutic agents requires a more profound understanding of the tumor-stroma interactions, including neural tissue, extracellular matrix, blood-brain barrier, astrocytes and microglia. Microphysiological brain tumor models offer physiologically relevant vascularized 'minitumors' that can help deciphering disease mechanisms, accelerating the drug discovery and predicting patient's response to anticancer treatments. This article reviews progress in tumor-on-a-chip platforms that are designed to comprehend the particular roles of stromal cells in the brain tumor microenvironment.

Evidence for residual SARS-CoV-2 in glioblastoma tissue of a convalescent patient.

Since coronavirus disease 2019 (COVID-19) swept all over the world, several studies have shown the susceptibility of a patient with cancer to COVID-19. In this case, the removed glioblastoma multiforme (GBM)-adjacent (GBM-A), GBM-peritumor and GBM-central (GBM-C) tissues from a convalescent patient of COVID-19, who also suffered from glioblastoma meanwhile, together with GBM-A and GBM tissues from a patient without COVID-19 history as negative controls, were used for RNA ISH, electron microscopy observing and immunohistochemical staining of ACE2 and the virus antigen (N protein). The results of RNA ISH, electron microscopy observing showed that SARS-CoV-2 directly infects some cells within human GBM tissues and SARS-CoV-2 in GBM-C tissue still exists even when it is cleared elsewhere. Immunohistochemical staining of ACE2 and N protein showed that the expressions of ACE2 are significantly higher in specimens, including GBM-C tissue from COVID-19 patient than other types of tissue. The unique phenomenon suggests that the surgical protection level should be upgraded even if the patient is in a convalescent period and the pharyngeal swab tests show negative results. Furthermore, more attention should be paid to confirm whether the shelter-like phenomenon happens in other malignancies due to the similar microenvironment and high expression of ACE2 in some malignancies.

The white matter is a pro-differentiative niche for glioblastoma.

Glioblastomas are hierarchically organised tumours driven by glioma stem cells that retain partial differentiation potential. Glioma stem cells are maintained in specialised microenvironments, but whether, or how, they undergo lineage progression outside of these niches remains unclear. Here we identify the white matter as a differentiative niche for glioblastomas with oligodendrocyte lineage competency. Tumour cells in contact with white matter acquire pre-oligodendrocyte fate, resulting in decreased proliferation and invasion. Differentiation is a response to white matter injury, which is caused by tumour infiltration itself in a tumoursuppressive feedback loop. Mechanistically, tumour cell differentiation is driven by selective white matter upregulation of SOX10, a master regulator of normal oligodendrogenesis. SOX10 overexpression or treatment with myelination-promoting agents that upregulate endogenous SOX10, mimic this response, leading to niche-independent pre-oligodendrocyte differentiation and tumour suppression in vivo. Thus, glioblastoma recapitulates an injury response and exploiting this latent programme may offer treatment opportunities for a subset of patients.

Titanium Dioxide, but Not Zinc Oxide, Nanoparticles Cause Severe Transcriptomic Alterations in T98G Human Glioblastoma Cells.

Titanium dioxide and zinc oxide are two of the most widely used nanomaterials. We assessed the effects of noncytotoxic doses of both nanomaterials on T98G human glioblastoma cells by omic approaches. Surprisingly, no effects on the transcriptome of T98G cells was detected after exposure to 5 µg/mL of zinc oxide nanoparticles during 72 h. Conversely, the transcriptome of the cells exposed to 20 µg/mL of titanium dioxide nanoparticles during 72 h revealed alterations in lots of biological processes and molecular pathways. Alterations to the transcriptome suggests that exposure to titanium dioxide nanoparticles might, potentially, compromise the integrity of the blood brain barrier integrity and cause neuroinflammation. The latter issue was further confirmed phenotypically with a proteomic analysis and by recording the release of interleukin 8. Titanium dioxide also caused autophagy, which was demonstrated through the increase in the expression of the autophagy-related 3 and microtubule associated protein 1 light chain 3 alpha genes. The proteomic analysis revealed that titanium dioxide nanoparticles might have anticancerigen properties by downregulating genes involved in the detoxication of anthracyclines. A risk assessment resulting from titanium dioxide exposure, focusing on the central nervous system as a potential target of toxicity, is necessary.

[Pt(O,O'-acac)(γ-acac)(DMS)]: Alternative Strategies to Overcome Cisplatin-Induced Side Effects and Resistance in T98G Glioma Cells.

Cisplatin (CDDP) is one of the most effective chemotherapeutic agents, used for the treatment of diverse tumors, including neuroblastoma and glioblastoma. CDDP induces cell death through different apoptotic pathways. Despite its clinical benefits, CDDP causes several side effects and drug resistance.[Pt(O,O'-acac)(γ-acac)(DMS)], namely PtAcacDMS, a new platinum(II) complex containing two acetylacetonate (acac) and a dimethylsulphide (DMS) in the coordination sphere of metal, has been recently synthesized and showed 100 times higher cytotoxicity than CDDP. Additionally, PtAcacDMS was associated to a decreased neurotoxicity in developing rat central nervous system, also displaying great antitumor and antiangiogenic activity both in vivo and in vitro. Thus, based on the knowledge that several chemotherapeutics induce cancer cell death through an aberrant increase in [Ca2+]i, in the present in vitro study we compared CDDP and PtAcacDMS effects on apoptosis and intracellular Ca2+ dynamics in human glioblastoma T98G cells, applying a battery of complementary techniques, i.e., flow cytometry, immunocytochemistry, electron microscopy, Western blotting, qRT-PCR, and epifluorescent Ca2+ imaging. The results confirmed that (i) platinum compounds may induce cell death through an aberrant increase in [Ca2+]i and (ii) PtAcacDMS exerted stronger cytotoxic effect than CDDP, associated to a larger increase in resting [Ca2+]i. These findings corroborate the use of PtAcacDMS as a promising approach to improve Pt-based chemotherapy against gliomas, either by inducing a chemosensitization or reducing chemoresistance in cell lineages resilient to CDDP treatment.

Dihydroartemisinin initiates ferroptosis in glioblastoma through GPX4 inhibition.

It has been demonstrated from previous studies about the killing effect of dihydroartemisinin (DHA) on glioblastoma, which involves multiple aspects: cytotoxicity, cell cycle arrest and invasion inhibition. DHA has the advantages of low cytotoxicity to normal cells, selective killing effect and low drug resistance, making it one of the popular anti-tumor research directions. Ferroptosis is a newly discovered form of cell death characterized by iron dependence and lipid reactive oxygen species (ROS) accumulation. In the present study, we found differences in the expression of transferrin receptors in normal human astrocytes (NHA) and glioblastoma cells (U87 and A172), which may be one of the mechanisms of DHA selective killing effect. Through the determination of ferroptosis-related protein expression, we found that the significant decrease of GPX4, accompanied by the constant expression of xCT and ACSL4, suggesting GPX4 was a pivotal target for DHA-activated ferroptosis in glioblastoma. Total and lipid ROS levels were increased and all these results could be reversed by the ferroptosis inhibitor, ferrostatin-1. These findings demonstrated ferroptosis would be a critical component of cell death caused by DHA and GPX4 was the main target. All these results provide a novel treatment direction to glioblastoma. The association between ferroptosis and polyamines is also discussed, which will provide new research directions for ferroptosis caused by DHA in glioblastoma.

A New Pathway Promotes Adaptation of Human Glioblastoma Cells to Glucose Starvation.

Adaptation of glioblastoma to caloric restriction induces compensatory changes in tumor metabolism that are incompletely known. Here we show that in human glioblastoma cells maintained in exhausted medium, SHC adaptor protein 3 (SHC3) increases due to down-regulation of SHC3 protein degradation. This effect is reversed by glucose addition and is not present in normal astrocytes. Increased SHC3 levels are associated to increased glucose uptake mediated by changes in membrane trafficking of glucose transporters of the solute carrier 2A superfamily (GLUT/SLC2A). We found that the effects on vesicle trafficking are mediated by SHC3 interactions with adaptor protein complex 1 and 2 (AP), BMP-2-inducible protein kinase and a fraction of poly ADP-ribose polymerase 1 (PARP1) associated to vesicles containing GLUT/SLC2As. In glioblastoma cells, PARP1 inhibitor veliparib mimics glucose starvation in enhancing glucose uptake. Furthermore, cytosol extracted from glioblastoma cells inhibits PARP1 enzymatic activity in vitro while immunodepletion of SHC3 from the cytosol significantly relieves this inhibition. The identification of a new pathway controlling glucose uptake in high grade gliomas represents an opportunity for repositioning existing drugs and designing new ones.

Ultrastructural evidence for presenсe of gap junctions in rare case of pleomorphic xanthoastrocytoma.

The phenomenon of unstable expression of gap junction's proteins connexins remains a "visiting card" of astrocytic tumors with various degrees of malignancy. At the same time, it stays unclear what is detected by the positive expression of connexins in astrocytic tumors: gap junctions, hemi-channels, or connexin proteins in cytosol. In the present work, for the first time, we demonstrate an ultrastructural evidence of gap junctions in pleomorphic xanthoastrocytoma, a rare primary brain tumor, the intercellular characteristics of which are poorly studied and remain very discursive and controversial. The primary tumor mass was resected during craniotomy from a 57-old patient diagnosed with pleomorphic xanthoastrocytoma Grade II based on the histopathological analysis. The immunohistochemical study was conducted with primary antibodies: Neurofilament, Myelin basic protein, Glial fibrillary acidic protein, and Synaptophysin. For electron microscopic examination fragments of tumor tissue were fixed in a glutaraldehyde, postfixed in a 1% OsO4, dehydrated and embedded into resin. After the detailed clinical, histological, and immunohistochemical study we revealed some ultrastructural characteristics of the tumor, as well as the first evidence of direct intercellular connection between the tumor cells via gap junctions. Regularly arranged gap junctions connected the somas of xanthastrocytes with dark cytoplasm containing lipid drops. Besides the localization between the cell bodies, from one to several gap junctions were found between the branches of xanthoastrocytoma in tumor intercellular space in close proximity to tumor cell. Our results may indicate gap junctions as a possible structure for intercellular communication between pleomorphic xanthoastrocytoma cells.

Establishment and Characterization of Cell Lines from Primary Culture of Hemangioblastoma Stromal Cells.

CONTEXT: A well-established cell line of hemangioblastomas (HBs) is still lacking. AIM: This study aims to explore a stable way to establish primary cell lines of HB stromal cells and investigate the morphological and molecular features of these cells. PATIENTS AND METHODS: Specimens of HBs from 13 patients were collected for establishment of primary cell lines of stromal cells. The details on cell culture were described, and the characterizations of cultured cells were conducted by morphological observation, immunocytochemical staining of inhibin-α, brachyury, CD133, CD34, GFAP, CD31, NeuN, CD45, Oligo2, and transmission electron microscopy. RESULTS: Eleven cases were successfully cultured with a success rate of 84.6%. The cultured cells survived for 10 generations with an estimated doubling time of 77.2 ± 5.89 h. Light microscopy revealed that these cells showed vigorous growth status and presented as polygons or trigons with significant heterogeneity. The immunocytochemical staining showed that inhibin-α, brachyury, CD133, and CD34 were expressed in all the cultured cells, whereas the expression of GFAP, CD31, NeuN, CD45, and Oligo2 was all negative. Transmission electron microscopy confirmed that the cultured cells were stromal cells with typical lipid droplets. The phenomenon of lysosomal autophagy was commonly observed without apoptotic cells in late stage. CONCLUSION: Appropriate selection of tumor specimens, short duration of devascularization, ideal digestion time, and nutritious medium are critical points for establishment of primary cell line of HB stromal cells. Stromal cells from both von Hippel-Lindau disease-related HBs and sporadic HBs might originate from embryologically arrested hemangioblasts.

A role for caveola-forming proteins caveolin-1 and CAVIN1 in the pro-invasive response of glioblastoma to osmotic and hydrostatic pressure.

In solid tumours, elevated interstitial fluid pressure (osmotic and hydrostatic pressure) is a barrier to drug delivery and correlates with poor prognosis. Glioblastoma (GBM) further experience compressive force when growing within a space limited by the skull. Caveolae are proposed to play mechanosensing roles, and caveola-forming proteins are overexpressed in GBM. We asked whether caveolae mediate the GBM response to osmotic pressure. We evaluated in vitro the influence of spontaneous or experimental down-regulation of caveola-forming proteins (caveolin-1, CAVIN1) on the proteolytic profile and invasiveness of GBM cells in response to osmotic pressure. In response to osmotic pressure, GBM cell lines expressing caveola-forming proteins up-regulated plasminogen activator (uPA) and/or matrix metalloproteinases (MMPs), some EMT markers and increased their in vitro invasion potential. Down-regulation of caveola-forming proteins impaired this response and prevented hyperosmolarity-induced mRNA expression of the water channel aquaporin 1. CRISPR ablation of caveola-forming proteins further lowered expression of matrix proteases and EMT markers in response to hydrostatic pressure, as a model of mechanical force. GBM respond to pressure by increasing matrix-degrading enzyme production, mesenchymal phenotype and invasion. Caveola-forming proteins mediate, at least in part, the pro-invasive response of GBM to pressure. This may represent a novel target in GBM treatment.

Ependymoma in a dwarf goat.

Ependymomas are relatively rare neuroglial tumours that derive from ependymal cells, lining the ventricles of the brain and the central canal of the spinal cord. They occur particularly in dogs, while reports in goats are extremely scarce. A 15-year-old female dwarf goat was found in lateral recumbency, developed opisthotonus and was killed humanely. Necropsy revealed a well-demarcated, non-encapsulated mass in the diencephalon at the level of the interthalamic adhesion. Histologically, the neoplasm showed highly cellular sheets of tumour cells with occasional perivascular pseudorosettes and true rosettes. Immunohistochemistry revealed an extensive and perivascularly accentuated expression of S100 protein and glial fibrillary acidic protein, while vimentin expression was observed to a minor extent. Tumour cells were negative for cytokeratin and CNPase. Ultrastructurally, intercellular junctions were present, but cilia and blepharoblasts were lacking. The presented findings are consistent with a cellular subtype of an ependymoma. Ependymomas should be regarded as a rare cause of central nervous signs in goats.

Extracellular vesicles originating from glioblastoma cells increase metalloproteinase release by astrocytes: the role of CD147 (EMMPRIN) and ionizing radiation.

BACKGROUND: Glioblastoma multiforme is an aggressive primary brain tumor that is characterized by local invasive growth and resistance to therapy. The role of the microenvironment in glioblastoma invasiveness remains unclear. While carcinomas release CD147, a protein that signals for increased matrix metalloproteinase (MMP) release by fibroblasts, glioblastoma does not have a significant fibroblast component. We hypothesized that astrocytes release MMPs in response to CD147 contained in glioblastoma-derived extracellular vesicles (EVs) and that ionizing radiation, part of the standard treatment for glioblastoma, enhances this release. METHODS: Astrocytes were incubated with EVs released by irradiated or non-irradiated human glioblastoma cells wild-type, knockdown, or knockout for CD147. Levels of CD147 in glioblastoma EVs and MMPs secreted by astrocytes were quantified. Levels of proteins in the mitogen activated protein kinase (MAPK) pathway, which can be regulated by CD147, were measured in astrocytes incubated with EVs from glioblastoma cells wild-type or knockdown for CD147. Immunofluorescence was performed on the glioblastoma cells to identify changes in CD147 localization in response to irradiation, and to confirm uptake of the EVs by astrocytes. RESULTS: Immunoblotting and mass spectrometry analyses showed that CD147 levels in EVs were transiently increased when the EVs were from glioblastoma cells that were irradiated with γ rays. Specifically, the highly-glycosylated 45 kDa form of CD147 was preferentially present in the EVs relative to the cells themselves. Immunofluorescence demonstrated that astrocytes incorporate glioblastoma EVs and subsequently increase their secretion of active MMP9. The increase was greater if the EVs were from irradiated glioblastoma cells. Testing MAPK pathway activation, which also regulates MMP expression, showed that JNK signaling, but not ERK1/2 or p38, was increased in astrocytes incubated with EVs from irradiated compared to non-irradiated glioblastoma cells. Knockout of CD147 in glioblastoma cells blocked the increased JNK signaling and the rise in secreted active MMP9 levels. CONCLUSIONS: The results support a tumor microenvironment-mediated role of CD147 in glioblastoma invasiveness, and reveal a prominent role for ionizing radiation in enhancing the effect. They provide an improved understanding of glioblastoma intercellular signaling in the context of radiotherapy, and identify pathways that can be targeted to reduce tumor invasiveness. Video abstract.

Intracranial Low-Grade Fibromyxoid Sarcoma: Findings on Electron Microscopy and Histologic Analysis.

BACKGROUND: Low-grade fibromyxoid sarcoma is a rare tumor that manifests as a deep soft tissue mass characterized by a benign histology, but with potentially aggressive clinical behavior and a high rate of recurrence; primary intracranial sarcomas are even rarer. We present a case of primary intracranial low-grade fibromyxoid sarcoma, emphasizing its clinical, radiologic, and histologic features. CASE DESCRIPTION: A 39-year-old woman presented with a recent history of headache and seizures. A right parietal mass was detected on computed tomography and magnetic resonance imaging, and she was subsequently operated on to remove the intracranial mass. Histologic examination of the resected tumor revealed mildly atypical fibroblastic cells embedded within a myxoid matrix. The diagnosis was confirmed by electron microscopy and cytogenetic analyses. CONCLUSIONS: This report describes electron microscopic evaluation of intracranial low-grade fibromyxoid sarcoma, which has an extremely rare occurrence.

Telomerase activity, TERT expression, hTERT promoter alterations, and alternative lengthening of the telomeres (ALT) in meningiomas - a systematic review.

Telomerase activity and (human) Telomerase Reverse Transcriptase (hTERT) expression are considered hallmarks in oncogenesis of neoplasms and are upregulated by alterations of the hTERT promoter. In meningiomas, numerous studies investigated hTERT expression, telomerase activity, promoter mutations, and methylations. Moreover, reports about hTERT-targeted chemotherapy in meningiomas have recently been published. We provide a systematic review of the literature about the role of hTERT in meningiomas. TERT expression and telomerase activity is found in benign and high-grade meningiomas and increase with WHO grade. Remarkably, rates of TERT expression/telomerase activity usually exceed mutation frequency and both telomerase activity and TERT expression have also been found in hTERT promoter wildtype meningiomas, indicating further mechanisms of TERT upregulation. Although hTERT promoter methylation has been reported in the vast majority of meningiomas, correlation with TERT expression remains controversial. Rates of promoter mutations, and methylation were shown to increase with rising WHO grade. Moreover, promoter methylation and mutations strongly correlate with prognosis. Although mutations predicted malignant progression, de novo mutations in high-grade recurrences of former benign lesions were also observed. Retroviral transduction of the TERT gene enabled immortalization in several grade I-III meningioma cell lines. In vitro analyses revealed significant effects on viability in hTERT-mutated meningioma cells after targeted treatment. Alternative mechanisms of telomere lengthening are usually absent in meningiomas. TERT and hTERT promoter alterations play a major role during oncogenesis of meningiomas with implications for prognosis and potentially treatment.

Reduced expression of proteolipid protein 2 increases ER stress-induced apoptosis and autophagy in glioblastoma.

Proteolipid protein 2 (PLP2) is an integral ion channel membrane protein of the endoplasmic reticulum. The protein has been shown to be highly expressed in many cancer types, but its importance in glioma progression is poorly understood. Using publicly available datasets (Rembrandt, TCGA and CGGA), we found that the expression of PLP2 was significantly higher in high-grade gliomas than in low-grade gliomas. We confirmed these results at the protein level through IHC staining of high-grade (n = 56) and low-grade glioma biopsies (n = 16). Kaplan-Meier analysis demonstrated that increased PLP2 expression was associated with poorer patient survival. In functional experiments, siRNA and shRNA PLP2 knockdown induced ER stress and increased apoptosis and autophagy in U87 and U251 glioma cell lines. Inhibition of autophagy with chloroquine augmented apoptotic cell death in U87- and U251-siPLP2 cells. Finally, intracranial xenografts derived from U87- and U251-shPLP2 cells revealed that loss of PLP2 reduced glioma growth in vivo. Our results therefore indicate that increased PLP2 expression promotes GBM growth and that PLP2 represents a potential future therapeutic target.

A gain of function paradox: Targeted therapy for glioblastoma associated with abnormal NHE9 expression.

Glioblastoma (GBM) is the most frequent and inevitably lethal primary brain cancer in adults. It is recognized that the overexpression of the endosomal Na+ /H+ exchanger NHE9 is a potent driver of GBM progression. Patients with NHE9 overexpression have a threefold lower median survival relative to GBM patients with normal NHE9 expression, using available treatment options. New treatment strategies tailored for this GBM subset are much needed. According to the prevailing model, NHE9 overexpression leads to an increase in plasma membrane density of epidermal growth factor receptors (EGFRs) which consequently enhances GBM cell proliferation and migration. However, this increase is not specific to EGFRs. In fact, the hallmark of NHE9 overexpression is a pan-specific increase in plasma membrane receptors. Paradoxically, we report that this gain of function in NHE9 can be exploited to effectively target GBM cells for destruction. When exposed to gold nanoparticles, NHE9 overexpressing GBM cells accumulated drastically high amounts of gold via receptor-mediated endocytosis, relative to control. Irradiation of these cells with near-infrared light led to apoptotic tumour cell death. A major limitation for delivering therapeutics to GBM cells is the blood-brain barrier (BBB). Here, we demonstrate that macrophages loaded with gold nanoparticles can cross the BBB, deliver the gold nanoparticles and effect the demise of GBM cells. In combination with receptor tyrosine kinase inhibition, we show this approach holds great promise for a new GBM-targeted therapy.

Hydroxychloroquine potentiates the anti-cancer effect of bevacizumab on glioblastoma via the inhibition of autophagy.

Bevacizumab (BEV) is widely used for the treatment of patients with recurrent glioblastoma (GBM), but recent evidence demonstrated that BEV induced cytoprotective autophagy, which allows tumor cells to survive. Hydroxychloroquine (HCQ) inhibits lysosomal acidification and blocks autophagy via influencing autophagosome fusion and degradation. HCQ is often used to enhance the efficacy of chemoradiotherapy. However, whether HCQ sensitizes GBM cells to BEV and the molecular mechanism of this effect are not clear. We showed that high concentrations of BEV increased the LC3-II/LC3-I ratio and caused the degradation of Beclin1 in the LN18 and LN229 cell lines, indicating that high concentrations of BEV induced the autophagy of the LN18 and LN229 cells. However, BEV (100 µg/ml) did not influence the autophagy of the LN18 and LN229 cells, and HCQ at less than 5 µg/ml significantly accumulated LC3B-II and p62 proteins and blocked the autophagy process. Importantly, we found that HCQ (5 µg/ml) potentiated the anti-cancer effect of BEV (100 µg/ml). Therefore, HCQ is a novel strategy that may augment the efficacy of BEV for GBM via the inhibition of autophagy.

Synaptic proximity enables NMDAR signalling to promote brain metastasis.

Metastasis-the disseminated growth of tumours in distant organs-underlies cancer mortality. Breast-to-brain metastasis (B2BM) is a common and disruptive form of cancer and is prevalent in the aggressive basal-like subtype, but is also found at varying frequencies in all cancer subtypes. Previous studies revealed parameters of breast cancer metastasis to the brain, but its preference for this site remains an enigma. Here we show that B2BM cells co-opt a neuronal signalling pathway that was recently implicated in invasive tumour growth, involving activation by glutamate ligands of N-methyl-D-aspartate receptors (NMDARs), which is key in model systems for metastatic colonization of the brain and is associated with poor prognosis. Whereas NMDAR activation is autocrine in some primary tumour types, human and mouse B2BM cells express receptors but secrete insufficient glutamate to induce signalling, which is instead achieved by the formation of pseudo-tripartite synapses between cancer cells and glutamatergic neurons, presenting a rationale for brain metastasis.

Glutamatergic synaptic input to glioma cells drives brain tumour progression.

A network of communicating tumour cells that is connected by tumour microtubes mediates the progression of incurable gliomas. Moreover, neuronal activity can foster malignant behaviour of glioma cells by non-synaptic paracrine and autocrine mechanisms. Here we report a direct communication channel between neurons and glioma cells in different disease models and human tumours: functional bona fide chemical synapses between presynaptic neurons and postsynaptic glioma cells. These neurogliomal synapses show a typical synaptic ultrastructure, are located on tumour microtubes, and produce postsynaptic currents that are mediated by glutamate receptors of the AMPA subtype. Neuronal activity including epileptic conditions generates synchronised calcium transients in tumour-microtube-connected glioma networks. Glioma-cell-specific genetic perturbation of AMPA receptors reduces calcium-related invasiveness of tumour-microtube-positive tumour cells and glioma growth. Invasion and growth are also reduced by anaesthesia and the AMPA receptor antagonist perampanel, respectively. These findings reveal a biologically relevant direct synaptic communication between neurons and glioma cells with potential clinical implications.

Evaluation of serum extracellular vesicles as noninvasive diagnostic markers of glioma.

Rationale: Glioma is the most common malignant primary brain tumor in the central nervous system (CNS). The lack of reliable noninvasive diagnostic and prognostic methods is one of the main reasons for the high mortality of glioma. Serum has become a useful biomarker for the diagnosis and prognosis prediction of glioma because extracellular vesicles (EVs) carry molecular components from their parental cells. Methods: To detect EVs and perform molecular analysis of serum EVs, we established and optimized a microbead-assisted method based on flow cytometry and estimated the efficacy of EGFR protein expression and NLGN3 and PTTG1 mRNA in serum EVs from glioma patients (n=23) and healthy individuals (n=12). We evaluated the ability of EGFR+ EVs to differentiate high-grade and low-grade glioma patients and checked the correlation between EGFR in EVs and the ki-67 labeling index (LI) in the tumor tissue. Results: We demonstrated that EGFR+ EVs are effective diagnostic and prognostic markers of glioma. The expression of EGFR in serum EVs can accurately differentiate high-grade and low-grade glioma patients, and EGFR in EVs positively correlates with ki-67 LI in the tumor tissue. We also showed the potential of NLGN3 and PTTG1 mRNA in EVs for detecting glioma patients. Conclusions: We demonstrate that the protein expression of EGFR in serum EVs is an effective diagnostic marker of glioma. EGFR in EVs highly correlates with the malignancy of glioma. We also show the potential of NLGN3 and PTTG1 in EVs for detecting glioma. The optimized flow cytometry with the aid of microbead-based EV enrichment show its potential as a noninvasive method for the detection of glioma and will be beneficial to the management of glioma.