Mast cell accumulation in glioblastoma with a potential role for stem cell factor and chemokine CXCL12

Glioblastoma multiforme (GBM) is the most common and malignant form of glioma with high mortality and no cure. Many human cancers maintain a complex inflammatory program triggering rapid recruitment of inflammatory cells, including mast cells (MCs), to the tumor site. However, the potential contribution of MCs in glioma has not been addressed previously. Here we report for the first time that MCs infiltrate KRas+Akt-induced gliomas, using the RCAS/TV-a system, where KRas and Akt are transduced by RCAS into the brains of neonatal Gtv-a- or Ntv-a transgenic mice lacking Ink4a or Arf. The most abundant MC infiltration was observed in high-grade gliomas of Arf-/- mice. MC accumulation could be localized to the vicinity of glioma-associated vessels but also within the tumor mass. Importantly, proliferating MCs were detected, suggesting that the MC accumulation was caused by local expansion of the MC population. In line with these findings, strong expression of stem cell factor (SCF), i.e. the main MC growth factor, was detected, in particular around tumor blood vessels. Further, glioma cells expressed the MC chemotaxin CXCL12 and MCs expressed the corresponding receptor, i.e. CXCR4, suggesting that MCs could be attracted to the tumor through the CXCL12/CXCR4 axis. Supporting a role for MCs in glioma, strong MC infiltration was detected in human glioma, where GBMs contained significantly higher MC numbers than grade II tumors did. Moreover, human GBMs were positive for CXCL12 and the infiltrating MCs were positive for CXCR4. In conclusion, we provide the first evidence for a role for MCs in glioma.     

The In Vivo Therapeutic Efficacy of the Oncolytic Adenovirus Delta24-RGD Is Mediated by Tumor-Specific Immunity

The oncolytic adenovirus Delta24-RGD represents a new promising therapeutic agent for patients with a malignant glioma and is currently under investigation in clinical phase I/II trials. Earlier preclinical studies showed that Delta24-RGD is able to effectively lyse tumor cells, yielding promising results in various immune-deficient glioma models. However, the role of the immune response in oncolytic adenovirus therapy for glioma has never been explored. To this end, we assessed Delta24-RGD treatment in an immune-competent orthotopic mouse model for glioma and evaluated immune responses against tumor and virus. Delta24-RGD treatment led to long-term survival in 50% of mice and this effect was completely lost upon administration of the immunosuppressive agent dexamethasone. Delta24-RGD enhanced intra-tumoral infiltration of F4/80+ macrophages, CD4+ and CD8+ T-cells, and increased the local production of pro-inflammatory cytokines and chemokines. In treated mice, T cell responses were directed to the virus as well as to the tumor cells, which was reflected in the presence of protective immunological memory in mice that underwent tumor rechallenge. Together, these data provide evidence that the immune system plays a vital role in the therapeutic efficacy of oncolytic adenovirus therapy of glioma, and may provide angles to future improvements on Delta24-RGD therapy.     

Hot spot formation in the nuclear envelope of oocytes in response to steroids.

A Glucocorticoid-sensitive cell rapidly responds to hormone stimulation with bidirectional exchange of specific macromolecules between cytosol and nucleus. Glucocorticoid-initiated macromolecules (GIMs) must overcome the nuclear envelope (NE) to enter or leave the nucleus. GIM translocation occurs through nuclear pore complexes (NPCs) that span the NE. We investigated the question whether transport of GIMs through NPCs occurs random or involves selected groups of NPCs (hot spots). Glucocorticoid receptors were expressed in Xenopus laevis oocytes and GIM transport was activated by triamcinolone acetonide, a potent synthetic glucocorticoid analogon. Glucocorticoid receptors associated with the NE and the chromatin were identified using western blot analysis and, at single molecule level, atomic force microscopy. Fluorescence-labeled dextran was used to describe passive NE permeability. We observed that after hormone injection (i) small GIMs, most likely GRs, localize within seconds on both sides of the NE. (ii) large GIMs, most likely ribonucleoproteins, localize within minutes on NPCs at the nucleoplasmic side (iii) both small and large GIMs accumulate on selected NPC clusters (iv) NE permeability transiently decreases when GIMs attach to NPCs. We conclude that GIM transport across the nuclear barrier does not randomly take place but is carried out by a selected population of NPCs.     

Antitumour and pro-apoptotic actions of highly unsaturated fatty acids in glioma

The highly unsaturated fatty acids (HUFA) of the n-6 and n-3 series are involved in cell signalling in normal and transformed cells and have recently been associated with pathways leading to tumour cell death. The antitumour activity of three HUFA (arachidonic acid, gamma linolenic acid and eicosapentaenoic acid) were studied in glioma cells and tissue. Using five glioma models, including primary cell suspensions prepared from 46 human glioma samples and an in vivo rat C6 glioma model, we obtained evidence that, following exposure to HUFA, either administered into the medium surrounding human glioma cells or in 16 preparations of multicellular spheroids derived from human and rodent glioma cell lines (C6, MOG, U87, U373) or administered intra-tumourally by infusion using osmotic mini-pumps in 48 rats, glioma regression and apoptosis were detected. Additionally, synergy between gamma irradiation and HUFA administration was observed in 13 experiments analyzing C6 glioma cell apoptosis in vitro. These pro-apoptotic and antiproliferative activities were observed using both C18 and C20 fatty acids of the n-6 and n-3 series, but not when saturated and monounsaturated C18 and C20 fatty acid preparations were used. In the glioma infusion model, in addition to the apoptosis detected in glioma tissue infused with HUFA for 3-7 days, preservation of normal neural tissue and vasculature in adjacent brain was observed. Also, there was little evidence of acute inflammatory infiltration in regressing tumours. Our findings suggest that intraparenchymal infusion of HUFA may be effective in stimulating glioma regression.     

FDPS promotes glioma growth and macrophage recruitment by regulating CCL20 via Wnt/β‐catenin signalling pathway

Glioma is one of the most lethal tumours and common malignant in the central nervous system (CNS), which exhibits diffuse invasion and aggressive growth. Several studies have reported the association of FDPS to tumour development and progression. However, the role of FDPS in progression of glioma and macrophage recruitment is not well‐elucidated. In the current study, a remarkable enhancement in FDPS level was observed in glioma tissues and associated with poor prognosis, contributed to tumour growth. FDPS was correlated with macrophage infiltration in glioma and pharmacological deletion of macrophages largely abrogated the oncogenic functions of FDPS in glioma. Mechanistically, FDPS activated Wnt/β‐catenin signalling pathway and ultimately facilitates macrophage infiltration by inducing CCL20 expression. In conclusion, overexpressed FDPS exhibits an immunomodulatory role in glioma. Therefore, targeting FDPS may be an effective therapeutic strategy for glioma.     

ITGB3BP is a potential biomarker associated with poor prognosis of glioma

Despite the growing recognition of ITGB3BP as an essential feature of various cancers, the relationship between ITGB3BP and glioma remains unclear. The main aim of this study was to determine the prognostic and diagnostic value of ITGB3BP in glioma. RNA‐Seq and microarray data from 2222 glioma patients were included, and we found that the expression level of ITGB3BP in glioma tissues was significantly higher than that in normal brain tissues. Moreover, ITGB3BP can be considered an independent risk factor for poor prognosis and has great predictive value for the prognosis of glioma. Gene Set Enrichment Analysis results showed that ITGB3BP contributes to the poor prognosis of glioma by activating tumour‐related signalling pathways. Some small‐molecule drugs were identified, such as hexestrol, which may specifically inhibit ITGB3BP and be useful in the treatment of glioma. The TIMER database analysis results revealed a correlation between the expression of ITGB3BP and the infiltration of various immune cells in glioma. Our findings provide the first evidence that the up‐regulation of ITGB3BP correlates with poor prognosis in human glioma. Thus, ITGB3BP is a potential new biomarker that can be used for the clinical diagnosis and treatment of glioma.     

The pro-migratory and pro-invasive role of the procoagulant tissue factor in malignant gliomas

During the infiltration process, glioma cells are known to migrate along preexisting anatomical structures such as blood vessels, axonal fiber tracts and the subependymal space, thereby widely invading surrounding CNS tissue. This phenomenon represents a major obstacle for the clinical treatment of these tumours. Several extracellular key factors and intracellular signaling pathways have been previously linked to the highly aggressive, invasive phenotype observed in malignant gliomas. The glioblastoma (GBM) which is the most malignant form of these tumors, is histologically characterized by areas of tumor necroses and pseudopalisading cells, the latter likely representing tumor cells actively migrating away from the hypoxic-ischemic core of the tumor. It is believed that intravascular thromboses play a major role in the emergence of hypoxia and intratumoral necroses in GBMs. One of the most highly upregulated prothrombotic factor in malignant gliomas is tissue factor (TF), a 47 kDa type I transmembrane protein belonging to the cytokine receptor superfamily. In a recent study, we provided evidence that TF/FVIIa signaling via the protease-activated receptor 2 (PAR-2) promotes cell growth, migration and invasion of glioma cells. In this point of view article we outline the key molecular players involved in migration and invasion of gliomas, highlight the potential role of TF for the pro-migratory and pro-invasive phenotype of these tumors and discuss the underlying mechanisms on the cellular level and in the tumor microenvironment.     

Interaction of brain fatty acid-binding protein with the polyunsaturated fatty acid environment as a potential determinant of poor prognosis in malignant glioma

Malignant gliomas are the most common adult brain cancers. In spite of aggressive treatment, recurrence occurs in the great majority of patients and is invariably fatal. Polyunsaturated fatty acids are abundant in brain, particularly ω-6 arachidonic acid (AA) and ω-3 docosahexaenoic acid (DHA). Although the levels of ω-6 and ω-3 polyunsaturated fatty acids are tightly regulated in brain, the ω-6:ω-3 ratio is dramatically increased in malignant glioma, suggesting deregulation of fundamental lipid homeostasis in brain tumor tissue. The migratory properties of malignant glioma cells can be modified by altering the ratio of AA:DHA in growth medium, with increased migration observed in AA-rich medium. This fatty acid-dependent effect on cell migration is dependent on expression of the brain fatty acid binding protein (FABP7) previously shown to bind DHA and AA. Increased levels of enzymes involved in eicosanoid production in FABP7-positive malignant glioma cells suggest that FABP7 is an important modulator of AA metabolism. We provide evidence that increased production of eicosanoids in FABP7-positive malignant glioma growing in an AA-rich environment contributes to tumor infiltration in the brain. We discuss pathways and molecules that may underlie FABP7/AA-mediated promotion of cell migration and FABP7/DHA-mediated inhibition of cell migration in malignant glioma.     

MicroRNA-326 Functions as a Tumor Suppressor in Glioma by Targeting the Nin One Binding Protein (NOB1)

Malignant glioma is the most common type of primary brain tumor in adults, characterized by rapid tumor growth and infiltration of tumor cells throughout the brain. Alterations in the activity of the 26S proteasome have been associated with malignant glioma cells, although the specific defects have not been identified. Recently, microRNA-326 (miR-326) was shown to play an important role in glioblastoma and breast cancer, but the underlying molecular mechanisms remain unclear. In the present study, the human Nin one binding protein (NOB1) was identified as a direct target of miR-326 and a potential oncogene in human glioma. Similar to NOB1 silencing by shRNA, overexpression of miR-326 in human glioma cell lines (A172 and U373) caused cell cycle arrest at the G1 phase, delayed cell proliferation and enhanced apoptosis. MiR-326 inhibited colony formation in soft agar and decreased growth of a xenograft tumor model, suggesting that miR-326 and NOB1 are required for tumorigenesis in vitro and in vivo. Furthermore, these processes were shown to involve the MAPK pathway. NOB1 overexpression in human glioma samples was detected by Affymetrix array analysis, and NOB1 mRNA and protein levels were shown to be increased in high-grade glioma compared to low-grade glioma and normal brain tissue. Furthermore, high levels of NOB1 were associated with unfavorable prognosis of glioma patients. Taken together, these results indicate that miR-326 and NOB1 may play an important role in the development of glioma.     

Ferroptosis-related gene signature correlates with the tumor immune features and predicts the prognosis of glioma patients

Background: Glioma is a malignant intracranial tumor and the most fatal cancer. The role of ferroptosis in the clinical progression of gliomas is unclear.Method: Univariate and least absolute shrinkage and selection operator (Lasso) Cox regression methods were used to develop a ferroptosis-related signature (FRSig) using a cohort of glioma patients from the Chinese Glioma Genome Atlas (CGGA), and was validated using an independent cohort of glioma patients from The Cancer Genome Atlas (TCGA). A single-sample gene set enrichment analysis (ssGSEA) was used to calculate levels of the immune infiltration. Multivariate Cox regression was used to determine the independent prognostic role of clinicopathological factors and to establish a nomogram model for clinical application.Results: We analyzed the correlations between the clinicopathological features and ferroptosis-related gene (FRG) expression and established an FRSig to calculate the risk score for individual glioma patients. Patients were stratified into two subgroups with distinct clinical outcomes. Immune cell infiltration in the glioma microenvironment and immune-related indexes were identified that significantly correlated with the FRSig, the tumor mutation burden (TMB), copy number alteration (CNA), and immune checkpoint expression was also significantly positively correlated with the FRSig score. Ultimately, an FRSig-based nomogram model was constructed using the independent prognostic factors age, World Health Organization (WHO) grade, and FRSig score.Conclusion: We established the FRSig to assess the prognosis of glioma patients. The FRSig also represented the glioma microenvironment status. Our FRSig will contribute to improve patient management and individualized therapy by offering a molecular biomarker signature for precise treatment.     

The pro-migratory and pro-invasive role of the procoagulant tissue factor in malignant gliomas

During the infiltration process, glioma cells are known to migrate along preexisting anatomical structures such as blood vessels, axonal fiber tracts and the subependymal space, thereby widely invading surrounding CNS tissue. This phenomenon represents a major obstacle for the clinical treatment of these tumors. Several extracellular key factors and intracellular signaling pathways have been previously linked to the highly aggressive, invasive phenotype observed in malignant gliomas. The glioblastoma (GBM), which is the most malignant form of these tumors, is histologically characterized by areas of tumor necroses and pseudopalisading cells, the latter likely representing tumor cells actively migrating away from the hypoxic- ischemic core of the tumor. It is believed that intravascular thromboses play a major role in the emergence of hypoxia and intratumoral necroses in GBMs. One of the most highly upregulated prothrombotic factor in malignant gliomas is tissue factor (TF), a 47 kDa type I transmembrane protein belonging to the cytokine receptor superfamily. In a recent study, we provided evidence that TF/FVIIa signaling via the protease-activated receptor 2 (PAR-2) promotes cell growth, migration and invasion of glioma cells. In this Commentary & View, we outline the key molecular players involved in migration and invasion of gliomas, highlight the potential role of TF for the pro-migratory and pro-invasive phenotype of these tumors and discuss the underlying mechanisms on the cellular level and in the tumor microenvironment.Key words: brain tumor, blood coagulation, hypoxia, MAP kinase, cancer stem cells, tumor invasion     

Prognostic value and immune cell infiltration of hypoxic phenotype‐related gene signatures in glioblastoma microenvironment

Glioblastoma (GBM) is a malignant intracranial tumour with the highest proportion and lethality. It is characterized by invasiveness and heterogeneity. However, the currently available therapies are not curative. As an essential environmental cue that maintains glioma stem cells, hypoxia is considered the cause of tumour resistance to chemotherapy and radiation. Growing evidence shows that immunotherapy focusing on the tumour microenvironment is an effective treatment for GBM; however, the current clinicopathological features cannot predict the response to immunotherapy and provide accurate guidance for immunotherapy. Based on the ESTIMATE algorithm, GBM cases of The Cancer Genome Atlas (TCGA) data set were classified into high‐ and low‐immune/stromal score groups, and a four‐gene tumour environment‐related model was constructed. This model exhibited good efficiency at forecasting short‐ and long‐term prognosis and could also act as an independent prognostic biomarker. Additionally, this model and four of its genes (CLECL5A, SERPING1, CHI3L1 and C1R) were found to be associated with immune cell infiltration, and further study demonstrated that these four genes might drive the hypoxic phenotype of perinecrotic GBM, which affects hypoxia‐induced glioma stemness. Therefore, these might be important candidates for immunotherapy of GBM and deserve further exploration.     

Bioinformatics analysis of the prognostic value of NEK8 and its effects on immune cell infiltration in glioma

Glioma is the most common malignancy of the nervous system with high rates of recurrence and mortality, even after surgery. The 5-year survival rate is only about 5%. NEK8 is involved in multiple biological processes in a variety of cancers; however, its role in glioma is still not clear. In the current study, we evaluated the prognostic value of NEK8, as well as its role in the pathogenesis of glioma. Using a bioinformatics approach and RNA-seq data from public databases, we found that NEK8 expression is elevated in glioma tissues; we further verified this result by RT-PCR, Western blotting and immunochemistry using clinical samples. Functional enrichment analyses of genes with correlated expression indicated that elevated NEK8 expression is associated with increased immune cell infiltration in glioma and may affect the tumour microenvironment via the regulation of DNA damage/repair. Survival analyses revealed that high levels of NEK8 are associated with a poorer prognosis; higher WHO grade, IDH status, 1p/19q codeletion, age and NEK8 were identified as an independent prognostic factor. These findings support the crucial role of NEK8 in the progression of glioma via effects on immune cell infiltration and suggest that it is a new prognostic biomarker.     

Adenoviral-mediated interleukin-18 expression in mesenchymal stem cells effectively suppresses the growth of glioma in rats

Glioma is the most common primary intracranial malignant tumor. Despite advances in surgical techniques and adjuvant radio- and chemotherapies, the prognosis for patients with glioma remains poor. We have explored the effects of using genetically modified mesenchymal stem cells (MSCs) to treat malignant glioma in rats. Mesenchymal stem cells isolated from Sprague-Dawley rats can directly suppress the growth of C6 cells in vitro. MSCs transplanted intratumorally can also significantly inhibit the growth of glioma and prolong survival in C6 glioma-bearing models. MSCs producing Interleukin-18 infected by adenoviral vector inhibited glioma growth and prolonged the survival of glioma-bearing rats. Transplantation of IL-18 secreting MSCs was associated with enhanced T cell infiltration and long-term anti-tumor immunity. Thus, IL-18 may be an effective adoptive immunotherapy for malignant glioma. When used in conjunction with MSCs as targeting vehicles in vivo, IL-18 may offer a promising new treatment option for malignant glioma.     

Immunohistochemical analysis of glioma-infiltrating leucocytes after peripheral therapeutic immunization with interferon-γ-transfected glioma cells

We have shown previously that rejection of preinduced rat brain tumours is possible following therapeutic immunizations with interferon-γ (IFNγ)-transfected glioma cells (N32-IFNγ). In the present study we have used the same model to evaluate whether quantitative differences in tumour-infiltrating lymphocytes can be detected between animals receiving therapeutic immunizations with either IFNγ-transfected glioma cells, wild-type glioma cells or no treatment. Since leucocyte transpedesis into the tumour can be anticipated to depend on the state of vascularization, we have mapped the development of microvessels in the tumour in parallel with the leucocyte infiltration. Our results show that microvessels start to form at day 7 and then gradually increase in number and size, indicating the establishment of an extensive vascularization by day 24. Leucocyte infiltration displays a biphasic pattern after tumour grafting. We have therefore studied the infiltration kinetics after an early immunization (1 day after intracerebral isografting) and compared the effects with those of a late immunization (10 days after intracerebral isografting) with N32-IFNγ or wild-type N32. Our results show (1) an early infiltration of granulocytes 3 days after isografting; (2) a T-cell-receptor-positive (TCR⁺) T-cell infiltration starting on day 10; (3) a macrophage infiltration starting on day 13; (4) a CD8⁺ cell infiltration starting on day 13. The proportions of TCR⁺ T cells, CD8⁺ cells and natural killer cells differs significantly between animals immunized with N32-IFNγ and those receiving wild-type N32, when analysed 14 days after immunization at day 10. This difference can only be detected when animals are immunized at later stages of tumour growth. We propose that this could depend on an early-immunization-independent leucocyte infiltration during tumour establishment. This has to be considered when evaluating studies of leucocyte infiltration in experimental tumours. Received: 7 October 1999 / Accepted: 10 December 1999     

Glioma Cell Death: Cell–Cell Interactions and Signalling Networks

The prognosis for patients with malignant gliomas is poor, but improvements may emerge from a better understanding of the pathophysiology of glioma signalling. Recent therapeutic developments have implicated lipid signalling in glioma cell death. Stress signalling in glioma cell death involves mitochondria and endoplasmic reticulum. Lipid mediators also signal via extrinsic pathways in glioma cell proliferation, migration and interaction with endothelial and microglial cells. Glioma cell death and tumour regression have been reported using polyunsaturated fatty acids in animal models, human ex vivo explants, glioma cell preparations and in clinical case reports involving intratumoral infusion. Cell death signalling was associated with generation of reactive oxygen intermediates and mitochondrial and other signalling pathways. In this review, evidence for mitochondrial responses to stress signals, including polyunsaturated fatty acids, peroxidising agents and calcium is presented. Additionally, evidence for interaction of glioma cells with primary brain endothelial cells is described, modulating human glioma peroxidative signalling. Glioma responses to potential therapeutic agents should be analysed in systems reflecting tumour connectivity and CNS structural and functional integrity. Future insights may also be derived from studies of signalling in glioma-derived tumour stem cells.     

Profiles of immune-related genes and immune cell infiltration in the tumor microenvironment of diffuse lower-grade gliomas

The tumor microenvironment is highly correlated with tumor occurrence, progress, and prognosis. We aimed to investigate the immune-related gene (IRG) expression and immune infiltration pattern in the tumor microenvironment of lower-grade glioma (LGG). We employed the Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data (ESTIMATE) algorithm to calculate immune and stromal scores and identify prognostic IRG based on The Cancer Genome Atlas data set. The potential molecular functions of these genes were explored with the help of functional enrichment analysis and the protein–protein interaction network. Remarkably, three cohorts that were downloaded from the Chinese Glioma Genome Atlas database were analyzed to further verify the prognostic values of these genes. Moreover, the Tumor IMmune Estimation Resource (TIMER) algorithm was used to estimate the abundance of infiltrating immune cells and explore the immune infiltration pattern in LGG. And unsupervised cluster analysis determined three clusters of the immune infiltration pattern and indicated that CD8⁺ T cells and macrophages were significantly associated with LGG outcomes. Altogether, our study identified a list of prognostic IRGs and provided a perspective to explore the immune infiltration pattern in LGG.     

Role of tenascins in the ECM of gliomas

Tenascins are a family of extracellular matrix molecules that are mainly expressed in embryonic development and down-regulated in adulthood. A re-expression in the adult occurs under pathological conditions such as inflammation, regeneration or neoplasia. As the most prominent member of the tenascin family, TN-C, is highly expressed in glioma tissue and rising evidence suggests that TN-C plays a crucial role in cell migration or invasion – the most fatal characteristics of glioma – also the other members of this protein family have been investigated with regard to their impact on glioma biology. For all tenascins correlations between the expression levels of the different family members and the degree of malignancy and invasiveness of glial tumors could be detected. Overall, the former and recent results in the research on glioma and tenascins point at distinct roles of each of the molecules in glioma biology and the devastating properties of these tumors.     

CX3CR1 promotes recruitment of human glioma-infiltrating microglia/macrophages (GIMs)

The transmembrane chemokine CX3CL1 and its receptor CX3CR1 are thought to be involved in the trafficking of immune cells during an immune response and in the pathology of various human diseases including cancer. However, little is known about the expression and function of CX3CR1 in human glioma-infiltrating microglia/macrophages (GIMs), representing the major cellular stroma component of highly malignant gliomas. Here, we show that CX3CR1 is overexpressed at both the mRNA and protein level in solid human astrocytomas of different malignancy grades and in glioblastomas. CX3CR1 was localized in ionized calcium-binding adapter molecule 1 (Iba1) and CD11b/c positive GIMs in situ as shown by fluorescence microscopy. In accordance with this, freshly isolated human GIM-enriched fractions separated by CD11b MACS technology displayed high Iba1 and CX3CR1 mRNA expression levels in vitro. Moreover, cultured human GIMs responded to CX3CL1-triggered activation of CX3CR1 with adhesion and migration in vitro. Besides an increase in motility, CX3CL1 also enhanced expression of matrix metalloproteases 2, 9, and 14 in GIM fractions in vitro. These data indicate that the CX3CL1/CX3CR1 system has a crucial tumor-promoting role in human glioblastomas via its impact on glioma-infiltrating immune subsets.