Inhibition of purinergic P2X receptor 7 (P2X7R) decreases granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in U251 glioblastoma cells.

Glioblastoma is the most aggressive form of primary brain cancer, with a median survival of 12-15 months. The P2X receptor 7 (P2X7R) is upregulated in glioblastoma and is associated with increased tumor cell proliferation. The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is also upregulated in glioblastoma and has been shown to have both pro- and anti-tumor functions. This study investigates the potential mechanism linking P2X7R and GM-CSF in the U251 glioblastoma cell line and the therapeutic potential of P2X7R antagonism in this setting. P2X7R protein and mRNA was demonstrated to be expressed in the U251 cell line as assessed by immunocytochemistry and qPCR. Its channel function was intact as demonstrated by live cell confocal imaging using a calcium indicator Fluo-4 AM. Inhibition of P2X7R using antagonist AZ10606120, decreased both GM-CSF mRNA (P < 0.05) and protein (P < 0.01) measured by qPCR and ELISA respectively. Neutralization of GM-CSF with an anti-GM-CSF antibody did not alter U251 cell proliferation, however, P2X7R antagonism with AZ10606120 significantly reduced U251 glioblastoma cell numbers (P < 0.01). This study describes a novel link between P2X7R activity and GM-CSF expression in a human glioblastoma cell line and highlights the potential therapeutic benefit of P2X7R inhibition with AZ10606120 in glioblastoma.

P2X7 receptor antagonism inhibits tumour growth in human high-grade gliomas.

Gliomas, the most common primary brain cancer, are highly infiltrative and extremely difficult to treat. Despite advancements, current treatment is limited, with patients surviving for a median of 14-15 months post-diagnosis. Previous research has demonstrated the upregulation of a purinergic receptor, P2X7R, in human gliomas. P2X7R is expressed on both glioma cells and microglia within the glioma microenvironment. It is hypothesized that P2X7R contributes to tumour growth and proliferation via immune-mediated mechanisms involving tumour cells and surrounding microglia. We sought to elucidate the role of P2X7R in a human glioblastoma cell line (U251) and on surgically resected human glioma samples. We treated U251 and human glioma cultures for 72 h with P2X7R antagonists, Brilliant Blue G (BBG), oxidized ATP (oATP) and AZ10606120. Cell counting via fluorescence confocal microscopy was conducted to assess tumour proliferation. We observed no significant reductions in tumour cell numbers following P2X7R antagonism with BBG (20 µM) and oATP (250 µM) in both U251 cells and human glioma samples. Interestingly, there was a significant reduction in tumour cell number in both U251 cells (p = 0.0156) and human glioma samples (p = 0.0476) treated with varying concentrations of AZ10606120. When compared with the conventional chemotherapeutic agent, temozolomide, AZ10606120 was also found to more effectively inhibit tumour proliferation in U251 cells (p < 0.0001). Our pilot results demonstrate a potential trophic role of P2X7R where its inhibition by AZ10606120, a potent antagonist, hinders glioma growth directly or through the inactivation of microglia. This sheds new light on P2X7R as a therapeutic target for human gliomas.

A Simple and Reliable Protocol for the Preparation and Culturing of Fresh Surgically Resected Human Glioblastoma Tissue.

Glioblastoma is a heterogeneous glial cell malignancy with extremely high morbidity and mortality. Current treatment is limited and provide minimal therapeutic efficacy. Previous studies were reliant on cell lines that do not accurately reflect the heterogeneity of the glioma microenvironment. Developing reliable models of human glioblastoma is therefore essential. Direct culture of human brain tumours is often difficult and there is a limited number of protocols available. Hence, we have developed an effective method for the primary culture of human glioblastoma samples obtained during surgical resection. Culturing tumour tissue direct from human brain is advantageous in that cultures (1) more closely resemble true human disease, relative to the use of cell lines; (2) comprise a range of cellular components present in the natural tumour microenvironment; and (3) are free of added antibodies and reagents. Additionally, primary glioblastoma cultures are valuable in studies examining the effects of anti-cancer pharmaceuticals and therapeutic agents, and can be further used in live cell imaging, immunocytochemistry, flow cytometry and immunoassay experiments. Via this protocol, cells are maintained in supplemented medium at 37 °C (5% CO2) and are expected to achieve sufficient confluency within 7 days of initial culture.

Potential biomarkers and challenges in glioma diagnosis, therapy and prognosis.

Gliomas are the most common central nervous system malignancies and present with significant morbidity and mortality. Treatment modalities are currently limited to surgical resection, chemotherapy and radiotherapy. Increases in survival rate over the previous decades are negligible, further pinpointing an unmet clinical need in this field. There is a continual struggle with the development of effective glioma diagnostics and therapeutics, largely due to a multitude of factors, including the presence of the blood-brain barrier and significant intertumoural and intratumoural heterogeneity. Importantly, there is a lack of reliable biomarkers for glioma, particularly in aiding tumour subtyping and measuring response to therapy. There is a need for biomarkers that would both overcome the complexity of the disease and allow for a minimally invasive means of detection and analysis. This is a comprehensive review evaluating the potential of current cellular, proteomic and molecular biomarker candidates for glioma. Significant hurdles faced in glioma diagnostics and therapy are also discussed here.

The role of microglia and P2X7 receptors in gliomas.

Gliomas are the most prevalent tumours of the central nervous system and present with high morbidity and mortality. The most common and most aggressive form of glioma is glioblastoma multiforme, of which patients have a median survival time of only 12 to 15 months. Current treatment options are limited and have a small impact on clinical outcome and prognosis. There is accumulating evidence that microglia, the immunocompetent cells of the central nervous system, and the purinergic P2X7 receptor (P2X7R) may contribute to tumour progression and pathology. Importantly, P2X7R on both tumour cells and infiltrating microglia is overexpressed in animal and human glioma cultures. Factors released by glioma cells and P2X7R activation recruit microglia into the largely immunosuppressive tumour microenvironment where they have been demonstrated to contribute to either tumour proliferation or tumour suppression. It is likely that P2X7R mediates a range of microglia effector functions in the glioma setting, potentially increasing tumour growth and proliferation. This review evaluates current evidence on the roles of microglia and P2X7R in glioma pathogenesis. Understanding the nature, mechanisms and outcomes of microglia and P2X7R activation in gliomas is necessary for the development of more therapies with increased efficacy and specificity.