Brain tumor initiating cells (BTICs) are self-renewing, tumorigenic cells that often reside in a necrotic and hypoxic niche in the brain. Here the authors show that BTICs can become more tumorigenic upon glucose restriction and compensate for this cellular stress by upregulating their capacity to take up glucose. Like all cancers, brain tumors require a continuous source of energy and molecular resources for new cell production. In normal brain, glucose is an essential neuronal fuel, but the blood-brain barrier limits its delivery. We now report that nutrient restriction contributes to tumor progression by enriching for brain tumor initiating cells (BTICs) owing to preferential BTIC survival and to adaptation of non-BTICs through acquisition of BTIC features. BTICs outcompete for glucose uptake by co-opting the high affinity neuronal glucose transporter, type 3 (Glut3, SLC2A3). BTICs preferentially express Glut3, and targeting Glut3 inhibits BTIC growth and tumorigenic potential. Glut3, but not Glut1, correlates with poor survival in brain tumors and other cancers; thus, tumor initiating cells may extract nutrients with high affinity. As altered metabolism represents a cancer hallmark, metabolic reprogramming may maintain the tumor hierarchy and portend poor prognosis.

Low-passage, serum-free cell lines cultured from patient tumour tissue are the gold-standard for preclinical studies and cellular investigations of glioblastoma (GBM) biology, yet entrenched, poorly-representative cell line models are still widely used, compromising the significance of much GBM research. We submit that greater adoption of these critical resources will be promoted by the provision of a suitably-sized, meaningfully-described reference collection along with appropriate tools for working with them. Consequently, we present a curated panel of 12 readily-usable, genetically-diverse, tumourigenic, patient-derived, low-passage, serum-free cell lines representing the spectrum of molecular subtypes of IDH-wildtype GBM along with their detailed phenotypic characterisation plus a bespoke set of lentiviral plasmids for bioluminescent/fluorescent labelling, gene expression and CRISPR/Cas9-mediated gene inactivation. The cell lines and all accompanying data are readily-accessible via a single website, Q-Cell (qimrberghofer.edu.au/q-cell/) and all plasmids are available from Addgene. These resources should prove valuable to investigators seeking readily-usable, well-characterised, clinically-relevant, gold-standard models of GBM.

Studies in cell culture and mouse models of cancer have indicated that the soluble sphingolipid metabolite sphingosine 1-phosphate (S1P) promotes cancer cell proliferation, survival, invasiveness, and tumor angiogenesis. In contrast, its metabolic precursor ceramide is prodifferentiative and proapoptotic. To determine whether sphingolipid balance plays a significant role in glioma malignancy, we undertook a comprehensive analysis of sphingolipid metabolites in human glioma and normal gray matter tissue specimens. We demonstrate, for the first time, a systematic shift in sphingolipid metabolism favoring S1P over ceramide, which increases with increasing cancer grade. S1P content was, on average, 9-fold higher in glioblastoma tissues compared with normal gray matter, whereas the most abundant form of ceramide in the brain, C18 ceramide, was on average 5-fold lower. Increased S1P content in the tumors was significantly correlated with increased sphingosine kinase 1 (SPHK1) and decreased sphingosine phosphate phosphatase 2 (SGPP2) expression. Inhibition of S1P production by cultured glioblastoma cells, using a highly potent and selective SPHK1 inhibitor, blocked angiogenesis in cocultured endothelial cells without affecting VEGF secretion. Our findings validate the hypothesis that an altered ceramide/S1P balance is an important feature of human cancers and support the development of SPHK1 inhibitors as antiangiogenic agents for cancer therapy.

The ability of cancer cells to sense external mechanical forces has emerged as a significant factor in the promotion of cancer invasion. Currently there are conflicting reports in the literature with regard to whether glioblastoma (GBM) brain cancer cell migration and invasion is rigidity-sensitive. In order to address this question we have compared the rigidity-response of primary patient-derived GBM lines. Cells were plated on polyacrylamide gels of defined rigidity that reflect the diversity of the brain tissue mechanical environment and cell morphology and migration were analysed by time-lapse microscopy. Invasiveness was assessed in multicellular spheroids embedded in 3D matrigel cultures. Our data reveal a range of rigidity-dependent responses between the patient-derived cell lines, from reduced migration on the most compliant tissue stiffness to those that are insensitive to substrate rigidity and are equally migratory irrespective of the underlying substrate stiffness. Notably, the rigidity-insensitive GBM cells show the greatest invasive capacity in soft 3D matrigel cultures. Collectively our data confirm both rigidity-dependent and independent behaviour in primary GBM patient-derived cells.

The role of astrocytes is becoming increasingly important to understanding how glioblastoma (GBM) tumor cells diffusely invade the brain. Yet, little is known of the contribution of extracellular vesicle (EV) signaling in GBM/astrocyte interactions. We modeled GBM-EV signaling to normal astrocytes in vitro to assess whether this mode of intercellular communication could support GBM progression. EVs were isolated and characterized from three patient-derived GBM stem cells (NES⁺/CD133⁺) and their differentiated (diff) progeny cells (NES⁻/CD133⁻). Uptake of GBM-EVs by normal primary astrocytes was confirmed by fluorescence microscopy, and changes in astrocyte podosome formation and gelatin degradation were measured. Quantitative mass spectrometry-based proteomics was performed on GBM-EV stimulated astrocytes. Interaction networks were generated from common, differentially abundant proteins using Ingenuity® (Qiagen Bioinformatics) and predicted upstream regulators were tested by qPCR assays. Podosome formation and Cy3-gelatin degradation were induced in astrocytes following 24-h exposure to GBM-stem and -diff EVs, with EVs released by GBM-stem cells eliciting a greater effect. More than 1700 proteins were quantified, and bioinformatics predicted activations of MYC, NFE2L2, FN1, and TGFβ1 and inhibition of TP53 in GBM-EV stimulated astrocytes that were then confirmed by qPCR. Further qPCR studies identified significantly decreased Δ133p53 and increased p53β in astrocytes exposed to GBM-EVs that might indicate the acquisition of a pro-inflammatory, tumor-promoting senescence-associated secretory phenotype (SASP). Inhibition of TP53 and activation of MYC signaling pathways in normal astrocytes exposed to GBM-EVs may be a mechanism by which GBM manipulates astrocytes to acquire a phenotype that promotes tumor progression.