We characterized GBM patients' tumor and systemic immune contexture with aim to reveal the mechanisms of immunological escape, their impact on patient outcome, and identify targets for immunotherapy. Increased CD3(+) T-cell infiltration was associated with prolonged survival independent of age, MGMT promoter methylation and post-operative treatment that implies potential for immunotherapy for GBM. Several mechanisms of escape were identified: within the tumor microenvironment: induced CD8(+)CD28(-)Foxp3(+) Tregs that may tolerize antigen presenting cells, elevated CD73 and CD39 ectonucleotidases that suppress T-cell function, and at the systemic level: elevated IL-10 levels in serum, diminished helper T-cell counts, and upregulated inhibitory CTLA-4.

Glioblastoma multiforme (GBM) is the most common primary brain tumour in adults and one of the most aggressive cancers in man. Despite technological advances in surgical management, combined regimens of radiotherapy with new generation chemotherapy, the median survival for these patients is 14.6 months. This is largely due to a highly deregulated tumour genome with opportunistic deletion of tumour suppressor genes, amplification and/or mutational hyper-activation of receptor tyrosine kinase receptors. The net result of these genetic changes is augmented survival pathways and systematic defects in the apoptosis signalling machinery. The only randomised, controlled phase II trial conducted targeting the epidermal growth factor receptor (EGFR) signalling with the small molecule inhibitor, erlotinib, has showed no therapeutic benefit. Survival signalling and apoptosis resistance in GBMs can be viewed as two sides of the same coin. Targeting increased survival is unlikely to be efficacious without at the same time targeting apoptosis resistance. We have critically reviewed the literature regarding survival and apoptosis signalling in GBM, and highlighted experimental, preclinical and recent clinical trials attempting to target these pathways. Combined therapies simultaneously targeting apoptosis and survival signalling defects might shift the balance from tumour growth stasis to cytotoxic therapeutic responses that might be associated with greater therapeutic benefits.

In this work, highly infiltrative brain tumors with a stem-like phenotype were established by xenotransplantation of human brain tumors in immunodeficient nude rats. These tumors coopted the host vasculature and presented as an aggressive disease without signs of angiogenesis. The malignant cells expressed neural stem cell markers, showed a migratory behavior similar to normal human neural stem cells, and gave rise to tumors in vivo after regrafting. Serial passages in animals gradually transformed the tumors into an angiogenesis-dependent phenotype. This process was characterized by a reduction in stem cells markers. Gene expression profiling combined with high throughput immunoblotting analyses of the angiogenic and nonangiogenic tumors identified distinct signaling networks in the two phenotypes. Furthermore, proinvasive genes were up-regulated and angiogenesis signaling genes were down-regulated in the stem-like tumors. In contrast, proinvasive genes were down-regulated in the angiogenesis-dependent tumors derived from the stem-like tumors. The described angiogenesis-independent tumor growth and the uncoupling of invasion and angiogenesis, represented by the stem-like cancer cells and the cells derived from them, respectively, point at two completely independent mechanisms that drive tumor progression. This article underlines the need for developing therapies that specifically target the stem-like cell pools in tumors.

It is well known that in vitro subculture represents a selection pressure on cell lines, and over time this may result in a genetic drift in the cancer cells. In addition, long‐term cultures harbor the risk of cross‐contamination with other cell lines. The consequences may have major impact on experimental results obtained in various laboratories, where the cell lines no longer reflect the original tumors that they are supposed to represent. Much neglected in the scientific community is a close monitoring of cell cultures by regular phenotypic and genetic characterization. In this report, we present a thorough characterization of the commonly used glioblastoma (GBM) model U‐251, which in numerous publications has been wrongly identified as U‐373, due to an earlier cross‐contamination. In this work, the original U‐251 and three subclones of U‐251, commonly referred to as U‐251 or U‐373, were analyzed with regard to their DNA profile, morphology, phenotypic expression, and growth pattern. By array comparative genomic hybridization (aCGH), we show that only the original low‐passaged U‐251 cells, established in the 1960s, maintain a DNA copy number resembling a typical GBM profile, whereas all long‐term subclones lost the typical GBM profile. Also the long‐term passaged subclones displayed variations in phenotypic marker expression and showed an increased growth rate in vitro and a more aggressive growth in vivo. Taken together, the variations in genotype and phenotype as well as differences in growth characteristics may explain different results reported in various laboratories related to the U‐251 cell line.

Natural killer (NK) cells are innate lymphoid cells that hold tremendous potential for effective immunotherapy for a broad range of cancers. Due to the mode of NK cell killing requiring one–to-one target engagement and site directed release of cytolytic granules, the therapeutic potential of NK cells has been most extensively explored in hematological malignancies. However, their ability to precisely kill antibody coated cells, cancer stem cells (CSCs) and genotoxically altered cells, while maintaining tolerance to healthy cells makes them appealing therapeutic effectors for all cancer forms, including metastases. Due to their release of pro-inflammatory cytokines, NK cells may potently reverse the anti-inflammatory tumor microenvironment (TME) and augment adaptive immune responses by promoting differentiation, activation and/ or recruitment of accessory immune cells to sites of malignancy. Nevertheless, integrated and coordinated mechanisms of subversion of NK cell activity against the tumor and its microenvironment exist. Although our understanding of the receptor ligand interactions that regulate NK cell functionality has evolved remarkably, the diversity of ligands and receptors is complex, as is their mechanistic foundations in regulating NK cell function. In this article, we review the literature and highlight how the TME manipulates the NK cell phenotypes, genotypes and tropism to evade tumor recognition and elimination. We discuss counter strategies that may be adopted to augment the efficacy of NK cell anti-tumor surveillance, the clinical trials that have been undertaken so far in solid malignancies, critically weighing the challenges and opportunities with this approach.

Glioblastoma (GBM) is a highly aggressive brain tumour, where patients respond poorly to radiotherapy and exhibit dismal survival outcomes. The mechanisms of radioresistance are not completely understood. However, cancer cells with an immature stem-like phenotype are hypothesised to play a role in radioresistance. Since the progenitor marker neuron-glial-2 (NG2) has been shown to regulate several aspects of GBM progression in experimental systems, we hypothesised that its expression would influence the survival of GBM patients. Quantification of NG2 expression in 74 GBM biopsies from newly diagnosed and untreated patients revealed that 50% express high NG2 levels on tumour cells and associated vessels, being associated with significantly shorter survival. This effect was independent of age at diagnosis, treatment received and hypermethylation of the O⁶-methylguanine methyltransferase (MGMT) DNA repair gene promoter. NG2 was frequently co-expressed with nestin and vimentin but rarely with CD133 and the NG2 positive tumour cells harboured genetic aberrations typical for GBM. 2D proteomics of 11 randomly selected biopsies revealed upregulation of an antioxidant, peroxiredoxin-1 (PRDX-1), in the shortest surviving patients. Expression of PRDX-1 was associated with significantly reduced products of oxidative stress. Furthermore, NG2 expressing GBM cells showed resistance to ionising radiation (IR), rapidly recognised DNA damage and effectuated cell cycle checkpoint signalling. PRDX-1 knockdown transiently slowed tumour growth rates and sensitised them to IR in vivo. Our data establish NG2 as an important prognostic factor for GBM patient survival, by mediating resistance to radiotherapy through induction of ROS scavenging enzymes and preferential DNA damage signalling.