There is strong evidence for the existence of cancer stem cells (CSCs) in the aggressive brain tumor glioblastoma multiforme (GBM). These cells have stem-like self-renewal activity and increased tumor initiation capacity and are believed to be responsible for recurrence due to their resistance to therapy. Several techniques have been used to enrich for CSC, including growth in serum-free defined media to induce sphere formation, and isolation of a stemlike cell using exclusion of the fluorescent dye Hoechst 33342, the side population (SP). We show that sphere formation in GBM cell lines and primary GBM cells enriches for a CSC-like phenotype of increased self-renewal gene expression in vitro and increased tumor initiation in vivo. However, the SP was absent from all sphere cultures.Direct isolation of the SP from the GBM lines did not enrich for stem-like activity in vitro, and tumor-initiating activity was lower in sorted SP compared with non-SP and parental cells. Transient exposure to doxorubicin enhanced both CSC and SP frequency. However, doxorubicin treatment altered the cytometric profile and obscured the SP demonstrating the difficulty of identifying SP in cells under stress. Doxorubicin-exposed cells showed a transient increase in SP, but the doxorubicin-SP cells were still not enriched for a stem-like self-renewal phenotype. These data demonstrate that the GBM SP does not necessarily contribute to self-renewal or tumor initiation, key properties of a CSC, and we advise against using SP to enumerate or isolate CSC. STEM CELLS 2011;29:452-461 Disclosure of potential conflicts of interest is found at the end of this article.

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Purpose: The prognosis for patients with glioblastoma multiforme (GBM) remains extremely poor despite recent treatment advances. There is an urgent need to develop novel therapies for this disease.Experimental Design: We used the implantable GL261 murine glioma model to investigate the therapeutic potential of a vaccine consisting of intravenous injection of irradiated whole tumor cells pulsed with the immuno-adjuvant a-galactosylceramide (a-GalCer).Results: Vaccine treatment alone was highly effective in a prophylactic setting. In a more stringent therapeutic setting, administration of one dose of vaccine combined with depletion of regulatory T cells (Treg) resulted in 43% long-term survival and the disappearance of mass lesions detected by MRI. Mechanistically, the a-GalCer component was shown to act by stimulating "invariant" natural killer-like T cells (iNKT cells) in a CD1d-restricted manner, which in turn supported the development of a CD4 þ T-cell-mediated adaptive immune response. Pulsing a-GalCer onto tumor cells avoided the profound iNKT cell anergy induced by free a-GalCer. To investigate the potential for clinical application of this vaccine, the number and function of iNKT cells was assessed in patients with GBM and shown to be similar to agematched healthy volunteers. Furthermore, irradiated GBM tumor cells pulsed with a-GalCer were able to stimulate iNKT cells and augment a T-cell response in vitro.Conclusions: Injection of irradiated tumor cells loaded with a-GalCer is a simple procedure that could provide effective immunotherapy for patients with high-grade glioma.

Glioblastoma multiforme (GBM) is a highly malignant brain tumor with an extremely short time to relapse following standard treatment. Since recurrent GBM is often resistant to subsequent radiotherapy and chemotherapy, immunotherapy has been proposed as an alternative treatment option. Although it is well established that GBM induces immune suppression, it is currently unclear what impact prior conventional therapy has on the ability of GBM cells to modulate the immune environment. In this study, we investigated the interaction between immune cells and glioma cells that had been exposed to chemotherapy or irradiation in vitro. We demonstrate that treated glioma cells are more immunosuppressive than untreated cells and form tumors at a faster rate in vivo in an animal model. Cultured supernatant from in vitro-treated primary human GBM cells were also shown to increase suppression, which was independent of accessory suppressor cells or T regulatory cell generation, and could act directly on CD4 1 and CD8 1 T cell proliferation. While a number of key immunosuppressive cytokines were overexpressed in the treated cells, including IL-10, IL-6 and GM-CSF, suppression could be alleviated in a number of treated GBM lines by inhibition of prostaglandin E2. These results reveal for the first time that conventional therapies can alter immunosuppressive pathways in GBM tumor cells, a finding with important implications for the combination of immunotherapy with standard treatment.Despite aggressive upfront therapy for glioblastoma multiforme (GBM), which includes surgical resection, radiotherapy and temozolomide-based chemotherapy, the median time to recurrence is only 6.9 months after standard treatment.1

BackgroundThe heterogeneity and tumourigenicity of metastatic melanoma is attributed to a cancer stem cell model, with CD133 considered to be a cancer stem cell marker in melanoma as well as other tumours, but its role has remained controversial.MethodsWe iteratively sorted CD133+ and CD133- cells from 3 metastatic melanoma cell lines, and observed tumourigenicity and phenotypic characteristics over 7 generations of serial xeno-transplantation in NOD/SCID mice.ResultsWe demonstrate that iterative sorting is required to make highly pure populations of CD133+ and CD133- cells from metastatic melanoma, and that these two populations have distinct characteristics not related to the cancer stem cell phenotype. In vitro, gene set enrichment analysis indicated CD133+ cells were related to a proliferative phenotype, whereas CD133- cells were of an invasive phenotype. However, in vivo, serial transplantation of CD133+ and CD133- tumours over 7 generations showed that both populations were equally able to initiate and propagate tumours. Despite this, both populations remained phenotypically distinct, with CD133- cells only able to express CD133 in vivo and not in vitro. Loss of CD133 from the surface of a CD133+ cell was observed in vitro and in vivo, however CD133- cells derived from CD133+ retained the CD133+ phenotype, even in the presence of signals from the tumour microenvironment.ConclusionWe show for the first time the necessity of iterative sorting to isolate pure marker-positive and marker-negative populations for comparative studies, and present evidence that despite CD133+ and CD133- cells being equally tumourigenic, they display distinct phenotypic differences, suggesting CD133 may define a distinct lineage in melanoma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2759-2) contains supplementary material, which is available to authorized users.

The switch from oxidative phosphorylation to glycolytic metabolism results in cells that generate fewer reactive oxygen species (ROS) and are resistant to the intrinsic induction of apoptosis. As a consequence, glycolytic cancer cells are resistant to radiation and chemotherapeutic agents that rely on production of ROS or intrinsic apoptosis. Further, the level of glycolysis correlates with tumor invasion, making glycolytic cancer cells an important target for new therapy development. We have synthesized a novel redox-active quinone phloroglucinol derivative, PMT7. Toxicity of PMT7 was in part due to loss of mitochondrial membrane potential in treated cells with subsequent loss of mitochondrial metabolic activity. Mitochondrial gene knockout ρ0 cells, a model of highly glycolytic cancers, were only half as sensitive as the corresponding wild-type cells and metabolic pathways downstream of MET were unaffected in ρ0 cells. However, PMT7 toxicity was also due to a block in autophagy. Both wild-type and ρ0 cells were susceptible to autophagy blockade, and the resistance of ρ0 cells to PMT7 could be overcome by serum deprivation, a situation where autophagy becomes necessary for survival. The stress response class III deacetylase SIRT1 was not significantly involved in PMT7 toxicity, suggesting that unlike other chemotherapeutic drugs, SIRT1-mediated stress and survival responses were not induced by PMT7. The dependence on autophagy or other scavenging pathways makes glycolytic cancer cells vulnerable. This can be exploited by induction of energetic stress to specifically sensitize glycolytic cells to other stresses such as nutrient deprivation or potentially chemotherapy.

Abstract. The transmembrane glycoprotein CD133 is a marker commonly used for isolation and analysis of putative cancer stem-like cells. However, analysis of CD133 expression is potentially confounded by the fact that two of the commonly used anti-CD133 antibodies, AC133 and 293C, only recognize CD133 that has undergone glycosylation. Therefore, our aim was to thoroughly examine antibody recognition and mRNA expression of CD133 in glioblastoma multiforme. Glioblastoma cell lines and primary cultures obtained from resected tumor tissue were analyzed by real-time PCR, Western blot analysis, and flow cytometry for CD133, and immunofluorescence was used to determine cellular localization. The AC133 and 293C antibodies did not detect any CD133 on the surface of the glioblastoma cells despite the fact that a protein was detected using C24B9, an anti-CD133 antibody that recognizes an unglycosylated epitope. This CD133 variant was truncated (~16 kDa) and, unlike typical expression of full-length CD133 protein, was found throughout the cytoplasm instead of localized to the plasma membrane. Levels of mRNA and protein for the variant increased with stress, indicating potential for it to be a functional molecule. Because AC133 and 293C antibodies do not detect all CD133 variants in glioblastoma cells, alternate detection methods need to be utilized for complete analysis of CD133 expression and for accurately determining the relationship between CD133 and cancer stem-like cells.

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