Quantifying fear effects on prey demography in nature.

In recent years, it has been argued that the effect of predator fear exacts a greater demographic toll on prey populations than the direct killing of prey. However, efforts to quantify the effects of fear have primarily relied on experiments that replace predators with predator cues. Interpretation of these experiments must consider two important caveats: (1) the magnitude of experimenter-induced predator cues may not be realistically comparable to those of the prey's natural sensory environment, and (2) given functional predators are removed from the treatments, the fear effect is measured in the absence of any consumptive effects, a situation which never occurs in nature. We contend that demographic consequences of fear in natural populations may have been overestimated because the intensity of predator cues applied by experimenters in the majority of studies has been unnaturally high, in some instances rarely occurring in nature without consumption. Furthermore, the removal of consumption from the treatments creates the potential situation that individual prey in poor condition (those most likely to contribute strongly to the observed fear effects via starvation or reduced reproductive output) may have been consumed by predators in nature prior to the expression of fear effects, thus confounding consumptive and fear effects. Here, we describe an alternative treatment design that does not utilize predator cues, and in so doing, better quantifies the demographic effect of fear on wild populations. This treatment substitutes the traditional cue experiment where consumptive effects are eliminated and fear is simulated with a design where fear is removed and consumptive effects are simulated through the experimental removal of prey. Comparison to a natural population would give a more robust estimate of the effect of fear in the presence of consumption on the demographic variable of interest. This approach represents a critical advance in quantifying the mechanistic pathways through which predation structures ecological communities. Discussing the merits of both treatments will motivate researchers to go beyond simply describing the existence of fear effects and focus on testing their true magnitude in wild populations and natural communities.

Iterative sorting reveals CD133+ and CD133- melanoma cells as phenotypically distinct populations.

BACKGROUND: The 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. METHODS: We 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. RESULTS: We 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. CONCLUSION: We 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.

Enhanced immunosuppression by therapy-exposed glioblastoma multiforme tumor cells.

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(+) and CD8(+) 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.

Side population is not necessary or sufficient for a cancer stem cell phenotype in glioblastoma multiforme.

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 stem-like 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.

The novel phloroglucinol PMT7 kills glycolytic cancer cells by blocking autophagy and sensitizing to nutrient stress.

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.

Density-dependent space use affects interpretation of camera trap detection rates.

Camera traps (CTs) are an increasingly popular tool for wildlife survey and monitoring. Estimating relative abundance in unmarked species is often done using detection rate as an index of relative abundance, which assumes that detection rate has a positive linear relationship with true abundance. This assumption may be violated if movement behavior varies with density, but the degree to which movement behavior is density-dependent across taxa is unclear. The potential confounding of population-level relative abundance indices by movement would depend on how regularly, and by what magnitude, movement rate and home-range size vary with density. We conducted a systematic review and meta-analysis to quantify relationships between movement rate, home-range size, and density, across terrestrial mammalian taxa. We then simulated animal movements and CT sampling to test the effect of contrasting movement scenarios on CT detection rate indices. Overall, movement rate and home-range size were negatively correlated with density and positively correlated with one another. The strength of the relationships varied significantly between taxa and populations. In simulations, detection rates were related to true abundance but underestimated change, particularly for slower moving species with small home ranges. In situations where animal space use changes markedly with density, we estimate that up to thirty percent of a true change in relative abundance may be missed due to the confounding effect of movement, making trend estimation more difficult. The common assumption that movement remains constant across densities is therefore violated across a wide range of mammal species. When studying unmarked species using CT detection rates, researchers and managers should explicitly consider that such indices of relative abundance reflect both density and movement. Practitioners interpreting changes in camera detection rates should be aware that observed differences may be biased low relative to true changes in abundance. Further information on animal movement, or methods that do not depend on assumptions of density-independent movement, may be required to make robust inferences on population trends.

Pharmacological concentrations of ascorbate radiosensitize glioblastoma multiforme primary cells by increasing oxidative DNA damage and inhibiting G2/M arrest.

Glioblastoma multiforme (GBM) has a very poor prognosis because of its chemo- and radiation therapy resistance. Here we investigated the ability of pharmacological concentrations of ascorbate to radiosensitize primary cells isolated from six GBM patients, mouse astrocytoma cells, and mouse astrocytes. We measured cell viability by trypan blue exclusion, generation of double-stranded DNA breaks by H2AX phosphorylation using fluorescently labeled antibodies and FACS analysis, apoptosis by annexin V/propidium iodide staining, inhibition of autophagy by 3-methyladenine, and cell cycle progression by propidium iodide staining of permeabilized cells. We showed that 5 mM ascorbate in combination with 6 Gy radiation killed more GBM primary cells by generating significantly more double-stranded breaks than either treatment alone (p<0.05). Combined treatment affected viability and double-stranded break generation in normal astrocytes to a much smaller extent. Radiation, but not 5 mM ascorbate, caused G2/M arrest in GBM cells and ascorbate prevented radiation-induced G2/M arrest in combined treatment. Cell death in response to 5 mM ascorbate or combination treatment was not mediated by apoptosis or autophagy. In conclusion, pharmacological concentrations of ascorbate radiosensitize GBM primary cells to a much greater extent than astrocytes; this large therapeutic ratio may be of clinical significance in radiation-resistant cancers.

Vaccination with irradiated tumor cells pulsed with an adjuvant that stimulates NKT cells is an effective treatment for glioma.

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 α-galactosylceramide (α-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 α-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(+) T-cell-mediated adaptive immune response. Pulsing α-GalCer onto tumor cells avoided the profound iNKT cell anergy induced by free α-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 age-matched healthy volunteers. Furthermore, irradiated GBM tumor cells pulsed with α-GalCer were able to stimulate iNKT cells and augment a T-cell response in vitro. CONCLUSIONS: Injection of irradiated tumor cells loaded with α-GalCer is a simple procedure that could provide effective immunotherapy for patients with high-grade glioma.

Glioblastoma cells negative for the anti-CD133 antibody AC133 express a truncated variant of the CD133 protein.

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 ( approximately 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.