Identification and isolation of slow-cycling glioma stem cells.

Cancer stem cells are defined as low-abundance, quiescent cells and are considered a major cellular source of tumor recurrence following therapy, which identifies these cells as important therapeutic targets for difficult-to-treat cancers, including high-grade gliomas. By contrast to the highly proliferative bulk tumor cells, glioma stem cells (GSC) are slow-cycling, and therefore less sensitive to DNA damaging cytotoxic drugs. GSC are also less reliant on aerobic glycolytic metabolism, leading to inadequate clearing of GSC by chemotherapy and radiotherapy. The definition of GSC is based on the expression of specific stem cell protein markers. This method of GSC isolation is successful in isolating cell populations that can reliably recapitulate the tumor. However, cell populations that lack stem marker expression may also be capable of tumor recapitulation. Therefore, robust, reproducible methods for isolating GSC are required to identify and isolate cells with stem cell characteristics. Here, we provide a comprehensive and reproducible protocol for the isolation of slow-cycling GSC. Using this method, GSC isolated retain key characteristics of the cells in situ, including expression of genes associated with cell quiescence and invasive potential, compared to non-quiescent cell populations. Thus, isolation of GSC gated on cell proliferation offers a reliable alternative method for in vitro GSC identification, that adequately mirrors the physiological properties of GSC seen in vivo.

Major elective abdominal surgery acutely impairs lower limb muscle pyruvate dehydrogenase complex activity and mitochondrial function.

BACKGROUND & AIMS: This post hoc study aimed to determine whether major elective abdominal surgery had any acute impact on mitochondrial pyruvate dehydrogenase complex (PDC) activity and maximal mitochondrial ATP production rates (MAPR) in a large muscle group (vastus lateralis -VL) distant to the site of surgical trauma. METHODS: Fifteen patients undergoing major elective open abdominal surgery were studied. Muscle biopsies were obtained after the induction of anesthesia from the VL immediately before and after surgery for the determination of PDC and maximal MAPR (utilizing a variety of energy substrates). RESULTS: Muscle PDC activity was reduced by >50% at the end of surgery compared with pre-surgery (p < 0.05). Muscle MAPR were comprehensively suppressed by surgery for the substrate combinations: glutamate + succinate; glutamate + malate; palmitoylcarnitine + malate; and pyruvate + malate (all p < 0.05), and could not be explained by a lower mitochondrial yield. CONCLUSIONS: PDC activity and mitochondrial ATP production capacity were acutely impaired in muscle distant to the site of surgical trauma. In keeping with the limited data available, we surmise these events resulted from the general anesthesia procedures employed and the surgery related trauma. These findings further the understanding of the acute dysregulation of mitochondrial function in muscle distant to the site of major surgical trauma in patients, and point to the combination of general anesthesia and trauma related inflammation as being drivers of muscle metabolic insult that warrants further investigation. CLINICAL TRIAL REGISTRATION: Registered at (NCT01134809).

Cell quiescence correlates with enhanced glioblastoma cell invasion and cytotoxic resistance.

Glioblastoma (GBM) tumor cells exhibit drug resistance and are highly infiltrative. GBM stem cells (GSCs), which have low proliferative capacity are thought to be one of the sources of resistant cells which result in relapse/recurrence. However, the molecular mechanisms regulating quiescent-specific tumor cell biology are not well understood. Using human GBM cell lines and patient-derived GBM cells, Oregon Green dye retention was used to identify and isolate the slow-cycling, quiescent-like cell subpopulation from the more proliferative cells in culture. Sensitivity of cell subpopulations to temozolomide and radiation, as well as the migration and invasive potential were measured. Differential expression analysis following RNAseq identified genes enriched in the quiescent cell subpopulation. Orthotopic transplantation of cells into mice was used to compare the in vivo malignancy and invasive capacity of the cells. Proliferative quiescence correlated with better TMZ resistance and enhanced cell invasion, in vitro and in vivo. RNAseq expression analysis identified genes involved in the regulation cell invasion/migration and a three-gene signature, TGFBI, IGFBP3, CHI3L1, overexpressed in quiescent cells which correlates with poor GBM patient survival.

Repair mechanisms help glioblastoma resist treatment.

Glioblastoma multiforme (GBM) is a malignant and incurable glial brain tumour. The current best treatment for GBM includes maximal safe surgical resection followed by concomitant radiotherapy and adjuvant temozolomide. Despite this, median survival is still only 14-16 months. Mechanisms that lead to chemo- and radio-resistance underpin treatment failure. Insights into the DNA repair mechanisms that permit resistance to chemoradiotherapy in GBM may help improve patient responses to currently available therapies.

Suppressing a charge density wave by changing dimensionality in the ferecrystalline compounds ([SnSe]1.15)1(VSe2)n with n = 1, 2, 3, 4.

The compounds, ([SnSe]1.15)1(VSe2)n with n = 1, 2, 3, and 4, were prepared using designed precursors in order to investigate the influence of the thickness of the VSe2 constituent on the charge density wave transition. The structure of each of the compounds was determined using X-ray diffraction and scanning transmission electron microscopy. The charge density wave transition observed in the resistivity of ([SnSe]1.15)1(VSe2)1 was confirmed. The electrical properties of the n = 2 and 3 compounds are distinctly different. The magnitude of the resistivity change at the transition temperature is dramatically lowered and the temperature of the resistivity minimum systematically increases from 118 K (n = 1) to 172 K (n = 3). For n = 1, this temperature correlates with the onset of the charge density wave transition. The Hall-coefficient changes sign when n is greater than 1, and the temperature dependence of the Hall coefficient of the n = 2 and 3 compounds is very similar to the bulk, slowly decreasing as the temperature is decreased, while for the n = 1 compound the Hall coefficient increases dramatically starting at the onset of the charge density wave. The transport properties suggest an abrupt change in electronic properties on increasing the thickness of the VSe2 layer beyond a single layer.

Raman spectroscopy insights into the size-induced structural transformation in SnSe nanolayers.

Raman spectroscopy is used to probe the structural changes in [SnSe]m[MoSe2]n ferecrystal thin films as a function of m, the number of bilayers of SnSe. In spite of the interleaved structure in the intergrowths, Raman spectra can be described as a superposition of spectra from the individual components, indicating that the interaction at the interface between the components is relatively weak. Analysis of room-temperature Raman spectra indicate that the MoSe2 layers separating the SnSe layers are nanocrystalline in all of the samples studied, with little change as the number of Se-Mo-Se trilayers (n) or SnSe bilayers (m) increases, reflecting the rotational disorder between adjacent trilayers. A thickness-dependent, continuous transition occurs in the SnSe layer as m is increased, from a pseudotetragonal structure when the layers are thin to a bulk-like orthorhombic SnSe structure when the SnSe layer thickness is increased. Polarization analysis of the Raman scattering from these materials allows the symmetry evolution of the SnSe layers through this transition to be determined.

Resistance to aerobic exercise training causes metabolic dysfunction and reveals novel exercise-regulated signaling networks.

Low aerobic exercise capacity is a risk factor for diabetes and a strong predictor of mortality, yet some individuals are "exercise-resistant" and unable to improve exercise capacity through exercise training. To test the hypothesis that resistance to aerobic exercise training underlies metabolic disease risk, we used selective breeding for 15 generations to develop rat models of low and high aerobic response to training. Before exercise training, rats selected as low and high responders had similar exercise capacities. However, after 8 weeks of treadmill training, low responders failed to improve their exercise capacity, whereas high responders improved by 54%. Remarkably, low responders to aerobic training exhibited pronounced metabolic dysfunction characterized by insulin resistance and increased adiposity, demonstrating that the exercise-resistant phenotype segregates with disease risk. Low responders had impaired exercise-induced angiogenesis in muscle; however, mitochondrial capacity was intact and increased normally with exercise training, demonstrating that mitochondria are not limiting for aerobic adaptation or responsible for metabolic dysfunction in low responders. Low responders had increased stress/inflammatory signaling and altered transforming growth factor-ß signaling, characterized by hyperphosphorylation of a novel exercise-regulated phosphorylation site on SMAD2. Using this powerful biological model system, we have discovered key pathways for low exercise training response that may represent novel targets for the treatment of metabolic disease.

Regulation of glycogen synthase kinase-3 beta (GSK-3ß) by the Akt pathway in gliomas.

Gliomas are aggressive brain tumours that, despite advances in multimodal therapies, continue to portend a dismal prognosis. Glioblastoma multiforme (GBM) represents the most aggressive glioma and patients have a median survival of 14 months, even with the best available treatments. The phosphoinositide 3-kinase/Akt/glycogen synthase kinase-3 beta (GSK-3ß) and Wnt/ß-catenin pathways are dysregulated in a number of cancers, and these two pathways share a common node protein, GSK-3ß. This protein is responsible for the regulation/degradation of ß-catenin, which reduces ß-catenin's translocation to the nucleus and influences the subsequent transcription of oncogenes. The non-specific small-molecule GSK-3ß inhibitor, lithium chloride (LiCl), and the specific Akt inhibitor, AktX, were used to treat U87MG and U87MG.Δ2-7 human glioma cell lines. LiCl treatment significantly affected cell morphology of U87MG and U87MG.Δ2-7 cells, while also increasing levels of phospho-GSK-3ß in a dose-dependent manner. Increased cell proliferation was observed at low-to-mid LiCl concentrations as determined by MTT cell growth assays. Treatment of U87MG and U87MG.Δ2-7 cells with AktX resulted in reduced levels of phospho-GSK-3ß through its inhibition of Akt, in addition to decreased levels of phosphorylated (active) Akt in a dose-dependent fashion. We have shown in this study that GSK-3ß regulation by phosphorylation is important for cell morphology and growth, and that LiCl enhances growth of U87MG and U87MG.Δ2-7 cells by inhibiting GSK-3ß through its phosphorylation, whereas AktX reduces growth via activation of GSK-3ß by inhibiting Akt's kinase activity.