This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Tumors, like all tissues, rely on metabolism to generate energy and to produce the macromolecules needed for cell survival and growth. It has long been hypothesized that rapid growth of aggressive tumors is propelled by specific, essential metabolic pathways that differentiate the tumor from surrounding tissues. These concepts have stimulated hope that a broader understanding of tumor metabolism will pave the way for better imaging techniques and novel therapies to curb tumor growth. So far, however, progress towards these goals has been hampered by the lack of accurate analytical tools to develop a systems-level view of tumor metabolism. Most previous studies have used simple assays to analyze individual components of the metabolic network (e.g. lactate production) rather than probing the entire network at once. As a result, we have developed a firm grasp of how some pathways (e.g. glycolysis) are regulated in tumor cells, but only a rudimentary understanding of how these pathways are integrated together into a metabolic platform that favors cell growth. An alternative approach, which we have used for several years, is to introduce nutrients labeled with stable isotopes C3C, 15N, etc) and then to measure distribution of the isotope into metabolite pools using NMR spectroscopy and mass spectrometry. We are now extending these methods to analyze metabolic fluxes in live tumors to identify the activities that are essential for tumor growth. There are three ongoing projects. Project 1: Identifying novel metabolic regulators of tumor cell survival and growth. Our previous work in glioblastoma cells identified a metabolic network featuring a robust Warburg effect complemented by mitochondrial metabolism of carbon from both glucose and glutamine. Utilization of glutamine in this pathway was essential in that it provided a net source of 5-carbon units to the tricarboxylic acid (TCA) cycle, enabling the cycle to function continuously even as intermediates were withdrawn for use in biosynthetic pathways to support cell growth and proliferation. Cells withdrawn from glutamine could not sustain viability unless an alternative source of TCA cycle intermediates was provided, confirming glutamine's crucial role in anaplerosis during cell growth. We are now performing loss of function experiments to identify the key regulators of growth from this glutamine-dependent pathway. Glutaminase (GLS), the rate-limiting enzyme in glutamine oxidation, will be suppressed using RNA interference, reducing cell proliferation in two-dimensional culture, anchorage-independent colony formation and growth of subcutaneous xenografts. We are establishing cell lines with stable knockdown of other targets and these will be analyzed using methods analogous to those used for other metabolic pathways. Project 2: Probing tumor metabolism in vivo. Developing rational strategies to manipulate tumor metabolism for therapeutic purposes will require a detailed knowledge of the metabolic activities at work in live tumors. We developed a method to probe tumor metabolism in an orthotopic model that captures many of the biological features of human glioblastoma. Tissue from human glioblastoma patients undergoing surgical resection can be dissociated and cells were introduced by stereotactic injection into the brains of NOD-SCID mice. When tumor-bearing mice develop neurological signs after 3-4 months, they will euthanized and tumor tissue was harvested for analysis and implantation into new hosts. The tumors displayed nuclear atypia, diffuse infiltration and a high proliferative index, similar to the parental tumors. In this project we will use dynamic and static FDG-PETCT using a Siemens Inveon PET-CT multimodality scanner to examine glucose uptake (or 18FDG signal). To identify pathways of glucose metabolism, mice will be catheterized and infused with D-[U-13C]glucose. Analysis of tumor and surrounding brain tissue will be performed by 13C NMR spectroscopy and isotopomer analysis. Project 3: Monitoring tumor metabolism in real-time using 13C hyperpolarization. We hope to build on the detailed view of tumor metabolism emerging from Projects 1 and 2 to develop new methods to image and quantify relevant metabolic fluxes in real time. The major obstacle to extending 13C-based imaging into clinical practice has historically been low abundance and sensitivity of this nucleus, resulting in an impractically low signal-to-noise ratio. This problem was dramatically addressed with recent advances in dynamic nuclear polarization (DNP, """"""""hyperpolarization""""""""). Work from other centers has validated hyperpolarization-based imaging in animal models of cancer. Most studies have focused on pyruvate, because the long T1 of pyruvare's carboxyl carbon (C1) results in a relatively long duration of enhanced polarization. Pyruvate is also positioned at a pivotal intersection between oxidative and non-oxidative metabolism, and its utilization in cancer cells is regulated by tumor suppressors, oncogenes, and hypoxia. Thus, the ability to observe and quantify routes of pyruvate metabolism in real time could provide outstanding information about tumor genetics and microenvironmental influences. We propose to examin the metabolism of hyperpolarized [1_13C]-pyruvate in cultured GBM cells, whose metabolism we had previously characLerized using conventional methods. Of interest will the hypothesis that although these cells exhibit a prominent Warburg effect, they also convert pyruvate to acetyl-CoA. Using a commercial polarizer [1-13C]-pyruvate will be hyperpolarized and introduced to GBM cells in a 10-mm NMR tube. Spectra will be acquired every 1.5 seconds for 2 minutes and mathematical models will be developed to measure fluxes through lactate dehydrogenase and other key reactions. At the same time, cells will be freeze-clamped and extracted for isotopomer analyss. Over the long term we believe hyperpolarization can be used to image metabolism in real time, and can be integrated with other techniques to provide an accurate and quantitative view of cancer cell metabolism.
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