Malignancy cells use alternate energetic pathways; however, malignancy stem cell (CSC) metabolic dynamic pathways are unknown. plasma samples from healthy controls (mean age 52.4 8.25) were obtained from ProteoGenex for validation studies (Table 1 and Supporting Information; Table H5). Metabolomic Profiling Sample Preparation Samples were sent to Metabolon, Inc. (Durham, NC) for metabolomic profiling studies. Briefly, samples were prepared using the automated MicroLab STAR system from the Hamilton Company (Reno, NV). A recovery standard was added prior to the first step in the extraction process for Quality Control (QC) purposes. To remove protein, dissociate small molecules bound to protein or caught in the precipitated protein matrix, and to recover chemically diverse metabolites, protein were precipitated with methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000) followed by centrifugation. The producing extract was divided into five fractions: one for analysis by Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS) 151533-22-1 manufacture with positive ion mode electrospray ionization, one for analysis by UPLC-MS/MS with unfavorable ion mode electrospray ionization, one for analysis by UPLC-MS/MS polar platform (unfavorable ionization), one for analysis by Gas 151533-22-1 manufacture ChromatographyCMass Spectroscopy (GC-MS), and one sample was reserved for backup. Samples were placed briefly on a TurboVap (Zymark) to remove the organic solvent. For LC, the samples were stored overnight under nitrogen before preparation for analysis. For GC, each sample 151533-22-1 manufacture was dried under vacuum overnight before preparation for analysis. For a complete description of quality assurance and quality control steps please BZS review Supporting Information reports (tissue and saliva); Report H1. Validation of Glutamate and Glutamine by GC-MS The levels of glutamate and glutamine were assessed in tissues, cell lines, saliva, and plasma samples using isotype dilution GC-MS as described previously.28 In brief, frozen tissues, saliva, plasma, or cell lysate pellets were homogenized in methanol after spiking with labeled internal standards (= 432) and glutamine (= 431) to that corresponding to spiked isotope-labeled glutamate (= 437) and glutamine (= 436), respectively. The levels of glutamate and glutamine were normalized to the tissue weight. Cell Lines and Culture The human HNSCC cell lines were as follows: UM-SCC-17B (supraglottis/soft tissue-neck) and UM-SCC-14A (floor of mouth; both provided by Thomas Carey, University of Michigan, Ann Arbor, MI);29 HSC-3 (tongue; provided by Randall Kramer, University of California, San Francisco, CA);30 OSCC-3 (tongue; provided by Mark Lingen, University of Chicago, Chicago, IL). HNSCC cell line authentication and origin were provided by their sources. HNSCC cells were maintained in Dulbeccos altered Eagles medium made up of 10% fetal bovine serum, 1% penicillin, and 1% streptomycin. Primary human oral keratinocytes (ScienCell, Carlsbad, CA) were maintained in oral keratinocyte medium (OKM) (ScienCell, Carlsbad, CA). Immunohistochemical Staining Standard immunohistochemical analyses were used to evaluate glutaminase manifestation in normal and tumor tissue sections using a glutaminase primary antibody (AP8809b, Abgent, San Diego, CA). Staining intensities [1 (poor), 2 (moderate), and 3 (strong)] for glutaminase were graded and analyzed in a blinded manner by a pathologist. Low manifestation was defined as intensity 1, and high manifestation was defined as intensity 2 or 3. Immunoblot Analysis To evaluate the protein manifestation levels of glutaminase and ALDH1, standard Western blot analyses were performed using an antiglutaminase primary antibody (ab607709, Abcam, San Francisco, CA) or an ALDH1 primary antibody (61195, BD Transduction, Franklin Lakes, NJ). test (saliva) were used to identify metabolites that differed significantly between experimental groups ( 0.05 and 0.05 < < 0.10). Analysis by two-way ANOVA with repeated steps identified features exhibiting the main effects of the group experimental parameter. An estimate of the false finding rate ( 0.05 (a potential for 5% false observations). However, where metabolites move in a consistent direction across a.