Patent Application
20120237937
Although measurements of gene expression and protein expression provide useful clues to metabolic dysfunctions in some cancers, they may not give a complete picture of metabolic changes that result in the cancer (e.g., malignant) phenotype. Posttranslational modifications, protein inhibitors, allosteric regulation by effector metabolites, alternative gene functions, or compartmentalization are some examples that result in metabolic changes that may not be reflected by gene expression or protein expression determination. Therefore, metabolic profiling (or metabolomic) investigations (which can be supplemented with protein expression or gene expression measurements) can be useful to study and detect cancer and cancer phenotypes.
Some embodiments of the invention include methods for detecting the presence of cancer in an animal that was administered an administered labeled molecule. In some instances the method comprises (i) obtaining a first NMR spectrum of a first non-cancer cell extract, and obtaining a second NMR spectrum of a first cancer cell extract, (ii) obtaining a first MS spectrum of a second non-cancer cell extract, and obtaining a second MS spectrum of a second cancer cell extract, or both. The first non-cancer cell extract was obtained from a first set of non-cancer cells removed from a tissue of the animal; the first cancer cell extract was obtained from a first set of cancer cells removed from the tissue of the animal; the second non-cancer cell extract was obtained from a second set of non-cancer cells removed from the tissue of the animal, and the second cancer cell extract was obtained from a second set of cancer cells removed from the tissue of the animal. A first amount of at least one resultant labeled molecule is determined from the first NMR spectrum, from the first MS spectrum, or from both. A second amount of at least one resultant labeled molecule is determined from the second NMR spectrum, from the second MS spectrum, or from both. Cancer can be detected by comparing the first amount of at least one resultant labeled molecule with the second amount of at least one resultant labeled molecule.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the description of specific embodiments presented herein.
Some embodiments of the invention include methods for detecting the presence of cancer in an animal that was administered an administered labeled molecule. In some instances the method comprises (i) obtaining a first NMR spectrum of a first non-cancer cell extract, and obtaining a second NMR spectrum of a first cancer cell extract, (ii) obtaining a first MS spectrum of a second non-cancer cell extract, and obtaining a second MS spectrum of a second cancer cell extract, or both. The first non-cancer cell extract was obtained from a first set of non-cancer cells removed from a tissue of the animal; the first cancer cell extract was obtained from a first set of cancer cells removed from the tissue of the animal; the second non-cancer cell extract was obtained from a second set of non-cancer cells removed from the tissue of the animal, and the second cancer cell extract was obtained from a second set of cancer cells removed from the tissue of the animal. A first amount of at least one resultant labeled molecule is determined from the first NMR spectrum, from the first MS spectrum, or from both. A second amount of at least one resultant labeled molecule is determined from the second NMR spectrum, from the second MS spectrum, or from both. Cancer can be detected by comparing the first amount of at least one resultant labeled molecule with the second amount of at least one resultant labeled molecule.
In some embodiments of the invention, labeled molecules and isotopomer approaches can be used to study animals with cancer. For example, metabolic differences can be investigated by infusing labeled molecules (e.g., labeled metabolites) into animals with cancer, followed by removal and processing of paired non-cancerous cells and cancerous cells of the animal's tissue. NMR, MS, or both can be used for isotopomer-based metabolomic analysis of the extracts of tissues. Labeled molecules can be, for example, administered intravenously into an animal prior to removal (e.g., surgical resection) of the cancer (e.g., primary tumor) cells and non-cancerous cells in the tissue. In some embodiments, the non-cancerous cells are taken from cells surrounding the cancerous cells in the tissue. In some instances, differences in metabolic transformations between the non-cancerous cells and cancer cells can be determined using NMR, MS, or both. In some embodiments, this approach can be used to detect cancer (e.g., in some instances including analysis of metabolic traits of cancer cells) without interferences from either intrinsic (e.g. genetic) or external environmental factors (e.g. diet) because the patient's own non-cancerous cells serve as an internal control.
As used herein, the term “metabolite” refers to the reactants (e.g., precursors), intermediates, and products of metabolic transformations.
Some embodiments of the invention include methods for detecting the presence of cancer in an animal that was administered an administered labeled molecule.
The route of administration of the administered labeled compound may be of any suitable route including, but are not limited to an oral route, a parenteral route, a cutaneous route, a nasal route, a rectal route, a vaginal route, or an ocular route. The choice of administration route can depend on the compound identity, such as the physical and chemical properties of the compound, as well as the age and weight of the animal, the particular cancer, degree of localization or encapsulation of the cancer, or the severity of the cancer. Of course, combinations of administration routes can be administered, as desired.
The administered labeled molecule can be any suitable labeled molecule, including but not limited to a 13C isotopomer of glucose, a 13C isotopomer of pyruvate, a 13C isotopomer of Ala, an 15N isotopomer of Ala, a 13C isotopomer of acetate, a 13C isotopomer of glutamine, an 15N isotopomer of glutamine, glucose (C1, C2, C3, C4, C5, C6, or any combination thereof that are 13C labeled; e.g., all glucose carbons are 13C labeled, 13C1-glucose, 13C2-glucose, 13C3-glucose, 13C4-glucose, or 13C5-glucose), pyruvate (C1, C2, C3 or any combination thereof are 13C labeled; e.g., C1, C2, and C3 are all 13C labeled, 13C1-pyruvate, or 13C2-pyruvate), acetyl CoA (C1, C2 or both are 13C labeled; e.g., 13C1-acetyl CoA), and Ala (C1, C2, C3 or any combination thereof are 13C labeled; e.g., C1, C2, and C3 are all 13C labeled, 13C1-Ala, or 13C2-Ala), 13C labeled glycerol, 13C labeled fatty acids (e.g., octanoic acid), 13C labeled amino acids (e.g., glutamine, serine, tryptophan that have one or more labels), or 15N labeled amino acids (e.g., glutamine, serine, tryptophan that have one or more labels). The administered labeled molecule can also include molecules (e.g., amino acids) that have one or more 15N labels, including but not limited to, 15N labeled amino acids. Of course, the administered labeled molecule can be labeled with one or more 13C labels, one or more 15N labels, one or more 2H, one or more 3H, one or more 77Se, one or more 31P, or combinations thereof. The designation “13Cx” indicates that x of the molecule's carbons are 13C labeled, but when x is less than the total number of the molecule's carbons the specific labeling locations are not designated and 13Cx refers to a set of molecules. For example, 13C5-glucose is the set of five glucose molecules that have 5 of the 6 carbons 13C labeled. When all carbons in a molecule are 13C-labeled that is designated as being uniformly labeled and is indicated by [U-13C]. In some embodiments, the administered labeled molecule is of uniformly 13C-labeled glucose ([U-13C]-Glc), 13C1-glucose, 13C2-glucose, 13C3-glucose, 13C4-glucose, 13C5-glucose, [U-13C]-pyruvate, 13C1-pyruvate, 13C2-pyruvate, [U-13C]-acetyl CoA, 13C1-acetyl CoA, [U-13C]-Ala, 13C1-Ala, or 13C2-Ala.
The amount of administered labeled molecule administered to the animal can be any suitable amount, including but not limited to about 10 mmol, about 25 mmol, about 50 mmol, about 75 mmol, about 100 mmol, about 125 mmol, about 150 mmol, about 175 mmol, about 200 mmol, about 400 mmol, about 600 mmol, about 700 mmol, or about 1000 mmol. The administered labeled molecule can be administered to the animal in a bolus administration or over a period of time. The amount of time the administered labeled molecule can be administered to the animal can be any suitable time, including but not limited to about 1 min., about 3 min., about 5 min., about 10 min., about 20 min., about 30 min., about 40 min., about 50 min., about 1 hour, about 2 hours, or about 5 hours. The amount and time of administration can, in some embodiments, depend upon one or more of the administered label molecule, the resultant labeled molecule, the animal, the tissue, the cancer to be detected, the health of the animal, the age and weight of the animal, the enzyme or metabolic pathway to be analyzed, or any other relevant factor.
Animals include but are not limited to primates, canine, equine, bovine, porcine, ovine, avian, or mammalian. In some embodiments, the animal is a human, dog, cat, horse, cow, pig, sheep, chicken, turkey, mouse, or rat.
The cancer (including cancerous tumors) can include but is not limited to carcinomas, sarcomas, hematologic cancers, neurological malignancies, basal cell carcinoma, thyroid cancer, neuroblastoma, ovarian cancer, melanoma, renal cell carcinoma, hepatocellular carcinoma, breast cancer, colon cancer, lung cancer, pancreatic cancer, brain cancer, prostate cancer, chronic lymphocytic leukemia, acute lymphoblastic leukemia, rhabdomyosarcoma, Glioblastoma multiforme, meningioma, bladder cancer, gastric cancer, Glioma, oral cancer, nasopharyngeal carcinoma, kidney cancer, rectal cancer, lymph node cancer, bone marrow cancer, stomach cancer, uterine cancer, leukemia, basal cell carcinoma, cancers related to epithelial cells, or cancers that can alter the regulation or activity of PC. Cancerous tumors include, for example, tumors associated with any of the above mentioned cancers.
In some embodiments, cancerous tissue cells and non-cancerous tissue cells are removed from the animal. In some embodiments, the non-cancerous cells are taken from cells that are nearby or surrounding the cancerous cells in the tissue. In still other embodiments, the non-cancerous cells are taken from the same tissue from a different part of the animal (e.g., from a contralateral lung or breast). The removed cancerous tissue cells and the removed non-cancerous cells can be each extracted using the same or different extraction methods or solutions. The amount of at least one resultant molecule (e.g., a molecule resulting from the animal's body transforming the administered labeled molecule) is determined in the cancer cell extract and the non-cancer cell extract using NMR, MS or both. The presence of cancer can be determined by comparing the amount of at least one resultant molecule in the cancer cell extract with the amount of at least one resultant molecule in the non-cancer cell extract. Of course, extraction methods and solutions used for preparing NMR samples may or may not be different from extraction methods and solutions used for preparing MS samples.
The tissue can be any animal tissue including (e.g., mammalian tissues), such as but not limited to connective tissue, muscle tissue, nervous tissue, adipose tissue, endothelial tissue, or epithelial tissue. The tissue can be at least part of an organ or part of an organ system. Organs can include, but are not limited to heart, blood, blood vessels, salivary glands, esophagus, stomach, liver, gallbladder, pancreas, large intestines, small intestines, rectum, anus, colon, endocrine glands (e.g., hypothalamus, pituitary, pineal body, thyroid, parathyroids and adrenals), kidneys, ureters, bladder, urethraskin, hair, nails, lymph, lymph nodes, lymph vessels, leukocytes, tonsils, adenoids, thymus, spleen, muscles, brain, spinal cord, peripheral nerves, nerves, sex organs (e.g., ovaries, fallopian tubes, uterus, vagina, mammary glands, testes, vas deferens, seminal vesicles, prostate and penis), pharynx, larynx, trachea, bronchi, lungs diaphragm, bones, cartilage, ligaments, or tendons. Organ systems can include, but are not limited to circulatory system, digestive system, endocrine system, excretory system, integumentary system, lymphatic system, muscular system, nervous system, reproductive system, respiratory system, or skeletal system.
The tissue has cells that are cancerous and cells that are non-cancerous. Tissue cells can be removed from the animal by any suitable methods, including but not limited to surgical methods (e.g., resection), biopsy methods, or animal sacrifice followed by organ removal and dissection. The non-cancerous tissue cells are removed from any suitable distance from the cancerous portion of the tissue (e.g., the tumor margin) and can be, but is not limited to at least about 1 mm, at least about 3 mm, at least about 5 mm, at least about 1 cm, at least about 2 cm, or at least about 3 cm from the cancerous portion of the tissue. In some instances, the non-cancerous tissue cells are taken from the same tissue from a different part of the animal (e.g., from a contralateral lung or breast). In some embodiments, the non-cancerous tissue cells are completely free from or substantially (e.g., 99.9%, 99%, 95%, or 90%) free from cancerous cells. Removed tissue cells can be frozen in liquid nitrogen. Preparation of the removed tissue cells can be performed in any suitable manner (e.g., including pulverizing or grinding the tissue) to obtain the resultant labeled molecule and can include one or more extractions with solutions comprising any suitable solvent or combinations of solvents, such as, but not limited to acetonitrile, water, chloroform, methanol, butylated hydroxytoluene, trichloroacetic acid, or combinations thereof.
In some embodiments, tissue cells are removed at a certain time after administration of the administered labeled molecule. The time between the last administration of the administered labeled molecule and the removal of the tissue cells can be any suitable time, including but not limited to about 1 min., about 5 min., about 10 min., about 15 min., about 20 min., about 30 min., about 40 min., about 50 min., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 6 hours, about 9 hours, about 12 hours, or about 20 hours. For example, the time can be at least about 1 min., at least about 5 min., at least about 10 min., no more than about 20 min., no more than about 30 min., no more than about 40 min., no more than about 1 hour, no more than about 5 hours, or no more than about 20 hours. The amount of time can, in some embodiments, depend upon one or more of the administered labeled molecule, the resultant labeled molecule, the animal, the tissue, the cancer to be detected, the health of the animal, the enzyme or metabolic pathway, or any other relevant factor.
The resultant labeled molecule can result from a transformation of the administered labeled molecule. In some embodiments, this transformation occurs by enzymatic action or by action via a metabolic pathway or an anaplerotic pathway. Such metabolic pathways can include, but are not limited to Krebs cycle (also known as the citric acid cycle), glycolysis, pentose phosphate pathway (oxidative and non-oxidative) (PPP), gluconeogenesis, lipid biosynthesis, amino acid syntheses (e.g., synthesis of non-essential amino acids), catabolic pathways, urea cycle, Cori cycle, or glutamate/glutamine cycle. Enzymes involved in the transformation can include, but are not limited to pyruvate carboxylase (PC), succinyl CoA synthetase (SCS), phosphoenolpyruvate carboxykinase (PEPCK), transketolase, transaldolase, pyruvate dehydrogenase (PDH), a dehydrogenase (DH), glutaminase (GLS), isocitrate dehydrogenase (IDH), α-ketoglutarate dehydrogenase (OGDH), mitochondrial malate dehydrogenase (MDH), succinate dehydrogenase (SDH), fumarate hydratase (FH), hexokinase II (HKII), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase 1 (PGK-1), lactate dehydrogenase 5 (LDH-5), phosphofructokinase 1 and 2 (PFK-1 and PFK-2), glutathione peroxidase (GPx), or glutathione-5-transferase (GST). In some embodiments, the resultant labeled molecule is an isotopomer of any of lactate, alanine (Ala), arginine (Arg), serine (Ser), proline (Pro), asparagine (Asn), Glycine (Gly), glutamate (Glu), oxidized glutathione (GSSG), Glu-GSSH, Glu-GSH, glutamine (Gln), γ-aminobutyrate (GAB), succinate, citrate, isocitrate, fumarate, malate, aspartate (Asp), creatine (Cr), oxaloacetate: (OAA), α-ketoglutarate: (αKG), phosphocholine (P-choline), N-methyl-phosphocholine, taurine, glycogen, phenylalanine (Phe), tyrosine (Tyr), myo-inositol, α- and β-glucose, NAD+, cytosine nucleotides (CXP), uracil nucleotides (UXP), guanine nucleotides (GXP), or adenine nucleotides (AXP). In other embodiments, the resultant labeled molecule is uniformly 13C labeled lactate ([U-13C]-lactate), [U-13C]-Ala, 13C-3-Glu, 13C-3-Gln, 13C-3-glutamyl residue of oxidized glutathione (Glu-GSSG), 13C-2-Glu-GSSG, 13C-2-Glu, 13C-2-Asp, of 13C-3-Ala, 13C-3-lactate, 13C-3-Glu, 13C-3-Gln, 13C-3-Asp, 13C-2,3-succinate, 13C-2,4-citrate, 13C-1′-ribose-5′AXP, 13C-1-α- and -β-glucose, 13C-2,3-lactate, 13C-2 to 4-Glu, 13C-4-Gln, 13C-4-GSSG, 13C-4-GSG, 13C-2,4-citrate, 13C-2,3-Asp, 13C-1′,4′,5′-5′-AXP, 13C-1′,4′-5′-UXP, 13C-2-γ-aminobutyrate, 13C-3-γ-aminobutyrate, 13C-4-γ-aminobutyrate, 13C-3-Pro, or 13C-4-Pro.
Measurements using mass spectrometry system can result in the detection of collections of isotopomers with the same molecular mass, termed isotopologues. In some embodiments, at least one resultant labeled molecule will represent collections of isotopologues. Isotopologues can include but are not limited to any collection of isotopomers resulting from the transformation of an administered labeled molecule. For example, at least one resultant labeled molecule can be, but is not limited to 13C2-lactate, 13C3-lactate, 13C2-Ala, 13C3-Ala, 13C2-succinate, 13C3-succinate, 13C4-succinate, 13C2-Asp, 13C3-Asp, 13C4-Asp, 13C2-Glu, 13C3-Glu, 13C4-Glu, 13C5-Glu, 13C2-Gln, 13C3-Gln, 13C4-Gln, 13C5-Gln, 13C2-fumarate, 13C3-fumarate, 13C4-fumarate, 13C2-malate, 13C3-malate, 13C4-malate, 13C2-Pro, 13C3-Pro, 13C4-Pro, 13C5-Pro, 13C2-Gly, 13C3-Gly, 13C2-Ser, 13C3-Ser, 13C2-pyruvate, 13C3-pyruvate, 13C2-citrate, 13C3-citrate, 13C2-isocitrate, or 13C3-isocitrate.
The amount of one or more molecules of at least one resultant molecule can be any amount detectable including but not limited to about 0.001 μmol/g dry tissue weight, 0.01 μmol/g dry tissue weight, 0.1 μmol/g dry tissue weight, about 1 μmol/g dry tissue weight, about 2 μmol/g dry tissue weight, about 5 μmol/g dry tissue weight, about 10 μmol/g dry tissue weight, about 50, μmol/g dry tissue weight, about 100 μmol/g dry tissue weight, about 200 μmol/g dry tissue weight, about 300 μmol/g dry tissue weight, or about 500 μmol/g dry tissue weight. The amount of one or more of at least one resultant molecule can, in some embodiments, depend upon one or more of the administered label molecule, the resultant labeled molecule, the animal, the tissue, the cancer to be detected, the health of the animal, the enzyme or metabolic pathway, or any other relevant factor.
Using NMR, the spectrum of a cancer cell extract and a spectrum of an extract of a non-cancer cell extract are obtained. The type of NMR spectrum can be any suitable spectrum type to determine the amount of at least one resultant molecule, including but not limited to, 1-D 1H, 1-D 13C, 1-D 15N, total correlation spectroscopy (TOCSY) (e.g., 2-D 1H TOCSY), COSY (and any COSY variants), NOESY, EXSY, or heteronuclear correlation scalar coupling experiments, such as, but not limited to, heteronuclear single quantum coherence spectroscopy (HSQC) (e.g., 1H-13C2-D HSQC, 1H1-D HSQC), SE-HSQC, CT-HSQC, HSQC-TOCSY (e.g., 1H-13C2-D HSQC-TOCSY), TROSY, HETCOR, COLOC, SECSY, FOCSY, J-resolved, INADEQUATE, HMQC, HMBC, HCACO, HCC, or CC-TOCSY. NMR spectra can include those collected, for example, using 1-D, 2-D, 3-D, or 4-D NMR techniques. NMR spectra can include those that are based on scalar coupling, dipolar coupling, or both. NMR spectra can also include spectra obtained using solid state techniques, including but not limited to those using magic angle spinning.
Using a mass spectrometry system, the MS spectrum of a cancer cell extract and a spectrum of a non-cancer cell extract are obtained. The mass spectrometry system can comprise the usual components of a mass spectrometer (e.g., ionization source, ion detector, mass analyzer, vacuum chamber, and pumping system) and other components, including but not limited to interface chromatography systems. The mass spectrometer can be any suitable mass spectrometer for determining the at least one resultant molecule. The mass analyzer system can include any suitable system including but not limited to, time of flight analyzer, quadrupole analyzer, magnetic sector, Orbitrap, linear ion trap, or fourier transform ion cyclotron resonance (FTICR). The ionization source can include, but is not limited to electron impact (EI), electrospray ionization (ESI), chemical ionization (CI), collisional ionization, natural ionization, thermal ionization, fast atom bombardment, inductively coupled plasma (ICP), or matrix-assisted laser desorption/ionization (MALDI). Interfaced chromatography systems can include any suitable chromatography system, including but not limited to gas chromatography (GC), liquid chromatography (LC), or ion mobility (which can be combined with LC or GC methods). In some instances, direct infusion can be used. In some instances the mass spectrometry system is GC/MS or LC/MS.
Once the NMR spectra, MS spectra, or both are obtained, the spectra are analyzed to determine the amount (e.g., the presence) of at least one resultant labeled molecule in the cancer cell extract and to determine the amount (e.g., the presence) of at least one resultant labeled molecule in the non-cancer cell extract.
For NMR spectra, analysis can include any suitable analysis to determine the amount of one or more resultant labeled molecule (such as, the number and position of labels in the resultant labeled molecule) including the determination of one or more NMR spectral characteristics, which include but are not limited to chemical shift, coupling patterns (e.g., dipolar coupling or spin-spin coupling, such as J-coupling), covalent linkage patterns, peak intensities, peak integrations (e.g., in a 1-D spectrum, in a projection spectrum of a 2-D spectrum, or cross peak integration in a 2-D spectrum) and the presence, extent, and quantification (e.g., peak intensity or peak integration) of satellite peaks (e.g., as a result of the splitting of 1H spectra by 13C). In some instances, the analysis can include a comparison of one or more NMR spectral characteristics with that of a database (e.g., a database of standards).
For MS spectra, analysis can include any suitable analysis to determine the amount of one or more resultant labeled molecule (such as, the number and position of labels in the resultant labeled molecule) including analysis of one or more characteristics, which include but are not limited to chromatographic retention times (e.g., for GC/MS or LC/MS), and mass fragmentation patterns. In some instances, the analysis can include a comparison of characteristics with that of a database (e.g., a database of standards).
The method can further comprise the determination of the protein expression, gene expression, or both of proteins or their genes. Any suitable protein (or its gene) expression can be determined, including but not limited to PC, SCS, PEPCK, transketolase, transadolase, PDH, DH, GLS, IDH, OGDH, MDH, SDH, FH, HKII, GAPDH, PGK-1, LDH-5, PFK-2, GST, or proteins associated with metabolic pathways such as, but are not limited to Krebs cycle (also known as the citric acid cycle), glycolysis, pentose phosphate pathway (oxidative and non-oxidative), gluconeogenesis, lipid biosynthesis, amino acid syntheses (e.g., synthesis of non-essential amino acids), catabolic pathways, urea cycle, Cori cycle or glutamate/glutamine cycle. Protein expression can be determined by any suitable technique including, but not limited to techniques comprising gel electrophoresis techniques (e.g., Western blotting), chromatographic techniques, antibody-based techniques, centrifugation techniques, or combinations thereof. Gene expression can be determined by any suitable technique including, but not limited to techniques comprising PCR based techniques (e.g., real-time PCR), gel electrophoresis techniques, chromatographic techniques, antibody-based techniques, centrifugation techniques, or combinations thereof. Methods for measuring gene expression can comprise measuring amounts of cDNA made from tissue-isolated RNA.
In some embodiments of the invention, some metabolites can be found at higher levels in cancer cells than their surrounding non-cancerous cells. In other embodiments, some metabolites can be found at lower levels in cancer cells than their surrounding non-cancerous cells. For example, a 13C-enrichment in lactate, Ala, succinate, Glu, Asp, and citrate that is higher in the cancer cells can suggest more active glycolysis and Krebs cycle in the cancer cells. In other examples, enhanced production of the Asp isotopomer with three 13C-labeled carbons and the buildup of 13C-2,3-Glu isotopomer in cancer tissues can be observed. This enhanced production can be consistent with the transformations of glucose into Asp or Glu via glycolysis, anaplerotic pyruvate carboxylation (PC), and the Krebs cycle.
In still other embodiments, PC activation in cancer tissues can be found. Without wishing to be bound by theory, such PC activation may assist in replenishing the Krebs cycle intermediates which can be diverted to lipid, protein, and nucleic acid biosynthesis to fulfill the high anabolic demands for growth in lung tumor tissues. The metabolites, if so produced from such diversions, may be detected using the methods of the present invention.
A. Human Studies
Patient Treatment and Sample Collection
Lung cancer patients were recruited based on the criteria of surgical eligibility and no history of diabetes. Each patient was consented in accordance with the U.S. HIPAA regulations. The following patient protocol was approved by the Internal Review Board at the University of Louisville. Ten grams uniformly 13C labeled glucose ([U-13C]-Glc or 13C6-Glc or 13C6-Glucose) in sterile saline solution were infused i.v. over a 30 min time period into each patient in the pre-op room approximately 3 or 12 hr prior to resection. Whole blood samples were collected into a vacutainer containing the anticoagulant K3EDTA or K3-EDTA before and after [U-13C]-Glc infusion as well as after the surgery. Additional blood samples were collected 3 and 12 hr after the [U-13C]-Glc infusion for the 12 hr treatment cases. To minimize metabolic changes, all tissue and blood samples were collected at the operating table with a comparable delay before liquid N2 freezing. The blood samples were placed on ice immediately after collection and centrifuged at 4° C. at 3,500×g for 15 min to recover the plasma fraction. All blood samples were aliquoted and flash-frozen in liquid N2 within 30 min of collection. Potassium EDTA was used as an anti-coagulant to minimize metabolic artifacts associated with anti-coagulation; it also served to remove the influence of interfering cations such as paramagnetic Fe3+ and Cu2+ for NMR analysis.
All timings from first incision to cutting arteries and veins to the lung were recorded so that the period of ischemia could be determined immediately after tissue resection, excess blood was blotted from the tissue, and small pieces of non-cancerous and tumor tissue were cut by the surgeon and freeze-clamped in liquid N2 within 5 minutes of removal from the chest cavity. The freezing process arrested metabolic changes almost instantaneously. In all cases, the tumors were well differentiated so that the extent of the tumor was assessed visually and by palpation. Non-cancerous tissue was removed at least 2 cm from the tumor margin and certified to be tumor-free by trained pathologists. Subsequent pathological evaluation also confirmed tumor status and provided the tumor stage. All samples were stored at −80° C. until further processing for analysis.
Tissue Processing and Extraction
Frozen tissue samples were pulverized into <10 μm particles in liquid N2 using a Spex freezer mill (Spex CertiPrep, Inc., Metuchen, N.J.) to maximize efficiency for subsequent extraction while maintaining biochemical integrity. An aliquot of the frozen powder was lyophilized before extraction for metabolites.
Water-soluble or polar metabolites were extracted from lyophilized tissue powders (4-48 mg) in ice-cold 10% trichloroacetic acid (TCA) (v/w minimum 40/1) while leaving proteins, nucleic acids, and polysaccharides behind, as described previously (Kim et al. Antioxidants & Redox Signaling 2001, 3:361-373). The extraction was performed twice for quantitative recovery. An aliquot (150 μl) of plasma samples were made to final 10% TCA concentration to precipitate proteins and recover metabolites in the supernatant. TCA was then removed from tissue or plasma extracts by lyophilization. The dry pellet was dissolved in nanopure water and two small aliquots were lyophilized for silylation and GC-MS analysis while the remaining bulk was passed through a Chelex 100 resin column (Bio-Rad Laboratories, Inc., Hercules, Calif.) to neutralize and remove interfering multivalent cations for NMR analysis.
NMR Analysis
The 1H reference standard, DSS (2,2-Dimethyl-2-silapentane-5-sulfonate sodium salt) was added (30 or 50 nmoles) to the TCA extracts of tissue or plasma samples, respectively. NMR analysis of the TCA extracts was performed at 20° C. on a Varian Inova 14.1 T NMR spectrometer (Varian, Inc., Palo Alto, Calif.) equipped with a 5-mm HCN triple resonance cold probe. The following NMR experiments were conducted for the determination of metabolite and 13C positional isotopomers: 1-D 1H, 2-D 1H TOCSY, 2-D 1H-13C HSQC and HSQC-TOCSY. 1H and 13C chemical shifts of the TCA extracts were referenced to DSS at 0.00 ppm and indirectly to the 1H shift, respectively. Various metabolites were identified based on their 1H and 13C chemical shifts, 1H coupling patterns, as well as 1H-1H and 1H-13C covalent linkage patterns (acquired from the TOCSY and HSQC experiments, respectively), in comparison with those in our in-house standard database. See Fan et al. (2008) Prog. NMR Spectrosc. 52, 69-117; Fan (1996) Prog. NMR Spectrosc 28, 161-219.
For metabolite and 13C-isotopomer quantification, selected 1H peaks in the 1-D NMR spectra were deconvoluted and integrated using MacNuts software (Acorn NMR, Inc., Livermore, Calif.). The resulting intensity of peaks of interest was calibrated by the peak intensity of DSS for absolute quantification. Percentage 13C abundance of labeled metabolites at specific carbon positions was quantified by integrating appropriate 13C satellite peaks in 1-D 1H or 2-D TOCSY spectra, as previously described (Lane et al. Metabolomics 2007, 3:79-86; Fan et al. Progress in NMR Spectroscopy 2008, 52:69-117.).
GC-MS Analysis
The same extracts from NMR analysis were subjected to GC-MS analysis for quantifying total and 13C-labeled mass isotopomers, as described in full previously (Fan et al. Metabolomics Journal 2005, 1:325-339). Briefly, the lyophilized extract was derivatized in MTBSTFA (N-methyl-N-[tert-butyldimethylsilyl]trifluoroacetamide) (Regis Chemical, Morton Grove, Ill.) and the tert-butyldimethylslylyl derivatives were separated and quantified on a PolarisQ GC-ion trap MSn (ThermoFinnigan, Austin, Tex.) equipped with a 50 m×0.15 mm i.d. open tubular column with 0.4 μm coat BPX-5 (5% phenyl/methyl equivalent) (SGE, Austin, Tex.). Metabolites were identified based on their GC retention times and mass fragmentation patterns by comparison with those of the standards. Absolute quantification of metabolites was done by calibrating the response of selected ions characteristic of a given metabolite from sample runs with that from standard runs (Fan et al. Metabolomics Journal 2005, 1:325-339). Relative metabolite abundances were calculated using Xcalibur (ThermoFinnigan, San Jose, Calif.) or Met-IDEA software to extract peak areas of individual ions characteristic of each component. For Met-IDEA, default program settings for ion trap mass spectrometer were used in the data analysis except for a mass accuracy m/z set at 0.001 and a mass range set on either side of the target m/z at ±0.6. For Xcalibur, peak detection, identification, background subtraction, and quantification was performed using parameters custom-tuned to each analyte. Quantification of mass isotopomers was conducted by subtraction of the isotopic profile at natural-abundance (determined empirically from the analyses of standards) from that of the sample. This procedure was repeated for each series of mass isotopomers to arrive at the 13C-enriched profiles (Fan et al. Metabolomics Journal 2005, 1:325-339). In all cases, the pseudo-molecular ion cluster, characteristic of MTBSTFA-derivatives, was used to ensure that true mass isotopomers were quantified.
Gene Expression Analysis
From an initial gene microarray analysis of a separate set of six paired tissue samples, a number of statistically significant gene expression differences were discerned between lung cancer tissues and their non-cancerous counterparts. This included an over expression of the pyruvate carboxylase (PC) gene in lung cancer versus paired non-cancerous tissues. Due to sample limitation, the array analysis was not performed for patients #6-10.
Real time (RT)-PCR was used instead to probe PC gene expression in patients #6-10. Non-cancerous and cancer tissue RNA was isolated using RNeasy minikit (Qiagen) primarily following the manufacturer's instruction. The only modification was that Trizol Reagent (invitrogen), instead of Buffer RLT provided by the kit, was used to disrupt and homogenize the pulverized frozen tissues at the first step of RNA isolation. The modified procedure was found to provide a higher RNA yield and better purity. The integrity of RNA was confirmed by 1% agarose gel electrophoresis. RNA was reverse transcribed into first strand cDNA using oligo(dT)18 and SuperScript II reverse transcriptase (Invitrogen). Specifically, 1 μg RNA was added into a 40 μl reaction mixture containing 2 μl 500 μg/ml Oligo(dT)18, 2 μl dNTP mix (10 mM each), 8 μl 5× first-strand buffer, 4 μl 0.1 M DTT, 2 μl RNaseOUT™ (40 units/μl) and 2 μl SuperScript II reverse transcriptase (200 units/μl). The reaction mixture was incubated at 42° C. for 50 min and then heated at 70° C. for 15 min to terminate the reaction.
RT-PCR amplification was performed with SYBR green dye using a Mastercycler ep Realplex 4S (Eppendorf). For each run, 20 μl 2.5×Real Master Mix (Qiagen), 0.3 μM of forward and reverse primers along with 2 μl first strand cDNA were mixed. The thermal cycling conditions included an initial denaturation step at 95° C. for 2 min, 50 cycles at 95° C. for 15 s, 55° C. for 15 s and 72° C. for 20 s. Each reaction was performed in duplicates. The efficiency of the amplification was close to 2.0 (i.e. 100%) for all primer pairs. Relative expression level of each gene was calculated using the Livak method as described previously (see, Livak et al. Methods 2001, 25:402-408.) with 18S ribosomal RNA as the internal control gene. The primer sequences used were designed by Beacon Designer 5.0 (Premier Biosoft International, Palo Alto, Calif.) as shown in Table 1.
APC: pyruvate carboxylase; GLS: glutaminase; IDH3: isocitrate dehydrogenase; OGDH: α-ketoglutarate dehydrogenase; FH: fumarate hydratase; MDH2: mitochondrial malate dehydrogenase.
Western Blotting of Pyruvate Carboxylase
Pulverized and lyophilized lung tissues were extracted twice in chloroform:methanol (2:1) plus 1 mM butylated hydroxytoluene to remove lipids, which interfered with the extraction of PC. The delipidated tissue powder was then extracted in 62.5 mM Tris-HCl plus 2% sodium dodecyl sulfate (SDS) and 5 mM dithiothreitol and heated at 95° C. for 10 min. to denature proteins. The protein extract was analyzed by SDS-PAGE using a 10% polyacrylamide gel and separated proteins were transferred to a PVDF membrane (Immobilon™-P, Millipore, Bedford, Mass.), and blotted against an anti-PC rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.) overnight at 4° C. PC was visualized with incubation in a secondary anti-rabbit antibody linked to horseradish peroxidase (HRP) (Thermo Scientific, Rockford, Ill.), followed by reaction with chemiluminescent HRP substrates (Supersignal® West Dura Extended Duration substrate, Thermo Scientific), and exposure to X-ray film. The film was digitized using a high-resolution scanner and the image density of appropriate bands (130 kDa for PC and 50 kDa for α-tubulin) was analyzed using Image J (NIH, Bethesda, Md.). The image density of the PC protein band was normalized to that of the α-tubulin protein band.
Statistical Analysis
GC-MS analysis was performed in triplicate and % RSD (relative standard deviation) of analysis was calculated for each reported metabolite (Table 4). The paired t-test was performed on each pair of tumor and non-cancerous tissues for the real-time PCR data (Table 6) and for the 1H-TOCSY data (Table 3). Linear regression calculations were done for pairs of metabolites in Table 5.
Time Course Analysis of 13C-Metabolites in Plasma
In order to determine the optimal time to sample tissue after infusing [U-13C]-Glc into lung cancer patients, we used 1H NMR to analyze the profiles of 13C-labeled products in plasma samples at several time points. Other than the administered tracer itself ([U-13C]-Glc), the major 13C-labeled metabolite in plasma was lactate. Plasma samples were collected 0, 3, and 12 hr after [U-13C]-Glc infusion, as described in the Patient Treatment and Sample Collection section above. Unlabeled glucose and lactate concentrations were quantified by 1-D 1H NMR using DSS as the calibration standard. Their % 13C enrichment was determined from the respective 13C satellite peaks in 1-D 1H NMR spectra. Panels A-D illustrate the time course changes in glucose and lactate concentrations as well as their % 13C enrichment. Panel E shows the 13C satellite pattern of the 3-methyl group of lactate (13C-CH3-lac) in the 1-D 1H NMR spectrum of patient #6 after 3 hr of [U-13C-Glc] infusion. The chemical shifts of lactate and Ala reflected the acidic pH of the TCA extract.
As shown in
AValues in excess of natural abundance as determined from GC-MS analysis;
BLactate + 1, lactate + 2, lactate + 3 refer to lactate with one, two, or three 13C-labeled carbons, respectively.
GC-MS analysis also revealed a significant presence of mass isotopomers of lactate with one or two 13C labels for patients #8-10, which is consistent with an active Cori cycle. Based on this time-course analysis of 13C-isotopomers of metabolites in human plasma samples, we chose a duration of 3-4 h between 13C-glucose infusion and surgical resection for patients #6-10 in order to optimize 13C incorporation from [U-13C]-Glc into various metabolites.
Of the twelve subjects undergoing 13C glucose infusion, the median age was 63 (range 52-76), 67% were male, 67% had squamous cell carcinoma, and 33% adeno- or adenosquamous carcinoma, all of grade II or III. All subjects had a history of heavy smoking.
Detection of Selective 13C Enrichment in Specific Carbon Positions of Lung Tissue Metabolites
Comparison of metabolite profiles in TCA extracts of paired normal and cancerous lung tissues of patient #6. Metabolites in the 13C HSQC projection (panel F) and 1-D 1H NMR spectra (panel G) were assigned as in
Selective 13C enrichment over natural abundance was revealed as follows. For example, in the 1H spectra (
A similar reasoning applies to the 13C enrichment in the 2,3 carbons of succinate (succinate-C2,3), 2,4 carbons of citrate and the 1′-carbon of the ribosyl residue of adenine nucleotides (5′AXP-C1') in patient #6. The unlabeled succinate, citrate and AXP levels in #6 were lower than those in #2B and 4B (as estimated from the intensities of 5′AXP-H1′, citrate and succinate 1H resonances in
In addition, the 13C enrichment in the C-1 carbons of α- and β-glucose was clearly demonstrated by the higher intensity and doublet nature of the 13C resonances (due to 13C-1 and 13C-2 spin coupling), in contrast to the lower intensity singlet for #2B and 4B (
Metabolite and 13C-Isotopomer Profiling of Lung Tissue Extracts by NMR
An example of the assignment of metabolites and their 13C-isotopomers in the TCA extracts by 2-D 1H TOCSY and high-resolution 1-D NMR spectra is illustrated in
Some metabolites observed in lung tissue extracts include isoleucine (Ile), leucine (Leu), valine (Val), lactate, alanine (Ala), arginine (Arg), proline (Pro), glutamate (Glu), oxidized glutathione (GSSG), glutamine (Gln), succinate, citrate, aspartate (Asp), creatine (Cr), phosphocholine (P-choline), taurine, glycine (Gly), phenylalanine (Phe), tyrosine (Tyr), myo-inositol, α- and β-glucose, NAD+, cytosine nucleotides (CXP), uracil nucleotides (UXP), guanine nucleotides (GXP), and adenine nucleotides (AXP). The 1H TOCSY assignment of metabolites was complemented by the 2-D 1H-13C HSQC analysis of the same extract, as shown in
In addition to metabolite identification, the TOCSY and HSQC analyses provided 13C positional isotopomer information for several metabolites in the lung TCA extracts. The 1H TOCSY data (See, ) of 3-methyl and 2-methine protons of lactate (lactate-H3 and H2) and Ala (Ala-H3 and H2) (traced by dashed rectangles in
) and 11-13 () (traced by dashed lines in
The 13C-positional isotopomer information obtained from the TOCSY analysis was confirmed by the HSQC analysis, including the positional isotopomers of 13C-3-Ala, 13C-3-lactate, 13C-3-Glu, 13C-3-Gln 13C-3-Glu-GSSG, 13C-2-Glu-GSSG, 13C-2-Glu, and 13C-2-Asp (See, respectively Ala-C3, Lactate-C3, Glu-C3, Gln-C3, GSSG-Glu-C3, Glu-GSSG-C2, Glu-C2, and Asp-C2 in
Based on the metabolite assignment in
AValues are average of seven patients; % 13C-Ala, lactate, and Glu were determined from 2-D 1H TOCSY data using appropriate cross-peaks; % [U-13C]-glucose was obtained from 1-D 1H spectra;
BNot detected or below detection limit;
CStandard deviation;
Dfrom paired t-test.
The extent of 13C enrichment in glucose (as [U-13C]-Glc) for non-cancerous tissues was determined from the 1-D 1H spectra (See,
To quantify the 13C abundance of metabolites at specific carbon positions, the 2-D HSQC spectra were utilized for the better resolution than the 1-D 13C projection spectra (See,
Metabolite and 13C-Isotopomer Profiling of Lung Tissue Extracts by GC-MS
Parallel analysis of the lung TCA extracts by GC-MS served to verify key findings in the metabolite profile obtained by NMR while providing absolute quantification of a subset of metabolites and their 13C mass isotopomers. Table 4 shows the quantification of selected metabolites and their total 13C enrichment (in excess of natural abundance) by GC-MS. Also shown is the quantification of the m+3 mass isotopomer of Asp (13C3-Asp or Asp with three of its carbons labeled in 13C).
13C-Ala C
0.28
0.34
0.57
0.79
1.60
13C-Asp C
0.59
0.82
13C3-Asp C, D
0.058
0.009
0.024
0.020
13C-Cit B, C
0.10
0.05
0.12
0.11
0.12
13C-Glu C
0.16
0.22
0.36
0.36
13C-Gln C
0.34
0.21
0.04
13C-Lac C
0.66
0.63
0.88
1.44
0.94
13C-Succ C
0.08
0.03
0.11
0.14
0.19
A in μmole/g dry weight as determined by GC-MS; the % RSD was generally <3% in triplicate analyses with the exception of Gln (11%) and 13C3-Asp (27%);
B sqC: squamous cell carcinoma; adenoC: adenocarcinoma; N: normal; C: cancer; Cit: citrate; Lac: lactate; Succ: succinate;
C
13C enrichment in excess of natural abundance; values in bold represent enhanced enrichment in cancer over its normal counterpart;
D
13C mass isotopomer of Asp with three carbons labeled; detection limit was 0.004 μmole/g dry weight. Values in parentheses are the percentages of 13C3 isotopomers of the total aspartate. This was much greater than 5 × 10−4% at natural abundance.
For patient #6, the GC-MS data revealed excess total 13C enrichment in tumor over non-cancerous tissues for Ala, Asp, Glu, Gln, lactate, citrate, and succinate. An enhanced production of 13C3-Asp in the tumor compared with the paired non-cancerous tissue was also evident. This is consistent with the NMR observation (See,
Quantitative Correlation of 13C-Metabolites in Human Lung Tissues
Utilizing the GC-MS data, pairs of biosynthetically-related 13C-labeled metabolites in tumor and non-cancerous tissues were tested for precursor-product relationships, as illustrated in
AR2 for the linear regression fit was calculated using Excel for patients #6-10 (n = 5);
BObtained from GC-MS analysis; [metabolite]: total concentration in μmole/g dry weight of metabolite; [13C-metabolite]: μmole/g dry weight of 13C-labeled metabolites
A linear correlation in the concentration of 13C-succinate with that of 13C-Ala, 13C-Glu, or 13C-citrate (
Glycolysis and Krebs Cycle in Human Lung Tumors is Activated
Based on the 13C isotopomer analysis by NMR (e.g.
The increased conversion of 13C carbons from glucose into the Krebs cycle intermediates (citrate and succinate) or related metabolites (Glu and Asp) in tumor compared to non-cancerous tissues (See,
Metabolomic Data Show Anaplerotic Pathway in Human Lung Tumor is Activated
The display of lung tumor tissues in the enhanced production of the [13C3]-Asp mass isotopomer (Table 4) is intriguing. Given the enhanced expression of PC, this may be explained with the activation of pyruvate carboxylation, i.e. carboxylation of [U-13C]-pyruvate to generate [13C3]-oxaloacetate, which is transaminated to produce [13C3]-Asp (
Under the low enrichment conditions, the PDH activity produces 13C-4,5-αKG (and thus 13C-4,5-Glu) and [13C2]-Asp (m0+2) through the Krebs cycle (See,
Gene Expression Patterns of PC, Glutaminase, and Krebs Cycle Dehydrogenases in Human Lung Tumors
The distinct 13C labeling patterns in the Krebs cycle metabolites in tumor tissues described above indicates the possibility of altered gene expression in relevant enzymes. This was examined by real-time PCR analysis as described in the “Gene Expression Analysis” section. Some mitochondrial dehydrogenases (DH) along with the anaplerotic pyruvate carboxylase (PC) and glutaminase for tumor and surrounding non-tumorous tissues are shown in Table 6.
A Tumor over surrounding non-tumor tissues;
B PC: pyruvate carboxylase; GLS: glutaminase; IDH3: isocitrate dehydrogenase; OGDH: α-ketoglutarate dehydrogenase; SDH: succinate dehydrogenase; FH: fumarate hydratase; MDH2: mitochondrial malate dehydrogenase;
C For patients #6-10;
D Obtained from paired t-test.
Increased expression of the two isoforms of PC gene was evident in patients #6-10 with an average fold change of 3.36±1.13 i.e. higher in tumors (p<0.01). In contrast, the expression of another anaplerotic enzyme gene, glutaminase (GLS) was lower in the tumors relative to the surrounding non-cancerous tissues, with an average fold change of 0.52±0.16. For Krebs cycle DH, there was a decrease of isocitrate DH (IDH) and α-ketoglutarate DH (OGDH) in contrast to a modest activation of malate DH (MDH) expression, while succinate DH (SDH) and fumarate hydratase (FH) showed no statistically significant changes in expression in tumor tissues.
PC Protein Expression Patterns in Human Lung Tumors
The in vivo 13C isotopomer profile and gene expression data (see above) indicate increased PC activity in the NSCLC tumors compared with non-tumorous lung tissue. To determine whether PC gene activation leads to an enhanced expression of the enzyme, Western blotting was performed on paired tumor and non-cancerous tissues from the five patients.
The PC response normalized to that of α-tubulin is shown in
Panel C shows ratios of α-tubulin-normalized Western Blot image analysis of tumor and non-cancerous tissue for patients #11-21 and #22b-28b. The horizontal dotted line represents a ratio of 1. Patient 25b had a lesion on the upper lobe (UL) and a lesion on the lower lobe (LL). Both lesions were at Stage I, but the UL lesion appeared to be in an earlier stage than the LL lesion, because (1) the LL lesion had a higher PET (positron emission tomography) SUV (standardized uptake value), (2) the LL lesion responded to Erlotinib whereas the UL lesion did not, and (3) EGFR analysis was consistent with the UL lesion being in an earlier stage. See also, Fan et al. (2009) Exp. Molec. Pathol. 87, 83-86. Three regions of patient 28b's lung tumor were analyzed; these data suggest that PC expression is consistent for the three regions analyzed.
Gene and Expression Data Show Anaplerotic Pathway in Human Lung Tumor is Activated
The metabolomic data were corroborated by measurements of enhanced gene and protein expression of PC in lung tumors relative to their non-cancerous counterparts.
These effects of PC may be mediated through its ability to replenish the Krebs cycle intermediates, thereby enhancing energy metabolism and fulfilling biosynthetic demands from proliferating cells. Thus, PC activation may play a role in the transformation of lung primary cells into a more highly proliferative state.
B. SCID Mouse Studies
The severe combined immunodeficient (SCID) mouse was used to study the biology of human tumors. The SCID mouse has an impaired ability to make either B or T lymphocytes, or activate some components of the complement system and as such do not reject foreign tissue such as a xenografted human tumor. The mouse tissue fluid volume is relatively small: a 20 g mouse contains ca. 2 ml blood, and a total of ca. 14 ml tissue water. Thus, smaller amounts of isotope enriched precursor metabolites are needed to raise the concentration to a measurable initial value.
We have determined the time course of [U-13C]-glucose utilization and metabolic rate in 20 g SCID mice using tail vein injection. Incorporation of 13C into metabolites extracted from different tissues have been used to demonstrate the major differences in tissue-dependent metabolism and help ascertain optimal sampling times for different target tissues.
Mouse Handling
The murine tumor therapy protocols were conducted in compliance with all guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Louisville. Fox Chase ICR severe combined immunodeficient (SCID) mice were purchased from Taconic (Hudson, N.Y.) and maintained in a barrier facility at the University of Louisville according to institutional protocols.
Orthotopic Lung Tumor
Human PC14PE6 and A549 cells were grown in RPMI medium as previously described (Fan et al. (2005) Metabolomics 1(4): 1-15), mixed with matrigel (total 100 μl/106 tumor cells) and injected into the left lung. An equal volume of normal saline was injected into the right lung. Mice were allowed to feed ad libitum, and were monitored daily for signs of distress. Mice were sampled at 10 days post injection. Additional naïve SCID mice were used as controls (Onn et al. (2003). Clinical Cancer Research 9(15): 5532-5539).
[U-13]C Glucose Infusion
A 20% solution of glucose in normal saline was sterile-filtered. 100 μL of this solution, or a 1:10 dilution (2%) were injected into the tail vein of a restrained mouse without anesthesia.
Approximately 50 μL samples of blood were taken intraorbitally at timed intervals, chilled and separated into plasma and whole blood by centrifugation at 4° C. at 3,000 g for 5 minutes. Plasma was flash frozen in liquid N2 for storage prior to workup.
Tissue Harvesting
Mice were sacrificed at different times post glucose injection, and the following organs were dissected sequentially: lung, heart, liver, kidney, brain, spleen, and thigh muscle. Dissected tissue was flash frozen in liquid N2 within 1 minute of killing the animal.
Plasma Extraction
Twenty to thirty μl of plasma was made to 10% trichloroacetic acid (TCA) and centrifuged at 4° C., at ≧22,000 g for 20 minutes to remove denatured proteins. The polar supernatant was lyophilized to remove TCA before preparation for NMR and GC-MS analysis.
Tissue Extraction
Frozen tissues were ground in liquid N2 to <10 μm particles in a 6750 Freezer/Mill (Retsch, Inc., Newtown, Pa.) and extracted for soluble and lipidic metabolites as follows. Up to 20 mg of frozen tissue powder in 15 ml polypropylene conical centrifuge tube (Sarstedt, Newton, N.C.) containing 3 mm diameter glass beads was vigorously mixed with 2 ml of cold acetonitrile (mass spectrometry grade, stored at −20° C.) to denature proteins, followed by addition of 1.5 ml nanopure water, and 1 ml HPLC-grade chloroform (Fisher Scientific). The mixture was shaken vigorously until achieving a milky consistency followed by centrifugation at 3,000 g for 20 minutes at 4° C. to separate the polar (top), lipidic (bottom), and tissue debris layers (interface). The polar and lipidic layers were recovered sequentially and the remaining cell debris was extracted again with 0.5 ml chloroform:methanol:butylated hydroxytoluene (BHT) (2:1:1 mM) which was pooled with the lipidic fraction. All three fractions were vacuum-dried in a speedvac device (Vacufuge, Eppendorf, New York, N.Y.) and/or by lyophylization. The dry weight of tissue debris was obtained for normalization of metabolite content. The polar extracts were redissolved in 100% D2O containing 30 nmol perdeuterated DSS (2,2-dimethyl-2-silapentane-5-sulfonate, Cambridge Isotope Laboratories, Andover, Mass.) as internal chemical shift and concentration reference for NMR measurement.
NMR Spectroscopy
NMR spectra were recorded at 14.1 T on a Varian Inova spectrometer equipped with a 5 mm inverse triple resonance cold probe, at 20° C. 1D NMR spectra were recorded with an acquisition time of 2 s and a recycle time of 5 sec. Concentrations of metabolites and 13C incorporation were determined by peak integration of the 1H NMR spectra referenced to the DSS methyl groups, with correction for differential relaxation, as previously described (See, Lane et al. (2008) Biophysical Tools for Biologists. 84: 541-588; Fan et al. (2008) Progress in NMR Spectroscopy 52: 69-117; Lane et al. (2007) Metabolomics 3: 79-86.). 1H Spectra were typically processed with zero filling to 131 k points, and apodized with an unshifted Gaussian and a 0.5 Hz line broadening exponential.
13C profiling was achieved using 1D 1H -{13C} HSQC spectra recorded with a recycle time of 1.5 s, with 13C GARP decoupling during the proton acquisition time of 0.15 s.
TOCSY and HSQC-TOCSY spectra were recorded with a mixing time of 50 ms and a B1 field strength of 8 kHz with acquisition times of 0.341 s in t2 and 0.05 s in t1. The fids were zero filled once in t2, and linear predicted and zero filled to 4096 points in t2. The data were apodized using an unshifted Gaussian and a 1 Hz line broadening exponential in both dimensions. Positional 13C incorporation into labeled metabolites was quantified as previously described (Lane et al. (2007) Metabolomics 3: 79-86.; Lane et al. (2008) Biophysical Tools for Biologists. 84: 541-588)
Time Course Changes of Plasma 13C-Glucose and 13C-Lactate
The two SCID mice were injected via the tail vein with 107 mmol [U-13C]-glucose. Blood was sequentially taken intraorbitally, and analyzed by NMR as described in the NMR Spectroscopy section. To assess the optimal time for metabolizing the 13C6-glucose in mice, plasma samples were taken at 15-minute intervals and processed for NMR analysis.
Once taken up by tissues, labeled glucose is metabolized principally via glycolysis, which generates labeled lactate and to a lesser extent alanine Lactate is then exported into the blood and ultimately to the liver to be reconverted into glucose by gluconeogenesis (Cori Cycle). 13C-lactate in plasma therefore represents lactate newly synthesized since the 13C glucose bolus, and is an indicator of systemic metabolism. This is consistent with the observed 13C enrichment in lactate (
Tissue-Dependent Metabolism: GC-MS Analysis
Individual tissues may take up and metabolize glucose at different rates, which will be reflected in the distribution of 13C labeled metabolites in various tissues.
Furthermore, an appreciable amount of singly and doubly 13C labeled lactate (lactate+1 and +2 or 13C1- and 13C2-lactate) was observed in all six tissues (
In addition to lactate, 13C-labeled isotopologue series for a number of metabolites were observed by GC-MS analysis.
The 13C— isotopologue series of succinate, Asp, Glu, and Gln were clearly present and some of which reached high levels, e.g. 13C1-/13C2-/13C3-Asp in brain, 13C1-/13C2-/13C3-/13C4-Glu in brain, kidney, and lung, as well as 13C1-/13C2-Gln in brain and heart. 13C2-succinate, 13C2-Asp, 13C2-Glu, and 13C2-Gln can be derived from 13C6-glucose via glycolysis plus the 1st turn of the Krebs cycle (See,
Tissue-Dependent Metabolism: NMR Analysis
The same extracts from
Lac measured at C3, G1c measured at C1α by NMR
More extensive 13C metabolite labeling was determined by two-dimensional NMR experiments.
Once the labeled metabolites were assigned by 2-D HSQC methods, the 1-D HSQC analysis of 1H directly attached to 13C provided a semi-quantitative estimate of the abundance of various 13C-labeled metabolites in the six tissues (
Metabolism in Normal Versus Cancerous Lung Tissues
13C6-glucose was used as a tracer to track changes in metabolic pathways induced by lung tumor development in SCID mice. An SCID mouse was injected with 1 million PC14PE6 lung cancer cells in matrigel in one of the lung lobes while the other lung lobe received saline only. After lung tumor establishment, the animal received 13C6-glucose 15 minutes prior to tissue dissection, extraction, and NMR analysis as described above.
A separate set of tumorous and normal lung tissue extracts were quantified by GC-MS, as shown in
It is noted that terms like “preferably,” “commonly,” and “typically” are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment of the present invention.
Detailed descriptions of one or more embodiments are provided herein. It is to be understood, however, that the present invention may be embodied in various forms. Therefore, specific details disclosed herein (even if designated as preferred or advantageous) are not to be interpreted as limiting, but rather are to be used as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner.
This application claims the benefit of U.S. Provisional Application No. 61/186,572, filed Jun. 12, 2009, which is herein incorporated by reference in its entirety.
This invention was made in part with government support under the National Cancer Institute (grant numbers 1R01CA101199-01 and 1R01CA118434-01A2), the National Center for Research Resources (NIH Grant Number 5P20RR018733), and the National Science Foundation EPSCoR (grant numbers EPS-0132295 and EPS-0447479). The U.S. government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US10/38425 | 6/11/2010 | WO | 00 | 6/4/2012 |
| Number | Date | Country | |
|---|---|---|---|
| 61186572 | Jun 2009 | US |
