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Stable Isotope Resolved Metabolomics Studies in ex vivo TIssue Slices
体外组织切片的稳定同位素分解代谢组学研究   

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Abstract

An important component of this methodology is to assess the role of the tumor microenvironment on tumor growth and survival. To tackle this problem, we have adapted the original approach of Warburg (Warburg, 1923), by combining thin tissue slices with Stable Isotope Resolved Metabolomics (SIRM) to determine detailed metabolic activity of human tissues. SIRM enables the tracing of metabolic transformations of source molecules such as glucose or glutamine over defined time periods, and is a requirement for detailed pathway tracing and flux analysis. In our approach, we maintain freshly resected tissue slices (both cancerous and non- cancerous from the same organ of the same subject) in cell culture media, and treat with appropriate stable isotope-enriched nutrients, e.g., 13C6-glucose or 13C5, 15N2-glutamine. These slices are viable for at least 24 h, and make it possible to eliminate systemic influence on the target tissue metabolism while maintaining the original 3D cellular architecture. It is therefore an excellent pre-clinical platform for assessing the effect of therapeutic agents on target tissue metabolism and their therapeutic efficacy on individual patients (Xie et al., 2014; Sellers et al., 2015).

Keywords: Tissue slices(组织切片), SIRM(惠普), Metabolic pathway tracing(代谢通路追踪), Preclinical testing(临床前研究), Cancer metabolism(肿瘤代谢)

Materials and Reagents

  1. 25 ml T Flasks NC vent cap (SARSTEDT AG & Co, catalog number: 83.1810.002 )
  2. Portable container for liquid nitrogen (Nalgene plastic dewar) (Thermo Fisher Scientific, catalog number: S34074C )
  3. Sterile syringes and needles (Thermo Fisher Scientific, catalog number: 10142534 )
  4. Disposable transfer pipets (Samco fine tip, 1 ml) (VWR International, catalog number: 16001192 )
  5. Aerosol barrier tips for 1 ml and 1-200 μl (Thermo Fisher Scientific, catalog number: 02-707-42 )
  6. Screw cap plastic vials (2 ml) color coded caps (yellow, blue, green and red) (USA Scientific, catalog number: 1420-8706 , 1420-8701 , 1420-8702 and 1420-9704 )
  7. Snap top plastic vials (1.5 ml) (USA Scientific, catalog number: 1615-5510 )
  8. 15 ml Falcon tubes (SARSTEDT AG & Co, catalog number: 62.554.205 )
  9. Dialyzed, sterile filtered fetal bovine serum (FBS) (free of serum metabolites),10-12 kDa (Atlanta Biochemical, catalog number: S12650 )
  10. Tracer examples: 13C6-glucose, 13C2-1, 2-glucose, 13C5,15N2-glutamine
  11. Sources: 13C6-glucose/D-glucose ([U-13C], 99%) (Cambridge Isotope Laboratories, catalog number: CLM-1396-CTM), 13C2-1, 2 glucose/D-glucose (1, 2-13C2, 99%) (Cambridge Isotope Laboratories, catalog number: CLM-504), 13C5, 15N2-glutamine/ L-glutamine (13C5, 99%; 15N2, 99%) (Cambridge Isotope Laboratories, catalog number: CNLM-1275 ) or
    Isotec: D-13C6-glucose (Sigma-Aldrich, catalog number: 660663 ), 13C2-1, 2 glucose (Sigma-Aldrich, catalog number: 661422 ), L-Glutamine-13C5, 15N2 (Sigma-Aldrich, catalog number: 607983 )
  12. Penicillin + Streptomycin: GE Healthcare PEN/STREP/FUNGIZONE 100 ml (Thermo Fisher Scientific, catalog number: SV3007901 )
  13. ProtocolTM 10% Neutral buffered formalin (Thermo Fisher Scientific, catalog number: 032-059 )
  14. 25% (w/v) sterile filtered 13C glucose (0.2 μm) in PBS (Stock solution can be frozen, aliquoted, and stored at 4 °C)
  15. Liquid nitrogen
  16. 70% ethanol (v/v)
  17. 60% acetonitrile in water (v/v) (Sigma-Aldrich, catalog number: L010400 )
  18. Sodium chloride (NaCl) (Thermo Fisher Scientific, catalog number: S271-1 )
  19. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  20. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S0876 )
  21. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P9791 )
  22. Amino acids: Glycine, L-Arginine, L-Asparagine, L-Aspartic acid, L-Cystine 2HCl, L-Glutamic Acid, L-Glutamine, L-Histidine, L-Hydroxyproline, L-Isoleucine, L-Leucine, L-Lysine hydrochloride, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine disodium salt dehydrate, L-Valine
  23. Vitamins: Biotin, Choline chloride, D-Calcium pantothenate, Folic Acid, i-Inositol, Niacinamide, Para-Aminobenzoic Acid, Pyridoxine hydrochloride, Riboflavin, Thiamine hydrochloride, Vitamin B12
  24. Calcium nitrate [Ca(NO3)2.4H2O]
  25. Magnesium sulfate (MgSO4)
  26. Glutathione (reduced)
  27. Phenol Red
  28. Relevant medium (e.g. DMEM, RPMI, other defined medium) which lacks the tracer of interest:
    a. Dulbecco′s Modified Eagle′s Medium (DMEM) is a powder formula, free of glucose, glutamine, pyruvate bicarbonate, and phenol red, giving considerable flexibility in formulation for SIRM studies (Sigma-Aldrich, catalog number: D5030 ) (see Recipes)
    b. RPMI 1640 is a liquid medium free of glucose and glutamine, but contains bicarbonate and phenol red (MP Biomedicals, catalog number: 091646854 ) (see Recipes)
  29. 0.2 µm sterile filtered Phosphate Buffered Saline (PBS) (see Recipes)
  30. Medium composition for 0.2% 13C6-glucose, 2 mM 12C-Gln (100 ml) (see Recipes)
  31. Medium composition for 0.2% 12C glucose, 2 mM 13C5, 15N2-Gln (100 ml) (see Recipes)

Equipment

  1. Class II Biosafety Hood
  2. Trigas incubator with oxygen sensor and CO2 sensor (Thermo Fisher Scientific, model: Hera cell 150i )
  3. Sterilized rocker (Rotoshake Genie) (Scientific Industries, model: SI-1100 )
  4. Liquid nitrogen freezer for storage
  5. K2-EDTA vacutainers (“purple top”) (BD, catalog number: 366643 )
  6. Refrigerated centrifuge with swing out rotor that can accept vacutainers [e.g. Sorvall Legend X1R (Thermo Fisher Scientific, catalog number: 75-004-261 ) with a rotor (Thermo Fisher Scientific, catalog number: 75003181 )]
  7. Pipettors (variable size ranges) (USA Scientific ErgoOne)
  8. Weck Knife/Dermatome (George Tiemann & Co., catalog number: 222-5-523 )
  9. Weigh boats (Thermo Fisher Scientific, catalog number: 08732113 and 08732115 )
  10. 4-place balance (Thermo Fisher Scientific, Mettler-Toledo, catalog number: 0133525 )
  11. Ice bucket (Thermo Fisher Scientific, catalog number: 02-591-44 )
  12. Sharp dissecting scissors (Thermo Fisher Scientific, catalog number: 08940 )
  13. Excelta™ Plastic Tweezers (Thermo Fisher Scientific, catalog number: 17-456-066 )
  14. Digital camera

Procedure

  1. Tissue procurement
    All tissue must be procured under an IRB approved protocol. As live human tissue is handled, all personnel must undergo and maintain biosafety, HIPAA and CITI certifications.
    An overview of the whole process is given in Scheme 1.


    Scheme 1. FlowChart. Preop: Preoperative room; OR: Operating Room. The number of thin slices to be taken depends on the size of the tumor. A piece of tissue is also flash frozen in the OR, and additional tissue is placed in formalin for pathological analysis.

    1. Blood samples provide overall information about the metabolic status of the individual subjects, and the buffy coat can be used for extracting DNA or RNA for sequence analysis.
    2. A 10 ml sample of blood should be drawn preoperatively into a purple top vacutainer (K2-EDTA) preoperatively. Other anticoagulants such as citrate or heparin should not be used as they interfere with metabolic assays. A blood sample should also be drawn perioperatively after resection. The blood is inverted twice to ensure dissolution of the EDTA, and kept on ice immediately after blood draw. The blood should be separated into packed red cells, buffy coat and plasma within 30 min by centrifuging at 3,500 x g for 15 min at 4 °C in a swing out rotor.
      Subsequent operations should be carried out in a BSL2+ biosafety cabinet.
      Note: We use the following color codes for storage: Red = whole blood, yellow = plasma, green = buffy coat, blue = urine.
    3. Plasma is aspirated into prechilled sterile 2 ml screw cap vials at 1 ml aliquots and flash frozen in liquid N2. Buffy coat is aspirated using a wide mouth plastic pipette into a 2 ml screwcap vial and flash frozen in liquid N2.
    4. These experiments have been carried out on fresh slices of paired cancerous (CA) and non-cancerous (NC) lung tissues resected from non-small cell lung cancer (Xie et al., 2014) and pancreatic cancer patients. Upon resection, thin slices (0.5-1 mm thick) of tissue are excised from the surface of visually non-necrotic or fibrotic tumor regions using a Weck microtome in the Operating Room (OR), within approximately 5-10 min of resection. Roughly 1 cm2 tissue is targeted (see Figure 1 A). Control non-cancerous tissue from a distant (>10 cm) region is obtained similarly. A pathologist on-site inspects the CA and NC tissue specimens. Highly necrotic tissue is discarded.
    5. At the same time, a small piece of bulk CA tissue should be placed in DMEM or other appropriate medium kept room temperature for implantation into a recipient NSG mouse as patient-derived xenograft or PDX. A small piece each of CA and NC tissues is soaked in formalin for pathological examination or flash frozen in liquid N2 for image-based metabolic analysis.
    6. Where tissue acquisition in the OR is impractical (such as colorectal or breast cancer resections), the slices can be prepared in the pathology laboratory located close to the OR. For comparison with freshly resected tissue, speed is essential as metabolism is rapidly changing. Whenever feasible, tissue freezing should be performed in the OR.
    7. The slices are placed into a drop of sterile PBS on two sterilized weigh boats to prevent sticking and for spreading slices evenly. Each weight boat is pre-numbered for CA or NC slices. The tissues on weight boats are then photographed. Each slice is rinsed briefly with sterile PBS, blotted (twice) and then carefully placed into pre-numbered (using ethanol-resistant marker pen) pre-tared (tare weight recorded) T25-flasks containing 10 ml DMEM (or other relevant medium) with the appropriate tracer (e.g. 10 mM 13C6-glucose or 2 mM 13C5, 15N2-glutamine), 10% dialyzed FBS (as needed), and 1x penicillin + streptomycin. The flasks with slices are brought to the culture room as soon as possible and sprayed with 70% ethanol, and wiped dry before placing them in the Biosafety hood.
    8. Pipet 200 μl culture media from each flask into 1.5 ml snap-cap tubes (t0-time zero media samples). Centrifuge for 10 min at 10,000 x g at 4 °C to remove tissue debris. Transfer 100 μl to tared 1.5 μl snap-cap tube for metabolite extraction and weigh the media transferred. Transfer the remaining media to a 1.5 ml screw-cap tube for long-term storage at -80 °C.
    9. Weigh flasks in a 2-place balance inside the Biosafety hood and record weight on the flask.
    10. Transfer flasks to a CO2 incubator containing a rocker set to low amplitude rocking (sufficient to ensure that the liquid moves over the slices without non-laminar flow) Figure 1B) set to 37 °C and 5% CO2, with oxygen set to the desired level (e.g. 20%, 1%).
    11. The flasks are continuously and gently rocked for 24 to 48 h for gas exchange and to maintain constant nutrient supplies at the tissue surface, while avoiding local buildup of waste products such as acids.
    12. As needed, the medium can be refreshed every 12 h, and sampled at 0, 6, 12, 24…. h for analysis of nutrient uptake and waste production. The flasks are weighed before and after each medium sampling point and flask weights are recorded on the flasks.

  2. Tissue Harvesting
    1. After 24 h incubation, weigh flasks.
    2. Place flasks on ice immediately after removing from the incubator to minimize further metabolism. Up to 6 flasks can be harvested at a time. Keep tissue slices on ice as much as possible during harvest.
    3. Using a transfer pipet, aspirate and transfer the conditioned media into 15-ml conical centrifuge tubes.
    4. Centrifuge media for 15 min at 3,500 to 4,690 x g, 4 °C to remove any particulates and debris.
    5. Pipet 100 μl T24 media supernatant into 1.5-ml snap-cap tubes for metabolite extraction.
    6. Pipet 1 ml media aliquot into 2-ml screw-cap tubes for long-term storage at -80 °C.
    7. The remaining medium is stored separately in a 7 ml vial at -80 °C for purposes such as exosome isolation.
    8. Invert and tap the flask to move the tissue slices into the cap or neck region of the flask for retrieval. Keep flask inverted on ice.
    9. Wash tissue slices 3x consecutively in ice-cold 10 ml cold PBS each in a 50 ml beaker.
    10. Blot dry the tissue slices on Kimwipe and photograph the flattened slice on a small weigh boat.
    11. Weigh the whole tissue slice on small weigh boats and record the weight.
    12. Split a very small piece for preservation in 1 ml buffered formalin in a 1.5 ml snap-cap tube for histology. The remaining tissue slice is split evenly and each piece should weigh no more than 20-30 mg by wet weight to facilitate tissue homogenization and extraction efficiency. Immediately after weighing, each piece is flash-frozen in liq. N2 and placed in a pre-liq. N2 chilled 1.5-ml snap-cap tube for long-term storage at -80 °C.
    13. After 6-8 h in formalin, replace the formalin with 70% ethanol for the tissue pieces prepared for histology.
    14. Homogenize tissues in cold 60% acetonitrile (v/v) and extract tissue homogenates for metabolite analyses according to standardized protocols (Fan, 2012; Fan, 2010) before analyses using stable isotope-resolving analytical techniques (e.g. NMR and MS) (Fan, 2012) (Figure 1C) (Lane et al., 2008).
    15. This Protocol describes the procedure for stable isotope labeling of thin tissue slices. SIRM analysis involves the quantification of isotopomers (by NMR) and isotopologues (MS) that result from metabolic transformations of source molecules (e.g. 13C glucose or 13C, 15N Glutamine) in cells or tissue (Figure 1C). The techniques of SIRM analysis by NMR and mass spectrometry are described in detail in Sellers et al. (2015); Lane et al. (2008) and Fan et al. (2012).

Representative data


Figure 1 Example ex vivo tissue slice experiment. A. Example thin slices of non-cancerous lung tissue (NC, left) adjacent to a lung adenocarcinoma (CA, right); B. T25-flasks on a rocker inside a CO2 incubator; C. Representative 1D 1H{13C} HSQC NMR spectra (recorded at 14.1 T, 15 °C) of extracts of CA versus NC lung slices from an non small cell lung cancer (NSCLC) patient incubated for 24 h in the presence of 10 mM 13C6-glucose. The tissue slices were pulverized and extracted as described (Sellers et al., 2015; Fan, 2012) which produces three phases- an upper aqueous phase containing polar metabolites, a lower organic phase containing non-polar metabolites (mainly lipids) and an interfacial phase that contains protein. Here the upper phase was lyophilized and redissolved in a phosphate buffer containing 50% D2O and 25 nmol DSS-d6 that serves both as a chemical shift reference and a concentration standard (Fan and Lane, 2013). The HSQC spectrum detects protons attached directly to 13C, and thus gives a readout of the metabolites that have incorporated 13C from the source molecule (glucose in this instance). The spectra of cancer and non-cancerous tissues are recorded under identical conditions, and the absolute intensities are normalized to the tissue protein weight. Peak areas were determined using peak fitting functions in MNOVA (Mestrelab Research, Santiago de Compostela, Spain)

Enhanced production of various 13C labeled metabolites in the CA tissue slice is evident, including 13C-lactate (Lac), which is consistent with the Warburg effect or accelerated glycolysis in tumor tissues (Warburg, 1956).

Notes

  1. These procedures have been tested on freshly resected NSCLC and pancreatic cancer, as well as in patient derived mouse xenografts. Other tissues may need experimentation with the composition of the medium and length of incubation period for metabolic viability.
  2. Larger or inflammatory tumors may have substantial areas of necrosis or fibrosis that need to be avoided.
  3. A Weck microtome is hand held, but with practice the surgeons can reproducibly produce slices < 1 mm thick. Slice thickness is easily estimated by measuring the area of the slice from a photograph and from the wet weigh as the average thickness h = weight/area.
  4. Some tumors are highly mucilaginous and are more difficult to slice reproducibly.
  5. An alternative to a Weck microtome is a vibrating microtome, which can reproducibly generate thinner slices from firm but not soft tissues (cf10), but is much slower. Very thin slices (100-200 μm) may show a proportionately larger wounding response and are more fragile.
  6. As many tumors are heterogeneous not only in the cancer/stromal content in different regions of the tumor but also in terms of genetics, it is advisable to obtain multiple slices from the tumor to cover this heterogeneity. This also makes histopathological examination of each slice critically important.
  7. The margins of some tumors are not obvious without pathological examination. Tissue proximal to the tumor as well as distal from the tumor should be sampled for comparison.

Recipes

  1. DMEM and RPMI Medium 1640
    For SIRM studies, the glutamine or glucose free version of the medium should be used, with supplementation of the appropriate concentration of 13C-enriched precursors in the base medium.

    COMPONENTS
    Molecular Weight
    Concentration  (mg/L)
    Molarity (mM) RPMI
    Molarity (mM) DMEM
    Amino Acids
    Glycine
    75
    10
    0.133
    0.40
    L-Arginine
    174
    200
    1.15
    0.483
    L-Asparagine
    132
    50
    0.379
    -
    L-Aspartic acid
    133
    20
    0.150
    -
    L-Cystine 2HCl
    313
    65
    0.208
    0/0.2
    L-Glutamic Acid
    147
    20
    0.136
    -
    L-Glutamine
    146
    300
    2.05
    2
    L-Histidine
    155
    15
    0.0968
    0.27
    L-Hydroxyproline
    131
    20
    0.153
    -
    L-Isoleucine
    131
    50
    0.382
    0.8
    L-Leucine
    131
    50
    0.382
    0.8
    L-Lysine hydrochloride
    146
    40
    0.274
    1.0
    L-Methionine
    149
    15
    0.101
    0.2
    L-Phenylalanine
    165
    15
    0.0909
    0.4
    L-Proline
    115
    20
    0.174
    -
    L-Serine
    105
    30
    0.286
    0.4
    L-Threonine
    109
    20
    0.168
    0.8
    L-Tryptophan
    204
    5
    0.0245
    0.078
    L-Tyrosine disodium salt dihydrate
    261
    29
    0.111
    0.4
    L-Valine
    117
    20
    0.171
    0.8
    Vitamins
    Biotin
    244
    0.2
    0.000820

    Choline chloride
    140
    3
    0.0214
    0.0285
    D-Calcium pantothenate
    477
    0.25
    0.000524
    0.008
    Folic Acid
    441
    1
    0.00227
    0.009
    i-Inositol
    180
    35
    0.194
    .04
    Niacinamide
    122
    1
    0.00820
    0.033
    Para-Aminobenzoic Acid
    137
    1
    0.00730

    Pyridoxine hydrochloride
    206
    1
    0.00485
    .019
    Riboflavin
    376
    0.2
    0.000532
    .001
    Thiamine hydrochloride
    337
    1
    0.00297
    .012
    Vitamin B12
    1,355
    0.005
    0.0000037
    -
    Inorganic Salts
    Calcium nitrate (Ca(NO3)2.4H2O)
    236
    100
    0.424

    Magnesium Sulfate (MgSO4) (anhyd.)
    120
    48.84
    0.407

    Potassium Chloride (KCl)
    75
    400
    5.33

    Sodium Bicarbonate (NaHCO3)
    84
    2,000
    23.81
    44
    Sodium Chloride (NaCl)
    58
    6,000
    103.45

    Sodium Phosphate dibasic (Na2HPO4.7H2O)
    268
    1,512
    5.64

    Other Components
    Glutathione (reduced)
    307
    1
    0.00326

    Phenol Red
    376.4
    5
    0.0133


  2. 10x PBS
    Sources of reagents are given in the Materials Section
    80 g NaCl
    2 g KCl
    14.4 g Na2HPO4 anhydrous
    2.4 g KH2PO4 anhydrous
    Dissolve in 950 ml 18 MOhm water, pH to 7.4, make to 1 L, sterile filter (0.2 μm)
  3. Medium composition for 0.2% 13C6-glucose, 2 mM 12C-Gln (100 ml)
    89.2 ml base medium minus tracer (e.g. glucose-free version) (89% concentration of nutrients)
    10 ml sterile filtered dialyzed FBS (10% FBS)
    0.8 ml 25% sterile filtered 13C6 glucose (0.2 µm) in PBS (10.75 mM glucose final)
    1 ml 100x streptomycin/penicillin stock
  4. Medium composition for 0.2% 12C glucose, 2 mM 13C5, 15N2-Gln (100 ml)
    88 ml base medium minus tracer (glutamine-free version) (88% concentration of all nutrients)
    10 ml sterile filtered dialyzed FBS (10% FBS)
    1 ml 0.2 M sterile filtered 13C5, 15N2-glutamine (0.2 µm) in PBS (2 mM final)
    1 ml 100x streptomycin/penicillin stock
    For hormone sensitive tissues, activated carbon-stripped FBS may be used.
    For other concentrations of FBS, adjust the volumes of the FBS and base medium accordingly.
    Note: Glutamine stock should be made fresh or stored at -20 °C in small aliquots to avoid repeated freeze and thawing. It forms pyroglutamate on storage in solution even at neutral pH at higher temperatures.

Acknowledgments

This work was supported in part by the following grants: NIH P01 CA163223-01A1, NIH 5R01ES022191-04, NIH 3R01ES022191-04S1, NIH 1U24DK097215-01A1, and the Kentucky Challenge for Excellence. This protocol has been developed based on work described in Xie et al. (2014); Sellers et al. (2015) and Bousamra et al. (2012). The authors declare no conflicts of interest.

References

  1. Bousamra, M., Day, J., Fan, T. W., Higashi, R. M., Kloecker, G., Lane, A. N. and Miller, D. M. (2012). Clinical aspects of metabolomics. In: The Handbook of Metabolomics. Humana, Vol. 17.
  2. Fan, T. W. (2012). Considerations of sample preparation for metabolomics investigation. Handbook of Metabolomics 17.
  3. Fan, T. W. (2010). Metabolomics-edited transcriptomics analysis (meta). In: McQueen, C. A. (ed). Comprehensive Toxicology. Academic Press Vol. 2 685-706.
  4. Fan, T. W. and Lane, A. N. (2013). Assignment strategies for NMR resonances in metabolomics research. In: Lutz, N., Sweedler, J. V. and Weevers, R. A. (eds). Methodologies for Metabolomics: Experimental Strategies and Techniques. Cambridge University Press.
  5. Fan, T. W., Lorkiewicz, P. K., Sellers, K., Moseley, H. N., Higashi, R. M. and Lane, A. N. (2012). Stable isotope-resolved metabolomics and applications for drug development. Pharmacol Ther 133(3): 366-391.
  6. Lane, A. N., Fan, T. W. and Higashi, R. M. (2008). Isotopomer-based metabolomic analysis by NMR and mass spectrometry. Methods Cell Biol 84: 541-588.
  7. Sellers, K., Fox, M. P., Bousamra, M., 2nd, Slone, S. P., Higashi, R. M., Miller, D. M., Wang, Y., Yan, J., Yuneva, M. O., Deshpande, R., Lane, A. N. and Fan, T. W. (2015). Pyruvate carboxylase is critical for non-small-cell lung cancer proliferation. J Clin Invest 125(2): 687-698.
  8. Warburg, O. (1923). Versuche an überlebendem Carcinomgewebe (Methoden). Biochem Zeitschr 142, 317-333.
  9. Warburg, O. (1956). On the origin of cancer cells. Science 123(3191): 309-314.
  10. Xie, H., Hanai, J., Ren, J. G., Kats, L., Burgess, K., Bhargava, P., Signoretti, S., Billiard, J., Duffy, K. J., Grant, A., Wang, X., Lorkiewicz, P. K., Schatzman, S., Bousamra, M., 2nd, Lane, A. N., Higashi, R. M., Fan, T. W., Pandolfi, P. P., Sukhatme, V. P. and Seth, P. (2014). Targeting lactate dehydrogenase--a inhibits tumorigenesis and tumor progression in mouse models of lung cancer and impacts tumor-initiating cells. Cell Metab 19(5): 795-809.
  11. Zimmermann, M., Sebastian Lange, S., Lampe, J., Smirnow, I., Bitzer, M. and Lauer, U. M. (2012). Precision-cut slices of normal and tumorous liver tissues generated with the Leica VT1200 S vibrating blade microtome. In: Tübingen, M. U. (ed). LeicaBiosystems, Vol. 95.8807 Rev B - Order no. 1495.8807 Tübingen.

简介

这种方法的一个重要组成部分是评估肿瘤微环境对肿瘤生长和存活的作用。为了解决这个问题,我们采用Warburg(Warburg,1923)的原始方法,通过将薄组织切片与稳定同位素解析代谢组学(SIRM)组合来确定人体组织的详细代谢活性。 SIRM使得能够在定义的时间段内跟踪诸如葡萄糖或谷氨酰胺的来源分子的代谢转化,并且是详细的途径追踪和通量分析的要求。在我们的方法中,我们保持在细胞培养基中新鲜切除的组织切片(来自同一受试者的相同器官的癌性和非癌性),并用适当的稳定的富含同位素的营养物,例如葡萄糖或13 C 15葡萄糖或13 C 15葡萄糖或13 C 15葡萄糖, - 谷氨酰胺。这些切片可存活至少24小时,并使得有可能消除对靶组织代谢的系统影响,同时保持原始的3D细胞结构。因此,它是用于评估治疗剂对靶组织代谢的影响及其对个体患者的治疗功效的极好的临床前平台(Xie等人,2014; Sellers等人,/em>,2015)。

关键字:组织切片, 惠普, 代谢通路追踪, 临床前研究, 肿瘤代谢

材料和试剂

  1. 25ml T烧瓶NC通气帽(SARSTEDT AG& Co,目录号:83.1810.002)
  2. 液氮便携式容器(Nalgene塑料杜瓦瓶)(Thermo Fisher Scientific,目录号:S34074C)
  3. 无菌注射器和针(Thermo Fisher Scientific,目录号:10142534)
  4. 一次性移液管(Samco细尖,1ml)(VWR International,目录号:16001192)
  5. 1ml和1-200μl的气溶胶阻挡尖端(Thermo Fisher Scientific,目录号:02-707-42)
  6. 螺旋盖塑料小瓶(2ml)彩色编码帽(黄色,蓝色,绿色和红色)(USA Scientific,目录号:1420-8706,1420-8701,1420-8702和1420-9704)
  7. 顶端塑料小瓶(1.5ml)(USA Scientific,目录号:1615-5510)
  8. 15ml Falcon管(SARSTEDT AG& Co,目录号:62.554.205)
  9. 透析的,无菌过滤的胎牛血清(FBS)(不含血清代谢物),10-12kDa(Atlanta Biochemical,目录号:S12650)
  10. 示踪剂实例:葡萄糖,葡萄糖,葡萄糖,葡萄糖,葡萄糖,葡萄糖,葡萄糖, 13 5 , 15 N <2> - 谷氨酰胺
  11. 来源:13 C葡萄糖/D-葡萄糖([U- 13 C],99%)(Cambridge Isotope Laboratories,目录号:CLM-1396-CTM),葡萄糖/D-葡萄糖(1→2),葡萄糖/D-葡萄糖> 2%,99%)(Cambridge Isotope Laboratories,目录号:CLM-504),13 C ,15 N谷氨酰胺/L-谷氨酰胺( 13 5,99%; 15 N 2 /99%)(Cambridge Isotope Laboratories,目录号:CNLM-1275)或
    葡萄糖(Sigma-Aldrich,目录号:660663),SEQ ID NO:13,SEQ ID NO:13,SEQ ID NO: > -1,2-葡萄糖(Sigma-Aldrich,目录号:661422),L-谷氨酰胺 - 13 C 15 S,15 S - > 2(Sigma-Aldrich,目录号:607983)
  12. 青霉素+链霉素:GE Healthcare PEN/STREP/FUNGIZONE 100ml(Thermo Fisher Scientific,目录号:SV3007901)
  13. 方案 10%中性缓冲福尔马林(Thermo Fisher Scientific,目录号:032-059)
  14. 在PBS中的25%(w/v)无菌过滤的13 C葡萄糖(0.2μm)(储备溶液 - 可以冷冻,分装并在4℃下储存)
  15. 液氮
  16. 70%乙醇(v/v)
  17. 60%乙腈水溶液(v/v)(Sigma-Aldrich,目录号:L010400)
  18. 氯化钠(NaCl)(Thermo Fisher Scientific,目录号:S271-1)
  19. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  20. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S0876)
  21. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P9791)
  22. 氨基酸:甘氨酸,L-精氨酸,L-天冬酰胺,L-天冬氨酸,L-胱氨酸2HCl,L-谷氨酸,L-谷氨酰胺,L-组氨酸,L-羟脯氨酸,L-异亮氨酸,L-亮氨酸, L-赖氨酸盐酸盐,L-甲硫氨酸,L-苯丙氨酸,L-脯氨酸,L-丝氨酸,L-苏氨酸,L-色氨酸,L-酪氨酸二钠盐,L-缬氨酸
  23. 维生素:生物素,氯化胆碱,D-泛酸钙,叶酸,异肌醇,烟酰胺,对氨基苯甲酸,盐酸吡哆醇,核黄素,盐酸硫胺素,维生素B12
  24. 硝酸钙[Ca(NO 3)2] 2 SiO 4 [O]
  25. 硫酸镁(MgSO 4)
  26. 谷胱甘肽(还原)
  27. 酚红
  28. 缺少感兴趣的示踪剂的相关介质(例如,DMEM,RPMI,其他定义的介质):
    一个。 Dulbecco改良Eagle培养基(DMEM)是不含葡萄糖,谷氨酰胺,丙酮酸盐碳酸氢盐和酚红的粉末配方,在用于SIRM研究的制剂中提供相当大的灵活性(Sigma-Aldrich,目录号:D5030)(参见Recipes )
    b。 RPMI 1640是不含葡萄糖和谷氨酰胺但含有碳酸氢盐和酚红的液体培养基(MP Biomedicals,目录号:091646854)(参见配方)。
  29. 0.2微米无菌过滤的磷酸盐缓冲盐水(PBS)(见配方)
  30. 用于0.2%13 C 6 - 葡萄糖,2mM 12 C-Gln(100ml)的培养基组成(参见配方)
  31. 用于0.2%葡萄糖,2mM葡萄糖,2mM葡萄糖,2mM葡萄糖,2mM葡萄糖,2mM葡萄糖,2mM葡萄糖,/100ml> -Gln(100ml)(参见配方)

设备

  1. 第二类生物安全罩
  2. 具有氧传感器和CO 2传感器(Thermo Fisher Scientific,型号:Hera cell 150i)的Trigas培养箱
  3. 灭菌摇床(Rotoshake Genie)(Scientific Industries,型号:SI-1100)
  4. 液氮冷冻仓
  5. ("紫色顶部")(BD,目录号:366643)
  6. 带有旋转转子的冷冻离心机,其可使用转子(Thermo Fisher Scientific,目录号:75003181)接受真空容器[例如 Sorvall Legend X1R(Thermo Fisher Scientific,目录号:75-004-261)
  7. 移液器(可变尺寸范围)(USA Scientific ErgoOne)
  8. Weck Knife/Dermatome(George Tiemann& Co.,目录号:222-5-523)
  9. 称量船(Thermo Fisher Scientific,目录号:08732113和08732115)
  10. 4位平衡(Thermo Fisher Scientific,Mettler-Toledo,目录号:0133525)
  11. 冰桶(Thermo Fisher Scientific,目录号:02-591-44)
  12. 尖锐的解剖剪刀(Thermo Fisher Scientific,目录号:08940)
  13. Excelta?塑料镊子(Thermo Fisher Scientific,目录号:17-456-066)
  14. 数码相机

程序

  1. 组织采购
    所有组织必须根据IRB批准的方案购买。随着活人体组织的处理,所有人员必须接受和维持生物安全性,HIPAA和CITI认证。
    方案1中给出了整个过程的概述。


    计划1. FlowChart 。Preop:术前房间;或:手术室。要采取的薄切片的数量取决于肿瘤的大小。一片组织也在OR中快速冷冻,并将另外的组织置于福尔马林中用于病理分析
    1. 血样提供关于代谢状态的总体信息 的个体受试者,并且可以使用血沉棕黄层 提取DNA或RNA用于序列分析
    2. 取10ml样品 血液应该在术前被抽成紫色的顶部真空采血管 (K 2 2 -EDTA)。其他抗凝剂如柠檬酸盐或 不应使用肝素,因为它们干扰代谢测定。一个 切除后应手术切除血液样本。的 将血液倒置两次以确保EDTA的溶解,并保持 冰抽血后立即。血液应分离 包装的红细胞,血沉棕黄层和血浆在30分钟内通过离心 ?3,500 x g 在4°C下在摇摆转子中保持15分钟 后续操作应在BSL2 + 生物安全柜中进行 注意:我们使用以下颜色代码存储:红色=全血,黄色=血浆,绿色=血沉棕黄色,蓝色=尿液。
    3. 将血浆在1℃下吸入预冷的无菌2ml螺旋盖小瓶中 ?ml等分试样并在液氮中快速冷冻。吸入面膜 使用广口塑料吸管插入2ml螺旋盖小瓶和闪光 冷冻在液体N 2中。
    4. 这些实验已经进行 新鲜切片的成对癌(CA)和非癌(NC)肺 从非小细胞肺癌切除的组织(Xie et al。,2014)和 胰腺癌患者。切除后,薄切片(0.5-1 mm厚) ?的组织从视觉上非坏死的表面切除 纤维化肿瘤区域使用Weck切片机在手术室 (OR),在大约5-10分钟内切除。大约1cm 2组织 (参见图1A)。控制非癌组织从a 类似地获得远(> 10cm)区域。病理学家现场 检查CA和NC组织标本。高度坏死组织 舍弃。
    5. 同时,一小块大块CA组织 应置于DMEM或其他适当介质保存室内 植入受体NSG小鼠的温度 患者衍生的异种移植物或PDX。每个CA和NC的小片 将组织浸泡在福尔马林中用于病理检查或闪光 冷冻在液体N 2中用于基于图像的代谢分析。
    6. 哪里 组织采集在OR是不切实际的(如结肠直肠或 乳腺癌切除术),可以在病理学中制备切片 实验室靠近OR。与新鲜切除的比较 ?组织,速度是必不可少的,因为新陈代谢正在快速变化。每当 可行,应在OR中进行组织冷冻
    7. 的 将切片置于两次灭菌的称重的一滴无菌PBS中 以防止粘附和均匀铺展切片。每个重量 船预先编号为CA或NC切片。重量船上的组织 然后被拍摄。每个切片用无菌PBS简单冲洗, 印迹(两次),然后小心地置于预编号(使用 耐乙醇标记笔)预配(记录皮重) T25-含有10ml DMEM(或其他相关培养基)的烧瓶 合适的示踪剂(例如10mM,13 C,6葡萄糖或2mM,13 C,15 C, 10%透析的FBS(根据需要)和1×青霉素+胰蛋白酶+ 链霉素。带切片的烧瓶被带到培养室 尽快并用70%乙醇喷雾,并擦干 将它们放在生物安全罩中。
    8. 移取200微升培养基 从每个烧瓶中取出至1.5ml snap-cap管(t 0次 - 零时间培养基样品)。 ?在4℃下以10,000×g离心10分钟以除去组织碎片。 转移100微升到去皮重的1.5微升snap-cap管代谢物提取 并称量转移的介质。将剩余的介质转移到1.5 ml螺旋盖管,在-80℃下长期保存
    9. 在生物安全罩内的2位置平衡中称重烧瓶,并在烧瓶上记录重量。
    10. 将烧瓶转移到含有摇床设置为低的CO 2培养箱中 振幅摇摆(足以确保液体移动) 没有非层流的切片)图1B)设置为37℃和5%CO 2, 氧气设置为所需水平(例如 20%,1%)。
    11. 烧瓶 连续轻轻摇动24至48小时进行气体交换 以在组织表面维持恒定的营养物供应,同时 避免废物如酸的局部积聚。
    12. 如 需要的,介质可以每12小时刷新,并在0,6,12, ?24 ...。 h用于分析营养物吸收和废物产生。烧瓶 ?在每个培养基取样点和烧瓶之前和之后称重 在烧瓶上记录重量。

  2. 组织收获
    1. 孵育24小时后,称重烧瓶。
    2. 将烧瓶置于冰上 立即从培养箱中取出以进一步最小化 代谢。一次可以收获多达6个烧瓶。保持组织 在收获期间尽可能多地在冰上切片
    3. 使用移液管,吸出并将条件培养基转移到15-ml锥形离心管中
    4. 将介质以3,500至4,690 x g,4℃离心15分钟,以除去任何颗粒和碎屑。
    5. 吸取100μlT sub 24培养基上清液到1.5-ml帽盖管中用于代谢物提取。
    6. 吸取1毫升培养基等分到2毫升螺旋盖管长期储存在-80℃。
    7. 将剩余的培养基单独储存在-80℃的7ml小瓶中,用于外来体分离等目的
    8. 倒置和轻拍烧瓶移动组织切片到盖或 颈部区域用于取出。保持瓶子在冰上倒置。
    9. 在冰冷的10 ml冷PBS中,连续洗涤组织切片3次,每次在50 ml烧杯中。
    10. 将干净的Kimwipe的组织切片和照片在一个小称量船上的扁平切片
    11. 称重整个组织切片在小称重船上,并记录重量。
    12. 分开一小块保存在1ml缓冲福尔马林中 ?在1.5ml snap-cap管中用于组织学。剩余的组织切片 分开均匀,每片应重量不超过20-30毫克湿 重量以促进组织匀浆和提取效率。 称重之后,立即将每片以1L速冻。 N 2和 放置在前liq。 N 2冷冻1.5-ml帽盖管长期使用 储存于-80℃。
    13. 在福尔马林中6-8小时后,用70%乙醇替代福尔马林用于组织学制备的组织片
    14. 在冷的60%乙腈(v/v)和提取物中均质化组织 组织匀浆根据标准进行代谢物分析 协议(Fan,2012; Fan,2010) 同位素解析分析技术(例如,NMR和MS)(Fan,2012) (图1C)(Lane ,2008)。
    15. 本协议描述 ?用于薄组织切片的稳定同位素标记的方法。 SIRM 分析涉及同位素异构体的定量(通过NMR)和 同位素异质体(MS),源自源的代谢转化 在细胞或组织中的分泌(例如,13 C葡萄糖或13 C,15 N谷氨酰胺) (图1C)。通过NMR和质量的SIRM分析的技术 光谱测定法在Sellers等人的(2015)中有详细描述; Lane等 al。(2008)和Fan 等人(2012)。

代表数据


图1.肺实质细胞实验 。A.肺腺癌(CA,右)附近的非癌性肺组织(NC,左) B.T25-烧瓶在CO 2培养箱内的摇床上; C.来自非小细胞的CA对NC肺切片的提取物的代表性1D H(<13 C)HSQC NMR谱(在14.1 T,15℃记录)肺癌(NSCLC)患者在10mM 13 C 16 - 葡萄糖存在下温育24小时。如(Sellers等人,2015; Fan,2012)所述粉碎和提取组织切片,其产生三个阶段 - 含有极性代谢物的上层水相,含有非极性代谢物的下层有机相(主要是脂质)和含有蛋白质的界面相。这里,将上层相冻干并再溶解于含有50%D 2 O和25nmol DSS-d 6的磷酸盐缓冲液中,其用作化学位移参考和浓度标准(Fan和Lane,2013)。 HSQC光谱检测直接连接到13 C的质子,并且因此给出从来源分子(在这种情况下为葡萄糖)引入了13 C的代谢物的读出。在相同条件下记录癌症和非癌性组织的光谱,并将绝对强度相对于组织蛋白质重量标准化。使用MNOVA(Mestrelab Research,Santiago de Compostela,Spain)中的峰拟合函数确定峰面积。
在CA组织切片中增强各种 13 C标记的代谢物的产生是明显的,包括13 C-乳酸盐(Lac),其与Warburg效应或加速糖酵解一致肿瘤组织(Warburg,1956)。

笔记

  1. 这些程序已在新鲜切除的NSCLC和胰腺癌以及患者来源的小鼠异种移植物中测试。其他组织可能需要对培养基的组成和代谢活力的潜伏期长度进行实验
  2. 较大或炎性肿瘤可能具有需要避免的相当大的坏死或纤维化区域。
  3. Weck切片机是手持的,但是在实践中, 1mm厚。通过从照片测量切片的面积容易地估计切片厚度,并且从湿重测量平均厚度h =重量/面积。
  4. 一些肿瘤是高度粘液的,并且更难以可再现地切片
  5. Weck切片机的替代物是振动切片机,其可以从坚硬而不是软组织中再现地产生较薄的切片(参见 10 ),但是更慢。非常薄的切片(100-200μm)可能显示出相对较大的伤口反应,并且更脆弱
  6. 由于许多肿瘤不仅在肿瘤的不同区域中的癌症/基质含量方面是异质的,而且在遗传学方面也是如此,因此建议从肿瘤获得多个切片以覆盖这种异质性。这也使每个切片的组织病理学检查至关重要
  7. 没有病理检查,一些肿瘤的边缘不明显。应当对接近肿瘤以及远离肿瘤的组织进行取样用于比较。

食谱

  1. DMEM和RPMI Medium 1640
    对于SIRM研究,应使用不含谷氨酰胺或葡萄糖的培养基,在基本培养基中补充合适浓度的富含13 C的前体。

    组件
    分子量
    集中(mg/L)
    摩尔浓度(mM)RPMI
    摩尔浓度(mM)DMEM
    氨基酸
    甘氨酸
    75
    10
    0.133
    0.40
    L-精氨酸
    174
    200
    1.15
    0.483
    L-天冬酰胺
    132
    50
    0.379
    -
    L-天冬氨酸 133
    20
    0.150
    -
    L-胱氨酸2HCl
    313
    65
    0.208
    0/0.2
    L-谷氨酸
    147
    20
    0.136
    -
    L-谷氨酰胺
    146
    300
    2.05
    2
    L-组氨酸
    155
    15
    0.0968
    0.27
    L-羟基脯氨酸
    131
    20
    0.153
    -
    L-异亮氨酸
    131
    50
    0.382
    0.8
    L-亮氨酸
    131
    50
    0.382
    0.8
    L-赖氨酸盐酸盐 146
    40
    0.274
    1.0
    L-甲硫氨酸
    147
    15
    0.101
    0.2
    L-苯丙氨酸
    15
    0.0909
    0.4
    L-Proline
    115
    20
    0.174
    -
    L-丝氨酸 105
    30
    0.286
    0.4
    L-苏氨酸
    109
    20
    0.168
    0.8
    L-色氨酸
    204
    5
    0.0245
    0.078
    L-酪氨酸二钠盐二水合物 261
    29
    0.111
    0.4
    L-缬氨酸
    117
    20
    0.171
    0.8
    维生素
    生物素
    244
    0.2
    0.000820

    氯化胆碱
    140
    3
    0.0214
    0.0285
    D-泛酸钙
    477
    0.25
    0.000524
    0.008
    叶酸
    441
    1
    0.00227 / 0.009
    i-肌醇
    180
    35
    0.194
    .04
    烟酰胺
    122
    1
    0.00820
    0.033
    对氨基苯甲酸
    137
    1
    0.00730

    盐酸吡哆醇
    206
    1
    0.00485
    .019
    核黄素
    376
    0.2
    0.000532
    .001
    盐酸硫胺素 337
    1
    0.00297
    .012
    维生素B12
    1,355
    0.005
    0.0000037
    -
    无机盐
    硝酸钙(Ca(NO 3)2)2·6H 2 O·6H 2 O)·/· 236
    100
    0.424

    硫酸镁(MgSO 4)(无水)
    120
    48.84
    0.407

    氯化钾(KCl)
    75
    400
    5.33

    碳酸氢钠(NaHCO 3)
    84
    2,000
    23.81
    44
    氯化钠(NaCl)
    58
    6,000
    103.45

    磷酸氢二钠(Na 2 HPO 4)2/7H 2 O 3)
    268
    1,512
    5.64

    其他组件
    谷胱甘肽(还原)

    1
    0.00326

    酚红
    376.4
    5
    0.0133


  2. 10x PBS
    试剂来源在材料部门
    中给出 80克NaCl
    2克KCl
    14.4g Na 2 HPO 4无水
    2.4g KH 2 PO 4水溶液
    溶于950ml 18Mohm水中,pH至7.4,换至1L,无菌过滤器(0.2μm)
  3. 0.2%的13 C 6葡萄糖,2mM的12 C-Gln(100ml)的培养基组成。
    89.2ml基本培养基减去示踪剂(例如无葡萄糖的版本)(89%营养物浓度)
    10ml无菌过滤的透析的FBS(10%FBS) 在PBS(最终10.75mM葡萄糖)中的0.8ml 25%无菌过滤的13 C 6葡萄糖(0.2μm)
    1ml 100x链霉素/青霉素原液
  4. 用于0.2%葡萄糖,2mM葡萄糖,2mM葡萄糖,2mM葡萄糖,2mM葡萄糖,2mM葡萄糖,2mM葡萄糖,/sub> -Gln(100ml)
    88 ml基础培养基减去示踪剂(不含谷氨酰胺的版本)(所有营养物的88%浓度) 10ml无菌过滤的透析的FBS(10%FBS) 在PBS中的1ml 0.2M无菌过滤的<15> 15,<15>,15 N 2 - 谷氨酰胺(0.2μm) 2 mM final)
    1ml 100x链霉素/青霉素原液
    对于激素敏感组织,可以使用活性炭剥离的FBS。
    对于其他浓度的FBS,相应地调整FBS和基础培养基的体积 注意:应将谷氨酰胺储备液制成新鲜或以-20℃小份储存,以避免反复冻融。它在溶液中储存时形成焦谷氨酸,甚至在中性pH和更高的温度下

致谢

这项工作部分由以下授权支持:NIH P01 CA163223-01A1,NIH 5R01ES022191-04,NIH 3R01ES022191-04S1,NIH 1U24DK097215-01A1和肯塔基挑战挑战赛。该协议是基于Xie et al。(2014)中描述的工作开发的;卖方(2015)和Bousamra (2012)。作者声明没有利益冲突。

参考文献

  1. Bousamra,M.,Day,J.,Fan,T.W.,Higashi,R.M.,Kloecker,G.,Lane,A.N.and Miller,D.M。(2012)。代谢组学的临床方面。在: Humana,Vol。
  2. Fan,T.W。(2012)。 代谢组学调查样品制备的注意事项 代谢组学手册 17.
  3. Fan,T.W。(2010)。代谢组学编辑的转录组学分析(meta)。参见:McQueen,C.A。(ed)。综合毒理学。学术出版社。 2 685-706。
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  5. Fan,T.W.,Lorkiewicz,P.K.,Sellers,K.,Moseley,H.N.,Higashi,R.M。和Lane,A.N。(2012)。 稳定的同位素解析代谢组学和药物开发应用药物治疗/em> 133(3):366-391。
  6. Lane,A.N.,Fan,T.W.and Higashi,R.M。(2008)。 通过NMR和质谱法进行基于同位素的代谢组学分析。方法细胞生物 84:541-588。
  7. Sellers,K.,Fox,MP,Bousamra,M.,2nd,Slone,SP,Higashi,RM,Miller,DM,Wang,Y.,Yan,J.,Yuneva,MO,Deshpande,和Fan,TW(2015)。 丙酮酸羧化酶对非小细胞肺癌增殖至关重要。 J Clin Invest 125(2):687-698
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  9. Warburg,O.(1956)。 关于癌细胞的起源 科学 123( 3191):309-314。
  10. Xie,H.,Hanai,J.,Ren,JG,Kats,L.,Burgess,K.,Bhargava,P.,Signoretti,S.,Billiard,J.,Duffy,KJ,Grant,这些研究结果表明,这些研究结果表明,这些研究结果表明,该方法能够有效地提高生产效率。 靶向乳酸脱氢酶 - 抑制肺癌小鼠模型中的肿瘤发生和肿瘤进展,启动细胞。细胞Metab 19(5):795-809。
  11. Zimmermann,M.,Sebastian Lange,S.,Lampe,J.,Smirnow,I.,Bitzer,M.and Lauer,U.M。(2012)。用Leica VT1200 S振动刀片切片机产生的正常和肿瘤肝组织的精确切片。 In:Tübingen,M.U。(ed)。 LeicaBiosystems,Vol。 95.8807 Rev B - 订货号1495.8807Tübingen。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Fan, T. W., Lane, A. N. and Higashi, R. M. (2016). Stable Isotope Resolved Metabolomics Studies in ex vivo TIssue Slices. Bio-protocol 6(3): e1730. DOI: 10.21769/BioProtoc.1730.
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