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13C Tracer Studies of Metabolism in Mouse Tumor Xenografts
13C 示踪研究老鼠肿瘤异种移植中的新陈代谢   

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Abstract

Mice are widely used for human tumor xenograft studies of cancer development and drug efficacy and toxicity. Stable isotope tracing coupled with metabolomic analysis is an emerging approach for assaying metabolic network activity. In mouse models there are several routes of tracer introduction, which have particular advantages and disadvantages that depend on the model and the questions addressed. This protocol describes the bolus i.v. route via repeated tail vein injections of solutions of stable isotope enriched tracers including 13C6-glucose and 13C5,15N2-glutamine. Repeated injections give higher enrichments and over longer labeling periods than a single bolus. Multiple injections of glutamine are necessary to achieve adequate enrichment in engrafted tumors.

Keywords: SIRM(惠普), Mouse PDX model(PDX模型小鼠), NOD/SCID/Gamma mouse(NOD/SCID小鼠/), Isotopomer distribution analysis(同位素分布分析)

Materials and Reagents

  1. 0.5 ml K2-EDTA collection tubes (BD, catalog number: 363706 )
  2. Sterile syringes and gauge 30 needles (Thermo Fisher Scientific, catalog number: 10142534 )
  3. 5-inch squares Al foil
  4. Indelible marker pens (black)-Sharpie fine tip
  5. Pasteur pipet
  6. Lancets (MEDIpoint, Inc., model: Goldenrod 5 mm )
  7. Mice bearing tumor xenograft
    Note: The strain of immunocompromised recipient mouse depends on type of implant. Young NOD/SCID Gamma (NSG) mice are especially favorable for the patient derived xenograft (PDX) model.
  8. Ketamine for anesthesia if needed, Schedule III controlled substance (Sigma-Aldrich, catalog number: K2753 )
  9. Tracer [e.g., 13C6-glucose, 13C2-1, 2-glucose (60 mg/mouse), or 13C5, 15N2-glutamine (22 mg/mouse)]
  10. Sources
    1. D-Glucose-13C6 (Cambridge Isotope Laboratories, Inc., catalog number: CLM-1396-CTM )
    2. D-Glucose-1, 2-13C2 (Cambridge Isotope Laboratories, Inc., catalog number: CLM-504 )
    3. L-Glutamine-13C5, 15N2 (Cambridge Isotope Laboratories, Inc., catalog number: CNLM-1275 )
    Or
    1. D-Glucose-13C6 (Sigma-Aldrich, catalog number: 660663 )
    2. D-Glucose-1, 2-13C2 (Sigma-Aldrich, catalog number: 661422 )
    3. L-Glutamine-13C5, 15N2 (Sigma-Aldrich, catalog number: 607983 )
  11. Liquid nitrogen
  12. 70% ethanol
  13. 10% Neutral buffered formalin (Thermo Fisher Scientific, ProtocolTM, catalog number: 032-059 )
  14. 25% w/v sterile filtered (0.2 µm) 13C glucose in PBS
    Note: Stock solution can be frozen and stored at -25 °C.
  15. Sterile wipes (Uline, catalog number: S-16183 )
  16. Sodium Chloride (NaCl) (Thermo Fisher Scientific, catalog number: S271-1 )
  17. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  18. Sodium phosphate dibasic (Na2HPO4) anhydrous (Sigma-Aldrich, catalog number: S-0876 )
  19. Potassium phosphate monobasic (KH2PO4) anhydrous (Sigma-Aldrich, catalog number: P9791 )
  20. 10x Phosphate Buffered Saline (PBS) (see Recipes)
  21. Stock solution for 0.2% 13C6-glucose (see Recipes)
  22. Stock solution for 13C5, 15N2-Gln (10 ml) (see Recipes)

Equipment

  1. Mouse restraining system (Plas-Labs, Inc., model: 541-RR )
  2. FirstHand Surgical Instrument Kits for Mice and Rats (Kent Scientific Corporation, model: INSMOUSEKIT )
  3. Blue wax dissection tray (VWR International, catalog number: 10060-188 )
  4. Warming blanket (Kent Scientific Corporation, model: DCT-15 )
  5. Water bottle with DI water (Thermo Fisher Scientific, catalog number: 02-897-11 )
  6. Polystyrene Weigh boats (USA Scientific Inc., catalog number: 2347-1427 )
  7. Calipers for measuring tumor size (Thermo Fisher Scientific, catalog number: 15-077-957 )
  8. 2-place Balance for weighing tissues (VWR International, Adventurer Ohaus®, catalog number: 10153-744 )
  9. Refrigerated Microfuge (Eppendorf, model: 5417R with rotor)
  10. Dewar/bucket (Nalgene plastic dewar) for lN2 (Thermo Fisher Scientific, catalog number: S34074C )
  11. Large Styrofoam box

Procedure

All experiments must be performed under an IACUC approved protocol. For human tissue xenografts, the appropriate IRB approvals must also be met.

  1. Schedule
    1. Allow tumor in mice to develop to at least 0.5 cm and up to 1.5 cm according to specific protocols for ectopic or orthotopic xenografts of cells or fresh tissue (PDX model) as well as other tumor models (e.g., transgenic tumor model).
    2. Preparations
      The day before Stable Isotope Resolved Metabolomics (SIRM) experiment:
      1. Pre-label Al squares with date, mouse number and tissue (e.g., tumor), and blood collection tubes (2 per mouse for collection immediately after tracer injection, and at the time of necropsy) with date, mouse number, collection time.
      2. Label storage box for -80 °C freezer.
      3. Prepare tracer solutions e.g., 25% 13C6-glucose (1.344 M) or 36.2 mg/ml 13C5, 15N2-Gln (0.2 M) in sterile PBS, sterile filter, and store in aliquots at < -20 °C.
      4. Label formalin bottles with date, mouse number and tumor source.
      5. Assemble all components in sealable boxes for transport.
      6. Prepare schedule sheet (see example below, Table 1).
    3. Tracer Injection
      When mice are ready, restrain the mouse in the mouse restrainer, sterilize the injection area of the tail vein using a sterile wipe. Inject stable isotope tracers through the tail vein (or other convenient vein, such as submandibular vein) into individual immobilized mice*. Placing the mouse on a warming blanket or gently heating the tail visibly dilates the vein to make injection easier.
      1. Record time of injection.
      2. Immediately take blood sample (see step 5 below).
      3. For 13C6-glucose (can be purchased from Sigma Isotec or Cambridge Isotopes Laboratories, see above): Inject 80 µl (20 mg) each 25% (w/v) stock solution (in PBS and 0.2 µm sterile filtered) at 15 min intervals 3 times (total = 322 µmol).
      4. For 13C5, 15N2-Gln: Same as above except for injecting 200 µl (7.2 mg) each 36.2 mg/ml stock solution (in PBS and 0.2 µm sterile filtered) at 15 min intervals 3 times (total = 142 µmol).
      5. The bolus injections take a few seconds. The extremely high heart rate of mice (500-600 beats per minute for resting adult mice) ensures that the tracer is systemically distributed very rapidly, therefore approximating a pulse.
        Note: *We have tried injection into ketamine-anesthetized mice. Since anesthesia slows metabolism, the timing has to be lengthened. Anesthesia can also alter metabolism. With the physical restraint method, it is important to minimize stress to the restrained mice during injection. Black mice are more difficult to inject via tail vein. The ability to do timed injections reproducibly takes considerable practice.
    4. Tissue harvest
      1. At 45 min after the first injection (15 min after the last injection), take photographs of the mouse and the tumor.
      2. Take blood sample before killing mice by cervical dislocation (do not use CO2 asphyxiation or lethal injection of barbiturates as they interfere with metabolism)
      3. Measure tumor size using calipers (Figure 1).
      4. Dissect relevant tissues, e.g., tumor, lung, liver, heart, kidney, brain, skeletal muscle, adipose, lower bowel and flash freeze tissues immediately (within 5 min of necropsy) in liquid N2, followed by placing frozen tissues in screw-cap 2 ml microfuge tubes pre-equilibrated in liquid N2 or in aluminum foil (made into a boat shape) floating on liquid N2, followed by wrapping**.
        Note: **Use non-erasable (solvent-resistant) permanent ink Sharpie pens to label sample tubes or aluminum foil to avoid label washout.
      5. The order of dissection is dictated mainly by the major organ of interest, which should be removed first if possible. Dissected tissue is rinsed briefly in cold PBS, blotted and flash-frozen in liquid N2 within 2-6 min of euthanasia.
      6. Tumor should be rinsed in cold PBS, blotted and weighed before flash freezing in lN2 (Figure 1) and photographed.
      7. Typically at least three people are needed for this work.
      8. With multiple injections, it is practical to do three mice as a group, timing injections 5 min apart.


        Figure 1. Mouse necropsy. A. NSG mouse with implanted tumors. Measuring size using electronic calipers; B. Dissection to reveal ectopic tumor. Blood tubes in the orange rack; C. Tumor pieces being weighed after blotting.

    5. Blood sample processing
      1. Blood should be collected***immediately into K2-EDTA microtubes ['purple top'] (designed for mice) after tracer injection (1st) and just prior to necropsy (2nd).
      2. Initial blood collection should be no more than 150 µl; collect as much blood as possible for the 2nd collection.
      3. Let blood stand at room temperature for 5 min before storage on ice****.
      4. Centrifuge blood at 3,500 x g for 15 min at 4 °C.
      5. Separate plasma from blood cells and flash freeze the separated components in liquid N2.
    Notes:
    1. EDTA anticoagulant is preferred over citrate, heparin etc. as EDTA interferes least with metabolism
    2. ***We collect blood intraorbitally or via the submandibular vein using a lancet which is generally preferred (Golde et al., 2005); other methods may be applicable (e.g., from other vein) and at sacrifice, cardiac punch for maximal blood collection.
    3. ****It is important to keep blood at RT for 5 min to reduce hemolysis but store blood on ice thereafter until centrifugation.
    4. All samples are stored at -80 °C or colder.
    5. If necessary, samples for analysis should be shipped overnight on dry ice.
    6. Metabolites are extracted from tissues and blood according to established protocols, and then analyzed by high resolution NMR, GC-MS, and FT-ICR-MS to establish not only the content of metabolites, but also their labeled isotopomer and isotopologue distributions, which represents metabolic transformation from the source tracer to the observed metabolites in the interval between injection and necropsy (Fan et al., 2011; Fan, 2012a; Fan et al., 2012; Lane et al., 2011).

Representative data

Qualitatively similar 13C metabolite profiles of individual organs have been obtained from these experiments at comparable time points, as estimated from 1-D 1H{13C}-HSQC spectra (e.g., Figure 2) (Fan et al., 2011; Yuneva et al., 2012; Sellers et al., 2015; Xie et al., 2014). The amounts of 13C in each metabolite per mg can be calculated from the intensity of the lactate methyl resonance compared with its concentration independently determined by either 1H NMR or GC-MS. These methods also provide the fractional enrichments of each observable metabolite, as described in Lane et al. (2008). Detailed descriptions of data reduction and analysis are provided in Fan et al. (2011) and Lane et al. (2008).


Figure 2. 1D 1H{13C} HSQC spectra of different organs of an NSG mouse infused with 13C6-glucose. The NSG mouse received three boluses of 20 mg each 13C6-glucose via tail vein injection at 15 min interval. Heart, kidney, liver, brain and lung tissues were dissected within 5 min of necropsy, flash-frozen in liq. N2, pulverized in liq. N2, and extracted with acetonitrile: H2O: CHCl3 (4:3:2 v/v) for polar metabolites as described in (10). The NMR spectra were normalized to the residual dry residual weight of the extract. Extracts are lyophilized prior to preparation of the NMR sample, as described (Fan, 2012b).

Table 1. Example record sheet
Title:
Date:
Experiment: Tumor from patient # (Date) implanted into 2 NSG mice (name of researcher).
2 mice, each piece subQ implanted on both flanks
mouse 1 F tumor sizes=   ear tag #
mouse 2 M tumor sizes=  ear tag #
SIRM/harvesting Date
Treatment: 3 x 80 µl 25% 13C6-glucose tail vein 15 min intervals (M1) or 3 x 200 µl, 36 mg/ml 13C5, 15N2-Gln (M2).
Anesthetic Y/N.
Organs extracted sequentially:
Blood, tumor, lung, heart, liver, kidney, brain…

Time
Process
Time post injection / min
comments
M1
80 µl Glc
0


Blood



80 µl Glc



80 µl Glc



blood



sac



Tumor

Mass: g

Lung



Heart



Liver



kidney



brain






M2
200 µl Gln
0


Blood



200 µl Gln



200 µl Gln



blood



sac



Tumor

Mass: g

Lung



Heart



Liver



kidney



brain


Put sample into formalin for pathology. Place small samples into medium for further propagation in NSG mice and cell culturing.

Notes

  1. The injections can be achieved with restraints, which is compromised by release of stress hormones. Alternatively the mice can be anesthetized with ketamine, which can slow general metabolism while they are unconscious and alter certain metabolic pathway(s) due to the non-intended effect of the anesthetic agent.
  2. Tail vein injection of black mice can be challenging. Alternatives are intraperitoneal injection (which has a different time course), feeding or direct infusion via the jugular vein or carotid artery (de Graaf et al., 2011) (http://jaxmice.jax.org/preconditioned/surgical/pricing.html).
  3. For xenografts a variety of immune compromised strains are available, which can be used according to the study design (http://jaxmice.jax.org/findmice/index.html). Xenografts can be formed after injection of established cancer cells (Fan et al., 2011; Lane et al., 2011), primary cell lines, and fresh tumor tissue (PDX model) (Sellers et al., 2015) either subcutaneously or orthotopically. The amounts tissue needed and the number of cells depends greatly on the source of the tumor or cell line characteristics.
  4. Blood analysis is crucial to monitoring the amount of tracer delivered and the overall utilization over the period of the experiment. The transient nature of the experiments provides both information about pathways that are operative during the short labeling time period and the relative rates of uptake and utilization of different tissues. This can be refined by time course analysis of a group of mice harvested at different times post injection. Different numbers of injections also can be used to extend or decrease the degree of labeling in different groups of metabolites. Examples of blood metabolite time courses in mice injected with 13C-glucose are in Fan et al. (2011).
  5. With bolus injections isotopic steady state cannot be reached (de Graaf et al., 2011; Maher et al., 2012), and metabolites with slow turnover do not become significantly labeled. For labeling these metabolites, continuous infusion via an appropriate vein for a longer period (Marin-Valencia et al., 2012) or longer-term tracer feeding can be employed.
  6. Due to some variability of mice age, handling, and tumor status, it may not be practical to average metabolite values from different individuals. 5 or more mice/group are needed depending on the effect size. Preferably all the same gender should be used, and whenever possible littermates. With PDX models we usually match the mouse gender to that of the tumor donor.

Recipes

  1. 10x PBS
    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)
  2. Stock solution for 13C6-glucose (10 ml)
    2.5 g 13C6-glucose
    1x PBS to a total of 10 ml
    Sterile filter (0.2 mm) into sterile screw cap container
    Stored at -4 °C
  3. Stock solution for 13C5, 15N2-Gln (10 ml)
    0.362 g 13C5, 15N2-Gln
    1x PBS to a total of 10 ml
    Sterile filter (0.2 mm) 1 ml aliquots into sterile screw cap vials
    Stored at -80 °C
    Note: Glutamine stock should be made fresh or stored at -20 °C in small aliquots to avoid repeated freezing and thawing. It forms pyroglutamate on storage in solution at neutral pH at higher temperatures.

Acknowledgments

This protocol was modified from previously published studies ( Fan et al., 2011). The work has been supported by NIH Grants 1R01CA118434-01A2, NIH 5R01ES022191-04, NIH 3R01ES022191-04S1 (to TWMF), R01CA-086412 and RO1 CA150947 (to JY), NIH R21CA133688 (to ANL), NIH P01 CA163223 (to ANL, TWMF and JY), and NIH 1U24DK097215-01A1 (to TWMF, ANL), the Kentucky Challenge for Excellence, and the Susan G. Komen Foundation BCTR0503648.

References

  1. Golde, W. T., Gollobin, P. and Rodriguez, L. L. (2005). A rapid, simple, and humane method for submandibular bleeding of mice using a lancet. Lab Anim (NY) 34(9): 39-43.
  2. Fan, T. W., Lane, A. N., Higashi, R. M. and Yan, J. (2011). Stable isotope resolved metabolomics of lung cancer in a SCID mouse model. Metabolomics 7(2): 257-269.
  3. 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.
  4. Fan, T. W. (2012a). Considerations of sample preparation for metabolomics investigation. Handbook of Metabolomics, 17.
  5. Lane, A. N., Fan, T. W., Bousamra II, M., Higashi, R. M., Yan, J. and Miller, D. M. (2011). Clinical applications of stable isotope-resolved metabolomics (SIRM) in non-small cell lung cancer. Omics, 15, 173-182.
  6. Yuneva, M. O., Fan, T. W., Allen, T. D., Higashi, R. M., Ferraris, D. V., Tsukamoto, T., Mates, J. M., Alonso, F. J., Wang, C., Seo, Y., Chen, X. and Bishop, J. M. (2012). The metabolic profile of tumors depends on both the responsible genetic lesion and tissue type. Cell Metab 15(2): 157-170.
  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. 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.
  9. 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.
  10. Fan, T. W. M. (2012b). In: Fan, T. W. M., Lane, A. N. and Higashi, R. M. (eds). The handbook of metabolomics: Pathway and flux analysis, methods in pharmacology and toxicology. Springer Science, 17: 7-27.
  11. de Graaf, R. A., Rothman, D. L. and Behar, K. L. (2011). State of the art direct 13C and indirect 1H-[13C] NMR spectroscopy in vivo. A practical guide. NMR Biomed 24(8): 958-972.
  12. Maher, E. A., Marin-Valencia, I., Bachoo, R. M., Mashimo, T., Raisanen, J., Hatanpaa, K. J., Jindal, A., Jeffrey, F. M., Choi, C., Madden, C., Mathews, D., Pascual, J. M., Mickey, B. E., Malloy, C. R. and DeBerardinis, R. J. (2012). Metabolism of [U-13C]glucose in human brain tumors in vivo. NMR Biomed 25(11): 1234-1244.
  13. Marin-Valencia, I., Yang, C., Mashimo, T., Cho, S., Baek, H., Yang, X. L., Rajagopalan, K. N., Maddie, M., Vemireddy, V., Zhao, Z., Cai, L., Good, L., Tu, B. P., Hatanpaa, K. J., Mickey, B. E., Mates, J. M., Pascual, J. M., Maher, E. A., Malloy, C. R., Deberardinis, R. J. and Bachoo, R. M. (2012). Analysis of tumor metabolism reveals mitochondrial glucose oxidation in genetically diverse human glioblastomas in the mouse brain in vivo. Cell Metab 15(6): 827-837.

简介

小鼠广泛用于癌症发展和药物功效和毒性的人肿瘤异种移植研究。 稳定同位素追踪与代谢物组学分析是一种新兴的方法来测定代谢网络活动。 在小鼠模型中,有几种示踪剂引入途径,其具有取决于模型和所解决的问题的特定优点和缺点。 该协议描述了bolus i.v. 通过重复尾静脉注射稳定的同位素富集的示踪剂的溶液的路径,所述示踪剂包括13 C 13 - 葡萄糖和13 C - 13 C - ,N 15 - ,N 15 - 谷氨酰胺。 与单次推注相比,重复注射给予更高的富集和更长的标记周期。 多次注射谷氨酰胺对于在移植肿瘤中实现充分富集是必需的。

关键字:惠普, PDX模型小鼠, NOD/SCID小鼠/, 同位素分布分析

材料和试剂

  1. 0.5ml K 2 -EDTA收集管(BD,目录号:363706)
  2. 无菌注射器和30号针头(Thermo Fisher Scientific,目录号:10142534)
  3. 5英寸方形铝箔
  4. 不可消失的记号笔(黑色) - 尖锐的细尖
  5. 巴斯德移液器
  6. 柳叶刀(MEDIpoint,Inc.,型号:Goldenrod5mm)
  7. 携带肿瘤异种移植物的小鼠
    注意:免疫受损小鼠的株系取决于植入物的类型。年轻NOD/SCIDγ(NSG)小鼠特别有利于患者来源的异种移植(PDX)模型。
  8. 如果需要,用氯胺酮麻醉,表III控制物质(Sigma-Aldrich,目录号:K2753)
  9. Tracer [例如 13 6 < - >葡萄糖, 13 2 < 2-葡萄糖(60mg /小鼠)或者13 C 15 S - 15 N - 谷氨酰胺(22) mg /小鼠)]
  10. 来源
    1. (Cambridge Isotope Laboratories,Inc。,目录号:CLM-1396-CTM)。< br />
    2. (Cambridge Isotope Laboratories,Inc。,目录号:CLM-504),其中所述抗体包含SEQ ID NO:2所示的氨基酸序列。
    3. (Cambridge Isotope Laboratories,Inc。,目录编号:12),其包含SEQ ID NO:2,SEQ ID NO:2,SEQ ID NO:2,SEQ ID NO: CNLM-1275)

    1. D-葡萄糖 - 13 C(Sigma-Aldrich,目录号:660663)
    2. D-葡萄糖-1,2-二醇(Sigma-Aldrich,目录号:661422)
    3. (Sigma-Aldrich,目录号:607983)中的L-谷氨酰胺和L-谷氨酰胺的混合物,
  11. 液氮
  12. 70%乙醇
  13. 10%中性缓冲福尔马林(Thermo Fisher Scientific,Protocol TM ,目录号:032-059)
  14. 在PBS中的25%w/v无菌过滤(0.2μm)13 C葡萄糖 注意:储备液可以冷冻保存在-25°C。
  15. 无菌抹布(Uline,目录号:S-16183)
  16. 氯化钠(NaCl)(Thermo Fisher Scientific,目录号:S271-1)
  17. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  18. 无水磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S-0876)
  19. 无水磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P9791)
  20. 10x磷酸盐缓冲盐水(PBS)(见配方)
  21. 0.2%的13 C 6 - 葡萄糖的储备溶液(参见配方)
  22. (10毫升)的储备溶液(见Recipes)
    />

设备

  1. 鼠标约束系统(Plas-Labs,Inc.,型号:541-RR)
  2. 用于小鼠和大鼠的第一手术器械套件(Kent Scientific Corporation,型号:INSMOUSEKIT)
  3. 蓝蜡清除盘(VWR International,目录号:10060-188)
  4. 加温毯(Kent Scientific Corporation,型号:DCT-15)
  5. 使用去离子水(Thermo Fisher Scientific,目录号:02-897-11)的水瓶
  6. 聚苯乙烯称重船(USA Scientific Inc.,目录号:2347-1427)
  7. 用于测量肿瘤尺寸的卡尺(Thermo Fisher Scientific,目录号:15-077-957)
  8. 用于称量组织的2位天平(VWR International,Adventurer Ohaus ?,目录号:10153-744)
  9. 冷藏微量离心机(Eppendorf,型号:5417R带转子)
  10. 用于LN2(Thermo Fisher Scientific,目录号:S34074C)的杜瓦瓶/桶(Nalgene plastic杜瓦尔)
  11. 大型发泡胶盒

程序

所有实验必须在IACUC批准的方案下进行。对于人体组织异种移植物,还必须满足适当的IRB批准。

  1. 排定
    1. 允许小鼠中的肿瘤发育至至少0.5cm和至多1.5cm 根据异位移植或原位异种移植的具体方案 细胞或新鲜组织(PDX模型)以及其他肿瘤模型(例如转基因肿瘤模型)。
    2. 准备
      稳定同位素分析代谢组学(SIRM)实验前一天:
      1. 带有日期,小鼠数量和组织(例如肿瘤)和采血管(每只小鼠2只,用于收集)的预标记Al方块 在示踪剂注射后立即和在尸体剖检时) 日期,鼠标数量,收集时间。
      2. -80°C冰箱的标签储存盒。
      3. 制备示踪剂溶液例如 25% 13 - 葡萄糖(1.344 M)或36.2 在无菌PBS,无菌过滤器中的0.1mg/ml的13 C 15 S,15 S/N 2 -Gln(0.2M) ,并存储 以等分试样, -20℃。
      4. 标签福尔马林瓶与日期,鼠标数量和肿瘤来源。
      5. 将所有组件装在可密封的盒子中进行运输
      6. 准备计划表(见下面的例子,表1)。
    3. 示踪剂注射
      当小鼠准备好时,将小鼠限制在小鼠限制器中, 使用无菌擦拭物对尾静脉的注射区域进行灭菌。 通过尾静脉注入稳定同位素示踪剂(或其他方便 ?静脉,例如下颌下静脉)植入个体固定化的小鼠中。 将鼠标放在保暖毯子上或轻轻加热尾部 明显扩张静脉,使注射更容易。
      1. 记录注射时间。
      2. 立即取血样(见下面的步骤5)。
      3. 对于13 C 16 - 葡萄糖(可购自Sigma Isotec或Cambridge 同位素实验室,参见上文):每次注射80μl(20mg)25%(w/v) 储备溶液(在PBS和0.2μm无菌过滤),间隔15分钟 ?次(总计=322μmol)。
      4. 对于 13 C 5 , 15 N <2> -Gln: 除了注射200μl(7.2mg)各36.2mg/ml储备溶液(in PBS和0.2μm无菌过滤)以15分钟间隔3次(总数= 142 μmol)。
      5. 大剂量注射需要几秒钟。极端 高心率(500-600次/分钟,静息成人 小鼠)确保示踪剂系统地非常快速地分布, 因此近似一个脉冲。
        注意:*我们尝试注射到氯胺酮麻醉的小鼠。由于麻醉减缓了新陈代谢,所以时间必须加长。麻醉也可以改变代谢。使用物理约束方法,重要的是在注射过程中使受约束的小鼠的应力最小化。黑色小鼠更难以通过尾静脉注射。重复进行定时注射的能力需要相当多的实践。
    4. 组织收获
      1. 在第一次注射后45分钟(最后一次注射后15分钟),拍摄小鼠和肿瘤的照片
      2. 在通过颈椎脱位杀死小鼠前取血样(do 不使用CO 2窒息或致死性注射巴比妥类药物 干扰新陈代谢)
      3. 使用卡尺测量肿瘤大小(图1)
      4. 解剖相关组织,例如肿瘤,肺,肝,心脏,肾脏, 脑,骨骼肌,脂肪,下肠和快速冷冻组织 立即(在尸检5分钟内)在液体N 2中,然后放置 ?冷冻组织在预平衡的螺旋盖2ml微量离心管中 液体N 2或在铝箔(制成船形)上漂浮 液体N 2,随后包裹**。
        注意:**使用不可擦除(耐溶剂)永久性墨水清洁笔用于标记样品管或铝箔,以避免标签被冲洗。
      5. 解剖的顺序是 主要由感兴趣的主要器官决定,应当移除 如果可能的话。在冷PBS中简单冲洗解剖组织, 在安乐死的2-6分钟内在液体N 2中吸干和快速冷冻
      6. 肿瘤应该在冷的PBS中冲洗,吸干和称重,然后在1NN 2(图1)中快速冷冻并拍照。
      7. 这项工作通常需要至少三个人。
      8. 多次注射,实际上做三个小鼠作为一组,定时注射相隔5分钟。


        图1.小鼠尸检。 A.植入肿瘤的NSG小鼠。测量 ?尺寸使用电子卡尺B.解剖显示异位肿瘤。 血液管在橙色架; C.肿瘤片后称重 印迹
    5. 血样处理
      1. 应立即收集血液至K 2 EDTA-EDTA微管['紫色 ?顶部"](为小鼠设计)在示踪剂注射(1次)之后和之前 到尸检(第2次)。
      2. 初始采血量不应超过150μl;收集尽可能多的血液为2 nd 集合。
      3. 让血液在室温下放置5分钟,然后储存在冰上。
      4. 在4℃下将血液以3500xg离心15分钟。
      5. 将血浆与血细胞分离并快速冷冻液体N 2中的分离的组分。
    注意:
    1. EDTA抗凝剂优于柠檬酸盐,肝素等,因为EDTA对新陈代谢的影响最小。
    2. 我们通过眶内或通过下颌下静脉收集血液 使用通常优选的柳叶刀(Golde等人,2005);其他 方法可以适用(例如从其他静脉)并且在牺牲时, 心脏冲洗以最大限度收集血液。
    3. ****很重要 保持血液在RT 5分钟以减少溶血,但将血液储存在冰上 ?之后直到离心。
    4. 所有样品都储存在-80°C或更冷的地方。
    5. 如有必要,分析样品应在干冰上过夜运输。
    6. 根据组织和血液提取代谢物 建立方案,然后通过高分辨率NMR,GC-MS, 和FT-ICR-MS不仅确定代谢物的含量,而且确定 ?它们的标记同位素和同位素分布 代表从源示踪剂到代谢转化 观察到的注射和尸检之间的间隔代谢产物(Fan ?et al。,2011; Fan,2012a; Fan等人,2012; Lane et al。,2011)。

代表数据

已经从这些实验在可比较的时间点获得了各个器官的定性相似的13 C代谢物概况,如从1-D sup-1 > C} -HSQC光谱(例如图2)(Fan等人,2011; Yuneva等人,2012; Sellers > et al。,2015; Xie et al。,2014)。每个代谢物中每mg的 13 C的量可以从乳酸盐甲基共振的强度计算,与通过1 H NMR或GC-MS独立测定的其浓度相比较。这些方法还提供每种可观察到的代谢物的部分富集,如Lane等人(2008)中所述。 Fan等人(2011)和Lane 等人(2008)提供了数据简化和分析的详细说明。


图2.注射了13 C标记的NSG小鼠的不同器官的1D图像1 H C C 6葡萄糖。 NSG小鼠通过尾静脉注射以15分钟的间隔接受三次20mg的每个<13 C 6葡萄糖。在尸检5分钟内解剖心脏,肾脏,肝脏,脑和肺组织,快速冷冻于液体。 N 2粉末。并且对于如所述的极性代谢物用乙腈:H 2 O:CHCl 3(4:3:2v/v)萃取(10)。将NMR光谱归一化为提取物的残余干残余重量。在制备NMR样品之前将提取物冻干,如(Fan,2012b)所述
表1.示例记录表
标题:
日期:
实验:将来自患者#(日期)的肿瘤植入2个NSG小鼠(研究者名称)。
2只小鼠,每片subQ植入两侧腹部
小鼠1F肿瘤大小=  耳标#
小鼠2 M肿瘤大小=耳标#
SIRM /收获日期
处理:3×80μl25%13 C 16 - 葡萄糖尾静脉15分钟间隔(M1)或3×200μl,36mg/ml < (M2)中的一个或多个来形成。
麻醉Y/N 依次提取器官:
血液,肿瘤,肺,心脏,肝,肾,脑...

时间
处理
时间后注射/分钟
评论
M1
80μlGlc
0






80μlGlc



80μlGlc



血液







肿瘤

质量:g





心脏



肝脏







大脑






M2
200μlGln
0






200μlGln



200μlGln



血液







肿瘤

质量:g





心脏



肝脏







大脑


将样品放入福尔马林进行病理学。将小样品放入培养基中用于在NSG小鼠中进一步繁殖和用于细胞培养。

笔记

  1. 注射可以用约束实现,其受到应激激素释放的影响。或者,可以用氯胺酮麻醉小鼠,其可以减缓一般代谢,同时它们无意识并且由于麻醉剂的非预期的作用而改变某些代谢途径。
  2. 黑色小鼠的尾静脉注射可能是具有挑战性的。替代方案是腹膜内注射(其具有不同的时间过程),通过颈静脉或颈动脉进食或直接输注(de Graaf等人,2011)(http://jaxmice.jax.org/preconditioned/surgical/pricing.html )。
  3. 对于异种移植物,可获得多种免疫受损菌株,其可根据研究设计使用( http: //jaxmice.jax.org/findmice/index.html )。可在注射建立的癌细胞(Fan等人,2011; Lane等人,2011),原代细胞系和新鲜肿瘤组织(PDX)后形成异种移植物模型)(Sellers等人,2015),皮下或原位。所需的组织量和细胞数量很大程度上取决于肿瘤或细胞系特征的来源
  4. 血液分析对于监测递送的示踪剂的量和实验期间的总体利用是至关重要的。实验的瞬时性质提供关于在短标记时间段期间操作的途径的信息和不同组织的摄取和利用的相对速率。这可以通过对在注射后不同时间收获的一组小鼠的时间过程分析来改进。不同数量的注射也可以用于延长或降低不同代谢物组中的标记程度。在注射了13 C - 葡萄糖的小鼠中的血液代谢物时程的实例在Fan等人(2011)。
  5. 通过推注,不能达到同位素稳定状态(de Graaf等人,2011; Maher等人,2012),并且具有缓慢更新的代谢物不会变得显着标记。为了标记这些代谢物,可以使用通过适当的静脉较长时间的连续输注(Marin-Valencia等人,2012)或更长期的示踪物喂养。
  6. 由于小鼠年龄,处理和肿瘤状态的一些变异性,使来自不同个体的代谢物值平均可能不实用。根据效果大小,需要5只或更多的小鼠/组。优选地,应当使用所有相同的性别,并且任何可能的同窝幼仔。使用PDX模型,我们通常将小鼠性别与肿瘤捐赠者的性别相匹配

食谱

  1. 10x PBS
    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)
  2. lt; 13> 6 6 - 葡萄糖(10ml)的储备溶液。
    2.5g 13 C - 葡萄糖
    1x PBS至总共10ml ml
    无菌过滤器(0.2 mm)装入无菌螺帽容器
    储存在-4°C
  3. 用于 13 15 N 2 -Gln(10ml)的储液。
    0.362g 13 , 15 N <2> -Gln
    1x PBS至总共10ml ml
    无菌过滤器(0.2毫米)1毫升等分到无菌螺帽小瓶
    储存于-80°C
    注意:应将谷氨酰胺储备液制成新鲜或在-20°C下小份储存,以避免反复冻融。它在中性pH和更高温度下在溶液中储存形成焦谷氨酸。

致谢

该方案从以前发表的研究中修改(Fan et al。,2011)。该工作已由NIH Grants 1R01CA118434-01A2,NIH 5R01ES022191-04,NIH 3R01ES022191-04S1(至TWMF),R01CA-086412和RO1 CA150947(至JY),NIH R21CA133688(至ANL),NIH P01 CA163223(至ANL ,TWMF和JY),以及NIH 1U24DK097215-01A1(TWMF,ANL),肯塔基挑战挑战赛和Susan G.Komen基金会BCTR0503648。

参考文献

  1. Golde,W.T.,Gollobin,P。和Rodriguez,L.L。(2005)。 使用柳叶刀的小鼠下颌下出血的快速,简单和人道的方法。实验室动画(NY) 34(9):39-43。
  2. Fan,T.W.,Lane,A.N.,Higashi,R.M。和Yan,J。(2011)。 在SCID小鼠模型中稳定同位素解析的肺癌代谢组学。代谢组学 7(2):257-269
  3. 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。
  4. Fan,T.W。(2012a)。代谢组学调查样品制备的考虑。
  5. Lane,A.N.,Fan,T.W.,Bousamra II,M.,Higashi,R.M.,Yan,J.and Miller,D.M。(2011)。稳定同位素解析代谢组学(SIRM)在非小细胞肺癌中的临床应用。 ,15,173-182
  6. Yuneva,MO,Fan,TW,Allen,TD,Higashi,RM,Ferraris,DV,Tsukamoto,T.,Mates,JM,Alonso,FJ,Wang,C.,Seo, JM(2012)。 肿瘤的代谢特征取决于负责任的遗传性病变和组织类型。 em> Cell Metab 15(2):157-170。
  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。
  8. 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。
  9. 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。
  10. Fan,T. W. M.(2012b)。 In:Fan,T.W.M.,Lane,A.N.and Higashi,R.M。(eds)。代谢组学手册:途径和通量分析,药理学和毒理学方法。 Springer Science,17:7-27。
  11. de Graaf,R.A.,Rothman,D.L。和Behar,K.L。(2011)。 最先进的 13 C和间接 1 H - [13 C] NMR光谱。实用指南。 NMR Biomed 24(8):958-972。
  12. Maher,EA,Marin-Valencia,I.,Bachoo,RM,Mashimo,T.,Raisanen,J.,Hatanpaa,KJ,Jindal,A.,Jeffrey,FM,Choi,C.,Madden,C.,Mathews, D.,Pascual,JM,Mickey,BE,Malloy,CRand DeBerardinis,RJ(2012)。 人脑肿瘤中[U- 13 C]葡萄糖的代谢< em> in vivo 。 NMR Biomed 25(11):1234-1244。
  13. Marin-Valencia,I.,Yang,C.,Mashimo,T.,Cho,S.,Baek,H.,Yang,XL,Rajagopalan,KN,Maddie,M.,Vemireddy,V.,Zhao, 。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。 肿瘤代谢分析揭示了小鼠脑中遗传多样的人胶质母细胞瘤中的线粒体葡萄糖氧化vivo 。 15(6):827-837。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Lane, A. N., Yan, J. and Fan, T. W. (2015). 13C Tracer Studies of Metabolism in Mouse Tumor Xenografts. Bio-protocol 5(22): e1650. DOI: 10.21769/BioProtoc.1650.
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