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, Mouse PDX model, NOD/SCID/Gamma mouse, Isotopomer distribution analysis
Materials and Reagents
Equipment
Procedure
All experiments must be performed under an IACUC approved protocol. For human tissue xenografts, the appropriate IRB approvals must also be met.
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…
Put sample into formalin for pathology. Place small samples into medium for further propagation in NSG mice and cell culturing.
Notes
Recipes
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
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