Mice
All experiments conducted in this study were approved by the MIT Committee on Animal Care or the MGH Institutional Animal Care and Use Committee. Female B6 and female NSG mice 6–10 weeks of age were used. Animals were housed at ambient temperature and humidity (18–23 °C, 40–60% humidity) on a 12-h light–12-h dark cycle and co-housed with littermates with ad libitum access to food and water. All experimental groups were age matched, numbered and randomly assigned to treatment or control groups to minimize bias. Randomization was performed using a randomized numbering sequence at the time of injection or treatment. Investigators were not blinded during animal injections or treatments; however, researchers were blinded during data acquisition and quantification. No statistical methods were used to pre-determine sample size. Animal studies were performed once per condition unless otherwise specified in the figure legends. Data were collected from distinct animals, where n represents biologically independent samples. Mice were euthanized when total tumour burden reached 2 cm in diameter or earlier if humane end point criteria (for example, more than 20% weight loss, body condition score of less than 2 or signs of distress) were observed. These institutional limits were not exceeded in any experiment.
Cell lines and culture
Human cell lines used in this study were obtained from the American Type Culture Collection (HCC1806: CRL-2335, from a female patient; MDA-MB-231: HTB-26, from a female patient). Mouse EO771 breast cancer cells were obtained from CH3 BioSystems (94A001, from B6 mice). Cell lines obtained from ATCC and CH3 BioSystems are authenticated by the suppliers; no additional authentication was performed by the authors. Lines were regularly tested for mycoplasma contamination using the MycoAlert PLUS Mycoplasma Detection Kit (LT07-710, Lonza BioSciences). All cells were cultured in a Heracell humidified incubator (Thermo Fisher Scientific) at 37 °C and 5% CO 2 . Cell lines were routinely maintained in RPMI-1640 (10-040-CV, Corning Life Sciences) supplemented with 10% heat-inactivated fetal bovine serum (10437-028, Gibco), and for cell culture experiments, 10% dialysed fetal bovine serum (26400-044, Gibco) was supplemented.
RPMI-1640 medium lacking serine or arginine was made using a previous method61. In brief, enough of all amino acids and sodium phosphate dibasic (S5136, Sigma-Aldrich) of RPMI-1640 media except for serine or arginine were weighed out to make 25 l of media, then the resulting powders were homogenized using an electric blade coffee grinder (80365, Hamilton Beach) that had been washed with methanol then water. The resulting powders were resuspended in water along with sodium bicarbonate (S5761, Sigma-Aldrich) and RPMI-1640 medium without amino acids and sodium phosphate (R8999-04A, US Biological) to make RPMI-1640 medium lacking serine or arginine. Serine or arginine was reconstituted in water to generate stock solutions and added back as needed to reach 286 µM serine or 1.15 mM arginine, reflecting standard RPMI-1640 concentrations.
DMEM medium lacking serine or arginine was prepared using a base powder formulation lacking all amino acids and pyruvate (D9800-13, US Biological). To this base, glucose, sodium pyruvate and sodium bicarbonate were added to match standard DMEM composition. Amino acids were weighed out and homogenized as described above for RPMI, then dissolved into the reconstituted DMEM base. Serine or arginine was added back as needed to reach 400 µM serine or 398 µM arginine, reflecting standard DMEM concentrations.
Isolation of interstitial fluid, CSF and plasma
Tissue interstitial fluid was collected from mouse tissues using an adapted protocol based on previous published work52. Female NSG or female B6 mice on ad libitum diet 6–9 weeks of age were used for all tissue interstitial fluid isolations. Organs from five mice were combined per interstitial fluid sample, and each pooled sample was treated as an individual data point for the liquid chromatography–mass spectrometry (LC–MS) measurements and analysis. All mice were euthanized at the same time of day to account for any effects of time of day on metabolism, and were euthanized by cervical dislocation to minimize metabolic artefacts associated with CO 2 exposure62. Tissues were kept on a chilled metal block on ice throughout the harvest and when ready to pool, were briefly rinsed in ice-cold saline (16005, Azer Scientific), excess liquid removed by blotting on filter paper (28298-020, VWR), then placed in a 50-ml conical vial lined with a 20-μm nylon mesh filter (148134, Spectrum Labs) and 0.25 μl of 0.5 M EDTA, pH 8.0 at the bottom. The tissues were centrifuged at 400g for 10 min at 4 °C. The flow through was collected and centrifuged again at 400g for 10 min at 4 °C before flash freezing and storage in −80 °C until further analysis. Before tissue harvest, plasma from each live mouse was collected by facial cheek bleed into EDTA-coated tubes (41.1395.105, Sarstedt), then centrifuged at 800g for 10 min at 4 °C. Supernatant containing plasma was flash frozen and stored at −80 °C until further analysis. An equal volume of each plasma sample was pooled from each mouse cohort before analysis by LC–MS.
CSF was isolated from the mouse brain as previously described63. In brief, female NSG or female B6 mice were anaesthetized with ketamine (90 mg kg−1) and xylazine (9 mg kg−1) via intramuscular injection, and CSF was collected by gently inserting a sharpened capillary (inner diameter of 0.75 mm and outer diameter of 1.0 mm) in the cisterna magna. Collected fluid was visually monitored for blood contamination. CSF was centrifuged at 800g for 10 min at 4 °C, and the supernatant was flash frozen and stored at −80 °C until further analysis. Following CSF collection, animals were euthanized via cervical dislocation and blood was collected via cardiac puncture and immediately placed in EDTA tubes (41.1395.105, Sarstedt), then centrifuged at 800g for 10 min at 4 °C. Plasma was flash frozen in liquid nitrogen and stored at −80 °C until further analysis.
To evaluate the stability of metabolite levels during sample handling, we performed a time-course experiment in female B6 mice. For plasma, blood was collected via cheek bleed directly into EDTA-coated tubes and either processed immediately as described above or held on ice for 5, 10 or 30 min before processing. For liver interstitial fluid, one liver was collected from each mouse and either processed immediately or placed on a chilled metal block on ice for 5, 10 or 30 min before interstitial fluid isolation using the standard centrifugation protocol described above. All samples were analysed by LC–MS to quantify time-dependent changes in metabolite levels.
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