Fully relaxed 1 H MR spectra of the water-soluble and lipid extracts were acquired on a Bruker Avance 500 nuclear magnetic resonance spectrometer operating at 11.7 T using a 5-mm HX inverse probe as previously described. Lipid cell extracts were dissolved in 0.6 ml of CDCl 3 /CD 3 OD (2:1, vol/vol) containing tetramethylsilane (TMS) as an internal concentration and chemical shift standard (CDCl 3 and CD 3 OD were premixed with TMS by the manufacturer Cambridge Isotope Laboratories, Inc, Andover, MA). The lyophilized water-soluble cell extracts were dissolved in D 2 O containing the concentration and chemical shift reference 3-(trimethylsilyl) propionic-2, 2, 3, 3-d 4 acid (TSP Sigma-Aldrich). Treated cancer cells were washed, centrifuged, and separated into lipid and water-soluble extracts using a dual-phase extraction method based on methanol/chloroform/water (1:1:1, vol/vol/vol) as previously described. Cells under control or experimental conditions were incubated with regular cell culture medium containing all supplements as described above. As a third condition, hypoxia was mimicked by treating cells for 24 hours with 200 μ M CoCl 2. Approximately 10 MDA-MB-231-HRE-tdTomato cells were exposed for 24 hours to standard normoxic conditions in a regular incubator (control) or to hypoxic conditions in a hypoxia cell culture chamber containing P O 2 less than 1% (Biospherix, Lacona, NY). The necrotic region was also determined by edge detection of the necrosis boundaries in the H&E-stained images. The tumor area was determined by edge detection of the tumor boundaries in three-dimensional MRI RARE images. In both of these two quantification methods, the hypoxic region was determined by set- ting a threshold of the highest 5% of the area under the histogram in the fluorescence images.
The average metabolite concentrations in normoxic, hypoxic, and necrotic regions were calculated by averaging all voxel values of a given metabolite within each of these three regions. To select this threshold, the histogram of a given metabolite image was calculated, and the threshold was automatically set to include the highest 5% of the area under the histogram. This quantification method depends on the threshold of signal intensity, which was set to capture tumor regions of the highest metabolite intensities.
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