CAL-120 cells were grown as 2D monolayers, and cell extracts for metabolomics were prepared as described above. Cells were extracted in 400 μl of ice-cold extraction solution. For each of the three independent experiments, cell extracts of three wells per condition were combined, and the protein content was determined in three wells incubated in parallel. All cell extracts were normalized to 2.16 μl of extraction solution per microgram of protein before LC-MS/MS analysis.

CAL-120 cells were seeded in DMEM-F12 or Plasmax (both supplemented with 2.5% FBS) at 4 × 106 cells per 200 ml of culture medium in glass spinner flasks (40 rev/min; cat. no. F7689; Techne; Cole-Parmer Ltd., UK) to allow sphere formation. After 72 hours (day 3), the cell cultures were supplemented with 100 ml of the respective medium to prevent nutrient exhaustion. On day 4, spheres were collected and embedded in paraffin for IHC or used for extraction of metabolites. Spheres were allowed to sediment, and culture medium volume was reduced to 1 ml. After a brief centrifugation step (1 min, 600g), the supernatant was removed, spheres were washed quickly with 1 ml of ice-cold PBS and centrifuged for 10 s, and the pellet was extracted in 1 ml of ice-cold extraction solution (vortexed briefly, and incubated for 5 min at 4°C). After centrifugation (10 min at 16,000g), the supernatant (cell extract) was collected for LC-MS/MS analysis, and the pellet was used to determine the protein content with a modified Lowry protein assay. Before LC-MS/MS analysis, cell extracts were diluted to 2.16 μl of extraction solution per microgram of protein, normalizing the samples to the extracts obtained from the 2D monolayers.

Female severe combined immunodeficient mice (n = 3; 7 to 8 weeks old; Charles River Laboratories) were injected in the mammary fat pad with 4 × 106 CAL-120 cells (cell suspension in 50 μl of Matrigel). Tumors were allowed to establish for 160 days, after which mice were humanely euthanized using schedule 1 methods. Tumors were quickly resected, divided into fragments, and either fixed in 10% neutral-buffered formalin for IHC or flash frozen on dry ice for extraction of metabolites. Frozen tumor fragments (two to four fragments per tumor; nine fragments in total) were weighed and dissociated in cold extraction solution (40 mg/ml) using ceramic beads and a Precellys homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France). After centrifugation (10 min, 16,000g), the supernatant was used for LC-MS/MS analysis.

Samples were analyzed with the same LC/MS setup described above. However, for this experiment, metabolites were separated over a 30-min mobile phase gradient, decreasing the acetonitrile content to 20%, at a flow rate of 100 μl/min. The total analysis time was 40 min, and metabolites were detected using the mass spectrometer at a resolution of 70,000 (at 200 m/z). A pooled sample comprising a mixture of all sample extracts was analyzed using the same HPLC conditions but running the mass spectrometer in single ionization mode to acquire the positive and negative ions in separate runs and using data-dependent fragmentation (ddMS2) to improve the confidence in metabolite identification. Data were analyzed using Compound Discoverer software (Thermo Scientific v2.1). Retention times were aligned across all sample data files (maximum shift, 3 min; mass tolerance, 5 ppm). Unknown compound detection (minimum peak intensity of 3 × 106) and grouping of compound adducts were carried out across all samples (mass tolerance, 5 ppm; retention time tolerance, 0.7 min). Missing values were filled using the software’s Fill Gap feature (mass tolerance, 5 ppm; signal/noise tolerance, 1.5). Compound identification was achieved by matching the mass and retention time of observed peaks to an in-house database generated using metabolite standards (mass tolerance, 5 ppm; RT tolerance, 0.5 min). Peak annotations were further confirmed using mzCloud (ddMS2) database search (precursor and fragment mass tolerance, 10 ppm; match factor threshold, 50). Within Compound Discoverer, data were normalized using a constant median strategy, and PCA was also performed using only features identified by the above strategy (in-house database and mzCloud). The weighted distance of in vitro conditions to tumor samples was calculated using the following equationEmbedded Imagewhere PC1repn is the PC1 value of each in vitro replicate and PC1tummean is the mean PC1 value of all tumor samples.

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