Five patient-derived organotypic cultures from patients with treatment-naïve primary ER⁺/PR⁺/HER2^- tumors while one came from a patient with neoadjuvant therapy for locally metastatic ER^low/PR^-/HER2^- tumor. They all exhibited tissue outgrowth in one month with some CA OTC harboring isolatable organoids and fibroblasts. We interrogated reprogrammed metabolism in CA versus paired NC OTC with dual ²H₇-glucose/¹³C₅,¹⁵N₂-Gln tracers coupled with Stable Isotope-Resolved Metabolomic analysis. The data are provided as Excel spreadsheets for each patient. The entries comprise reduced data from FT-MS analyses in terms of identified compounds, their ion intensities for each isotopologue, injection volumes and derived normalized concentrations. These data can therefore be reanalyzed as desired.

Dual stable isotope tracer treatment

Each patient’s OTC was replaced with a stable isotope tracer medium 2-3 weeks after culturing in BCOM. The tracer medium was composed of 0.68% (w/v) D₇ (²H₇)-glucose + 7.44 mM ¹³C₅,¹⁵N₂-Gln in BCOM and the treatment lasted for 2 (CZ016-017) or 3 days (CZ019-022). At tissue harvest, two small pieces of CZ016-017 OTC were cut for histology and live fluorescence spectroscopy while the rest were metabolically quenched in liquid N₂ before further processing for SIRM analysis. The CZ019-022 OTC were processed similarly for histology and SIRM analysis.

Tissue and medium extraction

Frozen OTC were pulverized into 10 µm particles in liquid N₂ with a SPEX 6775 Freezer/Mill (SPEX SamplePrep, Metuchen, NJ) to maximize metabolite extraction. Polar/non-polar metabolites and proteins were extracted simultaneously using the acetonitrile:H₂O:chloroform (2:1.5:1, v/v) solvent partitioning method as described previously (16). Polar metabolite extracts were split and lyophilized for NMR and IC-UHRFTMS analysis described below. For medium extraction, a 50 µL aliquot was mixed with 200 µl cold acetone (-20°C) (80% acetone final) and kept at -80°C for 20-30 min before centrifugation at 21,100xg for 20 min at 4°C to remove protein precipitates. The extracts were lyophilized before NMR analysis.

Stable Isotope-Resolved Metabolomic (SIRM) analysis

Lyophilized polar extracts were dissolved in  H₂O for anionic ion chromatography coupled with ultra high-resolution Fourier transform mass spectrometric (IC-UHRFTMS) analysis. The D, ¹³C, and/or ¹⁵N labeling patterns (total abundance in µmole/g residue and fractional enrichment) of relevant metabolites were used to reconstruct various catabolic and anabolic pathways.

Isotopologue nomenclature

As three stable isotopes were used, the number of transformed isotopologues was too large and unnecessary to display individually. For example, Asp with four ¹³C, three ¹⁵N, and one D (C₄N₃D) may have up to 5*2*4=40 isotopologues. We thus summed up relevant isotopologues for each metabolite for meaningful interpretation. For example, C3Dx is the sum of isotopologues having three ¹³C atoms and any number of D, whereas C*Dx is the sum of isotopologues with ≥ one ¹³C and any number of D. Total* is the sum of all labeled isotopologues observed. The unlabeled isotopologue is C0N0D0, i.e., no ¹³C, ¹⁵N, or D.