DOI: 10.5061/dryad.4qrfj6qnm
title: Patient-derived organotypic tissue cultures as a platform to evaluate metabolic reprogramming in breast cancer patients

description: 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 ERlow/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.
description: <strong>Dual stable isotope tracer
 treatment</strong> 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)
 D<sub>7</sub>
 (<sup>2</sup>H<sub>7</sub>)-glucose + 7.44 mM
 <sup>13</sup>C<sub>5</sub>,<sup>15</sup>N<sub>2</sub>-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 N<sub>2</sub> before further processing for SIRM analysis. The CZ019-022 OTC were processed similarly for histology and SIRM analysis. <strong>Tissue and medium extraction</strong><em> </em> Frozen OTC were pulverized into 10 µm particles in liquid N<sub>2</sub> 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:H<sub>2</sub>O: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. <strong>S</strong><strong>table Isotope-Resolved Metabolomic (SIRM) analysis</strong> Lyophilized polar extracts were dissolved in  H<sub>2</sub>O for anionic ion chromatography coupled with ultra high-resolution Fourier transform mass spectrometric (IC-UHRFTMS) analysis. The D, <sup>13</sup>C, and/or <sup>15</sup>N labeling patterns (total abundance in µmole/g residue and fractional enrichment) of relevant metabolites were used to reconstruct various catabolic and anabolic pathways. <strong><em>Isotopologue nomenclature</em></strong> As three stable isotopes were used, the number of transformed isotopologues was too large and unnecessary to display individually. For example, Asp with four <sup>13</sup>C, three <sup>15</sup>N, and one D (C<sub>4</sub>N<sub>3</sub>D) 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 <sup>13</sup>C atoms and any number of D, whereas C*Dx is the sum of isotopologues with ≥ one <sup>13</sup>C and any number of D. Total* is the sum of all labeled isotopologues observed. The unlabeled isotopologue is C0N0D0, i.e., no <sup>13</sup>C, <sup>15</sup>N, or D. 
description: # Patient-derived organotypic tissue cultures as a platform to evaluate
 metabolic reprogramming in breast cancer patients Dataset DOI:
 [10.5061/dryad.4qrfj6qnm](10.5061/dryad.4qrfj6qnm) ## Description of the
 data and file structure 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
 ERlow/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. ### Files and variables
 #### **1. File: CZ16\_17.xlsx.zip** **Description:** Reduced FT MS data
 for OTCs from patients 16 & 17 #### **2. File: CZ19.xlsx.zip**
 **Description:** Reduced FT MS data for OTCs from patient 19 #### **3.
 File: CZ20.xlsx.zip** **Description:** Reduced FT MS data for OTCs from
 patient 20 #### **4. File: CZ21.xlsx.zip** **Description:** Reduced FT MS
 data for OTCs from patient 21 #### **5. File: CZ22.xlsx.zip**
 **Description:** Reduced FT MS data for OTCs from patient 22 &gt;
 **Note:** All files have the same column structure. --- ### **Column
 Definitions (in .xlsx files)** * **Inf = infinity:** divide by zero in the
 normalization * **NA = not available** | Column | Name | Description | |
 :----- | :-------------------------------------------------- |
 :-------------------------------------------------------------------- | |
 A | Compound | Compound name | | B | Mol\_Formula | Molecular formula | |
 C | C\_isomers | Number of 13C atoms present | | D | N\_isomers | Number
 of 15N atoms | | E | H\_isomers | Number of 2H (D) atoms | | F | CND |
 Isotopologue descriptor (summary of columns C–E) | | G | SamplID | File
 name containing tissue type | | H | Intensity | Ion intensity: Raw peak
 area as ion counts | | I | Original.Intensity | Original ion intensity.
 Same as column H | | J | Corrected.Intensity | Corrected ion intensity
 from column H | | K | Flags | Ignore: Comment on whether ion was detected
 (not needed for analysis) | | L | Peak-Index | Peak number in mass
 spectrum | | M | Quantified\_uM\_ratio | Quantification in µM | | N |
 Quantified\_uM\_sequence\_ratio | Sequenced quantification in µM | | O |
 reconstitution\_volume\_uL | Volume of reconstituted sample (µL) | | P |
 injection\_volume\_uL | Volume injected (µL) | | Q | protein\_mg | Protein
 quantity in sample extracted (mg) | | R | icms\_split\_ratio | Fraction of
 total sample analyzed by IC-FTMS | | S |
 Amount\_ProteinAdj\_uMol\_g\_protein\_RatioBased | Analyte amount
 normalized to protein (µmol/g), ratio-based | | T |
 Amount\_ProteinAdj\_uMol\_g\_protein\_SequenceBased | Analyte amount
 normalized to protein (µmol/g), sequence-based | | U |
 CommentQuantification | Comments | | V | Fraction | Mole fraction of ion |
 | W | Normalized\_intensity | Corrected peak intensity normalized to
 protein | ## Code/software Microsoft Excel: Apart from natural abundance
 corrections, all calculations can be performed within Excel. ## Access
 information Data was derived from the following sources: IC-UHR-FTMS was
 performed following published procedure. Briefly, metabolites were
 separated on a Thermo Scientific Dionex™ IonPac AG11-HC analytical column
 paired with a Dionex IonPac AS11-HC guard column in a Dionex ICS-5000+ DP
 Ion Chromatography system. Eluted metabolites were detected by a Thermo
 Fisher Orbitrap Fusion™ Tribrid™ mass spectrometer (Waltham, MA, USA) run
 in both negative MS1 and MS/MS (Data-Independent Analysis or DIA) modes.
 The MS1 scan was acquired with a resolving power of 500,000 at *m/z* 200
 and an *m/z* range of 80 to 800, while the DIA scan was obtained with a
 resolution of 60,000 and a precursor *m/z* range of 280–440 with a wide
 isolation window of 200 m/z. TraceFinder™ software and an in-house
 database were used for data analysis. MS1 peak areas of assigned
 metabolites and isotopologues were corrected for natural abundance and
 quantified against a calibration standard mixture. Metabolite amounts thus
 obtained were normalized by dry residue weight. ## Human subjects data
 Ethics Approval and Consent to Participate All patients were consented
 prior to surgery for their tissue collection under the approved protocol
 of the University of Kentucky Institutional Review Board (IRB#44224, Total
 Cancer Care). The studies in this work abide by the Declaration of
 Helsinki principles.