Summary of Study ST002976

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001852. The data can be accessed directly via it's Project DOI: 10.21228/M8B424 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST002976
Study Title	Metabolomics Insights into Doxorubicin and 5-Fluorouracil Combination Therapy in Triple-Negative Breast Cancer: A Xenograft Model Study (Part 2)
Study Type	LC/MS/MS
Study Summary	Background: Breast cancer is one of the most prevalent malignancies and a leading cause of death among women worldwide. Among its subtypes, triple-negative breast cancer (TNBC), which poses significant clinical challenges due to its aggressive behavior and limited treatment options. Aim: This study explored the effects of doxorubicin (DOX) and 5-fluorouracil (5-FU) as monotherapies and in combination on MDA-MB-231 xenograft model. Employing advanced metabolomics analysis, the study was designed to investigate molecular alterations triggered by these treatments. Methods: State-of-the-art metabolomics analysis using Ultra-high-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-MS) was conducted including comprehensive plasma and tumor tissue sample profiling. Results: The study explored alterations induced by DOX, 5-FU, and their combination treatment. Each treatment group exhibited unique metabolic profiles in plasma and tumor analysis. Univariate and enrichment analyses identified alterations in metabolic pathways, including glycine and serine metabolism, spermidine and spermine biosynthesis, and purine and pyrimidine pathways. The combination of DOX and 5-FU significantly influenced plasma and tumor metabolites. The comprehensive metabolic profiling of both plasma and tumor samples shed light on the intricate changes within the tumor microenvironment and their systemic implications. Conclusion: The study findings offer insights into the metabolic vulnerabilities of TNBC in vivo induced by the studied chemotherapeutics. These findings highlight the involved metabolites and metabolic pathways in the response of MDA-MB-231 cells to DOX, 5-FU, and their combination which advance our understanding of TNBC treatment strategies, offering new possibilities for enhancing therapeutic outcomes. This part of study involves comprehensive metabolomic profiling of the tumor tissue samples specifically and tumor growth assessment provide valuable insights into these treatments' efficacy and potential synergistic effects in TNBC.
Institute	Sharjah Institute for Medical Research
Last Name	Facility
First Name	Core
Address	M32, SIMR, College of Pharmacy, Health Sciences, University of Sharjah, Sharjah, UAE, Sharjah, 000, United Arab Emirates
Email	tims-tof@sharjah.ac.ae
Phone	+971 6 5057656
Submit Date	2023-11-08
Raw Data Available	Yes
Raw Data File Type(s)	d
Analysis Type Detail	LC-MS
Release Date	2024-05-08
Release Version	1

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Project:

Project ID:	PR001852
Project DOI:	doi: 10.21228/M8B424
Project Title:	Metabolomics Insights into Doxorubicin and 5-Fluorouracil Combination Therapy in Triple-Negative Breast Cancer: A Xenograft Model Study
Project Type:	LC-MS/MS
Project Summary:	Background: Breast cancer is one of the most prevalent malignancies and a leading cause of death among women worldwide. Among its subtypes, triple-negative breast cancer (TNBC), which poses significant clinical challenges due to its aggressive behavior and limited treatment options. Aim: This study explored the effects of doxorubicin (DOX) and 5-fluorouracil (5-FU) as monotherapies and in combination on MDA-MB-231 xenograft model. Employing advanced metabolomics analysis, the study was designed to investigate molecular alterations triggered by these treatments. Methods: State-of-the-art metabolomics analysis using Ultra-high-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-MS) was conducted including comprehensive plasma and tumor tissue sample profiling. Results: The study explored alterations induced by DOX, 5-FU, and their combination treatment. Each treatment group exhibited unique metabolic profiles in plasma and tumor analysis. Univariate and enrichment analyses identified alterations in metabolic pathways, including glycine and serine metabolism, spermidine and spermine biosynthesis, and purine and pyrimidine pathways. The combination of DOX and 5-FU significantly influenced plasma and tumor metabolites. The comprehensive metabolic profiling of both plasma and tumor samples shed light on the intricate changes within the tumor microenvironment and their systemic implications. Conclusion: The study findings offer insights into the metabolic vulnerabilities of TNBC in vivo induced by the studied chemotherapeutics. These findings highlight the involved metabolites and metabolic pathways in the response of MDA-MB-231 cells to DOX, 5-FU, and their combination which advance our understanding of TNBC treatment strategies, offering new possibilities for enhancing therapeutic outcomes.
Institute:	Sharjah Institute for Medical Research
Last Name:	Facility
First Name:	Core
Address:	M32, SIMR, College of Pharmacy, Health Sciences, University of Sharjah, Sharjah, UAE, Sharjah, 000, United Arab Emirates
Email:	tims-tof@sharjah.ac.ae
Phone:	+971 6 5057656

Subject:

Subject ID:	SU003089
Subject Type:	Mammal
Subject Species:	BALB/C nude mice

Factors:

Subject type: Mammal; Subject species: BALB/C nude mice (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment
SA323219	2B1-01-10481	5-fluorouracil
SA323220	2A2-02-10480	5-fluorouracil
SA323221	2A2-01-10479	5-fluorouracil
SA323222	2B1-02-10482	5-fluorouracil
SA323223	2B2-02-10484	5-fluorouracil
SA323224	2B3-02-10486	5-fluorouracil
SA323225	2B3-01-10485	5-fluorouracil
SA323226	2A1-01-10477	5-fluorouracil
SA323227	2B2-01-10483	5-fluorouracil
SA323228	2A1-02-10478	5-fluorouracil
SA323229	1A2-02-10467	Doxorubicin
SA323230	1A3-01-10468	Doxorubicin
SA323231	1A2-01-10466	Doxorubicin
SA323232	1A1-01-10464	Doxorubicin
SA323233	1A1-02-10465	Doxorubicin
SA323234	1B3-02-10475	Doxorubicin
SA323235	1A3-02-10469	Doxorubicin
SA323236	1B3-01-10474	Doxorubicin
SA323237	1B1-01-10470	Doxorubicin
SA323238	1B2-02-10473	Doxorubicin
SA323239	1B1-02-10471	Doxorubicin
SA323240	1B2-01-10472	Doxorubicin
SA323241	3B2-02-10495	Doxorubicin + 5-fluorouracil
SA323242	3B3-01-10496	Doxorubicin + 5-fluorouracil
SA323243	3B2-01-10494	Doxorubicin + 5-fluorouracil
SA323244	3B3-02-10497	Doxorubicin + 5-fluorouracil
SA323245	3A1-01-10488	Doxorubicin + 5-fluorouracil
SA323246	3B1-02-10493	Doxorubicin + 5-fluorouracil
SA323247	3A2-01-10490	Doxorubicin + 5-fluorouracil
SA323248	3A1-02-10489	Doxorubicin + 5-fluorouracil
SA323249	3A2-02-10491	Doxorubicin + 5-fluorouracil
SA323250	3B1-01-10492	Doxorubicin + 5-fluorouracil
SA323251	4B2-01-10507	Positive Control
SA323252	4B2-02-10508	Positive Control
SA323253	4B3-01-10509	Positive Control
SA323254	4B1-02-10506	Positive Control
SA323255	4B3-02-10510	Positive Control
SA323256	4A4-01-10503	Positive Control
SA323257	4A1-02-10500	Positive Control
SA323258	4A1-01-10499	Positive Control
SA323259	4A2-01-10501	Positive Control
SA323260	4A2-02-10502	Positive Control
SA323261	4A4-02-10504	Positive Control
SA323262	4B1-01-10505	Positive Control

Showing results 1 to 44 of 44

Showing results 1 to 44 of 44

Collection:

Collection ID:	CO003082
Collection Summary:	MDA-MB-231 cell line was cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Sigma Aldrich, St. Louis, MO, USA) at 37 °C in a humidified atmosphere containing 5% CO2. This study was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Sharjah (Ethical Approval Number: ACUC-06-08-2022). Standard ethical guidelines were followed in all animal procedures conducted. Thirty female BALB/C nude mice (8-10 weeks age), with an average weight of 26-27 g, were included in the study and maintained in our institutional animal facility in accordance with following the animal care guidelines. The mice were randomized into five experimental groups (6 per group). Twenty-four mice were selected for the (CDX) model, and one group served as the negative control that didn’t receive any treatment. MDA-MB-231 cells (2 × 106 in 50 μL PBS & 50 μL Matrigel) were injected subcutaneously into the neck region of the mice. The mice were allowed to reach a tumor volume of 150-200 mm3 before further treatment. Then, tumor-bearing mice were randomized into the following groups: untreated xenografts (positive controls) and three treatment groups, DOX, 5-FU, and a combination of DOX and 5-FU. The mice in the DOX group were administered 1mg/kg of DOX once weekly , and the mice in the 5-FU group received 50 mg/kg of 5-FU daily for five consecutive days both treatments administered via an intraperitoneal (i.p.) route. The mice in the combination therapy group received DOX & 5-FU as separate injections, and the treatment duration was two weeks. An overview of the experiment flow. The body weight of the mice was measured at the start of the study and twice weekly from the beginning of the treatments. Tumor growth and progression were monitored by palpation and measurement of tumor size periodically using a digital vernier caliper. The tumor volume in cm3 was determined using the formula (volume = π/6 × length × width2). All mice were anesthetized and euthanized via cardiac puncture at the end of the treatment period. Tumors were excised and weighed, and their sizes were measured. Also, blood serum was collected from each mouse. Both tumor and serum samples were stored at -80°C for further analysis.
Sample Type:	Blood (plasma)

Treatment:

Treatment ID:	TR003098
Treatment Summary:	Then, tumor-bearing mice were randomized into the following groups: untreated xenografts (positive controls) and three treatment groups, DOX, 5-FU, and a combination of DOX and 5-FU. The mice in the DOX group were administered 1mg/kg of DOX once weekly , and the mice in the 5-FU group received 50 mg/kg of 5-FU daily for five consecutive days both treatments administered via an intraperitoneal (i.p.) route [32]. The mice in the combination therapy group received DOX & 5-FU as separate injections, and the treatment duration was two weeks. An overview of the experiment flow. The body weight of the mice was measured at the start of the study and twice weekly from the beginning of the treatments. Tumor growth and progression were monitored by palpation and measurement of tumor size periodically using a digital vernier caliper. The tumor volume in cm3 was determined using the formula (volume = π/6 × length × width2) [33]. All mice were anesthetized and euthanized via cardiac puncture at the end of the treatment period. Tumors were excised and weighed, and their sizes were measured. Also, blood serum was collected from each mouse. Both tumor and serum samples were stored at -80°C for further analysis.

Treatment ID:

TR003098

Treatment Summary:

Then, tumor-bearing mice were randomized into the following groups: untreated xenografts (positive controls) and three treatment groups, DOX, 5-FU, and a combination of DOX and 5-FU. The mice in the DOX group were administered 1mg/kg of DOX once weekly , and the mice in the 5-FU group received 50 mg/kg of 5-FU daily for five consecutive days both treatments administered via an intraperitoneal (i.p.) route [32]. The mice in the combination therapy group received DOX & 5-FU as separate injections, and the treatment duration was two weeks. An overview of the experiment flow. The body weight of the mice was measured at the start of the study and twice weekly from the beginning of the treatments. Tumor growth and progression were monitored by palpation and measurement of tumor size periodically using a digital vernier caliper. The tumor volume in cm3 was determined using the formula (volume = π/6 × length × width2) [33]. All mice were anesthetized and euthanized via cardiac puncture at the end of the treatment period. Tumors were excised and weighed, and their sizes were measured. Also, blood serum was collected from each mouse. Both tumor and serum samples were stored at -80°C for further analysis.

Sample Preparation:

Sampleprep ID:	SP003095
Sampleprep Summary:	Each collected serum sample (100 µL) was mixed with 300 µL of methanol (≥99.9 %, LC-MS CHROMASOLV) in Eppendorf tubes, followed by vortex and incubation at -20°C for 2 hours. After vortex and centrifugation at 14,000 rpm for 15 minutes, the supernatant was evaporated at 35-40°C using speed vacuum evaporation (EZ-2 Plus (GeneVac, Ipswich, UK). The extracted samples were resuspended in 200 µL of 0.1% formic acid in Deionized Water-LC-MS CHROMASOLV. Subsequently, the supernatant was filtered through a 0.45 µm pore size hydrophilic nylon syringe filter for LC-MS/MS analysis and collected in inserts within LC glass vials. All samples were placed in the autosampler at the temperature set at 4℃ to proceed with the analysis by Q-TOF MS. A pooled QC sample was created to ensure analysis reproducibility by mixing 10 µL from each sample.

Sampleprep ID:

SP003095

Sampleprep Summary:

Each collected serum sample (100 µL) was mixed with 300 µL of methanol (≥99.9 %, LC-MS CHROMASOLV) in Eppendorf tubes, followed by vortex and incubation at -20°C for 2 hours. After vortex and centrifugation at 14,000 rpm for 15 minutes, the supernatant was evaporated at 35-40°C using speed vacuum evaporation (EZ-2 Plus (GeneVac, Ipswich, UK). The extracted samples were resuspended in 200 µL of 0.1% formic acid in Deionized Water-LC-MS CHROMASOLV. Subsequently, the supernatant was filtered through a 0.45 µm pore size hydrophilic nylon syringe filter for LC-MS/MS analysis and collected in inserts within LC glass vials. All samples were placed in the autosampler at the temperature set at 4℃ to proceed with the analysis by Q-TOF MS. A pooled QC sample was created to ensure analysis reproducibility by mixing 10 µL from each sample.

Combined analysis:

Analysis ID	AN004886
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Bruker Elute
Column	Hamilton Intensity Solo 2 C18 (100 x 2.1mm, 1.8um)
MS Type	ESI
MS instrument type	QTOF
MS instrument name	Bruker timsTOF
Ion Mode	POSITIVE
Units	AU

Chromatography:

Chromatography ID:	CH003687
Chromatography Summary:	Mobile phases A (water with 0.1% formic acid) and B (acetonitrile with 0.1% formic acid) were used with the following gradient elusion mode: 0 to 2 min, 1% B; 2 to 17 min, 1–99% B; 17 to 20 min, 99% B; 20 to 20.1 min, 99–1% B; 20.1 to 30 min, 1% B. The flow rate was 0.25 mL/min from 0 to 20 min, 0.35 mL/min from 20 min to 28.3 min, and 0.25 mL/min from 28.3 to 30 min. The sample injection volume was 10 μl, and separation occurred on a Hamilton® Intensity Solo 2 C18 column (2.1 × 100 mm, 1.8 µm) (Bruker Daltonik) at 35°C.
Instrument Name:	Bruker Elute
Column Name:	Hamilton Intensity Solo 2 C18 (100 x 2.1mm, 1.8um)
Column Temperature:	35
Flow Gradient:	gradient elusion mode: 0 to 2 min, 1% B; 2 to 17 min, 1–99% B; 17 to 20 min, 99% B; 20 to 20.1 min, 99–1% B; 20.1 to 30 min, 1% B.
Flow Rate:	0.25 mL/min from 0 to 20 min, 0.35 mL/min from 20 min to 28.3 min, and 0.25 mL/min from 28.3 to 30 min.
Solvent A:	Water (0.1% Formic Acid)
Solvent B:	ACN (0.1% Formic Acid)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS004630
Analysis ID:	AN004886
Instrument Name:	Bruker timsTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	The ESI source conditions were set with a capillary voltage of 4500 V, drying gas flow rate of 10.0 l/min at 220°C, nebulizer pressure of 2.2 bar, and the End Plate offset at 500 V. Sodium formate (10 mM) was injected at the beginning of each sample run and used as a calibrant for internal calibration during data processing. The MS acquisition process consisted of two phases. First, an auto MS scan lasting from 0 to 0.3 minutes was utilized for calibrating sodium formate. The second phase encompassed auto MS/MS scanning with CID acquisition, including fragmentation, which extended from 0.3 to 30 minutes. Both acquisition phases were conducted in positive mode at a rate of 12 Hz. The automatic mass scan range within each run spanned from 50 to 1300 m/z, with a precursor ion width of ±0.5, a cycle time of 0.5 seconds, and a threshold of 400 counts. Active exclusion was initiated after three spectra and lifted after 0.2 minutes. For MS2 acquisition, a data-dependent acquisition (DDA) approach was employed, with collision energy settings varying between 100% and 250% and being set at 20 eV. TRX-2101/RT-28-calibrants from Nova Medical Testing Inc. for the Bruker T-ReX LC-QTOF were injected before sample analysis to assess the column's performance, reversed-phase liquid chromatography (RPLC) separation, multipoint retention time calibration, and the mass spectrometer. Additionally, TRX-3112-R/MS Certified Human serum solution for Bruker T-ReX LC-QTOF (provided by Nova Medical Testing Inc.) was prepared from pooled human blood and administered before sample analysis to validate the performance of the LC-MS instruments. The analysis followed a randomized sequence order, commencing with five injections of solvent A (0.1% formic acid in deionized water) to facilitate apparatus equilibration. Subsequently, five injections of the pooled QC sample were carried out. Furthermore, one QC injection was conducted every (9-10 samples) to assess the consistency of the analysis.
Ion Mode:	POSITIVE