Summary of Study ST002010

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 PR001274. The data can be accessed directly via it's Project DOI: 10.21228/M81Q4Z This work is supported by NIH grant, U2C- DK119886.

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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.

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Study IDST002010
Study TitleChemoresistant Ovarian Cancer Global Metabolomics
Study SummaryChemoresistance remains the major barrier to effective ovarian cancer treatment. The molecular features and associated biological functions of this phenotype remain poorly understood. We developed carboplatin resistant cell line models using OVCAR5 and CaOV3 cell lines with the aim of identifying chemoresistance-specific molecular features. Mass spectrometry analysis was used to analyse the metabolome of these cell lines and was able to separate these populations based on their molecular features. It revealed signaling and metabolic perturbations in chemoresistant cell lines. A comprehensive analysis of a larger patient cohort, including advanced in vitro and in vivo models, promises to help better understand the molecular mechanisms of chemo-resistance and associated enhancement of migration and invasion.
Institute
University of South Australia
Last NameAcland
First NameMitchell
AddressCnr North Terrace and Morphett Street, Adelaide SA 5000
Emailmitch.acland@gmail.com
Phone0425460869
Submit Date2021-12-05
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2021-12-22
Release Version1
Mitchell Acland Mitchell Acland
https://dx.doi.org/10.21228/M81Q4Z
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001274
Project DOI:doi: 10.21228/M81Q4Z
Project Title:Chemoresistant Ovarian Cancer Global Metabolomics
Project Summary:Chemoresistance remains the major barrier to effective ovarian cancer treatment. The molecular features and associated biological functions of this phenotype remain poorly understood. We developed carboplatin resistant cell line models using OVCAR5 and CaOV3 cell lines with the aim of identifying chemoresistance-specific molecular features. Mass spectrometry analysis was used to analyse the metabolome of these cell lines and was able to separate these populations based on their molecular features. It revealed signaling and metabolic perturbations in chemoresistant cell lines. A comprehensive analysis of a larger patient cohort, including advanced in vitro and in vivo models, promises to help better understand the molecular mechanisms of chemo-resistance and associated enhancement of migration and invasion.
Institute:University of South Australia
Last Name:Acland
First Name:Mitchell
Address:Cnr North Terrace and Morphett Street, Adelaide SA 5000
Email:mitch.acland@gmail.com
Phone:0425460869

Subject:

Subject ID:SU002091
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Chemoresistance Status
SA188294QC_ov_2N/A
SA188295DMEM_IST_2N/A
SA188296Blank_1N/A
SA188297QC_ov_3N/A
SA188298RPMI_IST_1N/A
SA188299Blank_2N/A
SA188300QC_ov_1N/A
SA188301RPMI_IST_2N/A
SA188302Blank_3N/A
SA188303DMEM_IST_1N/A
SA188304CaOV3_CBPR_3Resistant
SA188305OVCAR5_CBPR_2Resistant
SA188306OVCAR5_CBPR_3Resistant
SA188307CaOV3_CBPR_1Resistant
SA188308CaOV3_CBPR_2Resistant
SA188309OVCAR5_CBPR_1Resistant
SA188310CaOV3_Parent_3Sensitive
SA188311OVCAR5_Parent_1Sensitive
SA188312OVCAR5_Parent_2Sensitive
SA188313OVCAR5_Parent_3Sensitive
SA188314CaOV3_Parent_1Sensitive
SA188315CaOV3_Parent_2Sensitive
Showing results 1 to 22 of 22

Collection:

Collection ID:CO002084
Collection Summary:Two ovarian cancer cell lines were used to generate carboplatin resistant pairs. These were then grown in parallel before untargeted MS metabolomics analysis was applied in the pursuit of characterising metabolome mediated resistance mechanism
Sample Type:Cultured cells

Treatment:

Treatment ID:TR002103
Treatment Summary:The human OC cell line: CaOV3, was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and the OVCAR-5 cell line obtained from Dr. Thomas Hamilton (Fox Chase Cancer Centre, Philadelphia, PA). Both cell lines were authenticated by short tandem repeat (STR) DNA profile in 2020. OVCAR-5 cells were grown in RPMI 1640 media (Sigma Aldrich, St. Louis, MO, USA). Recent reports indi-cate that OVAR-5 might originate from metastatic gastrointestinal cancer and were potentially wrong fully labelled as Ovarian cancer [25]. CaOV3 cells were grown in DMEM media (Sigma Aldrich, St. Louis, MO, USA). Both were cultured with the addi-tion of 10% foetal bovine serum (Bovogen Biologicals, East Keilor, VIC, AUS) supple-mented with 1% penicillin/streptomycin (Sigma Aldrich, St. Louis, MO, USA) and 1% L-glutamine (Sigma Aldrich, St. Louis, MO, USA). OVCAR-5 and CaOV3 cells were made resistant to CBP after treatment with 6-8 cycles of CBP (50μM, Hospira Austral-ia, Pty Ltd) [26,27]. Resistance to CBP was measured regularly and CBPR cell lines were seen to be at least two-fold more resistant to CBP than their parental partners through the following experiments.
Treatment Compound:Carboplatin

Sample Preparation:

Sampleprep ID:SP002097
Sampleprep Summary:Cells were maintained at 60-80% confluence for 3 passages before being plated in 10cm dishes. Cell numbers were estimated from an additional dish with the same number of cells at seeding. Media was aspirated and cells were washed three times with 3mL warmed PBS. Metabolic arrest was achieved through the addition of ap-proximately 2 mL of liquid nitrogen directly to cells ensuring that the surface of the plate was covered before plates were placed onto dry ice. Metabolite extraction was achieved through the addition of 1mL 100% ice cold methanol. Cells were lifted off of the plate using an ice cold cell scraper and transferred to a 2mL Eppendorf. An addi-tional 1mL of 100% ice cold methanol was added before snap freezing by immersion in liquid nitrogen for 3 minutes. This was followed by thawing on dry ice and vortexing to resuspend contents. Freeze/thaw process was repeated 5 times to ensure full extrac-tion of metabolites. Samples were centrifuged at 16000g at -9⁰C for 5 minutes and the supernatant was retained. The pellet was resuspended in 500uL of 100% ice cold methanol and freeze/thaw in liquid nitrogen was repeated 5 times. This sample was centrifuged at 16000g at -9⁰C for 5 minutes and the supernatant was retained and combined with previously retained supernatant. The samples were then dried in a SpeedVac Vacuum Concentrator (John Morris Scientific, RVC 2-18) at room tempera-ture, with vacuum of 40mbar and rotor speed of 1000min-1. Before data acquisition samples were resuspended in volumes of 20mM ammonium carbonate and acetonitrile to achieve identical concentration of biological material based on cell number estimate.

Combined analysis:

Analysis ID AN003276
Analysis type MS
Chromatography type HILIC
Chromatography system Thermo Dionex Ultimate 3000 RS
Column SeQuant ZIC-HILIC (150 x 4.6mm, 5um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Orbitrap
Ion Mode POSITIVE
Units Intensity

Chromatography:

Chromatography ID:CH002418
Chromatography Summary:LCMS data was acquired on Q-Exactive Orbitrap mass spectrometer (Thermo Fisher) coupled with high-performance liquid chromatography (HPLC) system Dionex Ulti-mate® 3000 RS (Thermo Fisher). Chromatographic separation was performed on a ZIC-pHILIC column (5 µm, polymeric, 150 × 4.6 mm, SeQuant®, Merck). The mobile phase (A) was 20 mM ammonium carbonate and (B) acetonitrile. The gradient pro-gram started at 80% B and was reduced to 50% B over 15 min, then reduced from 50% B to 5% B over 3 min, followed by wash with 5% B for another 3 min, and finally 8 min re-equilibration with 80% B. The flow rate was 0.3 mL/min and column compartment temperature was 25 ºC. The total run time was 32 min with an injection sample vol-ume of 10 µL. Mass spectrometer operated in full scan mode with positive and nega-tive polarity switching at 35000 resolution at 200 m/z with detection range of 85 to 1, 275 m/z in full scan mode. Electro-spray ionization source (HESI) was set to 3.5 kV voltage for positive mode and 4.0 kV for negative mode, sheath gas was set to 50 and aux gas to 20 arbitrary units, capillary temperature 300 °C, probe heater temperature 120 °C.
Instrument Name:Thermo Dionex Ultimate 3000 RS
Column Name:SeQuant ZIC-HILIC (150 x 4.6mm, 5um)
Chromatography Type:HILIC

MS:

MS ID:MS003048
Analysis ID:AN003276
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Mass spectrometer operated in full scan mode with positive and negative polarity switching at 35000 resolution at 200 m/z with detection range of 85 to 1, 275 m/z in full scan mode. Electro-spray ionization source (HESI) was set to 3.5 kV voltage for posi-tive mode and 4.0 kV for negative mode, sheath gas was set to 50 and aux gas to 20 ar-bitrary units, capillary temperature 300 °C, probe heater temperature 120 °C. Mixtures of pure authentic standards containing over 320 metabolites were acquired as separate injections and used to confirm retention times. Metabolites confirmed with authentic standards were given the highest confidence MSI level 1. The acquired LCMS data was processed in untargeted fashion using the open-source software IDEOM [30,31]. Default IDEOM parameters were used to elimi-nate unwanted noise and artefact peaks. Putative identification of metabolites was achieved by accurate mass within 3 ppm mass error searching against the Kyoto Ency-clopedia of Genes and Genomes (KEGG), MetaCyc, and LIPIDMAPS databases and others. Despite the washing steps performed in sample preparation it is expected
Ion Mode:POSITIVE
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