Summary of Study ST002104

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 PR001333. The data can be accessed directly via it's Project DOI: 10.21228/M8DM7G 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 IDST002104
Study TitleChemoresistant Cancer Cell Lines are Characterized by Migratory, Amino Acid Metabolism, Protein Catabolism and IFN1 Signalling Perturbations
Study TypeBiomedical research
Study SummaryOur analysis was able to separate chemoresistant cells from their parental cells based on their metabolomic and proteomic features and identified altered biological processes and pathways which are of further interest. Preliminary investigation of patient-derived cells highlighted the need to perform broad biological and molecular analyses, compre-hensive in vitro and in vivo studies, using a larger patient cohort to achieve a deeper and clinically relevant characterization of the molecular drivers of chemoresistance.
Institute
Future Industries Institute
LaboratoryManuela Klingler-Hoffmann
Last NameAcland
First NameMitchell
AddressX Building, Mawson Lakes South Australia 5095
Emailmitch.acland@gmail.com
Phone0448671141
Submit Date2022-02-06
Num Groups4
Total Subjects12
Num MalesNA
Num FemalesNA
Study CommentsOVCAR5 and CaOV3 cell lines and their carboplatin resistant cell lines
PublicationsChemoresistant Cancer Cell Lines are Characterized by Migra-tory, Amino Acid Metabolism, Protein Catabolism and IFN1 Signalling Perturbations
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2022-03-29
Release Version1
Mitchell Acland Mitchell Acland
https://dx.doi.org/10.21228/M8DM7G
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001333
Project DOI:doi: 10.21228/M8DM7G
Project Title:Chemoresistant Cancer Cell Lines are Characterized by Migratory, Amino Acid Metabolism, Protein Catabolism and IFN1 Signalling Perturbations
Project Type:untargeted LCMS metabolomics of ovarian cell lines OVCAR5 and CaOV3 and their respective carboplatin resistant GO lines
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. Chemotaxis and CAM invasion assays revealed enhanced migratory and invasive potential in OVCAR5 resistant, compared to parental cells lines. Mass spectrometry analysis was used to analyse the proteome and metabolome of these cell lines and was able to separate these populations based on their molecular features. It revealed signalling and metabolic perturbations in chemoresistant cell lines. Comparison with the proteome of patient derived primary ovarian cancer cells grown in culture showed a shared dysregulation of cytokine and type 1 interferon signalling, potentially revealing a common molecular feature of chemoresistance. 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 chemoresistance and associated enhancement of migration and invasion.
Institute:Future Industries Institute
Laboratory:Manuela Klingler-Hoffmann
Last Name:Acland
First Name:Mitchell
Address:X Building, Mawson Lakes South Australia 5095
Email:mitch.acland@gmail.com
Phone:48671141
Publications:Chemoresistant Cancer Cell Lines are Characterized by Migratory, Amino Acid Metabolism, Protein Catabolism and IFN1 Signalling Perturbations
Contributors:Mitchell Acland, Noor A. Lokman, Clifford Young, Dovile Anderson, Mark Condina, Chris Desire, Tannith M. Noye, Wanqi Wang, Carmela Ricciardelli, Darren J. Creek, Martin K. Oehler, Peter Hoffmann and Manuela Klingler-Hoffmann

Subject:

Subject ID:SU002189
Subject Type:Cultured cells
Subject Species:Homo sapiens
Genotype Strain:OVCAR-5 and CaOV3 cell lines
Gender:Not applicable
Cell Biosource Or Supplier:CaOV3 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). OVCAR-5 cell line obtained from Dr. Thomas Hamilton (Fox Chase Cancer Centre, Philadelphia, PA).

Factors:

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

mb_sample_id local_sample_id conditions
SA201528CaOV3_Parent_2CaOV3 cell line
SA201529CaOV3_Parent_3CaOV3 cell line
SA201530CaOV3_Parent_1CaOV3 cell line
SA201531CaOV3_CBPR_3CaOV3 cell line CBPR mutation
SA201532CaOV3_CBPR_1CaOV3 cell line CBPR mutation
SA201533CaOV3_CBPR_2CaOV3 cell line CBPR mutation
SA201543DMEM_IST_2culture media for CaOV3
SA201544DMEM_IST_1culture media for CaOV3
SA201545RPMI_IST_1culture media for OVCAR5
SA201546RPMI_IST_2culture media for OVCAR5
SA201534Blank_1Extraction blank
SA201535Blank_2Extraction blank
SA201536Blank_3Extraction blank
SA201540OVCAR5_Parent_3OVCAR5_cell line
SA201541OVCAR5_Parent_2OVCAR5_cell line
SA201542OVCAR5_Parent_1OVCAR5_cell line
SA201537OVCAR5_CBPR_2OVCAR5 cell line CBPR mutation
SA201538OVCAR5_CBPR_3OVCAR5 cell line CBPR mutation
SA201539OVCAR5_CBPR_1OVCAR5 cell line CBPR mutation
SA201547QC_ov_3pooled QC
SA201548QC_ov_2pooled QC
SA201549QC_ov_1pooled QC
Showing results 1 to 22 of 22

Collection:

Collection ID:CO002182
Collection 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 approximately 2 mL of liquid nitrogen directly to cells ensuring that the surface of the plate was covered before plates were placed onto dry ice.
Sample Type:Cultured cells
Storage Conditions:-80℃

Treatment:

Treatment ID:TR002201
Treatment Summary:OVCAR-5 and CaOV3 cells were made resistant to carboplatinumv (CBP) after treatment with 6-8 cycles of CBP (50μM, Hospira Australia, Pty Ltd). 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:50μM carboplatinum, Hospira Australia, Pty Ltd
Treatment Route:in culture media
Treatment Dose:6-8 cycles of CBP 50μM

Sample Preparation:

Sampleprep ID:SP002195
Sampleprep Summary:Media was aspirated and cells were washed three times with 3mL warmed PBS. Metabol-ic arrest was achieved through the addition of approximately 2 mL of liquid nitrogen di-rectly 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 additional 1mL of 100% ice cold methanol was added be-fore 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 repeat-ed 5 times to ensure full extraction 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 temperature, with vacuum of 40mbar and rotor speed of 1000min-1. Before data acquisition samples were resuspended in appropriate volumes of methanol as per cell number.
Processing Storage Conditions:-80℃
Extraction Method:MeOH
Extract Storage:-80℃
Sample Resuspension:MeOH

Combined analysis:

Analysis ID AN003439 AN003440
Analysis type MS MS
Chromatography type HILIC HILIC
Chromatography system Thermo Dionex Ultimate 3000 RS Thermo Dionex Ultimate 3000 RS
Column SeQuant ZIC-pHILIC (150 x 4.2 mm, 5 um) SeQuant ZIC-pHILIC (150 x 4.2 mm, 5 um)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode POSITIVE NEGATIVE
Units peak height peak height

Chromatography:

Chromatography ID:CH002541
Chromatography Summary:LCMS data was acquired on Q-Exactive Orbitrap mass spectrometer (Thermo Fisher) coupled with high-performance liquid chromatography (HPLC) system Dionex Ultimate® 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 program 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 40ºC. The total run time was 32 min with an injection sample volume of 10 µL.
Methods Filename:Metabolomics_pHILIC_Parkville_v1.meth
Instrument Name:Thermo Dionex Ultimate 3000 RS
Column Name:SeQuant ZIC-pHILIC (150 x 4.2 mm, 5 um)
Column Pressure:60 bar at starting conditions
Column Temperature:25 C
Flow Gradient:0 min - 80%B, 15 min - 50%B, 18 min - 5%B, 21 min - 5%B, 24 min - 80%B, 32 min - 80%B
Flow Rate:0.3 ml/min
Injection Temperature:4 C
Internal Standard:CAPS, CHAPS, PIPES
Sample Injection:10 uL
Solvent A:20 mM ammonium formate
Solvent B:acetonitrile
Analytical Time:32 min
Capillary Voltage:3.5 kV
Oven Temperature:25 C
Washing Buffer:syringe wash 50% IPA
Weak Wash Solvent Name:10% MeOH
Strong Wash Solvent Name:10% MeOH
Sample Loop Size:25 uL
Sample Syringe Size:25 uL
Chromatography Type:HILIC

MS:

MS ID:MS003202
Analysis ID:AN003439
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 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.
Ion Mode:POSITIVE
Capillary Temperature:300 C
Capillary Voltage:4 kV
Collision Energy:NA
Collision Gas:NA
Ion Source Temperature:120 C
Mass Accuracy:1-2 ppm
Automatic Gain Control:1e6
Dataformat:profile
Acquisition Parameters File:Metabolomics_pHILIC_Parkville_v1.pdf
Analysis Protocol File:PQMS3-MBPF-WIN-0501_analysis.pdf
  
MS ID:MS003203
Analysis ID:AN003440
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 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.
Ion Mode:NEGATIVE
Capillary Temperature:300 C
Capillary Voltage:3.5 kV
Collision Energy:NA
Collision Gas:NA
Ion Source Temperature:120 C
Mass Accuracy:1-2 ppm
Automatic Gain Control:1e6
Dataformat:profile
Acquisition Parameters File:Metabolomics_pHILIC_Parkville_v1.pdf
Analysis Protocol File:PQMS3-MBPF-WIN-0501_analysis.pdf
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