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.
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 | ST002104 |
| Study Title | Chemoresistant Cancer Cell Lines are Characterized by Migratory, Amino Acid Metabolism, Protein Catabolism and IFN1 Signalling Perturbations |
| Study Type | Biomedical research |
| Study Summary | Our 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 |
| Laboratory | Manuela Klingler-Hoffmann |
| Last Name | Acland |
| First Name | Mitchell |
| Address | X Building, Mawson Lakes South Australia 5095 |
| mitch.acland@gmail.com | |
| Phone | 0448671141 |
| Submit Date | 2022-02-06 |
| Num Groups | 4 |
| Total Subjects | 12 |
| Num Males | NA |
| Num Females | NA |
| Study Comments | OVCAR5 and CaOV3 cell lines and their carboplatin resistant cell lines |
| Publications | Chemoresistant Cancer Cell Lines are Characterized by Migra-tory, Amino Acid Metabolism, Protein Catabolism and IFN1 Signalling Perturbations |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2022-03-29 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
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 |
|---|---|---|
| SA201528 | CaOV3_Parent_2 | CaOV3 cell line |
| SA201529 | CaOV3_Parent_3 | CaOV3 cell line |
| SA201530 | CaOV3_Parent_1 | CaOV3 cell line |
| SA201531 | CaOV3_CBPR_3 | CaOV3 cell line CBPR mutation |
| SA201532 | CaOV3_CBPR_1 | CaOV3 cell line CBPR mutation |
| SA201533 | CaOV3_CBPR_2 | CaOV3 cell line CBPR mutation |
| SA201543 | DMEM_IST_2 | culture media for CaOV3 |
| SA201544 | DMEM_IST_1 | culture media for CaOV3 |
| SA201545 | RPMI_IST_1 | culture media for OVCAR5 |
| SA201546 | RPMI_IST_2 | culture media for OVCAR5 |
| SA201534 | Blank_1 | Extraction blank |
| SA201535 | Blank_2 | Extraction blank |
| SA201536 | Blank_3 | Extraction blank |
| SA201540 | OVCAR5_Parent_3 | OVCAR5_cell line |
| SA201541 | OVCAR5_Parent_2 | OVCAR5_cell line |
| SA201542 | OVCAR5_Parent_1 | OVCAR5_cell line |
| SA201537 | OVCAR5_CBPR_2 | OVCAR5 cell line CBPR mutation |
| SA201538 | OVCAR5_CBPR_3 | OVCAR5 cell line CBPR mutation |
| SA201539 | OVCAR5_CBPR_1 | OVCAR5 cell line CBPR mutation |
| SA201547 | QC_ov_3 | pooled QC |
| SA201548 | QC_ov_2 | pooled QC |
| SA201549 | QC_ov_1 | pooled 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 4.2mm,5um) | SeQuant ZIC-pHILIC (150 x 4.2mm,5um) |
| 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 4.2mm,5um) |
| 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: | 100% water; 20 mM ammonium formate |
| Solvent B: | 100% 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 |
