Summary of Study ST003305
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 PR002055. The data can be accessed directly via it's Project DOI: 10.21228/M8052C 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 | ST003305 |
| Study Title | Comparative analysis of breast cancer metabolomes highlights fascin's central role in regulating key pathways related to disease progression |
| Study Summary | Omics technologies provide useful tools for the identification of novel biomarkers in many diseases, including breast cancer, which is the most diagnosed cancer in women worldwide. We and others have reported a central role for the actin-bundling protein (fascin) in regulating breast cancer disease progression at different levels. However, whether fascin expression promotes metabolic molecules that could predict disease progression has not been fully elucidated. Here, fascin expression was manipulated via knockdown (fascinKD+NORF) and rescue (fascinKD+FORF) in the naturally fascin-positive (fascinpos+NORF) MDA-MB-231 breast cancer cells. Whether fascin dysregulates metabolic profiles that are associated with disease progression was assessed using untargeted metabolomics analyses via liquid chromatography-mass spectrometry. An overall of 12,226 metabolites were detected in the tested cell pellets. Fascinpos+NORF cell pellets showed 2,510 and 3,804 significantly dysregulated metabolites compared to their fascinKD+NORF counterparts. Fascin rescue (fascinKD+FORF) revealed 2,710 significantly dysregulated cellular metabolites compared to fascinKD+NORF counterparts. 101 overlapped cellular metabolites between fascinKD+FORF and fascinpos+NORF were significantly dysregulated in the fascinKD+NORF cells. Analysis of the significantly dysregulated metabolites by fascin expression revealed their involvement in the metabolism of sphingolipid, phenylalanine, tyrosine and tryptophan biosynthesis, and pantothenate and CoA biosynthesis, which are critical pathways for breast cancer progression. Our findings of fascin-mediated alteration of metabolic pathways could be used as putative poor prognostic biomarkers and highlight other underlying mechanisms of fascin contribution to breast cancer progression. |
| Institute | King Faisal Specialist Hospital and Research Centre (KFSHRC) |
| Last Name | AlMalki |
| First Name | Reem |
| Address | King Fahad road, Riyadh, KSA, 00000, Saudi Arabia |
| rgalmalki@kfshrc.edu.sa | |
| Phone | +966534045397 |
| Submit Date | 2024-07-02 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Waters) |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-07-30 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002055 |
| Project DOI: | doi: 10.21228/M8052C |
| Project Title: | Comparative analysis of breast cancer metabolomes highlights fascin's central role in regulating key pathways related to disease progression |
| Project Summary: | Omics technologies provide useful tools for the identification of novel biomarkers in many diseases, including breast cancer, which is the most diagnosed cancer in women worldwide. We and others have reported a central role for the actin-bundling protein (fascin) in regulating breast cancer disease progression at different levels. However, whether fascin expression promotes metabolic molecules that could predict disease progression has not been fully elucidated. Here, fascin expression was manipulated via knockdown (fascinKD+NORF) and rescue (fascinKD+FORF) in the naturally fascin-positive (fascinpos+NORF) MDA-MB-231 breast cancer cells. Whether fascin dysregulates metabolic profiles that are associated with disease progression was assessed using untargeted metabolomics analyses via liquid chromatography-mass spectrometry. An overall of 12,226 metabolites were detected in the tested cell pellets. Fascinpos+NORF cell pellets showed 2,510 and 3,804 significantly dysregulated metabolites compared to their fascinKD+NORF counterparts. Fascin rescue (fascinKD+FORF) revealed 2,710 significantly dysregulated cellular metabolites compared to fascinKD+NORF counterparts. 101 overlapped cellular metabolites between fascinKD+FORF and fascinpos+NORF were significantly dysregulated in the fascinKD+NORF cells. Analysis of the significantly dysregulated metabolites by fascin expression revealed their involvement in the metabolism of sphingolipid, phenylalanine, tyrosine and tryptophan biosynthesis, and pantothenate and CoA biosynthesis, which are critical pathways for breast cancer progression. Our findings of fascin-mediated alteration of metabolic pathways could be used as putative poor prognostic biomarkers and highlight other underlying mechanisms of fascin contribution to breast cancer progression. |
| Institute: | King Faisal Specialist Hospital and Research Centre (KFSHRC) |
| Last Name: | AlMalki |
| First Name: | Reem |
| Address: | Al Mather road, Riyadh, KSA, 11211, Saudi Arabia |
| Email: | rgalmalki@kfshrc.edu.sa |
| Phone: | +966534045397 |
Subject:
| Subject ID: | SU003426 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Factors:
Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Group |
|---|---|---|---|
| SA358614 | CON_1_S | Breast cancer cells | Control |
| SA358615 | CON_2_P | Breast cancer cells | Control |
| SA358616 | CON_2_S | Breast cancer cells | Control |
| SA358617 | CON_1_P | Breast cancer cells | Control |
| SA358618 | CON_3_P | Breast cancer cells | Control |
| SA358619 | CON_3_S | Breast cancer cells | Control |
| SA358624 | 40N_1_S | Breast cancer cells | knockdown |
| SA358625 | 40N_3_S | Breast cancer cells | knockdown |
| SA358626 | 40N_2_S | Breast cancer cells | knockdown |
| SA358627 | 40N_3_P | Breast cancer cells | knockdown |
| SA358628 | 40N_1_P | Breast cancer cells | knockdown |
| SA358629 | 40N_2_P | Breast cancer cells | knockdown |
| SA358620 | QC_S_MDA_2 | Breast cancer cells | QC |
| SA358621 | QC_S_MDA_1 | Breast cancer cells | QC |
| SA358622 | QC_P_MDA_2 | Breast cancer cells | QC |
| SA358623 | QC_P_MDA_1 | Breast cancer cells | QC |
| SA358630 | 40F_1_P | Breast cancer cells | restore |
| SA358631 | 40F_2_S | Breast cancer cells | restore |
| SA358632 | 40F_1_S | Breast cancer cells | restore |
| SA358633 | 40F_3_P | Breast cancer cells | restore |
| SA358634 | 40F_2_P | Breast cancer cells | restore |
| SA358635 | 40F_3_S | Breast cancer cells | restore |
| Showing results 1 to 22 of 22 |
Collection:
| Collection ID: | CO003419 |
| Collection Summary: | Cell pellet metabolites were extracted after removing the media and washing the cells with chilled 1x cold PBS as previously described [20]. The plates were dipped in liquid nitrogen to quench the metabolism and reduce the experimental variations. 1 mL of cold 80% methanol: water was added to each plate for metabolites extraction, and cells were detached using a cell scraper and transferred to 1.5 ml Eppendorf tubes. The mixtures were vortexed at 4°C, 600 rpm for 1h in Thermomixer (Eppendorf, Germany). The samples were spun down at 4°C, 16000 rpm for 10 min (Eppendorf, Germany). The supernatants (secretomes) were transferred to new Eppendorf tubes. Similarly, 900 µl of extraction solvent 1:1 (v/v) acetonitrile: methanol (ACN: MeOH) was added to 100 µl of media for secretome metabolites extraction. The mixtures were vortexed in Thermomixer (Eppendorf, Germany) at 600 rpm, 4ºC for 1 h. The samples were spun down at 16000 rpm, 4ºC for 10 min (Eppendorf, Germany), and then the secretomes were transferred to new Eppendorf tubes. The cell pellets and secretome extracts were completely evaporated in a SpeedVac (Christ, Germany) and stored at -80°C until LC-MS analysis [21,22] . |
| Sample Type: | Breast cancer cells |
Treatment:
| Treatment ID: | TR003435 |
| Treatment Summary: | Gene knockdown and restoration MDA-MB-231 breast cancer cells are naturally fascin-positive. The establishment of stable fascin knockdown (fascinKD) and control (fascinpos) in MDA-MB-231 cells using fascin and scrambled shRNA, respectively, was previously described [doi:10.1371/journal.pone.0027339]. Furthermore, the rescue of fascin expression in the fascinKD MDA-MB-231 cells using fascin ORF (fascinKD+FORF) was also previously described [doi:10.1002/ijc.32183]. For transfection control, empty ORF was used in fascinKD (fascinKD+NORF) and fascinpos (fascinpos+NORF) cells. Fascin expression or knockdown was routinely checked at the RNA and protein levels. |
Sample Preparation:
| Sampleprep ID: | SP003433 |
| Sampleprep Summary: | Cell pellet metabolites were extracted after removing the media and washing the cells with chilled 1x cold PBS as previously described [doi:10.3390/ijms24044219]. The plates were dipped in liquid nitrogen to quench the metabolism and reduce the experimental variations. 1 mL of cold 80% methanol: water was added to each plate for metabolites extraction, and cells were detached using a cell scraper and transferred to 1.5 ml Eppendorf tubes. The mixtures were vortexed at 4°C, 600 rpm for 1h in Thermomixer (Eppendorf, Germany). The samples were spun down at 4°C, 16000 rpm for 10 min (Eppendorf, Germany). The supernatants (secretomes) were transferred to new Eppendorf tubes. Similarly, 900 µl of extraction solvent 1:1 (v/v) acetonitrile: methanol (ACN: MeOH) was added to 100 µl of media for secretome metabolites extraction. The mixtures were vortexed in Thermomixer (Eppendorf, Germany) at 600 rpm, 4ºC for 1 h. The samples were spun down at 16000 rpm, 4ºC for 10 min (Eppendorf, Germany), and then the secretomes were transferred to new Eppendorf tubes. The cell pellets and secretome extracts were completely evaporated in a SpeedVac (Christ, Germany) and stored at -80°C until LC-MS analysis [21,22] . |
Combined analysis:
| Analysis ID | AN005415 | AN005416 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Waters Acquity | Waters Acquity |
| Column | Waters XSelect HSS C18 (100 × 2.1mm, 2.5um) | Waters XSelect HSS C18 (100 × 2.1mm, 2.5um) |
| MS Type | ESI | ESI |
| MS instrument type | QTOF | QTOF |
| MS instrument name | Waters Xevo-G2-XS | Waters Xevo-G2-XS |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | peak area | peak area |
Chromatography:
| Chromatography ID: | CH004105 |
| Chromatography Summary: | The dried extract samples were reconstituted in a 1:1 mobile phase (A: 0.1% formic acid in dH2O and B: 0.1% formic acid in (1:1) (v/v) MeOH: ACN) for an LC-MS metabolomics analysis [20]. First, 5 µL of the sample was introduced to the inlet technique, where the metabolites were separated in reversed-phase liquid chromatography using an ACQUITY UPLC XSelect (100 × 2.1 mm × 2.5 μm) column (Waters Ltd., Elstree, UK). The mobile phase flow rate was set at 300 μL/min, the column temperature maintained at 55 °C and the samples were maintained at 4 °C in the autosampler. Mobile phases A and B were pumped to the column in a gradient mode (0–16 min 95–5% A, 16–19 min 5% A, 19–20 min 5–95% A, and 20–22 min 5–95% A). The molecules eluted from the LC were positively or negatively ionized using an electrospray ionization source (ESI) and separated in the gas phase based on m/z using a Xevo G2-S QTOF mass spectrometer (Waters Ltd., Elstree, UK). The metabolites were ionized in the ESI source, where the source temperature was 150 °C, the desolvation temperature was 500 °C, and the capillary voltages were kept at 3.20 kV (ESI+) or 3 kV (ESI−). The cone voltage was 40 V. the desolvation gas flow was 800.0 L/h, and the cone gas flow was 50 L/h. The collision energies of the low and high functions were set to off and 10–50 V, respectively, in the MSE data-independent acquisition (DIA) mode. The mass spectrometer was calibrated, as recommended by the vendor, with sodium formate in the range of 100–1200 Da in both ionization modes. The lock mass compound, leucine-enkephaline (an external reference to the ion m/z 556.2771 in (ESI+) and 554.2615 (ESI−)), was injected continuously, switching between the sample and the reference every 45 and 60 s for ESI+ and ESI−, respectively, for a 0.5 s scan time, a flow rate of 10 µL/min, a cone voltage of 30 V, and a collision energy of 4 V. DIA were collected in continuum mode with a Masslynx™ V4.1 workstation (Waters Inc., Milford, MA, USA). Quality control samples (QCs) were performed by pooling 10 µL from each study sample and extracted, after that, introduced to the instrument with randomization to validate the system's stability. |
| Instrument Name: | Waters Acquity |
| Column Name: | Waters XSelect HSS C18 (100 × 2.1mm, 2.5um) |
| Column Temperature: | 55 |
| Flow Gradient: | 0–16 min 95–5% A, 16–19 min 5% A, 19–20 min 5–95% A, and 20–22 min 5–95% A |
| Flow Rate: | 0.300 mL/min |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 50% methanol:50% acetonitrile; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH004106 |
| Chromatography Summary: | The dried extract samples were reconstituted in a 1:1 mobile phase (A: 0.1% formic acid in dH2O and B: 0.1% formic acid in (1:1) (v/v) MeOH: ACN) for an LC-MS metabolomics analysis [20]. First, 5 µL of the sample was introduced to the inlet technique, where the metabolites were separated in reversed-phase liquid chromatography using an ACQUITY UPLC XSelect (100 × 2.1 mm × 2.5 μm) column (Waters Ltd., Elstree, UK). The mobile phase flow rate was set at 300 μL/min, the column temperature maintained at 55 °C and the samples were maintained at 4 °C in the autosampler. Mobile phases A and B were pumped to the column in a gradient mode (0–16 min 95–5% A, 16–19 min 5% A, 19–20 min 5–95% A, and 20–22 min 5–95% A). The molecules eluted from the LC were positively or negatively ionized using an electrospray ionization source (ESI) and separated in the gas phase based on m/z using a Xevo G2-S QTOF mass spectrometer (Waters Ltd., Elstree, UK). The metabolites were ionized in the ESI source, where the source temperature was 150 °C, the desolvation temperature was 500 °C, and the capillary voltages were kept at 3.20 kV (ESI+) or 3 kV (ESI−). The cone voltage was 40 V. the desolvation gas flow was 800.0 L/h, and the cone gas flow was 50 L/h. The collision energies of the low and high functions were set to off and 10–50 V, respectively, in the MSE data-independent acquisition (DIA) mode. The mass spectrometer was calibrated, as recommended by the vendor, with sodium formate in the range of 100–1200 Da in both ionization modes. The lock mass compound, leucine-enkephaline (an external reference to the ion m/z 556.2771 in (ESI+) and 554.2615 (ESI−)), was injected continuously, switching between the sample and the reference every 45 and 60 s for ESI+ and ESI−, respectively, for a 0.5 s scan time, a flow rate of 10 µL/min, a cone voltage of 30 V, and a collision energy of 4 V. DIA were collected in continuum mode with a Masslynx™ V4.1 workstation (Waters Inc., Milford, MA, USA). Quality control samples (QCs) were performed by pooling 10 µL from each study sample and extracted, after that, introduced to the instrument with randomization to validate the system's stability. |
| Instrument Name: | Waters Acquity |
| Column Name: | Waters XSelect HSS C18 (100 × 2.1mm, 2.5um) |
| Column Temperature: | 55 |
| Flow Gradient: | 0–16 min 95–5% A, 16–19 min 5% A, 19–20 min 5–95% A, and 20–22 min 5–95% A |
| Flow Rate: | 0.300 mL/min |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 50% methanol:50% acetonitrile; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS005141 |
| Analysis ID: | AN005415 |
| Instrument Name: | Waters Xevo-G2-XS |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | The DIA data were collected with a Masslynx™ V4.1 workstation in continuum mode (Waters Inc., Milford, MA, USA). The raw MS data were processed following a standard pipeline using the Progenesis QI v.3.0 software. |
| Ion Mode: | POSITIVE |
| MS ID: | MS005142 |
| Analysis ID: | AN005416 |
| Instrument Name: | Waters Xevo-G2-XS |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | The DIA data were collected with a Masslynx™ V4.1 workstation in continuum mode (Waters Inc., Milford, MA, USA). The raw MS data were processed following a standard pipeline using the Progenesis QI v.3.0 software. |
| Ion Mode: | NEGATIVE |
