Summary of Study ST002967

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 PR001846. The data can be accessed directly via it's Project DOI: 10.21228/M83H81 This work is supported by NIH grant, U2C- DK119886.

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Study IDST002967
Study TitleLipidomics study of FASN inhibition in HT-29 and HCT 116 spheroids
Study SummaryCancerous cells synthesize most of their lipids de novo to keep up with their rapid growth and proliferation. Fatty acid synthase (FAS) is a key enzyme in the lipogenesis pathway that is upregulated in many cancers and has gained popularity as a druggable target of interest for cancer treatment. The first FAS inhibitor discovered, cerulenin, initially showed promise for chemotherapeutic purposes until it was observed that it had adverse side effects in mice. TVB2640 (Denifanstat), is part of the newer generation of inhibitors. With multiple generations of FAS inhibitors being developed, it is vital to understand their distinct molecular downstream effects to elucidate potential interactions in the clinic. Here, we profile the lipidome of two different colorectal cancer (CRC) spheroids treated with a generation 1 inhibitor (cerulenin) or a generation 2 inhibitor (TVB-2640). We observe that the cerulenin causes drastic changes to the spheroid morphology as well as alterations to the lipid droplets found within CRC spheroids. TVB-2640 causes higher abundances of polyunsaturated fatty acids (PUFAs) whereas cerulenin causes decreased abundance of PUFAs. The increase in PUFAs in TVB-2640 exposed spheroids indicates it is causing cells to die via a ferroptotic mechanism rather than a conventional apoptotic or necrotic mechanism.
Institute
The Ohio State University
DepartmentChemistry and Biochemistry
LaboratoryAmanda Hummon Lab
Last NameFries
First NameBrian
Address460 W 12th Ave, Columbus, OH 43210
Emailfries.94@osu.edu
Phone9375221195
Submit Date2023-11-08
Raw Data AvailableYes
Raw Data File Type(s)d
Analysis Type DetailLC-MS
Release Date2024-04-02
Release Version1
Brian Fries Brian Fries
https://dx.doi.org/10.21228/M83H81
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR001846
Project DOI:doi: 10.21228/M83H81
Project Title:Lipidomics study of FASN inhibition in HT-29 and HCT 116 spheroids
Project Type:MS Quantitative Analysis
Project Summary:Cancerous cells synthesize most of their lipids de novo to keep up with their rapid growth and proliferation. Fatty acid synthase (FAS) is a key enzyme in the lipogenesis pathway that is upregulated in many cancers and has gained popularity as a druggable target of interest for cancer treatment. The first FAS inhibitor discovered, cerulenin, initially showed promise for chemotherapeutic purposes until it was observed that it had adverse side effects in mice. TVB-2640 (Denifanstat), is part of the newer generation of inhibitors. With multiple generations of FAS inhibitors being developed, it is vital to understand their distinct molecular downstream effects to elucidate potential interactaCancerous cells synthesize most of their lipids de novo to keep up with their rapid growth and proliferation. Fatty acid synthase (FAS) is a key enzyme in the lipogenesis pathway that is upregulated in many cancers and has gained popularity as a druggable target of interest for cancer treatment. The first FAS inhibitor discovered, cerulenin, initially showed promise for chemotherapeutic purposes until it was observed that it had adverse side effects in mice. TVB2640 (Denifanstat), is part of the newer generation of inhibitors. With multiple generations of FAS inhibitors being developed, it is vital to understand their distinct molecular downstream effects to elucidate potential interactions in the clinic. Here, we profile the lipidome of two different colorectal cancer (CRC) spheroids treated with a generation 1 inhibitor (cerulenin) or a generation 2 inhibitor (TVB-2640). We observe that the cerulenin causes drastic changes to the spheroid morphology as well as alterations to the lipid droplets found within CRC spheroids. TVB-2640 causes higher abundances of polyunsaturated fatty acids (PUFAs) whereas cerulenin causes decreased abundance of PUFAs. The increase in PUFAs in TVB-2640 exposed spheroids indicates it is causing cells to die via a ferroptotic mechanism rather than a conventional apoptotic or necrotic mechanism.ions in the clinic. Here, we profile the lipidome of two different colorectal cancer (CRC) spheroids treated with a generation 1 inhibitor (cerulenin) or a generation 2 inhibitor (TVB-2640). We observe that the cerulenin causes drastic changes to the spheroid morphology as well as alterations to the lipid droplets found within CRC spheroids. TVB-2640 causes higher abundances of polyunsaturated fatty acids (PUFAs) whereas cerulenin causes decreased abundance of PUFAs. The increase in PUFAs in TVB-2640 exposed spheroids indicates it is causing cells to die via a ferroptotic mechanism rather than a conventional apoptotic or necrotic mechanism.
Institute:The Ohio State University
Department:Chemistry and Biochemistry
Laboratory:Amanda Hummon Lab
Last Name:Fries
First Name:Brian
Address:460 W 12th Ave, 470, Columbus, Ohio, 43210, USA
Email:fries.94@osu.edu
Phone:9375221195

Subject:

Subject ID:SU003080
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 Treatment
SA32296825HCT116_CER4Cerulenin
SA32296932HCT116_CER5Cerulenin
SA32297007HT29_CER1Cerulenin
SA32297111HCT116_CER2Cerulenin
SA32297214HT29_CER2Cerulenin
SA32297318HCT116_CER3Cerulenin
SA32297435HT29_CER5Cerulenin
SA32297521HT29_CER3Cerulenin
SA32297628HT29_CER4Cerulenin
SA32297704HCT116_CER1Cerulenin
SA32297822HT29_CTRL3Control
SA32297929HT29_CTRL4Control
SA32298005HCT116_CTRL1Control
SA32298115HT29_CTRL2Control
SA32298236HT29_CTRL5Control
SA32298319HCT116_CTRL3Control
SA32298408HT29_CTRL1Control
SA32298526HCT116_CTRL4Control
SA32298633HCT116_CTRL5Control
SA32298712HCT116_CTRL2Control
SA32298801POOLSamplePool1
SA32298902POOLSamplePool2
SA32299003POOLSamplePool3
SA32299110POOLSamplePool4
SA32299217POOLSamplePool5
SA32299324POOLSamplePool6
SA32299431POOLSamplePool7
SA32299538POOLSamplePool8
SA32299623HT29_TVB3TVB-2640
SA32299720HCT116_TVB3TVB-2640
SA32299827HCT116_TVB4TVB-2640
SA32299934HCT116_TVB5TVB-2640
SA32300013HCT116_TVB2TVB-2640
SA32300106HCT116_TVB1TVB-2640
SA32300230HT29_TVB4TVB-2640
SA32300316HT29_TVB2TVB-2640
SA32300409HT29_TVB1TVB-2640
SA32300537HT29_TVB5TVB-2640
Showing results 1 to 38 of 38

Collection:

Collection ID:CO003073
Collection Summary:HCT 116 and HT-29 spheroids were grown by using the liquid overlay method. Spheroids were seeded at 70000 cells/well and allowed to sit undisturbed for 4 days. On day 4, half of the media was changed every other day until dosing with therapeutics on day on day 12. Spheroids were washed in PBS and stored at negative eighty degrees Celsius.
Sample Type:Cultured cells
Storage Conditions:-80℃

Treatment:

Treatment ID:TR003089
Treatment Summary:On day 12, each spheroid was treated with their respective IC50 of either cerulenin or TVB-2640 and allowed to incubate with the therapeutic for 48 hours.

Sample Preparation:

Sampleprep ID:SP003086
Sampleprep Summary:Spheroids were lysed in 1X PBS using a probe sonicator followed by protein determination using the Pierce bicinchoninic assay (BCA) Protein Assay Kit (Thermo). 200 μg of protein was aliquoted from each sample and subjected to lipid extraction. 5 μL of equiSPLASH LIPIDOMIX internal standard (Avanti Polar Lipids, Alabaster, AL, USA) was added to each sample followed by lipid extraction as previously described. In brief, 300 μL of cold methanol was added to each sample and vortexed. Next, 1 mL of cold MTBE was added followed by vortexing and bath sonication. Then, 250 μL of LC-MS grade water was then added to induce phase separation followed by vortexing and bath sonication for 5 minutes. Each sample was then centrifuged at 14,000 rpm for 2 minutes. The upper organic phase of each sample was collected for lipidomics using a Hamilton syringe, rinsing with MTBE between every sample. 1 mL of cold MTBE was added a second time and this process was repeated to collect a second extraction of lipids from each sample. Lipid fractions were dried using a vacuum centrifuge and stored at -80 ⁰C until LC-MS analysis

Combined analysis:

Analysis ID AN004875
Analysis type MS
Chromatography type Reversed phase
Chromatography system 1290 Infinity II UHPLC (Agilent)
Column Waters ACQUITY UPLC BEH C18 (100 x 2.1mm, 1.7um)
MS Type ESI
MS instrument type QTOF
MS instrument name Agilent 6545 QTOF
Ion Mode UNSPECIFIED
Units pmol/ug of Protein

Chromatography:

Chromatography ID:CH003678
Chromatography Summary:Dried lipid extracts were resuspended in 9:1 in methanol/toluene to obtain a protein equivalent concentration of 2 µg/µL. At the same time, a pooled sample was created by taking an equal volume from each sample in the study. Diluted samples were injected on a 1290 Infinity II UHPLC System (Agilent Technologies Inc., Santa Clara, California, USA) onto an ACQUITY PREMIER BEH C18 column (1.7 μm, 2.1 x 100 mm, Waters Corporation, Belford, MA, USA) for reversedphase chromatography which was maintained at 50 °C with a constant flow rate at 0.400 ml/min, using a gradient of mobile phase A (50:50 acetonitrile/H2O with 5 mM ammonium formate) and mobile phase B (90:10 isopropanol/acetonitrile with 5 mM ammonium formate). The gradient program was as follows: 0 – 5 min, 15 – 40 %B; 5 – 6 min, 40 – 50 %B; 6 – 30 min, 50 – 95 %B; 30 – 32 min, hold 95 %B. A 10-minute post time was enabled which held the solvent composition at 15 %B before analyzing the next sample.
Instrument Name:1290 Infinity II UHPLC (Agilent)
Column Name:Waters ACQUITY UPLC BEH C18 (100 x 2.1mm, 1.7um)
Column Temperature:50
Flow Gradient:: 0 – 5 min, 15 – 40 %B; 5 – 6 min, 40 – 50 %B; 6 – 30 min, 50 – 95 %B; 30 – 32 min, hold 95 %B. A 10-minute post time was enabled which held the solvent composition at 15 %B before analyzing the next sample
Flow Rate:0.400 mL/min
Solvent A:50:50 ACN:H20 with 5 mM ammonium formate
Solvent B:90:10 IPA/ACN with 5 mM ammonium formate
Chromatography Type:Reversed phase

MS:

MS ID:MS004619
Analysis ID:AN004875
Instrument Name:Agilent 6545 QTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:e. "MS-Only", positive and negative ion mode acquisitions were conducted on the samples on an Agilent 6545 quadrupole time-of-flight mass spectrometer equipped with a JetStream ionization source. The source conditions were as follows: Gas Temperature, 300 °C; Drying Gas flow, 12 L/ min; Nebulizer, 30 psi; Sheath Gas Temperature, 350 °C; Sheath Gas Flow, 10 L/ min; Vcap, 3000 V; Fragmentor, 125 V; Skimmer, 45 V; and Oct 1 RF, 750 V. The acquisition rate in MS-Only mode was 4 spectra/second, utilizing m/z 121.050873 and m/z 922.009798 as reference masses in positive ion mode, and m/z 112.985587 and m/z 1033.988109 in negative ion mode. The acquisition parameters for Auto MS/MS are similar to MS-Only mode, with the addition of Collision Energy of 10, 20, and 40; isolation width, narrow (1.3 m/z); top 6 precursors per cycle; Precursor Threshold, 2000 counts and 0.01 %; active exclusion was enabled for 1 spectrum and released after 0.12 minutes. Iterative Auto MS/MS was also utilized, where 5 consecutive injections of the pooled sample were analyzed to generate a rolling exclusion list throughout the 5 injections, where a top 6 precursor per cycle and a collision energy of 20 was used. Data Analysis Lipidomics data were analyzed using MS-DIAL (version 4.9.221218) and visualized using the R Packages SCOPE and LipidR. The curated Alignment Table from MS-DIAL can be found in the Supplemental Information. Venn diagrams were generated using the Venny 2.1 website. Total triglyceride content data were analyzed using Microsoft Excel. The data analysis parameters for MS-DIAL can be found in the Supplemental Information
Ion Mode:UNSPECIFIED
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