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.

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	ST002967
Study Title	Lipidomics study of FASN inhibition in HT-29 and HCT 116 spheroids
Study 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. 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
Department	Chemistry and Biochemistry
Laboratory	Amanda Hummon Lab
Last Name	Fries
First Name	Brian
Address	460 W 12th Ave, Columbus, OH 43210
Email	fries.94@osu.edu
Phone	9375221195
Submit Date	2023-11-08
Raw Data Available	Yes
Raw Data File Type(s)	d
Analysis Type Detail	LC-MS
Release Date	2024-04-02
Release Version	1

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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

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