Summary of Study ST001707

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 PR001092. The data can be accessed directly via it's Project DOI: 10.21228/M8JM5Z 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.

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Study ID	ST001707
Study Title	Lipid Profiling of Mouse Intestinal Organoids for studying APC Mutations
Study Summary	Inactivating mutations including both germline and somatic mutations in the adenomatous polyposis coli (APC) gene drives most familial and sporadic colorectal cancers. Understanding the metabolic implications of this mutation will aid to establish its wider impact on cellular behaviour and potentially inform clinical decisions. However, to date, alterations in lipid metabolism induced by APC mutations remain unclear. Intestinal organoids have gained widespread popularity in studying colorectal cancer and chemotherapies, because their three-dimensional structure more accurately mimics an in vivo environment. Here, we aimed to investigate intra-cellular lipid disturbances induced by APC gene mutations in intestinal organoids using a reversed-phase ultra-high-performance liquid chromatography mass spectrometry (RP-UHPLC-MS)-based lipid profiling method. Lipids of the organoids grown from either wildtype (WT) or mice with Apc mutations (Lgr5–EGFP-IRES-CreERT2 Apcfl/fl) were extracted and analysed using RP-UHPLC-MS. Concentrations of phospholipids (e.g. PC(16:0/16:0), PC(18:1/20:0), PC(38:0), PC(18:1/22:1)), ceramides (e.g. Cer(d18:0/22:0), Cer(d42:0), Cer(d18:1/24:1)) and hexosylceramide (e.g. HexCer(d18:1/16:0), HexCer(d18:1/22:0)) were higher in Apcfl/fl organoids, whereas levels of sphingomyelins (e.g. SM(d18:1/14:0), SM(d18:1/16:0) ) were lower compared to WT. These observations indicate that cellular metabolism of sphingomyelin was upregulated, resulting in the cellular accumulation of ceramides and production of HexCer due to the absence of Apcfl/fl in the organoids. Our observations demonstrated lipid profiling of organoids and provided an enhanced insight into the effects of the APC mutations on lipid metabolism, making for a valuable addition to screening options of the organoid lipidome.
Institute	Imperial College London
Last Name	Li
First Name	Jia
Address	Imperial College London, UK
Email	jia.li@imperial.ac.uk
Phone	00442075943230
Submit Date	2021-02-17
Raw Data Available	Yes
Raw Data File Type(s)	cdf
Analysis Type Detail	LC-MS
Release Date	2021-02-22
Release Version	1

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Sample Preparation:

Sampleprep ID:	SP001790
Sampleprep Summary:	Following aqueous extraction using cold methanol and water (v:v, 1:1), 1 ml of pre-chilled dichloromethane (DCM)/methanol (v:v, 3:1) was added to the organoid samples. Samples were bead-beaten for 40 seconds followed by five minutes of chilling on dry ice. This procedure was repeated three times before being centrifuged for 10 mins at 21,000 rcf at 4ºC. A total of 600 μl of supernatant from each sample was transferred to a glass vial. Another 200 μl of supernatant from each sample was pooled into a 15-ml Falcon tube to form a quantity control (QC) sample and split into several aliquots of 600 μl each. An extraction blank sample was included to control for any potential contaminant introduced throughout the extraction process. Samples were dried by evaporation over night at room temperature and stored at -40˚C until further analysis. The dried extracts were reconstituted in 100 μl of water/acetonitrile (ACN)/isopropanol (IPA), (v:v:v, 1:1:3,). The lipids were dissolved by vigorous vortexing for five minutes, followed by five minutes of sonication. This step was repeated three times to allow the dry extracts to thoroughly dissolve in the solvent. Samples were subsequently centrifuged at 21,000 rcf for 10 minutes at 4ºC and transferred to 150-μl glass inserts placed in glass vials (Waters).

Sampleprep ID:

SP001790

Sampleprep Summary:

Following aqueous extraction using cold methanol and water (v:v, 1:1), 1 ml of pre-chilled dichloromethane (DCM)/methanol (v:v, 3:1) was added to the organoid samples. Samples were bead-beaten for 40 seconds followed by five minutes of chilling on dry ice. This procedure was repeated three times before being centrifuged for 10 mins at 21,000 rcf at 4ºC. A total of 600 μl of supernatant from each sample was transferred to a glass vial. Another 200 μl of supernatant from each sample was pooled into a 15-ml Falcon tube to form a quantity control (QC) sample and split into several aliquots of 600 μl each. An extraction blank sample was included to control for any potential contaminant introduced throughout the extraction process. Samples were dried by evaporation over night at room temperature and stored at -40˚C until further analysis. The dried extracts were reconstituted in 100 μl of water/acetonitrile (ACN)/isopropanol (IPA), (v:v:v, 1:1:3,). The lipids were dissolved by vigorous vortexing for five minutes, followed by five minutes of sonication. This step was repeated three times to allow the dry extracts to thoroughly dissolve in the solvent. Samples were subsequently centrifuged at 21,000 rcf for 10 minutes at 4ºC and transferred to 150-μl glass inserts placed in glass vials (Waters).