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 IDST001707
Study TitleLipid Profiling of Mouse Intestinal Organoids for studying APC Mutations
Study SummaryInactivating 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 NameLi
First NameJia
AddressImperial College London, UK
Emailjia.li@imperial.ac.uk
Phone00442075943230
Submit Date2021-02-17
Raw Data AvailableYes
Raw Data File Type(s)cdf
Analysis Type DetailLC-MS
Release Date2021-02-22
Release Version1
Jia Li Jia Li
https://dx.doi.org/10.21228/M8JM5Z
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR001092
Project DOI:doi: 10.21228/M8JM5Z
Project Title:Lipid Profiling of Mouse Intestinal Organoids for studying APC Mutations
Project 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 Road, South Kensington, London, London, SW7 2AZ, United Kingdom
Email:jia.li@imperial.ac.uk
Phone:02075943230

Subject:

Subject ID:SU001784
Subject Type:Cultured cells
Subject Species:Mus musculus
Taxonomy ID:10090

Factors:

Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Genotype
SA158943POS_APC_m830_1APC
SA158944POS_APC_m862_8APC
SA158945POS_APC_m862_6APC
SA158946NEG_APC_m862_7APC
SA158947POS_APC_m862_7APC
SA158948NEG_APC_m862_5APC
SA158949NEG_APC_m862_2APC
SA158950NEG_APC_m862_3APC
SA158951NEG_APC_m862_4APC
SA158952POS_APC_m862_5APC
SA158953NEG_APC_m862_6APC
SA158954POS_APC_m862_3APC
SA158955POS_APC_m830_5APC
SA158956POS_APC_m830_4APC
SA158957POS_APC_m830_3APC
SA158958POS_APC_m830_2APC
SA158959POS_APC_m830_6APC
SA158960POS_APC_m830_7APC
SA158961NEG_APC_m862_1APC
SA158962POS_APC_m862_2APC
SA158963POS_APC_m862_1APC
SA158964POS_APC_m830_8APC
SA158965POS_APC_m862_4APC
SA158966NEG_APC_m862_8APC
SA158967NEG_APC_m830_3APC
SA158968NEG_APC_m830_1APC
SA158969NEG_APC_m830_8APC
SA158970NEG_APC_m830_4APC
SA158971NEG_APC_m830_2APC
SA158972NEG_APC_m830_5APC
SA158973NEG_APC_m830_6APC
SA158974NEG_APC_m830_7APC
SA158975NEG_Blankblank
SA158976POS_Blankblank
SA158977POS_QC3quality control
SA158978POS_QC2quality control
SA158979POS_QC4quality control
SA158980POS_QC1quality control
SA158981NEG_QC4quality control
SA158982NEG_QC2quality control
SA158983NEG_QC3quality control
SA158984POS_QC5quality control
SA158985NEG_QC5quality control
SA158986NEG_QC1quality control
SA158987POS_Dilution1_2_2quality control dilutions
SA158988POS_Dilution1_8_2quality control dilutions
SA158989POS_Dilution1_2_1quality control dilutions
SA158990POS_Dilution1_8_3quality control dilutions
SA158991POS_Dilution1_8_1quality control dilutions
SA158992POS_Dilution1_4_3quality control dilutions
SA158993POS_Dilution1_4_1quality control dilutions
SA158994POS_Dilution1_4_2quality control dilutions
SA158995POS_Dilution1_2_3quality control dilutions
SA158996NEG_Dilution1_4_3quality control dilutions
SA158997NEG_Dilution1_2_3quality control dilutions
SA158998NEG_Dilution1_2_2quality control dilutions
SA158999NEG_Dilution1_2_1quality control dilutions
SA159000NEG_Dilution1_4_1quality control dilutions
SA159001NEG_Dilution1_4_2quality control dilutions
SA159002NEG_Dilution1_8_2quality control dilutions
SA159003NEG_Dilution1_8_1quality control dilutions
SA159004NEG_Dilution1_8_3quality control dilutions
SA159005POS_WT_m1641_1wildtype
SA159006NEG_WT_m1663_3wildtype
SA159007NEG_WT_m1663_2wildtype
SA159008NEG_WT_m1663_4wildtype
SA159009NEG_WT_m1663_5wildtype
SA159010NEG_WT_m1663_7wildtype
SA159011NEG_WT_m1663_6wildtype
SA159012NEG_WT_m1663_1wildtype
SA159013NEG_WT_m1641_8wildtype
SA159014NEG_WT_m1641_3wildtype
SA159015NEG_WT_m1641_2wildtype
SA159016NEG_WT_m1641_4wildtype
SA159017NEG_WT_m1641_5wildtype
SA159018NEG_WT_m1641_7wildtype
SA159019NEG_WT_m1641_6wildtype
SA159020NEG_WT_m1663_8wildtype
SA159021NEG_WT_m1641_1wildtype
SA159022POS_WT_m1663_3wildtype
SA159023POS_WT_m1663_2wildtype
SA159024POS_WT_m1663_4wildtype
SA159025POS_WT_m1663_5wildtype
SA159026POS_WT_m1663_7wildtype
SA159027POS_WT_m1663_6wildtype
SA159028POS_WT_m1663_1wildtype
SA159029POS_WT_m1641_8wildtype
SA159030POS_WT_m1641_3wildtype
SA159031POS_WT_m1641_2wildtype
SA159032POS_WT_m1641_4wildtype
SA159033POS_WT_m1641_5wildtype
SA159034POS_WT_m1641_7wildtype
SA159035POS_WT_m1641_6wildtype
SA159036POS_WT_m1663_8wildtype
Showing results 1 to 94 of 94

Collection:

Collection ID:CO001777
Collection Summary:Control (Lgr5–EGFP-IRES-CreERT2 Apc+/+) or experimental (Lgr5–EGFP-IRES-CreERT2 Apcfl/fl) adult mice were administered tamoxifen (80 mg/kg) daily via intraperitoneal injection for 4 consecutive days to induce Cre expression. Fourteen days following induction mice were sacrificed (cervical dislocation) and their intestinal cells were harvested for organoid culture of WT or Apc deficient intestinal stem cells.
Sample Type:Intestine

Treatment:

Treatment ID:TR001797
Treatment Summary:no treatment was applied

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).

Combined analysis:

Analysis ID AN002780 AN002781
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Acquity UPLC Acquity UPLC
Column Waters Acquity C18 CSH (10 x 2.1mm,1.7um) Waters Acquity C18 CSH (10 x 2.1mm,1.7um)
MS Type ESI ESI
MS instrument type QTOF QTOF
MS instrument name Waters Synapt G2 Waters Synapt G2
Ion Mode POSITIVE NEGATIVE
Units peak area peak area

Chromatography:

Chromatography ID:CH002057
Instrument Name:Acquity UPLC
Column Name:Waters Acquity C18 CSH (10 x 2.1mm,1.7um)
Chromatography Type:Reversed phase

MS:

MS ID:MS002576
Analysis ID:AN002780
Instrument Name:Waters Synapt G2
Instrument Type:QTOF
MS Type:ESI
MS Comments:refer to Methodology.docx
Ion Mode:POSITIVE
Analysis Protocol File:jli_20210217_034113_PR_MS_Methodology.docx
  
MS ID:MS002577
Analysis ID:AN002781
Instrument Name:Waters Synapt G2
Instrument Type:QTOF
MS Type:ESI
MS Comments:refer to Methodology.docx
Ion Mode:NEGATIVE
Analysis Protocol File:jli_20210217_034113_PR_MS_Methodology.docx
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