Return to study ST000644 main page
MB Sample ID: SA036404
Local Sample ID: | ms6138-8 |
Subject ID: | SU000667 |
Subject Type: | Human |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Species Group: | Human |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU000667 |
Subject Type: | Human |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Species Group: | Human |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
ms6138-8 | SA036404 | FL007860 | PSAT A13 + Dox (PKM1) | experiment |
ms6138-8 | SA036404 | FL007860 | 13C-glucose | tracer |
ms6138-8 | SA036404 | FL007860 | 48 hr | time |
ms6138-8 | SA036404 | FL007860 | PSAT | group ID |
Collection:
Collection ID: | CO000661 |
Collection Summary: | Pancreatic ductal adenocarcinoma cell line with PHGDH, PSAT, and PSPH KO. These cells have been incubated with no label, 13C-glucose or 13C-glutamine label for 48 hours. |
Sample Type: | Pancreas |
Treatment:
Treatment ID: | TR000681 |
Treatment Summary: | Pancreatic cancer cells exhibit altered metabolism, which is mediated in part by expressing the proliferation-supportive M2 isoform of pyruvate kinase (PK). Unlike normal embryonic cells that stop proliferating when forced to express the proliferation-incompatible M1 isoform of PK, pancreatic cancer cells can proliferate just as rapidly with either the M1 or M2 isoform. During forced PKM1 expression, pancreatic cancer cells upregulate their serine biosynthesis pathway. The three enzymes in the serine biosynthesis pathway include phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSPH). Each of the genes encoding the three enzymes in the serine biosynthesis pathway have successfully been knocked out from pancreatic cancer cells using the CRISPR/Cas9 system in our laboratory, with multiple confirmed clones for each knockout ready for metabolic characterization. |
Sample Preparation:
Sampleprep ID: | SP000674 |
Sampleprep Summary: | In this proposal, we will interrogate the metabolic pathways that support pancreatic cancer proliferation by performing isotope enrichment analysis on the CRISPR/Cas9 knockout pancreatic cancer cell lines. Each cell line expressing PKM1 or PKM2 will be seeded on 6-well plates so that they are ~70% confluent at the time of labeled media addition. Unlabeled media will be aspirated, and cell will be washed with PBS. Media labeled with 13C-glucose, 13C-glutamine, or 13C-serine labeled media will be added, and cells will be incubated for 1 hour or 24 hours. Polar metabolites and fatty acids will be extracted at the end of the incubation period using methanol, water, and chloroform. The methanol/water fraction containing polar metabolites will be separated from the chloroform fraction containing fatty acids, and each fraction will be dried down under nitrogen. Dried down samples will be sent to the Mayo Clinic Metabolomics Resource Core for analysis of TCA cycle intermediates, amino metabolites, free fatty acids, and sphingolipids. TCA cycle intermediates and amino metabolites (polar metabolites) should become labeled faster than fatty acids and sphingolipids; therefore, polar metabolites are proposed to be analyzed at 1 hour and 24 hours after 13C-label addition. Fatty acids are proposed to be analyzed for the 24 hr time point only, due to slow labeling. Statistical analysis will be performed by the Mayo Clinic Metabolomics Resource Core. |
Combined analysis:
Analysis ID | AN000976 |
---|---|
Analysis type | MS |
Chromatography type | Reversed phase |
Chromatography system | Waters Acquity |
Column | Waters Acquity BEH C8 (150 x 2mm,1.7um) |
MS Type | ESI |
MS instrument type | QTOF |
MS instrument name | Agilent 6550 QTOF |
Ion Mode | POSITIVE |
Units | % enrichment: MPE |
Chromatography:
Chromatography ID: | CH000701 |
Instrument Name: | Waters Acquity |
Column Name: | Waters Acquity BEH C8 (150 x 2mm,1.7um) |
Chromatography Type: | Reversed phase |
MS:
MS ID: | MS000871 |
Analysis ID: | AN000976 |
Instrument Name: | Agilent 6550 QTOF |
Instrument Type: | QTOF |
MS Type: | ESI |
Ion Mode: | POSITIVE |