Metabolomic Profiling from Formalin-Fixed, Paraffin-Embedded Tumor Tissue Using Targeted LC/MS/MS: Application in Sarcoma

Abstract

The relatively new field of onco-metabolomics attempts to identify relationships between various cancer phenotypes and global metabolite content. Previous metabolomics studies utilized either nuclear magnetic resonance spectroscopy or gas chromatography/mass spectrometry, and analyzed metabolites present in urine and serum. However, direct metabolomic assessment of tumor tissues is important for determining altered metabolism in cancers. In this respect, the ability to obtain reliable data from archival specimens is desirable and has not been reported to date. In this feasibility study, we demonstrate the analysis of polar metabolites extracted directly from ten formalin-fixed, paraffin-embedded (FFPE) specimens, including five soft tissue sarcomas and five paired normal samples. Using targeted liquid chromatography-tandem mass spectrometry (LC/MS/MS) via selected reaction monitoring (SRM), we detect an average of 106 metabolites across the samples with excellent reproducibility and correlation between different sections of the same specimen. Unsupervised hierarchical clustering and principal components analysis reliably recovers a priori known tumor and normal tissue phenotypes, and supervised analysis identifies candidate metabolic markers supported by the literature. In addition, we find that diverse biochemical processes are well-represented in the list of detected metabolites. Our study supports the notion that reliable and broadly informative metabolomic data may be acquired from FFPE soft tissue sarcoma specimens, a finding that is likely to be extended to other malignancies.

Gain of Glucose-Independent Growth upon Metastasis of Breast Cancer Cells to the Brain

“…To validate whether the brain metastatic cancer cells carried out gluconeogenesis in the absence of glucose using amino acids as substrates, we first cultured MDA-MB-231, MDA-MB-231Br3 and MDA-MB-361 cells in glucose free medium for 6 h, then added 13 C-glutamine into the glucose free cell culture medium and cultured cells for another 12 h before extraction of metabolites. The 13 C labeled metabolites were measured by tendem mass spectrometry (LC-MS) (37, 38, 41, 42) with a focus on tricarboxylic acid cycle (TCA), gluconeogenesis, pentose phosphate pathway (PPP), and metabolisms of purine and pyrimidine. Levels of intracellular intermediate metabolites fluctuate with metabolic activities of upstream and downstream pathways as well as with cell proliferative status, thus increase or decrease of a metabolite does not necessary represent the activity of the metabolic pathway in which the metabolite is produced.…”

Pharmacological Inhibition of Nicotinamide Phosphoribosyltransferase (NAMPT), an Enzyme Essential for NAD+ Biosynthesis, in Human Cancer Cells

“…The altered metabolite levels in tumors including, but not limited to glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, glyceraldehyde 3-phosphate, pentose phosphates, and sedoheptulose 7-phosphate or 2-phosphoglycerate, 3-phosphoglycerate, phosphoenolpyruvate, phosphoserine, and ␣-ketoglutarate can be used to assess effects of NAMPT inhibitors on their intended target in the clinic. First, the LC-MS method has been successfully used to detect and quantify many metabolites derived from several metabolic pathways, including glycolysis, the pentose phosphate pathway, the TCA cycle, and glutamine metabolism from fresh, frozen, and formalin-fixed paraffin-embedded human tumors (48,49). Among the 119 different metabolites analyzed, several of them, including glucose 6-phosphate, fructose 1,6-bisphosphate, glyceraldehyde 3-phosphate, and ribose 5-phosphate, which we show are significantly elevated in FK866-treated cancer cells and tumor xenografts, were reported to be robustly detected from human tumor tissues (48,49).…”

“…First, the LC-MS method has been successfully used to detect and quantify many metabolites derived from several metabolic pathways, including glycolysis, the pentose phosphate pathway, the TCA cycle, and glutamine metabolism from fresh, frozen, and formalin-fixed paraffin-embedded human tumors (48,49). Among the 119 different metabolites analyzed, several of them, including glucose 6-phosphate, fructose 1,6-bisphosphate, glyceraldehyde 3-phosphate, and ribose 5-phosphate, which we show are significantly elevated in FK866-treated cancer cells and tumor xenografts, were reported to be robustly detected from human tumor tissues (48,49). In addition, the amount (10 -15 mg or a 40-m formalinfixed paraffin-embedded slice) of materials required for this type of analysis may be obtained via needle biopsy.…”

“…Therefore, it is possible that a needle-based biopsying approach could obtain a sufficient amount of material for analysis. Finally, the tumor samples used in these two studies were frozen for 3-5 years on average (48,49), suggesting that these metabolites are stable. Second, the LC-MS method reported is feasible, and the changes in these metabolites represent the most direct and robust response of tumor cells to NAMPT inhibition.…”

Discovery of a Highly Selective NAMPT Inhibitor That Demonstrates Robust Efficacy and Improved Retinal Toxicity with Nicotinic Acid Coadministration

“…In addition, altered metabolite levels in tumors can be used as PD biomarkers for assessing effects of LSN3154567 on its intended target in the clinic. LC-MS methods have been successfully used to detect and quantify many metabolites derived from several metabolic pathways, including glycolytic pathway from fresh, frozen, and formalin-fixed paraffin-embedded human tumors (42,43). Thus, the LC-MS method developed in this study may be applicable to clinical use.…”