Determination of fatty acid uptake and desaturase activity in mammalian cells by NMR-based stable isotope tracing.

BACKGROUND: Mammalian cells both import exogenous fatty acids and synthesize them de novo. Palmitate, the end product of fatty acid synthase (FASN) is a substrate for stearoyl-CoA desaturases (Δ-9 desaturases) that introduce a single double bond into fatty acyl-CoA substrates such as palmitoyl-CoA and stearoyl-CoA. This process is particularly upregulated in lipogenic tissues and cancer cells. Tracer methodology is needed to determine uptake versus de novo synthesis of lipids and subsequent chain elongation and desaturation. Here we describe an NMR method to determine the uptake of 13C-palmitate from the medium into HCT116 human colorectal cancer cells, and the subsequent desaturation and incorporation into complex lipids. RESULTS: Exogenous 13C16-palmitate was absorbed from the medium by HCT116 cells and incorporated primarily into complex glycerol lipids. Desaturase activity was determined from the quantification of double bonds in acyl chains, which was greatly reduced by ablation of the major desaturase SCD1. SIGNIFICANCE: The NMR approach requires minimal sample preparation, is non-destructive, and provides direct information about the level of saturation and incorporation of fatty acids into complex lipids.

Mass Spectrometry Imaging and Histology for the Analysis of Budding Intestinal Organoids.

Three-dimensional (3D) organoids have been at the forefront of regenerative medicine and cancer biology fields for the past decade. However, the fragile nature of organoids makes their spatial analysis challenging due to their budding structures and composition of single layer of cells. The standard sample preparation approaches can collapse the organoid morphology. Therefore, in this study, we evaluated several approaches to optimize a method compatible with both mass spectrometry imaging (MSI) and immunohistological techniques. Murine intestinal organoids were used to evaluate embedding in gelatin, carboxymethylcellulose (CMC)-gelatin-CMC-sucrose, or hydroxypropyl methylcellulose (HPMC) and polyvinylpyrrolidone (PVP) solutions. Organoids were assessed with and without aldehyde fixation and analyzed for lipid distributions by MSI coupled with hematoxylin and eosin (H&E) staining and immunofluorescence (IF) in consecutive sections from the same sample. While chemical fixation preserves morphology for better histological outcomes, it can lead to suppression of the matrix-assisted laser desorption/ionization (MALDI) lipid signal. By contrast, leaving organoid samples unfixed enhanced MALDI lipid signal. The method that performed best for both MALDI and histological analysis was embedding unfixed samples in HPMC and PVP. This approach allowed assessment of cell proliferation by Ki67 while also identifying putative phosphatidylethanolamine (PE(18:0/18:1)), which was confirmed further by tandem MS approaches. Overall, these protocols will be amenable to multiplexing imaging mass spectrometry analysis with several histological assessments and help advance our understanding of the biological processes that take place in district subsets of cells in budding organoid structures.