Single Cell Metabolic Profiling of Tumor Mimics

Abstract

Chemical cytometry employs modern analytical methods to study the differences in composition between single cells to better understand development, cellular differentiation, and disease. Metabolic cytometry is a form of chemical cytometry wherein cells are incubated with and allowed to metabolize fluorescently labeled small molecules. Capillary electrophoresis with laser-induced fluorescence detection is then used to characterize the extent of metabolism at the single cell level. To date, all metabolic cytometry experiments have used conventional two-dimensional cell cultures. HCT 116 spheroids are a three-dimensional cell culture system, morphologically and phenotypically similar to tumors. Here, intact HCT 116 multicellular spheroids were simultaneously incubated with three fluorescently labeled glycosphingolipid substrates, GM3-BODIPY-FL, GM1-BODIPY-TMR, and lactosylceramide-BODIPY-650/665. These substrates are spectrally distinct, and their use allows the simultaneous probing of metabolism at three different points in the glycolipid metabolic cascade. Beginning with intact spheroids, a serial trypsinization and trituration procedure was used to isolate single cells from spatially distinct regions of the spheroid. Cells from the distinct regions showed unique metabolic patterns. Treatment with the lysosomal inhibitor and potential chemotherapeutic chloroquine consistently decreased catabolism for all substrates. Nearly 200 cells were taken for analysis. Principal component analysis with a multivariate measure of precision was used to quantify cell-to-cell variability in glycosphingolipid metabolism as a function of cellular localization and chloroquine treatment. While cells from different regions exhibited differences in metabolism, the heterogeneity in metabolism did not differ significantly across the experimental conditions.

Drug penetration and metabolism in 3D cell cultures treated in a 3D printed fluidic device: assessment of irinotecan via MALDI imaging mass spectrometry

“…Cells in the outer layers have ready access to oxygen and nutrients and are proliferative, while cells in the inside layers have decreased access to nutrients and oxygen. These inner layers of cells become quiescent, while those in the center of the spheroid develop into a core of necrotic cells [7,[9][10][11][12][13].…”

Quantitative Determination of Irinotecan and the Metabolite SN-38 by Nanoflow Liquid Chromatography-Tandem Mass Spectrometry in Different Regions of Multicellular Tumor Spheroids

“…Cells from these MCTS were separated layer by layer using serial trypsinization [22-24]. A solution of 0.05% trypsin/EDTA at room temperature was added, and the MCTS were gently rotated for 3 min on a rotary shaker at approximately 60 rpm.…”

“…In a previous study employing serial trypsinization, the isolated fractions of cells from surface layers, the intermediate region, and the necrotic core of MCTS were analyzed with quantitative iTRAQ chemistry to examine differences in the protein expression profiles in distinct spatial areas of MCTS [23]. We also previously employed serial trypsinization in combination with single cell capillary electrophoresis to study the metabolism of glycosphingolipids in different regions of MCTS [24]. …”

Chemometric analysis of MALDI mass spectrometric images of three-dimensional cell culture systems

“…When plated under specific growth conditions, these cells grow into spheres that can reach up to 1 mm in diameter. At this size, these models develop nutrient, oxygen and transport gradients that generate biologically distinct microenvironments within the spheroids 3, 13 . Importantly, these microenvironments closely resemble different populations of cells found in large tumors in vivo .…”