Single-Cell Metabolic Profiling of Macrophages Using 3D OrbiSIMS: Correlations with Phenotype

Abstract

Macrophages are important immune cells that respond to environmental cues acquiring a range of activation statuses represented by pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes at each end of their spectrum. Characterizing the metabolic signature (metabolic profiling) of different macrophage subsets is a powerful tool to understand the response of the human immune system to different stimuli. Here, the recently developed 3D OrbiSIMS instrument is applied to yield useful insight into the metabolome from individual cells after in vitro differentiation of macrophages into naïve, M1, and M2 phenotypes using different cytokines. This analysis strategy not only requires more than 6 orders of magnitude less sample than traditional mass spectrometry approaches but also allows the study of cell-to-cell variance. Characteristic metabolites in macrophage subsets are identified using a targeted lipid and data-driven multivariate approach highlighting amino acids and other small molecules. The diamino acids alanylasparagine and lipid sphingomyelin SM(d18/16:0) are uniquely found in M1 macrophages, while pyridine and pyrimidine are observed at increased intensity in M2 macrophages, findings which link to known biological pathways. The first demonstration of this capability illustrates the great potential of direct cell analysis for in situ metabolite profiling with the 3D OrbiSIMS to probe functional phenotype at the single-cell level using molecular signatures and to understand the response of the human body to implanted devices and immune diseases.

Omics and systems view of innate immune pathways

“…Using a targeted approach to analyze the lipid palette (matching lipid ion peaks to LIPID MAPS database), it was found that M1 macrophages had the highest lipid counts and different lipid composition compared to M2 or M0. 38 The amino acid composition and other metabolites showed notable differences as well. Overall, the study represents a novel approach for in-situcharacterization of metabolic profiles to assess phenotypical differences between closely related cell types with single cell resolution.…”

Small molecule metabolites: discovery of biomarkers and therapeutic targets

“…Single-cell metabolomics technologies will reveal new insights into modulating metabolism with small molecules. 198 , 214 , 413 , 638 , 639 It can provide meaningful cell phenotype, enabling us to analyze cell status and obtain the overall biological information.…”

Omics and systems view of innate immune pathways

“…They can adopt stimulus induced phenotypes because of their functional plasticity. This allows macrophages to appropriately respond to diverse pathogens and aid in tissue repair following acute damage [70]. M0 monocytes represent the common progenitor lineage for the pro-inflammatory M1 phenotype and the anti-inflammatory M2 phenotype.…”

“…However, when coupled with the high mass resolving power and mass accuracy of an Orbitrap, characterizing metabolic profiles at the single-cell level is possible. Using a targeted approach to analyze the lipid palette (matching lipid ion peaks to the LIPID MAPS database), it was found that M1 macrophages had the highest lipid counts and dif-ferent lipid composition compared to M2 or M0 [70]. The amino acid composition and other metabolites showed notable differences as well.…”