SCENITH: A Flow Cytometry-Based Method to Functionally Profile Energy Metabolism with Single-Cell Resolution, 2020, Argüello et al.

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SCENITH: A Flow Cytometry-Based Method to Functionally Profile Energy Metabolism with Single-Cell Resolution
Rafael J. Argüello; Alexis J. Combes; Remy Char; Julien-Paul Gigan; Ania I. Baaziz; Evens Bousiquot; Voahirana Camosseto; Bushra Samad; Jessica Tsui; Peter Yan; Sebastien Boissonneau; Dominique Figarella-Branger; Evelina Gatti; Emeline Tabouret; Matthew F. Krummel; Philippe Pierre

Energetic metabolism reprogramming is critical for cancer and immune responses. Current methods to functionally profile the global metabolic capacities and dependencies of cells are performed in bulk.

We designed a simple method for complex metabolic profiling called SCENITH, for single-cell energetic metabolism by profiling translation inhibition. SCENITH allows for the study of metabolic responses in multiple cell types in parallel by flow cytometry. SCENITH is designed to perform metabolic studies ex vivo, particularly for rare cells in whole blood samples, avoiding metabolic biases introduced by culture media. We analyzed myeloid cells in solid tumors from patients and identified variable metabolic profiles, in ways that are not linked to their lineage or their activation phenotype.

SCENITH’s ability to reveal global metabolic functions and determine complex and linked immune-phenotypes in rare cell subpopulations will contribute to the information needed for evaluating therapeutic responses or patient stratification.

Link | PDF (Cell Metabolism)

 

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This technique was used in Transcriptional reprogramming from innate immune functions to a pro-thrombotic signature by monocytes in COVID-19 (2022)

 

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Cells can produce ATP either by oxidative phosphorylation (OXPHOS) or by performing glycolysis. Aerobic glycolysis supports not only proliferation but also cell survival in hypoxic conditions. Immune cells are specially equipped to migrate into peripheral tissues and to adapt to the change in microenvironment. [...] Activation of T cells is generally linked to a metabolic switch from OXPHOS to aerobic glycolysis.

Current methods to determine metabolic profiles can be classified into three groups. The first group, epitomized by Seahorse (Agilent Technologies), uses metabolic inhibitors (i.e., 2-deoxyd-glucose [DG] and oligomycin A [O]) while monitoring the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). This method requires a large amount of purified cells to be incubated with special culture media for several hours

The second group quantifies the maximal activities of enzymes, such as the maximal dehydrogenase activity, by adding high concentrations of substrates to single cells using histochemistry. Finally, the third group uses mass spectrometry (MS) and MS imaging to measure the levels of metabolites produced by metabolic pathways

Cell sorting and incubation with cell culture media can change the metabolic activity of the cells, and thus they cannot be applied to establish metabolic profiles of heterogenous and scarce living cell populations obtained from human blood samples or biopsies, ex vivo.

We aimed to complement the toolbox for metabolic studies in the single-cell era by developing a method that functionally determines the overall metabolic capacities and dependencies of cells independent of their phenotype.

Approximatively half of the total energy that mammalian cells produce by degrading glucose, amino acids, and/or lipids is immediately consumed by the protein synthesis (PS) machinery. The tremendous energetic cost associated with this essential metabolic process offers a methodological opportunity

Our results demonstrate that our method is ideal for analyzing heterogenous samples, from which the details of metabolism, particularly among rare immune cell subsets, have remained inaccessible.

 

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SCENITH is a simple method for complex immuno-metabolic profiling. It allows the user to simultaneously establish the phenotype and extract the global metabolic profile of multiple cell types in parallel, ex vivo.

This rapid and sensitive method is consistent and comparable to other established techniques, including Seahorse. As the treatments with inhibitory drugs are performed in parallel, SCENITH can be used to monitor cellular responses to a combination of multiple metabolites and inhibitors.

The metabolic profile of immune subsets in the blood correlates with migratory capacity and effector functions in the body. Neutrophils and monocytes migrate into hypoxic/ damaged tissues and are already engaged in aerobic glycolysis in the blood. We confirmed this in human monocytes and neutrophils in the blood and within the tumor.

The metabolic profile of innate and adaptive immune cells correlates with the type of cytokines they produce; thus, the metabolic profile represents a universal readout for functional state