Background/Purpose: Several tissue resident populations undergo metabolic reprogramming during tissue fibrosis as a phenotypic adaptation to their changing metabolic demands. However, these shifts in metabolic profiles in fibrotic tissues have not been previously studied at single cell level with spatial resolution. Furthermore, the spatial cellular organization within metabolic niches in fibrotic tissues was not explored until now.Here we aimed to identify and map distinct metabolically-defined fibroblast, macrophage and endothelial cell subsets in SSc skin, characterize their cellular metabolic microenvironments, and evaluate their association with progression of skin fibrosis in SSc.

Methods: Sections from skin biopsies of nine SSc patients and seven controls were prepared. Imaging mass cytometry (IMC), a spatial proteomics technique, was employed. An IMC panel of 38 metal-labelled antibodies, of which 22 antibodies targeting key enzymes and signalling molecules in several metabolic pathways was designed and validated in human skin. Data analysis was performed as described. The glycolysis or TCA/OXPHOS scores calculated with this approach robustly correlate with the activities of the respective pathways, as determined by functional assays.

Results: We analyzed the expression of key regulators of metabolic pathways such as glycolysis, tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), fatty acid oxidation and synthesis, aminoacid internalization, pentose phosphate pathway, hypoxia, reactive oxygen species production as well as of regulators of mitochondrial dynamics, collectively termed as metabolic regulome, in SSc and control skin.Dermal cells of SSc patients with progressive skin fibrosis had a remarkably distinct metabolic regulome compared with patients with stable skin fibrosis and to controls, with upregulation of glycolysis and TCA/OXPHOS scores.Unbiased fibroblast clustering identified eight distinct fibroblasts subsets defined by their metabolic regulome. Of these, a subpopulation of fibroblasts with high expression of glycolysis and TCA/OXPHOS enzymes (termed Methi Fib) was upregulated in SSc patients with progressive skin fibrosis, and expressed high levels of markers of activated fibroblasts, such as αSMA and FAP. The metabolically-defined fibroblast subsets formed niches with endothelial cells and macrophages with similar levels of metabolic pathway activities, e.g. the Methi fibroblasts were located in spatial proximity to endothelial cells and macrophages that expressed high levels of glycolysis and TCA/OXPHOS enzymes.

Conclusion: We identified distinct metabolically-defined subsets fibroblasts in SSc and control skin, with an enrichment of metabolically highly active fibroblasts in progressive skin fibrosis. This fibroblast subset expressed myofibroblast markers and formed niches with endothelial cells and macrophages with a similar metabolic profile. Thus, metabolic phenotyping of fibroblasts and their niches by IMC-based spatial proteomics might identify SSc patients at risk for progression of skin fibrosis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/mapping-metabolic-changes-in-skin-fibrosis-in-systemic-sclerosis-by-spatial-proteomics/