Omic evaluation of G-CSF-induced changes in bone marrow: A focus on lipid metabolism Free

Introduction: Granulocyte Colony Stimulating Factor (G-CSF) is a mobilizing factor of hematopoietic stem cells (HSC). Protein and lipid metabolism play important roles in HSC mobilization. The importance of proteases, membrane lipid layers, and the enzyme phospholipase C (PLC) in G-CSF-induced mobilization has been previously demonstrated. However, there are no sufficient studies about the effect of lipid metabolism on mobilization. This study aims to elucidate the metabolic pathways occurring within the bone marrow (BM) niche during G-CSF-induced mobilization, with a particular focus on lipid metabolism.

Materials and Methods: In this study,we examined the bone marrow plasma samples of 30 healthy individuals who were healthy donors for HSC transplantation. Metabolomic-lipidomic analyses and PLC enzyme activity analysis were performed on samples from individuals with (2 or 3 days) and without in vivo G-CSF exposure before HSC collection. Metabolomic profiling was conducted using gas chromatography-mass spectrometry (GC-MS), while lipidomic profiling utilized liquid chromatography–quadrupole time-of-flight mass spectrometry (LC-qTOF-MS). PLC enzyme activity was assessed using the Amplex Red phosphatidylcholine-specific phospholipase C (PC-PLC) assay kit (A12218; Molecular Probe, Eugene, OR, USA) according to the protocol. Statistical comparisons between groups were performed using Student's t-test and one-way analysis of variance (ANOVA), with p < 0.05 considered statistically significant. Enrichment and pathway analysis were carried out using the MetaboAnalyst and Lion Web platform.

Results: Metabolomic analysis revealed statistically significant increases in 3-hydroxypropanoic acid, fucose, glutamic acid, O-phospho-L-serine, phosphoric acid, ribitol, trimethyllysine, myo-inositol, and oxaloacetic acid in the group exposed to G-CSF. Conversely, levels of alpha-glucosamine 1-phosphate, proline, and threonine were decreased in the G-CSF-exposed group. The most prominent changes observed in the lipidomic analysis involved ether-linked phosphatidylcholines, which are essential components of the cell membrane and lipid rafts. Although no statistically significant changes were detected in PLC enzyme activity, mean activity levels decreased after G-CSF exposure. Notably, increases in myo-inositol and phosphoric acid were also observed in the G-CSF-exposed group. Enrichment and pathway analysis revealed that the highest pathway impact was observed in lipid-related pathways (including carnitine synthesis, mitochondrial transport of acetyl groups, and inositol metabolism), whereas energy-related pathways (including glycolysis, the Warburg effect, the pentose phosphate pathway, the citric acid cycle, pyruvate metabolism, the malate-aspartate shuttle, and galactose metabolism) were the most frequently altered pathways based on the total pathway number.

Discussion: Our findings support the hypothesis that G-CSF contributes to HSC mobilization through multiple mechanisms. Although alterations related to protein metabolism were identified following G-CSF exposure in our study data, prominent changes were predominantly observed in energy and lipid metabolism. The observed increase in fucose levels is likely related to alterations in the fucosylation pathway, suggesting suppression of adhesion within the BM niche and promotion of mobilization. The decrease in essential amino acids may reflect increased cellular proliferation and protein synthesis demands following G-CSF exposure. Increased levels of myo-inositol and phosphoric acid may point to a reduction in glycosylphosphatidylinositol (GPI) anchor formation after G-CSF exposure. Alterations in lipid rafts and ether-linked phosphatidylcholines are indicative of G-CSF-related immunoregulatory and inflammatory responses. In this respect, the data obtained in our study is unique and will contribute to the development of bone marrow-targeting treatments that will affect healthy and pathological hematopoiesis.