High-throughput single-cell lipidomics reveals stage-specific phospholipid remodeling and ferroptosis-related signatures in the Evolution of plasma cell dyscrasia

Abstract Introduction: Multiple myeloma (MM) evolves through a well-defined clinical continuum from monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM) to active MM, with plasma cell leukemia (PCL) representing the most aggressive terminal stage. A critical feature driving this evolution is the inter- and intra-tumoral heterogeneity of myeloma cells, which dictates disease risk and prognosis. Metabolic reprogramming, particularly lipid metabolic remodeling, represents a hallmark of oncogenesis that regulates tumor progression and shapes the microenvironment.​The accompanying remodeling of lipid metabolism, especially at the structural and isomeric levels, remains poorly understood in MM, despite its critical roles in membrane organization, signaling, and oxidative stress pathways such as ferroptosis. We applied a novel high-throughput single-cell lipidomics platform integrating mobility-modulated sequential dissociation (MMSD), to systematically characterize the lipid metabolic profiles of plasma cells derived from bone marrow across MGUS, SMM, MM, and PCL. We further investigated changing trends of differentially expressed lipids and explored their potential association with ferroptosis susceptibility, aiming to provide new metabolic insights for early intervention and targeted therapy in MM. Methods: Bone marrow samples from MGUS, SMM, MM, and PCL patients were collected. Mononuclear cells were enriched by density gradient centrifugation, followed by fluorescence-activated cell sorting to purify CD38+CD138+ plasma cells. Single-cell lipid profiling was performed using a high-throughput platform, allowing accurate resolution of lipid isomers and isobars. Briefly, individual cells were introduced into mass spectrometer via a custom three-layered capillary system. Ion mobility separation was carried out under alternating low- and high-energy frames, and IM-MS together with wide-window IM-MS/MS spectra were deconvoluted to establish fragment–precursor relationships, enabling precise lipid identification at the single-cell level. Lipidomic differences among disease stages were visualized using UMAP and PLS-DA, and differential lipid features were further examined. Results: We enrolled a total of 1 MGUS, 1 SMM, 3 MM, and 2 PCL patients. UMAP analysis revealed clear clustering of plasma cells corresponding to respective stages, indicating robust intra-group consistency and clear inter-stage differences. We further focused on the changes of specific lipid isomers across disease groups. In PCL relative to MM, PE 38:4, identified by MMSD as PE 18:0_20:4, was significantly downregulated, while PS 38:4 (PS 18:0_20:4) was upregulated, suggesting a potential shift in PE-to-PS balance that may reduce lipid peroxidation substrates and attenuate ferroptosis sensitivity. Structural analysis of PE 36:4 revealed a progressive decrease in the ratio of PE 16:0_20:4 to PE 18:2_18:2 from MM to PCL, with marked inter-patient heterogeneity among MM cases. This variability may reflect differences in metabolic states, suggesting that PE isomer ratios could serve as potential biomarkers of front-line response. A similar decreasing trend was observed in PC 36:4 (PC 16:0_20:4 vs. PC 18:2_18:2), reinforcing the notion of coordinated phospholipid remodeling. Furthermore, PE 40:6 (comprising PE 18:0_22:6 and PE 18:1_22:5) exhibited a marked reduction in the 18:0_22:6 isomer during early transition from MGUS to SMM, which may be associated with disrupted immune homeostasis and altered membrane composition. Collectively, these results reveal dynamic reorganization of phospholipid species and isomeric distributions during alteration of clonal plasma cells, with implications for ferroptosis regulation. Conclusions： Our high-throughput single-cell lipidomics approach provides the first structural lipidomic atlas in different stages of plasma cell disorders, highlighting stage-specific remodeling of PE, PS, and PC species as well as their isomeric diversity. The observed PE-to-PS shift and progressive changes in isomer ratios underscore a potential metabolic mechanism for ferroptosis evasion and refractoriness, particularly in active MM and PCL. These results offer new insights into lipid-driven mechanisms of myeloma development and provide a foundation for exploring lipid-based biomarkers and therapeutic targets.