Single-cell profiling uncovers extracellular vesicle-associated malignant plasma cell subpopulations driving multiple...

Multiple myeloma (MM) is a heterogeneous hematological malignancy characterized by the clonal proliferation of plasma cells in the bone marrow, with distinct subtypes including smoldering MM (SMM), newly diagnosed MM (NDMM), and relapsed/refractory MM (RRMM). Despite therapeutic advances, outcomes remain unsatisfactory, especially for RRMM, due to unclear heterogeneity, progression mechanisms, and crosstalk between tumor cells and the bone marrow microenvironment (BMME) via direct interactions or extracellular vesicles (EVs).

Single-cell RNA sequencing (scRNA-seq) was performed on bone marrow samples from 12 MM patients, and major cell types were identified. Plasma cells were subjected to re-clustering to explore subpopulation heterogeneity. Functional enrichment analysis of differentially expressed genes (DEGs) was conducted. Cellular stemness was evaluated by CytoTRACE, and developmental trajectories were inferred via Monocle and Slingshot. Cell-cell communication was analyzed by CellChat, while transcription factor (TF) regulatory networks were identified through SCENIC analysis. Metabolic pathway activity was also assessed. Finally, loss-of-function experiments (siRNA-mediated ASS1 knockdown) were conducted to validate its functional role.

The analysis identified 7 major cell types. Plasma cells were further stratified into 6 subpopulations (C0-PCSK1N+, C1-IGHGP+, C2-IGHA1+, C3-ASS1+, C4-CD27+, C5-STMN1+). C3 and C5 were enriched in RRMM, while C1 and C4 were dominant in SMM. C3 exhibited hyperactive EV signature and higher stemness-like scores. Pseudotime trajectory analysis identified C3 as poorly differentiated malignant progenitors driving disease progression. C3 showed strong crosstalk with monocytes/macrophages/conventional dendritic cells (cDCs) via MIF and ICAM signaling. Key TFs regulating C3 included ATF5, TP73, MYB, CEBPB, and NFIA. Metabolic pathway analysis indicated enhanced vitamin B6 metabolism, phenylalanine metabolism, and oxidative phosphorylation in C3 and RRMM. ASS1 silencing inhibited proliferation, clonogenic capacity and migration, while promoting apoptosis in MM cells.

Our study delineates MM heterogeneity, developmental dynamics, and regulatory networks at the single-cell level. ASS1+ plasma cells represent a highly malignant subpopulation associated with RRMM, driving disease progression through unique TF regulatory networks, metabolic reprogramming, and crosstalk with the BMME. These findings provide novel insights into MM pathogenesis and identify ASS1 as a potential therapeutic target for MM patients, particularly those with RRMM.