(PA-522) Glycosylation Single-Cell Transcriptomic Profiling Decodes Driver Mechanism and Genetic Characteristics of Circulating Plasma Cells in Multiple Myeloma

Multiple myeloma (MM) is a hematologic malignancy involving clonal plasma cells in the bone marrow (BM). Circulating plasma cells (CPCs) in peripheral blood (PB) are detectable in newly diagnosed MM (NDMM) patients and serve as a prognostic marker. However, the biological mechanisms behind CPC formation remain unclear. This study explored CPC heterogeneity, genetic features, and migration mechanisms.

Methods:

We comprehensively analyzed transcriptomes and glycoproteomics in paired BM and PB samples from 4 NDMM patients using glycosylation single-cell RNA sequencing (scRNA-seq). Subsequently, we included paired samples from an additional 10 NDMM patients for further analysis by CyTOF.

Results:

Single cell RNA-seq revealed distinct transcriptomic and glycan profiles in CPCs versus BM plasma cells (BMPCs). CPCs overexpressed genes linked to migration (EMP3, AHNAK), adhesion (TYROBP, CD44), angiogenesis (LGALS1), and osteoclastogenesis (ANXA2), while BMPCs upregulated MPO. CPCs showed lower therapeutic target (BCMA, FcRH5) but higher drug-resistance gene expression. Glycosylation was more abundant in BMPCs.

We categorized BMPCs and CPCs into 14 distinct cell clusters (C1-C14). Only the C12 subcluster was present in BM and PB with a relatively higher glycosylation burden. RNA velocity analysis revealed C12 exhibited a strong directional flow toward other subclusters. Survival analysis based on the MMRF CoMMpass dataset (n = 858) showed that patients with high C12 had an inferior OS compared to low C12 (p < 0.0001). These findings indicate that C12 is the CPC-initiating cell cluster, essential for CPC production and a prognostic factor for survival. Next, we identified a total of 11 gene modules by hotspot analysis. We found that module 9, which highly expressed 21 genes (including PTTG1, CDC20, STMN1, RRM2, and HMGB1), was shared by both BMPCs and CPCs. KEGG enrichment analysis showed genes in module 9 were enriched for the cell cycle. Differentially expressed gene analysis between C12 in BM and PB showed that SPP1 was upregulated in medullary CPC-initiating cells. The high expression of  module 9 gene set in BM is a prerequisite for the production of CPCs, and on this basis, upregulation of SPP1 is the key to PB migration of MM cells. Furthermore, CyTOF analysis substantiated the presence of a subpopulation in CPC-initiating cells, which exhibited a characteristic MM stem cell-like phenotype of CD19+CD27-CD138-.

Mean Spearman correlation analysis revealed MM cells positively correlated with T/NK cells (R = 0.87, p < 0.001). Proliferating T cells exhibited metabolic activation and communicated with CPC-initiating cells via SPP1 signaling.

 

Conclusions:

In this study, we employed multi-omics techniques to precisely analyze the heterogeneity of CPCs from multiple perspectives of the transcriptome and proteome. Our results demonstrated that the circulation of MM was driven by specific CPC-initiating cells with unique genetic profiles and interaction with immune microenvironment cells.