Single-Cell Multi-Omics Analysis Reveals Heterogeneity of Malignant Plasma Cell during Progression of Multiple Myeloma
Authors: Feng Fangshuo , Sun Hao , Zhang Xiaoyu , Sun Xiyue , Yu Tengteng , Xiong Haiqing , An Gang , Qiu Lu-Gui , Hao Mu 
Published: December 2024

Multiple myeloma is a malignant plasma cell disease of the bone marrow, MM have a relatively benign stage of MGUS, which further progresses to SMM and eventually progresses to active MM, MM undergoes extramedullary invasion evolving into PCL, Heterogeneity analysis of plasma cells in these four phases facilitates our understanding of this evolution and the targeting of therapies.

A total of bone marrow samples from healthy controls (n=7) and patients with MGUS (n=2), SMM (n=5), MM (n=6), and PCL (n=2) were enrolled in this study for bone marrow single-cell multi-omics sequencing of bone marrow mononuclear cells (BMNCs), Based on the expression of genes characterizing each cell subpopulation, we define 10 cell types including plasma cells. Firstly, in order to elucidate the molecular expression characteristics of plasma cells and MM cells in each stage, we Identify malignant plasma cells from all plasma cells using inferCNV, which led to the identification of malignant plasma cells in each stage. Further subcluster analysis revealed significant disease stage heterogeneity of MM cells in patients with different disease stages such as MGUS, SMM, MM, and PCL, and MM cell heterogeneity gradually increased with disease progression. We also found that the proportion of malignant plasma cells with similar transcriptional characteristics to normal plasma cells gradually decreased from MGUS to PCL. GSVA analysis of the 4 stages of MM cells confirmed that the molecular features of MM cells in the MGUS and SMM stages are similar, molecular features in the MM and PCL stages are similar too. The UAMS70 gene set allows scoring of the degree of malignancy of MM cells, the degree of malignancy is determined based on the score, we observe a slow increase in the malignancy of plasma cells from MGUS to MM stage, but from MM to PCL stage showed a sharp increase, and, the SMM stage has a relatively similar proportion of malignant plasma cells with transcriptional similarity to normal plasma cells. Cytotrace shows a gradual increase in the differentiation capacity of malignant plasma cells from MGUS to PCL stage. In a cell trajectory analysis of malignant plasma cells from MGUS to PCL, it is found that cell development is divided into nine states, the more important of which are state 1, state 2, and state 8, and that there are significant differences in the distribution of the stages from MGUS to PCL both in terms of disease stage and between patients. State 2 has cells with low differentiation capacity, state 1 and state 8 have high differentiation capacity, MGUS and SMM are mainly distributed in state 2 and state 8, and PCL is distributed in state 8 and state 1. It is noteworthy that one of the 2 patients with PCL has cells completely distributed in state 1, and the other completely in state 8, indicating that even though there are differences in transcriptome expression, the cell trajectory of PCL cells from the same patient are similar. This indicates that even though there are differences in transcriptome expression, the cell trajectory distribution of the cells of the same patient in PCL is similar, and from the MGUS to the PCL, the cells of each patient tend to be more and more distributed mainly in one branch. Next, the HALLMARK gene set is used to score each state, and it is found that DNA damage repair, G2M checkpoint, immune response, and glycolysis pathways are enriched in all three states, suggesting that malignant plasma cells are activated with proliferative capacity and stimulate the immune response at all stages, and that glycolytic metabolism plays an important role. Notably, two PCL patients showed different states, one with activation of the p53 pathway and IL6-JAK-STAT3 pathway, and the other with activation of the MYC pathway and NOTCH pathway, suggesting the diversity of mechanisms driving PCL invasion in the patients. Moreover, it is found that some cells at the MGUS, SMM, and MM stages are already in the PCL state branch, suggesting that even at the early stage of the disease, some cells have already reached a more malignant state. VDJ analysis shows that the proportion of clonal plasma cells increases from the MGUS to the PCL stage, and single clone appears in multiple clusters, suggesting that even malignant plasma cells of the same clone differ greatly at the level of gene expression.

In summary, we find that the heterogeneity of plasma cells is evident in all aspects.