Abstract
Myelomatous effusion (ME) is a rare manifestion of extramedullary multiple myeloma (MM), characterized by the presence of neoplastic plasma cells (PCs) in bodily fluids, including pleural effusion and ascites. ME occurs in less than 1% of MM cases with limited therapeutic options, the median survival of patients with ME is only 4 months. The pathogenesis of ME has not been fully elucidated. This study performed single-cell RNA sequencing of malignant PCs and other immune cells in three MM patients relapsed with ME, aiming to identify PC clones able to migrate and survive in extramedullary sites.
We found ME contained a higher percentage of cytotoxic T cells, whereas peripheral blood composed a higher proportion of naive T cells through investigating the immune microenvironment intra-/extra-BM. T cells in ME are proliferating and promoting myeloma cell survival independent of the bone marrow microenvironment.
Malignant cells varied within and between sites and patients in their expression of signatures, suggesting the intertumoral heterogeneity of PCs. To investigate cell migration of PCs, we found that a group of gene modules was highly expressed in intramedullary and extramedullary PC clusters using CNMF analysis, suggesting that these modules are associated with cell viability and migration. We speculate that the gene set is a key factor in the migration of myeloma cells into the serous cavities, therefore, we defined cell clusters expressing such gene sets as extramedullary-initiating cells (EMICs). These gene sets were associated with increased cellular proliferation, involving in p53 signaling, and related to poor prognosis in MM. The transcriptional regulators essential for the survival and growth of MM tumors, such as E2F1, YY1, and SMAD1, were activated in EMICs.
We further examined the genetic differences between intramedullary and extramedullary EMICs. The expression of leukocyte immunoglobulin-like family proteins LILRB4, the S100 family protein (S100A8/A9), and long noncoding RNA (MALAT1 and NEAT1) were upregulated in extramedullary EMICs. LILRB4, an immune inhibitory receptor that contains putative immunoreceptor tyrosine-based inhibitory (ITIM) motifs, was highly expressed in the PCs of ME than those in the bone marrow (Wilcoxon, p<2.2e-16).
To verify the role of LILRB4 on extramedullary infiltrating, we used CRISPR-Cas9 strategy to knock-out LILRB4 (LILRB4-KO), and overexpressed LILRB4 (LILRB4-OE) by lentiviral transfection in MM cell line (NCI-H929). The cleaved PARP and CASPASE3 upregulated in LILRB4-KO cells and downregulated in LILRB4-OE cells. LILRB4-OE induced arrest in the G1 phase, while the percentage of cell in G1 phase was strikingly decreased in LILRB4-KO cells. The effect of LILRB4 on myeloma cell migration was evaluated by trans-well chamber testing. LILRB4 overexpression enhanced cell migration. Meanwhile, we observed decreased trans-endothelial migration in LILRB4-KO cell lines. LILRB4 increased the mRNA expression of angiopoietin 1, integrin αv, and integrin β3, indicating LILRB4 may promoted PC invasion through integrin family.
In conclusion, these data suggested EMICs and LILRB4 associated with extramedullary development and provided insight into evolutionary mechanisms of ME.
Disclosures
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
