1033 - Single-Cell Transcriptome Profiling Reveals Neoplastic Effects of Exosomal S100A4 in Chronic Lymphocytic Leukemia

Introduction
The crosstalk between chronic lymphocytic leukemia (CLL) cells and immunocytes favors the progression of CLL by promoting the survival of tumor cells and exhaustion of immunocytes. Illustrating the characteristics of CLL cells and immunocytes is an attractive strategy for the treatment of CLL patients. Profiling the peripheral blood mononuclear cells (PBMCs) in CLL patients and healthy donors with single-cell RNA sequencing (scRNA-seq) could provide a detailed molecular and cellular map of PBMCs of CLL patients and healthy donors.

Methods
In the current study, the PBMCs from CLL patients and healthy donors were studied at a single-cell resolution. scRNA-seq was performed on the PBMCs of CLL patients and healthy donors. Flow cytometry assays, and quantitative real-time polymerase chain reaction were performed to verify cellular identity and function.

Results
This study presented a large-scale single-cell transcriptomic atlas of human PBMCs and identified the subclusters of CLL cells, T cells, and natural killer (NK) cells (Figure 1A-D). CLL cells exhibited unique genetic markers compared with normal B cells, including cell proliferation, cell cycle progression and extracellular exosome (Figure 1E). Genes related to the secretion of exosome including VPS28, CD63 and CD81 were upregulated in CLL cells (Figure 1F-H). Exosome inhibitor GW4869 induced cell apoptosis and G0/G1 phase arrest in CLL cells.

T cells of CLL patients were investigated to elucidate the molecular mechanisms of T-cell dysfunction in the progression of CLL. T cells of CLL patients presented the hallmarks of exhaustion and the proportion of exhausted CD8+ T cells was upregulated (Figure 2A-C). Exosome from CLL cells promoted the exhaustion of T cells. The expression of S100A4 was upregulated in the exosome secreted by CLL cells (Figure 2D). Knocking down of S100A4 promoted the apoptosis of CLL cells (Figure 2E). Furthermore, knocking down of S100A4 reserved the exhaustion of CD8+ T cells induced by exosome from CLL cells.

Conclusions
The present study revealed the transformation of immune cells during the progression of CLL. The interactions between CLL cells and immune cells were investigated to elucidate the cell communications which accelerated the progression of CLL. Conclusively, this study elucidated the mechanisms of CLL progression through illustrating the characteristics of PBMCs of CLL patients and presenting the cell communications between CLL cells and immunocytes.