System-wide analysis of Extracellular Vesicle-Binding Cells in vivo by Abseq and sc-RNA-Seq
Background
We have developed very sensitive Phosphatidylserine (PS)-binding reagents that can detect the great majority (>90%) of all extracellular vesicles (EVs) in the blood, even those with little PS on their surface. When administered in vivo into mice, we also detect EV-binding cells in different tissues (Kranich et al., J Extracellular Vesicles, 2020; Rausch et al., J Extracellular Vesicles, 2021; Rausch et al., PNAS, 2023). As there is little known about cell-associated EVs and their target cells in different tissues, we combined our in vivo EV-labeling approach with single cell RNAseq. This generated a landscape of EV-binding cells in various organs and tissues and revealed gene expression pattern common to various cellular subset associated with EVs.
Methods
To detect EV-binding cells in scRNAseq experiments we injected mice with our highly sensitive PS-specific reagent. After isolation of cells we used the Abseq approach to identify cells bound to the PS-binding reagent. Abseq was combined with whole transcriptome sequencing to reveal the transcriptomes of EV-positive and EV-negative cells.
Results
Our scRNA-seq data revealed that macrophages, granulocytes, B cells and effector CD8 T cells are the main cell types that bind EVs. Enrichment analyses showed that especially genes involved in cell activation are differentially expressed in EV-binding cells.
Conclusion
Our approach reliably detects EV binding cells and allows whole transcriptome comparison between EV positive and EV negative cells. With this method we will identify signaling pathways that are triggered by EV binding and changes in gene expression that may allow cells to bind EVs. We are currently expanding this approach to more tissues and will also apply it to different disease models, such as inflammation, infection, autoimmunity and cancer to detect changes in EV-binding by different target cells.
Keywords
Phosphatidylserine, EV-binding cells, C1 tetramers
Funding/Acknowledgments
DFG-CRC 1054 (B03, Z01, Z02)
Authors
Jan Kranich1 (Corresponding Author: jkranich[at]bmc.med.lmu[dot]de), Franziska Prummer1, Tijana Kandic1, Christine Ried1, Thomas Brocker1
1LMU Munich, Institute for Immunology, Munich, Germany