Aptamer-Assisted Traceless Isolation of PD-L1-Positive Small Extracellular Vesicles for Dissecting Their Subpopulation Signature and Function

Abstract

Small extracellular vesicles (sEVs) are heterogeneous membrane-bound vesicles that carry numerous bioactive molecules. Studies have reported that sEVs carrying PD-L1 on the surface could contribute to immunosuppression; however, the precise mechanisms are unclear. To fully dissect their mode of action, it requires qualified methods to specifically isolate natural PD-L1-positive sEVs from heterogeneous sEVs. This study reported an aptamer-assisted capture-and-release strategy for traceless isolation of PD-L1-positive sEVs. The PD-L1 aptamer-anchored magnetic microspheres enable the specific capture of PD-L1-positive sEVs. The traceless release of captured PD-L1-positive sEVs was triggered by competition of complementary oligonucleotides, endowing the obtained label-free PD-L1-positive sEVs with natural properties. Benefited from this traceless isolation strategy, the distinct molecule profiles in adhesion and immuno-regulation between PD-L1-positive and PD-L1-negative sEVs were revealed. Compared to PD-L1-negative sEVs, PD-L1-positive sEVs were much more concentrated in cadherin binding, accompanied by increased adhesion to lymphatic endothelial cells and T cells but decreased adhesion to the extracellular matrix. Moreover, PD-L1-positive sEVs could transfer their enriched immunosuppressive “synapse”-related proteins to antigen-presenting cells, thereby inducing a tolerogenic-like phenotype. In summary, the present work dissects the subpopulation signature and action mode of PD-L1-positive sEVs for the first time and provides a general approach to the traceless isolation of sEV subpopulations.

Reliable Detection of Extracellular PD-L1 by DNA Computation-Mediated Microfluidics

“…A myriad of methods have been developed for EV PD-L1 detection. − High-speed centrifugation can isolate EV PD-L1 from soluble PD-L1 protein by precipitation. , However, this is too time-consuming and cumbersome for clinical applications. Hence, a more convenient microfluidic affinity sorting method has been developed.…”

Extracellular vesicles in tumor angiogenesis and resistance to anti‐angiogenic therapy

“…As a critical intercellular communicator, EVs carry different bioactive molecules (lipids, nucleic acids, and proteins) and can deliver those cargoes to receipt cells to participate in many biological processes, particularly in the development of cancers 4 . A growing body of evidence now suggest that tumor cell‐derived T‐EVs directly transfer bioactive cargoes to ECs to promote tumor angiogenesis 5,6 . Moreover, T‐EVs exert pro‐angiogenic effects via other cells, such as fibroblasts and immune cells.…”

Aptamers Targeting the PD-1/PD-L1 Axis: A Perspective

“…14 Their biological behavior has been studied in more detail. 14 Such PD-L1 + EVs were able to better bind cadherin with increased adhesion to lymphatic endothelial cells and T cells but with decreased adhesion to the extracellular matrix. They are also able to transfer immunosuppressive properties to antigen-presenting cells.…”

“…This way, they may contribute to T cell inactivation. PD-L1 + EVs have been isolated with microfluidic systems or in pure form from sample media with magnetic beads coated with PD-L1 aptamer to separate bound EVs from the bulk, followed by competitive binding to the aptamer to release the EVs . Their biological behavior has been studied in more detail .…”

“…PD-L1 + EVs have been isolated with microfluidic systems 13 or in pure form from sample media with magnetic beads coated with PD-L1 aptamer to separate bound EVs from the bulk, followed by competitive binding to the aptamer to release the EVs. 14 Their biological behavior has been studied in more detail. 14 Such PD-L1 + EVs were able to better bind cadherin with increased adhesion to lymphatic endothelial cells and T cells but with decreased adhesion to the extracellular matrix.…”