Opportunities and challenges of natural killer cell-derived extracellular vesicles

Abstract

Extracellular vesicles (EVs) are increasingly recognized as important intermediaries of intercellular communication. They have significant roles in many physiological and pathological processes and show great promise as novel biomarkers of disease, therapeutic agents, and drug delivery tools. Existing studies have shown that natural killer cell-derived EVs (NEVs) can directly kill tumor cells and participate in the crosstalk of immune cells in the tumor microenvironment. NEVs own identical cytotoxic proteins, cytotoxic receptors, and cytokines as NK cells, which is the biological basis for their application in antitumor therapy. The nanoscale size and natural targeting property of NEVs enable precisely killing tumor cells. Moreover, endowing NEVs with a variety of fascinating capabilities via common engineering strategies has become a crucial direction for future research. Thus, here we provide a brief overview of the characteristics and physiological functions of the various types of NEVs, focusing on their production, isolation, functional characterization, and engineering strategies for their promising application as a cell-free modality for tumor immunotherapy.

Keywords: cancer immunotherapy; engineering strategy; extracellular vesicles; natural killer cell; tumor microenvironment.

FIGURE 1

The cell sources of NEVs could be mainly divided into peripheral blood mononuclear cells (PBMC) and other cell lines. In the purification process of NEVs, the more mature isolation methods include ultracentrifugation (UC), ultrafiltration, and size exclusion chromatography (SEC). Purified NEVs have anti-tumor activity and immunomodulatory effects, and engineered modifications can confer new functions on NEVs. Common NEVs engineering techniques can be classified as exogenous and endogenous modifications. Exogenous modifications: (A) NEVs can be used to prepare the PTX-NEVs drug delivery system through electroporation (Han et al., 2020). (B) The therapeutic potential of doxorubicin-loaded NEVs shows promising antitumor activity in vivo against the MCF-7 induced tumor model (Pitchaimani et al., 2018). (C) Light-activatable silencing NK-derived exosomes (LASNEO) are orchestrated by engineering the NEVs with hydrophilic small interfering RNA (siRNA) and hydrophobic photosensitizer Ce6 (Zhang et al., 2022). (D) NEVs are used as a versatile toolkit to synergistically improve adoptive T-cell therapy for solid tumors (Nie et al., 2021). (E) NEVs are in combination with their biomimetic core–shell nanoparticles for tumor-targeted therapy (Wang et al., 2019). Endogenous modifications: (F) The NK cell is lentivirally transduced to express and load BCL-2 siRNAs (siBCL-2) into NEVs (20).

FIGURE 2

The surface receptors of NEVs, loaded cytotoxic proteins, and functional miRNAs induce apoptosis in tumor cells. In addition to stimulating the polarization of macrophages towards M1 and activating T cells directly or via activated monocytes, NEVs can also activate resting NK cells, thereby augmenting their tumor-killing ability.

FIGURE 3

(A) NEVs activate monocytes. Flow cytometry analysis of CD80–CD86 geo mean fluorescence intensity (gMFI) of gated CD14⁺ cells in PBMCs cultured in the presence or absence of NEVs, and/or lipopolysacharide (LPS) for 24 h. Upper panels: Representative dot plots showing CD80–CD86 expression in the presence of NEVs, lower panel: Flow cytometry of human leukocyte antigen DR isotype (HLA-DR) gMFI of CD14⁺ gated monocytes (Federici et al., 2020). (B) Flow cytometry analysis of CD25 expression by CD3⁺ gated T cells in PBMCs evaluated after 72 h of culture with NEVs (Federici et al., 2020). (C) The graph shows the results obtained with PBMCs of different healthy donors (n = 3), in the presence or absence of transforming growth factor beta (TGFβ)/interleukin (IL)-10 (10 ng/ml each) (Federici et al., 2020). (D) NEVs affect the interaction between monocytes and T cells. Flow cytometry analysis of 72 h proliferation and CD25 expression by CD3, CD4, and CD8 T cells cultured in the presence of monocytes (medium), monocytes preconditioned with NEVs (Federici et al., 2020). (E) NEVs induce the release of cytokines by PBMCs. Cytometric bead array-measured cytokine production of 72 h PBMCs cultured (Federici et al., 2020). (F–H) Activation of resting NK cells by NEVs affects the expression of natural cytotoxicity receptors on their surface and tumor-killing viability (Shoae-Hassani et al., 2017). (F) NK cells were stained with specific NCRs monoclonal antibodies, a resting NK cell expresses different levels of NCRs. (G) The NEVs induce the expression of NCRs especially NKp44 similar to cytokine-activated NK. (H) In vitro cytotoxicity of peripheral blood natural killer cells against neuroblastoma (NB) cells. NEVs strongly stimulated NK activity in the presence of IL-21.

FIGURE 4

(A) Schematic illustration of light-activatable silencing NK-derived exosomes (LASNEO) mediated synergetic tumor eradication. (B) Binding of NEVs to CLTs via click chemistry reaction. (C) Schematic design of the NN/NKEXO cocktail for tumor targeting and drug delivery. (D) Loading of BCL-2 siRNAs (siBCL-2) in NK-92MI-derived EVs by lentiviral transfection.