Proteome Analysis of Human Natural Killer Cell Derived Extracellular Vesicles for Identification of Anticancer Effectors

Abstract

Cancer immunotherapy is a clinically validated therapeutic modality for cancer and has been rapidly advancing in recent years. Adoptive transfer of immune cells such as T cells and natural killer (NK) cells has emerged as a viable method of controlling the immune system against cancer. Recent evidence indicates that even immune-cell-released vesicles such as NK-cell-derived exosomes also exert anticancer effect. Nevertheless, the underlying mechanisms remain elusive. In the present study, the anticancer potential of isolated extracellular vesicles (EVs) from expanded and activated NK-cell-enriched lymphocytes (NKLs) prepared by house-developed protocol was evaluated both in vitro and in vivo. Moreover, isolated EVs were characterized by using two-dimensional electrophoresis (2-DE)-based proteome and network analysis, and functional study using identified factors was performed. Our data indicated that the EVs from expanded and active NKLs had anticancer properties, and a number of molecules, such as Fas ligand, TRAIL, NKG2D, β-actin, and fibrinogen, were identified as effector candidates based on the proteome analysis and functional study. The results of the present study suggest the possibility of NK-cell-derived EVs as a viable immunotherapeutic strategy for cancer.

Natural Killer Cell-Derived Extracellular Vesicles: Novel Players in Cancer Immunotherapy

“…In addition to expressing typical exosome markers (e.g., CD63, Rab5b, and TSG101), most studies have used CD56 as a marker to identify NKEVs isolated from CD56 + NK cells. Additionally, receptors of NK cells such as natural killer cell P30-related protein (NKp30), NKp46, NKp44, NKG2D, and DNAM1 were shown to be expressed by NKEVs in some studies and may be useful for their identification (6,12,14,17). However, definitive markers have yet to be established as CD56 is not exclusive to NK cells and a subset of NK cells are CD56 − .…”

“…For example, breast cancer cells (SKBR3) and melanoma cells (501mel) were shown to resist lysis of exosomes released by resting or activated NK cells even over a long period of coculture (6). However, it was later shown that exosomes derived from NK-92 MI cells had antitumor effects on melanoma both in vitro (B16F10 cells) and in vivo (8), and those isolated from IL-2/IL-15-activated NK cells showed cytotoxicity in breast cancer cell lines (MDA-MB-231/F and MCF-7) (12,15,16). The heterogeneous content of exosomes from different cellular sources may account for these inconsistent observations.…”

“…(14). NKEVs may transfer cargo to recipient cells via plasma membrane fusion, clathrin-mediated endocytosis, or receptor-mediated internalization (12,32). The cytotoxic effects of NKEVs were shown to be partly abolished by antibodies against Fas or Fas ligand (FasL), suggesting that ligandreceptor interaction is another mechanism by which NKEVs are targeted to tumor cells (6,8), although this has yet to be demonstrated experimentally.…”

“…NKEVs were found to reduce the expression of programmed death (PD)-1-a major immune checkpoint molecule that is expressed by a variety of immune cells and plays an important role in immune escape by tumors (54)-on CD3/CD28-stimulated T cells (7). Additionally, NKEVs contain many molecules related to the immune response including IFN-g, TNF-a, IL-10, MHC-I, and MHC-2, although their effects have not been well studied (10,12,14).…”

Natural Killer Cells: The Linchpin for Successful Cancer Immunotherapy

“…However, NK cells not only directly kill compromised cells, but when properly activated, can be potent producers of TNF-a and IFN-g, the last one being a known inducer of PD-L1 expression. Alternative mechanisms by which NK cells were shown to carry out their anti-tumor function involve expression of death receptor ligands FasL and/or TRAIL (9)(10)(11)(12) and release of extracellular vesicles, such as exosomes, with cytotoxic activity (13) that contain effector miRNAs [reviewed in (14)], cytokines, and display NK cell surface receptors (15)(16)(17). In addition to direct killing, NK cells secrete chemokines and cytokines to recruit and coordinate responses by other immune cells, such as T cells (7) and dendritic cells (DCs), in the tumor microenvironment or site of infection and can prime the adaptive immune response for better viral or tumor control (5)(6)(7)(18)(19)(20)(21)(22)(23) [reviewed in (24)] (Figure 1).…”