Extracellular vesicles derived from natural killer cells use multiple cytotoxic proteins and killing mechanisms to target cancer cells

Abstract

Extracellular vesicles (EVs) are secreted membrane vesicles, which play complex physiological and pathological functions in intercellular communication. Recently, we isolated natural killer (NK) cell-derived EVs (NK-EVs) from ex vivo expansion of NK cell cultures. The isolated NK-EVs contained cytotoxic proteins and several activated caspases, and they induced apoptosis in target cells. In this report, the protein levels of cytotoxic proteins from NK-EV isolates were analysed by ELISA. The mean values of perforin (PFN, 550 ng/mL), granzyme A (GzmA, 185 ng/mL), granzyme B (GzmB, 23.4 ng/mL), granulysin (GNLY, 56 ng/mL), and FasL (2.5 ng/mL) were obtained from >60 isolations using dot plots. The correlation between cytotoxicity and cytotoxic protein levels was examined by linear regression. PFN, GzmA, GzmB, GNLY all had a positive, moderate correlation with cytotoxicity, suggesting that there is not a single cytotoxic protein dominantly involved in killing and that all of these proteins may contribute to cytotoxicity. To further explore the possible killing mechanisms, cells were treated with NK-EVs, proteins extracted and lysates assessed by Western blotting. The levels of Gzm A substrates, SET and HMG2, were diminished in targeted cells, indicating that GzmA may induce a caspase-independent death pathway. Also, cytochrome C was released from mitochondria, a central hallmark of caspase-dependent death pathways. In addition, several ER-associated proteins were altered, suggesting that NK-EVs may induce ER stress resulting in cell death. Our results indicate that multiple killing mechanisms are activated by NK-derived EVs, including caspase-independent and -dependent cell death pathways, which can mediate cytotoxicity against cancer cells. Abbreviations : NK: natural killer cells; aNK: activated NK cells; EV: extracellular vesicles; ER: endoplasmic reticulum; ALL: acute lymphoblastic leukaemia; FBS: foetal bovine serum. GzmA: granzyme A; GzmB: granzyme B; GNLY: granulysin; PFN: perforin

Characterization of Human NK Cell-Derived Exosomes: Role of DNAM1 Receptor in Exosome-Mediated Cytotoxicity against Tumor

“…No difference in surface antigen expression was found in exosomes from IL15-stimulated NK cells as shown in Figure 2B. Importantly, NK exosomes carried cytotoxicity-related proteins such as Perforin1, Granzyme A and B [29]. As for the case of surface antigens, their expression levels were similar in IL2 and IL15 NK cell-derived exosomes ( Figure 2C).…”

“…This could favor the exosome-binding and internalization, thus participating to the NK exosome-mediated cytotoxicity. Since apoptosis represents one of the mechanisms by which NK exosomes exert their effect on target cells, we further investigated whether blocking of DNAM1/DNAM1-L interaction could interfere with the induction of apoptosis by NK exosomes in target cells [29]. To this end, caspase activity was analyzed in tumor target cells after 2 hours incubation with NK exosomes in the presence or absence of blocking antibodies to DNAM1-ligands.…”

“…This latter may exert a cytotoxic effect on tumor target cells with consequent inhibition of tumor growth [25][26][27][28]. Tumor cell killing by NK exosomes has been suggested to act by multiple mechanisms involving Perforin/Granzymes and Fas-Fas Ligand [29,30].…”

Natural-Killer-Derived Extracellular Vesicles: Immune Sensors and Interactors

“…NKEVs express multiple cytotoxic proteins, such as perforin, FasL, granzyme A and B, granulysin (22,25), as well as molecules that activate NK-mediated cytolysis, i.e., CD40L. Indeed, reports indicate that NKEVs can trigger multiple killing mechanisms (52). NK cells and probably also NKEVs may possess regulatory functions (6).…”

The cancer exosomes: Clinical implications, applications and challenges

“…Zhu et al 77 explored that NK-92MI cells produce exosomes with two functional NK proteins, perforin, FasL and secreted tumor necrosis factor (TNF)-alpha, which affected the cell proliferation, to exert cytotoxic effects on melanoma cells without side effects. Furthermore, Wu et al 78 studied NK cell-derived exosomes and their result revealed that at NK-derived exosome presented perforin, granzyme A, granzyme B, granulysin and FasL could mediate cytotoxicity against cancer cells via caspase-independent and caspase-dependent cell death signaling pathways. Not only proteins presented on NK cells, exosomal tumor suppressor miR-186 also showed cytotoxicity to the amplified neuroblastoma.…”