Novel alternatives to extracellular vesicle-based immunotherapy – exosome mimetics derived from natural killer cells

Abstract

Exosomes are endogenous nanocarriers that can deliver biological information between cells. They are secreted by all cell types, including immune cells such as natural killer (NK) cells. However, mammalian cells release low quantities of exosomes, and the purification of exosomes is difficult. Here, nanovesicles were developed by extrusion of NK cells through filters with progressively smaller pore sizes to obtain exosome mimetics (NK-EM). The anti-tumour effect of the NK-EM was confirmed in vitro and in vivo. The morphological features of the NK-EM were revealed by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and Western blot. In vitro, the cytotoxicity of the NK-EM to cancer cells (glioblastoma, breast carcinoma, anaplastic thyroid cancer and hepatic carcinoma) was assessed using bioluminescence imaging (BLI) and CCK-8 assay. For in vivo study, a xenograft glioblastoma mouse model was established. The anti-tumour activity of NK-EM was confirmed in vivo by the significant decreases of BLI, size and weight (all p < .001) of the tumour compared with the control group. Moreover, NK-EM cytotoxicity for glioblastoma cells that related with decreased levels of the cell survival markers p-ERK and p-AKT, and increased levels of apoptosis protein markers cleaved-caspase 3, cytochrome-c and cleaved-PARP was confirmed. All those results suggest that NK-EM exert stronger killing effects to cancer cells compared with the traditional NK-Exo, at the same time, the tumour targeting ability of the NK-EM was obtained in vivo. Therefore, NK-EM might be a promising immunotherapeutic agent for treatment of cancer.

NK Cell-Based Glioblastoma Immunotherapy

“…NK cells are defined by the expression of CD56 (neural cell adhesion molecule) and the absence of the T-cell co-receptor CD3. Regarding the surface expression of the CD56 protein, there are two sets of NK cells: the CD56 bright subset, that comprises 10% of the circulating NK cells, is more proliferative but shows lower cytotoxicity toward cancer cells; and the CD56 dim subset, that comprises the majority of circulating NK cells (90%) and has a higher capacity to recognize and kill target cells [39,40]. Although CD56 dim NK cells are predominant in the blood, CD56 bright are the most abundant NK subset in the human body because of their concentration in lymphoid and non-lymphoid tissues.…”

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

“…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].…”

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

“…As expected, we showed that activation of caspase −3, −7 and −9 was detected in cancer cells incubated with NK‐EVs, and caspase inhibitors (−2, −3, −6, −8, −9, −10, −12) blocked NK‐EV‐induced cytotoxicity, suggesting that NK‐EVs activate caspase pathways in target cells [19]. The ability to isolate highly cytotoxic NK‐EVs on a large scale may lead to new clinical applications [24,25]. In this scenario, one needs to determine whether the yield of EVs and levels of the cytotoxic proteins in separate isolations vary depending on sources and environmental factors.…”