Bioactivity of Exosomes Derived from Trained Natural Killer Cells versus Non‐Trained One: More Functional and Antitumor Activity

Abstract

Background. Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system, capable of killing viral-infected and cancerous cells. NK cell-mediated immunotherapy has remarkably changed the current paradigm of cancer treatment in recent years. It emerged as a safe and effective therapeutic approach for patients with advanced-stage leukemia. Several immune-escape mechanisms can be enacted by cancer cells to avoid NK-mediated killing. Exosomes released by NK cells that carry proteins and miRNAs can exert an antitumor effect. In the present study, we hypothesized that maybe exosomes derived from trained natural killer cells show more antitumor effect in comparison to non-trained one. Methods. PBMC was separated by the Ficoll method and cultured with IL-2 for 21 days to expand NK cells. The NK cells were co-cultured with K562 for 72 hours and exosome-derived co-cultured (as trained) and natural killer cell-derived exosomes (as non-trained) were extracted by Exo kit. The exosomes were confirmed by dynamic light scattering (DLS), transmission electron microscopy (TEM), flow cytometry, and western blotting. The K562 cells were separately treated by trained and non-trained exosomes and MTT assay, apoptosis, and real-time PCR were performed. Results. Based on flow cytometry, CD56 marker was 89.7% and 40.1% for NK cells and NK-derived exosomes, respectively. CD63 and CD9 were positive for exosomes by western blotting. The morphology of exosome was confirmed by TEM. Treated K562 cells by trained exosomes indicated the diminished cell viability and higher apoptosis. Furthermore, the trained exosomes showed up-regulation in both P53 and caspase3 genes as compared with non-trained sample. Discussion. Trained Exos showed a potent inhibitory effect on proliferation and induced apoptosis on K562 cell lines compared to the same dose of non-trained Exos. According to the results of qRT-PCR, trained Exos exerted an antitumor activity through up-regulation of caspase 3 and P53 in the apoptotic signaling pathway in tumor cells. Our findings indicate an effective action of trained Exos against cancer cells.

Application of exosomes in tumor immunity: recent progresses

“…As an important part of innate immune responses, exosomes secreted by NK not only carry the characteristic markers of exosomes, but also the markers of NK, such as NKG2D, perforin, granzyme, and CD40L ( Federici et al, 2020 ). Some studies have found that exosomes extracted from NK after co-culture with K562 cells can upregulate the expression of caspase three and p53 genes in tumor cells apoptotic signaling pathway to exert anti-tumor activity and exert rapid cytotoxic activity on target cells, which is more effective than intact NK ( Mohammadi et al, 2022 ). T lymphocytes can include CD4 + T cells and CD8 + T cells because of the different CD molecules they express.…”

Immunoregulatory functions and therapeutic potential of natural killer cell-derived extracellular vesicles in chronic diseases

“…This orchestrated response is notably accompanied by a marked increase in the production of NK-EVs ( 47 ). Furthermore, EVs produced by memory-like NK cells exhibit greater toxicity towards cancer cells ( 48 ).…”

“…EVs derived from NK cells previously exposed to neuroblastoma cells (NB), which express NCRs and activation signals, can activate resting human NK cells, enhancing their NK-mediated anti-NB tumor response ( 49 ). NK cells exposed in advance to EVs derived from CML cells exhibit higher gene expression levels of caspase 3 and P53 compared to the untreated EVs group, showing stronger cytotoxic effects on tumor cells ( 48 ).…”

Cytochalasin B‐Induced Membrane Vesicles of NK Cells for Efficient Tumor Immunotherapy

“…[13] Because NK vesicles express typical surface markers and contain similar biological components as parental NK cells, active vesicles released from NK cells can induce apoptosis in tumor cells and have therefore been developed as promising anticancer agents. [14][15][16] Despite their therapeutic potential, the clinical application of EVs has been hindered by their low productivity and an incomplete understanding of their functional mechanisms. To overcome these limitations of EVs, we artificially induced EVmimicking vesicles on NK cells by chemical treatment and mechanical stimulation.…”