Exosomes Derived From Natural Killer Cells Exert Therapeutic Effect in Melanoma

Abstract

Objective: Exosomes are nanovesicles that are released from normal and tumor cells and are detectable in cell culture supernatant and human biological fluids. Although previous studies have explored exosomes released from cancer cells, little is understood regarding the functions of exosomes released by normal cells. Natural killer (NK) cells display rapid immunity to metastatic or hematological malignancies, and efforts have been undertaken to clinically exploit the antitumor properties of NK cells. However, the characteristics and functions of exosomes derived from NK cells remain unknown. In this study, we explored NK cell-derived exosome-mediated antitumor effects against aggressive melanoma in vitro and in vivo.Methods: B16F10 cells were transfected with enhanced firefly luciferase (effluc) and thy1.1 genes, and thy1.1-positive cells were immunoselected using microbeads. The resulting B16F10/effluc cells were characterized using reverse transcriptase polymerase chain reaction (RT-PCR), western blotting, and luciferase activity assays. Exosomes derived from NK-92MI cells (NK-92 Exo) were isolated by ultracentrifugation and density gradient ultracentrifugation. NK-92 Exo were characterized by transmission electron microscopy and western blotting. We also performed an enzyme-linked immunosorbent assay to measure cytokines retained in NK-92 Exo cells. The in vitro cytotoxicity of NK-92 Exo against the cancer cells was determined using a bioluminescence imaging system (BLI) and CCK-8 assays. To investigate the possible side effects of NK-92 Exo on healthy cells, we also performed the BLI and CCK-8 assays using the human kidney Phoenix™-Ampho cell line. Flow cytometry and western blotting confirmed that NK-92 Exo induced apoptosis in the B16F10/effluc cells. In vivo, we used a B16F10/effluc cell xenograft model to detect the immunotherapeutic effect of NK-92 Exo. We injected NK-92 Exo into tumors, and tumor growth progression was monitored using the IVIS Lumina imaging system and ultrasound imaging. Tumor mass was monitored after in vivo experiments.Results: RT-PCR and western blotting confirmed effluc gene expression and protein levels in B16F10/effluc cells. B16F10/effluc activity was found to increase with increasing cell numbers, using BLI assay. For NK-92 Exo characterization, western blotting was performed on both ultracentrifuged and density gradient-isolated exosomes. The results confirmed that NK cell-derived exosomes express two typical exosome proteins, namely CD63 and ALIX. We demonstrated by western blot analysis that NK-92 Exo presented two functional NK proteins, namely perforin and FasL. Moreover, we confirmed the membrane expression of FasL. The enzyme-linked immunosorbent assay results indicated that NK-92 Exo can secrete tumor necrosis factor (TNF)-α, which affected the cell proliferation signaling pathway. The antitumor effect of NK-92 Exo against B16F10/effluc cells in vitro was confirmed by BLI (p < 0.001) and CCK-8 assays (p < 0.001). Furthermore, in normal healthy cells, even after 24 h of ...

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

“…Their therapeutic effect was also studied using NK cell lines as exosome producers to facilitate cell expansion for vesicle retrieval. Exosomes from the NK-92MI cell line showed remarkable cytolytic activity against melanoma (23) and glioblastoma (24), while EVs from in vitro expanded NK cells cocultured with K562.mbIL21 cells displayed antitumor activities against acute lymphoblastic leukemia and breast carcinoma (25). Apart from a consistent equipment of molecules aimed at target killing, NKEVs were also found to contain miR-186, a tumor suppressor micro-RNA that contributes to their cytostatic effects in neuroblastoma and prevents the inhibitory effects on NK cells of transforming growth factor beta (TGFβ) (26).…”

Exosomes in chronic inflammatory skin diseases and skin tumors

“…Previous studies have discovered that natural killer (NK) cells may be associated with the immune response against tumors . NK‐92 cell line‐derived exosomes exert cytotoxic effects against melanoma, which are partly associated with the functional protein, FasL . These findings suggest that interaction between melanoma and other immune cells can also influence the pathogenesis of melanoma, both beneficially and pathologically.…”

The cancer exosomes: Clinical implications, applications and challenges

“…NK cells exert rapid immune effect to metastatic or hematological malignancies, which have been confirmed and clinically exploited the antitumor characteristics. Zhu et al . 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.…”

“…NK cells exert rapid immune effect to metastatic or hematological malignancies, which have been confirmed and clinically exploited the antitumor characteristics. 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.…”