Comparison of characteristics and tumor targeting properties of extracellular vesicles derived from primary NK cells or NK-cell lines stimulated with IL-15 or IL-12/15/18

Abstract

NK cell-based therapies have shown promise for hematological cancer forms, but their use against solid tumors is hampered by their poor ability to infiltrate the tumor. NK cells release extracellular vesicles (EVs) containing cytolytic proteins, indicating that NK-cell derived EVs may have therapeutic potential. In this study, we compared the tumor-targeting potential of EVs derived from either primary NK cells or the NK cell lines NK-92 and KHYG-1 cultured in IL-15 alone or in combination with IL-12 and IL-18. Primary NK cells were also stimulated through the activating receptor CD16. Tumor cell apoptosis was measured using a panel of human colon, melanoma, glioblastoma, prostate, breast, and ovarian tumor cell line spheroids. NK cells or NK-92 cells stimulated with IL-12, IL-15, and IL-18 generated EVs with higher efficiency than EVs from resting cells, although similar amounts of EVs were produced under both conditions. Proteomic analysis indicated similar distribution of cytolytic proteins in EVs from primary NK cells and NK-92, but lower levels in KHYG-1 EVs that translated into poor capacity for KHYG-1 EVs at targeting tumor cell lines. Further, we show that CD16-stimulated NK cells release low amounts of EVs devoid of cytolytic proteins. Importantly, EVs from cytokine-stimulated NK cells penetrate into the spheroid core, and tumor spheroid susceptibility to NK-cell derived EVs was linked to differential expression of the NKG2D ligands MICA/B, which was blocked with an anti-NKG2D antibody. We conclude that EVs from activated primary NK cells or NK-92 cells has the best potential to infiltrate and target solid tumors.

Isolation of a cytolytic subpopulation of extracellular vesicles derived from NK cells containing NKG7 and cytolytic proteins

“…Fraction 7 were enriched for EVs with more translucent EVs, indicating a separate subpopulation of EVs. We previously reported enrichment of CD63, CD81, granzyme B and perforin in fraction 4 of SEC-separated EVs ( 14 ). TEM analysis demonstrated similar morphology of vesicles separated by SEC ( Figure 1G ).…”

“…Furthermore, the microvesicle/ectosome marker CD99 ( 22 ), was exclusively detected in DG-UC fraction 5 with our mass spectrometry analysis, further confirming the enrichment of microvesicles within this fraction. Interestingly, we previously showed that the NK cell line KHYG-1 predominantly released EVs containing CD81 and not CD63, and that these vesicles contained low amounts of cytolytic proteins and lacked in killing activity towards tumor targets ( 14 ). These findings support the view that CD81 is present on two distinct vesicle subtypes, where CD81 + EVs may derive mainly from the plasma membrane, whereas a distinct population of CD81 + CD63 + vesicles is predominantly endosomally derived.…”

“…We and others have shown that bulk NK-EVs induce tumor cell apoptosis (9)(10)(11)(12)(13)(14). We hypothesized that the fractionation process would enrich for cytolytic EVs, and therefore next compared apoptosis induced by EVs within the different fractions obtained by DG-UC or SEC.…”

“…However, NK cell therapies for solid tumors have been more difficult to achieve, partly due to their low ability to infiltrate the tumor ( 7 , 8 ). NK-cell derived extracellular vesicles (EVs) could circumvent this problem, and previous studies have shown that bulk EVs from NK cells (NK-EVs) contain cytolytic proteins, and can induce apoptosis of diverse tumor cell lines ( 9 – 14 ). The exact mechanism for how tumor cell death is induced by NK-EVs is still unclear, but the EVs appear to express several activating receptors such as DNAM-1 and NKG2D, as well as FasL, that could mediate contact with the tumor cells and induce apoptosis ( 9 , 13 , 14 ).…”

Cytokine-Induced Memory-Like NK Cells: From the Basics to Clinical Applications

“…peripheral blood and umbilical cord blood). Interestingly, a recent report revealed that extracellular vesicles derived from IL-12/15/18-stimulated NK-92 cells showed higher ability to induce apoptosis in HCT116 colon cancer cell spheroids in comparison with IL-15-stimulated controls ( 86 ). These results suggests that NK-92 cells are somehow sensitive to IL-12/15/18 stimulation and thus it would be interesting to explore if memory-like behavior could be imprinted in NK cell lines.…”