(I-335) Natural Killer Cell Derived Small Extracellular Vesicles for Glioblastoma Therapy

Glioblastoma multiforme (GBM) is an aggressive form of brain cancer that is difficult to treat due to the selectivity of the blood brain barrier (BBB). Natural killer (NK) cells are immune cells that have anti-tumorigenic properties. However, various components in the GBM tumor microenvironment inhibit proper cell function, including tumor macrophages and high TGF-β, a cytokine that would inhibit NK cell activity. NK cell-derived small extracellular vesicles (NK-sEVs) have been shown to exhibit similar characteristics as their parent cells like the NKG2D receptor and apoptotic proteins such as Granzyme B and FasL while also being small enough to pass through the BBB [1]. Considering NK-sEV localization to GBM cells remains low, this project aims to improve GBM localization and efficacy of the NK-sEVs by developing and integrating a targeting peptide onto the sEV membrane and to demonstrate NK-sEV anti-cancer properties against tumor cells.

NK-92MI and U87 cells were incubated in exosome-depleted media before sEVs were isolated from collected culture media through differential centrifugation, filtration, and ultracentrifugation. sEVs were characterized using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) to confirm sEV size. NK-sEV Granzyme B and FasL proteins were quantified using an enzyme-linked immunosorbent assay (ELISA), while anti-cancer microRNA was quantified using quantitative real time polymerase chain reactions (qRT-PCR). U87 cells were incubated with IL-13αD micelles and NK-sEVS modified with an IL-13αD targeting peptide in order to demonstrate enhanced binding and cytotoxicity. MTS assays were used to detect BBB and GBM-associated cell viability after NK-sEV treatment to confirm safety against brain cells and toxicity against tumor cells. A Transwell model was used to further confirm NK-sEV penetration capabilities through an in vitro BBB model.

TEM and NTA confirmed that the NK-sEVs were within the expected size range of 100-150 nm for both the unmodified and the targeting samples. The IL-13αD peptide was found to significantly enhance both micelle and NK-sEV binding onto U87 cells, and it did not inhibit the ability for the NK-sEVs to be taken up by the U87 cells. Granzyme B and FasL ELISAs (n=4) demonstrated significantly higher quantities of the apoptotic proteins compared to U87 cells and sEVs as well (p< 0.01 and p< 0.0001, respectively). qRT-PCR showed higher quantities of anti-cancer microRNAs as well compared to the U87 cell and U87-sEV controls. MTS assays on astrocyte, pericyte, and BBB endothelial cells confirmed full cell viability after treatment with NK-sEV and U87-sEV samples. An MTS assay was also used to evaluate therapeutic potential of NK-sEVs against U87 cells. IL-13αD NK-sEVs, NK-sEVS, and U87-sEVs were incubated with U87 cells for 24 hours and analyzed for cell viability through colorimetry. Overall, the targeting NK-sEVs demonstrated the highest toxicity against U87 cells, while both NK-sEV samples showed increased toxicity against U87 cells compared to the U87-sEV control. Lastly, a Transwell model confirmed that NK-sEVs had enhanced BBB penetration and localization onto U87 cells compared to non-treated control groups.

[1] Federici, C., et. al. Natural-killer-derived extracellular vesicles: Immune sensors and Interactors. Frontiers in Immunology, 2020.