NK cells have four antitumor pathways: activated receptors/antitumor molecules, the antibody-dependent cellular cytotoxicity (ADCC) effect, cytolytic granule release, and cytokine secretion[2]. Many cell surface and intracellular factors are involved in these four pathways, and their activation induces cell injury through various signaling pathways. The major activated receptors/antitumor molecules were NKp30, NKp44, NKp46, TRAIL, FASL, CD226, and NKG2D. NKp30, NKp44, and NKp46 are natural cytotoxic receptors. They regulate cytotoxic and cytokine-secreting functions through downstream signal activation following binding with FcεRIγ and/or CD3ζ (NKp46 and NKp30) and DAP12 (NKp44) after binding to their ligands[25], [26]. After TRAIL binds to its death receptors (DR4 and DR5) and FasL binds to Fas, they activate the extrinsic and intrinsic apoptosis pathways and induce transcriptional events leading to NF-κB-dependent proinflammatory cytokine expression [27–30]. CD226 activates downstream signaling cascades that activate phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-2 (PLCγ2), ERK, and AKT downstream and remove the negative regulator of NK cell activation through phosphorylation of the forkhead box protein O1 transcription factor via activated AKT [31, 32]. NKG2D binding with its ligand promotes cytotoxicity, granule release, and cytokine release through activation of the DAP10 signaling molecule and the following signals: PLCγ2, c-Jun-NH (2)-terminal kinase, phosphatidylinositol 3-hydroxy kinase (PI3K), and Janus kinase 2-signal transducer and activator of transcription 5 (JAK-STAT5) pathway[33, 34]. CD16 (IgG-activated Fc receptor III) recruits SYK family kinases via crosslinking by immune complexes and induces ADCC effects by activating several other signaling molecules and their downstream signals, including the PI3K and SOS pathways[35].
NK cells secrete cytolytic granules, including the pore-forming protein, perforin, and the serine protease, granzyme B, which synergistically mediate the apoptosis of target cells [36]. NK cells also secrete various cytokines, such as TNFα, IFNα, and IFNγ. TNFα induces apoptosis and necroptosis through the kinase receptor-interacting serine/threonine-protein kinase 1 after binding to TNFR1, which is associated with the death domain (TRADD)[37, 38]. IFNα and IFNγ can bind to their respective receptors and activate several pathways, including the JAK-STAT pathway, to coordinate different cell functions, such as immune regulation, leukocyte transportation, cell proliferation, apoptosis, and antimicrobial, antitumor, and pro-tumor effects [39, 40].
We analyzed these cell surface and intracellular cytotoxic factors expressed on eNK cells. Antitumor-related molecules (TRAIL, CD226, and NKG2D) and an ADCC-inducing molecule (CD16) were more highly expressed in these cells than in NK-92 cells (Fig. 3, Supplementary Table 1). In addition, perforin, granzyme B, TNFα, and IFNγ were highly expressed (Fig. 4, Supplementary Table 2). The eNK cells are derived from genetically transfected iPSCs through hematopoietic progenitor cells (HPCs) to NK cells. During the process of generating NK cells from HPCs, the surface molecules expressed during their maturation fluctuate, and their functions differ depending on the maturation stage [41–43]. The high expression of factors involved in all four antitumor pathways of NK cells indicates that eNK cells have a high antitumor and mature NK cell phenotype.
In the activated receptor/antitumor molecular pathway, the expression of their ligands on cancer cells is necessary for efficient binding to the receptor and an effective immune response through NK cell activation. Cancer cell lines are widely used in in vitro studies. Although HepG2 and HuH7 are the most commonly used cell lines in hepatocarcinoma studies, approximately 40 liver cancer cell lines have been established from patients with different disease backgrounds[44]. We initially selected eight cell lines (HepG2, C3A, HuH7, PLC/PRF/5, SNU-387, SNU-423, SNU-449, and SK-HEP-1). SK-HEP-1 was derived from a patient with adenocarcinoma, whereas the other cell lines were derived from patients with HCC. Additionally, each cell line contained different mutated genes[44]. Although many studies using these cell lines have been conducted, the expression status of ligands for NK cell antitumor molecules in liver cancer cell lines has not yet been elucidated. In this study, we focused on the antitumor molecules (TRAIL, CD226, and NKG2D) that trigger different antitumor signals, and examined the expression status of these ligands in eight liver cancer cell lines. Our results revealed that each cell line could be characterized according to its sensitivity to TRAIL, NKG2D, CD47, and PD1. HuH7 barely expressed NKG2D ligands, whereas almost all other cell lines appeared to be sensitive to NKG2D (Fig. 5a, Supplementary Table 3a). The expression of DR5 varied between approximately 10% and 100% (Fig. 5b, Supplementary Table 3b). Furthermore, each cell line showed variable expression of CD47, PDL1, and PDL2 (Fig. 5d, Supplementary Table 3d). Even in patients, HCC shows heterogeneous features at both the molecular and morphological levels. Thus, the key to enhance the treatment efficacy of eNK cells is whether they can show antitumor effects against cancer cells with various phenotypes. We selected HepG2 (high sensitivity to NKG2D and TRAIL, low sensitivity to PD1), HuH7 (moderate sensitivity to TRAIL, low sensitivity to NKG2D and PD1), and SNU423 (low sensitivity to TRAIL, high sensitivity to NKG2D and PD1) cells (Fig. 5, Supplementary Table 3) for cytotoxicity assays. The eNK cells showed remarkably high cytotoxicity against all three cell lines compared with that of NK-92 cells (Fig. 6). We did not observe a significant difference in the cytotoxicity of eNK cells between the cell lines at E/T ratios = 5 and 10; however, a difference was observed in the cytotoxic activity between cells at an E/T ratio = 1 (Fig. 6a). This may be due to differences in the expression of ligands for the antitumor molecules of NK cells on the surfaces of each HCC cell line.
It has been reported that, in the process of serial killing, NK cells initially predominantly use the perforin/granzyme B pathway to rapidly kill tumor cells; however, they later switch to death receptor-mediated cytotoxicity, which requires a longer time to induce cell death when granules are reduced[45]. Our blocking assays revealed that the TRAIL and perforin/granzyme B pathways are largely involved in the cytotoxicity mechanisms of eNK cells (Fig. 7, Fig. 8), and the effects of perforin/granzyme B and TRAIL pathway inhibition were consistent with previous reports[45]. Thus, eNK cells also have the potential to switch killing mechanisms during the serial killing.
Although we focused on the in vitro cytotoxicity and mechanisms of action of eNK cells in the present study, their effects in in vivo models remain unclear and require further investigation.
In conclusion, eNK cells have a strong antitumor phenotype and high cytotoxic activity against HCC cell lines with various phenotypes. Therefore, eNK cells may represent a novel therapeutic strategy candidate for HCC.