Antitumor effects of natural killer cells derived from gene-engineered human induced pluripotent stem cells on hepatocellular carcinoma

Antitumor effects of natural killer cells derived from gene-engineered human induced pluripotent stem cells on hepatocellular carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Antitumor effects of natural killer cells derived from gene-engineered human induced pluripotent stem cells on hepatocellular carcinoma Mayuna Nakamura, Yuka Tanaka, Keishi Hakoda, Masahiro Ohira, Tsuyoshi Kobayashi, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4765613/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Feb, 2025 Read the published version in Cancer Immunology, Immunotherapy → Version 1 posted 13 You are reading this latest preprint version Abstract Mortality and recurrence rates of hepatocellular carcinoma (HCC) remain high despite the use of various treatment methods. Recently, cell-based immunotherapy using natural killer (NK) cells has attracted considerable attention in cancer immunotherapy. NK cells generated from induced pluripotent stem cells (iPSCs) are a new option for use as an NK cell resource. The eNK cells (HLCN061, developed by HEALIOS K.K.) are human iPSC-derived NK cells differentiated from clinical-grade iPSCs in which IL-15, CCR2B, CCL19, CD16a, and NKG2D have been introduced. In this study, we aimed to evaluate the potential of eNK cell therapy for HCC treatment. The analysis of eNK cells for cell surface and intracellular molecules revealed that antitumor-related surface molecules (TRAIL, CD226, and CD16) and intracellular cytotoxic factors (perforin, granzyme B, TNFα, and IFNγ) were highly expressed. In addition, eNK cells exhibited high cytotoxicity against HCC cell lines (HepG2, HuH7, and SNU-423), which are sensitive to NKG2D, TRAIL, and CD226. The TRAIL and perforin/granzyme B pathways are largely involved in this cytotoxic mechanism, as indicated by the reduction in cytotoxicity induced by TRAIL inhibitory antibodies and concanamycin A, which inhibits perforin/granzyme B-mediated cytotoxicity. Our data suggest that eNK cells, whose functions have been enhanced by genetic engineering, have the potential to improve HCC treatment. hepatocellular carcinoma iPS cells NK cells anti-tumor effect genetic engineering cell therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Cell-based immunotherapy has received considerable attention, and various cancer therapeutic approaches have been developed over the past several decades. Natural killer (NK) cells are innate immune cells that account for approximately 15% of circulating blood lymphocytes[ 1 ]. NK cells possess various functional factors and target abnormal cells, such as cancer and virus-infected cells, without prior sensitization. This feature has attracted attention because of its applications in cancer immunotherapy. NK cell products can be generated from multiple sources, such as peripheral and umbilical cord blood, NK cell lines, and induced pluripotent stem cells (iPSCs)[ 2 ]. Compared with blood-derived NK cells, the NK-92 cell line and NK cells derived from iPSCs can be cultured on a large scale and manufactured off-the-shelf. In addition, because these cells are more easily transduced than blood-derived NK cells are, the development of NK cells with enhanced functions, such as transgenic NK and chimeric antigen receptor (CAR)-NK cells, is currently underway. The iPSCs represent a new option for NK cell generation, and several generation methods have been described[ 3 , 4 ]. To enhance their functions, iPSC-derived NK cells expressing functional molecules and CARs have been developed using genetically engineered technologies[ 5 – 7 ]. In preclinical studies, iPSC-NK cells have shown effector cytotoxic responses in vitro against a variety of hematological and solid tumor cell lines, including lung, hepatocellular, and ovarian cancers, as well as myeloid leukemia and melanoma[ 3 , 8 ]. Clinical trials using iPSC-derived NK and CAR-NK cells, alone or in combination with other drugs, have been conducted, and some data have provided promising results[ 6 – 8 ]. Liver cancer is the sixth most common malignancy and third most common cause of cancer-related deaths worldwide[ 9 ]. Moreover, hepatocellular carcinoma (HCC) recurrence occurs in approximately 40–80% of patients within five years of hepatic resection or radiofrequency ablation[ 10 ]. Although there are various treatment options for HCC, such as surgical resection, liver transplantation, thermal ablation, transarterial chemoembolization, and drugs, new therapeutic options are needed to improve treatment effects and reduce recurrence. Recent studies have shown that targeting NK cells can help in the treatment of HCC, the restoration of normal liver function, and to subsequently increase survival rates of patients with HCC[ 11 ]. Initially, we focused on NK cell therapy to prevent HCC recurrence. We previously reported that activated donor liver-derived NK cells are effective in preventing the postoperative recurrence of liver cancer, and the main mechanisms involved were the high expression of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), perforin activity, and interferon (IFN)γ production[ 12 – 14 ]. However, because liver-derived NK cells can only be obtained from liver transplant donors, they are difficult to apply to liver cancer treatment. Other research groups have also reported that peripheral blood NK cells activated by various cytokines[ 15 ], glypican 3 (GPC3)-specific CAR-engineered NK cells[ 16 ], NK cells expressing the NKG2D-CD3ζ-DAP10 receptor[ 17 ], and NK cells with TLR7/TLR8 agonists[ 18 ] are effective for HCC treatment[ 11 , 19 ]. Clinical trials using NK cells alone or in combination with other drugs have been conducted for HCC but have not yet reached practical applications[ 20 ]. Therefore, the development of NK cells with high therapeutic efficacy for HCC is required. In this study, we aimed to evaluate the potential of eNK cells (HLCN061; developed by HEALIOS K.K.), which are human iPSC-derived NK cells differentiated from clinical grade iPSCs in which IL-15, CCR2B, CCL19, CD16a, and NKG2D have been introduced, in the treatment of HCC. 2. Materials and methods 2.1 Cell lines and cell culture HepG2, C3A, SK-HEP-1, PLC/PRF/5, SNU-387, SNU-423, SNU-449, and NK-92 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and HuH7 cells from the Japanese Cancer Research Resources Bank (JCRB, Osaka, Japan). HepG2, C3A, SK-HEP-1, and PLC/PRF/5 cells were cultured in high-glucose Dulbecco’s minimum essential medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Biological Industries, Israel), 1% HEPES buffer (Gibco), and 1% penicillin–streptomycin (PS; Gibco). SNU-387, SNU-423, and SNU-449 cells were cultured in RPMI-1640 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% FBS, 1% HEPES buffer, and 1% PS. HuH7 cells were cultured in low-glucose Dulbecco’s minimum essential medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FBS and 1% PS. NK-92 cells were cultured in α-minimum essential medium (Nacalai Tesque) composed of 20% FBS, 1% PS, 0.05 mM 2-mercaptoethanol (Nacalai Tesque), and 10 ng/mL interleukin (IL)-2 (Miltenyi Biotec, Bergisch Gladbach, Germany). The eNK cells were developed at Kobe Research Institute, HEALIOS K.K. (Hyogo, Japan), and provided as cryopreserved cells. The eNK cells were cultured in CTS AIM-V medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5% FBS (SAFC Biosciences, Lenexa, KS, USA), 50 ng/mL animal-derived-free human recombinant stem cell factor (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and human recombinant animal-derived-free IL-15 (PeproTech, Cranbury, NJ, USA) for three days. All the cell lines were cultured in 5% CO 2 at 37°C. 2.2 Flow cytometry All analyses were performed using a FACS Celesta or FACS Canto II cytometer (BD Biosciences, San Jose, CA, USA). The eNK cells, NK-92 cells, and liver cancer cell lines were stained with the following monoclonal antibodies. FITC-conjugated anti-CD56 (clone B159), APC-H7-conjugated anti-CD3 (clone SK7), APC-conjugated anti-programmed death 1 (PD1) (clone MIH4), anti-NKG2D (clone 1D11), anti-TRAIL (clone RIK-2), Alexa Fluor 647-conjugated anti-CD226 (clone DX11), BV480-conjugated anti-CD16 (clone 3G8), BV421-conjugated anti-NKp30 (clone p30-15), BV605-conjugated anti-NKp44 (clone p44-8), PE-conjugated anti-NKp46 (clone 9E2/Nkp46), anti-CD261 (clone S35-934), anti-CD47 (clone B6H12), anti-PDL1 (clone MIH1), and anti-PDL2 (clone MIH18) antibodies were obtained from BD Biosciences. APC-conjugated anti-MICA/B (clone 6D4), anti-CD112 (clone TX31), PE-conjugated anti-CD155 (clone SKll.4), Alexa Fluor 700-conjugated anti-NKG2A (clone S19004C), signal regulatory protein-α (SIRPα) (clone 15–414) were obtained from BioLegend (San Diego, CA, USA). PE-conjugated anti-CD262 (clone DJR2-4) and anti-CD263 (clone DJR3) antibodies were purchased from eBioscience (Carlsbad, CA, USA). APC-conjugated anti-ULBP1 (clone #170818), anti-GPC3 (clone #307801), PE-conjugated anti-ULBP2/5/6 (clone #165903), anti-ULBP3 (clone #166510), anti-ULBP4 (clone #709116), and anti-CD264 (clone #104918) antibodies were purchased from R&D Systems (Minneapolis, MN, USA). Dead cells were excluded from the analysis using forward scatter and 7-amino-actinomycin D (7-AAD; BD Biosciences). Data analyses were performed using FlowJo software ver.10 (BD Biosciences). 2.3 Intracellular flow cytometry Perforin, granzyme B, IFNα, IFNγ, and TNFα production in NK-92 and eNK cells were measured through intracellular staining, according to the manufacturer's instructions. Briefly, 4 h after treatment with a leukocyte activation cocktail (BD GolgiPlug; BD Biosciences), the eNK and NK-92 cells were stained with antibodies: FITC-conjugated anti-CD56 (clone B159) and APC-conjugated anti-CD3 (clone HIT3a) (BD Biosciences). The cells were then fixed, permeabilized with Cytofix/Cytoperm solution (BD Biosciences), and washed with Perm/Wash Buffer (BD Biosciences). Subsequently, the cells were stained with PE-conjugated monoclonal antibodies: anti-perforin (clone dG9) (BioLegend), anti-granzyme B (clone GB11), anti-IFNα (clone 7N4-1), anti-IFNγ (clone B27), and anti-TNFα (clone 6401.1111) (BD Biosciences), and thereafter analyzed using a FACS Canto II cytometer. Data analyses were performed using FlowJo software ver.10. 2.4 Cytotoxicity assay Cytotoxicity assays were conducted using a real-time cell analyzer (xCELLigence S16 system; Agilent Technologies, Santa Clara, CA, USA). All experiments were performed at 37°C with 5% CO 2 . Initially, 50 µL culture medium was added to a 16-well E-plate (Agilent Technologies) to measure the background. Then, HepG2, HuH7, or SNU423 cells were seeded onto an E-plate at 4 × 10 4 , 6 × 10 3 , or 5 × 10 3 cells suspended in 100 µL culture medium, respectively. To ensure uniform cell adhesion, the plates were incubated for 30 min at room temperature and cultured overnight. After incubation, 100 µL of the medium was removed, and NK-92 or eNK cells added at effector/target (E/T) ratios = 1, 5, and 10 (duplicates for each). The plate was allowed to stand for 30 min at room temperature and then set in the device. The cell index was measured every 15 min for 8 h. % Cytotoxicity was calculated using the following formula: % Cytotoxicity = [1 − (cell index of effector and target cells − cell index of effector only)/cell index of target only] × 100 2.5 Blocking assay Blocking assays were performed using the 51 Cr-release assay method, as previously described[ 21 ], using HepG2, HuH7, and SNU-423 tumor cells as targets. Briefly, 1 × 10 4 or 1 × 10 5 eNK cells were preincubated at 37℃ in round-bottomed 96-well microtiter plates in the presence of 50 ng/mL IL-15, 10 µg/mL anti-TRAIL mAb, and/or 25 nmol/L concanamycin A (CMA) (Sigma-Aldrich) for 30 min. Then, 1 × 10 4 51 Cr-labelled target tumor cells were added and co-cultured for 4 and 18 h. The percentage of specific 51 Cr release was calculated as follows: % Cytotoxicity = [(cpm of experimental release – cpm of spontaneous release)/(cpm of maximum release – cpm of spontaneous release)] × 100. All assays were performed in quadruplicate. 2.6 Statistical analysis Data were analyzed using a one-tailed Student’s t-test. Significance levels are indicated as * p < 0.05, ** p < 0.01, and *** p < 0.001. 3. Results 3.1 eNK cells highly express antitumor molecules Flow cytometry analysis of the eNK cells after recovery culture from cryopreservation revealed that the CD56 + CD3 − and CD56 dim CD3 − NK cell fractions were approximately 80% and 20%, respectively. NKT (CD56 + , CD3 + ) and T cells (CD56 − , CD3 + ) were virtually absent (Fig. 1 a). Both CD56 + and CD56 dim cells were relatively large lymphocytes that did not stain with 7-AAD, indicating that the viability of both fractions was well conserved (Fig. 1 ). The expression of major functional molecules characteristic of NK cells (NKG2D, TRAIL, CD226, CD16, NKp30, NKp44, NKp46, NKG2A, SIRPα, and PD1) was also analyzed on eNK and NK-92 cells via flow cytometry. The eNK cells unimodally expressed NKG2D, TRAIL, CD16, NKp30, and NKp44, whereas CD226 and NKp46 were bimodally expressed (Fig. 2 a). CD56 + and CD56 dim cells shared a similar expression pattern for these surface molecules and showed no differences in expression levels (Fig. 2 b). A marked difference from the key molecule expression pattern of NK-92 cells was the expression of TRAIL and CD16 and the decreased expression of NKG2A on eNK cells (Fig. 3 , Supplementary Table 1, Supplementary Fig. 1a). SIRPα and PD1 were hardly expressed in both eNK and NK-92 cells (Supplementary Table 1). These data indicate that eNK cells have a highly antitumor phenotype and are less susceptible to immunosuppression through inhibitory receptors and/or SIRPα and PD1 signaling. 3.2 eNK cells have a high capacity for the production of intracellular cytotoxic factors and cytokines Intracellular cytotoxic substances and cytokines are important factors in the antitumor activity of NK cells. We evaluated the expression of typical cytotoxic substances (granzyme B and perforin) and cytokines (TNFα, IFNα, and IFNγ) in eNK and NK-92 cells via intracellular flow cytometry. Granzyme B, perforin, IFNγ, and TNFα were highly expressed in both cell types (Fig. 4 , Supplementary Fig. 1b, Supplementary Table 2). The expression levels of granzyme B and perforin were similar in both cases; however, the MFI levels were the highest in NK-92 cells. In contrast, TNFα expression was higher in eNK cells than in NK-92 cells (Fig. 4 b). 3.3 Expression of ligands for antitumor molecules on natural killer cells differs among human liver cancer cell lines The binding of antitumor and immune checkpoint molecules to their ligands is an essential component of the therapeutic efficacy of immune cell-based therapy and is associated with various phenotypes [ 22 ]. Hence, sensitivity to NK cells may differ depending on the cancer cell phenotype. We analyzed the expression of ligands for antitumor (NKG2D, TRAIL, and CD226) and immune checkpoint molecules (PD1 and CD47) on eight liver cancer cell lines (HepG2, C3A, HuH7, PLC/PRF/5, SNU-387, SNU-423, SNU-449, and SK-HEP-1). Of the NKG2D ligands, consisting of ULBP1–6 and MICA/B, ULBP1 was expressed at different frequencies on different cell lines, but the expression intensity was low, regardless of the expression frequency (Fig. 5 a, Supplementary Table 3a). ULBP3 and ULBP4 were not expressed on any of the cell lines (Fig. 5 a). MICA/B was highly expressed, except on HuH7 cells, but its MFI differed depending on the cell line (Fig. 5 a, Supplementary Table 3a). Thus, all ligands for NKG2D were barely expressed on HuH7 cells. These data indicate that liver cancer cell lines, other than HuH7, are likely to be sensitive to NKG2D, albeit with varying expression levels. TRAIL receptors expressed on the cell surface can be divided into death (DR4 and DR5) and decoy receptors (DcR1 and DcR2). DR5 (CD262), the major TRAIL death receptor, was expressed on the cell surface of almost 100% of HepG2, C3A, SNU-449, and SK-HEP-1 cells, but only in approximately 10% of SNU423 cells (Fig. 5 b, Supplementary Table 3b). Decoy receptor expression was low in most cell lines. These data suggest that liver cancer cell lines are susceptible to varying degrees of TRAIL cytotoxicity, and that evasion of such cytotoxicity by decoy receptors is unlikely to occur. The binding of CD226 to CD112 or CD155 is known to result in NK cell effector function. CD112 and CD155 were expressed to varying degrees on almost 100% of the cell lines (Fig. 5 c, Supplementary Table 3c). These data indicate that liver cancer cell lines may induce activation signals in NK cells via CD226 to varying degrees. We also evaluated the expression of GPC3, an HCC-specific marker. GPC3 expression was high in HepG2 cells and moderate in C3A cells (Fig. 5 d). However, the other cell lines showed poor GPC3 expression. PDL1 and PDL2 expression was observed in SNU-387, SNU-423, and SNU-449 cells but not in the other cell lines (Fig. 5 d, Supplementary Table 3d). Expression of CD47, a ligand for SIRPα, varied widely among the cell lines (Fig. 5 d, Supplementary Table 3d). These results suggest that the impact of these two signaling pathways on immune regulatory mechanisms varies widely among the cell lines. 3.4 eNK cells exhibit high cytotoxicity against hepatocellular carcinoma cell lines Based on results of the phenotypic analysis of liver cancer cell lines (Fig. 5 ), we selected HepG2, HuH7, and SNU423 cells to investigate the antitumor effect of eNK cells by continuously observing cytotoxicity using a real-time cell analyzer. When the E/T ratio was 10, eNK cells showed over 90% cytotoxic activity in all three cell types (Fig. 6 a). No significant difference was observed between when the E/T values were 5 and 10 (Fig. 6 a). However, a difference in cytotoxic activity was observed between the cell lines at an E/T ratio = 1. To compare the cytotoxicity of eNK cells with other previously established NK cell lines, we additionally performed cytotoxicity testing with NK-92 cells, a well-characterized human NK cell line that has demonstrated promising anticancer activities in some clinical trials [ 23 ], [ 24 ]. NK-92 cells also showed differences in cytotoxicity to the cell lines (Fig. 6 b); however, eNK cells were more cytotoxic at all E/T ratios tested (Fig. 6 ). These results indicate that eNK cells have high antitumor activity in HCC and that their cytotoxicity can be influenced by the differential expression of ligands for NK cell antitumor molecules. 3.5 Perforin, granzyme B, and TRAIL are highly related to the cytotoxicity of eNK cells To investigate the mechanisms underlying the observed cytotoxicity of eNK cells, we examined the contribution of TRAIL and perforin/granzyme B, which are highly expressed on these cells. Cytotoxicity was significantly inhibited in all three cell lines in the presence of CMA (Fig. 7 ). At an E/T ratio = 10, the anti-TRAIL antibody inhibited injury to HepG2 cells in the presence of CMA, whereas the anti-TRAIL antibody alone had no effect (Fig. 7 A). At an E/T ratio = 1, the anti-TRAIL antibody alone inhibited cytotoxicity 18 h after the addition of eNK cells (Fig. 8 a). In addition, comparing the presence or absence of anti-TRAIL antibodies in the presence of CMA at an E/T ratio = 1, cytotoxicity tended to be lower in the former group, but not significantly so (Fig. 8 a). Cell damage of HuH7 cells was inhibited in the presence of CMA and/or anti-TRAIL antibodies 4 and 18 h after the addition of eNK cells (Fig. 7 b, Fig. 8 b). At an E/T ratio = 10, the anti-TRAIL antibody alone slightly suppressed cell injury against SNU-423 after 4 h, whereas no significant difference was observed at 18 h (Fig. 7 c). At an E/T ratio = 1, there was no significant difference observed between treatment with the anti-TRAIL antibody alone or with CMA alone at 18 h, but inhibition was observed in the presence of both (Fig. 8 c). These data suggest that TRAIL and perforin/granzyme B pathways are closely involved in the cytotoxicity of eNK cells against all three HCC cell lines. 4. Discussion 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. 5 a, Supplementary Table 3a). The expression of DR5 varied between approximately 10% and 100% (Fig. 5 b, Supplementary Table 3b). Furthermore, each cell line showed variable expression of CD47, PDL1, and PDL2 (Fig. 5 d, 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. 6 a). 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. Abbreviations 7-AAD 7-amino-actinomycin D ADCC antibody-dependent cellular cytotoxicity CAR chimeric antigen receptor CMA concanamycin A E/T effector/target GPC3 glypican 3 HCC hepatocellular carcinoma HPC hematopoietic progenitor cell IFN interferon iPSC induced pluripotent stem cell JAK-STAT Janus kinase-signal transducer and activator of transcription NK natural killer PD1 programmed death 1 PI3K phosphatidylinositol 3-hydroxy kinase PLCγ2 phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-2 SIRPα signal regulatory protein-α TNF tumor necrosis factor TRAIL tumor necrosis factor-related apoptosis-inducing ligand. Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Funding This study was partially supported in part by HEALIOS K.K. and AMED (Grant Number: JP23fk0210108). Author Contribution MN performed the experiments, data curation, and data analysis, and wrote the original draft.YT contributed to conceptualization, methodology, and writing(review & editing) and supervised this study.KH, MO, and TK contributed to writing (review & editing).HO contributed to conceptualization, methodology, funding acquisition, and writing (review & editing).KK manufactured eNK cells. KT supervised the manufacturing of eNK cells and contributed to funding acquisition.All authors reviewed the manuscript and approved the final version of the manuscript for submission. Acknowledgement We thank Editage (www.editage.jp) for the English language review. Data statement : The data supporting the findings of this study are available from the corresponding author upon reasonable request. References Smyth MJ, Hayakawa Y, Takeda K, Yagita H (2002) New aspects of natural-killer-cell surveillance and therapy of cancer, Nat Rev Cancer , vol. 2, no. 11, pp. 850–861, Nov. 10.1038/nrc928 Du N, Guo F, Wang Y, Cui J (2021) Nk cell therapy: A rising star in cancer treatment, Cancers (Basel) , vol. 13, no. 16, Aug. 10.3390/cancers13164129 Hsu LJ, Liu CL, Kuo ML, Shen CN, Shen CR (2021) An alternative cell therapy for cancers: Induced pluripotent stem cell (ipsc)-derived natural killer cells. Biomedicines 9(10). 10.3390/biomedicines9101323 Lupo KB, Il Moon J, Chambers AM, Matosevic S (2021) Differentiation of natural killer cells from induced pluripotent stem cells under defined, serum- and feeder-free conditions. Cytotherapy 23(10). 10.1016/j.jcyt.2021.05.001 Goldenson BH, Hor P, Kaufman DS (2022) iPSC-Derived Natural Killer Cell Therapies - Expansion and Targeting. Front Immunol 13. 10.3389/fimmu.2022.841107 Lin X, Sun Y, Dong X, Liu Z, Sugimura R, Xie G (2023) IPSC-derived CAR-NK cells for cancer immunotherapy. Biomed Pharmacotherapy 165. 10.1016/j.biopha.2023.115123 Cichocki F, van der Stegen SJC, Miller JS (2023) Engineered and banked iPSCs for advanced NK- and T-cell immunotherapies. Blood 141(8). 10.1182/blood.2022016205 Maddineni S, Silberstein JL, Sunwoo JB (2022) Emerging NK cell therapies for cancer and the promise of next generation engineering of iPSC-derived NK cells. J Immunother Cancer 10(5). 10.1136/jitc-2022-004693 International agency for research on cancer, world Health organization cancer today. Accessed: Dec. 08, 2023. [Online]. Available: https://gco.iarc.fr/today/fact-sheets-cancers Vitale A et al (2017) Personalized treatment of patients with very early hepatocellular carcinoma. J Hepatol 66. no. 210.1016/j.jhep.2016.09.012 Juengpanich S et al (2019) The role of natural killer cells in hepatocellular carcinoma development and treatment: A narrative review. Translational Oncol 12. no. 810.1016/j.tranon.2019.04.021 Ohira M et al (2009) Adoptive immunotherapy with liver allograft-derived lymphocytes induces anti-HCV activity after liver transplantation in humans and humanized mice. J Clin Invest 119(11). 10.1172/JCI38374 Ohira M et al (2012) Clinical-scale isolation of interleukin-2-stimulated liver natural killer cells for treatment of liver transplantation with hepatocellular carcinoma. Cell Transpl 21(7). 10.3727/096368911X627589 Ohira M et al (2022) Pilot study to determine the safety and feasibility of deceased donor liver natural killer cell infusion to liver transplant recipients with hepatocellular carcinoma. Cancer Immunol Immunother 71(3). 10.1007/s00262-021-03005-3 Tatsumi T, Takehara T (2016) Impact of natural killer cells on chronic hepatitis C and hepatocellular carcinoma. Hepatol Res 46. no. 510.1111/hepr.12619 Yu M et al (2018) Development of GPC3-Specific Chimeric Antigen Receptor-Engineered Natural Killer Cells for the Treatment of Hepatocellular Carcinoma. Mol Ther 26(2). 10.1016/j.ymthe.2017.12.012 Kamiya T, Chang YH, Campana D (2016) Expanded and activated natural killer cells for immunotherapy of hepatocellular carcinoma. Cancer Immunol Res 4(7). 10.1158/2326-6066.CIR-15-0229 Zhou Z, Yu X, Zhang J, Tian Z, Zhang C (2015) TLR7/8 agonists promote NK-DC cross-talk to enhance NK cell anti-tumor effects in hepatocellular carcinoma. Cancer Lett 369(2). 10.1016/j.canlet.2015.09.017 Sajid M, Liu L, Sun C (2022) The Dynamic Role of NK Cells in Liver Cancers: Role in HCC and HBV Associated HCC and Its Therapeutic Implications. Front Immunol 13. 10.3389/fimmu.2022.887186 Mantovani S, Oliviero B, Varchetta S, Mele D, Mondelli MU (2020) Natural killer cell responses in hepatocellular carcinoma: Implications for novel immunotherapeutic approaches, Cancers , vol. 12, no. 4. MDPI AG, Apr. 01, 10.3390/cancers12040926 Kajitani K, Tanaka Y, Arihiro K, Kataoka T, Ohdan H (2012) Mechanistic analysis of the antitumor efficacy of human natural killer cells against breast cancer cells, Breast Cancer Res Treat , vol. 134, no. 1, pp. 139–155, Jul. 10.1007/s10549-011-1944-x Choi JH, Thung SN (2023) Advances in Histological and Molecular Classification of Hepatocellular Carcinoma. Biomedicines 11 9. Multidisciplinary Digital Publishing Institute (MDPI). 10.3390/biomedicines11092582 Klingemann H (2023) The NK-92 cell line—30 years later: its impact on natural killer cell research and treatment of cancer. Cytotherapy 25. no. 510.1016/j.jcyt.2022.12.003 Zhang J, Zheng H, Diao Y (2019) Natural killer cells and current applications of chimeric antigen receptor-modified NK-92 cells in tumor immunotherapy. Int J Mol Sci 20(2). 10.3390/ijms20020317 Chen Y, Lu D, Churov A, Fu R (2020) Research Progress on NK Cell Receptors and Their Signaling Pathways, Mediators of Inflammation , vol. 2020. 10.1155/2020/6437057 Barrow AD, Martin CJ, Colonna M (2019) The natural cytotoxicity receptors in health and disease, Frontiers in Immunology , vol. 10, no. MAY. 10.3389/fimmu.2019.00909 Yamada A, Arakaki R, Saito M, Kudo Y, Ishimaru N (2017) Dual role of Fas/FasL-mediated signal in peripheral immune tolerance, Frontiers in Immunology , vol. 8, no. APR. 10.3389/fimmu.2017.00403 Strasser A, Jost PJ, Nagata S (2009) The Many Roles of FAS Receptor Signaling in the Immune System. Immunity 30. no. 210.1016/j.immuni.2009.01.001 Johnstone RW, Frew AJ, Smyth MJ (2008) The TRAIL apoptotic pathway in cancer onset, progression and therapy. Nat Rev Cancer 8(10). 10.1038/nrc2465 Picarda G, Trichet V, Téletchéa S, Heymann D, Rédini F (2012) TRAIL receptor signaling and therapeutic option in bone tumors: the trap of the bone microenvironment. Am J Cancer Res, 2, 1 Wu B et al (2021) Poliovirus receptor (PVR)-like protein cosignaling network: new opportunities for cancer immunotherapy. J Experimental Clin Cancer Res 40(1). 10.1186/s13046-021-02068-5 Yeo J, Ko M, Lee DH, Park Y, Jin HS (2021) Tigit/cd226 axis regulates anti-tumor immunity. Pharmaceuticals 14(3). 10.3390/ph14030200 Siemaszko J, Marzec-przyszlak A, Bogunia‐kubik K (2021) Nkg2d natural killer cell receptor—a short description and potential clinical applications. Cells 10(6). 10.3390/cells10061420 Wang J, Li CD, Sun L (2020) Recent advances in molecular mechanisms of the NKG2D pathway in hepatocellular carcinoma. Biomolecules 10(2). 10.3390/biom10020301 Nimmerjahn F, Ravetch JV (2008) Fcγ receptors as regulators of immune responses. Nat Rev Immunol 8(1). 10.1038/nri2206 Voskoboinik I, Whisstock JC, Trapani JA (2015) Perforin and granzymes: Function, dysfunction and human pathology. Nat Rev Immunol 15(6). 10.1038/nri3839 Annibaldi A, Meier P (2018) Checkpoints in TNF-Induced Cell Death: Implications in Inflammation and Cancer. Trends Mol Med 24(1). 10.1016/j.molmed.2017.11.002 Tiegs G, Horst AK (2022) TNF in the liver: targeting a central player in inflammation. Semin Immunopathol 44. no. 410.1007/s00281-022-00910-2 Ding H, Wang G, Yu Z, Sun H, Wang L (2022) Role of interferon-gamma (IFN-γ) and IFN-γ receptor 1/2 (IFNγR1/2) in regulation of immunity, infection, and cancer development: IFN-γ-dependent or independent pathway. Biomed Pharmacotherapy 155. 10.1016/j.biopha.2022.113683 Platanias LC (2005) Mechanisms of type-I- and type-II-interferon-mediated signalling. Nat Rev Immunol 5. no. 510.1038/nri1604 Abel AM, Yang C, Thakar MS, Malarkannan S (2018) Natural killer cells: Development, maturation, and clinical utilization, Frontiers in Immunology , vol. 9, no. AUG. 10.3389/fimmu.2018.01869 Bi J, Wang X (2020) Molecular Regulation of NK Cell Maturation. Front Immunol 11. 10.3389/fimmu.2020.01945 Wang D, Malarkannan S (2020) Transcriptional regulation of natural killer cell development and functions. Cancers 12(6). 10.3390/cancers12061591 Arzumanian VA, Kiseleva OI, Poverennaya EV (2021) The curious case of the HepG2 cell line: 40 years of expertise, International Journal of Molecular Sciences , vol. 22, no. 23. 10.3390/ijms222313135 Prager I et al (2019) NK cells switch from granzyme B to death receptor-mediated cytotoxicity during serial killing. J Exp Med 216(9). 10.1084/jem.20181454 Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterialNakamuraM.et.al.docx Cite Share Download PDF Status: Published Journal Publication published 04 Feb, 2025 Read the published version in Cancer Immunology, Immunotherapy → Version 1 posted Editorial decision: Revision requested 05 Aug, 2024 Reviews received at journal 05 Aug, 2024 Reviews received at journal 04 Aug, 2024 Reviewers agreed at journal 24 Jul, 2024 Reviews received at journal 23 Jul, 2024 Reviewers agreed at journal 22 Jul, 2024 Reviewers agreed at journal 22 Jul, 2024 Reviewers agreed at journal 21 Jul, 2024 Reviewers agreed at journal 20 Jul, 2024 Reviewers invited by journal 20 Jul, 2024 Editor assigned by journal 19 Jul, 2024 Submission checks completed at journal 19 Jul, 2024 First submitted to journal 18 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4765613","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":336074030,"identity":"b4ea015e-d96e-4a46-bb64-1885ddd7096f","order_by":0,"name":"Mayuna Nakamura","email":"","orcid":"","institution":"Department of Gastroenterological and Transplant Surgery, Graduate School of Biomedical and Health Sciences, Hiroshima University","correspondingAuthor":false,"prefix":"","firstName":"Mayuna","middleName":"","lastName":"Nakamura","suffix":""},{"id":336074031,"identity":"3fbb5893-389c-49cc-bc17-44be971174c5","order_by":1,"name":"Yuka Tanaka","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABA0lEQVRIiWNgGAWjYFACNjApZ8AMJBkbJODizIS0GJOuJXEDA1gLEc4yuJGW+PFHzZ307ey8Dx/+3GERzd/AY8Dwo4aB3Ry3lsPSPMee5e5sZjc25j0jkTvjAI8BY88xBmZLHFYa3EhvkGZgO5y74TAbmzRjm0Ruw/03Bgy8DQzMBgdwamn++ePf4XQDoBbJn0At80G2/MWrJe2YBG/b4QSQFiBDIncDUAszPlskzzxLs+btO2wIdBizMUjLxgNsBYdljkng9Avf8TTjmz++HZY3OH+M8eHPtrrceQeYNz58U2OTjCvEFLDaDhSUSDbAoUUeh+0MDHa4tIyCUTAKRsGIAwBOBFm7e6corwAAAABJRU5ErkJggg==","orcid":"","institution":"Department of Gastroenterological and Transplant Surgery, Graduate School of Biomedical and Health Sciences, Hiroshima University","correspondingAuthor":true,"prefix":"","firstName":"Yuka","middleName":"","lastName":"Tanaka","suffix":""},{"id":336074032,"identity":"e5368d08-ffc8-4711-93ef-45de025efa94","order_by":2,"name":"Keishi Hakoda","email":"","orcid":"","institution":"Department of Gastroenterological and Transplant Surgery, Graduate School of Biomedical and Health Sciences, Hiroshima University","correspondingAuthor":false,"prefix":"","firstName":"Keishi","middleName":"","lastName":"Hakoda","suffix":""},{"id":336074033,"identity":"7131f12b-43ee-412d-81ed-74808ef27757","order_by":3,"name":"Masahiro Ohira","email":"","orcid":"","institution":"Department of Gastroenterological and Transplant Surgery, Graduate School of Biomedical and Health Sciences, Hiroshima University","correspondingAuthor":false,"prefix":"","firstName":"Masahiro","middleName":"","lastName":"Ohira","suffix":""},{"id":336074034,"identity":"ecb43be6-501f-4484-8df0-f9ce8a013592","order_by":4,"name":"Tsuyoshi Kobayashi","email":"","orcid":"","institution":"Department of Gastroenterological and Transplant Surgery, Graduate School of Biomedical and Health Sciences, Hiroshima University","correspondingAuthor":false,"prefix":"","firstName":"Tsuyoshi","middleName":"","lastName":"Kobayashi","suffix":""},{"id":336074035,"identity":"cb403f13-da2f-4d52-b1fe-5ef6fcc2afa4","order_by":5,"name":"Kenji Kurachi","email":"","orcid":"","institution":"Research Division, Kobe Research Institute, HEALIOS K.K.","correspondingAuthor":false,"prefix":"","firstName":"Kenji","middleName":"","lastName":"Kurachi","suffix":""},{"id":336074036,"identity":"72b38d25-f60c-4358-b42f-af1453eda477","order_by":6,"name":"Kouichi Tamura","email":"","orcid":"","institution":"Research Division, Kobe Research Institute, HEALIOS K.K.","correspondingAuthor":false,"prefix":"","firstName":"Kouichi","middleName":"","lastName":"Tamura","suffix":""},{"id":336074037,"identity":"fe6ae8d0-762b-4e0b-8a10-6bd0bda57847","order_by":7,"name":"Hideki Ohdan","email":"","orcid":"","institution":"Department of Gastroenterological and Transplant Surgery, Graduate School of Biomedical and Health Sciences, Hiroshima University","correspondingAuthor":false,"prefix":"","firstName":"Hideki","middleName":"","lastName":"Ohdan","suffix":""}],"badges":[],"createdAt":"2024-07-19 02:08:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4765613/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4765613/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00262-025-03940-5","type":"published","date":"2025-02-04T15:56:52+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62345806,"identity":"9fb1929c-d6b8-431a-9d1c-627a47cabbce","added_by":"auto","created_at":"2024-08-13 07:20:01","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":163241,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe phenotype of eNK cells fraction.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe phenotypic characteristics of the eNK cells were analyzed via flow cytometry. (A) Representative dot plots of each fraction gated with 7-AAD/FSC, SSC/FSC, and CD56/CD3. (B) Comparison of each fraction gated with 7-AAD/FSC, SSC/FSC between CD56\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e−\u003c/sup\u003e and CD56\u003csup\u003edim\u003c/sup\u003eCD3\u003csup\u003e−\u003c/sup\u003e NK cell fractions in eNK cells.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4765613/v1/f248aa2b178eadf153fc88a3.jpg"},{"id":62345808,"identity":"36cc4476-e80f-4d2d-a3c8-ffafa06f157c","added_by":"auto","created_at":"2024-08-13 07:20:01","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":345254,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of functional molecules in eNK cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunctional molecules (NKG2D, TRAIL, CD226, CD16, NKp30, NKp44, NKp46, NKG2A, SIRPα, and PD1) were evaluated using flow cytometry. (A) The expression of functional molecules on whole eNK cells. (B) Comparison of the expression of these molecules between CD56\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e−\u003c/sup\u003e and CD56\u003csup\u003edim\u003c/sup\u003eCD3\u003csup\u003e−\u003c/sup\u003e NK cell fractions in eNK cells. Representative histograms are shown with comparisons against isotype controls (opened lines).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4765613/v1/27800840e64b6818cb74b97b.jpg"},{"id":62345804,"identity":"1ddc2a30-3e00-4156-ba8b-b168feb4da4d","added_by":"auto","created_at":"2024-08-13 07:20:01","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":61739,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of the expressions of functional molecules between eNK and NK92 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunctional molecules (NKG2D, TRAIL, CD226, CD16, NKp30, NKp44, NKp46, NKG2A, SIRPα, and PD1) on NK92 cells were evaluated using flow cytometry and compared to the expression of these functional molecules on eNK cells. The percentage of positive cells and MFI of eNK(A), and NK92 (B) cells are expressed as the mean ± SEM (n = 3).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4765613/v1/778f88ee7573e5b913835f54.jpg"},{"id":62346820,"identity":"d556e621-444e-436b-8427-2a30780ea097","added_by":"auto","created_at":"2024-08-13 07:28:01","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":172621,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of representative intracellular cytotoxic factors and cytokines in eNK cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A)The expression of granzyme B, perforin, IFNα, IFNγ, and TNFα that were evaluated using intracellular flow cytometry. Histograms are shown with comparisons against isotype controls (opened lines). \u0026nbsp;Comparison of the expression of these factors between eNK and NK92 cells. The percentage of positive cells (B) and MFI (C) are expressed as the mean ± SEM (n = 3).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4765613/v1/95f1a6b699af0fc63cc146ec.jpg"},{"id":62346821,"identity":"fbc208a7-efc9-4c45-87b0-e58addc29733","added_by":"auto","created_at":"2024-08-13 07:28:02","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":341636,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of NKG2D ligands, TRAIL receptors, CD226 ligands, GPC3, PDL1, PDL2, and CD47 in liver cancer cell lines.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Expression of NKG2D ligands (ULBP1~6 and MICA/B), (B) TRAIL receptors (DR4, DR5, DcR1, and DcR2), (C) CD226 ligands (CD112 and CD155), and (D) GPC3, PDL1, PDL2, and CD47 were evaluated using flow cytometry. The percentage of positive cells and MFI were calculated using FlowJo. All data are expressed as the mean ± SEM (n = 3). Each Fig. shows the following cell lines (from left to right): HepG2, C3A, HuH7, PLC/PRF/5, SNU-387, SNU-423, SNU-449, and SK-HEP-1.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4765613/v1/106001e53cd18cf9f6b7cd61.jpg"},{"id":62347661,"identity":"2286bbf9-09a9-4531-973c-7aea2a459eed","added_by":"auto","created_at":"2024-08-13 07:36:02","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":163218,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytotoxic effects of eNK\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eand NK-92 cells in hepatocellular carcinoma cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHepG2, HuH7, and SNU-423 cells were co-cultured with eNK cells (A) or NK-92 cells (B) at three different effector/target (E/T) ratios (E/T = 1, 5, and 10) for 12 h. The cytotoxic activity of eNK and NK-92 cells against hepatocellular carcinoma cells was evaluated using the xCELLigence software. All data are expressed as the mean ± SEM of three independent experiments. ● E/T = 1; □ E/T = 5; ▲ E/T = 10.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4765613/v1/304f6b199dbd514798d137d5.jpg"},{"id":62345813,"identity":"212d3af9-5b98-44a2-a1c7-7338c2118e88","added_by":"auto","created_at":"2024-08-13 07:20:02","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":301879,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechanisms of the cytotoxic effects of eNK\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003ecells in hepatocellular carcinoma cells (E/T ratio = 10).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) HepG2, (B) HuH7, and (C) SNU-423 cells were co-cultured with eNK cells with an effector/target (E/T) ratio = 10 for 4 and 18 h in the presence of 50 ng/mL IL-15, 10 μg/mL anti-TRAIL mAb, and/or 25 nmol/L concanamycin A (CMA). The cytotoxic activity of eNK cells in hepatocellular carcinoma cells was evaluated using a \u003csup\u003e51\u003c/sup\u003eCr-release assay. All data are expressed as the mean ± SEM of four replicates. Significance levels are presented as n.s. (not significant); *\u003cem\u003ep \u003c/em\u003e\u0026lt;0.05; **\u003cem\u003ep \u003c/em\u003e\u0026lt;0.01; and ***\u003cem\u003ep \u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4765613/v1/4213514f03a057d366f6ce89.jpg"},{"id":62347660,"identity":"00cdb5d6-d818-483a-845c-91b2606baac0","added_by":"auto","created_at":"2024-08-13 07:36:01","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":269046,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechanisms of the cytotoxic effects of eNK cells in hepatocellular carcinoma cells (E/T ratio = 1).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) HepG2, (B) HuH7, and (C) SNU-423 cells were co-cultured with eNK cells with an effector/target (E/T) ratio = 1 for 4 and 18 h in the presence of 50 ng/mL IL-15, 10 μg/mL anti-TRAIL mAb, and/or 25 nmol/L concanamycin A (CMA). The cytotoxic activity of eNK cells in hepatocellular carcinoma cells was evaluated using a 51Cr-release assay. All data are expressed as the mean ± SEM of four replicates. Significance levels are presented as n.s. (not significant); *p \u0026lt;0.05; **p \u0026lt;0.01; and ***p \u0026lt;0.001.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4765613/v1/d1b3bd67ca315ffbc18dc2dc.jpg"},{"id":75930438,"identity":"4498801d-4339-4bb0-aef8-adfd7c9fe4cd","added_by":"auto","created_at":"2025-02-10 16:11:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2822646,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4765613/v1/af8e1151-6a14-4416-936a-8e170adbac8e.pdf"},{"id":62345810,"identity":"f785d951-9ecf-4f98-94cb-0e387f511837","added_by":"auto","created_at":"2024-08-13 07:20:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6277569,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialNakamuraM.et.al.docx","url":"https://assets-eu.researchsquare.com/files/rs-4765613/v1/500564f4cd61b9fa2377d0f3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antitumor effects of natural killer cells derived from gene-engineered human induced pluripotent stem cells on hepatocellular carcinoma","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCell-based immunotherapy has received considerable attention, and various cancer therapeutic approaches have been developed over the past several decades. Natural killer (NK) cells are innate immune cells that account for approximately 15% of circulating blood lymphocytes[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. NK cells possess various functional factors and target abnormal cells, such as cancer and virus-infected cells, without prior sensitization. This feature has attracted attention because of its applications in cancer immunotherapy. NK cell products can be generated from multiple sources, such as peripheral and umbilical cord blood, NK cell lines, and induced pluripotent stem cells (iPSCs)[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Compared with blood-derived NK cells, the NK-92 cell line and NK cells derived from iPSCs can be cultured on a large scale and manufactured off-the-shelf. In addition, because these cells are more easily transduced than blood-derived NK cells are, the development of NK cells with enhanced functions, such as transgenic NK and chimeric antigen receptor (CAR)-NK cells, is currently underway.\u003c/p\u003e \u003cp\u003eThe iPSCs represent a new option for NK cell generation, and several generation methods have been described[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. To enhance their functions, iPSC-derived NK cells expressing functional molecules and CARs have been developed using genetically engineered technologies[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In preclinical studies, iPSC-NK cells have shown effector cytotoxic responses \u003cem\u003ein vitro\u003c/em\u003e against a variety of hematological and solid tumor cell lines, including lung, hepatocellular, and ovarian cancers, as well as myeloid leukemia and melanoma[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Clinical trials using iPSC-derived NK and CAR-NK cells, alone or in combination with other drugs, have been conducted, and some data have provided promising results[\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLiver cancer is the sixth most common malignancy and third most common cause of cancer-related deaths worldwide[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Moreover, hepatocellular carcinoma (HCC) recurrence occurs in approximately 40\u0026ndash;80% of patients within five years of hepatic resection or radiofrequency ablation[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Although there are various treatment options for HCC, such as surgical resection, liver transplantation, thermal ablation, transarterial chemoembolization, and drugs, new therapeutic options are needed to improve treatment effects and reduce recurrence. Recent studies have shown that targeting NK cells can help in the treatment of HCC, the restoration of normal liver function, and to subsequently increase survival rates of patients with HCC[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Initially, we focused on NK cell therapy to prevent HCC recurrence. We previously reported that activated donor liver-derived NK cells are effective in preventing the postoperative recurrence of liver cancer, and the main mechanisms involved were the high expression of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), perforin activity, and interferon (IFN)γ production[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, because liver-derived NK cells can only be obtained from liver transplant donors, they are difficult to apply to liver cancer treatment. Other research groups have also reported that peripheral blood NK cells activated by various cytokines[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], glypican 3 (GPC3)-specific CAR-engineered NK cells[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], NK cells expressing the NKG2D-CD3ζ-DAP10 receptor[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and NK cells with TLR7/TLR8 agonists[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] are effective for HCC treatment[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Clinical trials using NK cells alone or in combination with other drugs have been conducted for HCC but have not yet reached practical applications[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Therefore, the development of NK cells with high therapeutic efficacy for HCC is required.\u003c/p\u003e \u003cp\u003eIn this study, we aimed to evaluate the potential of eNK cells (HLCN061; developed by HEALIOS K.K.), which are human iPSC-derived NK cells differentiated from clinical grade iPSCs in which IL-15, CCR2B, CCL19, CD16a, and NKG2D have been introduced, in the treatment of HCC.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cell lines and cell culture\u003c/h2\u003e \u003cp\u003eHepG2, C3A, SK-HEP-1, PLC/PRF/5, SNU-387, SNU-423, SNU-449, and NK-92 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and HuH7 cells from the Japanese Cancer Research Resources Bank (JCRB, Osaka, Japan). HepG2, C3A, SK-HEP-1, and PLC/PRF/5 cells were cultured in high-glucose Dulbecco\u0026rsquo;s minimum essential medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Biological Industries, Israel), 1% HEPES buffer (Gibco), and 1% penicillin\u0026ndash;streptomycin (PS; Gibco). SNU-387, SNU-423, and SNU-449 cells were cultured in RPMI-1640 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% FBS, 1% HEPES buffer, and 1% PS. HuH7 cells were cultured in low-glucose Dulbecco\u0026rsquo;s minimum essential medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FBS and 1% PS. NK-92 cells were cultured in α-minimum essential medium (Nacalai Tesque) composed of 20% FBS, 1% PS, 0.05 mM 2-mercaptoethanol (Nacalai Tesque), and 10 ng/mL interleukin (IL)-2 (Miltenyi Biotec, Bergisch Gladbach, Germany).\u003c/p\u003e \u003cp\u003eThe eNK cells were developed at Kobe Research Institute, HEALIOS K.K. (Hyogo, Japan), and provided as cryopreserved cells. The eNK cells were cultured in CTS AIM-V medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5% FBS (SAFC Biosciences, Lenexa, KS, USA), 50 ng/mL animal-derived-free human recombinant stem cell factor (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and human recombinant animal-derived-free IL-15 (PeproTech, Cranbury, NJ, USA) for three days. All the cell lines were cultured in 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Flow cytometry\u003c/h2\u003e \u003cp\u003eAll analyses were performed using a FACS Celesta or FACS Canto II cytometer (BD Biosciences, San Jose, CA, USA). The eNK cells, NK-92 cells, and liver cancer cell lines were stained with the following monoclonal antibodies. FITC-conjugated anti-CD56 (clone B159), APC-H7-conjugated anti-CD3 (clone SK7), APC-conjugated anti-programmed death 1 (PD1) (clone MIH4), anti-NKG2D (clone 1D11), anti-TRAIL (clone RIK-2), Alexa Fluor 647-conjugated anti-CD226 (clone DX11), BV480-conjugated anti-CD16 (clone 3G8), BV421-conjugated anti-NKp30 (clone p30-15), BV605-conjugated anti-NKp44 (clone p44-8), PE-conjugated anti-NKp46 (clone 9E2/Nkp46), anti-CD261 (clone S35-934), anti-CD47 (clone B6H12), anti-PDL1 (clone MIH1), and anti-PDL2 (clone MIH18) antibodies were obtained from BD Biosciences. APC-conjugated anti-MICA/B (clone 6D4), anti-CD112 (clone TX31), PE-conjugated anti-CD155 (clone SKll.4), Alexa Fluor 700-conjugated anti-NKG2A (clone S19004C), signal regulatory protein-α (SIRPα) (clone 15\u0026ndash;414) were obtained from BioLegend (San Diego, CA, USA). PE-conjugated anti-CD262 (clone DJR2-4) and anti-CD263 (clone DJR3) antibodies were purchased from eBioscience (Carlsbad, CA, USA). APC-conjugated anti-ULBP1 (clone #170818), anti-GPC3 (clone #307801), PE-conjugated anti-ULBP2/5/6 (clone #165903), anti-ULBP3 (clone #166510), anti-ULBP4 (clone #709116), and anti-CD264 (clone #104918) antibodies were purchased from R\u0026amp;D Systems (Minneapolis, MN, USA). Dead cells were excluded from the analysis using forward scatter and 7-amino-actinomycin D (7-AAD; BD Biosciences). Data analyses were performed using FlowJo software ver.10 (BD Biosciences).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Intracellular flow cytometry\u003c/h2\u003e \u003cp\u003ePerforin, granzyme B, IFNα, IFNγ, and TNFα production in NK-92 and eNK cells were measured through intracellular staining, according to the manufacturer's instructions. Briefly, 4 h after treatment with a leukocyte activation cocktail (BD GolgiPlug; BD Biosciences), the eNK and NK-92 cells were stained with antibodies: FITC-conjugated anti-CD56 (clone B159) and APC-conjugated anti-CD3 (clone HIT3a) (BD Biosciences). The cells were then fixed, permeabilized with Cytofix/Cytoperm solution (BD Biosciences), and washed with Perm/Wash Buffer (BD Biosciences). Subsequently, the cells were stained with PE-conjugated monoclonal antibodies: anti-perforin (clone dG9) (BioLegend), anti-granzyme B (clone GB11), anti-IFNα (clone 7N4-1), anti-IFNγ (clone B27), and anti-TNFα (clone 6401.1111) (BD Biosciences), and thereafter analyzed using a FACS Canto II cytometer. Data analyses were performed using FlowJo software ver.10.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Cytotoxicity assay\u003c/h2\u003e \u003cp\u003eCytotoxicity assays were conducted using a real-time cell analyzer (xCELLigence S16 system; Agilent Technologies, Santa Clara, CA, USA). All experiments were performed at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Initially, 50 \u0026micro;L culture medium was added to a 16-well E-plate (Agilent Technologies) to measure the background. Then, HepG2, HuH7, or SNU423 cells were seeded onto an E-plate at 4 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e, 6 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e, or 5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells suspended in 100 \u0026micro;L culture medium, respectively. To ensure uniform cell adhesion, the plates were incubated for 30 min at room temperature and cultured overnight. After incubation, 100 \u0026micro;L of the medium was removed, and NK-92 or eNK cells added at effector/target (E/T) ratios\u0026thinsp;=\u0026thinsp;1, 5, and 10 (duplicates for each). The plate was allowed to stand for 30 min at room temperature and then set in the device. The cell index was measured every 15 min for 8 h. % Cytotoxicity was calculated using the following formula:\u003c/p\u003e \u003cp\u003e% Cytotoxicity = [1 \u0026minus; (cell index of effector and target cells\u0026thinsp;\u0026minus;\u0026thinsp;cell index of effector only)/cell index of target only] \u0026times; 100\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Blocking assay\u003c/h2\u003e \u003cp\u003eBlocking assays were performed using the \u003csup\u003e51\u003c/sup\u003eCr-release assay method, as previously described[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], using HepG2, HuH7, and SNU-423 tumor cells as targets. Briefly, 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e or 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e eNK cells were preincubated at 37℃ in round-bottomed 96-well microtiter plates in the presence of 50 ng/mL IL-15, 10 \u0026micro;g/mL anti-TRAIL mAb, and/or 25 nmol/L concanamycin A (CMA) (Sigma-Aldrich) for 30 min. Then, 1 \u0026times; 10\u003csup\u003e4 51\u003c/sup\u003eCr-labelled target tumor cells were added and co-cultured for 4 and 18 h. The percentage of specific \u003csup\u003e51\u003c/sup\u003eCr release was calculated as follows:\u003c/p\u003e \u003cp\u003e% Cytotoxicity = [(cpm of experimental release \u0026ndash; cpm of spontaneous release)/(cpm of maximum release \u0026ndash; cpm of spontaneous release)] \u0026times; 100.\u003c/p\u003e \u003cp\u003eAll assays were performed in quadruplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using a one-tailed Student\u0026rsquo;s t-test. Significance levels are indicated as *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 eNK cells highly express antitumor molecules\u003c/h2\u003e \u003cp\u003eFlow cytometry analysis of the eNK cells after recovery culture from cryopreservation revealed that the CD56\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e\u0026minus;\u003c/sup\u003e and CD56\u003csup\u003edim\u003c/sup\u003eCD3\u003csup\u003e\u0026minus;\u003c/sup\u003e NK cell fractions were approximately 80% and 20%, respectively.\u003c/p\u003e \u003cp\u003eNKT (CD56\u003csup\u003e+\u003c/sup\u003e, CD3\u003csup\u003e+\u003c/sup\u003e) and T cells (CD56\u003csup\u003e\u0026minus;\u003c/sup\u003e, CD3\u003csup\u003e+\u003c/sup\u003e) were virtually absent (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Both CD56\u003csup\u003e+\u003c/sup\u003e and CD56\u003csup\u003edim\u003c/sup\u003e cells were relatively large lymphocytes that did not stain with 7-AAD, indicating that the viability of both fractions was well conserved (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe expression of major functional molecules characteristic of NK cells (NKG2D, TRAIL, CD226, CD16, NKp30, NKp44, NKp46, NKG2A, SIRPα, and PD1) was also analyzed on eNK and NK-92 cells via flow cytometry. The eNK cells unimodally expressed NKG2D, TRAIL, CD16, NKp30, and NKp44, whereas CD226 and NKp46 were bimodally expressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). CD56\u003csup\u003e+\u003c/sup\u003e and CD56\u003csup\u003edim\u003c/sup\u003e cells shared a similar expression pattern for these surface molecules and showed no differences in expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA marked difference from the key molecule expression pattern of NK-92 cells was the expression of TRAIL and CD16 and the decreased expression of NKG2A on eNK cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Supplementary Table\u0026nbsp;1, Supplementary Fig.\u0026nbsp;1a). SIRPα and PD1 were hardly expressed in both eNK and NK-92 cells (Supplementary Table\u0026nbsp;1). These data indicate that eNK cells have a highly antitumor phenotype and are less susceptible to immunosuppression through inhibitory receptors and/or SIRPα and PD1 signaling.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 eNK cells have a high capacity for the production of intracellular cytotoxic factors and cytokines\u003c/h2\u003e \u003cp\u003eIntracellular cytotoxic substances and cytokines are important factors in the antitumor activity of NK cells. We evaluated the expression of typical cytotoxic substances (granzyme B and perforin) and cytokines (TNFα, IFNα, and IFNγ) in eNK and NK-92 cells via intracellular flow cytometry. Granzyme B, perforin, IFNγ, and TNFα were highly expressed in both cell types (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Supplementary Fig.\u0026nbsp;1b, Supplementary Table\u0026nbsp;2). The expression levels of granzyme B and perforin were similar in both cases; however, the MFI levels were the highest in NK-92 cells. In contrast, TNFα expression was higher in eNK cells than in NK-92 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3 Expression of ligands for antitumor molecules on natural killer cells differs among human liver cancer cell lines\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe binding of antitumor and immune checkpoint molecules to their ligands is an essential component of the therapeutic efficacy of immune cell-based therapy and is associated with various phenotypes [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Hence, sensitivity to NK cells may differ depending on the cancer cell phenotype. We analyzed the expression of ligands for antitumor (NKG2D, TRAIL, and CD226) and immune checkpoint molecules (PD1 and CD47) on eight liver cancer cell lines (HepG2, C3A, HuH7, PLC/PRF/5, SNU-387, SNU-423, SNU-449, and SK-HEP-1).\u003c/p\u003e \u003cp\u003eOf the NKG2D ligands, consisting of ULBP1\u0026ndash;6 and MICA/B, ULBP1 was expressed at different frequencies on different cell lines, but the expression intensity was low, regardless of the expression frequency (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, Supplementary Table\u0026nbsp;3a). ULBP3 and ULBP4 were not expressed on any of the cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). MICA/B was highly expressed, except on HuH7 cells, but its MFI differed depending on the cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, Supplementary Table\u0026nbsp;3a). Thus, all ligands for NKG2D were barely expressed on HuH7 cells. These data indicate that liver cancer cell lines, other than HuH7, are likely to be sensitive to NKG2D, albeit with varying expression levels.\u003c/p\u003e \u003cp\u003eTRAIL receptors expressed on the cell surface can be divided into death (DR4 and DR5) and decoy receptors (DcR1 and DcR2). DR5 (CD262), the major TRAIL death receptor, was expressed on the cell surface of almost 100% of HepG2, C3A, SNU-449, and SK-HEP-1 cells, but only in approximately 10% of SNU423 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, Supplementary Table\u0026nbsp;3b). Decoy receptor expression was low in most cell lines. These data suggest that liver cancer cell lines are susceptible to varying degrees of TRAIL cytotoxicity, and that evasion of such cytotoxicity by decoy receptors is unlikely to occur.\u003c/p\u003e \u003cp\u003eThe binding of CD226 to CD112 or CD155 is known to result in NK cell effector function. CD112 and CD155 were expressed to varying degrees on almost 100% of the cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, Supplementary Table\u0026nbsp;3c). These data indicate that liver cancer cell lines may induce activation signals in NK cells via CD226 to varying degrees.\u003c/p\u003e \u003cp\u003eWe also evaluated the expression of GPC3, an HCC-specific marker. GPC3 expression was high in HepG2 cells and moderate in C3A cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). However, the other cell lines showed poor GPC3 expression. PDL1 and PDL2 expression was observed in SNU-387, SNU-423, and SNU-449 cells but not in the other cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, Supplementary Table\u0026nbsp;3d). Expression of CD47, a ligand for SIRPα, varied widely among the cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, Supplementary Table\u0026nbsp;3d). These results suggest that the impact of these two signaling pathways on immune regulatory mechanisms varies widely among the cell lines.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.4 eNK cells exhibit high cytotoxicity against hepatocellular carcinoma cell lines\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on results of the phenotypic analysis of liver cancer cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), we selected HepG2, HuH7, and SNU423 cells to investigate the antitumor effect of eNK cells by continuously observing cytotoxicity using a real-time cell analyzer. When the E/T ratio was 10, eNK cells showed over 90% cytotoxic activity in all three cell types (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). No significant difference was observed between when the E/T values were 5 and 10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). However, a difference in cytotoxic activity was observed between the cell lines at an E/T ratio\u0026thinsp;=\u0026thinsp;1. To compare the cytotoxicity of eNK cells with other previously established NK cell lines, we additionally performed cytotoxicity testing with NK-92 cells, a well-characterized human NK cell line that has demonstrated promising anticancer activities in some clinical trials [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. NK-92 cells also showed differences in cytotoxicity to the cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb); however, eNK cells were more cytotoxic at all E/T ratios tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These results indicate that eNK cells have high antitumor activity in HCC and that their cytotoxicity can be influenced by the differential expression of ligands for NK cell antitumor molecules.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Perforin, granzyme B, and TRAIL are highly related to the cytotoxicity of eNK cells\u003c/h2\u003e \u003cp\u003eTo investigate the mechanisms underlying the observed cytotoxicity of eNK cells, we examined the contribution of TRAIL and perforin/granzyme B, which are highly expressed on these cells. Cytotoxicity was significantly inhibited in all three cell lines in the presence of CMA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAt an E/T ratio\u0026thinsp;=\u0026thinsp;10, the anti-TRAIL antibody inhibited injury to HepG2 cells in the presence of CMA, whereas the anti-TRAIL antibody alone had no effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). At an E/T ratio\u0026thinsp;=\u0026thinsp;1, the anti-TRAIL antibody alone inhibited cytotoxicity 18 h after the addition of eNK cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). In addition, comparing the presence or absence of anti-TRAIL antibodies in the presence of CMA at an E/T ratio\u0026thinsp;=\u0026thinsp;1, cytotoxicity tended to be lower in the former group, but not significantly so (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). Cell damage of HuH7 cells was inhibited in the presence of CMA and/or anti-TRAIL antibodies 4 and 18 h after the addition of eNK cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). At an E/T ratio\u0026thinsp;=\u0026thinsp;10, the anti-TRAIL antibody alone slightly suppressed cell injury against SNU-423 after 4 h, whereas no significant difference was observed at 18 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). At an E/T ratio\u0026thinsp;=\u0026thinsp;1, there was no significant difference observed between treatment with the anti-TRAIL antibody alone or with CMA alone at 18 h, but inhibition was observed in the presence of both (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec). These data suggest that TRAIL and perforin/granzyme B pathways are closely involved in the cytotoxicity of eNK cells against all three HCC cell lines.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eNK cells have four antitumor pathways: activated receptors/antitumor molecules, the antibody-dependent cellular cytotoxicity (ADCC) effect, cytolytic granule release, and cytokine secretion[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. 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[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. 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 [\u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. 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[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. 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[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNK cells secrete cytolytic granules, including the pore-forming protein, perforin, and the serine protease, granzyme B, which synergistically mediate the apoptosis of target cells [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. 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)[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Supplementary Table\u0026nbsp;1). In addition, perforin, granzyme B, TNFα, and IFNγ were highly expressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Supplementary Table\u0026nbsp;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 [\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003eIn 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 \u003cem\u003ein vitro\u003c/em\u003e 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[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. 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[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, Supplementary Table\u0026nbsp;3a). The expression of DR5 varied between approximately 10% and 100% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, Supplementary Table\u0026nbsp;3b). Furthermore, each cell line showed variable expression of CD47, PDL1, and PDL2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, Supplementary Table\u0026nbsp;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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Supplementary Table\u0026nbsp;3) for cytotoxicity assays. The eNK cells showed remarkably high cytotoxicity against all three cell lines compared with that of NK-92 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). We did not observe a significant difference in the cytotoxicity of eNK cells between the cell lines at E/T ratios\u0026thinsp;=\u0026thinsp;5 and 10; however, a difference was observed in the cytotoxic activity between cells at an E/T ratio\u0026thinsp;=\u0026thinsp;1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). 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.\u003c/p\u003e \u003cp\u003eIt 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[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Our blocking assays revealed that the TRAIL and perforin/granzyme B pathways are largely involved in the cytotoxicity mechanisms of eNK cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), and the effects of perforin/granzyme B and TRAIL pathway inhibition were consistent with previous reports[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Thus, eNK cells also have the potential to switch killing mechanisms during the serial killing.\u003c/p\u003e \u003cp\u003eAlthough we focused on the \u003cem\u003ein vitro\u003c/em\u003e cytotoxicity and mechanisms of action of eNK cells in the present study, their effects in \u003cem\u003ein vivo\u003c/em\u003e models remain unclear and require further investigation.\u003c/p\u003e \u003cp\u003eIn 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.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e7-AAD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e7-amino-actinomycin D\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eADCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eantibody-dependent cellular cytotoxicity\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCAR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003echimeric antigen receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCMA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003econcanamycin A\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eE/T\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eeffector/target\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGPC3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eglypican 3\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehepatocellular carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHPC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehematopoietic progenitor cell\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIFN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einterferon\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eiPSC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einduced pluripotent stem cell\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eJAK-STAT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eJanus kinase-signal transducer and activator of transcription\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNK\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enatural killer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePD1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eprogrammed death 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePI3K\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephosphatidylinositol 3-hydroxy kinase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePLCγ2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSIRPα\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esignal regulatory protein-α\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTNF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etumor necrosis factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTRAIL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etumor necrosis factor-related apoptosis-inducing ligand.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was partially supported in part by HEALIOS K.K. and AMED (Grant Number: JP23fk0210108).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMN performed the experiments, data curation, and data analysis, and wrote the original draft.YT contributed to conceptualization, methodology, and writing(review \u0026amp; editing) and supervised this study.KH, MO, and TK contributed to writing (review \u0026amp; editing).HO contributed to conceptualization, methodology, funding acquisition, and writing (review \u0026amp; editing).KK manufactured eNK cells. KT supervised the manufacturing of eNK cells and contributed to funding acquisition.All authors reviewed the manuscript and approved the final version of the manuscript for submission.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Editage (www.editage.jp) for the English language review.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eData statement\u003c/strong\u003e: The data supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSmyth MJ, Hayakawa Y, Takeda K, Yagita H (2002) New aspects of natural-killer-cell surveillance and therapy of cancer, \u003cem\u003eNat Rev Cancer\u003c/em\u003e, vol. 2, no. 11, pp. 850\u0026ndash;861, Nov. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nrc928\u003c/span\u003e\u003cspan address=\"10.1038/nrc928\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDu N, Guo F, Wang Y, Cui J (2021) Nk cell therapy: A rising star in cancer treatment, \u003cem\u003eCancers (Basel)\u003c/em\u003e, vol. 13, no. 16, Aug. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/cancers13164129\u003c/span\u003e\u003cspan address=\"10.3390/cancers13164129\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHsu LJ, Liu CL, Kuo ML, Shen CN, Shen CR (2021) An alternative cell therapy for cancers: Induced pluripotent stem cell (ipsc)-derived natural killer cells. Biomedicines 9(10). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/biomedicines9101323\u003c/span\u003e\u003cspan address=\"10.3390/biomedicines9101323\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLupo KB, Il Moon J, Chambers AM, Matosevic S (2021) Differentiation of natural killer cells from induced pluripotent stem cells under defined, serum- and feeder-free conditions. Cytotherapy 23(10). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jcyt.2021.05.001\u003c/span\u003e\u003cspan address=\"10.1016/j.jcyt.2021.05.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoldenson BH, Hor P, Kaufman DS (2022) iPSC-Derived Natural Killer Cell Therapies - Expansion and Targeting. Front Immunol 13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2022.841107\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2022.841107\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin X, Sun Y, Dong X, Liu Z, Sugimura R, Xie G (2023) IPSC-derived CAR-NK cells for cancer immunotherapy. Biomed Pharmacotherapy 165. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.biopha.2023.115123\u003c/span\u003e\u003cspan address=\"10.1016/j.biopha.2023.115123\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCichocki F, van der Stegen SJC, Miller JS (2023) Engineered and banked iPSCs for advanced NK- and T-cell immunotherapies. Blood 141(8). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1182/blood.2022016205\u003c/span\u003e\u003cspan address=\"10.1182/blood.2022016205\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaddineni S, Silberstein JL, Sunwoo JB (2022) Emerging NK cell therapies for cancer and the promise of next generation engineering of iPSC-derived NK cells. J Immunother Cancer 10(5). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1136/jitc-2022-004693\u003c/span\u003e\u003cspan address=\"10.1136/jitc-2022-004693\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eInternational agency for research on cancer, world Health organization cancer today. Accessed: Dec. 08, 2023. [Online]. Available: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gco.iarc.fr/today/fact-sheets-cancers\u003c/span\u003e\u003cspan address=\"https://gco.iarc.fr/today/fact-sheets-cancers\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVitale A et al (2017) Personalized treatment of patients with very early hepatocellular carcinoma. J Hepatol 66. no. 210.1016/j.jhep.2016.09.012\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJuengpanich S et al (2019) The role of natural killer cells in hepatocellular carcinoma development and treatment: A narrative review. Translational Oncol 12. no. 810.1016/j.tranon.2019.04.021\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOhira M et al (2009) Adoptive immunotherapy with liver allograft-derived lymphocytes induces anti-HCV activity after liver transplantation in humans and humanized mice. J Clin Invest 119(11). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1172/JCI38374\u003c/span\u003e\u003cspan address=\"10.1172/JCI38374\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOhira M et al (2012) Clinical-scale isolation of interleukin-2-stimulated liver natural killer cells for treatment of liver transplantation with hepatocellular carcinoma. Cell Transpl 21(7). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3727/096368911X627589\u003c/span\u003e\u003cspan address=\"10.3727/096368911X627589\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOhira M et al (2022) Pilot study to determine the safety and feasibility of deceased donor liver natural killer cell infusion to liver transplant recipients with hepatocellular carcinoma. Cancer Immunol Immunother 71(3). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00262-021-03005-3\u003c/span\u003e\u003cspan address=\"10.1007/s00262-021-03005-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTatsumi T, Takehara T (2016) Impact of natural killer cells on chronic hepatitis C and hepatocellular carcinoma. Hepatol Res 46. no. 510.1111/hepr.12619\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu M et al (2018) Development of GPC3-Specific Chimeric Antigen Receptor-Engineered Natural Killer Cells for the Treatment of Hepatocellular Carcinoma. Mol Ther 26(2). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ymthe.2017.12.012\u003c/span\u003e\u003cspan address=\"10.1016/j.ymthe.2017.12.012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKamiya T, Chang YH, Campana D (2016) Expanded and activated natural killer cells for immunotherapy of hepatocellular carcinoma. Cancer Immunol Res 4(7). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1158/2326-6066.CIR-15-0229\u003c/span\u003e\u003cspan address=\"10.1158/2326-6066.CIR-15-0229\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou Z, Yu X, Zhang J, Tian Z, Zhang C (2015) TLR7/8 agonists promote NK-DC cross-talk to enhance NK cell anti-tumor effects in hepatocellular carcinoma. Cancer Lett 369(2). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.canlet.2015.09.017\u003c/span\u003e\u003cspan address=\"10.1016/j.canlet.2015.09.017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSajid M, Liu L, Sun C (2022) The Dynamic Role of NK Cells in Liver Cancers: Role in HCC and HBV Associated HCC and Its Therapeutic Implications. Front Immunol 13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2022.887186\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2022.887186\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMantovani S, Oliviero B, Varchetta S, Mele D, Mondelli MU (2020) Natural killer cell responses in hepatocellular carcinoma: Implications for novel immunotherapeutic approaches, \u003cem\u003eCancers\u003c/em\u003e, vol. 12, no. 4. MDPI AG, Apr. 01, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/cancers12040926\u003c/span\u003e\u003cspan address=\"10.3390/cancers12040926\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKajitani K, Tanaka Y, Arihiro K, Kataoka T, Ohdan H (2012) Mechanistic analysis of the antitumor efficacy of human natural killer cells against breast cancer cells, \u003cem\u003eBreast Cancer Res Treat\u003c/em\u003e, vol. 134, no. 1, pp. 139\u0026ndash;155, Jul. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s10549-011-1944-x\u003c/span\u003e\u003cspan address=\"10.1007/s10549-011-1944-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoi JH, Thung SN (2023) Advances in Histological and Molecular Classification of Hepatocellular Carcinoma. Biomedicines 11 9. Multidisciplinary Digital Publishing Institute (MDPI). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/biomedicines11092582\u003c/span\u003e\u003cspan address=\"10.3390/biomedicines11092582\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKlingemann H (2023) The NK-92 cell line\u0026mdash;30 years later: its impact on natural killer cell research and treatment of cancer. Cytotherapy 25. no. 510.1016/j.jcyt.2022.12.003\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang J, Zheng H, Diao Y (2019) Natural killer cells and current applications of chimeric antigen receptor-modified NK-92 cells in tumor immunotherapy. Int J Mol Sci 20(2). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms20020317\u003c/span\u003e\u003cspan address=\"10.3390/ijms20020317\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Y, Lu D, Churov A, Fu R (2020) Research Progress on NK Cell Receptors and Their Signaling Pathways, \u003cem\u003eMediators of Inflammation\u003c/em\u003e, vol. 2020. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1155/2020/6437057\u003c/span\u003e\u003cspan address=\"10.1155/2020/6437057\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarrow AD, Martin CJ, Colonna M (2019) The natural cytotoxicity receptors in health and disease, \u003cem\u003eFrontiers in Immunology\u003c/em\u003e, vol. 10, no. MAY. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2019.00909\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2019.00909\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamada A, Arakaki R, Saito M, Kudo Y, Ishimaru N (2017) Dual role of Fas/FasL-mediated signal in peripheral immune tolerance, \u003cem\u003eFrontiers in Immunology\u003c/em\u003e, vol. 8, no. APR. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2017.00403\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2017.00403\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStrasser A, Jost PJ, Nagata S (2009) The Many Roles of FAS Receptor Signaling in the Immune System. Immunity 30. no. 210.1016/j.immuni.2009.01.001\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJohnstone RW, Frew AJ, Smyth MJ (2008) The TRAIL apoptotic pathway in cancer onset, progression and therapy. Nat Rev Cancer 8(10). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nrc2465\u003c/span\u003e\u003cspan address=\"10.1038/nrc2465\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePicarda G, Trichet V, T\u0026eacute;letch\u0026eacute;a S, Heymann D, R\u0026eacute;dini F (2012) TRAIL receptor signaling and therapeutic option in bone tumors: the trap of the bone microenvironment. Am J Cancer Res, 2, 1\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu B et al (2021) Poliovirus receptor (PVR)-like protein cosignaling network: new opportunities for cancer immunotherapy. J Experimental Clin Cancer Res 40(1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13046-021-02068-5\u003c/span\u003e\u003cspan address=\"10.1186/s13046-021-02068-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYeo J, Ko M, Lee DH, Park Y, Jin HS (2021) Tigit/cd226 axis regulates anti-tumor immunity. Pharmaceuticals 14(3). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ph14030200\u003c/span\u003e\u003cspan address=\"10.3390/ph14030200\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiemaszko J, Marzec-przyszlak A, Bogunia‐kubik K (2021) Nkg2d natural killer cell receptor\u0026mdash;a short description and potential clinical applications. Cells 10(6). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/cells10061420\u003c/span\u003e\u003cspan address=\"10.3390/cells10061420\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J, Li CD, Sun L (2020) Recent advances in molecular mechanisms of the NKG2D pathway in hepatocellular carcinoma. Biomolecules 10(2). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/biom10020301\u003c/span\u003e\u003cspan address=\"10.3390/biom10020301\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNimmerjahn F, Ravetch JV (2008) Fcγ receptors as regulators of immune responses. Nat Rev Immunol 8(1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nri2206\u003c/span\u003e\u003cspan address=\"10.1038/nri2206\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVoskoboinik I, Whisstock JC, Trapani JA (2015) Perforin and granzymes: Function, dysfunction and human pathology. Nat Rev Immunol 15(6). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nri3839\u003c/span\u003e\u003cspan address=\"10.1038/nri3839\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnnibaldi A, Meier P (2018) Checkpoints in TNF-Induced Cell Death: Implications in Inflammation and Cancer. Trends Mol Med 24(1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.molmed.2017.11.002\u003c/span\u003e\u003cspan address=\"10.1016/j.molmed.2017.11.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTiegs G, Horst AK (2022) TNF in the liver: targeting a central player in inflammation. Semin Immunopathol 44. no. 410.1007/s00281-022-00910-2\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing H, Wang G, Yu Z, Sun H, Wang L (2022) Role of interferon-gamma (IFN-γ) and IFN-γ receptor 1/2 (IFNγR1/2) in regulation of immunity, infection, and cancer development: IFN-γ-dependent or independent pathway. Biomed Pharmacotherapy 155. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.biopha.2022.113683\u003c/span\u003e\u003cspan address=\"10.1016/j.biopha.2022.113683\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePlatanias LC (2005) Mechanisms of type-I- and type-II-interferon-mediated signalling. Nat Rev Immunol 5. no. 510.1038/nri1604\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbel AM, Yang C, Thakar MS, Malarkannan S (2018) Natural killer cells: Development, maturation, and clinical utilization, \u003cem\u003eFrontiers in Immunology\u003c/em\u003e, vol. 9, no. AUG. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2018.01869\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2018.01869\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBi J, Wang X (2020) Molecular Regulation of NK Cell Maturation. Front Immunol 11. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2020.01945\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2020.01945\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang D, Malarkannan S (2020) Transcriptional regulation of natural killer cell development and functions. Cancers 12(6). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/cancers12061591\u003c/span\u003e\u003cspan address=\"10.3390/cancers12061591\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArzumanian VA, Kiseleva OI, Poverennaya EV (2021) The curious case of the HepG2 cell line: 40 years of expertise, \u003cem\u003eInternational Journal of Molecular Sciences\u003c/em\u003e, vol. 22, no. 23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms222313135\u003c/span\u003e\u003cspan address=\"10.3390/ijms222313135\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrager I et al (2019) NK cells switch from granzyme B to death receptor-mediated cytotoxicity during serial killing. J Exp Med 216(9). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1084/jem.20181454\u003c/span\u003e\u003cspan address=\"10.1084/jem.20181454\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"hepatocellular carcinoma, iPS cells, NK cells, anti-tumor effect, genetic engineering, cell therapy","lastPublishedDoi":"10.21203/rs.3.rs-4765613/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4765613/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMortality and recurrence rates of hepatocellular carcinoma (HCC) remain high despite the use of various treatment methods. Recently, cell-based immunotherapy using natural killer (NK) cells has attracted considerable attention in cancer immunotherapy. NK cells generated from induced pluripotent stem cells (iPSCs) are a new option for use as an NK cell resource. The eNK cells (HLCN061, developed by HEALIOS K.K.) are human iPSC-derived NK cells differentiated from clinical-grade iPSCs in which IL-15, CCR2B, CCL19, CD16a, and NKG2D have been introduced. In this study, we aimed to evaluate the potential of eNK cell therapy for HCC treatment. The analysis of eNK cells for cell surface and intracellular molecules revealed that antitumor-related surface molecules (TRAIL, CD226, and CD16) and intracellular cytotoxic factors (perforin, granzyme B, TNFα, and IFNγ) were highly expressed. In addition, eNK cells exhibited high cytotoxicity against HCC cell lines (HepG2, HuH7, and SNU-423), which are sensitive to NKG2D, TRAIL, and CD226. The TRAIL and perforin/granzyme B pathways are largely involved in this cytotoxic mechanism, as indicated by the reduction in cytotoxicity induced by TRAIL inhibitory antibodies and concanamycin A, which inhibits perforin/granzyme B-mediated cytotoxicity. Our data suggest that eNK cells, whose functions have been enhanced by genetic engineering, have the potential to improve HCC treatment.\u003c/p\u003e","manuscriptTitle":"Antitumor effects of natural killer cells derived from gene-engineered human induced pluripotent stem cells on hepatocellular carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-13 07:19:57","doi":"10.21203/rs.3.rs-4765613/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-05T09:59:52+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-05T09:00:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-04T10:37:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"64315903583484647292338293279679475121","date":"2024-07-25T01:11:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-23T20:37:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"257845778668752869811238593914329555892","date":"2024-07-23T03:51:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"126739248205461936648328058893282198549","date":"2024-07-22T08:38:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"188020750067020381518245407330842297369","date":"2024-07-21T05:29:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"225876638907515696840281228127063498150","date":"2024-07-21T02:03:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-20T22:01:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-19T04:34:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-19T04:33:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Immunology, Immunotherapy","date":"2024-07-19T02:07:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9706b9ad-42f7-4c32-9c57-7a1d87391047","owner":[],"postedDate":"August 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-10T16:02:57+00:00","versionOfRecord":{"articleIdentity":"rs-4765613","link":"https://doi.org/10.1007/s00262-025-03940-5","journal":{"identity":"cancer-immunology-immunotherapy","isVorOnly":false,"title":"Cancer Immunology, Immunotherapy"},"publishedOn":"2025-02-04 15:56:52","publishedOnDateReadable":"February 4th, 2025"},"versionCreatedAt":"2024-08-13 07:19:57","video":"","vorDoi":"10.1007/s00262-025-03940-5","vorDoiUrl":"https://doi.org/10.1007/s00262-025-03940-5","workflowStages":[]},"version":"v1","identity":"rs-4765613","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4765613","identity":"rs-4765613","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}