Cell culture
The liver cancer cell lines Huh-7 and Hepa1c1c7 and the NK cell line NK92MI were purchased from the Korean Cell Line Bank (Republic of Korea) and American Type Culture Collection (USA). Huh-7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; HyClone) and 1% penicillin‒streptomycin (P/S; Gibco, Grand Island, NY, USA). Hepa1c1c7 cells were cultured in minimum essential medium alpha (MEM-α; Gibco) supplemented with 10% FBS and 1% P/S. NK92MI cells were cultured in MEM-α supplemented with 2 mM L-glutamine (Gibco), 1% P/S, 0.1 mM 2-mercaptoethanol (Gibco), 0.02 mM folic acid (Sigma‒Aldrich, St. Louis, MO, USA), 0.2 mM inositol (Sigma‒Aldrich), and 12.5% FBS. All cells were incubated at 37 °C in a 5% CO2 atmosphere.
Following the manufacturer’s protocol, mouse primary NK cells (mNK) were extracted from mouse spleens using a commercially available mouse NK cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). The isolated mNK cells were then cultured in RPMI medium (HyClone) containing 10% FBS, 1% antibiotic/antimycotic solution (Gibco), 2 mM L-glutamine, 1% nonessential amino acids (Gibco), and 50 µM 2-mercaptoethanol. Mouse interleukin-2 (IL-2; PeproTech, Cranbury, NJ, USA) was also added to the culture.
In vitro PEF exposure and DAMP expression analysis
To assess the effects of PEF on liver cancer cells, 2 × 105 Huh-7 and Hepa1c1c7 cells were seeded into 12-well plates and treated using an ECM 830 electroporator (BTX, Holliston, MA, USA). After 12 h, the cells were washed with phosphate-buffered saline (PBS) and treated with a bipolar PEF setting in serum-free medium (voltage: 500 V, pulse duration: 100 µs, electrode spacing: 5 mm). After 2 h, the medium was collected to analyze the amount of ATP released. Analysis of the released ATP was performed using an ATP bioluminescence kit (Sigma‒Aldrich). HMGB1 release was assessed by collecting CM after 24 h of PEF treatment and performing an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocol (ABclonal Biotechnology, Woburn, MA, USA). To confirm the ablation zone caused by PEF, PEF-treated cancer cells were washed with PBS and stained with calcein-AM (Invitrogen, Waltham, MA, USA). After fixation with 4% paraformaldehyde (PFA), the area of living cells was observed using the area scan mode of a microplate reader (Synergy H1; BioTek, Winooski, VT, USA).
NK cell migration assay
The migration activity of NK cells was analyzed using a 24-well insert 8.0 μm Transwell chamber (SPL, Pocheon, Republic of Korea). Complete medium and 75% Con-CM or PEF-CM were added to the bottom chamber. A total of 1 × 105 NK cells suspended in serum-free medium and stained with 1 μM CellTrace CFSE (Invitrogen) were loaded into the upper transwell insert. After 16 h, the CFSE-labeled NK cells in the bottom chamber were imaged using a fluorescence microscope (TM-30iF; TAESHIN BIO, Namyangju, Republic of Korea) and quantified using a Luna Cell Counter (Logos, Anyang, Republic of Korea). Anti-CX3CL1 antibody (5 μg/mL, R&D Systems, Minneapolis, MN, USA) was added to PEF-CM to neutralize CX3CL1 released by PEF treatment.
NK cell-mediated cytotoxicity assay
To analyze NK-mediated cytotoxicity by flow cytometry, NK cells were first stained with CellTrace CFSE or Far-red dye (Invitrogen) and then cocultured with target cancer cells for 4 h at effector-to-target (E/T) ratios of 10:1, 5:1, 2.5:1, and 1.25:1. Total cells were subsequently stained with 7-aminoactinomycin D (7AAD; Thermo Fisher Scientific, Waltham, MA, USA), and cell lysis was determined using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA) and analyzed using FlowJo Software (BD Bioscience, Franklin Lakes, NJ, USA).
Coculture imaging of cancer cells with NK cells
To evaluate the targeting capability of NK cells in vitro, Huh-7 cells were stained with CellTrace CFSE, and 1 × 10³ Huh-7 cells per well were seeded into a 96-well confocal dish (SPL). After 24 h, NK cells were stained with CellTrace Far-Red and added to wells containing CFSE-labeled cancer cells at an E/T ratio of 2.5. Following a 4 h incubation, 4% PFA was added to each well for fixation. Images of NK cells bound to cancer cells were captured using a confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany) and analyzed using ZEN software (Carl Zeiss). The number of NK cells bound to cancer cells was counted at 10 cancer cells per well across a total of three wells. To detect the activation of apoptosis in cancer cells by NK cells, far-red-stained NK cells were cocultured with cancer cells. Cocultured samples were stained with CellEventTM Caspase-3/7 reagent (Invitrogen) according to the manufacturer’s protocol. Fluorescence images of stained samples were acquired using a confocal laser scanning microscope.
Immunoblotting
Cells were lysed with PRO-PREP lysis buffer (iNtRON Biotechnology, Seongnam, Republic of Korea) containing phosphatase inhibitors (Roche Applied Science, Penzberg, Germany) and then heated for 10 min at 95 °C. Cell lysates were resolved on sodium dodecyl sulfate‒polyacrylamide gels, and the separated proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). The membranes were blotted with primary antibodies against Caspase-3, Cleaved Caspase-3, and GAPDH (Cell Signaling); CD3ζ (Abcam, Cambridge, UK); and β-actin and HMGB1 (ABclonal) at a 1:1000 dilution. After washing, the blots were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody at RT for 1 h (1:1000; Abcam). Immunoreactivity was detected using an enhanced chemiluminescence solution (Thermo Fisher Scientific).
Chemokine and cytokine analysis
To screen for alterations in chemokine expression induced by IRE treatment in HCC tumors, tumors were mechanically disrupted and lysed with PRO-PREP lysis buffer. The lysates were centrifuged at 13,000 rpm for 30 min, and the resulting supernatants were pooled from at least three independent biological samples. The pooled samples were then processed for chemokine array according to the manufacturer’s protocol (R&D Systems). To determine the levels of human CX3CL1 and mouse CX3CL1 in PEF-CM from Huh-7 and Hepa1c1c7 cells, ELISA was performed using reagents from ABclonal Biotechnology. Huh-7 and Hepa1c1c7 cells (2 × 105) were seeded in 12-well plates. After 12 h, the cells were washed with PBS and treated with bipolar PEF pulses in serum-free medium. After 24 h, the CM was harvested and processed for ELISA according to the manufacturer’s protocol. To evaluate the local concentrations of IFN-γ and TNF-α within HCC tumors, ELISA was performed using reagents from R&D Systems. Tumors were mechanically disrupted and lysed with PRO-PREP lysis buffer, and the tissue lysates were centrifuged at 13,000 rpm for 30 min. The supernatants were then processed for ELISA according to the manufacturer’s protocol.
Imaging of ROS
To detect ROS levels by PEF, PEF-treated liver cancer cells were incubated with 20 μM 2′,7′-dichlorofluorescin diacetate solution (DCF-DA; Sigma‒Aldrich) for 30 min at 37 °C and washed twice with PBS. The fluorescence intensity was imaged under a fluorescence microscope TM-30iF and measured using ImageJ software.
Synthesis of DLNPs
Lipid components, including D-Lin-MC3-DMA (MC3; MedChemExpress, Monmouth Junction, NJ, USA), 1,2-dioleoyl-3-trimetylammonium-propane (DOTAP; Avanti Polar Lipids, Alabaster, AL, USA), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti), cholesterol (Sigma‒Aldrich), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethlene glycerol-2000 (PEG-DMG; Avanti), were individually solubilized in ethanol to generate stock solutions at concentrations of 25 mM for DOTAP, MC3, DSPC, and cholesterol and 12.5 mM for PEG-DMG. The mRNA encoding enhanced green fluorescent protein (EGFP) was purchased from TriLink Biotechnology (San Diego, CA, USA). To generate anti-hGPC3 CAR mRNA and anti-mGpc3 CAR mRNA, we used the mMESSAGE mMACHINETM T7 Ultra Kit (Thermo Fisher Scientific). The anti-hGPC3 CAR and anti-mGpc3 CAR constructs were designed as shown in Supplementary Figs. 10 and 11. The lipid solutions were equilibrated at 37 °C for 10 min before mixing. The lipids were mixed at a molar ratio of 30:35:7:26.95:1.05 (DOTAP: MC3: DSPC: Cholesterol: PEG-DMG). This lipid formulation was then combined with mRNA in 25 mM sodium acetate buffer (pH 5.2), maintaining a volume ratio of 1:3 and a weight ratio of 1:20 (mRNA to lipids). The synthesized DLNPs were dialyzed in PBS for 1 h to eliminate residual ethanol and equilibrate the pH to neutral before application.
Characterization of DLNPs
DLNPs were characterized using dynamic light scattering (DLS; Malvern Nano ZS, Malvern Instruments Ltd., Worcestershire, UK) to ascertain their diameter and surface zeta potential. Morphological assessments were conducted using cryo-electron microscopy (Cryo-EM; Glacios, Thermo Fisher Scientific). The encapsulation efficiency of mRNA within the DLNPs was quantified using a Quanti-itTM RiboGreen RNA assay kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. This procedure involved lysing the DLNPs with 0.5% Triton X-100, staining the released mRNA with Quant-it RiboGreen reagent, and quantifying the fluorescence using a Synergy H1 microplate reader. The encapsulation efficiency (%) was calculated using the following formula:
$${Encapsulation}\,{efficeincy}\,\left( \% \right)=\left(1-\frac{free\,mRNA\,concentration}{total\,mRNA\,concentration}\right)\times 100$$
Generation and expansion of HCC patient-derived organoids (HCC-PDOs)
Patient-derived HCC cells were isolated using a digestion method as described in Broutier et al.60,61 (SKKU2024-04-003). Briefly, the tumor tissue (1 ~ 3 mm tissue) was minced and washed using basal media (Advanced DMEM/F-12 (Gibco) with 1% P/S, 1% GlutaMAX (Gibco), and 1% HEPES (Gibco)). Minced tissue was added to prewarmed digestion solution, basal media supplemented with B-27 without vitamin A (Gibco), 1.25 mM NAC (Sigma‒Aldrich), 5% RSPO1-CM (Homemade), 2.5 mg/mL collagenase D (Roche), and 0.1 mg/mL DNase I (Sigma‒Aldrich). The minced tissues in the digestion solution were incubated using a shaking incubator at 37 °C for 30 min to 2 h (confirming the tissue conditions in the digestion solution every 30 min). After incubation, digested tissues were filtered through a 100 µm nylon cell strainer (SPL) with additional cold-basal media and spun down at 200 x g for 5 min at 4 °C, and the supernatant was discarded. If red blood cells remained in the pellet, the pellet was incubated in 3 mL of ACK buffer (Gibco) for 3 min at room temperature (RT). After washing, the cells were resuspended in 70% Matrigel (Corning, diluted basal media) and seeded in a prewarmed plate (seeding density: 1 × 105 cells per well in a 24-well plate). After incubation for 30–60 min, prewarmed media was added, and the media was replaced every 2–3 days. The organoid media was based on basal media supplemented with B-27 without vitamin A, 1.25 mM NAC, 10 nM gastrin (Sigma‒Aldrich), 50 ng/mL human EGF (R&D Systems), 15% RSPO1-CM, 100 ng/mL human FGF10 (Peprotech), 50 ng/mL human HGF (Peprotech), 3 µM CHIR99021 (TOCRIS, Bristol, UK), 100 ng/mL human FGF7 (Peprotech), 2 µM A83-01 (TOCRIS), 0.5 nM Wnt Surrogate FC Fusion protein (IPA), 10 µM Y-27632 (TOCRIS), and 10 µM TRULI (TOCRIS).62 The grown HCC-PDOs were passaged at a 1:2 ratio once every 1–2 weeks as described in previous studies.60,62
Immunofluorescence of HCC-PDOs
Whole-mount organoid staining was conducted using a method described in Dekkers et al.63 Briefly, HCC-PDOs were harvested from Matrigel using cold-basal media or cold-cell recovery solution (Corning) and fixed in 4% PFA on ice for 30 min. After incubation, the fixed HCC-PDOs were washed using 1X DPBS (Gibco) and transferred to a confocal plate. Fixed HCC-PDOs were blocked with blocking solution (1X DPBS supplemented with 0.1% Triton X-100 (Sigma‒Aldrich) and 0.2% bovine serum albumin (BSA; Sigma‒Aldrich)) at 4 °C for 1 h. After blocking, HCC-PDOs were incubated with primary antibodies (GPC3, Invitrogen, 1:200) at 4 °C overnight in blocking solution. After incubation, the samples were washed using blocking solution 3 times and incubated with secondary antibodies (anti-mouse Alexa 488, Invitrogen, 1:1000) and DAPI (Invitrogen, 1:1000) overnight in blocking solution at 4 °C. Stained HCC-PDOs were washed 3 times and stored at 4 °C in DPBS before imaging. The samples were confirmed using a fluorescence microscope, DM IL LED (Leica, Wetzlar, Germany).
Flow cytometry
Cells were harvested as single-cell suspensions. After washing with PBS, single cells were stained with Zombie (BioLegend, San Diego, CA, USA) for live/dead staining and blocked with FcBlocker (BD Bioscience) in FACS buffer (0.09% sodium azide and 2% FBS in PBS). Single cells were then incubated with a fluorophore-conjugated antibody (CD45, CD11b, CD3, NK1.1, CD4, CD8, Ly6G, CD11c, MHCII, CD80, CD86; BioLegend) in FACS buffer at RT for 2 h. After staining, the fluorescently labeled cells were washed twice with FACS buffer and fixed in 2% PFA. Fluorescently labeled cells were acquired on a MA900 (NFEC-2024-10-300262; Sony Biotechnology Inc., San Jose, CA, USA) flow cytometer. The gating strategy is shown in Supplementary Figs. 1 and 16.
Animal study
All in vivo experiments were conducted according to the guidelines of the protocol approved by the Sungkyunkwan University Institutional Animal Care and Use Committee (SKKUIACUC2023-04-41-1; Republic of Korea). The mice were kept in a controlled environment with a regulated temperature and 12 h light-dark cycle. Seven-week-old C57BL/6 and nude mice were obtained from Orient Bio (Republic of Korea). Seven-week-old NOD-Prkdcem1BaekIl2rgem1Baek (NSG) mice were obtained from JA Bio (Republic of Korea). Before the experimental procedures, the mice were acclimated for one week. To establish an orthotopic model of HCC, thioacetamide (TAA; 50 mg/kg, Sigma‒Aldrich) was intraperitoneally injected into the mice three times for 10 days to induce liver fibrosis. Subsequently, 2 × 107 Hepa1c1c7 cells/mL were prepared by mixing equal volumes of cells and Matrigel (Corning, Corning, NY, USA). Twenty microliters of this mixture was then inoculated into the livers of C57BL/6 or nude mice. After 10 days, IRE was directly administered to the tumor region of the liver (voltage: 500 V, pulse duration: 100 µs, electrode spacing: 1 mm), and then control mNK or CAR-mNK cells (2 × 106 cells/mouse) were injected intravenously.
Huh-7 cells (5 × 106) were mixed with Matrigel (DPBS/Matrigel ratio = 1) and then inoculated subcutaneously into the right flank of NSG or nude mice. When the diameter of the subcutaneous tumors reached 7 mm, IRE was applied to the tumor (voltage: 500 V, pulse duration: 100 µs, electrode spacing: 5 mm). After IRE treatment, 1 × 107 Con-NK92MI or CAR-NK92MI cells were intravenously injected into the tail vein. Animals were euthanized when tumors reached 1500 mm³ of calculated tumor volume.
Biochemical assessments and histological analysis
For histological and biochemical assessments, mice were euthanized at specific time points, after which organs and blood samples were collected. Blood samples were centrifuged at 4000 rpm for 20 min to separate serum. Key biochemical parameters, such as AST, ALT, TBIL, and LDH levels, were quantified using a Fujifilm DRI-CHEM NX500i automated chemistry analyzer (Tokyo, Japan). For histopathological examination, major organs were quickly excised, fixed in 4% PFA, and processed. Tissue samples were dehydrated, infiltrated, and embedded in Paraplast. Paraffin sections were cut using a microtome, stained with hematoxylin and eosin (H&E), and analyzed using a ScanScope CS2 system (Leica Biosystems, San Diego, CA, USA).
Immunofluorescence tissue imaging analysis
Paraffin sections were deparaffinized, rehydrated, and heat-treated in antigen retrieval buffer. After several PBS washes, sections were blocked with 5% BSA in PBS, incubated for 30 min, and incubated overnight with primary antibody (NKp46, ABclonal, 1:50) at 4 °C. After washing with PBS, the sections were incubated with HRP-conjugated secondary antibody at RT for 20 min (1:1000). After washing with PBS, signal amplification was conducted by TSA Multiplex Immunohistochemistry kits (TissueGnostics, Vienna, Austria). Immunofluorescence images were obtained using TissueFAXs i8 plus (TissueGnostics).
Antigen-specific CD8+ T-cell analysis
Single-cell suspensions of spleen from each mouse (5 × 10⁵ cells per well) were seeded in v-bottom 96-well plates and restimulated for 4 h with Hepa1c1c7 cell lysate (10 μg/ml). A cell stimulation cocktail containing protein transport inhibitors (Invitrogen) was added to the cells. For flow cytometry analysis, cells were fixed and permeabilized using the intracellular fixation & permeabilization buffer set (Invitrogen) according to the manufacturer’s protocol. Permeabilized cells were blocked using FcBlocker and stained with fluorescent-conjugated antibodies (CD45, CD3, CD11b, CD8, and IFN-γ) at RT for 2 h. Fluorescently labeled cells were acquired on a MA900 flow cytometer. The gating strategy is shown in Supplementary Fig. 19.
Statistical analysis
Statistical analyses were performed using GraphPad Prism V.10.5.0 software (GraphPad Software, Boston, MA, USA). Statistical significance was determined using an unpaired Student’s t test, two-way ANOVA, and log-rank test. Data are presented as the mean ± S.D. from at least three independent experiments, with P < 0.05 considered statistically significant.
