Background
Primary liver cancer is one of the most commonly diagnosed forms of cancer worldwide in 2020, with approximately 906,000 new cases and 830,000 deaths [
1]. The majority of these cases are hepatocellular carcinoma (HCC), with the highest rates recorded in Eastern Asia (Age-standardised incidence rate: 14.8) [
2]. When diagnosed early, the primary treatment for HCC is surgical resection; however, many patients are diagnosed at advanced stages, making systemic therapies the only option. Despite being potentially curable with hepatectomy, nearly 70% of patients relapse within five years [
3]. Recently, immune checkpoint inhibitors have revolutionized the management of HCC, yet the factors associated with adverse clinical reactions and molecular pathways to drug resistance remain largely unknown [
4]. Consequently, there is an urgent need for effective adjuvant therapies to reduce the recurrence for HCC post-curative treatments.
Immunotherapy has recently become a significant component of cancer treatment, with adoptive cell therapy (ACT) being one of its most important forms [
5]. ACT involves the infusion of tumor-infiltrating lymphocytes or peripheral blood-derived immune cells, such as T lymphocytes (T cells) and natural killer cells (NK cells), into patients. NK cells are generally known to be effective in the innate defense against tumor growth and metastases, with a 73% response rate being observed in clinical trials involving hematological malignancies [
6]. However, the response rate for NK cells adoptive transfer immunotherapy is much lower when it comes to solid tumors such as colorectal cancer, non-small cell lung cancer, melanoma, and hepatocellular carcinoma. This poses a considerable challenge for NK cell-based therapy to be used effectively against solid tumors.
It has been established that there is a positive correlation between the density of NK cells and the prognosis of HCC patients, indicating a critical role of NK cells in controlling tumor growth [
7]. Cytokine-induced memory-like (CIML) NK cells, which differentiate after being activated with interleukin-12 (IL-12), IL-15, and IL-18, have been observed to possess enhanced anti-tumor responses. Preclinical and clinical studies suggest that memory NK cell activities could be beneficial in tumor settings and may contribute to relapse prevention in the context of hematopoietic malignancies[
8,
9]. A key feature of memory-like NK cells is their extended lifespan and the ability to generate persistent responses. Despite this, CIML NK cells have not yet achieved significant clinical success in the treatment of solid tumors, unlike myeloid malignancies. This is due to factors that may impede the homing and penetration of NK cells into the deeper areas of tumor masses, such as hypoxia, lack of visible NK cell receptors, and desmoplastic stroma in the solid tumor setting[
10‐
13].
The iRGD peptide (sequence: CRGDKGPDC) mediates the permeability of tumor blood vessels, resulting in internalization and penetration into tumor tissues, thereby improving diagnostic accuracy and therapeutic efficacy [
14‐
16]. Our team has also reported that iRGD can enhance the infiltration of transferred T cells into tumors [
17]. Specifically, T cells that were coated with iRGD connected to 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-polyethylene glycol (DSPE-PEG) demonstrated a significantly increased ability to penetrate deep into tumor tissues. Furthermore, combining iRGD modification with PD-1 knockout lymphocytes has been shown to be highly effective in multiple xenograft mouse models.
In this research, we posited that combining two strategies to improve NK cell-mediated anti-HCC responses, namely memory-like differentiation and iRGD-modification, would be a feasible and efficient immunotherapeutic approach for HCC. Our results showed that IL-12, IL-15, and IL-18-activated NK cells had memory-like properties, which resulted in an increased production of IFN-γ and TNF-α upon restimulation with tumor cells and heightened cytotoxicity when compared to IL-15-maintained NK cells. Furthermore, we also established that CIML NK cells modified with iRGD had enhanced penetration capacity and killing capacity in MCSs and a HCC xenograft tumor model.
Materials and methods
Cell lines
The cell lines HepG2, SK-Hep-1, HUVEC and iRGD receptor-positive HGC27 were purchased from the Cell Bank of the Shanghai Institute of Biochemistry and Cell Biology. The cells were cultured in RPMI-1640 medium which was enriched with 10% fetal calf serum, 100 U/mL penicillin and 100 µg/mL streptomycin at 37 °C and 5% CO2. The cell identities were authenticated by phenotype or genotype and only Mycoplasma-free cells were employed. Human dendritic cells were generated as previously described [
18]. In brief, dendritic cells (DCs) were generated from monocytes enriched by adherence for 2 h, and then cultured in AIM-V medium containing human GM-CSF (500 U/ml, Peprotech) and IL-4 (500 U/mL, Peprotech) for 5 days. To obtain mature DCs, fresh complete medium containing LPS (10 ng /mL, Sigma) and IFN-γ (500 U/mL, Peprotech) was added to the culture on day 5. The culture was continued for an additional 24 h.
NK cells purification and generation of CIML NK cells
PBMCs were collected from patients with informed consent and used to isolate primary NK cells via CD56 positive selection (Miltenyi Biotec, Germany). For cytokine induced-memory-like and control NK cells, NK cells were cultured at 3–5 × 10
6 cells/mL and pre-activated for 16 h (on day 1) with a mixture of 10 ng/mL IL-12 (Amoytop, China), 50 ng/mL IL-15 (Amoytop, China), and 50 ng/mL IL-18 (Amoytop, China) or control conditions (IL-15, 3 ng/mL). After 16 h, the NK cell preparations were washed three times with PBS (Hyclone) to remove cytokines and placed in a complete AIM-V medium containing 10% fetal bovine serum (Gibico, USA) supplemented with IL-15 (3 ng/mL) to support survival. The medium was refreshed every 2 or 3 days, with additional IL-15 supplementation [
11,
12].
Flow cytometry analysis
A Cytoflex flow cytometer (Beckman Coulter, USA) was utilized to perform fluorescent expression analysis. For phenotypic NK cell characterization, the following antibody clones were employed: anti-CD16-PE (3G8, BD Biosciences, USA), anti-CD56-BB700(B159, BD Biosciences, USA), anti-CD56-APC (B159, BD Biosciences, USA), anti-CD3-FITC (HIT3a, BD Biosciences, USA), anti-KIR-APC (DX9, BD Biosciences, USA), anti-NKp30-APC-R700 (P30-15, BD Biosciences, USA), anti-NKG2A-BV650 (CD159a, BD Biosciences, USA). The frequency of cells that fell within the CD56+ gate was used to determine the cells expressing each antigen. Flow cytometry analysis of anti- CD25-APC (2A3, BD Biosciences, USA), and anti- CD137-PE (4B4-1, BD Biosciences, USA) was employed to assess CIML NK cells activated by cytokines. Anti-CD107a-PE (H4A3, BD Biosciences, USA) and anti-GranzymeB-PE (GB11, eBioscience, USA) were employed to assess CIML NK cells cytotoxicity when co-culture with HCC cells. Restimulation assays were performed by incubating NK cell preparations with target cells (K562, HepG2, HGC27, or SK-Hep-1 cells) at an effector-to-target ratio of 2:1 for 6 h or 24 h in complete medium (supplemented with 3 ng/mL IL-15) one week after initial stimulation. Cells were washed, and their expression of different markes was analyzed through antibody staining and flow cytometry, as previously described. Expression of iRGD receptors on HepG2, monocyte, DC and HUVEC was measured by staining cells with anti-αvβ3-FITC (LM6090, EMD Millipore), anti-αvβ5-FITC (P1F6, EMD Millipore), or anti-NRP-1-PE (AD5-17F6, Miltenyi Biotec GmbH). Monocytes were gated with anti-CD14-APC (M5E2, BD Biosciences, USA) and DCs were gated with anti-CD11c-APC (B-ly6, BD Biosciences, USA). Prior to analysis, all samples were suspended in flow cytometry staining buffer (FACS) buffer and stained with the indicated antibodies for 30 min at 4 °C in the dark, followed by two washes and resuspension in FACS buffer.
Intracellular cytokine staining
Sorted CD56+ NK cells were pre-activated with IL-12, IL-15, and IL-18 for a brief period. After a 7-day rest period, ICS was conducted and 1 × 106 CIML NK cells were re-stimulated with HepG2 cells at the designated effector to target cell ratios. Subsequently, GolgiStop (BD Biosciences) was administered to the stimulated NK cells in accordance with the manufacturer's instructions for 8 h. Following treatment, the NK cells were stained with a fixable live/dead stain (FVS780) and anti-CD56-BB700 for 30 min at room temperature. Cells were then fixed and permeabilized using the Fixation/Permeabilization Solution Kit (BD Biosciences). Intracellular cytokines were stained with anti-IFN-γ-PE (B27, BD Biosciences) and anti-TNF-α-APC (MAb11, BD Biosciences) for 1 h at 4 °C. After washing with permeabilization buffer, the cells were fixed with 1% paraformaldehyde solution (Sigma-Aldrich) and analyzed using a CytoFLEX.
CD107a and granzyme B assays
For CD107a and granzyme B assays, sorted NK cells were pre-activated with IL-12, IL-15, and IL-18 or a control condition for 16 h before being differentiated into memory-like or control NK cells over a 7-day period. Following re-stimulation with either HepG2 or SK-Hep-1 cells at the designated effector to target cell ratio of 2:1, CD107a-PE antibody was added for the first hour, followed by GolgiPlug (BD Biosciences) for a further five hours. The cells were then washed, stained with a fixable live/dead stain (FVS780) and surface antibodies for 30 min at room temperature before being analyzed using a CytoFLEX. For granzyme B assays, NK cells re-stimulation with target cells for the first hour, cells were cultured with protein transport inhibitor GolgiPlug (BD Biosciences) for a further five hours. Following treatment, samples were fixed, permeabilized (Fixation/Permeabilization Solution Kit, BD Biosciences), and stained with anti-Granzyme B-PE (GB11, eBioscience, USA). After washing with permeabilization buffer, the cells were fixed with 1% paraformaldehyde solution (Sigma–Aldrich) and analyzed using a CytoFLEX.
Cytometric bead array analysis of cytokines
For tumor cells restimulation, K562, HepG2 and SK-Hep-1 cells were added on day 7 to the respective NK cell preparation (E: T ratio of 2:1). After 6 h, the concentrations of cytokines in the culture supernatants were quantified using a cytometric bead array in accordance with the instructions provided by the manufacturer (BD Biosciences). The Human IFN-γ Flex Set (Bead B8) (BD Biosciences) was used to detect single-cytokine IFN-γ. The samples were analyzed by a Cytoflex flow cytometer and the data were analyzed with the help of FCAP version 3.0 array software (Soft Flow).
Cytotoxicity assay
The cytotoxicity of CIML NK cells was evaluated in vitro against HCC cell lines (HepG2 and SK-Hep-1) on day 7 using a carboxy fluorescein succinimidyl amino ester and propidium iodide (CFSE/PI) assay. Initially, target tumor cells were labeled with 4 μM CFSE in a 37 °C incubator with 5% CO2 for 10 min. Following labeling, the cells were washed with PBS and then seeded into 48-well plates. The CFSE-labeled target tumor cells were then incubated with NK cells at various effector-to-target ratios at 37 °C and 5% CO2 for 6 h. Wash the cells with PBS(Hyclone) (5 min, 300 × g, RT) and resuspend the pellet in 100 μL PBS (Hyclone). To assess the ratio of cell death, PI (Sigma) was added. Finally, the samples were examined via flow cytometry.
Synthesis of DSPE-PEG-iRGD
As previously described [
17], DSPE-PEG-Mal (Laysan Bio, Inc, USA) and C-iRGD (ZPC, china) were mixed at a 1:1 molar ratio in Hepes buffer (pH = 6.5) and allowed to react at room temperature for 48 h under a nitrogen atmosphere. The reaction mixture was then dialyzed in deionized water for 48 h to remove any free iRGD, and the resulting solution was lyophilized and analyzed using MALDI-TOF MS and
1H NMR spectroscopy.
The effect of CIML NK cells modified with DSPE -PEG -iRGD on multicellular spheroids (MCSs)
HGC27 cells were seeded in a 96-well plate (round bottom, ultra-low attachment surface, Corning, USA) containing RPMI1640 supplemented with 10% FBS (7500 cells/well). After attachment, half of the medium was replaced with RPMI1640 supplemented with 10% FBS. Daily observation under a light microscope was conducted to determine the formation of 'spherical' spheroids, and their diameters were measured using ImageJ software. When the spheroids had reached a size of around 500 µm, they were chosen for further research. In order to evaluate penetration, CIML NK cells and c-NK cells were cultivated for 7 days and then labelled with CFSE before being modified with DSPE-PEG-iRGD (20 µg modification reagent for every 1 × 106 cells). These modified NK cells were then added to the multicellular spheroids (MCSs) at an effector-to-target (E:T) ratio of 5:1 which was calculated on the initial number of spheroids inoculated. After 6 h of incubation at 37 °C, the spheroids were washed for the removal of free NK cells, fixed in 4% paraformaldehyde, and imaged using a ZEN 710 confocal microscope (Zeiss, Jena, Germany). Images were acquired at the midheight of the spheroids and surface plots were generated using ImageJ software. For killing assay, NK cell preparations were modified with DSPE-PEG-iRGD and co-cultured with MCSs. After incubation, MCSs were treated with Calcein AM and Ethidium homodimer III (EthD-III) solutions (Viability/Cytotoxicity Assay Kit, biotium, USA) as per the manufacturer's instructions. The MCTS were then washed with PBS twice and placed in confocal dishes (In Vitro Scientific, Austria). The excitation wave-lengths were 494 nm for Calcein AM, 532 nm for EthD- III and the fluorescence signals were collected in 517 nm for Calcein AM and 625 nm for EthD-III. The images were then processed using ImageJ software.
Xenograft mouse models
The Ethics Committee of Drum Tower Hospital (Nanjing, China) approved all experiments in this study. All animal procedures were conducted in accordance with the guidelines set by the Animal Care Committee at Drum Tower Hospital. The investigators were not blind to the animal studies. Every effort was made to decrease the number of animals used and to reduce their suffering. The mice were allocated randomly based on age and weight. For the subcutaneous tumor model, 6–8 weeks old male BALB/c nude mice were injected subcutaneously in the right axilla with HepG2 cells (5 × 106 suspended in 100 μl PBS).
In vivo near-infrared florescence imaging
To investigate the tumor targeting efficiency of CIML NK-iRGD in tumor-bearing mice, 107 CIML NK cells stained with near-infrared fluorescent probe DiR (Bridgen, China) were injected intravenously (HepG2 subcutaneous tumor model). At different time intervals, the mice were anesthetized and scanned using a CRi MaestroTM Automated In Vivo Imaging System (C.R. INTERNATIONAL INC, USA). We also created a single-cell suspension of tumors after 24h that had been treated with CIML NK or CIML NK-iRGD and evaluated the infiltration of adoptive transferred NK cells by flow cytometry.
In vivo antitumor efficacy
To create a subcutaneous tumor model, 6–8 weeks old male BALB/c nude mice were injected subcutaneously in the right axilla with HepG2 cells suspended in 100 μl PBS. Two weeks post tumor induction, the mice were treated with intravenous injection of 0.1 ml PBS, P–C-NK, P–C-NK-iRGD, P-CIML NK, or P-CIML NK-iRGD every seven days for a total of two times. Additionally, 50,000 U of human recombinant IL-2 was administered intraperitoneally every other day after cell transfer. At the time of adoptive cell transfer, the mice were weighed and checked for tumor development. The volume of the tumor was estimated by assuming an ellipsoid shape and calculated as length × width2 × 0.5. Lastly, major organs were collected, fixed in 4% paraformaldehyde, sectioned, and stained with H&E in order to assess safety. After retro-orbital blood was taken, Balb/c-nude mice with HepG2 tumors were put down, and surgical procedures were conducted to obtain subcutaneous tumor tissue and organs (heart, liver, spleen, lung, and kidney). These tissues were then fixed in 4% paraformaldehyde, sectioned, and stained with H&E to assess safety. Furthermore, mice blood samplings were analyzed for serum biochemical tests such as blood urea nitrogen (BUN), creatinine (Cr), alanine transaminase (ALT), aspartate transaminase (AST).
Statistical analysis
GraphPad Prism v.9.0 was used for graphical representation and statistical analysis with all statistical comparisons indicated in the figure legends. Data are represented showing mean ± standard error of the mean (s.e.m.). All comparisons used a two-sided an of 0.05 for significance testing.
Discussion
Hepatocellular carcinoma (HCC) is a major health concern with an increasing prevalence and poor prognosis [
31]. NK cells have been identified as potential targets for immunotherapeutic approaches in HCC treatment due to their anti-tumor activity. Studies have reported that the number and function of NK cells are significantly reduced in HCC patients, and this reduction of tumor-infiltrating NK cells is linked to poor survival in the advanced stages of HCC [
32]. This highlights the importance of intrahepatic NK cells in the immune response against HCC. Consequently, various strategies have been developed to improve NK cell function and restore NK cell activity in the fight against HCC. NK cell-based adoptive transfer therapy requires expanding NK cells ex vivo, long persistence in vivo, maximal in vivo activity, and NK cell killing specificity.
Numerous studies have highlighted the powerful antitumor effects and therapeutic potential of cytokine-induced memory-like NK cells [
9,
33‐
35]. pre-clinical studies have documented clinical responses in over half of patients with relapsed refractory acute myeloid leukemia [
9], without any apparent toxicity. Moreover, the transferred NK cells proliferated, expanded, and sustained enhanced antileukemia responses in patients [
8,
28]. Nevertheless, clinical studies on CIML NK cell-based immunotherapy for solid tumors are rare. The treatment of solid tumors by NK cells faces many difficulties due to tumor microenvironment (TME) and inhibitory immunity in solid tumors, leading to weak NK cell function, poor tumor trafficking, and infiltration of NK cells into the tumor. NK cells have been observed to infiltrate primary tumors of solid cancer, as well as metastases and tumor-infiltrating lymph nodes. However, most solid tumors usually have lower NK cell infiltration [
36], and most studies report a decline in NK cell infiltration in malignant tissues compared to corresponding non-malignant tissues [
37,
38].
We compared NK cells pre-exposed to IL-12, IL-15, and IL-18 with controls from the same donor in terms of memory-like phenotypes and effector function. Our data revealed a distinctive memory-like phenotype of CD56dimCD16dim CIML NK cells, characterized by lower levels of KIR and higher expression of NKp30 and NKG2A. Furthermore, IL-12, IL-15, and IL-18 induced CD25 and CD137 upregulation on CIML NK cells even after incubation with cancer cell lines (K562, HepG2, and SK-Hep-1 cells) after a 6-day interval. CIML NK cells were found to express TNF-α and IFN-γ at higher levels than control NK cells in the presence of HepG2 target cells. Additionally, CIML NK cells were able to produce large amounts of IFN-γ within 6 h of incubation with cancer targets, resulting in enhanced cytotoxicity against HCC cells in vitro. In accordance with augmented cytotoxicity, NK cells primed with IL-12, IL-15, and IL-18 show increased levels of granzyme B and CD107a when restimulated with the target cells HepG2 and SK-Hep-1. This was further validated by the fact that spheroids derived from HGC27 cell lines were efficiently penetrated and killed by iRGD-modified CIML NK cells. Finally, iRGD-modified IL-12, IL-15, and IL-18 primed PBMCs, which is mainly the CIML NK cells inside that had a higher cytotoxic potential in the xenograft model of HCC.
Previous studies have identified CIML NK cells by their increased expression of CD56, NKG2A, and NKp30, as well as a modest decrease in the median expression of CD16 [
9,
33]. Upon comparison of the phenotypes of CIML NK cells with sorted CD56
brightCD16
−, CD56
dimCD16
+, and CD56
dimCD16
dim subsets, a modest decrease in the percentage of cells expressing KIR were observed. In our experimental system, CIML NK cells contained a stable percentage of NKp30
+ NK cells (approximately 90%). Additionally, a significantly higher expression of NKG2A
+ NK cells was noted in CIML NK cells compared to controls from the same donor. Our data suggest that pre-activation appears to reduce the percentage of KIR
+CD56
dimCD16
dim NK cells and enrich NKp30
+NKG2A
+CD56
dimCD16
dim NK cells, which may be due to activation-induced shedding of CD16. Functional analysis of CIML NK cells demonstrated a much higher expression of cytokines and cytotoxicity when exposed to HCC cancer cells. Our results indicate that these memory-like NK cells are resting and have the capacity to be readily boosted, with effector memory-like NK cells being a major source of IFN-γ in the recall immune response, developing an immediate IFN-γ response.
The combination of CIML NK cells and iRGD may overcome intrinsic infiltration resistance in tumor tissues and facilitate NK cell-mediated cytotoxicity. Our research indicates that CLML NK cells modified with iRGD demonstrate greater accumulation and infiltration in the MCSs or subcutaneous hepatocellular carcinoma tumor model. Enhanced penetration, coupled with its memory characteristics, eventually translated into potent antitumor efficacy of CIML NK cells modified with iRGD, as demonstrated in xenograft mouse models. The modification of IL-12, IL-15, and IL-18 primed PBMCs with iRGD significantly reduced the growth of HepG2-cell bulk with ensured safety. However, to ensure safety, much effort is still required to verify the durability of CIML NK cells and to assess the efficacy of iRGD-modified CIML NK cells when administered through adoptive transfer. We have already conducted a clinical trial on the combination of activated T cells with iRGD-based adoptive immunotherapy (ChiCTR2200061306). Following this, we plan to initiate clinical trials of adoptive immunotherapy using iRGD-modified CIML NK cells.
Our experiment demonstrated that IL-12, IL-15, and IL-18 mainly activate NK cells, not T cells; however, our in vivo experiment utilized adoptive transfer of IL-12, IL-15, and IL-18-activated PBMCs, which has certain restrictions. To address this, we plan to use K562 cells that have been altered with CD137L to boost the CIML NK from PBMCs, thus guaranteeing that more than 90% of CD56+ CIML NK is transfused back in vivo.
Based on current clinical results, the potential of CIML NK cells as effective anti-tumor agents has been demonstrated [
28,
29,
35,
39]. However, these cells face the challenge of tumor-associated immune suppression, including the production of immunosuppressive molecules, low nutrient levels, and hypoxic conditions, as well as the presence of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). Several strategies have been proposed to combine CIML NK cells with immunotherapeutic monoclonal antibodies targeting checkpoint inhibitory receptors to address this. For instance, a combination therapy of CTLA-4 inhibitor (Ipilimumab), CIML NK cell infusion, and interleukin-15 superagonist (N-803) is currently being evaluated for the treatment of advanced head and neck cancer (Clinical trials. gov # NCT04290546). This regimen is based on the immunomodulatory role of CTLA-4 inhibitor (ipilimumab) in depleting tumor-infiltrating Tregs, followed by the adoptive transfer of CIML NK cells. Thus, it is important to consider immunomodulatory treatments in CIML NK-iRGD cell therapy to improve the efficacy of solid tumor management.
For the first time, we examined the use of iRGD in cytokine-induced memory-like NK-based adoptive immunotherapy. Our findings showed that CIML NK cells modified with iRGD had improved penetration and killing abilities in MCSs and HCC xenograft tumor models. This combination approach could greatly improve the effectiveness of adoptive NK cell immunotherapy for various solid tumor types.
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