Khalifa et al. Experimental Hematology & Oncology
https://doi.org/10.1186/s40164-024-00502-w(2024) 13:36

Experimental Hematology &Oncology

Open Access

RESEARCH

Vaccination with a combination of STING
agonist-loaded lipid nanoparticles and CpGODNs protects against lung metastasis via the
induction of CD11bhighCD27low memory-like
NK cells
Alaa M. Khalifa1, Takashi Nakamura1*, Yusuke Sato1 and Hideyoshi Harashima1*
Abstract
Background Natural killer (NK) cells are effective in attacking tumor cells that escape T cell attack. Memory NK cells
are believed to function as potent effector cells in cancer immunotherapy. However, knowledge of their induction,
identification, and potential in vivo is limited. Herein, we report on the induction and identification of memory-like NK
cells via the action of a combination of a stimulator of interferon genes (STING) agonist loaded into lipid nanoparticles(STING-LNPs) and cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG-ODNs), and the potential of theinducted memory-like NK cells to prevent melanoma lung metastasis.
Methods The antitumor effects of either the STING-LNPs, CpG-ODNs, or the combination therapy were evaluatedusing a B16-F10 lung metastasis model. The effect of the combined treatment was evaluated by measuring cytokine
production. The induction of memory-like NK cells was demonstrated via flow cytometry and confirmed throughtheir preventative effect.
Results The combination of STING-LNPs and CpG-ODNs tended to enhance the production of interleukin 12 (IL-12)
and IL-18, and exerted a therapeutic effect against B16-F10 lung metastasis. The combination therapy increased
the population of CD11bhighCD27low NK cells. Although monotherapies failed to show preventative effects, thecombination therapy induced a surprisingly strong preventative effect, which indicates that CD11bhighCD27low cells
could be a phenotype of memory-like NK cells.
Conclusion As far as could be ascertained, this is the first report of the in vivo induction, identification, andconfirmation of a phenotype of the memory-like NK cells through a prophylactic effect via the use of an
immunotherapeutic drug. Our findings provide novel insights into the in vivo induction of CD11bhighCD27low
memory-like NK cells thus paving the way for the development of efficient immunotherapies.

*Correspondence:
Takashi Nakamuratnakam@pharm.hokudai.ac.jp
Hideyoshi Harashimaharasima@pharm.hokudai.ac.jp
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Khalifa et al. Experimental Hematology & Oncology(2024) 13:36

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Keywords STING agonist, CpG-ODN, CD11bhighCD27low memory-like NK cells, Prophylactic cancer vaccine, Drugdelivery system
Graphical Abstract

Background
Substantial efforts have recently been directed towards
the advancement of tumor immune-based therapies
that result in reducing the burden of tumors, extending patient survival, and inducing complete remission insome cases [1]. Most immunotherapies are T-cell based
and are efficacious against many types of cancers [2–4].
However, because tumor cells can evade T-cell immune
surveillance, this is considered a limitation to their effectiveness [5, 6]. Therefore, utilizing innate immune cells
and empowering them to fight against cancer is critical,
particularly in tumors that lack major histocompatibility
complex class I (MHC-I) expression, which is considered
to be the main cause of T-cell resistance [7, 8]. MHC-I
expression is important for antigen presentation and, inturn, is indispensable for the effector function of T cells,
but it is an inhibitory receptor for natural killer (NK) cells[9, 10]. Therefore, the loss of MHC-I expression in tumor
cells is expected to allow NK cells to attack and kill thetumor cells.
Recent discoveries have led to a paradigm shift in thecharacterization of NK cells. Historically, NK cells wereassumed to be short-lived cells associated with innate
immune responses, whereas the existence of subsets of
NK cells with memory functions has now been revealed.
Although numerous issues remain to be addressed, NK
cells with memory functions can be roughly divided
into two types: memory NK cells and memory-like NK
cells [11]. Memory NK cells found in viral infected models are specific to viral antigens [12]. Memory-like NK
cells, however, are non-specific for certain antigens suchas cytokine-induced memory-like (CIML) NK cells thathave shown long-lasting effects even after the absence or

discontinuation of a stimulus [13]. However, the potential of using CIML NK cells in cancer therapy in vivo isunclear, and available information concerning its phenotype remains limited.
Research over the past few decades has highlighted
the great importance of innate immune memory in hostdefense as well as in the area of vaccinology [14, 15].
Memory-like NK cells were previously reported to be
induced by Bacillus Calmette–Guérin (BCG). Accordingly, BCG is known to induce heterologous protection
against various infections as well as many solid cancers[14, 16, 17]. In subsequent studies, researchers generated
the memory-like features of natural killer cells throughthe use of a combination of cytokines that has resulted
in enhanced functionality against many types of cancers[18, 19]. Many preclinical and very early stages of clinical trials have confirmed the safety and the promising
functional activity of these CIML NK cells following theiradoptive transfer [20–22]. Romee et al. reported that
CIML NK cells could reduce the burden of tumors andenhance the survival of tumor-bearing mice [23]. In addition, complete remissions have been observed in patientswith refractory acute myeloid leukemia following theinfusion of CIML NK cells [20].
Researchers are currently attempting to identify thephenotype of these CIML NK cells. This memory phenotypical characteristic of NK cells differs depending on
the nature of the induction and whether they are murineor human-derived [24]. Human CIML NK cells show anincrease in CD94, NKG2D, NKG2A, NKp46, NKp44, andCD25 [20]. Murine CIML NK cells also show an increasein CD25 and CD11b [19]. In addition, adoptively transferred cytokine-preactivated murine NK cells display aKhalifa et al. Experimental Hematology & Oncology(2024) 13:36

mature phenotype of CD11bhighCD27low, KLRG1high, andCD43high with a potent effector function, but this phenotype has not yet been confirmed to be associated withmemory responses [25].
For the effective induction of antitumor responses thatare mediated by NK cells in vivo, we successfully developed a lipid nanoparticle (LNP) loaded with the stimulator of interferon genes (STING) agonist (STING-LNPs)[7, 8, 26–28]. The STING pathway is a cytosolic innate
sensor for DNA and cyclic dinucleotides and is a potential candidate for use in cancer immunotherapy [7, 8].
STING-LNP treatment induced the production of systemic type I interferons (IFNs) leading to the activation of
NK cells [8, 27, 29]. However, STING agonists do not efficiently induce the production of IL-12 and IL-18, which
are indispensable for memory-like NK cell induction[30]. In contrast, CpG-ODN, an agonist of the toll-likereceptor 9 (TLR9), is a potent adjuvant for inducing Th1
cytokine responses such as IL-12 and IL-18 [31]. Given
the above information, we hypothesized that the use of a
combination of STING-LNPs and CpG-ODNs might efficiently induce the production of CIML NK cells.
In the present study, we aimed to tackle the challengeof the in vivo induction of memory-like NK cells by utilizing the benefits of a proper delivery system. We investigated the potential of combining STING-LNPs with
CpG-ODNs for the in vivo induction of the most matureand potent phenotype of NK cells, which we speculated
would be CD11bhighCD27low NK cells. The combination therapy tended to enhance the production of IL-12
and IL-18 while significantly increasing the antitumor
effect of the combination therapy against B16-F10 lungmetastasis. Flow cytometry (FCM) analyses revealed
that the combination therapy increased the mostmature phenotype of NK cells, which were identified as
CD11bhighCD27low NK cells. Two treatment cycles are
essential for the induction of CD11bhighCD27low NK cells.
Moreover, although monotherapies of either STINGLNPs or CpG-ODNs failed to show a preventative antitumor effect, the combination therapy induced a strong
and long-term preventative antitumor effect because of
the sustained presence of CD11bhighCD27low NK cells. As
far as we could ascertain, this is the first report to confirm that the CD11bhighCD27low NK cells induced by the
combination therapy of STING-LNPs and CpG-ODNs
induces the phenotypical characteristics of CIML NK
cells. These findings are the first to provide novel insights
regarding the in vivo induction and the phenotypicalcharacterization of CIML NK cells by using a combination of immunotherapeutic drugs that could not be
achieved without the aid of a proper delivery system.

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Methods
Reagents, cell lines, and mice

YSK12-C4, (6Z, 9Z, 28Z, 31Z)-19-(4-(dimethylamino)
butyl) heptatriaconta-6,9,28,31-tetraen-19-ol, was synthesized as previously reported [32–36]. Cholesterol was
obtained from Avanti Polar Lipids, Inc. (Alabaster, AL).
The 1,2-dimirystoyl-sn-glycerol methoxyethyleneglycol
2000 ether (DMG-PEG2000) was purchased from the NOF
Corporation (Tokyo, Japan). Cyclic di-GMP (c-di-GMP)
was purchased from Cayman Chemical (Ann Arbor, MI).
CpG-ODN (5’-ggGGTCAACGTTGAgggggg-3’, bases
in lower letters are phosphonothioate) was synthesizedby Hokkaido System Science (Sapporo, Japan). FITC
anti-mouse CD3, PE anti-mouse NK1.1, PE/Cyanine7
anti-mouse KLRG1, APC anti-mouse CD27, Alexa700
anti-mouse CD11b, and APC/Cy7 anti-mouse CD43
antibodies and their isotype controls as well as the purified anti-mouse CD16/32 antibody and 7-AAD viability
staining solution were purchased from Biolegend (San
Diego, CA). A VersaComp Antibody capture bead kit was
obtained from Beckman Coulter (Indianapolis, IN).
The B16-F10-luc2 cell line was purchased from Caliper
Life Science (Hopkinton, MA). B16-F10-luc2 cells were
cultured in RPMI-1640 (high glucose) containing 10%
fetal bovine serum (FBS), 1 mM sodium pyruvate and
10 mM HEPES. C57BL/6 N female mice (6–7 weeks old)
were purchased from Japan SLC Inc. (Shizuoka, Japan).
All animal experiments were approved by the Ethics ofPharmaceutical Science Animal Care Committee of Hokkaido University (approval number: 20-01226).
Preparation of the STING-LNPs

The STING-LNPs were prepared as previously reported[8]. In a typical preparation, 150 µL of 1 mM citratebuffer (pH 4.5) containing 500 nmol of c-di-GMP wasadded under vortexing to 300 µL of a lipid solution(YSK12-C4:cholesterol:DMG-PEG2000) = (85:15:1, molratio) in 90% t-butanol. The resultant mixture was added
to 1.6 mL of 1 mM citrate buffer (pH 4.5) with vortexing followed by adding 4 mL PBS. This solution was filtered using an Amicon Ultra (MWCO 100,000) and was
replaced with 4 mL of PBS. The particle size and zetapotential of the resultant STING-LNPs was measured
using a Zetasizer Nano ZS ZEN3600 (Malvern Instruments, UK). To assess the amount of c-di-GMP that was
encapsulated, the STING-LNPs were lysed with 10%
sodium deoxycholate, and the UV absorbance at 252 nm(ε = 24,700) was then measured via 264 UV/Vis spectroscopy. The required amounts of both the STING-LNPs
and the CpG-ODNs were mixed immediately prior to
injection. The resultant complex was characterized in asimilar manner.

Khalifa et al. Experimental Hematology & Oncology(2024) 13:36

Checking the cytokine levels in serum through ELISA

Mice were injected intravenously with either the STINGLNPs (4 µg), CpG-ODNs (150 µg), CpG-ODNs (250 µg),
or the combination therapy (a mixture of STING-LNPs
and CpG-ODNs). After 2 h, blood was collected, and thesera that was obtained was stored at −80°C. The concentrations of IFN-β, IL-12, IL-18, and IL-1β in the serum
were quantified using an ELISA kit (R&D systems, USA)
following the manufacturer’s instructions.
Evaluating antitumor and prophylactic activity via
luciferase assay

For the antitumor protocol, mice were challenged by
B16-F10-Luc2 cells (2 × 105) on day 0 to establish a pulmonary metastasis model. Following the intravenous
administration of either PBS, STING-LNPs (4 µg), CpGODNs (250 µg), or combined therapies for two cycles ondays 6 and 10, and the lungs were collected on day 14. In
the case of the depletion experiment, either anti-NK1.1
or its isotype control clone C1.18.4 (200 µg) and either
anti-CD8a or its isotype control clone LTF-2 (200 µg) was
intraperitoneally injected on days 5 and 9 followed by the
intravenous administration of either PBS or a combination of STING-LNPs and CpG-ODNs on days 6 and 10.
On day 14, lungs were collected and used in luciferase
activity measurements. For the prophylactic protocol,
mice were intravenously administered PBS as a control
and either the STING-LNPs (4 µg), CpG-ODNs (250 µg),
or the combined therapies on days 0 and 4 for two cycles,
and then the mice were inoculated with tumor cells
either on days 5, 14, 24 or 90, after which lungs werecollected on days 15, 28, 38, or 104, respectively. Tumor
metastasis was assessed as previously reported [7, 8].
The lung samples were completely homogenized in lysis
buffer, and luciferase activity was then quantified using aluminometer (Luminescencer-PSN, ATTO, Japan). In thecase of the immune cell depletion experiment, the valuesof relative light units (RLU) per whole lung for the control group (PBS-treated mice) were set to 1.
FCM analysis for evaluating NK cell markers in the spleen

Mice were challenged with B16-F10-Luc2 cells (2 × 105)
on day 0 to establish a pulmonary metastasis model.
This was followed by intravenous administrations ofeither PBS, STING-LNPs (4 µg), CpG-ODNs (250 µg),
or combined therapies on days 6 and 10 for two cycles.
A single-cell suspension from the spleen was prepared
for the FCM experiment on day 14. For the prophylactic
protocol, mice were intravenously administered PBS as acontrol, and either the STING-LNPs (4 µg), CpG-ODNs(250 µg) or combined therapies on days 0 and 4 for two
cycles, and tumor cells were inoculated either on days 5,
14, or 24 followed by spleen collection on days 15, 28, or
38, respectively. To track the induction and the functional

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lifetime of the memory-like NK cells, mice were intravenously administered PBS as a control or the combinationof STING-LNPs (4 µg) and CpG-ODNs (250 µg) on days
0 and 4 for two cycles followed by spleen collection ondays 7, 14, 21, and 28. To evaluate the effect of tumor cell
inoculation on the induction level of memory-like NK
cells, mice were intravenously administered either PBS
as a control or the combination of STING-LNPs (4 µg)
and CpG-ODNs (250 µg) on days 0 and 4 for two cycles,
and tumor cells were inoculated on day 24 in one groupof mice followed by spleen collection on day 28. Purified
anti-mouse CD16/32 antibody was added to the splenocytes for blocking. The splenocytes were then stainedwith the FITC anti-mouse CD3 Antibody, the PE antimouse NK1.1 Antibody, the PE/Cyanine7 anti-mouse
KLRG1 antibody, the APC anti-mouse CD27 antibody,the Alexa700 anti-mouse CD11b antibody, the APC/Cy7
anti-mouse CD43 antibody, and the isotype controls. The
splenocytes were stained with 7-AAD and then analyzed
using a CytoFlex (Beckman Coulter, Indianapolis, IN).
Data were analyzed using FlowJo software.
Statistical analysis

All statistical analyses were performed using GraphPad
Prism software. Comparisons between the two treatments were performed using an unpaired t-test. Comparisons between multiple treatments were performed
utilizing one-way analysis of variance (ANOVA) followed
by a Tukey test. Statistical significance was designated asfollows: ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05.

Results
Combining the STING-LNPs with CpG-ODNs enhanced the
production of cytokines, which was associated with theinduction of CIML NK cells

The STING-LNPs were prepared using YSK12-C4 as
the main component. YSK12-C4 is an ionizable lipid
with a high affinity for immune cells and is well known
for its high fusogenic activity that allows efficient cytosolic delivery [7, 8, 32, 33, 37]. The diameter, polydispersity index (PDI), and the zeta potential of the LNPs were
170 ± 15 nm, 0.048 ± 0.023, and 7.1 ± 0.4 mV, respectively(mean ± SD, n = 4). The required amounts of both STINGLNPs and CpG-ODNs were simply mixed and characterized before injecting the combination therapy. The
diameter, PDI, and zeta potential of the complex was
195 ± 10 nm, 0.082 ± 0.021, and − 7.2 ± 0.4 mV, respectively(mean ± SD, n = 3). We screened two in vivo doses of
CpG-ODNs for the induction of IFN-β, IL-12, IL-18, and
IL-1β, essential cytokines for the activation of NK cells
and their differentiation into CIML NK cells [19, 38].
Mice were intravenously administered either the STINGLNPs (4 µg), CpG-ODNs (150 or 250 µg), or the combination therapy. The monotherapy of the STING-LNPs

Khalifa et al. Experimental Hematology & Oncology(2024) 13:36

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drastically induced the induction of IFN-β. In contrast,
the use of only CpG-ODNs induced only low levels. The
use of a combination, however, induced much higher levels than that observed in the case of CpG-ODN monotherapy (Fig. S1A, B). The STING-LNP monotherapy
failed to induce IL-12, and, in a similar manner, only a
low level of IL-18 was observed. In addition, only low
levels of IL-12 and IL-18 were induced by the administration of 150 µg of CpG-ODNs (Fig. S1B and C). However, the combination of the STING-LNPs and 250 µg of
CpG-ODNs induced high levels of IL-12 and IL-18 (Fig.
S1C and D). Furthermore, IL-1β was similarly inducedby all groups (Fig. S1E). These results indicate the importance of combining CpG-ODNs with STING-LNPs forthe induction of IL-12 and IL-18. Accordingly, 250 µg of
CpG-ODNs combined with the STING-LNPs resulted
in the highest level of induced IL-12, and appears to be agood candidate for the induction of CIML NK cells.

(double negative: DN), CD11b− CD27+ cells (single positive: CD27+ SP), CD11b+ CD27+ cells (double positive:DP), and CD11b+ CD27− cells (CD11b+ SP). These fourdevelopmental stages from the most immature phenotype, have the ability to secrete cytokines and would havethe best ability to secrete cytokines until the most matureand potent NK cells with the highest cytolytic function had developed [39]. The dot plots shown in Fig. 1B
reveal that the NK cell population was changed drastically only after the combination therapy. The percentageof the DN population was comparable between all groups
except for the combination group, which revealed a drastic decrease (Fig. 1C). In a similar manner, the percentage of the CD27+ SP population was comparable among
all groups, but it was significantly reduced following the
combination group (Fig. 1C). Moreover, the percentageof DP population was similar for all groups except for
the STING-LNPs that showed a slight increase that was
nonetheless comparable to that of the combination group(Fig. 1C). In addition, the percentage of the CD11b+ SP
population was exclusively and drastically increased following the administration of the combination of the
STING-LNPs and CpG-ODNs by comparison with thatof all other groups (Fig. 1C). Though there was a drasticchange in the subset of NK cells following the four treatment groups, there was no significant change in the percentage of NK cells (Fig. S4). These results confirm thatthe combination therapy causes an increase in the numbers of the most mature and functional stage of NK cells
as CD11bhighCD27low with a high KLRG1 expression,
which is related to the phenotype of the memory NK cells
induced by a viral infection with KLRG1high and CD27low
expression [12].

The combination of STING-LNPs and CpG-ODNs stimulated
the production of memory-like NK cells

Next, we confirmed our hypothesis that the combination
therapy induces the activation of NK cells as well as promoting their differentiation into memory-like NK cells,
which is the most mature phenotype of NK cells that arecharacterized as CD11bhigh CD27low NK cells. Mice withlung metastasis were intravenously administered either
PBS, the STING-LNPs, CpG-ODNs, or the combination
therapy for two cycles on days 6 and 10. The spleens werecollected and checked for the activation and maturationof NK cells and to identify the presence of memory-like
NK cells by FCM. After gating the NK cell population(NK1.1+CD3−), the expressions of CD11b, CD27, CD43,
and KLRG1 were evaluated (Fig. S2). The combinationof the STING-LNPs and CpG-ODNs resulted in a significant increase in the level of expression of CD11b as
well as the percentage of CD11b+ NK cells by comparison with the other groups (Fig. 1A and S3). The level of
CD27 expression and the percentage of CD27+ NK cells
were significantly reduced following the combination
therapy compared with that of the other groups (Fig. 1A
and S3). In addition, the expression level of KLRG1 andthe percentage of KLRG1+ NK cells were drasticallyincreased after the combination therapy compared withthat of the other groups (Fig. 1A and S3). These results
indicate that the combination of the STING-LNPs and
CpG-ODNs induces both the maturation and activationof NK cells. We also checked the induction of memorylike NK cells based on the simultaneous expressions of
CD11b and CD27, because we speculated that producing NK cells with the most mature phenotype would leadto the generation of memory-like NK cells. The surface
densities of CD27 and CD11b divide the murine NK
cells into four subsets as follows: CD11b− CD27− cells

NK cells play a significant role in the enhancement of theantitumor effect of the combination of STING-LNPs and
CpG-ODNs

In the next step, we examined the ability of this combination therapy to induce therapeutic efficacy against a B16F10 lung metastasis model. As shown in Fig. 2A, micewith lung metastasis were intravenously administeredeither PBS, the STING-LNPs, CpG-ODNs, or a combination of both for two cycles on days 6 and 10. Lungs werecollected on day 14 to evaluate the antitumor effect. The
combination therapy clearly caused a reduction in thenumber of tumor colonies in the lungs (Fig. 2A). In addition, the combination therapy resulted in a significant
reduction in luciferase activity, indicating that the numbers of tumor cells had decreased (Fig. 2A). We subsequently examined the contribution of NK cells and CD8+
T cells on the antitumor effect via administration of the
combination therapy. An anti-NK1.1 antibody treatment abrogated the antitumor effect via the combination
therapy, whereas following the the anti-CD8a antibody

Khalifa et al. Experimental Hematology & Oncology(2024) 13:36

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Fig. 1 Combining STING-LNPs and CpG-ODNs can generate memory-like NK cells. Mice were intravenously injected with B16-F10-Luc2 cells (2 × 105).
PBS, STING-LNPs (4 µg/mouse of c-di-GMP), CpG-ODNs (250 µg), or the combination therapy was intravenously injected. (A) The FI (Median) of CD11b,
CD27, CD43, and KLRG1 expression in NK cells. (B) Dot plots show the expressions of CD11b and CD27 on splenic NK cells (gated on CD3ε−NK1.1+) on day
14. Numbers indicate percentages. (C) The percentages of DN (CD11b− CD27−), CD27+ SP (CD11b− CD27+), DP (CD11b+ CD27+), and CD11b+ SP (CD11b+
CD27−) NK cells. The values represent the mean ± SEM (n = 3), ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05). DN, double negative; SP, single positive;
DP, double positiveKhalifa et al. Experimental Hematology & Oncology(2024) 13:36

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Fig. 2 NK cells play a significant role in the enhancement of the antitumor effect of STING-LNPs and CpG-ODNs. (A) The antitumor effect of monotherapies and that of the combination therapy against B16-F10 lung metastasis. Mice were intravenously injected with B16-F10-Luc2 cells (2 × 105). Either PBS,
STING-LNPs (4 µg/mouse of c-di-GMP), CpG-ODNs (250 µg), or the combination therapy was intravenously injected for two cycles. The images represent
the lungs of different treatment groups and the quantitative analysis of lung metastasis based on the values of luciferase activities. The values represent
the mean ± SEM (n = 4, *P < 0.05). (B) The effect of the depletion of NK cells or cytotoxic T lymphocytes (CTL) on antitumor activity via the combination
therapy. B16-F10 lung metastasis-bearing mice were intraperitoneally injected with either anti-NK1.1 (200 µg), anti-CD8a (200 µg), or their isotype control in similar doses. The images represent the lungs of different treatment groups and quantitative analysis of lung metastasis based on the values of
luciferase activities. The RLU values per whole lung for the PBS-treated mice was set to 1. The values represent the mean ± SEM (n = 4–6, *P < 0.05). ns,
non-significant

Khalifa et al. Experimental Hematology & Oncology

Fig. 3 (See legend on next page.)(2024) 13:36

Page 8 of 17

Khalifa et al. Experimental Hematology & Oncology(2024) 13:36

Page 9 of 17(See figure on previous page.)
Fig. 3 Two cycles of STING-LNPs and CpG-ODNs are indispensable for tumor reduction and memory-like NK cell induction. (A) Antitumor effects ofmonotherapies and combination therapy against B16-F10 lung metastasis. Mice were intravenously injected with B16-F10-Luc2 cells (2 × 105). PBS, STINGLNPs (4 µg/mouse of c-di-GMP), CpG-ODNs (250 µg), or the combination therapy was intravenously injected for one cycle. Images show the lungs ofdifferent treatment groups, and the quantitative analysis of lung metastasis was based on the values of the luciferase activities. The values represent themean ± SEM (n = 4–6). ns, non-significant. (B) The FI (Median) values for CD11b, CD27, CD43, and KLRG1 expression in NK cells. (C) The percentages of DN(CD11b− CD27−), CD27+ SP (CD11b− CD27+), DP (CD11b+ CD27+), and CD11b+ SP (CD11b+ CD27−) NK cells (gated on CD3ε−NK1.1+) on day 10. The values
represent the mean ± SEM (n = 3, ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05). DN, double negative; SP, single positive; DP, double positive

treatment the antitumor effect was maintained (Fig. 2B).
This indicates that the antitumor effect caused by the
combination therapy is largely dependent on NK cells.

phenotype of NK cells that express CD11bhighCD27low,
which we speculate are memory-like NK cells.

Two treatment cycles are indispensable for achieving
tumor reduction and CIML NK cell induction

Long-term vaccination effect through the sustained
persistence of memory-like NK cells induced by the
combination of STING-LNPs and CpG-ODNs

The issue arose as to whether two cycles of this combination is actually required to produce a significant antitumor effect and CIML NK cell induction, or whether one
cycle would be sufficient. B16-F10 lung metastasis-bearing mice were intravenously administered either PBS, theSTING-LNPs, CpG-ODNs, or the combination therapy
for one cycle on day 6, and on day 10 the lungs were collected (Fig. 3A). Termination was performed on day 10,
but not day 14, to fix the four-day interval between thetreatment administration and termination as a two-cycle
administration protocol. After the one-cycle administration, no significant reduction in tumor burden wasobserved, but the burden was reduced slightly following the administration of STING-LNPs and the combination therapy (Fig. 3A). We also examined the issue of
whether or not one cycle of our combination therapy
could activate NK cells and induce their differentiation tomemory-like cells. Following a similar protocol, spleens
were collected and the surface markers were checked via
FCM. In the STING-LNPs and the combination therapy,
the levels of expression of CD11b and KLRG1 and thepercentages were drastically elevated and reached levels
similar to that following two cycles of the combination
therapy (Fig. 3B and S5). We then checked the simultaneous surface density of CD11b and CD27 on the NK cells.
The percentages of DN and CD27+ SP populations weresignificantly reduced in the case of the STING-LNPs andthe combination therapy (Fig. 3C). Additionally, the percentage of the DP population was drastically elevated following the administration of either STING-LNPs aloneor the combination therapy (Fig. 3C). However, the percentage of the CD11b+ SP was comparable among allgroups (Fig. 3C). Collectively, the percentages of DN
and CD27+ SP populations were similar after one andtwo cycles of the combination therapy. Although DP was
the highest population following one cycle, it failed toreach the population of CD11b+ SP that had been drastically increased following the two cycles (Fig. S6). These
results indicate that one cycle of the combination therapy
enhances the maturation and activation of NK cells, butshow that it was insufficient for inducing the most potent

Next, we examined the ability of combination therapy
to induce a prophylactic effect against the B16-F10 lungmetastasis model to confirm that CD11bhighCD27low
NK cells are, in fact, memory-like NK cells. As shownin Fig. 4A, we employed a prophylactic protocol settingin which two cycles of either PBS, STING-LNPs, CpGODNs, or the combination therapy were intravenously
administered on days 0 and 4, followed by tumor inoculation on day 5. All treatment groups resulted in a significant reduction in tumor burden (Fig. 4A). This finding
suggests that the anti-tumor activity could be due to theinstantaneous activation of an innate immune response
and is independent of memory-like NK cell responses.
Then, we checked the phenotype of the NK cells on
day 15 following a similar protocol setting, but we used
spleen samples instead. The level of expression of CD11b
and the percentage of CD11b+ NK cells was significantly
induced only by the combination therapy (Fig. 4B and
Fig. S7). In addition, the percentage of CD27+ NK cells
was significantly reduced only after administration of the
combination therapy (Fig. S7). The expression and percentage of KLRG1 were elevated following administration of the combination group (Fig. 4B and S7). Then, we
checked the simultaneous surface density of the CD11b
and CD27 expression on the NK cells per group. The
percentage of the DN population was similar among allgroups except for a slight decrease for the combinationgroup (Fig. 4C). The percentage of CD27+ SP was significantly reduced after administration of the combination therapy compared with that of each monotherapy(Fig. 4C). In addition, the percentage of the DP population was significantly decreased after the administrationsof CpG-ODN and the combination therapy comparedwith that of the PBS group (Fig. 4C). Most importantly,
the percentage of the CD11b+ SP population was drastically increased only for the group that received the combination therapy by comparison with that of all groups(Fig. 4C).
Accordingly, we changed the protocol setting to evaluate the memory function of the CD11bhighCD27low
NK cells that were retained on day 15 after two cycles

Khalifa et al. Experimental Hematology & Oncology

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Khalifa et al. Experimental Hematology & Oncology(2024) 13:36

Page 11 of 17(See figure on previous page.)
Fig. 4 Antitumor effect based on the innate activation of NK cells. (A) Antitumor effect of monotherapies compared with that of the combination
therapy against B16-F10 lung metastasis following a prophylactic protocol. Mice were intravenously injected with either PBS, STING-LNPs (4 µg/mouse ofc-di-GMP), CpG-ODNs (250 µg), or the combination therapy for two cycles. After one day, the mice were intravenously injected with 2 × 105 B16-F10-Luc2
cells. Representative photos show the lungs of the different treatment groups, and quantitative analysis of the lung metastasis was based on the values of
luciferase activities. The values represent the mean ± SEM (n = 4–6, *P < 0.05). (B) The FI (Median) values for CD11b, CD27, CD43, and KLRG1 expression in
NK cells. (C) The percentages of DN (CD11b− CD27−), CD27+ SP (CD11b− CD27+), DP (CD11b+ CD27+), and CD11b+ SP (CD11b+ CD27−) NK cells. The values
represent the mean ± SEM (n = 3, ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05). DN, double negative; SP, single positive; DP, double positiveof vaccination. A prophylactic protocol was initiated,
whereby two cycles of either PBS, the STING-LNPs,
CpG-ODNs, or the combination therapy was administered intravenously on days 0 and 4 followed by tumor
inoculation on day 14. The lungs were then collectedon day 28, and luciferase activity among the groups wasmeasured (Fig. 5A). Interestingly, only the combination
therapy resulted in a dramatic preventative effect, while
the other groups were comparable (Fig. 5A). This preventative effect confirmed that CD11bhighCD27low can be thephenotype of memory-like NK cells, and this was the first
confirmation of the phenotype of the memory-like NK
cells.
To track induction of the memory-like NK cells
and their prolonged existence following the combination therapy with and without tumor inoculation, we
employed a prophylactic protocol setting in which twocycles of either the PBS or the STING-LNPs and CpGODNs combination therapy was intravenously administered on days 0 and 4 (Fig. 5B). This was followed bychecking the phenotype of the NK cells via FCM in the
spleen on days 7, 14, 21, and 28. Other mice groups werechallenged with B16-F10 cells on day 24 and the phenotype of the memory-like NK cells was checked at 4 days
after inoculation. The population of SP CD11b+ NK cells
was induced following the vaccination starting fromday 7 and significantly in comparison to the PBS groupon day 14. The SP CD11b+ NK cells reached a peak on
day 21, and the level was maintained (Fig. 5C). Moreover, the expression of KLRG1, a differentiation marker,
was tracked among the different days. The SP CD11b+
KLRG1+ expression level was drastically elevated at day
7 following the two-cycle vaccination when comparedwith all days of the PBS group (Fig. 5D). However (andunexpectedly), challenging mice with tumor cells did
not affect the SP CD11b+ NK cells or the SP CD11b+
KLRG1+ expression levels (Fig. 5C and D). To examine
the prolonged prophylactic effect of the extended lifetime of the memory-like NK cells induced by two cyclesof the combination therapy, we employed a prophylactic
protocol setting in which two cycles of either the PBS,
the STING-LNPs, the CpG-ODNs, or the combination
therapy was intravenously administered on days 0 and
4 followed by tumor inoculation on day 24. The lungs
were then collected on day 38 and the luciferase activity
among the groups was then measured (Fig. 5E). Only the
combination of STING-LNPs and CpG-ODNs resulted

in a significant preventative antitumor effect, and one
sample even seemed to be completely protected against
tumor induction (Fig. 5E). This prolonged prophylactic effect continued for 3 months following the vaccination (Fig. 6). These findings indicate that the sustained
existence of CD11bhighCD27low memory-like NK cells
induced by the administration of STING-LNPs and CpGODNs vaccination results in a prolonged prophylactic
effect against lung metastasis.

Discussion
The concept of inducing the production of memory-like
NK cells for cancer immunotherapy has attracted theinterest of a number of researchers. However, all the currently available research has involved either a pathogenassociated molecular pattern (PAMPS) or a combinationof cytokines for the in vitro induction of the memory-like
NK cells [16, 25]. Our findings are the first report concerning the in vivo induction and phenotypic characterization of memory-like NK cells showing a memory
effect in cancer immunotherapy. The findings reported in
the present study indicate that two cycles of the combination of STING-LNPs and CpG-ODNs are indispensable
and tend to be sufficient for the release of cytokines thatare required for the induction of memory-like NK cells
that are, in turn, essential for the prevention of, and fightagainst, melanoma lung metastasis. Our findings have thepotential for use in the development of immunotherapies
that could harness the potential of NK cells for treating
and preventing cancer metastasis.
For the development of efficient NK cell-based immunotherapy or vaccinations against cancers, an immune
adjuvant that could induce the cytokines essential for
memory-like NK cell induction is essential. STING activation results in the robust induction of type 1 IFNs but
it also induces low levels of type-1 immune responses,
in addition to its type 2 immune responses [8, 40]. On
the other hand, CpG-ODNs are reported to induce highlevels of type-1 immune responses, but IFNs are known
to be only weakly induced [41, 42]. Therefore, to overcome this limitation, we combined the STING-LNPs
and CpG-ODNs in order to induce type-1 immune
responses, particularly those of IL-12, IL-18, and IFNβ. Concerning the mechanism responsible for how the
STING-LNPs and CpG-ODNs induce the production ofmemory-like NK cells, trained immunity has been found
to rely mainly on epigenetic and metabolic modifications.

Khalifa et al. Experimental Hematology & Oncology

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Khalifa et al. Experimental Hematology & Oncology(2024) 13:36

Page 13 of 17(See figure on previous page.)
Fig. 5 The sustained existence of the memory-like NK cells induced by the combination of STING-LNPs and CpG-ODNs resulted in prolonged protection against the development of melanoma lung metastasis. (A) The prophylactic effect of monotherapies compared with that of combination therapy
against B16-F10 lung metastasis. Mice were intravenously injected with either PBS, the STING-LNPs (4 µg/mouse of c-di-GMP), CpG-ODNs (250 µg), or
the combination therapy for two cycles. After 10 days, mice were intravenously injected with 2 × 105 B16-F10-Luc2 cells. Representative photos show thelungs of the different treatment groups, and quantitative analysis of the lung metastasis was based on the values of luciferase activities. The values represent the mean ± SEM (n = 3–5, *P < 0.05). (B) The sustained persistence of the memory-like NK cells induced by STING-LNPs and CpG-ODNs vaccination.
Mice were intravenously injected with either PBS or a combination of STING-LNPs (4 µg/mouse of c-di-GMP) and CpG-ODNs (250 µg) for two cycles. After20 days one group of mice was intravenously injected with (2 × 105) B16-F10-Luc2 cells. (C) The percentage of CD11b+ SP (CD11b+ CD27−) NK cells on theindicated days after vaccination. Upper graph: cell percentage among days for the SP CD11b+ NK vaccinated group. Bottom graph: comparison of the SP
CD11b+ NK cell percentage of the vaccinated group with that of the PBS group on each termination day. The values represent the mean ± SEM (n = 3–4,**p < 0.01). (D) The FI (Median) of KLRG1 expression on CD11b+ SP (CD11b+ CD27−) NK cells on the indicated days after vaccination. Upper graph: comparison of the KLRG1 expression level of SP CD11b+ NK cells for the vaccinated group among days. Bottom graph: KLRG1 expression levels of SP CD11b+
NK cells comparing the PBS and vaccinated groups on each termination day. The values represent the mean ± SEM (n = 3, ****p < 0.0001; **p < 0.01;*p < 0.05). (E) Prolonged vaccination effect following two cycles of the STING-LNPs and CpG-ODNs. Mice were intravenously injected with either PBS, theSTING-LNPs (4 µg/mouse of c-di-GMP), CpG-ODNs (250 µg), or the combination therapy for two cycles. After 20 days, mice were intravenously injectedwith B16-F10-Luc2 cells (2 × 105). Representative photos show the lungs of different treatment groups, and quantitative analysis of the lung metastasis
was based on the values of luciferase activities. The values represent the mean ± SEM (n = 4–5, *P < 0.05)

Epigenetic reprogramming includes DNA methylation,
histone modification and chromatin opening for enhancing gene transcription and translation [43, 44]. These epigenetic changes are known to be inherited from parent
to daughter cells [45]. Regarding the metabolic changes,
the CIML NK cells tend to exhibit elevated expressionsof nutrient transporters, and the metabolic profile isskewed towards glycolysis [44]. The glycolytic pathway is
known to be essential for the IFN-γ and granzyme B production that achieves efficient levels of cytotoxicity [46].
In addition to blocking negative regulators such as killer
immunoglobulin receptors (KIR) and to transforming
growth factor-β (TGF-β) while enhancing the expressionof CD25, IL-2 receptors are essential for the survival andproliferation of NK cells [47]. Accordingly, we expectedthat the first stimulus of this combination would activate
myeloid cells to secrete cytokines that would then activate NK cells. Upon restimulation, the NK cells would
acquire memory-like properties through epigenetic andmetabolic changes that would result in a robust andquicker production of specific inflammatory cytokines
and effector molecules thus harnessing the effect [44]. In
addition to the previously mentioned mechanisms, we
do believe that the delivery system utilized here plays apivotal role in enhancing the induction of memory-like
NK cells and effector molecules hence, the preventative
effect. Thus, we plan to investigate the mechanism deeply
via checking the genetic alteration in addition to the roleof the delivery system.
CIML NK cells have shown enhanced antitumor
responses particularly against melanoma cell lines [23].
The exact mechanism behind this is not yet completely
understood. However, CIML NK cells were found toincrease the production of IFN-γ, CD107a, and TNF [20,23]. In addition, the upregulation of activating receptors such as NKG2D, NKP46, and DNAM-1 which are
known to control NK cell cytotoxicity and cytokine production [20, 48]. The ligands of these activating receptors

are reported to be present on the melanoma cell lines
and allow for cytokine induction and degranulation in
response to melanoma targets [49]. Moreover, memorylike NK cells can block inhibitory regulatory receptors
and can even negate their signals [20, 47]. We therefore
conclude that once the resting and self-renewing memory-like NK cells are sensitized by tumor cells, they will
have the capacity to kill them, and, hence, prevent theirmetastasis and induction.
Herein we provide evidence that two cycles rather than
one cycle of STING-LNPs and CpG-ODNs is required
to induce the production of memory-like NK cells thatare able to produce a significant antitumor effect. Ourfindings are consistent with previous reports showingthat the first stimulus results in the activation of innate
immune cells followed by a resting state until the second stimulus occurs, which is essential for the induction of trained immune cells [50]. In addition, Romeeet al. reported that the functional activity of in vitro
IL-12 + IL-18-preactivated NK cells was significantly
increased compared with control NK cells only after
restimulation [22]. Our previous report also showed that
two cycles of STING-LNPs and anti-Programmed cell
death-1 (PD-1) antibody were essential and sufficientfor inducing an antitumor effect [7]. Our findings showthat two cycles, rather than one cycle, of the combination
therapy could result in significant tumor control following the induction of memory-like NK cells. One cycle of
the STING-LNPs and that of the combination therapy
induced only slight tumor control (Fig. 3A). Mechanistic findings have shown high expression levels of CD11b,
CD43, and KLRG1 indicating that a higher maturation
and activation of the NK cells occurred following one
stimulus with the STING-LNPs and that of the combination group (Fig. 3B). Although one cycle of either
the STING-LNPs or the combination therapy allowed
NK cells to differentiate and reach the DP population, a
CD11b+ SP population (memory-like NK cells) couldKhalifa et al. Experimental Hematology & Oncology(2024) 13:36

Page 14 of 17

Fig. 6 Effect of vaccination with the combination of STING-LNPs and CpG-ODNs against the development of melanoma lung metastasis after threemonths. (A) The prophylactic effect of the combination therapy against B16-F10 lung metastasis. Mice were intravenously injected with either PBS or
the combination of STING-LNPs (4 µg/mouse of c-di-GMP) and CpG-ODNs (250 µg) for two cycles. After 90 days, mice were intravenously injected with
2 × 105 B16-F10-Luc2 cells. (B) Representative photos show the lungs of different treatment groups, and (C) Quantitative analysis of the lung metastasis
was based on the values of luciferase activities. The values represent the mean ± SEM (n = 3–5, *P < 0.05)

not be achieved (Fig. 3C). This confirms the importanceof the administration of two cycles of the STING-LNPs
and CpG-ODNs for the induction of CD11bhighCD27low
memory-like NK cells and hence, good tumor control. If
the restimulation of innate immune cells could provide
enhanced responses compared with the initial stimulus,
reports have shown that the cells are considered to haveacquired trained immunity [51].
Long-lived memory or memory-like NK cells have beenreported, but the prolonged effect of the persistent memory-like NK cells has not yet been confirmed. Previously,
because of murine cytomegalovirus (MCMV), memory
NK cells were proven to have long lifetimes of around
90 days [12]. In addition, the in vitro preactivated CIML
NK cells could still be detected after 3 weeks following
its adoptive transfer [52]. Even though their lifetimes had
been prolonged, these memory or memory-like NK cells
were detected in very low percentages compared with
their initial induction [12, 18]. However, we report hereinthat memory-like NK cells, induced in vivo through the
combination of immunotherapeutic drugs, persist for atleast one month at their peak percentage, with no reduction, which results in a prolonged prophylactic effect(Fig. 5C). The long-term persistence of the memory-like
NK cells is controlled mainly by metabolic and epigenetic
changes that are induced by the cytokines in CIML NK

cells. IL-12, IL-18, and IL-15 are known to cause CIML
NK cells to undergo a metabolic shift toward glycolysis
that persists even after cytokine removal. This results inan enhancement of the proliferation, long-term persistence, and recall function of NK cells [53]. In addition,
IL-15 and IL-2 play an important role in the proliferation,
homeostasis, survival, expansion, and persistence of NK
cells [54]. Terren et al. reported that the effector function of CIML NK cells, IFN-γ production, was greatly
impaired following the inhibition of glycolysis [54]. In asimilar manner, effects such as the proliferation and cytotoxicity of NK cells were impaired in vitro when glucose
metabolism was inhibited, which resulted in susceptibility to MCMV infection in vivo [46]. Furthermore, previous studies have shown that memory-like NK cells havestability and heritable properties [18, 45, 55]. In otherwords, differentiation to memory-like NK cells throughpermanent or heritable changes at the epigenetic or metabolic level could result in a self-renewing population ofcells regardless of a continuous stimulus [18]. Therefore,
our findings concerning the prolonged persistence ofthe memory-like NK cells and the long-term vaccination
effect that maintains their enhanced functionality could
be related to their heritable properties.
Vaccines have historically shown great efficacy in preventing infections and/or decreasing their onset. Many

Khalifa et al. Experimental Hematology & Oncology(2024) 13:36

Page 15 of 17

vaccinations are available for the prevention of a variety of viral and bacterial infections, but researchers
have struggled to develop cancer vaccines. The conceptof trained immunity encouraged us to undertake thisresearch and it promises to pave the way for the further
development of prophylactic cancer vaccines. Vaccination with BCG has been reported to decrease the susceptibility towards many infections through the long-term
boosting of the formation of innate immune cells. BCG
was recently reported to protect against many cancers aswell as COVID-19 through the induction of memory-like
NK cells [56, 57]. Moreover, recent studies have shownthat re-engineering the BCG with a STING-agonist-augmented trained immunity and enhanced the antitumor
effect [58, 59]. Despite all these positive results regarding controlling the tumor burden through trained immunity, little effort has been directed towards developing
a prophylactic tumor vaccine that could protect against
cancers. We demonstrate herein that the combinationof STING-LNPs and CpG-ODNs has offered protection against a melanoma lung metastasis model throughinducing the formation of memory-like NK cells andextending their existence (Figs. 5 and 6). This drastic control of tumor metastasis and its prevention confirms theinduction and the phenotypical characterization of memory-like NK cells as CD11bhighCD27low and KLRG1high
NK cells.
These findings promise to pave the way for the development of many efficient cancer immunotherapies byharnessing the potential of producing memory-like NK
cells in vivo instead of via in vitro pre-activation andadoptive transfer. The phenotypical characterization of
the memory-like NK cells performed and confirmed here
will allow for easier identification later. In addition, thein vivo induction of memory-like NK cells will help inavoiding the consequences that sometimes occur during and after the infusion of memory NK cells to patientsin clinical trials. Out of 13 patients in a clinical trial, 3
deaths were reported after infusion because of bacteremia with septic shock [20]. In addition, it may also protect against the inflammation that can occur towards thepresence of foreign cells. Collectively, the in vivo induction of memory-like NK cells promises to be much safer
than adoptive transfer as well as saving time and loweringcosts.

combination of STING-LNPs and CpG-ODNs induced
sufficient levels of cytokines that are essential for theactivation and differentiation of NK cells to memory-likeCD11b+ SP NK cells, which could have extended lifetimes. This resulted in an enhanced antitumor effect andprotection against the development of a lung metastasis
model.

Conclusions
The findings of the present study provide novel
insights regarding the in vivo induction and the phenotypic characterization of memory-like NK cells as
CD11bhighCD27low and KLRG1high NK cells throughthe use of a combination of immune adjuvants. These
results highlight the importance of such cells in protecting against lung metastasis. In this study, two cycles of a

Declarations

Abbreviations
STING	Stimulator of interferon genes
LNP	Lipid nano particleIL	interleukin
MHC-I	Major histocompatibility complex-I
CIML	Cytokine induced memory-like
BCG	Bacillus Calmette Guerin
PD-1	Programmed cell death-1
FCM	Flow cytometry
DMG-PEG2000	1,2-dimirystoyl-sn-glycerol methoxyethyleneglycol
C-di-GMP	Cyclic di-GMP
PBS	Phosphate Buffered Saline
CTL	Cytotoxic T lymphocyte
RLU	Relative light units
DN	Double negative
SP	Single positiveDP	Double positive
KIR	Killer immunoglobulin receptor
TGF-β	Transforming growth factor-β
MCMV	Murine cytomegalovirus

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s40164-024-00502-w.
Supplementary Material 1
Acknowledgements
We appreciate the helpful advice from Dr. Milton S. Feather in reviewing theEnglish in this manuscript.
Author contributions
AMK, TK and HH conceived and designed the study, reviewed, and edited themanuscript. AMK performed the experiments, data analysis, investigation,
visualization and wrote the first draft. YS: performed the synthesis of YSK12-C4.
TN and HH: supervised and managed the project and acquired funding.
Funding
JSPS KAKENHI (Grant Number 20H03373) (T.N.), grant from Mochida Memorial
Foundation for Medical and Pharmaceutical Research (T.N.), the Special
Education and Research Expenses from the Ministry of Education, Culture,
Sports, Science and Technology (MEXT) scholarship (H.H.) and Hokkaido
University Support Program for Frontier Research (H.H.).
Data availability
The datasets used and/or analyzed during the current study are available fromthe corresponding author on reasonable request.

Ethical approval
All animal experiments were approved by the Ethics of Pharmaceutical
Science Animal Committee of Hokkaido University (approval number:
20–0126). All experiments were carried out in accordance with National
University Corporation Hokkaido University Regulations on Animal
Experimentation.

Khalifa et al. Experimental Hematology & Oncology(2024) 13:36

Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Author details
1
Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6,
Kita-ku, Sapporo 060-0812, Hokkaido, Japan
Received: 7 September 2023 / Accepted: 13 March 2024

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