Review

Harnessing NK Cells to Control Metastasis
Xuewen Deng 1,* and Hiroshi Terunuma 1,2
Biotherapy Institute of Japan, Inc., 2-4-8 Edagawa, Koto-ku, Tokyo 135-0051, Japan
N2 Clinic Yotsuya, 5F 2-6 Samon-cho, Shinjuku-ku, Tokyo 160-0017, Japan* Correspondence: tohxuewen@bij-net.com; Tel.: +81-3-5632-6080; Fax: +81-3-5632-6083
1
2

Abstract: In recent years, tumor immunotherapy has produced remarkable results in tumor treatment. Nevertheless, its effects are severely limited in patients with low or absent pre-existing T cell
immunity. Accordingly, metastasis remains the major cause of tumor-associated death. On the other
hand, natural killer (NK) cells have the unique ability to recognize and rapidly act against tumor
cells and surveil tumor cell dissemination. The role of NK cells in metastasis prevention is undisputable as an increase in the number of these cells mostly leads to a favorable prognosis. Hence, it
is reasonable to consider that successful metastasis involves evasion of NK-cell-mediated immunosurveillance. Therefore, harnessing NK cells to control metastasis is promising. Circulating tumor
cells (CTCs) are the seeds for distant metastasis, and the number of CTCs detected in the blood ofpatients with tumor is associated with a worse prognosis, whereas NK cells can eliminate highly
motile CTCs especially in the blood. Here, we review the role of NK cells during metastasis, particularly the specific interactions of NK cells with CTCs, which may provide essential clues on how toharness the power of NK cells against tumor metastasis. As a result, a new way to prevent or treat
metastatic tumor may be developed.
Keywords: tumor; metastasis; circulating tumor cells (CTCs); NK cells; immunotherapy

Citation: Deng, X.; Terunuma, H.

1. Introduction

Harnessing NK Cells to Control

Although tumor treatment has improved, most metastatic tumors remain incurable[1]. Metastasis is the major cause of tumor-associated death [2]. Hence, concerted efforts
have been made to develop therapies that can control metastasis in patients with tumor[3]. Tumors are complex ecosystems composed of neoplastic cells, extracellular matrix(ECM), and “accessory” non-neoplastic cells, which include resident mesenchymal cells,
endothelial cells, and immune cells. Accessory cells crosstalk with tumor cells, which fuels
and shapes tumor development [4]. Immune cells are essential players in tumor, impacting tumor fate in different stages and therapeutic interventions [4,5]. Over the past decades, immunotherapy has revolutionized tumor treatment [6]. The emergence of immune
checkpoint inhibitors (ICIs) targeting programmed cell death protein 1 (PD-1), cytotoxic
T lymphocyte-associated protein (CTLA-4), and programmed cell death ligand 1 (PD-L1)
has rendered many aggressive tumors treatable and even curable [6]. However, ICIs are
successfully used in only a fraction of patients. In particular, their effects are severely limited in patients with low or absent pre-existing T cell immunity [7]. Thus, the initiation of
a de novo tumor-specific immune response is required, a process that is dependent on theactions of innate immune cells, among which natural killer (NK) cells play essential roles
in tumor immunosurveillance and antitumor immunity owing to their unique ability toidentify and kill tumor cells [8]. Indeed, a higher infiltration of NK cells was found to be
associated with the better response to anti-PD-1 therapy [9]. NK cells might play an important role in the tumor subsets that exhibit loss of neoantigen presentation due to thedownregulation of major histocompatibility complex class I (MHC-I) molecules [10]. Currently, tumor immunotherapy based on NK cells is a very hot topic in oncology andMetastasis. Vaccines 2022, 10, 2018.
https://doi.org/10.3390/
vaccines10122018
Academic Editor: Matthew Blunt
Received: 2 November 2022
Accepted: 23 November 2022
Published: 25 November 2022
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Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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generates considerable interest from the scientific community and pharmaceutical industry. Consequently, there are many studies in which the therapeutic value of NK cells is
being evaluated [11,12]. However, the efficacy of NK cell therapy against solid tumor ishampered by inadequate tumor infiltration and immunosuppressive tumor microenvironment. Since NK cells play a more important role in the elimination of metastatic tumor
than primary solid tumors [13], we will focus on the interactions of NK cells with metastatic tumor cells, the prominent role of NK cells in the control of metastasis, and the evasion of metastatic tumor cells from NK-cell-mediated immunosurveillance. Understanding tumor cell resistance to NK cells, particularly the specific interactions of circulatingtumor cells (CTCs) with NK cells may provide essential clues on how to harness NK cell
power to maximize their antitumor potential and may pave the way to the developmentof novel therapeutic strategies for metastatic tumor.
2. Tumor Metastasis
Metastasis, which is the gravest stage of tumor, occurs when tumor cells acquire invasive features [14] and the ability to evade immunosurveillance [15]. For the successful
outgrowth of tumor cells to distant metastatic sites, several critical steps and obstacles
need to be overcome. This multistep process, known as the metastatic cascade, involves
the detachment of tumor cells from their neighbors and local invasion of surrounding tissues to enter the circulatory system as CTCs in the form of either single (monoclonal) or
multiple (polyclonal) tumor cells, until they lodge at secondary sites and enter into a preexisting or neo-formed vasculature and remain there as disseminated tumor cells (DTCs)
or micrometastatic sites (Figure 1). DTCs remain dormant until they resume proliferationand establish detectable metastatic lesions, giving rise to overt macrometastasis and organ
colonization [16]. During this process, mesenchymal-like phenotype tumor cells havehighly mobile and invasive properties, which occur during the so-called epithelial-to-mesenchymal transition (EMT) [17]. Activation of the EMT program impinges on the immunomodulatory properties and immunogenicity of tumor cells [18–20]. CTCs must perform
well in multiple events for successful metastasis, especially for successful in survival in
the circulation [21] (Figure 1). Most of the CTCs are shed from the primary tumor site,which has become an immunosuppressive microenvironment that protects them from immune attacks, and die during their transport in blood vessels because of hemodynamic
shear force, oxidative stress, and susceptibility to immune effector cells [22,23] (Figure 1).
Ultimately, only a very small fraction of CTCs survive and become seeds for metastasis.
Among tumor-extrinsic factors for metastasis, antitumor immunity, which is a major hindrance to metastatic colonization of CTCs and DTCs [22,23] (Figure 1). Polyclonal CTCs,
which have higher metastatic potential [24,25] because they are associated with decreased
expression levels of NK-cell-activating ligands that exhibit higher resistance to killing by
NK cells [26]. Conventionally, metastasis has been considered to occur in later stages oftumor progression. However, accumulating evidence has also shown metastatic tumor
cell dissemination during early tumor formation [27]. The metastatic cascade and immunosurveillance involving NK cells in metastasis are illustrated in Figure 1.

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Figure 1. Metastatic cascade and immunosurveillance. Primary tumor cells undergo epithelial-tomesenchymal transition (EMT) within tumor-cell-extrinsic factors in nutritional, stromal, and immunological microenvironments endowed with distinct metastatic potential. The EMT trans-differentiated cells detach from the primary tumor and interact with and remodel the extracellular matrix(ECM) for intravasation, leaving the protection of the immunosuppressive tumor microenvironment. They reach the circulation, which make them more vulnerable to attacks by immune effector
cells, particularly, NK cells, which can directly and indirectly interact with circulating tumor cells(CTCs) to control metastasis. However, they survive as polyclonal clusters of CTCs and cooperatewith platelets and neutrophils, which shield them and alter the function of NK cells as a mode ofescape from immunosurveillance until they encounter conditions that are permissive for extravasation and lodge at secondary sites. They can persist in a relatively silent state as disseminated tumor
cells (DTCs) or micrometastatic tumor cells for a highly variable period of time, until they resume
proliferation to establish a clinically detectable metastatic disease. Abbreviations: TAF, tumor-associated fibroblast; CTC, circulating tumor cell; CTL, cytotoxic T lymphocyte; DC, dendritic cell; DTC,
disseminated tumor cell; ECM, extracellular matrix; EMT, epithelial-mesenchymal transition;
MDSC, myeloid-derived suppressor cell; NK, natural killer cell; PLT, platelet; TAM, tumor-associated macrophage; Treg, regulatory T cell.

3. NK Cells Are Highly Involved in for the Immunosurveillance of Metastasis
NK cells are effector cells that constitute a key part of the innate immune system andrepresent up to 5–20% of circulating lymphocyte [28,29]. NK cells show an absence of CD3
molecule but highly express CD56 or CD16, are predominantly defined as CD3−CD56+.
There are two major types of NK cell: CD56dimCD16bright/+ (CD56dim) and CD56brightCD16dim/−(CD56bright) NK cells. The CD56dim NK cells are the major subset in peripheral blood, havehigh cytotoxicity, whereas CD56bright NK cells are predominantly in secondary lymphoidorgans as cytokine producing NK cells [28,29]. NK cells have the unique ability to differentiate between normal and transformed cells, and they can recognize and rapidly act
against malignant cells without prior sensitization [28,29]. They possess various activating
and inhibitory receptors, and the net functional outcome is a complex integration of signals among these activating and inhibitory receptors [28,29]. These inhibitory receptors,
such as the inhibitory isoforms of killer-cell immunoglobulin receptors (KIRs), andCD94/NKG2A heterodimers recognize various forms of MHC-I molecules. Thus, the decrease in the expression levels or the absence of MHC-I molecules on tumor cells reduces
the strength of inhibitory signals delivered to NK cells, thus promoting NK cell activation.
NK cell activation also results from the engagement of activating receptors, such as theactivating isoforms of KIRs, the signaling lymphocyte-activating molecule-related receptors NKG2D, DNAX accessory molecule-1 (DNAM-1), and the natural cytotoxicity receptors NKp30, NKp44, and NKp46, which recognize stress-inducible ligands on tumor cells
that are scarcely expressed in healthy cells. Therefore, NK cells have a well-documented

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antitumor effect, including antibody-dependent cellular cytotoxicity (ADCC) throughspecific IgG antibodies to target antigens [28–31]. Indeed, an epidemiological study has
shown that a reduced NK cell function was associated with an increased tumor incidence
in humans [32].
When NK cells recognize aberrant cells, such as tumor cells, they are activated andtransport specific lytic granules such as perforins and granzymes toward immunologicalsynapses to induce apoptosis of target cells [33]. Perforins are cytolytic proteins that are
inserted into the plasma membrane of a target cell and induce osmotic lysis in a Ca2+dependent manner [34]. Granzymes are serine proteases that activate caspase signaling,leading to the apoptosis of the target cell [35]. Perforins are critical in controlling tumor
metastasis [36,37]. NK cells form multiple contacts with target cells and can sequentiallykill several tumor cells in a time-dependent manner [38]. Interestingly, upon a single encounter, an NK cell releases only one tenth of its cytotoxic lytic granules, but it has been
determined that even a single granule is sufficient to induce tumor cell death [34]. NK
cells have been shown to shift from inducing fast granzyme B-mediated cell death to slow
death mediated by receptor ligands (Fas ligand and tumor necrosis factor (TNF)-related
apoptosis-inducing ligand (TRAIL)) in later stages and can serially kill up to more than 30
tumor cells [39].
Although the role of NK cells in the control of primary tumors remains controversial,
their robust antimetastatic effect in vivo has been demonstrated in various experimental
models [40–44]. Tumor stem cells (TSCs) [45] and EMT trans-differentiated cells [17] areconsidered responsible for tumor metastasis. Meanwhile, NK cells can target TSCs and
EMT cells [30,46–48]. Human and mouse breast TSCs induce NK cell activation and expansion in vivo, which correlate with the inhibition of TSCs metastatic spread [46]. EMT
induces E-cadherin- and cell adhesion molecule 1 (CADM1)-mediated NK cell susceptibility. Thus, a higher CADM1 expression level correlates with improved survival and reduced metastasis in patients with lung and breast adenocarcinomas [48]. In human tumors, there is accumulating evidence of correlations among the number of infiltrating NK
cells, metastasis, and the prognosis of various tumors, such as esophageal tumor (ET) [49],gastric carcinomas (GCs) [50], gastrointestinal sarcoma tumors [51,52], renal cell carcinomas (RCCs) [53], colorectal carcinomas (CRCs) [54], and prostate tumor (PT-1) [55]. In
addition, the presence of highly effective NK cells indicates a good prognosis in metastatic
PT-1 [56]. Overall, an increased number of NK cells is highly beneficial for the survival oftumor patients [8]. Importantly, however, CTCs are considered to be indicators of an increased risk of metastasis and poorer outcomes in tumor patients. In particular, NK cells
are present in blood and lymph nodes and may participate in the immunosurveillance of
CTCs [22,44]. In an experimental model, NK cells destroy CTCs before the extravasationand thus suppressing tumor metastasis [42,44,57]. Thus, a short period of an elevatednumber of NK cells is correlated with the reduction in the number of CTCs [58,59]. Indeed,
NK cells were found to eliminate disseminated tumor cells from the lungs within 24 h ofarrival, but not thereafter [44]. Moreover, the cytotoxicity of NK cells from CTC-positive
patients is lower than that from CTC-negative patients with metastatic breast tumor (BT),
CRC, and PT-1, showing a close correlation between peripheral blood CTC number andNK cell antitumor activity [60,61]. Hence, a decreased circulating NK cell activity is associated with increased risk of metastasis in patients with pharyngeal carcinoma [62]. In
addition, a decreased NK cell activity is a parameter for predicting distant metastasis following curative surgery for CRC [63–65]. Moreover, surgical stress induces NK cell dysfunction in animal models [66–68], and human tumors [69–73], leading to metastasis [66–68]. Thus, enhancing NK cell function by inhibiting the induced NK cell dysfunction can
prevent postoperative metastasis [66–68,74]. Taken together, these results indicate that
NK cells eliminate CTCs and play a prominent role in the control of metastasis [13].

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4. Evasion of NK-Cell-Mediated Immunosurveillance in Metastasis
The immune system plays a major role in every step of tumor progression [4,5]. The
escape of tumor cells from immunosurveillance is a prerequisite for metastasis [15,23].
Since NK cells are highly involved in for immunosurveillance in metastasis [13], it is therefore plausible that for successful metastasis, tumor cells have to evade NK-cell-mediatedimmunosurveillance. Indeed, several mechanisms of NK-cell-mediated tumor cell escapehave been described [23,26,75]. These mechanisms involve tumor cell intrinsic features
and tumor-driven extrinsic microenvironmental factors (Figure 2). Herein, we explore
these escape mechanisms of NK cells in metastasis. The intrinsic features endow tumor
cells with distinct metastatic potential through epigenetic and genetic alterations, including the downregulation of NK cell activating receptor ligands due to, for example, thereduction in the expression levels of MHC-I polypeptide-related sequence A(MICA)/MICB and UL16-binding protein 2 (ULBP2) caused by the aberrant expression of
oncogenic microRNA, miR-20a, and miR-34a/c in tumor cells [76,77]. MICA/MICB andULBP2 are the ligands of the NK cell activating receptor NKG2D. Along similar lines,
tumor cells expressing miR-296-3p inhibit the expression of intercellular adhesion molecule 1 (ICAM-1), which is the ligand of β2 integrin lymphocyte function-associated antigen 1 (LFA-1), providing activating signals to NK cells [58]. In addition, NK cell inhibitory
receptor ligands, such as human leukocyte antigen-G (HLA-G) and PD-L1, are up-regulated. HLA-G is the non-classic MHC-I gene and the ligand for the inhibitory receptor of
KIRs, which are highly expressed in numerous tumor cells [78]. HLA-G exerts its function
through its binding to immunoglobulin-like transcript 2 (ILT2) and KIRs on NK cells toprotect tumor cells from NK cell cytotoxicity [79]. Thus, HLA-G expression is associatedwith tumor metastasis [80]. Moreover, plasma soluble HLA-G (sHLA-G) in extracellular
vesicles was associated with the presence of CTCs and disease progression, indicating that
CTCs exploit sHLA-G in the blood to evade NK-cell-mediated immunosurveillance [81].
Moreover, NK cell functions are affected by the inhibitory ligand PD-L1 expressed on tumor cells, which provide inhibitory signals to NK cells through the PD-1/PD-L1 axis, andreduce NK cell cytotoxicity [82]. In addition, cell death receptor FAS expression on tumor
cells is down-regulated, leading to escape from NK-cell-related apoptosis [83].
In contrast, the tumor cell-driven extrinsic microenvironmental factors suppress NK
cell immunosurveillance through modulation of the recruitment, cell surface molecules,or the release of immunosuppressive soluble factors such as TGF-β1 [84–86] (Figure 2).
NK cells in lung, breast, or prostate tumor tissues displaying altered receptor expression
with impaired cytotoxicity compared with NK cells in control tissues [84–86]. These alterations have also been observed in peripheral NK cells and associated with disease progression [85,86]. The expression levels of activating receptors on NK cells were decreased:for example, such as CD16 in BT, multiple myeloma (MM), and CRC [86–88]; NKp46 inpancreatic tumor (PT-2), GC, CRC, acute myeloid leukemia (AML), and cervical tumor(CC-1) [89–91]; NKp44 in BT and AML [92,93]; NKp30 in BT, hepatocellular carcinoma(HCC), PT-2, GC, CRC, chronic lymphocytic leukemia, and CC-1 [86,89–91,94];
CD94/NKG2C in AML [91]; NKG2D in BT, lung tumor (LT), CRC, colon carcinoma (CC2), PT-2, GC, and CC-1 [86,89,90,95,96]; CD244 in AML [91]; DNAM-1 in BT, CC-2, andAML [86,91]. On the other hand, the expression levels of inhibitory receptors on NK cells
were increased: for example, KIR3DL1 in PT-2, GC, and CRC [84]; KIR2DL2/L3 in melanoma [97]; NKG2A in BT, LT, and CRC [86,98,99]; PD-1 in ET, HCC, CRC, GC, biliary
tumor, and LT [100,101]; TIM-3 in LT, melanomas, and GC [102–104]. TGF-β1 in the tumor
environment is involved in the phenotype alteration and functional impairment of NK
cells [85,86]. In addition, TGF-β1 and other immunosuppressive factors produced by tumor cells have been shown to suppress NK cell effector functions [105] (Figure 2) andrecruit immunosuppressive cells to tumor tissue, such as regulatory T cells (Tregs) [106],
myeloid-derived suppressor cells (MDSCs) [107], CD11b+Ly6G+ neutrophils [108], and indoleamine 2, 3-dioxygenase 1 (IDO1)-expressing dendritic cells (DCs) [109]. They also
contribute to the phenotype alteration and functional impairment of NK cells to prepareVaccines 2022, 10, 2018

6 of 21for distant microenvironments for metastatic niches (Figure 1). Moreover, neutrophils cancooperate with metastatic tumor cells to enhance both the dissemination of tumor cells
out of the primary tumor and their subsequent intravasation into the lung vasculature
through the secretion of G-CSF to attract neutrophils, which suppress NK cell activity
through ROS signaling and shield intraluminal metastatic tumor cells from being clearedby NK cells (Figure 2), and ultimately enhance metastatic outgrowth [109,110]. Furthermore, tumor cells may reprogram NK cells through DNA methyltransferases (Dnmt1,
Dnmt3a, and Dnmt3b), thereby increasing the expression level of inhibitory receptors
TIGIT and KLRG1 on NK cells and promoting metastatic outgrowth [111].
Platelets are small, anucleated cell fragments that have a characteristic discoid shapeand diameters ranging from 1 to 3μm [112]. The main roles of platelets are the maintenance of the hemostasis of the vascular system and the promotion of wound healing atsites of vascular injury [113]. However, having a high platelet count was identified as arisk factor for adverse outcomes in numerous different tumors [114]. Platelets may affectmany components of antitumor immunity [115]. An increased coagulability of platelets
facilitates vascular evasion and the establishment of metastasis [114,116], whereas the abrogation of platelet function results in reduced metastasis, depending on the presenceof NK cells [117]. Platelets help tumor cells evade NK cell immunosurveillance in the
blood stream, supporting CTCs to establish metastasis [114,116] (Figure 1). Platelets promote metastasis by coating CTCs traveling through the blood, thereby physically shielding them from shear force. Moreover, a ‘pseudonormal’ phenotype is conferred to CTCs
by the transfer of platelet-derived MHC-I molecules, which causes CTCs to mimic hostcells and protects them from recognition by NK cells. Moreover, platelet-induced NK cell
inhibitory ligands, such as TNF family members of glucocorticoid-induced TNF receptorrelated (GITR) ligands, and NKG2D ligands shedding, particularly MICA and MICB on
CTCs, protect CTCs from NK cell immunosurveillance [118–121] (Figure 2). In addition,platelets may play a prometastatic role by promoting thrombin activation resulting in
shedding the DNAM-1 ligand Necl5 from the metastatic tumor cells to evade NK cell surveillance [44]. Moreover, platelets release soluble factors upon activation when adhesion
to CTCs. TGF-β1 is one of the platelet-derived soluble factors that inhibit NK cell function
partially mediated by the downregulation of the activating receptor NKG2D on NK cells[122] (Figure 2). TGF-β1 initiates and maintains the EMT phenotype alteration of CTCs[123]. Therefore, platelets affect NK cell function on several different levels, and both soluble and membrane-bound factors are involved in the evasion of CTCs from NK cell immunosurveillance in vivo.

Figure 2. Circulating tumor cells (CTCs) evade surveilling NK cells during metastasis. For successfully metastasis, tumor cells have to evade from NK-cell-mediated immunosurveillance. TumorVaccines 2022, 10, 2018

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cells have several strategies involving tumor cell intrinsic features and tumor-driven extrinsic microenviroment factors to subvert their recognition and elimination by NK cells. Intrinsic features
endow tumor cells with distinct metastatic potential owing to epigenetic and genetic alterations that
include the downregulation of NK cell activating receptor ligands and/or the upregulation of NK
cell inhibitory receptor ligands. In contrast, the tumor-cell-driven extrinsic microenvironment suppresses NK cell immunosurveillance through modulation of activating receptors and/or inhibitory
receptors on NK cells. Platelets and neutrophils may favor the escape of CTCs from NK cell immunosurveillance, thereby supporting CTCs to establish metastasis. (Arrows indicate the change in
expression levels of surface receptors).

Taken together, these results exemplify the obstacles posed by NK cell immunosurveillance in metastasis (Figure 2). The characterization of diverse mechanisms contributing to the dysfunction of NK cells in different tumors may pave the way for the development of novel therapeutic strategies by harnessing NK cells in the control of metastasis.
5. Harnessing NK Cells in Control of Metastasis
A breakthrough in tumor immunotherapy comes from the clinical successes of ICI
and chimeric antigen receptor (CAR) T cell therapies, proving that these treatments havegreat promise for tumor patients [6,124]. NK-cell-based tumor therapy currently constitutes a major area of immunotherapy innovation and has grown exponentially [11,12,125].
To cure tumors, unleashing the full antitumor potential of NK cells is an attractive option(Figure 3) (Table 1), since NK cells play a more important role in the elimination of metastasis [13], which is the major cause of tumor-related death [2]. Most of these strategies arevery often reviewed in detail [11–13], and the advantage/disadvantages for targeting metastasis are summarized by Lorenzo-Herrero et al. [126].

Figure 3. Strategies to harness NK cells in control of metastasis. Each therapeutic strategy is evaluated in isolation or in combination, and even in combination with other strategies including standard treatment. Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; mAbs, monoclonal
antibodies. (Arrows indicate enhancement).
Table 1. Selected clinical trials of NK-cell-based tumor therapy. Data source ClinicalTrials.gov(www.clinicaltrials.gov, accessed on 18 November 2022).

Agent
N-803 (IL-15
superagonist)ApproachTumor Type
Cytokine-based therapy
Advanced-stage
Monotherapy
melanoma,

Phase

Trial IdentifierI

NCT01946789

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NSCLC, RCC,HNSCC
NIZ985 (solube ILIn combination with
Solid tumours,
15)/IL-15 receptor
spartalizumb (anti-PD- lymphoma or
α heterodimer
1 antibody)melanoma(hetIL-15)
Adoptive NK cell therapy
Solid and
Autologous NK After treatment withhaematologicalcells
bortezomib
tumor
In combination with
Allogeneic NK
Her2+ or EGFR+
trastuzumab or
cells
solid tumors
cetuximab
Before autologous
UCB NK cells
NHL
HSCT
NK-92 cells
Merkel cell
expressing high- Combined with N-803 carcinoma thataffinity variant ofand avelumab
has progressed
CD16
after ICICIML NK cells Combined with N-803
R/R AML
iC9/CD19-CAR- In combination with
CD19+ R/R B cellCD28-zeta-2A-ILlymphodepleting
lymphoma
15 NK cells
chemotherapy
Monoclonal antibody therapy
Haploidentical NK After treatment with
Neuroblastoma
cells
anti-GD2 mAb
Expanded
After treatment with
EGFR+ NPC or
autologous NK
cetuximab
HNSCC
cells
Expanded
After treatment with HER2+ breast or
autologous NK
trastuzumab
gastric cancer
cells
Receptor inhibitor therapy
In combination with
Lirilumab (antiMultiple
elotuzumab or
KIR antibody)
Myelomaurelumab
IPH2101 (anti-KIR In combination with
Multiple
antibody)
lenalidomide
MyelomaMonalizumb (anti- In combination with Advanced solid
NKG2A antibody)durvalumab
tumorsResectable StageIII orCobolimab (TIM-3 In combination with
Oligometastatic
Inhibitor)
dostarlimab
Stage IV
Melanoma
Domvanalimab
Monotherapy or in
Advanced solid(anti-TIGIT
combination withtumorsantibody)
zimberelimab
TGF-β1 inhibitor therapy

I /Ib

NCT04261439

I

NCT00720785

I

NCT03319459

I/II

NCT03579927

I

NCT03853317

II

NCT02782546

I/II

NCT03056339

I

NCT02650648

I/II

NCT02507154

I/II

NCT02030561

I

NCT02252263

I

NCT01217203

I/II

NCT02671435

II

NCT04139902

I

NCT03628677

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Galunisertib

In combination withnivolumab

Advanced
Refractory Solid
TumorsI/II

NCT02423343

Owing to the large number of trials in each category, example trials have been selected to illustrate
the research and trials mentioned in this Review. Abbreviations: AML, acute myeloid leukemia;
CIMI, cytokine-induced memory-like; HNSCC, head and neck squamous cell carcinoma; HSCT,
hematopoietic stem cell transplantation; ICI, immune checkpoint inhibitor; NHL, non-Hodgkin
lymphoma; NPC, nasopharyngeal carcinoma; NSCLC, non-small cell lung cancer; RCC, renal cell
carcinoma; R/R, relapsed and/or refractory; UCB, umbilical cord blood.

5.1. Cytokine-Based Therapy
Cytokines endow NK cells with enhanced effector functions in antitumor immunityand enhance their persistence in vivo [127] (Figure 3). IL-2 and IL-15 are key cytokinesthat upregulate the activity of NK cells [128]. IL-2 was the first cytokine employed in tumor treatment and the first reproducible and effective human tumor immunotherapy approved by the Food and Drug Administration (FDA) [129]. An improved survival of patients with metastatic RCC and melanomas was achieved by high-dose IL-2 treatment[130]. However, it caused life-threatening toxicities, including vascular leak syndrome[129,130]. In addition, IL-2 therapy induces the proliferation and activation of Tregs andtherefore immunosuppression [131]. Thus, it has been speculated that the limited efficacy
of IL-2 therapies in vivo is at least partially attributable to the inhibitory effect of Tregs.
For these reasons, there is a need for alternatives to IL-2 and a rationale for the evaluationof variant forms of recombinant IL-2 in order to gain a higher affinity to NK cells andlower affinity to Tregs [132]. Over the past decades, IL-15 has emerged as a promising
substitute for IL-2. IL-2 and IL-15 are closely related homeostatic cytokines, and both require CD132 and CD122 heterodimers for signaling [128]. Clinical trials with recombinant
IL-15 or IL-15/IL-15Rα complexes are being conducted in metastatic tumors [133] (Table1). In the first in-human phase I clinical trial of IL-15 in patients with metastatic malignant
melanoma and metastatic RCC, hyperproliferation and an increase in the number of circulating NK cells were observed [133]. Even though a preliminary antitumor evaluation
showed no objective responses, two patients showed clearance of lung metastasis [133].
Other cytokines that activate NK cells without stimulating Tregs, such as IL-12, IL-18, andIL-21, are now being studied [127]. However, cytokine-based-NK cell-activating strategies
in the treatment of metastasis remain to be established.
5.2. Adoptive NK Cell Therapy
An alternative approach to the systemic activation of NK cells is to directly introduce
activated NK cells to a patient, known as adoptive transfer (Figure 3) (Table 1). As reviewed by Myers et al. [11], a wide variety of sources of therapeutic NK cells are currently
being tested clinically, including autologous NK cells, allogenic NK cells, umbilical cord
blood (UCB) NK cells, NK cell lines, cytokine-induced memory-like (CIML) NK cells, andCAR NK cells. The adoptive transfer of a patients’ own NK cells (autologous transfer)enables ex vivo stimulation and expansion prior to re-administration as a therapeutic modality. Initial clinical results of adoptive transfer of expanded autologous NK cells in patients with metastatic tumor showed that this treatment strategy is well tolerated, but theclinical response was limited [129,134]. It was considered that the failure of autologous
NK cell therapies could be partially attributed to the inability of inhibitory KIRs to recognize self-MHC I on tumor cells. Thus, the adoptive transfer of ‘foreign’ NK cells (allogenic
NK cells) for therapy has been examined in patients with various tumors. Allogeneic NK
cells derived from healthy donor cells are advantageous because they have higher potential in antitumor activity. A complete remission and disease-free survival have been observed in patients with AML after the adoptive transfer of allogenic KIR-ligand mismatched donor NK cells [135]. The lack of engagement of inhibitory KIR receptors on allogenic NK cells with MHC-I ligands in these patients may be beneficial and contribute toVaccines 2022, 10, 201810 of 21the clinical response [135]. Thus, the transfer of allogeneic NK cells results in a reductionin the CTC number in patients with stage IV non-small cell lung cancer (NSCLC) [136]and recurrent BT [137]. However, the clinical response is limited in metastatic tumor patients in studies of adoptive allogenic NK cell therapy [138–143]. To expand the therapeutic use of allogenic NK cells, the use of UCB is considered. NK cells constitute up to 30%of the lymphocytes in UCB, which is a robust source of therapeutic NK cells [144]. The
therapeutic efficacy of UCB NK cells is currently being evaluated in clinical trial (Table 1).
Clonal NK cell lines, such as NK-92 and KHYG-1, are an alternative source of allogeneic
NK cells. The NK-92 cell line has received FDA approval for use in clinical trials and has
been extensively tested in clinical trials [145]. However, these cells are aneuploid andtherefore genetically unstable, which requires them to be irradiated prior to infusion. NK
cells have innate memory, that is, they can remember a prior activation event and consequently respond more robustly when restimulated. NK cell memory has been identified
following combined cytokine of IL-12, IL-15, and IL-18 preactivation [146]. The CIML NK
cells were shown to have a phenotype distinct from conventional NK cells. They have
improved effector functions and induce remission in AML patients [147]. Following theclinical successes achieved with CAR T cell therapies, the use of CAR NK cells represents
a promising immunotherapeutic strategy, and CAR NK cells might show greater advantages than CAR T cells, such as the induction of less severe side effects [148]. In thisregard, it has recently been shown that CAR NK cells do not cause any serious cytokine
storm in patients with lymphoid tumors [149]. In a murine model, CAR NK cell therapy
reduced lung metastasis in an RCC by targeting ERbB2/HER2, indicating its potential in
the control of disease dissemination [150].
5.3. Agonists of Activating and Inhibitory Receptors
NK cell immunosurveillance may be improved in metastasis by targeting NK-cellfunction-related activating and inhibitory receptors (Figure 3) (Table 1). Over the past two
decades, monoclonal antibodies (mAbs) have been widely used in tumor treatment, andNK cell activity has been increased by employing tumor-specific antibodies that ligate to
CD16 receptors on NK cells to promote ADCC [151]. The CD20-targeting mAb rituximab,
the epidermal growth factor receptor (EGFR)-targeting mAb cetuximab, and the erb-b2
receptor tyrosine kinase 2 (ERBB2)-targeting mAb trastuzumb are used in the treatmentof solid and hematological tumors [152]. The CD38-targeting mAb daratunumb [153] andthe CD139-targeting mAb elotuzumab [154] constitute the treatment arsenal against MM.
The clinical response is modulated by the polymorphism in the genes encoding CD16 receptors [155], indicating that ADCC plays a crucial role in the therapeutic activity of thesemAbs. Children with neuroblastoma are given allogeneic NK cells following administration of anti-GD2 mAb, which recognizes a surface molecule highly expressed in neuroblastoma cells. Administration of NK cells with ADCC enhanced by anti-GD2 mAb produced a partial or complete response in approximately 40% of patients [156,157].
Trastuzumab was successfully introduced in the treatment of HER2+ BT and GC. In patients with HER2+ BT, even trastuzumab monotherapy has produced a clinical response[158], whereas a clinical study of adoptive infusion of autologous NK cells in addition to
trastuzumab in a refractory patient with HER2+ BT showed the reversal of the resistanceto trastuzumab [159]. Clinical trials on the combinations of autologous NK cells withmAbs are ongoing, such as the combinations of autologous NK cells with trastuzumab in
HER2+ tumors [160] and with cetuximab in recurrent and/or metastatic nasopharyngeal
carcinoma [161] (Table 1). The combination of adoptive NK cells and mAbs targeting
CTCs via NK-cell-mediated ADCC is a promising therapeutic strategy for the control ofmetastasis, as this function appears to be poorly affected by the barrier of the tumor microenvironment. Moreover, CD16 can also be engaged with bispecific or trispecific killercell engagers (BiKE or TriKE), which bind the CD16 and tumor antigens simultaneously,
to trigger NK cell activation through the CD16 receptor, significantly increasing NK cellcytolytic activity and cytokine production against tumor targets [162].

Vaccines 2022, 10, 201811 of 21Blocking inhibitory receptors on NK cells is a suitable strategy to increase antitumor
activity [11] (Figure 3) (Table 1). The blockade of the anti-KIR antibody lirilumab enhanced NK cell activity through the blockade of NK cell interaction between inhibitory
receptors KIR2DL-1, KIR2DL-2, and KIR2DL-3 with HLA-C group 1 and 2 allotypes [163].
A phase I trial in patients with relapsed/refractory MM using lirilumab as a single agent
has shown enhanced ex vivo patient-derived NK cell cytotoxicity against MM (Table 1).
However, no objective responses were observed [164]. Therefore, the combination of
IPH2101 with lenalidomide is under clinical investigation [165] (Table 1). In addition, thehumanized anti-NKG2A antibody monalizumab, which blocks NKG2A–HLA-E interaction, enhanced NK cell activity against various tumor cells; its clinical trials are ongoing[166] (Table 1). Monalizumab monotherapy for recurrent metastatic squamous cell carcinoma of the head and neck was much less efficacious with no objective response, and thedisease was stable in only 23% of the patients [167]. On the other hand, checkpoint blockade targeting the PD-1/PD-L1 and CTLA-4 inhibitory axis has produced remarkable results in tumor treatment, and the clinical benefits are considered to mainly be based on
the reactivation of exhausted T cells [168]. However, the effect of ICIs on NK cells throughPD-1/PD-L1 blockade also mediates antitumor immunity [9]. Thus, targeting PD-1 byblocking the PD-1/PD-L1 signaling axis enhances the NK cell immune response against
MM [169]. Clinical studies have been conducted to investigate the effect of pembrolizumab (anti-PD-1) combined with autologous [170] or allogeneic [171] NK cells in patientswith NSCLC. These studies showed increased NK cell activity in patients receiving NK
cells and improved survival compared with patients receiving pembrolizumab alone[170,171]. Additionally, a fully humanized anti-PD-L1 mAb has been shown to block PD1/PD-L1 interactions and promote NK-cell-mediated ADCC against tumor cells [172]. Furthermore, TIM-3 blockade has been shown to reverse NK cell impairments and increase
NK cell antitumor activity in patients with advanced melanoma and lung adenocarcinoma [102,104]. A plethora of anti-TIGIT mAbs are in advanced clinical development forsolid tumors, such as domvanalimb, vibostolimb, tiragolumab, and ociperlimab [173] (Table 1). Further investigation, however, is required to elucidate the actual anti-metastatic
potential of blocking inhibitory receptors in humans.
TGF-β1 is one of the major suppressive cytokines produced by tumor cells and platelets. It inhibits NK cell effector functions and helps tumor cells evade NK cell immunosurveillance [84–86,123] (Figure 2). A pharmacological inhibitor of the TGF-β1 pathway(Figure 3), galunisertib, has been tested in patients with neuroblastoma [174] and HCC[175], and an increased NK cell activity against tumor cells was observed [174].
As platelets can protect tumor cells from NK cell immunosurveillance, targeting theprotective interaction of platelets with tumor cells has been suggested to improve the NK
cell antitumor activity [114] (Figure 3). The platelet inhibitor ticagrelor specifically inhibits
tumor-associated platelets and strongly reduces lung metastasis in a mammary carcinoma
mouse model [176]. In addition, silencing tumor-specific tissue factors (TFs) by nanoparticle-mediated delivery of siRNA resulted in reduced platelet adhesion and ultimately the
number of lung metastases [177]. Future research will show if targeting the interaction ofplatelets with tumor cells can improve the efficiency of NK cell immunosurveillance.
6. Conclusions and Future Direction
Tumor immunotherapy by revitalizing immune responses against tumor cells has
shifted the paradigm in tumor therapy [6,168]. Nevertheless, the metastatic spread of tumor cells remains the main cause of tumor-related death [2]. NK cells are innate immune
cells that can directly and rapidly kill tumor cells without antigen restriction, and they arehighly responsible for immunosurveillance in metastasis [13,44]. Hence, tumor cells haveto evade NK-cell-mediated immunosurveillance for successful metastasis (Figure 2).
Thus, it is promising to harness NK cells for the prevention or treatment of tumor metastasis (Figure 3). As the evading mechanisms involved NK cell immunosurveillance in themetastatic cascade, particularly the specific interactions of CTCs with NK cells inVaccines 2022, 10, 201812 of 21

metastasis (Figure 2), an optimal therapeutic window may exist to achieve maximal NK
cell antitumor activity. The experimental lung metastasis model showed that NK cells
eliminate CTCs from the lung within 24 h of arrival, but not thereafter. Half of NK–tumor
cell encounters lead to tumor cell death in the first 4 h after tumor cell arrival [44]. NK celltherapies would be mostly used in the adjuvant setting as adjuvants, such as after surgery[74] and following stem cell transplantation [178] targeting minimal residual disease. Specifically, NK cells may be most effective for targeting CTCs, as their function appears to
be poorly affected by the barrier of the tumor microenvironment [179]. Clinical trials areunderway to evaluate the efficacy of NK cell immunotherapy by each therapeutic strategy
alone or in combination (Figure 3), and further in combination with other strategies, including standard treatments, such as mAbs therapy [160,161] and chemoradiotherapy[180], with encouraging clinical results. Moreover, recent data suggest that molecularly
targeted agents [181] and radiotherapy [182] capable of inducing senescence in tumor cells
elicit NK-cell-mediated immunosurveillance, facilitating tumor regression, indicating that
NK cells are likely to be a key player in future multimodal strategies against tumors. Further studies should be performed in order to fully understand the antitumor and antimetastatic properties of NK cells, particularly when and at which steps of the metastaticcascade NK cells operate, and how many times NK cell immunotherapy is required, which
will pave the way to developing novel therapeutic strategies for the prevention or treatment of tumor metastasis.
Author Contributions: Conceptualization, X.D. and H.T.; literature review, X.D.; writing—originaldraft preparation, X.D.; writing—review and editing, X.D. and H.T. All authors have read andagreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data sharing not applicable.
Conflicts of Interest: X.D. and H.T. hold patents for feeder-cell-free NK cell expansion culture technology and are stockholders of Biotherapy Institution of Japan Inc., a biotech startup company developing NK-cell-based immunotherapy and Mesenchymal Stromal Cell (MSC)-based products,
such as adipose MSCs, and MSC-derived conditioned medium (CM) concentrates and extracellular
vesicle (EV) isolates (StemSup®) for clinical research.

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