Introduction

Glioblastoma (GBM) is the most common malignant tumor.1 The global incidence of GBM is approximately 10 cases per 100,000 people. The median survival for GBM is approximately 12 months, with estimated survival rates at 1, 2, and 5 years being 28.4%, 11.5%, and 3.4%, respectively.2Figure 1 illustrates various therapeutic approaches for GBM. Although surgical treatment, postoperative chemoradiotherapy and other treatments have made certain progress, the prognosis of GBM patients is still poor due to multiple factors such as postoperative recurrence and resistance to chemoradiotherapy. Given the poor prognosis of GBM, more effective treatment strategies are urgently needed.

Figure 1 Multiple approaches for GBM treatment.

Research on NK cells has progressed over the past 50 years (Figure 2).3Figure 3 presents several NK cell-based therapeutic strategies.4 NK cells possess several activating and inhibiting receptors on their surface, which play critical roles in identifying malignant cells. The activation of NK cells depends on the abundance and expression of these receptor ligands in glioma cells.5 Furthermore, numerous inhibitory factors within the tumor immune microenvironment (TIM) inhibit NK immune responses. Enhancing NK infiltration into glioma tissue and boosting their activity are key strategies in targeted NK cell-based therapy. Recent studies targeting the TIM and tumor cells, in combination with NK cell therapies, have shown significant potential for tumor treatment. Therefore, the review aims to provide an in-depth summary of the latest advancements in NK cell-based therapies for GBM.

Figure 2 History of NK cells.

Figure 3 NK cells-based therapies.

NK Cell Development, Functions, and Localizations

In mice and humans, NK cells undergo distinct stages of development (Figure 4).6–9 Many NK cells are not only cytotoxic but also produce various molecules, including perforin and granzyme, which can lead to stress-induced cell death. Additionally, NK cells express FASL and TRAIL, allowing them to induce apoptosis in target cells by binding to the corresponding receptors, FAS or TRAILR. Furthermore, NK cells secrete various cytokines, such as interferon IFN-γ, TNF-α, interleukin [IL]-10, granulocyte-macrophage colony-stimulating factor (GM-CSF), and chemokines (CCL3, CCL4, CCL5, XCL1).10

Figure 4 The development process of NK cells.

The regulation of the NK cell effector function is maintained by a dynamic equilibrium between inhibitory and activating cell surface receptors (Figure 5). Additionally, NK cells recognize tumor cells based on the balance between stimulatory and inhibitory receptors11–21 (Figure 6). These cells operate by killing cancerous cells in an antigen-independent manner (Figure 7).22–25

Figure 5 NK Cell Functions.

Figure 6 NK cells recognize tumor cells based on a balance between the stimulatory and inhibitory receptors above.

Figure 7 NK cell surface receptors and ligands on tumor cells are involved in tumor recognition. NK cells express a set of stimulatory (or activation) receptors as well as inhibitory receptors to recognize healthy cells and aberrant cells such as virus-infected or a potential tumorigenic cell through MHC-1 receptor appearance.

Some previous research shows that NK cells are present in various tissues and organs, with Figure 8 illustrating their localizations.26

Figure 8 The localization of NK Cells.

Sources of NK Cells for Immunotherapy

NK cells for clinical treatment can be obtained from umbilical cord blood (UCB),27,28 peripheral blood (PB),29,30 stem cells, and NK-92.31,32 Currently, most clinical trials involving NK cells focus on UCB-NK, PB-NK, and NK-92 cells. However, significant challenges exist in the manufacturing process for the final therapeutic cell doses.33–37Figure 9 depicts the specific workflow for isolating NK cells from various donor sources and their subsequent expansion for adaptive transfer.

Figure 9 Sources of NK cells for immunotherapy. (A) Allogeneic umbilical cord blood NK cells (CB-NK Cells); (B) NK cell cancer cell lines (NK-92); (C) hESC and iPSCs; (D) Merits and demerits with the different NK cell sources vary as described.

NK Cell Functionality in the TME

The regulation of NK cell metabolism is influenced by various factors.38,39 Regarding nutrition, NK cells are competitive with tumor cells and myeloid-derived suppressor cells (MDSCs).40 Tumor cells not only consume large amounts of glucose but also produce lactic acid, which is transported to NK cells by the transporters SLC16A1 and SLC16A, impairing ATP production.41 Tumor cells produce extracellular adenosine through the action of extracellular nucleotidases CD39 and CD73, which inhibit oxidative phosphorylation and glycolysis in NK cells.42 Additionally, TGF-β secreted by tumor cells and MDSCs disrupt the mTOR signaling pathway43–46 (Figure 10).

Figure 10 The functions of NK cell in the TME. (A) Tumor microenvironment shapes NK cell metabolism and effector functions; (B) A schematic representation of several metabolites and other factors present in the TME that limit NK cell effector functions.

NK Cells and Their Interactions in the Glioma Microenvironment Effect of Glioma on NK Cell Function

Gliomas are classified as immune “cold tumors” due to their low lymphocyte infiltration, and the glioma microenvironment has the capacity to suppress the immune response systematically and locally. MHC-I on the surface of normal cells binds to inhibitory receptors on the surface of NK cells, regulating their killing function. However, overexpression of MHC-I in central nervous system tumors inhibits NK cell activity. Most glioma cells exhibit high levels of MHC-I molecules, which interact with KIRs to inhibit NK cell activity. NKG2D, a non-MHC-I-specific activation receptor for NK cells, plays a crucial role in determining NK cell cytotoxicity against tumor cells, which is dependent on the abundance of NKG2D ligands present in glioma cells. Glioma cells can also regulate the expression of receptors on the surface of NK cells, thereby inhibiting their function. Mutations in the isocitrate dehydrogenase1 1/2 genes in diffuse glioma promote epigenetic reprogramming of various immune genes, including downregulation of NKG2D ligands and resistance to NK cell-mediated cytotoxicity. Studies show that IDH mutant gliomas are more prone to mutations that inactivate their own NKG2D ligands, allowing them to evade NK cell-mediated immune surveillance.47,48 However, other studies suggest that the IDH1-R132H mutation can enhance the recruitment of NK cells to the tumor site by modulating the TIM, potentially improving patient prognosis.49

Glioma can impair NK cell function not only by downregulating NKG2D ligands to inactivate NK cell receptors but also by the secretion of cytokines. Glioma stem cells (GSCs) in GBM significantly induce and secrete transforming growth factor β (TGF-β), which inhibits NK cell function in the glioma immune microenvironment.50 The interaction between αv integrin on GSCs and CD9 and CD103 on NK cells stimulates TGF-β production by GSCs. TGF-β, released by GSCs, then activates transforming growth factor beta receptor 2 on NK cells, blocking their antitumor activity. Combining donor-derived NK cells or allogeneic NK cells with inhibitors targeting αv integrin or TGF-β receptors enhances their antitumor activity.51 TGF-β also downregulates NKG2D expression in peripheral blood NK cells, inhibiting their function.11,12,52,53 Patients with GBM frequently exhibit lymphocytopenia due to gliomas activating signal transducer and activator of transcription 3. Hyperphosphorylation of STAT3 induces various soluble immunosuppressive factors, including interleukin-10 (IL-10), prostaglandin E-2, vascular endothelial growth factor, and TGF-β, which collectively inhibit CTL activity and proliferation.54 Additionally, gliomas may secrete soluble immunosuppressive factors such as carbohydrate-binding proteins and galectin-1, which reduce lymphocyte viability. Galectin1-deficient gliomas are more susceptible to lysis by NK cells and are cleared before they can adapt to the antitumor immune response. Inhibiting galectin-1 in gliomas can restore NK cell immune surveillance and improve survival rates of patients with glioma. Furthermore, glioma cells can upregulate indoleamine 2, 3-Dioxygenase (IDO) to inhibit antitumor immunity. As a key rate-limiting enzyme in tryptophan metabolism, IDO depletes tryptophan and generates kynuridine, a metabolite that inactivates NK cells and fosters the development of regulatory T cells (Tregs), thereby contributing to tumor immunosuppression.

In patients with glioma, the accumulation of MDSCs in peripheral blood is significantly higher than that in healthy individuals.55,56 MDSC interacts with macrophages to promote IL-10 expression and inhibit IL-12 secretion, leading to the conversion of macrophages into tumor-promoting M2-type cells. Additionally, other chemokines and cytokines, such as CX3CL1 and CCL5, contribute to abnormal tumor angiogenesis in the TME by recruiting glioma-associated macrophages.57,58 Furthermore, the tumor can gain a survival advantage by depriving surrounding normal cells of nutrients and oxygen, which may inhibit NK cell metabolism and antitumor activity.59

Role of NK Cells in Glioma Immunity

The number of NK cells is associated with the prognosis of glioma, as neurons produce the CX3CL1 chemokine, which promotes the accumulation of CX3CR1 + NK cells in the brain.60 A study shows that GBM exhibits the highest degree of NK cell infiltration among tumors when compared to that of breast cancer and melanoma, suggesting the important role NK cells play in glioma immune monitoring.61 NK cells are also present in meningiomas and metastatic brain tumors,62 and they can effectively lyse GBM and medulloblastoma in vitro.63 Killer cell immunoglobulin-like receptor 2DS2 and KIR2DS4 alleles are linked to the regulation of cytomegalovirus gene expression, which induces platelet-derived growth factor D by enhancing the recruitment of peripheral cells and promoting tumor angiogenesis. Thus, PDGFD expression further stimulates the growth of GBM.64 Most PDGFD in GBM can bind to the activated NKp44 receptor, enhancing the cytotoxic capability of NK cells to control tumor growth and improve survival rates in patients with GBM. B7-H6 (NKp30 ligand) is highly expressed in human gliomas, potentially inducing tumor development by specifically binding to NKP30 and upregulating IL-32 expression. These studies suggest that NK cells significantly influence the prognosis of patients with glioma.

As key effector cells of the innate immune system of the body, the infiltration of NK cells in the TME serves as a reliable indicator of tumor prognosis. Bioinformatics analyses indicate that patients with glioma with high expression levels of NK cell activation genes exhibit significantly improved prognosis. Compared to higher-grade gliomas, the number of activated NK cells is significantly higher in lower-grade gliomas, suggesting a marked decrease in activated NK cells during the progression from low to high-grade gliomas.65 Another study shows a significant correlation between the number of activated NK cells and the survival rate of patients with GBM.66 Therefore, NK cells can prevent the malignant transformation of brain gliomas. They can effectively eliminate undifferentiated GSCs. IFN-γ released by NK cells promotes the differentiation of GSCs, enhancing their sensitivity to chemotherapy but reducing their susceptibility to NK cell cytotoxicity.67 Enhancing NK cell-mediated destruction of GSCs while promoting IFN-γ release to induce GSC differentiation represents a potential direction for targeted therapy.68,69

Application of NK Cells in Treating Glioma Activate NK Cell Therapy

Central nervous system tumors typically exhibit poor immunogenicity and high immunosuppression levels, making immunotherapy less effective. The cellular activity of NK cells is influenced by the balance between inhibitory and activating receptors. Chemotherapeutic and molecularly targeted drugs for brain gliomas can upregulate the expression of NK cell-activating receptors and promote the release of cytokines or the induction of NKG2D ligand expression. Temozolomide (TMZ) and radiotherapy can increase NKG2D ligand expression in the glioma tissues of patients with GBM. Animal studies show that TMZ or radiation-induced NKG2D ligand expression prolongs survival in GBM mouse models. Activation of DNA damage repair (DDR) mechanisms can induce the production of NKG2D ligands, enhancing the antitumor function of NK cells. However, the proteasome inhibitor bortezomib (BTZ) can downregulate the expression of activating receptor NKG2D and DNAM-1 ligand by activating DDR pathways, thus promoting NK cell lethality against GBM. In a study involving experimental animals, the combination of autologous NK cells and BTZ significantly inhibits tumor growth and extends survival in 25% of cases.68 Triple therapy combining BTZ with oncolytic herpes simplex virus (OHSV) and NK cells has shown greater efficacy in treating GBM, as BTZ enhances the expression of the NK cell-activating receptor DNAM-1. OHSV infection can induce NK cells to secrete IFN-γ and TNF-α, resulting in increased tumor cell death and improving survival rates in transplanted tumors in nude mice. Additionally, the demethylating agent decitabine increases NKG2D ligand expression, thereby restoring the cytotoxicity capability of NK cells against IDH mutant glioma cells.69,70 The histone deacetylase inhibitor mucorin A induces the upregulation of NKG2D ligands, enhancing NK-cell-mediated cleavage of GBM cells.71

Recently, triple-functional antibodies that recognize tumor antigens while binding to NKp46 and CD16 have shown superior antitumor effects in vivo compared to that of traditional antibodies targeting tumor antigens.72 Proteins encoded by the MHC-I chain-related genes A and B (MICA and MICB) are classified as NKG2D ligands. Antibodies targeting MICA and MICB prevent their loss from the surface of tumor cells, promoting the binding between NKG2D and its ligands and inhibiting tumor growth. The antitumor effects of these antibodies are primarily mediated through the activation of NKG2D and CD16.73 KIRs can recognize MHC-I molecules on tumor cells, inhibiting NK cell activation and promoting tumor cell growth.74 However, KIRs from allogeneic NK cells do not recognize the MHC-I molecules of the recipient patient, resulting in a lack of inhibitory signals and activating NK cells to effectively kill tumor cells. Utilizing NK cells with KIR2DS2 immune genotypes has proven effective in killing GBM cells and prolonging survival in animal models.11,75,76

NK Cell-Based Immunotherapy Against GBM

Given the benefits of immunotherapy utilizing NK cells, this approach is promising for treating GBM. NK cells can target GBM heterogeneity without the need for specific tumor-associated antigen recognition.77–81 They effectively eliminate tumor cells that downregulate MHC-I expression on their surface,82 as the interaction between MHC-I and KIRs inhibits NK cell activity and protects healthy autologous cells.83,84 Given these advantages, Tables 1–3 summarizes current clinical studies investigating NK cell-based therapies for GBM.85,86 Recent studies show the efficacy of intratumoral G47∆ treatment for residual or recurrent GBM using a stereotactic surgical system.87,88 This method is particularly effective from a direct delivery standpoint, as it directly influences immune cells within the immunosuppressive GBMTME. Figure 11 illustrates various NK cell therapy approaches, including adoptive NK cell therapy, CAR-NK cell therapy, checkpoint-blocking therapy, and gene-edited NK therapy.

Table 1 Clinical Trials of CAR NK Cell Therapy Against Glioma

Table 2 Clinical Trials of Adoptive NK Cell Therapy Against Glioma

Table 3 Clinical Trials of LAK Cell Therapy Against Glioma

Figure 11 Multi-approaches of NK cell-based immunotherapy against GBM.

Combination Therapy

The primary factor contributing to the low efficiency of immunotherapy for solid tumors is the immunosuppressive effect of TME. Current research targeting TIM and tumor cells in conjunction with NK cell therapy shows effectiveness in tumor treatment. Recent studies indicate that combining NK cells with monoclonal antibodies or chemotherapeutic agents significantly enhances the survival rate of animals with GBM. Chondroitin sulfate proteoglycan 4, also known as neuron glial antigen 2 (NG2), plays a crucial role in intracellular signaling, activating pathways such as ERK and FAK, which are closely associated with tumor growth and metastasis. Research shows that combining activated NK cells with the NG2-targeting monoclonal antibody mAb9.2.27 can inhibit tumor deterioration and extend the median survival time in GBM models. Studies of GBM antigens show that NK cells regulate the pro-inflammatory environment in GBM primarily through IFN-γ secretion rather than through direct cytotoxic action. The mechanism of the combined treatment involving NK cells and mAb9.2.27 in rat GBM models is based on the conversion of glioma-associated macrophages from an anti-inflammatory M2 phenotype to a pro-inflammatory M1 phenotype. This transformation results in significantly increased levels of IFN-γ and TNF-α in the cerebrospinal fluid of treated rats, while immunosuppressive cytokines such as IL-10, IL-6, and IL-1β are significantly decreased. Cancer adjuvants, including CpG oligonucleotide, can enhance the antitumor immune response by mimicking pathogen-associated molecular patterns and inducing plasmacytoid dendritic cells to produce type I interferon (IFN-I). IFN-I further enhances the antitumor activity of NK cells. Although CpG oligonucleotide therapy has shown promising results in vitro, in vivo studies involving patients with primary and recurrent gliomas, have not shown significant therapeutic effects using CpG oligonucleotide alone. Research shows that NK cells serve as the primary antitumor effector cells in mouse glioma models treated with a repeated low dose of CpG oligonucleotide. Despite their potential, tumor-infiltrating NK cells remain susceptible to local and systemic inhibitory signals, necessitating more effective strategies to enhance NK cell functionality within the brain TME. Combined treatment with CpG oligonucleotide and Treg elimination can enhance the cytotoxic effects of NK cells against brain glioma.

Malignant glioma-induced binding of human leukocyte antigen E (HLA-E) and lectin-like transcript 1 (LLT1) to CD94/NKG2A and CD161 inhibits NK cell function. Blocking the interaction of HLA-E with CD94/NKG2A or LLT1 with CD161 using small interfering RNAs or blocking antibodies promotes NK cell-mediated lysis of glioma cells. Moreover, combining the humanized anti-NKG2A antibody monalizumab with cetuximab enhances antibody-dependent cellular cytotoxicity (ADCC) in head and neck squamous cell carcinoma.89 As EGFR serves as a critical target for various tumors, including gliomas, the ability of monalizumab to block NKG2A may augment NK cell-mediated ADCC against gliomas, especially TMZ-resistant gliomas.90

Combination therapy using anti-PD-1 and anti-cytotoxic T lymphocyte-associated protein 4 (CTLA-4) has increased NK cell and CD8+T cell infiltration in the CNS, improving survival rates in GBM mouse models. The blood-brain barrier (BBB), a selectively permeable membrane, protects the brain parenchyma from harmful substances but also limits the entry of immunotherapeutic agents and immune cells. Poly (β-L-malic acid) can covalently bind to anti-PD-1 or anti-CTLA-4 antibodies, facilitating trans-BBB transport and enhancing NK cell infiltration and survival in mice vaccinated with the intracellular GL261 model.91 Combining these techniques with cytolytic NK cells may help to overcome the challenges posed by BBB selective permeability and the immunosuppressive environment of the brain TME.92

Genetically Modified NK Cells

Chimeric antigen receptors (CARs) replace the α and β variable regions of the TCR with tumor-specific antibody single-chain variable regions (SCFV). These are fused to the CD3ζ signaling chain of T-cell or NK cell receptors through a transmembrane junction region. This enables the transduction of somatic toxic T lymphocytes (CAR-T) or NK cells (CAR-NK), allowing for the recognition of tumor-specific antigens through SCFVs, which facilitates targeted tumor cell killing (Figure 12). Adoptive transfer of NKG2D-based CAR T cells in combination with radiotherapy showed therapeutic synergy in a GL261 in situ glioma mouse model. Mice inoculated with GL261 intratuminal CAR T cells exhibited a higher survival rate than those injected with intravenous CAR T cells despite a lower number of CAR T cells being administered directly into the brain parenchyma. However, the continuous in vivo expansion of CAR-T cells can lead to cytokine release syndrome and subsequent graft-versus-host disease. These conditions significantly affect patient quality of life. CAR-NK cells mitigate these issues by targeting various tumor antigens, including EGFR and human epidermal growth factor receptor 2 (HER2, also known as ErbB2). These CAR-NK cells maintain natural cytotoxicity while targeting tumor antigens effectively.

Figure 12 CRA-NK molecule delivery of genetic cargo NK cells with CAR encoding RV, LV or AAV.

EGFR gene amplification and protein overexpression occur in 40%–60% of GBM cases, while these markers are rarely expressed in normal brain tissue. GBM typically co-expresses EGFR and EGFR type III mutants (EGFRvIII). Different CAR-NK constructs can be designed to specifically target EGFR and EGFRvIII in GBM, depending on the target.93 Studies confirm that CAR NK-92 cells, which exhibit high expression of NK cell-activating receptors, effectively inhibit the growth of xenografted gliomas that overexpress EGFR. By repeatedly injecting CAR NK-92 cells (which express high levels of NK cell-activating receptors) intracranially into GBM xenografted immunosuppressed mice (NSG mice) with EGFR or EGFRvIII expression, significant tumor growth inhibition and improved overall survival were observed compared to the control group treated with NK-92 cells alone.94 HER2, a member of the EGFR family, is not expressed in the central nervous system of healthy adults but is present in 80% of GBM cases, where its expression correlates with poor survival outcomes. CAR-NK cells targeting EGFR, EGFRvIII, and HER2 have shown potent cytotoxic effects against primary GBM tumor cells and cell lines in vitro.95 The injection of HER2-targeted NK cells into NSG mouse models with xenografted GBM cells effectively inhibits tumor progression and prolongs survival. HER2-NK-92/CAR-NK cells continue to exhibit tumor-killing abilities and retain the cytotoxic function of normal NK cells despite potential antigen loss. GSCs are susceptible to allogeneic NK cells, and specific targeting with HER2-CAR NK-92 cells can lyse HER2-positive GSCs. However, prolonged exposure may lead to the elimination of target cells by parental NK-92 cells that lack CAR. Oncolytic viruses (OVs) possess a dual mechanism of action, directly attacking cancer cells while also stimulating a tumor-specific immune response. Combining EGFR-CAR NK cells with OV-IL15C, which highly expresses a human IL15/IL15Rα sushi domain fusion protein, has been utilized to treat GBM in mouse models. The infiltration and activation levels of NK and CD8+ T cells in glioma tissue are significantly higher in the combined treatment group than in the single treatment group. Moreover, the effectiveness of the combined treatment is also significantly higher, resulting in a significant extension of survival time for the mice.96 Therefore, the activation of CAR-NK cells is determined by the integration of CAR signals and the intrinsic signals of NK cells.

Genome-Editing NK Cell Therapy

This section provides an overview of genome-edited NK cell therapy, excluding CAR-NK therapy. The combination of adoptive NK cell therapy with checkpoint inhibitors presents a promising strategy for treating GBM, but the effectiveness of checkpoint inhibitors is influenced by the timing of administration and delivery efficiency, especially in GBM. Genome-edited NK cells can knock out immunosuppressive receptor genes, continuously blocking the expression of inhibitory receptors.

The CRISPR/Cas9 genetic-editing system is widely used to modify T cells by targeting inhibitory genes such as PD-1 and CTLA-4.97–101 Conversely, CRISPR/Cas9 genome editing of NK cells has faced challenges, primarily due to low viral transduction efficiency; however, recent advances using Cas9/RNP electroporation have provided new insights.102–105 Rautela et al describe the CRISPR/Cas9 genome editing of major human NK cells, particularly targeting the CISH gene and the NKp46 receptor.105 Two groups were used to reveal the use of CRISPR/Cas9 through electroporation to create CD38 knockout NK cells.106 These CD38 knockout NK cells show an improved metabolic profile, enhancing ADCC against CD38+ multiple myeloma cell lines and patient-derived samples.106–108Table 4 presents the CRISPR/Cas9-mediated NK cell-based therapies mentioned above.

Table 4 CRISPR/Cas9 Electroporation Genome-Editing NK Cell-Based Immunotherapy Against GBM

For GBM therapy, several studies establish TIM-3 knockout major NK cells, which exhibit enhanced cytotoxicity against GBM.110 In the experimental design, 106 orders of NK cells per shot were subjected to electroporation. The researchers were able to obtain 4.7×108 genetically modified NK cells from 16 mL of blood in 2 weeks, providing sufficient quantities for clinical application. In comparison, a previous study reports an electroporation setting of 105 orders of NK cells per session with significantly lower expansion efficacy.105 Huang et al show that CRISPR/Cas9 knocks out multiple genes in major NK cells; however, these NK cells show no significant activation.109 While CRISPR/Cas9 genome editing in major NK cells currently lags behind that in T cells, ongoing advancement in this technology indicates that it may offer a potential strategy for combating GBM.

Technical Bottleneck and Future Direction of NK Cell Therapy for Glioma

NK cell-based adoptive immunotherapy for cancer is a rapidly evolving field with the potential to benefit patients with solid tumors. This approach includes identifying and effectively blocking NK cell immune checkpoints, as well as utilizing specific killer cell junctions or redirecting NK cell tumor-specific lysis through CAR loading. However, the transgenic efficiency of primary NK cells is only approximately 15%, presenting a significant technical bottleneck of CAR-NK applications. Improving the efficiency of NK cell transgene delivery is an important research focus in CAR-NK cell therapy.110 Currently, CAR-NK cells are entering clinical trials for the treatment of malignant gliomas.51 In addition to ongoing studies on engineered CAR NK-92 cells, other allogeneic sources—such as peripheral or umbilical blood NK cells and NK cells differentiated from induced pluripotent stem cells—are also preparing to enter clinical trials for brain tumors. Although GBM shows strong adaptability that allows evasion of immune attacks and counteraction of immunosuppression, the heterogeneous expression of CAR-targeted antigens presents a significant challenge for immune escape. For instance, HER2-CAR-T therapy for GBM has shown that these T cells can metastasize into tumors, but the highly effective T cells within the TME lead to rapid selection of antigen-losing variants.111 CAR-NK cells may encounter similar challenges. However, NK cells naturally exhibit diverse cytotoxic effects, with activated receptors interacting with various ligands expressed by tumor cells, including natural cytotoxic receptors (NKp46, NKp44, and NKp30), NKG2D, and DNAM-1 (CD226). These NK cell receptors typically recognize stress-inducing ligands expressed on immune or tumor cells exposed to prolonged therapeutic stress. Therefore, CAR-NK cells can inhibit glioma cells through CAR-dependent and NK cell receptor-dependent pathways, enabling the elimination of GBM cells that express low or no CAR-targeted antigens. The HER2-NK-92/5.28z CAR NK retains effector function despite potential antigen loss, thereby restoring the cytotoxic capacity of adoptive metastatic cells to that of baseline NK cells. Currently, HER2-CAR-NK therapy is undergoing clinical trials for GBM treatment (CAR2BRAIN; NCT0338978).

In comparison to CAR-T cells, CAR-NK cells exhibit a shorter lifespan and limited in vivo expansion. This results in higher safety during treatment; however, it also restricts long-term efficacy, necessitating repeated treatments to achieve significant effects. The ectopic expression of pro-inflammatory cytokines, such as IL-15 and CAR, enhances the persistence of CAR-NK cells and promotes T-cell penetration and activation. The strategy of inducing endogenous immune memory has emerged as a foundation for CAR-NK cell activation in GBM therapy.112 In patients with GBM, a strategic combination of adoptive NK cell transfer with conventional therapies—such as radiation therapy, immune checkpoint inhibitors, angiogenesis inhibitors, or myeloid intercellular regulators—can modulate the function of innate and adaptive immune cells in the TME. This approach may effectively harness the cytotoxic effects of CAR-NK cells against GBM.113–115

Bio-Material-Based NK Cell Surface Modification Technologies

Up to now, varieties of manipulation techniques for decorating functional moieties onto cell surface and heightening target recognition have been developed116 (Figure 13). A hydrophobic interaction-mediated ex-vivo cell surface engineering utilizing lipid-based bio-materials may be a most advanced technology that could accomplish efficient cell surface modification by a single way without influence of inherent properties of cells (especially, NK cells).116 In order to NK cells with target-specificity, synthetic CD30-specific aptamers are modified on cell surfaces to result in aptamer-engineered NK cells (ApEn-NK) without gene mutation or other injuries. Under surface-modified aptamer guide, ApEn-NK specifically bind to CD30-expressing lymphoma cells but do not react to off-target cells. The research results specific cell binding of ApEn-NK triggers higher apoptosis/death rates of lymphoma cells compared to parental NK cells. Additionally, experiments with primary human NK cells indicated the potential of ApEn-NK to specifically target and kill lymphoma cells, thus providing a novel method for targeted immunotherapy by NK cells.117 In addition, one study reported a chemoenzymatic glycocalyx editing way to introduce high-affinity and specific CD22 ligands onto NK-92MI and cytokine-induced NK cells to obtain tumor-specific CD22 targeting. These CD22-ligand modified cells proved effectively raised tumor cell binding and killing in vitro without damaging normal B cells.118 Another study, researcher developed synthetic lipid-folate conjugates, for steady modifying onto NK cell surfaces via hydrophobic interactions, thus increasing folate-mediated ligand-receptor immune interactions with target cancers.119 Similarly, lipid-PEG conjugated hyaluronic acid (HA) materials (HA-PEG-Lipid) for the simple ex-vivo surface coating of NK cells is developed for 1) lipid-mediated cellular membrane anchoring via hydrophobic interaction and thus 2) fully demonstration of the CD44 ligand (i e, HA) onto NK cells for cancer targeting, without the need for genetic manipulation.120 Therefore, with the continuous progress of biotechnology, we firmly believe bio-material-based NK cell surface modification will enhance NK cell therapy technologies for tumor treatment (especially, GBM)

Figure 13 Bio-material-based NK cell surface modification technologies.

Conclusions

NK cells serve as the first line of defense in the body and play a crucial role in combating cancer cells. NK cell infiltration in gliomas correlates with patient prognosis. Analysis of RNA sequencing data from the TCGA database indicates that NK cell infiltration scores in low-grade gliomas and GBMs are higher than those of T cells.121 However, the glioma microenvironment inhibits NK cell function, leading to their dysfunction. When NK cells are injected into a patient, they often become dysfunctional due to inhibitory signals released by tumor cells or tumor-associated mononuclear cells/macrophages and rapidly lose their ability to recognize and eliminate tumors. Currently, little is understood about the mechanisms underlying immunotherapy resistance in GBM or the intrinsic properties of these tumors. Therefore, an in-depth exploration of NK cell dysfunction and the glioma microenvironment is essential for identifying strategies to reverse NK cell impairment. Dysregulation of N6-methyladenosine (m6A) modification is closely linked to the onset, progression, and drug resistance of gliomas.122,123 Studies show that in pancreatic ductal adenocarcinoma with a high m6A score, T-cell dysfunction correlates with reduced NK cell presence. As an emerging field, the relationship between m6A modification status and NK cell function, as well as immune cell infiltration in gliomas, remains unclear.124

Current findings suggest that combining immunotherapy with other treatments may lead to breakthroughs in GBM therapy. Therefore, future glioma treatments are likely to focus on combining NK cell therapies with molecularly targeted drugs, including anti-angiogenic agents, EGFR inhibitors, and epigenetic modifiers. Currently, NK cell treatments are primarily in the animal experimental phase, and their application in patients is still a distance away. Ongoing clinical trials are expected to explore NK cell combination therapies further, necessitating a reevaluation of clinical trial designs to efficiently assess the potential clinical effect of these immunotherapy approaches. We believe that NK cell-based immunotherapy will exhibit improved efficacy in glioma treatment in the future.

Abbreviations

GBM, glioblastoma; TIM, tumor immune microenvironment; UCB, umbilical cord blood; MDSCs, myeloid-derived suppressor cells; GSCs, Glioma stem cells; IL-10, interleukin-10; IDO, indoleamine 2,3-Dioxygenase; Tregs, regulatory T cells; TMZ, Temozolomide; DDR, DNA damage repair; BTZ, bortezomib; OHSV, oncolytic herpes simplex virus; MICA, MHC-I chain-related genes A; MICB, MHC-I chain-related genes B; NG2, neuron glial antigen 2; IFN-I, type I interferon; HLA-E, human leukocyte antigen E; LLT1, lectin-like transcript 1; ADCC, antibody-dependent cellular cytotoxicity; CTLA-4, anti-cytotoxic T lymphocyte-associated protein 4; BBB, blood-brain barrier; CARs, Chimeric antigen receptors; SCFV, single-chain variable regions; HER2, human epidermal growth factor receptor 2; OVs, Oncolytic viruses.

Acknowledgments

1. The authors give their thanks to all those who have helped with this issue.

2. Hao Wu thanks his wife (Li Jiang) and his two lovely sons (Hua Wu and Xia Wu).

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

1. Shaanxi Provincial Key Research and Development Plan Project Support (2020ZDLSF01-02) 2. Doctor Foundation of the Second Affiliated Hospital of Xi’an Medical University (No. X2Y-R11).

Disclosure

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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