Natural Killer Cell-mediated Immunosurveillance Research Articles

Platelet-mediated circulating tumor cell evasion from natural killer cell killing via immune checkpoint CD155-TIGIT.

e14552 Background: Circulating tumor cells (CTCs) are precursors of cancer metastasis. However, how CTCs evade immunosurveillance during hematogenous dissemination remains unclear. The interaction between platelets and CTCs is the most extensively studied of all blood cells, and it is well known that CTCs are surrounded by platelets, which form microplots on their surface and play a crucial role in cancer metastasis. The precise mechanisms platelets employ to contribute to CTC immune evasion have not been fully elucidated. Our previous single-cell RNA sequencing data indicated high expression of platelet-related genes in CTCs. Here, our study demonstrated that platelet adhesion to CTCs is prevalent in multiple cancers and is indispensable for CTCs metastasis. Methods: CTC isolation was performed by our self-established ChimeraX-i120 platform. CTC-platelet adhesions were identified by single-cell RNA sequencing and multiplex immunofluorescence of blood samples from multiple cancer types. The cytotoxicity of natural killer (NK) cells was assessed by the reduction in tumor cell numbers measured using the xCELLigence system. In vitro and ex vivo co-culture systems were established by using platelets and NK cells isolated from the peripheral blood of patients for direct or indirect co-culturing with cell lines and patient-derived organoids (PDOs). The effects of platelets and NK cells on metastasis and immune responses were investigated in vivo by utilizing mouse models. Results: Clinically, CTC-platelet aggregates were associated with significantly shorter progression-free survival and overall survival in hepatocellular carcinoma patients. In vitro, ex vivo, and in vivo assays demonstrated direct platelet adhesions gifted cancer cells with an evasive ability from NK cell killing by upregulating inhibitory checkpoint CD155, therefore facilitating distant metastasis. Mechanistically, CD155 was transcriptionally regulated by the FAK/JNK/c-Jun cascade in a platelet contact-dependent manner. Further competition assays and cytotoxicity experiments revealed that CD155 on CTCs inhibited NK cell cytotoxicity only by engaging with immune receptor TIGIT, but not CD96 and DNAM1, another two receptors for CD155. Interrupting the CD155-TIGIT interactions with a TIGIT antibody restored NK cell immunosurveillance on CTCs and markedly attenuated tumor metastasis. Conclusions: Our results demonstrated CTC evasion from NK cell-mediated innate immunosurveillance mainly via immune checkpoint CD155-TIGIT, potentially offering an immunotherapeutic strategy for eradicating CTCs.

Platelet-mediated circulating tumor cell evasion from natural killer cell killing via immune checkpoint CD155-TIGIT.

Circulating tumor cells (CTCs) are precursors of cancer metastasis. However, how CTCs evade immunosurveillance during hematogenous dissemination remains unclear. We identified CTC-platelet adhesions by single-cell RNA sequencing and multiplex immunofluorescence of blood samples from multiple cancer types. Clinically, CTC-platelet aggregates were associated with significantly shorter progression-free survival and overall survival in hepatocellular carcinoma patients. In vitro, ex vivo, and in vivo assays demonstrated direct platelet adhesions gifted cancer cells with an evasive ability from natural killer (NK) cell killing by upregulating inhibitory checkpoint CD155, therefore facilitating distant metastasis. Mechanistically, CD155 was transcriptionally regulated by the FAK/JNK/c-Jun cascade in a platelet contact-dependent manner. Further competition assays and cytotoxicity experiments revealed that CD155 on CTCs inhibited NK cell cytotoxicity only by engaging with immune receptor TIGIT, but not CD96 and DNAM1, another two receptors for CD155. Interrupting the CD155-TIGIT interactions with a TIGIT antibody restored NK cell immunosurveillance on CTCs and markedly attenuated tumor metastasis. Our results demonstrated CTC evasion from NK cell-mediated innate immunosurveillance mainly via immune checkpoint CD155-TIGIT, potentially offering an immunotherapeutic strategy for eradicating CTCs.

SETDB1 Suppresses Interferon Responses and NK Cell-Mediated Immunosurveillance Specifically in Monocytic AML

Acute myeloid leukemia (AML) with monocytic differentiation (monocytic AML) responds poorly to current treatments, including venetoclax-based therapy. To identify novel therapeutic targets in monocytic AML, we conducted in vivo and in vitro CRISPR/Cas9 library screens using a pooled sgRNA library targeting epigenetic regulators with a mouse monocytic AML model driven by SETBP1 and ASXL1 mutations (cSAM: combined expression of SETBP1 and ASXL1 Mutations). We ranked all the epigenetic genes according to the in vivo specific essentiality, and selected several candidates which were more depleted in mice rather than in cell cultures. The top-ranked gene was Setdb1, which encodes a histone H3 lysine 9 (H3K9) methyltransferase. In addition, several H3K9 trimethylation (H3K9me3) regulators, ATF7IP and TRIM33, were also more important in vivo than in vitro in our screens. ATF7IP is a cofactor of SETDB1 to enhance its enzymatic activity. TRIM33 is also involved in H3K9me3 modification. These data suggest the critical role of the SETDB1-ATF7IP-TRIM33 H3K9 methyltransferase complex in the development of AML in vivo. To validate the results of our screenings and to investigate the role of immune cells in our transplantation assay, we next transplanted control and Setdb1-knockout (KO) cSAM cells into three types of mice with different immune systems: the immunocompetent C57BL/6 mice, the immunodeficient NSG mice lacking T, B and natural killer (NK) cells, and the C57BL/6J- Rag2-/- mice lacking mature T and B cells but possessing NK cells. Although Setdb1 depletion did not inhibit the growth of cSAM cells in NSG mice, it showed the strong growth-inhibitory effect in C57BL/6 mice and Rag2-/- mice (Fig 1A). Since both C57BL/6 mice and Rag2-/- mice have NK cells, these data suggest that SETDB1 promotes leukemogenesis by suppressing NK cell-mediated cytotoxicity in vivo. Mechanistically, loss of Setdb1 induced upregulation of NKG2D ligands, interferon-stimulated genes (ISGs), and endogenous retrovirus (ERV) sequences through H3K9me3 reduction, thereby rendering cSAM cells more susceptible to NK cell-mediated cytotoxicity. Setdb1 depletion also induced myeloid maturation of cSAM cells towards macrophages and downregulation of major histocompatibility complex class I (MHC-I), which contributed to the increased susceptibility of Setdb1-depleted cSAM cells to NK cell-mediated cytotoxicity. Next, we investigated whether SETDB1 inactivation also sensitizes human AML cells to NK cells using the co-culture assay with an IL-2 dependent NK cell line NK-92. Loss of SETDB1 rendered monocytic AML cell lines, NOMO-1, THP-1, OCI-AML3, U937, and NB4, more susceptible to NK-92 cell-mediated cytotoxicity. SETDB1 depletion also induced overactivation of interferon signaling and intrinsic apoptosis in these monocytic AML cells. In contrast, non-monocytic AML cells, such as K562, TF-1, and Kasumi-1, were not sensitive to SETDB1 depletion. SETDB1 depletion neither increased the susceptibility to NK cell-mediated cytotoxicity nor induced apoptosis in non-monocytic AML cells. Subsequent analyses revealed that SETDB1 depletion reduced H3K9me3 at the monocyte-specific enhancer regions with ERV sequences to upregulate NK cell-activating ligands and ISGs. Because the “ERV enhancers” are active only in monocyte lineage, SETDB1 is essential specifically in monocytic AML. Finally, we identified MNDA and its mouse counterpart Ifi203 as biomarkers to predict the sensitivity of each AML to SETDB1 depletion. MNDA is one of the ISGs and is involved in myeloid cell differentiation. Human monocytic AML cells that are dependent on SETDB1 expressed higher MNDA at both RNA and protein levels compared to other subtypes of AML. We also found that Ifi203-high mouse AML cells transformed by MLL-AF9 were sensitive to Setdb1 depletion, whereas Setdb1 was dispensable in Ifi203-low mouse AML cells transformed by RUNX1-RUNX1T1. In summary, our findings reveal the critical and selective role of SETDB1 in monocytic AML. SETDB1 mediates H3K9me3 at the monocyte-specific ERV enhancers to repress the expression of NK cell-activating ligands and ISGs, thereby suppressing immunogenicity and apoptosis in monocytic AML (Fig 1B). SETDB1 as well as its binding partners could be promising therapeutic targets to treat monocytic AMLs that are relatively resistant to the current standard therapies.

Viral escape from NK cell-mediated immunosurveillance: A lesson for cancer immunotherapy?

Natural killer (NK) cells are innate lymphocytes that participate in immune responses against virus-infected cells and tumors. As a countermeasure, viruses and tumors employ strategies to evade from NK cell-mediated immunosurveillance. In this review, we examine immune evasion strategies employed by viruses, focusing on examples from human cytomegalovirus (HCMV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We explore selected viral evasion mechanisms categorized into three classes: (1) providing ligands for the inhibitory receptor NKG2A, (2) down-regulating ligands for the activating receptor NKG2D, and (3) inducing the immunosuppressive cytokine transforming growth factor (TGF)-β. For each class, we draw parallels between immune evasion by viruses and tumors, reviewing potential opportunities for overcoming evasion in cancer therapy. We suggest that in-depth investigations of host-pathogen interactions between viruses and NK cells will not only deepen our understanding of viral immune evasion but also shed light on how NK cells counter such evasion attempts. Thus, due to the parallels of immune evasion by viruses and tumors, we propose that insights gained from anti-viral NK cell responses may serve as valuable lessons that can be leveraged for designing future cancer immunotherapies. This article is protected by copyright. All rights reserved.

NK Cells in Chronic Lymphocytic Leukemia and Their Therapeutic Implications.

Key features of chronic lymphocytic leukemia (CLL) are defects in the immune system and the ability of leukemic cells to evade immune defenses and induce immunosuppression, resulting in increased susceptibility to infections and disease progression. Several immune effectors are impaired in CLL, including T and natural killer (NK) cells. The role of T cells in defense against CLL and in CLL progression and immunotherapy has been extensively studied. Less is known about the role of NK cells in this leukemia, and data on NK cell alterations in CLL are contrasting. Besides studies showing that NK cells have intrinsic defects in CLL, there is a large body of evidence indicating that NK cell dysfunctions in CLL mainly depend on the escape mechanisms employed by leukemic cells. In keeping, it has been shown that NK cell functions, including antibody-dependent cellular cytotoxicity (ADCC), can be retained and/or restored after adequate stimulation. Therefore, due to their preserved ADCC function and the reversibility of CLL-related dysfunctions, NK cells are an attractive source for novel immunotherapeutic strategies in this disease, including chimeric antigen receptor (CAR) therapy. Recently, satisfying clinical responses have been obtained in CLL patients using cord blood-derived CAR-NK cells, opening new possibilities for further exploring NK cells in the immunotherapy of CLL. However, notwithstanding the promising results of this clinical trial, more evidence is needed to fully understand whether and in which CLL cases NK cell-based immunotherapy may represent a valid, alternative/additional therapeutic option for this leukemia. In this review, we provide an overview of the current knowledge about phenotypic and functional alterations of NK cells in CLL and the mechanisms by which CLL cells circumvent NK cell-mediated immunosurveillance. Additionally, we discuss the potential relevance of using NK cells in CLL immunotherapy.

Prospects for NK Cell Therapy of Sarcoma.

Natural killer (NK) cells are innate lymphoid cells with potent antitumor activity. One of the most NK cell cytotoxicity-sensitive tumor types is sarcoma, an aggressive mesenchyme-derived neoplasm. While a combination of radical surgery and radio- and chemotherapy can successfully control local disease, patients with advanced sarcomas remain refractory to current treatment regimens, calling for novel therapeutic strategies. There is accumulating evidence for NK cell-mediated immunosurveillance of sarcoma cells during all stages of the disease, highlighting the potential of using NK cells as a therapeutic tool. However, sarcomas display multiple immunoevasion mechanisms that can suppress NK cell function leading to an uncontrolled tumor outgrowth. Here, we review the current evidence for NK cells' role in immune surveillance of sarcoma during disease initiation, promotion, progression, and metastasis, as well as the molecular mechanisms behind sarcoma-mediated NK cell suppression. Further, we apply this basic understanding of NK-sarcoma crosstalk in order to identify and summarize the most promising candidates for NK cell-based sarcoma immunotherapy.

The Oncoprotein SKI Acts as A Suppressor of NK Cell-Mediated Immunosurveillance in PDAC.

Simple SummaryPancreatic ductal adeno carcinoma is one of the most lethal solid tumors and the survival rate has not improved significantly over the past decades. The disease is characterized by an immune-suppressive tumor microenvironment, which promotes the limited response to novel immunotherapies. The aim of our study was to contribute to a better understanding of the diminished Natural Killer (NK) cell-activity in pancreatic cancer. We showed that oncoprotein SKI, which is involved in CBP/p300-mediated acetylation, diminished the expression of activating ligands for the cytotoxicity receptor NKG2D on tumor cells, thereby counteracting NK cell-dependent cytotoxicity. Treatment of tumor cells with histone deacetylase inhibitors (HDACi) induced the expression of these ligands and improved NK cell-dependent killing. Thus, we unraveled a so far unknown role of SKI in NK cell-mediated immunosurveillance. Our results suggest that the combination of HDACi with NK cell-based immunotherapies may be beneficial for pancreatic cancer patients.Drugs targeting epigenetic mechanisms such as histone deacetylase inhibitors (HDACi) suppress tumor growth. HDACi also induce the expression of ligands for the cytotoxicity receptor NKG2D rendering tumors more susceptible to natural killer (NK) cell-dependent killing. The major acetylases responsible for the expression of NKG2D ligands (NKG2D-L) are CBP and p300. The role of the oncogene and transcriptional repressor SKI, an essential part of an HDAC-recruiting co-repressor complex, which competes with CBP/p300 for binding to SMAD3 in TGFβ signaling, is unknown. Here we show that the siRNA-mediated downregulation of SKI in the pancreatic cancer cell lines Panc-1 and Patu8988t leads to an increased target cell killing by primary NK cells. However, the higher cytotoxicity of NK cells did not correlate with the induction of NKG2D-L. Of note, the expression of NKG2D-L and consequently NK cell-dependent killing could be induced upon LBH589 (LBH, panobinostat) or valproic acid (VPA) treatment irrespective of the SKI expression level but was significantly higher in pancreatic cancer cells upon genetic ablation of SKI. These data suggest that SKI represses the inducible expression of NKG2D-L. The combination of HDACi with NK cell-based immunotherapy is an attractive treatment option for pancreatic tumors, specifically for patients with high SKI protein levels.

Lactate dehydrogenase-elevating virus enhances natural killer cell-mediated immunosurveillance of mouse mesothelioma development

BackgroundViral infections can reduce early cancer development through enhancement of cancer immunosurveillance. This study was performed to analyse this effect of viral infection in a mouse model of solid tumor.MethodsThe experimental model used was the effect of BALB/c mouse infection by lactate dehydrogenase-elevating virus on AB1 mesothelioma cancer development.ResultsAcute infection with lactate dehydrogenase-elevating virus strongly reduced in vivo early AB1 mesothelioma growth and death resulting from cancer development. This effect was not due to a direct cytolytic effect of the virus on AB1 cells, but to an in vivo activation of natural killer cells. Gamma-interferon production rather than cytotoxic activity against AB1 cells mediated this protective effect. This gamma-interferon production by natural killer cells was dependent on interleukin-12 production.ConclusionsTogether with other reported effects of infectious agents on cancer development, this observation may support the hypothesis that enhancement of innate immunosurveillance against tumors may result from infection with common infectious agents through modulation of the host immune microenvironment.

MicroRNA-130a enhances the killing ability of natural killer cells against non-small cell lung cancer cells by targeting signal transducers and activators of transcription 3

Non-small cell lung cancer (NSCLC) is a serious threat for human health and life. Natural killer (NK) cell-based immunotherapy is a promising anti-tumor strategy in various cancers including NSCLC. Emerging microRNA (miRNA) has been identified as vital regulators in NK cell-mediated immunosurveillance process. MicroRNA-130a (miR-130a) level and signal transducers and activators of transcription 3 (STAT3) mRNA level was measured by RT-qPCR assay. STAT3 protein level was determined by western blot assay. IFN-γ and TNF-α secretion was examined by corresponding ELISA kits. NK cell cytotoxicity was assessed by lactate dehydrogenase (LDH) assay. The interaction between miR-130a and STAT3 was explored by bioinformatics analysis, luciferase reporter assay and RNA immunoprecipitation (RIP) assay. We found that MiR-130a level was notably reduced and STAT3 expression was dramatically increased in primary NK cells isolated from NSCLC patients. But, miR-130a was highly expressed and STAT3 was low expressed in IL-2-activated NK-92 cells. Functional analysis revealed that miR-130a overexpression potentiated killing ability of NK cells against A549 cells. Further investigations unveiled that STAT3 was a target of miR-130a and STAT3 overexpression abrogated miR-130a-induced improvement in killing activity of NK cells against NSCLC cells. In conclusion, MiR-130a improved the killing capacity of NK cells against NSCLC cells by targeting STAT3, laying a foundation for future studies on the roles and molecular basis of miR-130a in NK cell-based immunotherapy against various cancers.

Hypersialylation Protects Multiple Myeloma Cells from NK Cell-Mediated Immunosurveillance and This Can be Overcome By Targeted Desialylation Using a Sialyltransferase Inhibitor

Introduction: Evading Natural Killer (NK) cell-mediated immunosurveillance is key to the development of Multiple Myeloma (MM). Recent attention has focused on the role of hypersialylation in facilitating immune-evasion of NK cells. Abnormal cell surface sialylation is considered a hallmark of cancer and we have implicated hypersialylation in MM disease progression. Certain sialylated glycans can act as ligands for the sialic acid-binding immunoglobulin-like lectin (Siglec) receptors expressed by NK cells (Siglec-7 and Siglec-9). These ITIM motif-containing inhibitory receptors transmit an inhibitory signal upon sialic acid engagement. We hypothesized that desialylation of MM cells or targeted interruption of Siglec expression could lead to enhanced NK cell mediated cytotoxicity of MM cells. Methodology: MM cells were treated with the sialidase neuraminidase prior to co-culture with primary NK (PNK) cells. MM cells were treated with 300µM 3Fax-Neu5Ac (sialyltransferase inhibitor) for 3 days prior to co-cultures with PNK cells. PNK cells were expanded, IL-2 activated (500U/ml) overnight, or naïve (resting). Primary MM samples/MM cell lines were screened with Siglec-7/9 chimeras (10µg/ml). PNK (IL-2 activated) cells were stained with anti-Siglec-7 and anti-Siglec-9 antibodies. Siglec-7 was targeted for knockout (KO) using the CRISPR/Cas9 system, a pre-designed guideRNA and the MaxCyteGT transfection system. MM cells were treated with 10µg/ml of Daratumumab prior to co-culture with expanded PNK cells. Results: Using recombinant Siglec-7/9 chimeras a panel of MM cell lines (MM1S, RPMI-8226, H929, JJN3 and U266) were shown to express ligands for Siglec-7 and Siglec-9 (>85%, n=3). Primary MM cells isolated from BM of newly diagnosed (n=3) and relapsed patients (n=2) were also shown to express Siglec-7 ligands (72.5±17.5%, 36.5% respectively). PNK cells express Siglec-7 and Siglec-9 (94.3±3.3% and 61±8.8% respectively, n=6). Desialylation of the MM cell lines JJN3 and H929 using neuraminidase significantly enhanced killing of MM cells by healthy donor (HD) derived PNK cells (expanded, IL-2 activated and naïve, n=7) at multiple effector:target (E:T) cell ratios. Furthermore, de-sialylation of JJN3 and H929 using neuraminidase resulted in increased NK cell degranulation (CD107α expression), compared to a glycobuffer control (n=7). De-sialylation, using 300µM 3Fax-Neu5Ac, resulted in strongly enhanced killing of MM1S by expanded HD-derived PNK cells at multiple E:T ratios (n=5, p<0.01 at 0.5:1, p<0.001 at 1:1, p<0.01 at 2.5:1). Furthermore, CD38 expression on H929 MM cells significantly increased after treatment with 300µM 3Fax-Neu5Ac for 3 days (p<0.01, n=3). In a cytotoxicity assay, expanded PNK cell-mediated antibody dependent cellular cytotoxicity (ADCC) of H929 MM cells pre-treated with Daratumumab (anti-CD38 moAb) and 3Fax-Neu5Ac was significantly higher than H929 cells pre-treated with Dara (p<0.05 at 0.5:1, p<0.01 at 1:1) or 3Fax-Neu5Ac (p<0.01 at 0.5:1, p<0.01 at 1:1) alone (n=5). Using CRISPR/Cas9, over 50% complete KO of Siglec-7 was observed on expanded PNK cells, yet did not result in enhanced NK cell-mediated cytotoxicity against either H929 or JJN3 (n=7). Siglec-9 KO using CRISPR/Cas9 is ongoing. Discussion: Hypersialylation of MM cells facilitates immune evasion and targeted removal of sialic acid strongly enhances the cytotoxicity of NK cells against MM. However, to date the role of Siglecs remains inconclusive. Nevertheless, our data suggest that targeted desialylation is a novel therapeutic strategy worth exploring in MM. In particular, upregulation of CD38 provides a strong rationale for combinatory strategies employing targeted desialylation with CD38 moAbs such as Daratumumab, with the goal of maximizing ADCC. Disclosures Sarkar: Onkimmune: Research Funding. O'Dwyer:Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; GlycoMimetics Inc: Research Funding; AbbVie: Consultancy.

"Acquired" NKG2D Ligand Stimulates NK Cell-mediated Tumor Immunosurveillance.

Engagement of activating receptor NKG2D to its ligand mediates natural killer (NK) cell activation and enhances cytotoxicity. NKG2D ligands (NKG2DLs) are frequently expressed on the tumor cell surface. However, the expression patterns of different NKG2DLs vary between tumor cells. Downregulation of certain ligand enables the tumor cells to escape NK cell-mediated immunosurveillance. By generating tumor cell lines with high expression of NKG2D ligand MULT1, we aimed to explore the function of NKG2DLs diversity on the activation and regulation of NKG2D signaling pathway. NK cells were potently activated by the "acquired" MULT1 expression on MOVCAR 5009 cells. Further, the progression of the tumor was significantly inhibited in mice inoculated with MULT1-expressing MOVCAR 5009 cells. Also, the pulmonary metastasis of MULT1-expressing B16-F0 cells was also significantly reduced in vivo. Our results implied that "acquired" NKG2D ligands enhance antitumor responses of NK cells, providing insights for designing novel therapeutic strategies and drugs to enhance NK cell surveillance over malignances.

Assessment of NK cell-mediated cytotoxicity by flow cytometry after rapid, high-yield isolation from peripheral blood.

Natural killer (NK) cells constitute the predominant innate lymphocyte subset that mediates the anti-viral and anti-tumor immune responses. NK cells use an array of innate receptors to sense their environment and to respond to infections, cellular stress and transformation. The resulting NK cell activation, including cytotoxicity and cytokine production, is a fundamental component of the early immune response. The most recent discoveries in NK cell biology have stimulated the translational research that has led to remarkable results for the treatment of human malignancies. Therefore, the rapid isolation of NK cells from the peripheral blood or tumor microenvironment and the subsequent assessment of cytolytic function are crucial to the study of their potency and NK cell-mediated immunosurveillance. Here, we provide protocols for NK cell isolation and the assessment of NK cell cytotoxicity using flow cytometry.

Sugar Free: Novel Immunotherapeutic Approaches Targeting Siglecs and Sialic Acids to Enhance Natural Killer Cell Cytotoxicity Against Cancer.

Natural Killer (NK) cells are cytotoxic lymphocytes that play a key role in the immune system, targeting and destroying invading pathogens and malignantly transformed cells. Evading NK cell-mediated immunosurveillance is therefore critical to facilitating cancer cell survival and metastasis. Signals from a range of inhibitory and activating receptors located on the NK cell surface regulate NK cell cytotoxicity. Recently, attention has turned to the role of hypersialylated tumor cell surfaces in mediating immune-evasion of NK cells. Two inhibitory sialic acid-binding immunoglobulin-like lectin (Siglec) receptors are expressed by NK cells: Siglec-7 and Siglec-9. The abundance of sialic acids on tumor cell surface is hypothesized to regulate NK cell-mediated cytotoxicity by interacting with Siglec-7 and Siglec-9, causing a dampening of NK cell activation pathways. Targeting Siglec-7 and Siglec-9, or the sialic acid coated tumor cell surface is therefore being investigated as a novel therapeutic approach to enhance the NK cell response against cancer. In this review we report on the currently published documentation of the role for Siglec-7 and Siglec-9 receptors on NK cells and their ligands expressed by tumor cells. We also discuss the strategies currently explored to target Siglec-7, Siglec-9 and the sialylated tumor cell surface as well as the impact abrogation of these interactions have on NK cell cytotoxicity against several cancer types.

NK Cell-Based Immunotherapy in Cancer Metastasis.

Metastasis represents the leading cause of cancer-related death mainly owing to the limited efficacy of current anticancer therapies on advanced malignancies. Although immunotherapy is rendering promising results in the treatment of cancer, many adverse events and factors hampering therapeutic efficacy, especially in solid tumors and metastases, still need to be solved. Moreover, immunotherapeutic strategies have mainly focused on modulating the activity of T cells, while Natural Killer (NK) cells have only recently been taken into consideration. NK cells represent an attractive target for cancer immunotherapy owing to their innate capacity to eliminate malignant tumors in a non-Major Histocompatibility Complex (MHC) and non-tumor antigen-restricted manner. In this review, we analyze the mechanisms and efficacy of NK cells in the control of metastasis and we detail the immunosubversive strategies developed by metastatic cells to evade NK cell-mediated immunosurveillance. We also share current and cutting-edge clinical approaches aimed at unleashing the full anti-metastatic potential of NK cells, including the adoptive transfer of NK cells, boosting of NK cell activity, redirecting NK cell activity against metastatic cells and the release of evasion mechanisms dampening NK cell immunosurveillance.

Mouse Models to Study Natural Killer Cell-Mediated Immunosurveillance and Metastatic Latency.

Metastatic latency is a major concern in the clinic, yet how these disseminated cancer cells survive and initiate metastases is unknown (Massagué and Obenauf, Nature 529:298-306, 2016). Here, we describe an approach to isolate latency competent cancer (LCC) cells from early stage human lung and breast carcinoma cell lines using mouse xenograft models (Malladi, Cell 165:45-60, 2016). Cancer cell lines labeled with GFP-luciferase and antibiotic selection markers were injected intracardially into athymic mice. Three months, post-injection, LCC cells were identified in situ and isolated. Upon reinjection, LCC cells retain their tumorigenic potential, enter a slow-cycling or quiescent state, and evade NK cell-mediated innate immune surveillance.

NKG2D Immunoligand rG7S-MICA Enhances NK Cell-mediated Immunosurveillance in Colorectal Carcinoma.

Colorectal carcinoma (CRC) is one of the most common malignant cancers worldwide. The poor response of CRC to chemotherapy has whipped up the interest in targeted therapy with monoclonal antibodies for its potential efficiency. However, cetuximab, as one of the first-line targeted drugs in the treatment of CRC, has drug resistance and poor prognosis in clinic. To address this, a novel bispecific protein with CRC targeting and natural killer (NK) cell triggering was used for treatment. NK cell-mediated immunosurveillance is normally activated by the activating receptor natural killer cell receptor NK group 2, member D (NKG2D), which binds its key ligand major histocompatibility complex (MHC) class I-related chain A (MICA) expressed on the tumor cells. To trigger NK cell-mediated cytotoxicity, we fused MICA portion to a single-chain antibody fragment rG7S targeting the tumor-associated antigen CD24. In vitro, flow cytometry, cytotoxicity assay, degranulation, and cytokines release assay revealed that the fusion protein rG7S-MICA could both binds to CD24 and NKG2D which enhances NK cell sensitivity and NKG2D-mediated immunosurveillance against CD24 CRC cells. Furthermore, in a CD24 CRC-bearing nude mice model, rG7S-MICA effectively recruits NK cell to the tumor site and increase the release of cytokines such as interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), and shows potential antitumor effects. In conclusion, rG7S-MICA provides a novel immunotherapeutic strategy for CRC, which could be further developed against other CD24 malignancies.

An overview of the potential strategies for NK cell-based immunotherapy for acute myeloid leukemia.

Patients with acute myeloid leukemia (AML) have relatively low survival rates compared to patients with other pediatric cancers. Relapse is frequent with conventional treatment and is a major cause of morbidity and mortality. Natural killer (NK) cells offer an alternative approach to chemotherapy that combats relapse by substantially eradicating AML blasts. New methods for enhancing NK cell activation and expression of the activating ligand on target malignant cells will increase the likelihood of success with this approach. We review these latest discoveries in NK cell-based therapy for AML and delineate recent advances in sensitizing AML cells to NK cell-mediated immunosurveillance.

Immunosenescence: limitations of natural killer cell-based cancer immunotherapy.

Cancer is primarily considered a disease of old age. Immunosenescence refers to the age-associated changes in the immune system, and its contribution to the increased risk of cancer in old individuals has been discussed for many years. Natural killer (NK) cells are cytotoxic innate immune cells specialized in defence against tumour and virus-infected cells. NK cell cytotoxicity is the result of a fine balance between activating and inhibitory receptors. Several activating receptors have been identified that recognize different ligands frequently found over-expressed on tumour cells or virus-infected cells. The most important NK cell inhibitory receptors interact with major histocompatibility complex class I molecules expressed on almost all nucleated cells preventing NK cell-mediated lysis of healthy cells. NK cell immunosenescence is characterized by a redistribution of NK cell subsets, a diminished expression of several activating receptors and lower per-cell cytotoxicity. Altered expression of activating receptors has also been described in young and elderly cancer patients probably due to chronic exposure to ligands on tumour cells. Thus, the effect of both age and cancer may act synergistically to diminish NK cell-mediated tumour immunosurveillance. Different strategies harnessing the power of NK cells to target tumour cells have been designed including adoptive therapy with autologous or allogeneic expanded NK cells. In addition, checkpoint blockade of inhibitory receptors and the use of agonist antibodies to stimulate activating receptors are emerging areas of research. In this context, the effect of immunosenescence should be considered to improve the efficiency of cancer immunotherapy.

Linking cancer-induced Nlrp3 inflammasome activation to efficient NK cell-mediated immunosurveillance

ABSTRACTWe have recently demonstrated that the Nlrp3 inflammasome can detect the presence of colorectal cancer (CRC) metastatic growth in the liver and limit its growth. Inflammasome signaling primed natural killer (NK) cells through Interleukin (IL)-18 and activated their ability to trigger FasL-induced apoptosis of the tumor.

Low expression of CD112 is associated with poor overall survival in patients with hepatocellular carcinoma.

CD112 as an important ligand of CD226 can stimulate the natural killer (NK) cell-mediated target cell lysis. Previous studies have reported that CD112 is involved in cancer initiation and progression. However, its expression and clinical significance in hepatocellular carcinoma (HCC) have never been investigated. In this study, we used immunohistochemistry to examine CD112 expression in cancer and pericancer tissues from 159 HCC cases. Western blot and immunofluorescence were used to detect CD112 expression in HCC cell lines. χ(2) Test was used to assess the association of CD112 expression with clinicopathological characteristics, whereas Kaplan-Meier survival function and Cox proportional hazards regression model were used to explore the association between CD112 expression and clinical outcome of patients with HCC. Overall, CD112 expression was significantly reduced in HCC tissues when compared with adjacent pericancer liver tissues (P < .001). Western blot and immunofluorescence analyses showed that most HCC cell lines had low CD112 expression level. Furthermore, low CD112 expression was significantly associated with high serum α-fetoprotein level (P = .004) in patients with HCC. Kaplan-Meier analysis showed that patients with low CD112 expression had poorer postsurgery overall survival than those with high CD112 expression (log-rank P = .045). In conclusion, our findings demonstrate that the down-regulation of CD112 may be an important mechanism through which HCC cells evade the natural killer cell-mediated immunosurveillance, and thus, CD112 may be a useful biomarker to assess the immunologic niche of HCC.