Enhanced Natural Killer Cell Cytotoxicity Research Articles

Combination Effect of Radiotherapy and Targeted Therapy with NK Cell-Based Immunotherapy in head and Neck Squamous Cell Carcinoma

ABSTRACT Background Head and neck squamous cell carcinoma (HNSCC) has a poor prognosis, and current treatments are limited by high toxicity and low survival rates, highlighting the need for new therapeutic approaches. Natural killer (NK) cells can identify and eliminate cancer cells without prior antigen exposure. Radiotherapy directly targets tumors and increases activating ligands on tumor cells, promoting NK cell interactions. Cetuximab, an EGFR-targeting antibody, enhances NK cell cytotoxicity. Additionally, anti-PD-1 antibodies may further boost NK cell function by blocking inhibitory signals. The study aimed to enhance HNSCC treatment efficacy by combining radiotherapy and targeted therapy with expanded NK cells. Methods NK cells were isolated, activated, and expanded from healthy donors. The FaDu and SCC-47 cell lines were inoculated into NOD/SCID mice. The mice were treated with PD-1 inhibitors, cetuximab, and radiation, followed by intravenous injection of NK cells. Results Radiation increased ligands that regulate NK cell sensitivity. The combination of cetuximab, radiotherapy, and expanded NK cells significantly suppressed cancer progression and improved survival rates. However, adding anti-PD-1 antibodies did not further enhance outcomes. Conclusion This study suggests that a multimodal approach combining cetuximab, radiotherapy, and NK cells can significantly improve HNSCC therapy efficacy, offering a novel and promising treatment strategy.

A PSMA-targeted Tri-specific Killer Engager enhances NK cell cytotoxicity against prostate cancer.

Natural killer (NK) cell tumor infiltration is associated with good prognosis in patients with metastatic castration-resistant prostate cancer (mCRPC). NK cells recognize and kill targets by a process called natural cytotoxicity. We hypothesized that promoting an antigen-specific synapse with co-activation may enhance NK cell function in mCRPC. We describe a Tri-specific Killer Engager (TriKE) construct that engages with the activating receptor CD16 on NK cells, prostate-specific membrane antigen (PSMA) on mCRPC cells, and has an interleukin (IL)-15 moiety that is essential for NK cell survival, proliferation, and priming. We show that the PSMA TriKE specifically binds to PSMA-expressing cells and significantly enhances expansion, degranulation and cytokine production of NK cells derived from healthy donors or prostate cancer patients. Bystander killing of PSMA-negative was also achieved with PSMA TriKE treatment when co-cultured with PSMA-positive cells, suggesting potential PSMA TriKE benefit in controlling tumor antigen escape. When tested under physiologic conditions recapitulating the mCRPC tumor microenvironment (TME), NK cells treated with PSMA TriKE and prolonged exposure to hypoxia or MDSCs maintained their potent function while IL-15 treated NK cells showed greatly impaired cytotoxicity. Finally, in vivo testing of PSMA TriKE showed improved tumor control and survival of mice as compared to IL-15 and untreated control groups. In conclusion, PSMA TriKE demonstrates potential as a new therapy for advanced prostate cancer by providing additional signals to NK cells to maximize their anti-tumor potential in prostate cancer, especially in the setting of a hostile TME.

Homeostatic self-MHC-I recognition regulates anti-metastatic function of mature lung natural killer cells

Natural killer (NK) cells are important innate immune effector cells for controlling tumor growth and metastasis. Differentiated mature NK cells preferentially reside in the peripheral tissues and express higher levels of self-major histocompatibility complex class I (MHC-I)-recognizing inhibitory receptors. MHC-I recognition by NK cells are known to be important for their development and maturation processes, however, the role of homeostatic MHC-I recognition in maintaining effector functions of mature NK cells in the peripheral tissues needs to be elucidated. In this study, we utilized a pan anti-MHC-I blocking monoclonal antibody (anti-MHC-I) to examine the role of homeostatic MHC-I recognition in the response of pulmonary mature NK cells in an experimental lung metastasis model of B16F10 melanoma. Anti-MHC-I treatment showed significant inhibition of the lung metastasis of B16F10 melanoma in NK cell- and IFN-γ-dependent mechanisms. The blockade of homeostatic MHC-I recognition increased mature lung NK cell responsiveness, such as direct cytotoxicity and IFN-γ production, rather than the number of lung NK cells. Mechanistically, the gene expression of activating receptors including DNAX accessory molecule-1 (DNAM-1) was upregulated in NK cells treated with anti-MHC-I, and further the enhanced NK cell cytotoxicity against B16F10 cells was DNAM-1-dependent. Collectively, homeostatic self-MHC-I recognition regulates anti-metastatic function of mature lung NK cells by restraining the expression of activating receptors.

Metabolic programs drive function of therapeutic NK cells in hypoxic tumor environments.

Limited oxygen (hypoxia) in solid tumors poses a challenge to successful immunotherapy with natural killer (NK) cells. NK cells have impaired cytotoxicity when cultured in hypoxia (1% oxygen) but not physiologic (>5%) or atmospheric oxygen (20%). We found that changes to cytotoxicity were regulated at the transcriptional level and accompanied by metabolic dysregulation. Dosing with interleukin-15 (IL-15) enhanced NK cell cytotoxicity in hypoxia, but preactivation with feeder cells bearing IL-21 and 4-1BBL was even better. Preactivation resulted in less perturbed metabolism in hypoxia; greater resistance to oxidative stress; and no hypoxia-induced loss of transcription factors (T-bet and Eomes), activating receptors, adhesion molecules (CD2), and cytotoxic proteins (TRAIL and FasL). There remained a deficit in CD122/IL-2Rβ when exposed to hypoxia, which affected IL-15 signaling. However, tri-specific killer engager molecules that deliver IL-15 in the context of anti-CD16/FcγRIII were able to bypass this deficit, enhancing cytotoxicity of both fresh and preactivated NK cells in hypoxia.

INTASYL self-delivering RNAi decreases TIGIT expression, enhancing NK cell cytotoxicity: a potential application to increase the efficacy of NK adoptive cell therapy against cancer

Natural killer (NK) cells are frontline defenders against cancer and are capable of recognizing and eliminating tumor cells without prior sensitization or antigen presentation. Due to their unique HLA mismatch tolerance, they are ideal for adoptive cell therapy (ACT) because of their ability to minimize graft-versus-host-disease risk. The therapeutic efficacy of NK cells is limited in part by inhibitory immune checkpoint receptors, which are upregulated upon interaction with cancer cells and the tumor microenvironment. Overexpression of inhibitory receptors reduces NK cell-mediated cytotoxicity by impairing the ability of NK cells to secrete effector cytokines and cytotoxic granules. T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), a well-known checkpoint receptor involved in T-cell exhaustion, has recently been implicated in the exhaustion of NK cells. Overcoming TIGIT-mediated inhibition of NK cells may allow for a more potent antitumor response following ACT. Here, we describe a novel approach to TIGIT inhibition using self-delivering RNAi compounds (INTASYL™) that incorporates the features of RNAi and antisense technology. INTASYL compounds demonstrate potent activity and stability, are rapidly and efficiently taken up by cells, and can be easily incorporated into cell product manufacturing. INTASYL PH-804, which targets TIGIT, suppresses TIGIT mRNA and protein expression in NK cells, resulting in increased cytotoxic capacity and enhanced tumor cell killing in vitro. Delivering PH-804 to NK cells before ACT has emerged as a promising strategy to counter TIGIT inhibition, thereby improving the antitumor response. This approach offers the potential for more potent off-the-shelf products for adoptive cell therapy, particularly for hematological malignancies.

Paulownin elicits anti-tumor effects by enhancing NK cell cytotoxicity through JNK pathway activation.

Paulownin, a natural compound derived from Paulownia tomentosa wood, exhibits various physiological functions, including anti-bacterial and anti-fungal effects. However, the impact of paulownin on natural killer (NK) cell immune activity remains largely unknown. In this study, we investigated the effect of paulownin on NK cell activity both in vitro and in vivo, and explored its potential mechanisms. NK-92 cells were used for in vitro experiments and a BALB/c mouse model with B16F10 cells injected subcutaneously were used for in vivo anti-tumor analysis. We found that paulownin enhanced the cytolytic activity of NK-92 cells against leukemia, human colon, and human lung cancer cell lines. Paulownin treatment increased the expression of the degranulation marker protein CD107a and cytolytic granules, including granzyme B and perforin in NK-92 cells. Moreover, these enhancements of cytotoxicity and the expression of cytolytic granules induced by paulownin were also observed in human primary NK cells. Signaling studies showed that paulownin promoted the phosphorylation of JNK. The increased perforin expression and elevated cytotoxic activity induced by paulownin were effectively inhibited by pre-treatment with a JNK inhibitor. In vivo studies demonstrated that the administration of paulownin suppressed the growth of B16F10 melanoma cells allografted into mice. Paulownin administration promoted the activation of NK cells in the spleen of mice, resulting in enhanced cytotoxicity against YAC-1 cells. Moreover, the anti-tumor effects of paulownin were reduced upon the depletion of NK cells. Therefore, these results suggest that paulownin enhances NK cell cytotoxicity by activating the JNK signaling pathway and provide significant implications for developing new strategies for cancer immunotherapy.

NK cell line modified to express a potent, DR5 specific variant of TRAIL, show enhanced cytotoxicity in ovarian cancer models

ObjectiveOvarian cancer is a lethal gynaecological malignancy with unsatisfactory 5 year survival rates of 30–50 %. Cell immunotherapy is a promising new cancer treatment where immune cells, such as Natural Killer (NK) cells, are administered to enable the patient to fight cancer through direct cytotoxicity. NK cells orchestrate an adaptive immune response by enabling the release of tumour antigens. NK cell cytotoxicity and effector responses are largely driven by TRAIL engagement. In this study we investigated the cytotoxic potential of a human NK cell line that were modified to express a potent DR5 specific TRAIL variant. We hypothesised that this modification would enhance NK cell cytotoxicity against TRAIL sensitive and resistant ovarian cancer cell lines in vitro. MethodsKHYG-1 human NK cells were modified with a TRAIL variant targeting DR5 (TRAILv-KHYG-1). Human ovarian cancer cell lines, OVCAR-3 and SKOV-3, were cultured with modified or non-modified NK cells at different effector:target (E:T) ratios for 4 or 16 h. Apoptosis was assessed by Annexin-APC and 7-AAD and measured using flow cytometry. Apoptotic cells were defined as annexin V 7-AAD double positive. Cytokine expression was measured by multiplex ELISA, and analysed by flow cytometry. ResultsModified and non-modified NK cells significantly reduced OVCAR-3 cell viability as compared to OVCAR-3 cells that were cultured alone after 4 and 16 h treatment. OVCAR-3 cell viability was reduced after treatment with 1:1 E:T ratio with TRAILv-KHYG-1 cells after 16 h. On the contrary, neither NK cell line had any effect of SKOV-3 cell viability despite SKOV-3 cells having more DR5 surface expression compared to OVCAR-3 cells. ConclusionsTRAILv-KHYG-1 cells significantly reduced OVCAR-3 cell viability as compared to non-modified NK cells. However, no significant reduction in viability was observed when SKOV-3 cell were cultured with either NK cells, despite having more DR5 surface expression compared to OVCAR-3 cells. These data indicate that mechanisms other than DR5 expression drive TRAIL resistance in ovarian cancer.

A 5-ALA mediated photodynamic therapy increases natural killer cytotoxicity against oral squamous cell carcinoma cell lines.

Oral squamous cell carcinoma (OSCC) constitutes over 90% of oral cancers, known for its aggressiveness and poor prognosis. Photodynamic therapy (PDT) has emerged as a promising adjuvant therapy and is linked to immunogenic cell death, activating innate and adaptive anti-tumor responses. Natural Killer (NK) cells, key players in malignant cell elimination, have not been extensively studied in PDT. This study evaluates whether PDT increases OSCC cell lines' susceptibility to NK cell cytotoxicity. PDT, using 5-aminolevulinic acid (5-ALA) and LED irradiation, was applied to Ca1 and Luc4 cell lines. Results showed a dose-dependent viability decrease post-PDT. Gene expression analysis revealed upregulation of NK cell-activating ligands (ULBP1-4, MICA/B) and decreased MHC class I expression in Ca1, suggesting increased NK cell susceptibility. Enhanced NK cell cytotoxicity was confirmed in Ca1 but not in Luc4 cells. These findings indicate that PDT may enhance NK cell-mediated cytotoxicity in OSCC, offering potential for improved treatment strategies.

Abstract 2717: Bifunctional fusion protein PDL1sFv/MICAe for cancer immunotherapy

Abstract Many cancer cells upregulate the expression of programmed death ligand 1 (PD-L1) to surpass T-cell mediated immune response. Immune checkpoint blockers targeting PD-L1 or its receptor PD1 have been shown to suppress the inhibitory signals and enhance T cell function. As a new immunotherapy, immune checkpoint blockers have been used to treat multiple cancers. However, most clinically available blockers only engage with one type of immune cell: the T cells. Natural killer (NK) cells, on the other hand, play a significant role in anti-cancer immune responses. It will represent an advancement in anti-cancer immunotherapy if NK cells are engaged with immune checkpoint blockers. In this study, we designed a unique bifunctional fusion gene, PDL1sFv/MICAe, consisting of the variable regions of an anti-PD-L1 antibody and the extracellular domain of stress protein MICA and hypothesized that fusion protein PDL1sFv/MICAe will simultaneously bind to PD-L1 on tumor cells to block the inhibitory signal and extend T cells activity, and to NKG2D to activate NK Cells. cDNA sequence of fusion gene PDL1sFv/MICAe was designed based on the variable region of an established clinically used antibody, Atezolizumab, and the extracellular domain of MICA with linkers in between. After a computational confirmation of its structure and binding affinities, the fusion gene was synthesized and cloned into an expressing vector, and the fusion protein was produced for evaluation. Western blotting and fluorescent microscopic analyses confirmed that the protein binds to PD-L1 of human tumor cells, including MCF7 and MDAMB231, and NKG2D of KHYG1 cells. Preliminary results also show that the fusion protein enhances NK cell cytotoxicity against tumor cells. Studies on confirming the fusion protein’s activities on other tumor cells, NK cell IFN-γ production, T cell function, etc., are underway. In summary, fusion protein PDL1sFv/MICAe represents an improved version of the immune checkpoint blockers and could treat cancer patients quickly and effectively. Citation Format: Junyi Li, Yanzhang Wei. Bifunctional fusion protein PDL1sFv/MICAe for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2717.

Therapeutic approaches to enhance natural killer cell cytotoxicity.

Enhancing the cytotoxicity of natural killer (NK) cells has emerged as a promising strategy in cancer immunotherapy, due to their pivotal role in immune surveillance and tumor clearance. This literature review provides a comprehensive overview of therapeutic approaches designed to augment NK cell cytotoxicity. We analyze a wide range of strategies, including cytokine-based treatment, monoclonal antibodies, and NK cell engagers, and discuss criteria that must be considered when selecting an NK cell product to combine with these strategies. Furthermore, we discuss the challenges and limitations associated with each therapeutic strategy, as well as the potential for combination therapies to maximize NK cell cytotoxicity while minimizing adverse effects. By exploring the wealth of research on this topic, this literature review aims to provide a comprehensive resource for researchers and clinicians seeking to develop and implement novel therapeutic strategies that harness the full potential of NK cells in the fight against cancer. Enhancing NK cell cytotoxicity holds great promise in the evolving landscape of immunotherapy, and this review serves as a roadmap for understanding the current state of the field and the future directions in NK cell-based therapies.

Adoptive NK Cell Therapy - a Beacon of Hope in Multiple Myeloma Treatment.

Major advances in the treatment of multiple myeloma (MM) have been achieved by effective new agents such as proteasome inhibitors, immunomodulatory drugs, or monoclonal antibodies. Despite significant progress, MM remains still incurable and, recently, cellular immunotherapy has emerged as a promising treatment for relapsed/refractory MM. The emergence of chimeric antigen receptor (CAR) technology has transformed immunotherapy by enhancing the antitumor functions of T cells and natural killer (NK) cells, leading to effective control of hematologic malignancies. Recent advancements in gene delivery to NK cells have paved the way for the clinical application of CAR-NK cell therapy. CAR-NK cell therapy strategies have demonstrated safety, tolerability, and substantial efficacy in treating B cell malignancies in various clinical settings. However, their effectiveness in eliminating MM remains to be established. This review explores multiple approaches to enhance NK cell cytotoxicity, persistence, expansion, and manufacturing processes, and highlights the challenges and opportunities associated with CAR-NK cell therapy against MM. By shedding light on these aspects, this review aims to provide valuable insights into the potential of CAR-NK cell therapy as a promising approach for improving the treatment outcomes of MM patients.

NK cell exhaustion in the tumor microenvironment.

Natural killer (NK) cells kill mutant cells through death receptors and cytotoxic granules, playing an essential role in controlling cancer progression. However, in the tumor microenvironment (TME), NK cells frequently exhibit an exhausted status, which impairs their immunosurveillance function and contributes to tumor immune evasion. Emerging studies are ongoing to reveal the properties and mechanisms of NK cell exhaustion in the TME. In this review, we will briefly introduce the maturation, localization, homeostasis, and cytotoxicity of NK cells. We will then summarize the current understanding of the main mechanisms underlying NK cell exhaustion in the TME in four aspects: dysregulation of inhibitory and activating signaling, tumor cell-derived factors, immunosuppressive cells, and metabolism and exhaustion. We will also discuss the therapeutic approaches currently being developed to reverse NK cell exhaustion and enhance NK cell cytotoxicity in the TME.

Cordycepin Enhances the Cytotoxicity of Human Natural Killer Cells against Cancerous Cells.

Cancer treatment with natural killer (NK) cell immunotherapy is promising. NK cells can recognize and kill cancer cells without sensitization, making them a potential cancer treatment alternative. To improve clinical efficacy and safety, more research is needed. Enhancing NK cell function improves therapeutic efficacy. Due to its potent apoptosis induction, Cordycepin, a bioactive compound from Cordyceps spp., inhibits cancer cell growth. Cordycepin has immunoregulatory properties, making it a promising candidate for combination therapy with NK cell-based immunotherapy. Cordycepin may enhance NK cell function and have clinical applications, but more research is needed. In this study, cordycepin treatment of NK-92 MI cells increased THP-1 and U-251 cell cytotoxicity. Cordycepin also significantly increased the mRNA expression of cytokine-encoding genes, including tumour necrosis factor (TNF), interferon gamma (IFNG), and interleukin 2 (IL2). NK-92 MI cells notably secreted more IFNG and granzyme B. Cordycepin also decreased CD27 and increased CD11b, CD16, and NKG2D in NK-92 MI cells, which improved its anti-cancer ability. In conclusion, cordycepin could enhance NK cell cytotoxicity against cancerous cells for the first time, supporting its use as an alternative immunoactivity agent against cancer cells. Further studies are needed to investigate its efficacy and safety in clinical settings.

Fluid shear stress enhances natural killer cell's cytotoxicity toward circulating tumor cells through NKG2D-mediated mechanosensing.

Tumor cells metastasize to distant organs mainly via hematogenous dissemination, in which circulating tumor cells (CTCs) are relatively vulnerable, and eliminating these cells has great potential to prevent metastasis. In vasculature, natural killer (NK) cells are the major effector lymphocytes for efficient killing of CTCs under fluid shear stress (FSS), which is an important mechanical cue in tumor metastasis. However, the influence of FSS on the cytotoxicity of NK cells against CTCs remains elusive. We report that the death rate of CTCs under both NK cells and FSS is much higher than the combined death induced by either NK cells or FSS, suggesting that FSS may enhance NK cell's cytotoxicity. This death increment is elicited by shear-induced NK activation and granzyme B entry into target cells rather than the death ligand TRAIL or secreted cytokines TNF-α and IFN-γ. When NK cells form conjugates with CTCs or adhere to MICA-coated substrates, NK cell activating receptor NKG2D can directly sense FSS to induce NK activation and degranulation. These findings reveal the promotive effect of FSS on NK cell's cytotoxicity toward CTCs, thus providing new insight into immune surveillance of CTCs within circulation.

Novaferon gene modification promotes NK92 cell anti-tumor activity

With significant developments in chimeric antigen receptor T-cell therapy, adoptive immunotherapy has unlocked new levels of treatment for malignancies. Natural killer (NK) cells are promising alternative immune effector cells for this strategy. Multiple anti-tumor therapies are largely dependent on type I interferon (IFN) signaling. Type I IFNs enhance NK cell cytotoxicity. Novaferon (nova) is an unnatural, novel IFN-like protein produced by gene shuffling of IFN-α with strong biological activity. To augment the antitumor activity of NK cells, we generated NK92-nova cells that stably express nova. We found that NK92-nova cells mediated enhanced pan-cancer antitumor activity compared to NK92-vec cells. The increased antitumor activity was associated with the enhanced secretion of cytokines, such as IFN-γ, perforin, and granzyme B. Meanwhile, most of the activating receptors were upregulated in the NK92-nova cells. After co-culture with NK92-nova cells, the expression of NKG2D ligands on the HepG2 cells increased, resulting in an enhanced susceptibility of HepG2 cells to NK92 cell-mediated cytolysis. NK92-nova cells significantly inhibited HepG2 tumor growth in a xenograft model without systemic toxicity. Therefore, NK92-nova cells are a novel and safe strategy for cancer immunotherapy.

TIGIT immune checkpoint blockade enhances immunity of human peripheral blood NK cells against castration-resistant prostate cancer

Castration-resistant prostate cancer (CRPC) patients have a 14-month median survival, emphasizing the need for alternative treatments. Previously, we demonstrated that expanded high-dose natural killer (NK) cells derived from human peripheral blood exhibit therapeutic efficacy against CRPC. However, which immune checkpoint blockade promotes NK cell antitumor immunity against CRPC remains unknown. Here, we explored immune checkpoint molecule expression in NK and CRPC cells during their interactions, and identified that the T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domain (TIGIT) monoclonal antibody (mAb), vibostolimab, significantly enhanced NK cell cytotoxicity against CRPC cells and cytokine production in vitro, demonstrated by upregulation of degranulation marker CD107a and Fas-ligand (Fas-L) and increased interferon-gamma (IFN-γ) and tumor necrosis factor-alpha secretion. TIGIT blockade increased Fas-L expression and IFN-γ production via the NF-κB signaling pathway and restored degranulation via the mitogen-activated protein kinase ERK (extracellular signal-regulated kinase) kinase/ERK pathway in activated NK cells. Vibostolimab significantly enhanced NK cell antitumor effects against CRPC in two xenograft mouse models. Vibostolimab also increased T cell chemotaxis induced by activated NK cells in vitro and in vivo. Overall, blocking TIGIT/CD155 signaling enhances the antitumor effect of expanded NK cells against CRPC; this finding supports the translational application of TIGIT mAb and NK cell combination strategies from bench to bedside for CRPC treatment.

Successful Identification of a Novel Therapeutic Compound for Hepatocellular Carcinoma Through Screening of ADAM9 Inhibitors.

MHC-class I-related chain A (MICA) functions as a ligand for natural killer group D, an activating receptor on natural killer (NK) cells, and its expression correlates with the carcinogenesis and progression of hepatocellular carcinoma (HCC). Although membranous MICA (mMICA) activates NK cells, soluble forms of MICA (sMICA), shed by cleaving enzymes, such as A disintegrin and metalloprotease (ADAM) 9, suppress NK cells. Therefore, the prevention of MICA shedding through the inhibition of ADAM9 has the potential to activate cancer immunity. Although we have discovered several ADAM inhibitors, many did not sufficiently activate NK cells without being cytotoxic, and, thus, new ADAM9 inhibitor candidates are needed. To identify possible compounds for drug development, chemical library screening (a total of 741 compounds) was conducted using a fluorescence assay. Compounds with reduced fluorescence intensity were used as hit compounds in a subsequent analysis. Their impact on sMICA and mMICA in HCC cell lines was assessed using ELISA and flow cytometry, respectively. The cytotoxicity of NK cells was also evaluated by co-culturing NK cells with HCC cells. CCL347, a symmetrical compound with five benzene rings, was identified as a hit compound. CCL347 significantly reduced sMICA levels in the culture medium supernatant with negligible cytotoxicity. Although mMICA was also reduced, CCL347 successfully enhanced NK cell cytotoxicity in co-cultures of NK cells and HCC cells. CCL347 has potential as a novel therapeutic drug for HCC.

Anticancer potential of Marina Crystal Minerals (MCM) against the growth of murine mammary adenocarcinoma cells in vivo

In vitro studies have shown that Marina Crystal Minerals (MCM), a crystallized mixture of minerals and trace elements from sea water, possesses apoptotic and immune modulatory effects in human breast cancer cells MDA-MB-231. The current study aimed to evaluate MCM’s anticancer effect in vivo against murine mammary adenocarcinoma cells and to explore its underlying mechanisms. Mice were inoculated intramuscularly with Ehrlich ascites carcinoma (EAC) cells, a breast adenocarcinoma. Tumors became palpable within 9 days. Tumor-bearing mice were injected with MCM intraperitoneally (IP) or intratumorally (IT) at a dose of 40 mg/kg BW for 6 days/week until day 28 post-inoculation. Tumor growth, cell cycle progression, cell cycle regulatory proteins, apoptosis, apoptotic regulatory markers, mitochondrial membrane potential (MMP), natural killer (NK) cell activity, and histopathological effects were investigated. Treatment with MCM reduced tumor volume by 49.4% for IP and 59.5% for IT injection. MCM induced cancer cell apoptosis, as indicated by a sub-G1 peak and confirmed by Annexin V/PI assay and histopathological examination. This was mediated by increased Bax expression, caspase-3 activation, decreased Bcl-2 expression, and MMP disruption. Furthermore, MCM treatment induced G1 cell cycle arrest, mediated through significantly increased expression of p53, p21, and p27 and decreased expression of cyclin D1 and PCNA in cancer cells. Finally, MCM treatment markedly enhanced NK cell cytotoxicity. MCM possesses chemopreventive potential to reduce tumor growth by suppressing cell proliferation, inducing apoptosis in EAC cells via a mitochondrial dependent pathway, and activating the immune system. Our results suggest MCM’s beneficial potential for treating breast adenocarcinoma.

Enhancing Natural Killer Cell-Mediated Cancer Immunotherapy by the Biological Macromolecule Nocardia rubra Cell-Wall Skeleton.

The biological macromolecule Nocardia rubra cell-wall skeleton (Nr-CWS) has well-established immune-stimulating and anti-tumor activities. However, the role of Nr-CWS on natural killer (NK) cells remains unclear. Here, we explore the function and related mechanisms of Nr-CWS on NK cells. Using a tumor-bearing model, we show that Nr-CWS has slightly effect on solid tumor. In addition, using a tumor metastasis model, we show that Nr-CWS suppresses the lung metastasis induced by B16F10 melanoma cells in mice, which indicates that Nr-CWS may up-regulate the function of NK cells. Further investigation demonstrated that Nr-CWS can increase the expression of TRAIL and FasL on spleen NK cells from Nr-CWS treated B16F10 tumor metastasis mice. The spleen index and serum levels of TNF-α, IFN-γ, and IL-2 in B16F10 tumor metastasis mice treated with Nr-CWS were significantly increased. In vitro, the studies using purified or sorted NK cells revealed that Nr-CWS increases the expression of CD69, TRAIL, and FasL, decreases the expression of CD27, and enhances NK cell cytotoxicity. The intracellular expression of IFN-γ, TNF-α, perforin (prf), granzyme-B (GrzB), and secreted TNF-α, IFN-γ, IL-6 of the cultured NK cells were significantly increased after treatment with Nr-CWS. Overall, the findings indicate that Nr-CWS could suppress the lung metastasis induced by B16F10 melanoma cells, which may be exerted through its effect on NK cells by promoting NK cell terminal differentiation (CD27lowCD11bhigh), and up-regulating the production of cytokines and cytotoxic molecules.

MiR-30c facilitates natural killer cell cytotoxicity to lung cancer through targeting GALNT7.

MicroRNAs (miRNAs) have been reported to play important roles in regulating natural killer (NK) cell cytotoxicity to cancer cells. This study aimed to investigate the effects and potential mechanism of miR-30c in regulating NK cell cytotoxicity to lung cancer cells. Primary NK cells were derived from the peripheral blood of lung cancer and normal participants. Exosomes were isolated and validated via transmission electron microscopy and nanoparticle tracking analysis. The levels of miR-30c, polypeptide N-acetylgalactosaminyltransferase 7 (GALNT7) and proteins in PI3K/AKT pathway were determined using quantitative real-time polymerase chain reaction or western blot. Tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) levels and the cytotoxicity of effector NK cells to target lung cancer cells were measured via enzyme linked immunosorbent assay, cell apoptosis or xenograft experiments. The relationship between miR-30c and GALNT7 was analyzed by luciferase activity, RNA pull-down and RNA immunoprecipitation assays. And a xenograft mice model was established to verify the effect of miR-30c in regulating NK cell cytotoxicity to lung cancer cells in vivo. NK cell-derived exosomes carrying miR-30c, and miR-30c level was significantly downregulated in primary NK cells of lung cancer patients. MiR-30c overexpression promoted TNF-α and IFN-γ secretion and enhanced the cytotoxicity of interleukin 2 (IL-2)-treated NK cells to lung cancer cells, while knockdown of miR-30c played an opposite effect in regulating the cytotoxicity of NK cells to lung cancer cells. GALNT7 was a target of miR-30c and was negatively regulated by miR-30c. Besides, miR-30c targeted GALNT7 to exert its function in regulating NK cell cytotoxicity. Furthermore, GALNT7 prompted the activation of PI3K/AKT pathway in NK cells. Additionally, miR-30c overexpression enhanced NK cell cytotoxicity to lung cancer cells and inhibited tumor growth in vivo. miR-30c enhanced NK cell cytotoxicity to lung cancer cells via decreasing GALNT7 and inactivating the PI3K/AKT pathway, suggesting that regulating miR-30c expression maybe a promising approach for enhancing NK cell-based antitumor therapies.