scholarly journals Reovirus enhances cytotoxicity of natural killer cells against colorectal cancer via TLR3 pathway

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Shiqi Long ◽  
Yangzhuo Gu ◽  
Yuanyuan An ◽  
Xiaojin Lin ◽  
Xiaoqing Chen ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Nk Cells ◽  
Natural Killer ◽  
Signaling Pathway ◽  
Cell Function ◽  
Nk Cell ◽  
Antitumor Effects ◽  
Downstream Signaling ◽  
Egfr Expression ◽  
Downstream Signaling Pathway

Abstract Background Cetuximab has been approved for use for first-line treatment of patients with wild-type KRAS metastatic colorectal cancer (CRC). However, treatment with cetuximab has shown limited efficacy as a CRC monotherapy. In addition, natural killer (NK) cell function is known to be severely attenuated in cancer patients. The goal of this study was to develop a new strategy to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by NK cells, in combination with cetuximab against CRC cells. Methods Ex vivo expanded NK cells were stimulated with reovirus, and reovirus-activated NK cells mediated ADCC assay were performed on CRC cells in combination with cetuximab. The synergistic antitumor effects of reovirus-activated NK cells and cetuximab were tested on DLD-1 tumor-bearing mice. Finally, Toll-like receptor 3 (TLR3) knockdown in NK cells, along with chemical blockade of TLR3/dsRNA complex, and inhibition of the TLR3 downstream signaling pathway, were performed to explore the mechanisms by which reovirus enhances NK cell cytotoxicity. Results We first confirmed that exposure of NK cells to reovirus enhanced their cytotoxicity in a dose-dependent manner.We then investigated whether reovirus-activated NK cells exposed to cetuximab-bound CRC cells exhibited greater anti-tumor efficacy than either monotherapy. Co-culture of CRC cell lines with reovirus-activated NK cells indicated that NK cytotoxicity was significantly higher in combination with cetuximab, regardless of KRAS mutation status or EGFR expression level. We also found that reovirus activation of NK cells, in conjunction with cetuximab, resulted in significantly stronger anti-tumor efficacy.Finally, TLR3 knockdown, inhibition of TLR3/dsRNA complex or TBK1/IKKε demonstrated that activation of NK cells by reovirus was dependent on TLR3 and its downstream signaling pathway. Conclusions This study demonstrated that combination treatment of reovirus-activated NK cells with cetuximab synergistically enhances their anti-tumor cytotoxicity, suggesting a strong candidate strategy for clinical treatment of CRC.

scholarly journals Black Raspberries Suppress Colorectal Cancer by Enhancing Smad4 Expression in Colonic Epithelium and Natural Killer Cells

2020 ◽  
Vol 11 ◽  
Author(s):  
Yi-Wen Huang ◽  
Chien-Wei Lin ◽  
Pan Pan ◽  
Tianjiao Shan ◽  
Carla Elena Echeveste ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Nk Cells ◽  
Natural Killer ◽  
Dietary Intervention ◽  
Nk Cell ◽  
Good Prognosis ◽  
Colonic Epithelium ◽  
Antitumor Effects ◽  
Black Raspberries ◽  
Smad4 Expression

Innate immune cells in the tumor microenvironment have been proposed to control the transition from benign to malignant stages. In many cancers, increased infiltration of natural killer (NK) cells associates with good prognosis. Although the mechanisms that enable NK cells to restrain colorectal cancer (CRC) are unclear, the current study suggests the involvement of Smad4. We found suppressed Smad4 expression in circulating NK cells of untreated metastatic CRC patients. Moreover, NK cell-specific Smad4 deletion promoted colon adenomas in DSS-treated ApcMin/+ mice and adenocarcinomas in AOM/DSS-treated mice. Other studies have shown that Smad4 loss or weak expression in colonic epithelium associates with poor survival in CRC patients. Therefore, targeting Smad4 in both colonic epithelium and NK cells could provide an excellent opportunity to manage CRC. Toward this end, we showed that dietary intervention with black raspberries (BRBs) increased Smad4 expression in colonic epithelium in patients with FAP or CRC and in the two CRC mouse models. Also, benzoate metabolites of BRBs, such as hippurate, upregulated Smad4 and Gzmb expression that might enhance the cytotoxicity of primary human NK cells. Of note, increased levels of hippurate is a metabolomic marker of a healthy gut microbiota in humans, and hippurate also has antitumor effects. In conclusion, our study suggests a new mechanism for the action of benzoate metabolites derived from plant-based foods. This mechanism could be exploited clinically to upregulate Smad4 in colonic epithelium and NK cells, thereby delaying CRC progression.

scholarly journals 217 Cytotoxicity of nicotinamide enhanced natural killer cells GDA201 is based on metabolic modulation as demonstrated by artificial intelligence assisted analysis of NK cell transcriptome and metabolome

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A230-A230
Author(s):  
Dima Yackoubov ◽  
Aviad Pato ◽  
Julia Rifman ◽  
Sherri Cohen ◽  
Astar Hailu ◽  
...  
Keyword(s):  
Artificial Intelligence ◽  
Machine Learning ◽  
Informed Consent ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Function ◽  
Nk Cell ◽  
Medical Center ◽  
Enzymatic Reactions ◽  
Combined Algorithm

BackgroundNicotinamide (NAM), an allosteric inhibitor of NAD-dependent enzymes, has been shown to preserve cell function and prevent differentiation in ex vivo cell culture. GDA-201 is an investigational natural killer (NK) cell immunotherapy derived from allogeneic donors and expanded using IL-15 and NAM. In previous preclinical studies, NAM led to increased homing and cytotoxicity, preserved proliferation, and enhanced tumor reduction of NK cells. In a phase I clinical trial, treatment with GDA-201 showed tolerability and clinical responses in patients with refractory non-Hodgkin lymphoma (NHL) (Bachanova, et. al., Blood 134:777, 2019). While NAM is known to affect cellular metabolism and participate in 510 enzymatic reactions −in 66 as an inhibitor or activator− its mechanism of action and role in GDA-201 cytotoxicity is unknown.MethodsIn order to define the network of intracellular interactions that leads to the GDA-201 phenotype, flow-cytometry, next generation sequencing (NGS), and liquid chromatography–mass spectrometry (LC-MS)-based metabolite quantification were performed on NK cells cultured for 14 days with IL-15 and human serum in the presence or absence of NAM (7 mM). Artificial Intelligence (AI) machine learning analysis was applied by Pomicell in order to analyze the data using the Pomicell databases supporting data extracted from multiple origins including scientific articles organized using natural language processing tools. AI training was done using a combined algorithm designed to blindly explain and predict the transcriptomic and metabolomic (omics) profile.ResultsOmics analyses defined 1,204 differentially expressed genes, and 100 significantly modified metabolites in the presence of NAM. An in silico model was created that successfully predicted the experimental data in 83% of the cases. Upregulation of TIM-3 expression in GDA-201 was predicted to be mediated by inhibition of IL-10 and SIRT3, via CREB1/HLA-G signaling and adrenoceptor beta 2 (ADRB2) upregulation. Adenosine metabolite reduction supports this and suggests dopaminergic activation of NK cytotoxicity. Upregulation of CD62L in the presence of NAM was predicted to be mediated by transcription factor Dp-1 (TFDP1) via dihydrofolate reductase (DHFR) activation and intracellular folic acid reduction. Interferon-gamma and CASP3 modulation (via JUN and MCL1, respectively), via PPARa inhibition, support that finding.ConclusionsIn conclusion, AI machine learning of transcriptome and metabolome data revealed multiple pleiotropic metabolic pathways modulated by NAM. These data serve to further elucidate the mechanism by which NAM enhances cell function, leading to the observed cytotoxicity and potency of GDA-201.Ethics ApprovalWe hereby declare that the collection of the Apheresis units in the three participating institutes (sites) has been done under an approved clinical study that meets the following requirements:1. Ethics approval has been obtained from the local EC at each of the sites, prior to any study related activities.2. The working procedures of the EC at the sites for conduct of clinical studies are in due compliance with local regulations (Israeli Ministry of Health) and provisions of Harmonized International Guidelines for Good Clinical Practice, namely: ICH-GCP.3. Sites follow EC conditions & requirements in terms of submissions, notifications, and approval renewals. 4. Participants gave Informed Consent (approved by the EC) before taking part in the study.5. Informed Consent has been approved by the ECs. The Israeli template of Informed Consent is in used and it includes study specific information (e.g. study goal, design, method, duration, risks, etc.). Name of the Institute Name of the EC/IRB EC Study No.Hadassah Medical Center Helsinki Committee 0483-16-HMORambam Health Care Campus Helsinki Committee 0641-18-RMBIchilov Sourasky Medical Center Tel-Aviv Helsinki Committee 0025-17-TLV

scholarly journals Natural Killer Cells in the Malignant Niche of Multiple Myeloma

2022 ◽  
Vol 12 ◽  
Author(s):  
Ondrej Venglar ◽  
Julio Rodriguez Bago ◽  
Benjamin Motais ◽  
Roman Hajek ◽  
Tomas Jelinek
Keyword(s):  
Multiple Myeloma ◽  
Nk Cells ◽  
Natural Killer ◽  
Defense Mechanisms ◽  
Hematological Malignancies ◽  
Cell Function ◽  
Nk Cell ◽  
Current Knowledge ◽  
Suppressor Activity ◽  
Infected Cells

Natural killer (NK) cells represent a subset of CD3- CD7+ CD56+/dim lymphocytes with cytotoxic and suppressor activity against virus-infected cells and cancer cells. The overall potential of NK cells has brought them to the spotlight of targeted immunotherapy in solid and hematological malignancies, including multiple myeloma (MM). Nonetheless, NK cells are subjected to a variety of cancer defense mechanisms, leading to impaired maturation, chemotaxis, target recognition, and killing. This review aims to summarize the available and most current knowledge about cancer-related impairment of NK cell function occurring in MM.

scholarly journals The Ly-49D Receptor Activates Murine Natural Killer Cells

1996 ◽  
Vol 184 (6) ◽  
pp. 2119-2128 ◽  
Author(s):  
L.H. Mason ◽  
S.K. Anderson ◽  
W.M. Yokoyama ◽  
H.R.C. Smith ◽  
R. Winkler-Pickett ◽  
...  
Keyword(s):  
Gene Family ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Function ◽  
Nk Cell ◽  
Cell Subset ◽  
Class I ◽  
Target Cells ◽  
Color Flow ◽  
Functional Capabilities

Proteins encoded by members of the Ly-49 gene family are predominantly expressed on murine natural killer (NK) cells. Several members of this gene family have been demonstrated to inhibit NK cell lysis upon recognizing their class I ligands on target cells. In this report, we present data supporting that not all Ly-49 proteins inhibit NK cell function. Our laboratory has generated and characterized a monoclonal antibody (mAb) (12A8) that can be used to recognize the Ly-49D subset of murine NK cells. Transfection of Cos-7 cells with known members of the Ly-49 gene family revealed that 12A8 recognizes Ly-49D, but also cross-reacts with the Ly-49A protein on B6 NK cells. In addition, 12A8 demonstrates reactivity by both immunoprecipitation and two-color flow cytometry analysis with an NK cell subset that is distinct from those expressing Ly-49A, C, or G2. An Ly-49D+ subset of NK cells that did not express Ly49A, C, and G2 was isolated and examined for their functional capabilities. Tumor targets and concanovalin A (ConA) lymphoblasts from a variety of H2 haplotypes were examined for their susceptibility to lysis by Ly-49D+ NK cells. None of the major histocompatibility complex class I–bearing targets inhibited lysis of Ly-49D+ NK cells. More importantly, we demonstrate that the addition of mAb 12A8 to Ly-49D+ NK cells can augment lysis of FcγR+ target cells in a reverse antibody-dependent cellular cytotoxicity–type assay and induces apoptosis in Ly49D+ NK cells. Furthermore, the cytoplasmic domain of Ly-49D does not contain the V/IxYxxL immunoreceptor tyrosine-based inhibitory motif found in Ly-49A, C, or G2 that has been characterized in the human p58 killer inhibitory receptors. Therefore, Ly-49D is the first member of the Ly-49 family characterized as transmitting positive signals to NK cells, rather than inhibiting NK cell function.

CIP4 Targeted to Recruit GTP-Cdc42 Involving in Invadopodia Formation Through NF-κB Signaling Pathway Promotes Invasion and Metastasis of Colorectal Cancer

2021 ◽  
Author(s):  
Zhiyan Hu ◽  
Jiaxian Zhu ◽  
Yidan Ma ◽  
Ting Long ◽  
Lingfang Gao ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Signaling Pathway ◽  
Tumor Metastasis ◽  
Migration And Invasion ◽  
Downstream Signaling ◽  
And Function ◽  
Downstream Signaling Pathway ◽  
Invadopodia Formation

Abstract Background CIP4 (Cdc42-interacting protein 4), a member of the F-BAR family which plays an important role in regulating cell membrane and actin, has been reported to interact with Cdc42 and closely associated with tumor invadopodia formation. However, the specific mechanism of the interaction between CIP4 and Cdc42 as well as the downstream signaling pathway in response in colorectal cancer (CRC) remains unknown, which is worth exploring for its impact on tumor infiltration and metastasis. Methods Immunohistochemistry and western blot analyses were performed to detect the expression of CIP4 and Cdc42. Their relationship with CRC clinicopathological characteristics was further analyzed. Wound-healing, transwell migration and invasion assays tested the effect of CIP4 on cells migration and invasion ability in vitro, and the orthotopic xenograft colorectal cancer mouse mode evaluated the tumor metastasis in vivo. The invadopodia formation and function were assessed by immunofluorescence, scanning electron microscopy (SEM) and matrix degradation assay. The interaction between CIP4 and Cdc42 was confirmed by co-immunoprecipitation (co-IP) and GST-Pull down assays. Immunofluorescence was used to observed the colocalization of CIP4, GTP-Cdc42 and invadopodia. The related downstream signaling pathway was investigated by western blot and immunofluorescence. Results CIP4 expression was significantly higher in human colorectal cancer tissues and correlated with the CRC infiltrating depth and metastasis as well as the lower survival rate in patients. In cultured CRC cells, knockdown of CIP4 inhibited cell migration and invasion ability in vitro and the tumor metastasis in vivo, while overexpression of CIP4 confirmed the opposite situation by promoting invadopodia formation and matrix degradation ability. In addition, we identified GTP-Cdc42 as a directly interactive protein of CIP4, which was upregulated and recruited by CIP4 to participate in this process. Furthermore, activated NF-κB signaling pathway was found in CIP4 overexpression CRC cells contributing to invadopodia formation while inhibition of either CIP4 or Cdc42 led to suppression of NF-κB pathway resulted in decrease quantity of invadopodia. Conclusion Our findings suggested that CIP4 targets to recruit GTP-Cdc42 and directly combines with it to accelerate invadopodia formation and function by activating NF-κB signaling pathway, thus promoting CRC infiltration and metastasis.

IMMU-42. OPTIMIZING IMMUNOTHERAPY FOR GLIOMA USING CYTOKINE-ACTIVATED NATURAL KILLER CELLS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi101-vi102
Author(s):  
Amber Kerstetter-Fogle ◽  
Folashade Otegbeye ◽  
David Soler ◽  
Peggy Harris ◽  
Alankrita Raghavan ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Glioma Cell ◽  
Cell Function ◽  
Nk Cell ◽  
Cell Killing ◽  
Cellular Immunotherapy ◽  
Stereotactic Radiation ◽  
Tumor Bed

Abstract INTRODUCTION Glioblastoma multiforme (GBM) is the most common primary central nervous system malignancy associated with a 12-15 month survival after surgery and radio-chemotherapy. Utilizing adoptive cellular immunotherapy using natural killer (NK) cells has developed over the past two decades for a variety of hematologic malignancies. This approach in solid malignancies is limited by questions of cell dose versus tumor burden, insufficient tumor infiltration, and a tumor microenvironment that suppresses NK cell function. METHODS We isolated NK cells from healthy volunteers and activated them using IL-2, -15, -12, -18, then perform cytotoxic assays in the presence of glioma stem cells. We also tested the efficacy of the NK cells with intracranial delivery in a pre-clinical murine model of glioma. We tested various concentrations of IL-2 and IL-15 on the cytokine culture platform. RESULTS In this study, we demonstrate human NK cells, activated using a cytokine cocktail of interleukins-2, -15, -12, and -18, exert strong cytotoxic events against glioma cell lines. To further examine the efficacy of activated NK cells in vitro, we utilized intracranially xenografted glioma lines and demonstrated a survival benefit with tumor bed injections of these cytokine-activated NK cells (p=0.0089). We were able to confirm that NK cells cultured with low doses (200u IL2; 50ng/ml IL15) of both cytokines are just as effective as higher doses. This is important, as in vivoexhaustion of NK cells stimulated with high doses of either cytokine has been well validated. We also found that low-dose irradiation (4Gy) of glioma cells prior to co-culture with cytokine-activated NK cells promoted increased targeted glioma cell killing within 4 hours(32% cell killing). CONCLUSIONS These findings suggest that in a clinical study, injection of cytokine-activated NK cells into the glioblastoma tumor bed could be used as adjuvant treatment following either stereotactic radiation or surgical resection.

Contribution of Natural Killer Cells to Inhibition of Angiogenesis by Interleukin-12

Blood ◽  
1999 ◽  
Vol 93 (5) ◽  
pp. 1612-1621 ◽  
Author(s):  
Lei Yao ◽  
Cecilia Sgadari ◽  
Keizo Furuke ◽  
Eda T. Bloom ◽  
Julie Teruya-Feldstein ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Function ◽  
Nk Cell ◽  
Primary Cultures ◽  
Interleukin 12 ◽  
Ifn Γ

Abstract Interleukin-12 (IL-12) inhibits angiogenesis in vivo by inducing interferon-γ (IFN-γ) and other downstream mediators. Here, we report that neutralization of natural killer (NK) cell function with antibodies to either asialo GM1 or NK 1.1 reversed IL-12 inhibition of basic fibroblast growth factor (bFGF)-induced angiogenesis in athymic mice. By immunohistochemistry, those sites where bFGF-induced neovascularization was inhibited by IL-12 displayed accumulation of NK cells and the presence of IP-10–positive cells. Based on expression of the cytolytic mediators perforin and granzyme B, the NK cells were locally activated. Experimental Burkitt lymphomas treated locally with IL-12 displayed tumor tissue necrosis, vascular damage, and NK-cell infiltration surrounding small vessels. After activation in vitro with IL-12, NK cells from nude mice became strongly cytotoxic for primary cultures of syngeneic aortic endothelial cells. Cytotoxicity was neutralized by antibodies to IFN-γ. These results document that NK cells are required mediators of angiogenesis inhibition by IL-12, and provide evidence that NK-cell cytotoxicity of endothelial cells is a potential mechanism by which IL-12 can suppress neovascularization.

scholarly journals Natural killer (NK) cell function in paroxysmal nocturnal hemoglobinuria: a deficiency of NK cells, but not an NK cell deficiency

Blood ◽  
2015 ◽  
Vol 125 (8) ◽  
pp. 1351-1352 ◽  
Author(s):  
Yasser M. El-Sherbiny ◽  
Gina M. Doody ◽  
Richard J. Kelly ◽  
Anita Hill ◽  
Peter Hillmen ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Cell Function ◽  
Nk Cell

Hemostatic Factors Contribute to Tumor Cell Metastatic Potential by a Mechanism Linked to Natural Killer Cell Function.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 690-690 ◽  
Author(s):  
Joseph S. Palumbo ◽  
Kathryn E. Talmage ◽  
Jessica V. Massari ◽  
Christine M. La Jeunesse ◽  
Matthew J. Flick ◽  
...  
Keyword(s):  
Natural Killer Cell ◽  
Tumor Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Platelet Activation ◽  
Cell Function ◽  
Nk Cell ◽  
Killer Cell ◽  
Metastatic Potential ◽  
Natural Killer Cell Function

Abstract A linkage between hemostatic system components and tumor cell metastatic potential has been well established, but the underlying mechanism(s) by which various circulating and cell-associated coagulation factors and platelets promote tumor cell dissemination remains to be fully defined. One potential mechanism by which tumor cell-associated microthrombi might enhance metastatic potential is by interfering with the cytolytic elimination of tumor cell emboli by natural killer (NK) cells. In order to explore this hypothesis, we studied tumor dissemination in mice lacking either fibrinogen or Gαq, a G protein critical for platelet activation. Comparative studies of experimental lung metastasis in control and Gαq−/− mice showed that loss of platelet activation resulted in a two-orders-of-magnitude decrease in pulmonary metastatic foci formed by either Lewis lung carcinoma or B16 melanoma. The difference in metastatic success was not the result of differences in tumor growth rate, as tumors transplanted into the dorsal subcutis of Gαq−/− and wildtype animals grew at similar rates. Rather, tumor cell fate analyses using radiolabeled tumor cells showed that the survival of tumor cells within the lung was significantly improved in mice that retained platelet activation function relative to Gαq−/− mice with a profound platelet activation defect. In order to examine the potential interplay between platelet activation and natural killer cell function, we compared pulmonary tumor cell survival in cohorts of control and Gαq−/− mice immuno-depleted of NK cells with an anti-asialo GM1 antibody. Remarkably, platelet function was no longer a determinant of metastatic potential in mice lacking NK cells. Given that fibrin(ogen) is also an established determinant of metastatic success we explored whether the influence of this key hemostatic factor on tumor cell dissemination was also mechanistically-coupled to natural killer cell function. We interbred fibrinogen-deficient mice with Gz-Ly49A transgenic mice known to have a constitutive deficit in NK cells. In those cohorts of mice with normal NK cells, we affirmed the earlier finding that fibrinogen deficiency resulted in a significant diminution in metastatic potential. However, consistent with our findings in mice with defective platelet activation, fibrinogen was found to no longer be a determinant of metastatic potential in mice lacking NK cells. These data establish another important link between innate immune surveillance and the hemostatic system. Further, it appears that at least one mechanism by which tumor cell-associated microthrombi increase metastatic potential is by restricting NK cell-mediated tumor cell elimination. Given that NK cell cytotoxicity requires direct contact with any target cell, one attractive model presently being explored is that tumor cell-associated platelets physically block NK cell access to tumor cell emboli.

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