[The Three-dimensional Environment of Type â Collagen Gels With Varying Stiffness Modulates the Immunological Functions of NK Cells]. / 不同刚度â 型胶原凝胶三维环境调控NKç»†èƒžå ç–«åŠŸèƒ½.

Objective: To construct type Ⅰ collagen gels with different stiffness and to investigate the effects of three-dimensional (3D) culture environments of the gels on the morphology, free migration ability, and cell killing function of natural killer (NK) cells. Methods: Type Ⅰ collagen was isolated from the tails of Sprague Dawley (SD) rats and collagen gels with different levels of stiffnesses were prepared accordingly. The microstructure of the collagen gels was observed by laser confocal microscopy. The stiffness of the collagen gels was assessed by measuring the plateau modulus with a rheometer. NK-92MI cells were cultured in collagen gels with different levels of stiffness. The morphology of NK-92MI cells was observed by inverted microscope. High content imaging system was used to record the free migration process of NK-92MI cells and analyze the migration speed and distance. NK-92MI cells were cultured with type Ⅰ collagen gels with different levels of stiffness for 24 h and 48 h and, then, co-cultured with human colorectal DLD-1, a human adenocarcinoma epithelial cell line. CCK8 assay was performed to determine the proliferation rate of DLD-1 cells and analyze the cell killing ability of NK-92MI cells. Results: Low-stiffness type Ⅰ collagen gel and high-stiffness type Ⅰ collagen gel with the respective stiffness of (10.970±2.10) Pa and (114.50±3.40) Pa were successfully prepared. Compared with those cultured with the low-stiffness type Ⅰ collagen gel, the NK-92MI cells in the high-stiffness type Ⅰ collagen gel showed a more elongated shape (P<0.05), the mean area of the cells was reduced ([69.88±26.97] µm2 vs. [46.59±21.62] µm2, P<0.05), the roundness of the cells decreased (0.82±0.12 vs. 0.78±0.18, P<0.05), cell migration speed decreased ([2.50±0.91] µm/min vs. [1.70±0.72] µm/min, P<0.001) and the migration distance was shortened ([147.10±53.74] µm vs. [98.03± 40.95] µm, P<0.0001), with all the differences being statistically significant. Compared with those cultured with the low-stiffness type Ⅰ collagen gel, NK-92MI cells cultured with high-stiffness type Ⅰ collagen gel for 24 h could promote DLD-1 cell proliferation, with the proliferation rate being (46.39±12.79)% vs. (65.87±4.45)% (P<0.05) and reduce the cell killing ability. Comparison of the cells cultured for 48 h led to similar results, with the proliferation rates being (31.36±2.88)% vs. (74.57±2.16)% (P<0.05), and the differences were all statistically significant. Conclusion: The 3D culture environment of type Ⅰ collagen gels with different levels of stiffness alters the morphology, migration ability, and killing function of NK-92MI cells. This study provides the research basis for exploring and understanding the mechanisms by which the biomechanical microenvironment affects the immune response of NK cells, as well as laying the theoretical foundation for optimizing immunotherapy protocols.

Pirfenidone suppressed triple-negative breast cancer metastasis by inhibiting the activity of the TGF-ß/SMAD pathway.

Among breast cancer patients, metastases are the leading cause of death. Despite decades of effort, little progress has been made to improve the treatment of breast cancer metastases, especially triple-negative breast cancer (TNBC). The extracellular matrix plays an important role in tumour growth and metastasis by causing its deposition, remodelling, and signalling. As we know, the process of fibrosis results in excessive amounts of extracellular matrix being deposited within the cells. So, it will be interesting to study if the use of anti-fibrotic drugs in combination with conventional chemotherapy drugs can produce synergistic antitumor effects. In this study, we assessed the efficacy of Pirfenidone (PFD), an FDA-approved medication for the treatment of idiopathic pulmonary fibrosis, on TNBC cells as well as its anti-tumour effects in xenograft tumour model. PFD inhibited in a dose-dependent manner breast cancer cell proliferation, migration, and invasion, while promoted their apoptosis in vitro. PFD also suppressed TGF-ß-induced activation of Smad signalling pathway and expression level of EMT-inducing transcription factors (e.g. SNAI2, TWIST1, ZEB1) as well as the mesenchymal genes such as VIMENTIN and N-Cadherin. On the contrary, the expression level of epithelial marker gene E-Cadherin was up-regulated in the presence of PFD. In vivo, PFD alone exerted a milder but significant anti-tumour effect than the chemotherapy drug nanoparticle albumin-bound paclitaxel (nab-PTX) did in the breast cancer xenograft mouse model. Interestingly, PFD synergistically boosted the cancer-killing effect of nab-PTX. Furthermore, Our data suggest that PFD suppressed breast cancer metastasis by inhibiting the activity of the TGFß/SMAD pathway.

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

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.

Retraction Note to: MicroRNA-322 Cluster Promotes Tau Phosphorylation via Targeting Brain-Derived Neurotrophic Factor.

The Editors have retracted this article [1] following an investigation conducted by the journal. After publication concerns were raised regarding interpretation of the data presented in Fig. 4. The Editors requested additional data and clarification to confirm interpretation of data results. After further review, the Editors found that the additional data were not adequate to support the conclusion of the article and that P-values for the additional data were based on improper statistical analyses. With more appropriate statistical analysis, the reported effects for miR-322 and BDNF were not statistically significant. Dr. Chichu Xie agrees to this retraction. None of the other authors have responded to any correspondence from the publisher about this retraction.

Cytoglobin ameliorates the stemness of hepatocellular carcinoma via coupling oxidative-nitrosative stress signals.

Cancer stem cells (CSCs) account for tumor self-renewal and heterogeneity. Oxidative-nitrosative stress (ONS) is an independent etiologic factor throughout tumorigenesis. Emerging evidences indicated that the interaction of ONS with CSCs contributes to tumor progression and resistance to chemoradiotherapy. Cytoglobin (Cygb) is a member of human hexacoordinate hemoglobin family and acts as a dynamic mediator of redox homeostasis. We observed that Cygb is significantly deregulated in human hepatocellular carcinoma (HCC) tissue and its decrease aggravates the growth of liver cancer stem cells (LCSCs) and increases the subpopulation of CD133(+) LCSCs. Cygb restoration inhibits HCC proliferation and LCSC growth, and decreases the subpopulation of CD133 (+) LCSCs in vitro. We found that Cygb absence promotes LCSC phenotypes and PI3 K/AKT activation, whereas Cygb restoration inhibits LCSC phenotypes and PI3 K/AKT activation. Furthermore, exogenous antioxidants can eliminate the inhibitory effect of Cygb to LCSC growth and phenotypes, as well as PI3 K/AKT activation. Collectively, this study demonstrated that cytoglobin functions as a tumor suppressor and targets CSCs at an ONS-dependent manner. Thus, Cygb restoration could be a novel and promising therapeutic strategy against HCC with aberrant ROS/RNS accumulation.

Roles of genetic and microenvironmental factors in cancer epithelial-to-mesenchymal transition and therapeutic implication.

Epithelial-to-mesenchymal transition (EMT) is a process in which epithelial cells lose their cell-cell contacts resulting in the formation of mesenchymal cells with migratory properties. Increasing evidence indicate EMT plays a key role in the invasion, metastasis and therapeutic resistance of cancer and maintenance of the phenotype of cancer stem cells (CSCs), which makes the prognosis of patients worse. The progression of cancer from epithelial tissue towards a malignant phenotype is driven by multiple factors that remodel the tissue architecture. This review summarizes and analyzes current studies of genetic and microenvironmental factors in inducing and maintaining cancer EMT and therapeutic implications. This will enable a better understanding of the contribution of EMT-associated factors to cancer progression and highlights that genetic factors and tumor microenvironment responsible for EMT could be used as attractive targets for therapeutic intervention.

MicroRNA-322 Cluster Promotes Tau Phosphorylation via Targeting Brain-Derived Neurotrophic Factor.

Brain-derived neurotrophic factor (BDNF) is a crucial regulator to support synaptic plasticity and neuronal survival, its significant decrease is a pathophysiological hallmark in Alzheimer's disease (AD) brains and accounts for poor prognosis. MicroRNAs (miRNAs) interfere with the translation of target mRNAs and control a variety of physiological and pathological processes. MiR-322 is the rodent homologue of human miR-424, it is involved in the modulation of cell differentiation, proliferation, apoptosis and metabolic activities in diverse tissues and organs. However, the roles and potential mechanisms of miR-322 remain elusive in AD pathogenesis. Here we observed miR-322 is significantly increased along with BDNF decrease in AD mouse brain. Bioinformatics prediction implicated that BDNF 3'-untranslated region (3'-UTR) possesses the putative target sequence of miR-322. Luciferase reporter assay identified that miR-322 can directly conjugate to BDNF 3'-UTR. The functional research showed that MiR-322 input deregulates BDNF expression at either mRNA or protein levels, whereas miR-322 silence restores BDNF expression in vitro. Furthermore, we found miR-322 promotes Tau phosphorylation via negatively controlling BDNF-TrkB receptor activation, otherwise MiR-322 silence restores TrkB activation and attenuates tau phosphorylation. Collectively, this study demonstrated a novel miRNA-dependent manner of BDNF degradation in AD pathogenesis, it may drive a miRNAs- or BDNF based therapeutic strategies against Alzheimer's disease.

Large-scale Isolation of Exosomes Derived from NK Cells for Anti-tumor Therapy.

Exosomes are lipid bilayer-enclosed vesicles, actively secreted by cells, containing proteins, lipids, nucleic acids, and other substances with multiple biological functions after entering target cells. Exosomes derived from NK cells have been shown to have certain anti-tumor effects and potential applications as chemotherapy drug carriers. These developments have resulted in high demand for exosomes. Although there has been large-scale industrial preparation of exosomes, they are only for generally engineered cells such as HEK 293T. The large-scale preparation of specific cellular exosomes is still a major problem in laboratory studies. Therefore, in this study, we used tangential flow filtration (TFF) to concentrate the culture supernatants isolated from NK cells and isolated NK cell-derived exosomes (NK-Exo) by ultracentrifugation. Through a series of characterization and functional verification of NK-Exo, the characterization, phenotype, and anti-tumor activity of NK-Exo were verified. Our study provides a considerably time- and labor-saving protocol for the isolation of NK-Exo.

Combination of CTLA-4 blockade with MUC1 mRNA nanovaccine induces enhanced anti-tumor CTL activity by modulating tumor microenvironment of triple negative breast cancer.

The immunosuppressive tumor microenvironment (TME) is the main reason for the failure of many immunotherapies that directly stimulate anti-tumor immune response. Anti-CTLA-4 antibody may reduce effector regulatory T (Treg) cell numbers and their suppressive activity in the TME. We have previously reported that combination of anti-CTLA-4 antibody with MUC1 mRNA nanovaccine may mutually enhance each single treatment. But the enhancement mechanism of therapeutic efficacy of MUC1 mRNA nanovaccine plus anti-CTLA-4 monoclonal antibody (mAb) is unknown. In this study, anti-tumor CTL activity induced by combination of CTLA-4 Blockade with MUC1 mRNA nanovaccine and immunosuppressive factors in the TME of triple negative breast cancer were investigated. The results demonstrated that combined therapy with nanovaccine and anti-CTLA-4 mAb could induce stronger anti-tumor CTL response than each monotherapy, result in significantly decreased numbers of myeloid-derived suppressor cells (MDSC), Treg cells, tumor-associated fibroblasts (TAFs) and tumor vasculature in the TME, downregulated levels of interleukin-6, tumor necrosis factor-α and transforming growth factor-ß, and significantly upregulated levels of IFN-γ and interleukin-12 as well as increased number of CD8+ T cell, and appear more effective than either nanovaccine or anti-CTLA-4 mAb alone at increasing level of apoptosis in tumor cells. In addition, combination immunotherapy could significantly downregulated the signal transducer and activator of transcription 3 (STAT3) signal pathway. Therefore, it can be concluded that combination of CTLA-4 blockade with MUC1 mRNA nanovaccine enhances anti-tumor cytotoxic T-lymphocyte activity by reducing immunosuppressive TME and inhibiting tumor-promoting STAT3 signaling pathway.

CXCL12 derived from CD248-expressing cancer-associated fibroblasts mediates M2-polarized macrophages to promote nonsmall cell lung cancer progression.

Nonsmall cell lung cancer (NSCLC) is among the most prevalent malignant tumours threatening human health. In the tumour microenvironment (TME), cancer-associated fibroblasts (CAFs) induce M2-polarized macrophages, which strongly regulate tumour progression. However, little is known about the association between CAFs and M2 macrophages. CD248 is a transmembrane glycoprotein found in several cancer cells, tumour stromal cells, and pericytes. Here, we isolated CAFs from tumour tissues of NSCLC patients to detect the relationship between CD248 expression and patient prognosis. We knocked down the expression of CD248 on CAFs to detect CXCL12 secretion and macrophage polarization. We then examined the effects of CD248-expressing CAF-induced M2 macrophage polarization to promote NSCLC progression in vitro and in vivo. We found that CD248 is expressed mainly in NSCLC-derived CAFs and that the expression of CD248 correlates with poor patient prognosis. Blocking CXCL12 receptor (CXCR4) drastically decreased M2 macrophage chemotaxis. CD248 promotes CAFs secreting CXCL12 to mediate M2-polarized macrophages to promote NSCLC progression both in vitro and in vivo. Collectively, our data suggest that CD248-positive CAFs induce NSCLC progression by mediating M2-polarized macrophages.

NK cell-derived exosomes enhance the anti-tumor effects against ovarian cancer by delivering cisplatin and reactivating NK cell functions.

Exosomes are membranous vesicles actively secreted by almost all cells and they deliver certain intracellular molecules, including nucleic acids, proteins, and lipids, to target cells. They are also considered to be good carriers for drug delivery due to their biocompatibility, high permeability, low immunogenicity, and low toxicity. Exosomes from immune cells were also reported to have immunomodulatory activities. Herein we evaluated the application of exosomes derived from expanded natural killer cells (eNK-EXO) for the treatment of ovarian cancer (OC). We demonstrate that eNK-EXO express typical protein markers of natural killer (NK) cells, can be preferentially uptaken by SKOV3 cells, and display cytotoxicity against OC cells. Furthermore, eNK-EXO loaded with cisplatin could sensitize drug-resistant OC cells to the anti-proliferation effect of cisplatin. In addition, we show that eNK-EXO could activate NK cells from immunosuppressive tumor microenvironment, the mechanism of which is explored by transcriptional analysis. In summary, eNK-EXO exhibit anti-tumor activity against OC on its own, could be used to deliver cisplatin and enhance its cytotoxic effect against drug-resistant OC cells and also reverse the immunosuppression of NK cells, which may lead to great prospect of using eNK-EXO in the treatment of OC in the clinic. Our work also builds a strong foundation for further evaluation of eNK-EXO in other solid tumor therapies.

Oncolytic reovirus induces ovarian cancer cell apoptosis in a TLR3-dependent manner.

Globally, ovarian cancer is the seventh most common cancer and the eighth-most common cause of cancer death among women with a five-year survival rate of less than 45%. Although reovirus is known to be effective for treating ovarian cancer, some types of tumor cells still exhibit resistance to reovirus. In order to solve this resistance problem in the treatment of ovarian cancer, we selected the reovirus-resistant OV-90 ovarian cancer cells to study reovirus oncolytic effects. We found that the viability of OV-90 cells decreased after reovirus double-stranded RNA (dsRNA) genome transfection. Interestingly, we observed that chemical blockage of the Toll-like receptor 3 (TLR3)-dsRNA binding complex in OV-90 cells and the inhibition of downstream TLR3 signaling disrupted OV-90 apoptosis triggered by reovirus dsRNA. Together, these results demonstrate that reovirus dsRNA induces reovirus-resistant tumor cell apoptosis through the TLR3 signaling pathway.

Cucurbit[7]uril-anchored polymer vesicles enhance photosensitization in the nucleus.

Effective photosensitizers (PSs) are highly desirable in many applications, such as photodynamic therapy and catalytic chemistry. Here, we demonstrated that vesicles of cucurbit[7]uril (Q[7] or CB[7])-anchored polymers enhanced photosensitization in the nucleus. The polyacrylic acid chain spacer triggered Q[7] polymers on the surfaces of the vesicles at a regular distance, thus not only leading to efficient inhibition of the aggregation induced self-quenching of the porphyrin based cationic PS in aqueous solution but also maintaining the PS at high concentration on the nanoscale via stable host-guest interactions. Further experiments indicated that Q[7] polymer based vesicles as a PS loading vehicle had a high penetration depth, entering the nuclei of cancer cells. Therefore, highly enhanced photosensitization and efficient anticancer effects were achieved.

Correction: Cytokine-induced killer cell delivery enhances the antitumor activity of oncolytic reovirus.

[This corrects the article DOI: 10.1371/journal.pone.0184816.].

Cytokine-induced killer cell delivery enhances the antitumor activity of oncolytic reovirus.

Oncolytic viruses (OV) have recently emerged as a promising therapeutic modality in cancer treatment. OV selectively infect and kill tumor cells, while sparing untransformed cells. The direct cytotoxic effects combined with the capacity to trigger an immune response make OV an appealing combination partner in the burgeoning field of cancer immunotherapy. One of the leading OV therapeutic candidates is the double-stranded RNA virus reovirus. In order to improve the oncolytic activity of reovirus and allow for systemic administration despite the prevalence of neutralizing antibodies, cytokine-induced killer (CIK) cells were explored as cell carriers for reovirus delivery. In this study, CIK cells were successfully loaded with reovirus ex vivo, and viral replication was limited in CIK cells. Confocal microscopy and flow cytometry demonstrated that CIK cells retained reovirus on the surface. Moreover, CIK cells could promote reovirus infection of tumor cells in the presence of neutralizing antibodies; meanwhile, cytotoxicity of CIK cells was increased after loading with reovirus. These findings support further investigation of reovirus and CIK combination for antitumor therapy.

[Overexpression of NHE1 suppresses ABCA1 protein expression via increasing calpain activity in RAW264.7 cells].

Objective To investigate the effect of over-expressed Na+/H+ exchanger 1 (NHE1) on the protein expression of adenosine three phosphate binding cassette transporter A1 (ABCA1) in RAW264.7 cells. Methods RAW264.7 cells were infected with the adenoviral vector encoding NHE1-EGFP (AdNHE1). The infected RAW264.7 cells were subjected to Western blot analysis for NHE1-EGFP fusion protein. The subcellular localization of NHE1-EGFP fusion protein was observed by confocal laser scanning microscopy. NHE1 activity was measured by the method of pH recovery in response to an acute acid pulse. Furthermore, Western blotting was performed to determine ABCA1 protein levels and calpain activity in NHE1-overexpressing RAW264.7 cells. The effect of calpain inhibitor N-acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLN) on ABCA1 protein levels in the presence of TO-901317 was examined by Western blotting. Results NHE1-EGFP fusion protein was highly expressed and localized in cytoplasm and cell membrane of RAW264.7 cells infected with AdNHE1. NHE1-EGFP fusion protein reduced ABCA1 protein expression and increased calpain activity. The calpain inhibitor ALLN blocked the decrease of ABCA1 protein expression. Conclusion Overexpressed NHE1 suppresses the expression of ABCA1 protein via increasing the calpain activity in RAW264.7 cells.