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Showing papers on "Cell culture published in 2016"


Journal ArticleDOI
TL;DR: It is shown that LDHA-associated lactic acid accumulation in melanomas inhibits tumor surveillance by T and NK cells, and is a potent inhibitor of function and survival of T andNK cells leading to tumor immune escape.

948 citations


Journal ArticleDOI
TL;DR: 2D and 3D cultures in vitro as well as different versions of3D cultures are compared to allow for a better understanding of cancer biology and facilitate the study of biomarkers and targeting therapies.
Abstract: Cell culture is a widely used in vitro tool for improving our understanding of cell biology, tissue morphology, and mechanisms of diseases, drug action, protein production and the development of tissue engineering. Most research regarding cancer biology is based on experiments using two-dimensional (2D) cell cultures in vitro. However, 2D cultures have many limitations, such as the disturbance of interactions between the cellular and extracellular environments, changes in cell morphology, polarity, and method of division. These disadvantages led to the creation of models which are more closely able to mimic conditions in vivo. One such method is three-dimensional culture (3D). Optimisation of the culture conditions may allow for a better understanding of cancer biology and facilitate the study of biomarkers and targeting therapies. In this review, we compare 2D and 3D cultures in vitro as well as different versions of 3D cultures.

633 citations


Journal ArticleDOI
TL;DR: The finding that senescent cells can be eliminated pharmacologically paves the way to new strategies for the treatment of age-related pathologies.
Abstract: Senescent cells, formed in response to physiological and oncogenic stresses, facilitate protection from tumourigenesis and aid in tissue repair. However, accumulation of such cells in tissues contributes to age-related pathologies. Resistance of senescent cells to apoptotic stimuli may contribute to their accumulation, yet the molecular mechanisms allowing their prolonged viability are poorly characterized. Here we show that senescent cells upregulate the anti-apoptotic proteins BCL-W and BCL-XL. Joint inhibition of BCL-W and BCL-XL by siRNAs or the small-molecule ABT-737 specifically induces apoptosis in senescent cells. Notably, treatment of mice with ABT-737 efficiently eliminates senescent cells induced by DNA damage in the lungs as well as senescent cells formed in the epidermis by activation of p53 through transgenic p14(ARF). Elimination of senescent cells from the epidermis leads to an increase in hair-follicle stem cell proliferation. The finding that senescent cells can be eliminated pharmacologically paves the way to new strategies for the treatment of age-related pathologies.

610 citations


Journal ArticleDOI
01 Mar 2016-Gut
TL;DR: A 15 kDa protein with anti-inflammatory properties is produced by F. prausnitzii, a commensal bacterium involved in CD pathogenesis, and is able to inhibit the NF-κB pathway in intestinal epithelial cells and to prevent colitis in an animal model.
Abstract: Background Crohn’s disease (CD)-associated dysbiosis is characterised by a loss of Faecalibacterium prausnitzii, whose culture supernatant exerts an anti-inflammatory effect both in vitro and in vivo. However, the chemical nature of the anti-inflammatory compounds has not yet been determined. Methods Peptidomic analysis using mass spectrometry was applied to F. prausnitzii supernatant. Anti-inflammatory effects of identified peptides were tested in vitro directly on intestinal epithelial cell lines and on cell lines transfected with a plasmid construction coding for the candidate protein encompassing these peptides. In vivo, the cDNA of the candidate protein was delivered to the gut by recombinant lactic acid bacteria to prevent dinitrobenzene sulfonic acid (DNBS)-colitis in mice. Results The seven peptides, identified in the F. prausnitzii culture supernatants, derived from a single microbial anti-inflammatory molecule (MAM), a protein of 15 kDa, and comprising 53% of non-polar residues. This last feature prevented the direct characterisation of the putative anti-inflammatory activity of MAM-derived peptides. Transfection of MAM cDNA in epithelial cells led to a significant decrease in the activation of the nuclear factor (NF)-κB pathway with a dose-dependent effect. Finally, the use of a food-grade bacterium, Lactococcus lactis, delivering a plasmid encoding MAM was able to alleviate DNBS-induced colitis in mice. Conclusions A 15 kDa protein with anti-inflammatory properties is produced by F. prausnitzii, a commensal bacterium involved in CD pathogenesis. This protein is able to inhibit the NF-κB pathway in intestinal epithelial cells and to prevent colitis in an animal model.

536 citations


Journal ArticleDOI
TL;DR: This protocol describes a quick and reliable screening method that is suitable for the examination of the impact of chemotherapeutics or other compounds on cell survival and growth inhibition.
Abstract: Adherent cells detach from cell culture plates during cell death. This characteristic can be used for the indirect quantification of cell death and to determine differences in proliferation upon stimulation with death-inducing agents. One simple method to detect maintained adherence of cells is the staining of attached cells with crystal violet dye, which binds to proteins and DNA. Cells that undergo cell death lose their adherence and are subsequently lost from the population of cells, reducing the amount of crystal violet staining in a culture. This protocol describes a quick and reliable screening method that is suitable for the examination of the impact of chemotherapeutics or other compounds on cell survival and growth inhibition. However, characterization of the cause of reduced crystal violet staining requires additional methods detailed elsewhere.

531 citations


Journal ArticleDOI
TL;DR: In this paper, microglia-like cells from human embryonic stem (ES) and induced pluripotent stem (iPS) cells were derived using defined serum-free culture conditions.
Abstract: Microglia, the only lifelong resident immune cells of the central nervous system (CNS), are highly specialized macrophages that have been recognized to have a crucial role in neurodegenerative diseases such as Alzheimer's, Parkinson's and adrenoleukodystrophy (ALD). However, in contrast to other cell types of the human CNS, bona fide microglia have not yet been derived from cultured human pluripotent stem cells. Here we establish a robust and efficient protocol for the rapid production of microglia-like cells from human (h) embryonic stem (ES) and induced pluripotent stem (iPS) cells that uses defined serum-free culture conditions. These in vitro pluripotent stem cell-derived microglia-like cells (termed pMGLs) faithfully recapitulate the expected ontogeny and characteristics of their in vivo counterparts, and they resemble primary fetal human and mouse microglia. We generated these cells from multiple disease-specific cell lines and find that pMGLs derived from an hES model of Rett syndrome are smaller than their isogenic controls. We further describe a platform to study the integration and live behavior of pMGLs in organotypic 3D cultures. This modular differentiation system allows for the study of microglia in highly defined conditions as they mature in response to developmentally relevant cues, and it provides a framework in which to study the long-term interactions of microglia residing in a tissue-like environment.

465 citations


Journal ArticleDOI
16 Jun 2016-Oncogene
TL;DR: Preclinical evidence for the feasibility of novel therapies exploiting ASCT2 transporter activity in breast cancer is provided, particularly in the high-risk basal-like subgroup of TN breast cancer where there is not only high expression of AsCT2, but also a marked reliance on its activity for sustained cellular proliferation.
Abstract: Alanine, serine, cysteine-preferring transporter 2 (ASCT2; SLC1A5) mediates uptake of glutamine, a conditionally essential amino acid in rapidly proliferating tumour cells. Uptake of glutamine and subsequent glutaminolysis is critical for activation of the mTORC1 nutrient-sensing pathway, which regulates cell growth and protein translation in cancer cells. This is of particular interest in breast cancer, as glutamine dependence is increased in high-risk breast cancer subtypes. Pharmacological inhibitors of ASCT2-mediated transport significantly reduced glutamine uptake in human breast cancer cell lines, leading to the suppression of mTORC1 signalling, cell growth and cell cycle progression. Notably, these effects were subtype-dependent, with ASCT2 transport critical only for triple-negative (TN) basal-like breast cancer cell growth compared with minimal effects in luminal breast cancer cells. Both stable and inducible shRNA-mediated ASCT2 knockdown confirmed that inhibiting ASCT2 function was sufficient to prevent cellular proliferation and induce rapid cell death in TN basal-like breast cancer cells, but not in luminal cells. Using a bioluminescent orthotopic xenograft mouse model, ASCT2 expression was then shown to be necessary for both successful engraftment and growth of HCC1806 TN breast cancer cells in vivo. Lower tumoral expression of ASCT2 conferred a significant survival advantage in xenografted mice. These responses remained intact in primary breast cancers, where gene expression analysis showed high expression of ASCT2 and glutamine metabolism-related genes, including GLUL and GLS, in a cohort of 90 TN breast cancer patients, as well as correlations with the transcriptional regulators, MYC and ATF4. This study provides preclinical evidence for the feasibility of novel therapies exploiting ASCT2 transporter activity in breast cancer, particularly in the high-risk basal-like subgroup of TN breast cancer where there is not only high expression of ASCT2, but also a marked reliance on its activity for sustained cellular proliferation.

396 citations


01 Jan 2016
TL;DR: In this article, the authors showed that the B(a)P-treated populations consistently contained cells displaying a longer period of active growth in culture compared to the untreated control cells.
Abstract: Rapidly growing primary cultures of normal human mammary epithelial cells (HMEC) were exposed to 1 ,ug of benzo(a)pyrene (B(a)P) per ml for two or three 24-hr periods. The B(a)P-treated populations consistently contained cells displaying a longer period of active growth in culture compared to the untreated control cells. Widespread heteroge- neity in morphology and growth patterns was evidenced in these "extended life" (EL) cultures, with multiple sequential changes in these parameters occurring during the course of their life in culture. Two apparently immortal continuous cell lines have thus far emerged from these EL cultures. These lines have been characterized to be of human mammary epi- thelial origin and derived from the originally treated HMEC specimen. The continuous lines do not appear to be malignant- ly transformed as they do not cause tumor formation in nude mice and show little or no anchorage-independent growth. Nonetheless, they have acquired several properties character- istic of tumor-derived HMEC, which distinguish them from their normal progenitors. These cell lines, as well as the EL strains, may provide useful substrates for studies to determine what agents can induce further transforming events. Addition- ally, analysis of the multiple steps occurring in the EL cul- tures, as well as in the emergence of the continuous cell lines, could potentially elucidate the processes occurring during hu- man epithelial cell carcinogenesis and escape from senescence.

387 citations


PatentDOI
TL;DR: In this article, a simple, fast, effective and safe directed differentiation of embryonic stem cells into pancreatic beta-like cells secreting insulin in response to glucose levels is described.
Abstract: Methods are provided for the simple, fast, effective and safe directed differentiation of embryonic stem cells into pancreatic beta-like cells secreting insulin in response to glucose levels. The cells are useful transplant therapeutics for diabetic individuals.

340 citations


Journal ArticleDOI
TL;DR: It is shown that in these specific conditions individual inner cell mass cells grow into colonies that may then be expanded over multiple passages while retaining a diploid karyotype and naive properties.
Abstract: Conventional generation of stem cells from human blastocysts produces a developmentally advanced, or primed, stage of pluripotency. In vitro resetting to a more naive phenotype has been reported. However, whether the reset culture conditions of selective kinase inhibition can enable capture of naive epiblast cells directly from the embryo has not been determined. Here, we show that in these specific conditions individual inner cell mass cells grow into colonies that may then be expanded over multiple passages while retaining a diploid karyotype and naive properties. The cells express hallmark naive pluripotency factors and additionally display features of mitochondrial respiration, global gene expression, and genome-wide hypomethylation distinct from primed cells. They transition through primed pluripotency into somatic lineage differentiation. Collectively these attributes suggest classification as human naive embryonic stem cells. Human counterparts of canonical mouse embryonic stem cells would argue for conservation in the phased progression of pluripotency in mammals.

285 citations


Journal ArticleDOI
TL;DR: Understanding of PD-L1 functions is extended, nonimmune effects of anti-PD-L 1 immunotherapy are illustrated, and broader uses for PD- L1 as a biomarker for assessing cancer therapeutic responses are suggested.
Abstract: PD-L1 antibodies produce efficacious clinical responses in diverse human cancers, but the basis for their effects remains unclear, leaving a gap in understanding of how to rationally leverage the therapeutic activity. PD-L1 is widely expressed in tumor cells but its contributions to tumor pathogenicity are incompletely understood. In this study, we evaluated the hypothesis that PD-L1 exerts tumor cell-intrinsic signals that are critical for pathogenesis. Using RNAi methodology, we attenuated PD-L1 in the murine ovarian cell line ID8agg and the melanoma cell line B16 (termed PD-L1lo cells), which express basal PD-L1. We observed that PD-L1lo cells proliferated more weakly than control cells in vitro. As expected, PD-L1lo cells formed tumors in immunocompetent mice relatively more slowly, but unexpectedly, they also formed tumors more slowly in immunodeficient NSG mice. A comparative microarray analysis identified a number of genes involved in autophagy and mTOR signaling that were affected by PD-L1 expression. In support of a functional role, PD-L1 attenuation augmented autophagy and blunted the ability of autophagy inhibitors to limit proliferation in vitro and in vivo in NSG mice. PD-L1 attenuation also elevated mTORC1 activity and augmented the anti-proliferative effects of the mTORC1 inhibitor rapamycin. PD-L1 cells were also relatively deficient in metastasis to the lung and we found that anti-PD-L1 administration could block tumor cell growth and metastasis in NSG mice. This therapeutic effect was observed with B16 cells but not ID8agg cells, illustrating tumor- or tissue-specific effects in the therapeutic setting. Overall, our findings extend understanding of PD-L1 functions, illustrate non-immune effects of anti-PD-L1 immunotherapy and suggest broader uses for PD-L1 as a biomarker for assessing cancer therapeutic responses.

Journal ArticleDOI
TL;DR: Use of ΔΨm-based sorting to enrich for cells with superior metabolic features was observed in CD8(+), CD4(+) T cell subsets, and long-term hematopoietic stem cells, and this metabolism-based approach to cell selection may be broadly applicable to therapies involving the transfer of HSC or lymphocytes for the treatment of viral-associated illnesses and cancer.

Journal ArticleDOI
TL;DR: The Scavenger Receptor Class A family (SR-A) is identified as a novel monocyte/macrophage uptake receptor for EV and blockade of SR-A with dextran sulfate dramatically decreased EV liver clearance in mice, while enhancing tumor accumulation.

Journal ArticleDOI
TL;DR: The events following activation of third-generation CAR T cells specific for GD2 suggest that deletion also occurs in vivo and that PD-1-targeted combination therapy approaches may be useful to augment CAR T-cell efficacy and persistence in patients.

01 Jan 2016
TL;DR: In this article, a medium that supports long-term growth in culture of human primary mammary tumor cells, of normal epithelial cells from mammoplasty, and of mammary cancer cell lines was described.
Abstract: A medium is described that supports long- term growth in culture of human primary mammary tumor cells, of normal epithelial cells from mammoplasty, and of mammary tumor cell lines. Tumor cells are shown to be distinguishable from normal mammary epithelial cells by morphology, by growth requirements, and by two markers: preferential expression of the HMFG-2 epitope on tumor cells and preferential retention in tumor cell mitochondria of the lipophilic fluorescent dye rhodamine 123. Differential fluores- cence of HMFG-2 fluorescein-conjugated antibodies can be used as a basis for separation of normal and tumor cells in a fluorescence-activated cell sorter, as can differential retention of rhodamine 123.

Journal ArticleDOI
TL;DR: A protumorigenic subset of B cells that constitutively expressed higher levels of programmed cell death-1 (PD-1) and constituted ∼10% of all B cells in advanced-stage hepatocellular carcinoma (HCC) is identified, providing significant new insights for human cancer immunosuppression and anticancer therapies regarding PD-1/PD-L1.
Abstract: B cells consistently represent abundant cellular components in human tumors. Regulatory B cells that are functionally defined by their ability to produce IL-10 downregulate inflammation and control T cell immunity. Here we identified a protumorigenic subset of B cells that constitutively expressed higher levels of programmed cell death-1 (PD-1) and constituted ~10% of all B cells in advanced stage hepatocellular carcinoma (HCC). These PD-1high B cells exhibited a unique CD5highCD24−/+CD27high/+CD38dim phenotype different from the phenotype of conventional CD24highCD38high peripheral regulatory B cells. TLR4-mediated Bcl-6 upregulation was crucial for PD-1high B cell induction by HCC environmental factors, and that effect was abolished by IL-4-elicited STAT6 phosphorylation. Importantly, upon encountering PD-L1+ cells or undergoing PD-1 triggering, PD-1high B cells acquired regulatory functions that suppressed tumor-specific T cell immunity and promoted cancer growth via IL-10 signals. Our findings provide significant new insights for human cancer immunosuppression and anticancer therapies regarding PD-1/PD-L1.

Journal ArticleDOI
TL;DR: An easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y, which provides researchers with a more accurate translational model of human infection and disease.
Abstract: Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods(1-4) and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

Journal ArticleDOI
TL;DR: 3D bioprintedglioma model provides a novel alternative tool for studying gliomagenesis, glioma stem cell biology, drug resistance, and anticancer drug susceptibility in vitro.
Abstract: Glioma is still difficult to treat because of its high malignancy, high recurrence rate, and high resistance to anticancer drugs. An alternative method for research of gliomagenesis and drug resistance is to use in vitro tumor model that closely mimics the in vivo tumor microenvironment. In this study, we established a 3D bioprinted glioma stem cell model, using modified porous gelatin/alginate/fibrinogen hydrogel that mimics the extracellular matrix. Glioma stem cells achieved a survival rate of 86.92%, and proliferated with high cellular activity immediately following bioprinting. During the in vitro culture period, the printed glioma stem cells not only maintained their inherent characteristics of cancer stem cells (Nestin), but also showed differentiation potential (glial fibrillary acidic protein and β-tubulin III). In order to verify the vascularization potential of glioma stem cells, tumor angiogenesis biomarker, vascular endothelial growth factor was detected by immunohistochemistry, and its expression increased from week one to three during the culture period. Drug-sensitivity results showed that 3D printed tumor model was more resistant to temozolomide than 2D monolayer model at TMZ concentrations of 400-1600 μg ml-1. In summary, 3D bioprinted glioma model provides a novel alternative tool for studying gliomagenesis, glioma stem cell biology, drug resistance, and anticancer drug susceptibility in vitro.

01 Jan 2016
TL;DR: In this paper, a plasmid containing the long terminal repeat (LTR) sequence of human T-cell leukemia virus type I (HTLV-I) was constructed by constructing plasmids contain- ing the LTR sequence.
Abstract: Promoter function for gene expression of the long terminal repeat (LTR) of human T-cell leukemia virus type I (HTLV-I) was studied by constructing plasmids contain- ing the LTR sequence. The gene encoding chloramphenicol acetyltransferase (CATase) was linked to an HTLV-I LTR se- quence (pLTR-CAT) by replacing the simian virus 40 promot- er in plasmid pSV2-CAT with the LTR sequence. The tran- sient CATase activities of cells transfected with the plasmids were compared. The results are summarized as follows: (i) The HTLV LTR was active even in an epithelial cell line, with effi- ciency similar to that of the simian virus 40 promoter. (ii) pLTR-CAT expressed high CATase activity, 40-200 times that expressed by pSV2-CAT, in HTLV-I-infected T-cell lines, such as the human cell lines MT-2 and HUT-102, or in HTLV-I- infected rat cell lines. (iii) This enhanced activity of the LTR seems to be associated with HTLV gene expression, since only low activity of pLTR-CAT was observed in the HTLV-infected cell line MT-1, in which only a small percent of cells express viral antigens. (iv) In HTLV-infected rat cell lines, the pX-en- coded protein p40X was the only viral protein detected. Thus, we suggest that p40x is the factor associated directly or indi- rectly with the enhanced activity of the LTR.

Journal ArticleDOI
TL;DR: It is demonstrated that tumorigenic cancer cell lines, transformed primary human fibroblasts, and tumors in vivo respond similarly but that autophagy is insufficient for survival, and cancer cells die while their normal counterparts are spared.

Book
13 Jul 2016
TL;DR: The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions, validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions.
Abstract: Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions.

Journal ArticleDOI
TL;DR: 3D cell cultures demonstrate higher innate resistance to anti-cancer drugs compared to 2D cultures, which may be facilitated by the altered receptor proteins, drug transporters and metabolising enzyme activity.
Abstract: // Susan Breslin 1 , Lorraine O’Driscoll 1 1 School of Pharmacy and Pharmaceutical Sciences & Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland Correspondence to: Lorraine O’Driscoll, email: lodrisc@tcd.ie Keywords: 3D cell culture, monolayer culture, breast cancer, targeted drugs, drug resistance mechanisms Received: November 23, 2015 Accepted: May 28, 2016 Published: June 10, 2016 ABSTRACT Solid tumours naturally grow in 3D wherein the spatial arrangement of cells affects how they interact with each other. This suggests that 3D cell culture may mimic the natural in vivo setting better than traditional monolayer (2D) cell culture, where cells are grown attached to plastic. Here, using HER2-positive breast cancer cell lines as models (BT474, HCC1954, EFM192A), the effects of culturing cells in 3D using the poly-HEMA method compared to 2D cultures were assessed in terms of cellular viability, response/resistance to anti-cancer drugs, protein expression and enzyme activity. Scanning electron microscopy showed the morphology of cells in 3D to be substantially different to those cultured in 2D. Cell viability in 3D cells was substantially lower than that of cells in 2D cultures, while 3D cultures were more resistant to the effects of HER-targeted (neratinib) and classical chemotherapy (docetaxel) drugs. Expression of proteins involved in cell survival, transporters associated with drug resistance and drug targets were increased in 3D cultures. Finally, activity of drug metabolising enzyme CYP3A4 was substantially increased in 3D compared to 2D cultures. Together this data indicates that the biological information represented by 3D and 2D cell cultures is substantially different i.e. 3D cell cultures demonstrate higher innate resistance to anti-cancer drugs compared to 2D cultures, which may be facilitated by the altered receptor proteins, drug transporters and metabolising enzyme activity. This highlights the importance of considering 3D in addition to 2D culture methods in pre-clinical studies of both newer targeted and more traditional anti-cancer drugs.

Journal ArticleDOI
TL;DR: It is shown that Oas3, but not OAS1 or OAS2, is required to activate RNase L and to restrict the replication of four different human viruses, suggesting that OAS3 may provide a target for antiviral therapies and thatOAS1 and OAS 2 may have alternative roles.
Abstract: The 2′,5′-oligoadenylate (2-5A) synthetase (OAS)–RNase L system is an IFN-induced antiviral pathway. RNase L activity depends on 2-5A, synthesized by OAS. Although all three enzymatically active OAS proteins in humans—OAS1, OAS2, and OAS3—synthesize 2-5A upon binding dsRNA, it is unclear which are responsible for RNase L activation during viral infection. We used clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein-9 nuclease (Cas9) technology to engineer human A549-derived cell lines in which each of the OAS genes or RNase L is knocked out. Upon transfection with poly(rI):poly(rC), a synthetic surrogate for viral dsRNA, or infection with each of four viruses from different groups (West Nile virus, Sindbis virus, influenza virus, or vaccinia virus), OAS1-KO and OAS2-KO cells synthesized amounts of 2-5A similar to those synthesized in parental wild-type cells, causing RNase L activation as assessed by rRNA degradation. In contrast, OAS3-KO cells synthesized minimal 2-5A, and rRNA remained intact, similar to infected RNase L-KO cells. All four viruses replicated to higher titers in OAS3-KO or RNase L-KO A549 cells than in parental, OAS1-KO, or OAS2-KO cells, demonstrating the antiviral effects of OAS3. OAS3 displayed a higher affinity for dsRNA in intact cells than either OAS1 or OAS2, consistent with its dominant role in RNase L activation. Finally, the requirement for OAS3 as the major OAS isoform responsible for RNase L activation was not restricted to A549 cells, because OAS3-KO cells derived from two other human cell lines also were deficient in RNase L activation.

Journal ArticleDOI
TL;DR: The molecular and cellular validation of enzyme-instructed self-assembly (EISA) as a multiple step process for selectively killing cancer cells that overexpress alkaline phosphatases (ALPs) and the cell death modality likely associates with the cell type and the interactions between nanofibers and the death receptors are proved.
Abstract: Selective inhibition of cancer cells remains a challenge in chemotherapy. Here we report the molecular and cellular validation of enzyme-instructed self-assembly (EISA) as a multiple step process for selectively killing cancer cells that overexpress alkaline phosphatases (ALPs). We design and synthesize two kinds of D-tetrapeptide containing one or two phosphotyrosine residues and with the N-terminal capped by a naphthyl group. Upon enzymatic dephosphorylation, these D-tetrapeptides turn into self-assembling molecules to form nanofibers in water. Incubating these D-tetrapeptides with several cancer cell lines and one normal cell line, the unphosphorylated D-tetrapeptides are innocuous to all the cell lines, the mono- and diphosphorylated D-tetrapeptides selectively inhibit the cancer cells, but not the normal cell. The monophosphorylated D-tetrapeptides exhibit more potent inhibitory activity than the diphosphorylated D-tetrapeptides do; the cancer cell lines express higher level of ALPs are more susceptible to inhibition by the phosphorylated D-tetrapeptides; the precursors of D-tetrapeptides that possess higher self-assembling abilities exhibit higher inhibitory activities. These results confirm the important role of enzymatic reaction and self-assembly. Using uncompetitive inhibitors of ALPs and fluorescent D-tetrapeptides, we delineate that the enzyme catalyzed dephosphorylation and the self-assembly steps, together, result in the localization of the nanofibers of D-tetrapeptides for killing the cancer cells. We find that the cell death modality likely associates with the cell type and prove the interactions between nanofibers and the death receptors. This work illustrates a paradigm-shifting and biomimetic approach and contributes useful molecular insights for the development of spatiotemporal defined supramolecular processes/assemblies as potential anticancer therapeutics.

Journal ArticleDOI
01 Feb 2016-Gut
TL;DR: A robust and quasi-immortal 3D organoid model has been established, which is considered instrumental for future research aimed to understand the underlying mechanisms of infection, mucosal immunity and cancer of the human stomach.
Abstract: Background and aims Helicobacter pylori is the causative agent of gastric diseases and the main risk factor in the development of gastric adenocarcinoma. In vitro studies with this bacterial pathogen largely rely on the use of transformed cell lines as infection model. However, this approach is intrinsically artificial and especially inappropriate when it comes to investigating the mechanisms of cancerogenesis. Moreover, common cell lines are often defective in crucial signalling pathways relevant to infection and cancer. A long-lived primary cell system would be preferable in order to better approximate the human in vivo situation. Methods Gastric glands were isolated from healthy human stomach tissue and grown in Matrigel containing media supplemented with various growth factors, developmental regulators and apoptosis inhibitors to generate long-lasting normal epithelial cell cultures. Results Culture conditions were developed which support the formation and quasi-indefinite growth of three dimensional (3D) spheroids derived from various sites of the human stomach. Spheroids could be differentiated to gastric organoids after withdrawal of Wnt3A and R-spondin1 from the medium. The 3D cultures exhibit typical morphological features of human stomach tissue. Transfer of sheared spheroids into 2D culture led to the formation of dense planar cultures of polarised epithelial cells serving as a suitable in vitro model of H. pylori infection. Conclusions A robust and quasi-immortal 3D organoid model has been established, which is considered instrumental for future research aimed to understand the underlying mechanisms of infection, mucosal immunity and cancer of the human stomach.

Journal ArticleDOI
TL;DR: A three-dimensional differentiation protocol for generating proximal airway epithelial progenitor cell spheroids from CPM+ VAFECs is reported, which could be induced to generate MCACs and other airway lineage cells without alveolar epithelial cells.
Abstract: Multi-ciliated airway cells (MCACs) play a role in mucociliary clearance of the lung. However, the efficient induction of functional MCACs from human pluripotent stem cells has not yet been reported. Using carboxypeptidase M (CPM) as a surface marker of NKX2-1+-ventralized anterior foregut endoderm cells (VAFECs), we report a three-dimensional differentiation protocol for generating proximal airway epithelial progenitor cell spheroids from CPM+ VAFECs. These spheroids could be induced to generate MCACs and other airway lineage cells without alveolar epithelial cells. Furthermore, the directed induction of MCACs and of pulmonary neuroendocrine lineage cells was promoted by adding DAPT, a Notch pathway inhibitor. The induced MCACs demonstrated motile cilia with a “9 + 2” microtubule arrangement and dynein arms capable of beating and generating flow for mucociliary transport. This method is expected to be useful for future studies on human airway disease modeling and regenerative medicine.

Journal ArticleDOI
TL;DR: Exosomal miR‐24‐3p is involved in tumour pathogenesis by mediating T‐cell suppression via repression of FGF11, and may serve as a potential prognostic biomarker in NPC.
Abstract: Recent studies have shown that extracellular microRNAs are not only potential biomarkers but are also involved in cell interactions to regulate the intercommunication between cancer cells and their microenvironments in various types of malignancies. In this study, we isolated exosomes from nasopharyngeal carcinoma (NPC) cell lines and patient sera (T-EXOs), or control NP69 cells and healthy donor sera (HD-EXOs). We found that miR-24-3p was markedly enriched in T-EXOs as compared with HD-EXOs; the serum exosomal miR-24-3p level was correlated with worse disease-free survival of patients (p < 0.05). Knockdown of exosomal miR-24-3p (miR-24-3p-sponge-T-EXOs) by a sponge RNA targeting miR-24-3p restored the T-EXO-mediated (control-sponge-T-EXO) inhibition of T-cell proliferation and Th1 and Th17 differentiation, and the induction of regulatory T cells (Tregs). Mechanistic analyses revealed that administration of exosomal miR-24-3p increased P-ERK, P-STAT1 and P-STAT3 expression while decreasing P-STAT5 expression during T-cell proliferation and differentiation. Moreover, by in vivo and in vitro assessments, we found FGF11 to be a direct target of miR-24-3p. However, both miR-24-3p-sponge-T-EXOs and T-EXOs (control-sponge-T-EXOs) impeded proliferation and Th1 and Th17 differentiation, but induced Treg differentiation, of lenti-shFGF11-transfected T cells. The levels of phosphorylated ERK and STAT proteins were different in lenti-ScshRNA-transfected T cells and lenti-shFGF11-transfected T cells following administration of miR-24-3p-sponge-T-EXO. Interestingly, tumour FGF11 expression was positively correlated with the number of CD4+ and CD8+ T cells in vivo, and predicted favourable patient DFS (p < 0.05). Additionally, hypoxia increased cellular and exosomal miR-24-3p levels and enhanced the inhibitory effect of T-EXO on T-cell proliferation and differentiation. Collectively, our findings suggest that exosomal miR-24-3p is involved in tumour pathogenesis by mediating T-cell suppression via repression of FGF11, and may serve as a potential prognostic biomarker in NPC. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Journal ArticleDOI
TL;DR: HLA-F is established as a ligand of KIR3DS1 and cell-context-dependent expression of HLA- F is demonstrated that might explain the widespread influence of Kir3 DS1 in human disease.
Abstract: The activating natural killer (NK)-cell receptor KIR3DS1 has been linked to the outcome of various human diseases, including delayed progression of disease caused by human immunodeficiency virus type 1 (HIV-1), yet a ligand that would account for its biological effects has remained unknown. We screened 100 HLA class I proteins and found that KIR3DS1 bound to HLA-F, a result we confirmed biochemically and functionally. Primary human KIR3DS1(+) NK cells degranulated and produced antiviral cytokines after encountering HLA-F and inhibited HIV-1 replication in vitro. Activation of CD4(+) T cells triggered the transcription and surface expression of HLA-F mRNA and HLA-F protein, respectively, and induced binding of KIR3DS1. HIV-1 infection further increased the transcription of HLA-F mRNA but decreased the binding of KIR3DS1, indicative of a mechanism for evading recognition by KIR3DS1(+) NK cells. Thus, we have established HLA-F as a ligand of KIR3DS1 and have demonstrated cell-context-dependent expression of HLA-F that might explain the widespread influence of KIR3DS1 in human disease.

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TL;DR: Exosome-mimetic nanovesicles can be a platform for RNAi delivery to cell cytoplasm and are taken up by recipient cells, which resulted in attenuation of target gene expression.

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TL;DR: It is reported that rare SG ductal EpCAM+ cells express nuclear β-catenin, indicating active Wnt signaling, allowing extensive in vitro expansion and enabling restoration of SG function upon transplantation.
Abstract: Adult stem cells are the ultimate source for replenishment of salivary gland (SG) tissue. Self-renewal ability of stem cells is dependent on extrinsic niche signals that have not been unraveled for the SG. The ductal compartment in SG has been identified as the location harboring stem cells. Here, we report that rare SG ductal EpCAM(+) cells express nuclear β-catenin, indicating active Wnt signaling. In cell culture experiments, EpCAM(high) cells respond potently to Wnt signals stimulating self-renewal and long-term expansion of SG organoids, containing all differentiated SG cell types. Conversely, Wnt inhibition ablated long-term organoid cultures. Finally, transplantation of cells pre-treated with Wnt agonists into submandibular glands of irradiated mice successfully and robustly restored saliva secretion and increased the number of functional acini in vivo. Collectively, these results identify Wnt signaling as a key driver of adult SG stem cells, allowing extensive in vitro expansion and enabling restoration of SG function upon transplantation.