Showing papers on "Cell culture published in 2012"
TL;DR: It is found that mutant p53 depletion is sufficient to phenotypically revert breast cancer cells to a more acinar-like morphology, which implicate the mevalonate pathway as a therapeutic target for tumors bearing mutations in p53.
735 citations
TL;DR: It is demonstrated that a Rho kinase inhibitor (Y-27632), in combination with fibroblast feeder cells, induces normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro, without transduction of exogenous viral or cellular genes.
Abstract: We demonstrate that a Rho kinase inhibitor (Y-27632), in combination with fibroblast feeder cells, induces normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro, without transduction of exogenous viral or cellular genes. Primary prostate and mammary cells, for example, are reprogrammed toward a basaloid, stem-like phenotype and form well-organized prostaspheres and mammospheres in Matrigel. However, in contrast to the selection of rare stem-like cells, the described growth conditions can generate 2 × 106 cells in 5 to 6 days from needle biopsies, and can generate cultures from cryopreserved tissue and from fewer than four viable cells. Continued cell proliferation is dependent on both feeder cells and Y-27632, and the conditionally reprogrammed cells (CRCs) retain a normal karyotype and remain nontumorigenic. This technique also efficiently establishes cell cultures from human and rodent tumors. For example, CRCs established from human prostate adenocarcinoma displayed instability of chromosome 13, proliferated abnormally in Matrigel, and formed tumors in mice with severe combined immunodeficiency. The ability to rapidly generate many tumor cells from small biopsy specimens and frozen tissue provides significant opportunities for cell-based diagnostics and therapeutics (including chemosensitivity testing) and greatly expands the value of biobanking. In addition, the CRC method allows for the genetic manipulation of epithelial cells ex vivo and their subsequent evaluation in vivo in the same host.
672 citations
TL;DR: This review article focuses on current strategies and achievements in cell line development, mainly in vector engineering and cell engineering, for high and stable protein production in rCHO cells.
Abstract: Recombinant Chinese hamster ovary cells (rCHO) cells have been the most commonly used mammalian host for large-scale commercial production of therapeutic proteins. Recent advances in cell culture technology for rCHO cells have achieved significant improvement in protein production leading to titer of more than 10 g/L to meet the huge demand from market needs. This achievement is associated with progression in the establishment of high and stable producer and the optimization of culture process including media development. In this review article, we focus on current strategies and achievements in cell line development, mainly in vector engineering and cell engineering, for high and stable protein production in rCHO cells. The approaches that manipulate various DNA elements for gene targeting by site-specific integration and cis-acting elements to augment and stabilize gene expression are reviewed here. The genetic modulation strategy by “direct” cell engineering with growth-promoting and/or productivity-enhancing factors and omics-based approaches involved in transcriptomics, proteomics, and metabolomics to pursue cell engineering are also presented.
648 citations
TL;DR: It is demonstrated that multiwalled carbon nanotubes (MWCNTs) have the ability to enhance the growth of tobacco cell culture in a wide range of concentrations.
Abstract: Carbon nanotubes have shown promise as regulators of seed germination and plant growth. Here, we demonstrate that multiwalled carbon nanotubes (MWCNTs) have the ability to enhance the growth of tobacco cell culture (55-64% increase over control) in a wide range of concentrations (5-500 μg/mL). Activated carbon (AC) stimulated cell growth (16% increase) only at low concentrations (5 μg/mL) while dramatically inhibited the cellular growth at higher concentrations (100-500 μg/mL). We found a correlation between the activation of cells growth exposed to MWCNTs and the upregulation of genes involved in cell division/cell wall formation and water transport. The expression of the tobacco aquaporin (NtPIP1) gene, as well as production of the NtPIP1 protein, significantly increased in cells exposed to MWCNTs compared to control cells or those exposed to AC. The expression of marker genes for cell division (CycB) and cell wall extension (NtLRX1) was also up-regulated in cells exposed to MWCNTs compared to control cells or those exposed to activated carbon only.
566 citations
TL;DR: Although cells in different phases of the cell cycle were found to internalize nanoparticles at similar rates, after 24 h the concentration of nanoparticles in the cells could be ranked according to the different phases: G2/M > S > G0/G1.
Abstract: Nanoparticles are considered a primary vehicle for targeted therapies because they can pass biological barriers and enter and distribute within cells by energy-dependent pathways. So far, most studies have shown that nanoparticle properties, such as size and surface, can influence how cells internalize nanoparticles. Here, we show that uptake of nanoparticles by cells is also influenced by their cell cycle phase. Although cells in different phases of the cell cycle were found to internalize nanoparticles at similar rates, after 24 h the concentration of nanoparticles in the cells could be ranked according to the different phases: G2/M > S > G0/G1. Nanoparticles that are internalized by cells are not exported from cells but are split between daughter cells when the parent cell divides. Our results suggest that future studies on nanoparticle uptake should consider the cell cycle, because, in a cell population, the dose of internalized nanoparticles in each cell varies as the cell advances through the cell cycle.
559 citations
TL;DR: It is shown here that senescent cells induce a DNA damage response, characteristic for senescence, in neighbouring cells via gap junction‐mediated cell–cell contact and processes involving ROS, which can induce a bystander effect, spreading senescences towards their neighbours in vitro and, possibly, in vivo.
Abstract: Senescent cells produce and secrete various bioactive molecules including interleukins, growth factors, matrix-degrading enzymes and reactive oxygen species (ROS). Thus, it has been proposed that senescent cells can damage their local environment, and a stimulatory effect on tumour cell growth and invasiveness has been documented. However, it was unknown what effect, if any, senescent cells have on their normal, proliferation-competent counterparts. We show here that senescent cells induce a DNA damage response, characteristic for senescence, in neighbouring cells via gap junction-mediated cell-cell contact and processes involving ROS. Continuous exposure to senescent cells induced cell senescence in intact bystander fibroblasts. Hepatocytes bearing senescence markers clustered together in mice livers. Thus, senescent cells can induce a bystander effect, spreading senescence towards their neighbours in vitro and, possibly, in vivo.
553 citations
TL;DR: A potent and selective small molecule inhibitor, EI1, is developed, which inhibits the enzymatic activity of Ezh2 through direct binding to the enzyme and competing with the methyl group donor S-Adenosyl methionine, providing strong validation of EzH2 as a potential therapeutic target for the treatment of cancer.
Abstract: Ezh2 (Enhancer of zeste homolog 2) protein is the enzymatic component of the Polycomb repressive complex 2 (PRC2), which represses gene expression by methylating lysine 27 of histone H3 (H3K27) and regulates cell proliferation and differentiation during embryonic development. Recently, hot-spot mutations of Ezh2 were identified in diffused large B-cell lymphomas and follicular lymphomas. To investigate if tumor growth is dependent on the enzymatic activity of Ezh2, we developed a potent and selective small molecule inhibitor, EI1, which inhibits the enzymatic activity of Ezh2 through direct binding to the enzyme and competing with the methyl group donor S-Adenosyl methionine. EI1-treated cells exhibit genome-wide loss of H3K27 methylation and activation of PRC2 target genes. Furthermore, inhibition of Ezh2 by EI1 in diffused large B-cell lymphomas cells carrying the Y641 mutations results in decreased proliferation, cell cycle arrest, and apoptosis. These results provide strong validation of Ezh2 as a potential therapeutic target for the treatment of cancer.
528 citations
TL;DR: This protocol to isolate and culture pyramidal neurons from the early postnatal mouse hippocampus and cortex is presented and can be used to culture and maintain neurons for a variety of applications including immunocytochemistry, biochemical studies, shRNA-mediated knockdown and live imaging studies.
Abstract: The ability to culture and maintain postnatal mouse hippocampal and cortical neurons is highly advantageous, particularly for studies on genetically engineered mouse models. Here we present a protocol to isolate and culture pyramidal neurons from the early postnatal (P0-P1) mouse hippocampus and cortex. These low-density dissociated cultures are grown on poly-L-lysine–coated glass substrates without feeder layers. Cultured neurons survive well, develop extensive axonal and dendritic arbors, express neuronal and synaptic markers, and form functional synaptic connections. Further, they are highly amenable to low- and high-efficiency transfection and time-lapse imaging. This optimized cell culture technique can be used to culture and maintain neurons for a variety of applications including immunocytochemistry, biochemical studies, shRNA-mediated knockdown and live imaging studies. The preparation of the glass substrate must begin 5 d before the culture. The dissection and plating out of neurons takes 3–4 h and neurons can be maintained in culture for up to 4 weeks.
526 citations
TL;DR: In this paper, the authors developed K562-based artificial antigen-presenting cells with membrane-bound IL-21 (mbIL21) and assessed their ability to support human NK-cell proliferation.
Abstract: NK cells have therapeutic potential for a wide variety of human malignancies. However, because NK cells expand poorly in vitro, have limited life spans in vivo, and represent a small fraction of peripheral white blood cells, obtaining sufficient cell numbers is the major obstacle for NK-cell immunotherapy. Genetically-engineered artificial antigen-presenting cells (aAPCs) expressing membrane-bound IL-15 (mbIL15) have been used to propagate clinical-grade NK cells for human trials of adoptive immunotherapy, but ex vivo proliferation has been limited by telomere shortening. We developed K562-based aAPCs with membrane-bound IL-21 (mbIL21) and assessed their ability to support human NK-cell proliferation. In contrast to mbIL15, mbIL21-expressing aAPCs promoted log-phase NK cell expansion without evidence of senescence for up to 6 weeks of culture. By day 21, parallel expansion of NK cells from 22 donors demonstrated a mean 47,967-fold expansion (median 31,747) when co-cultured with aAPCs expressing mbIL21 compared to 825-fold expansion (median 325) with mbIL15. Despite the significant increase in proliferation, mbIL21-expanded NK cells also showed a significant increase in telomere length compared to freshly obtained NK cells, suggesting a possible mechanism for their sustained proliferation. NK cells expanded with mbIL21 were similar in phenotype and cytotoxicity to those expanded with mbIL15, with retained donor KIR repertoires and high expression of NCRs, CD16, and NKG2D, but had superior cytokine secretion. The mbIL21-expanded NK cells showed increased transcription of the activating receptor CD160, but otherwise had remarkably similar mRNA expression profiles of the 96 genes assessed. mbIL21-expanded NK cells had significant cytotoxicity against all tumor cell lines tested, retained responsiveness to inhibitory KIR ligands, and demonstrated enhanced killing via antibody-dependent cell cytotoxicity. Thus, aAPCs expressing mbIL21 promote improved proliferation of human NK cells with longer telomeres and less senescence, supporting their clinical use in propagating NK cells for adoptive immunotherapy.
506 citations
TL;DR: This review suggests that an energy density value of 0.5 to 4.0 J/cm2 and a visible spectrum ranging from 600 to 700 nm of LLLT are very helpful in enhancing the proliferation rate of various cell lines.
Abstract: The aim of this work is to review the available literature on the details of low-level laser therapy (LLLT) use for the enhancement of the proliferation of various cultured cell lines including stem cells. A cell culture is one of the most useful techniques in science, particularly in the production of viral vaccines and hybrid cell lines. However, the growth rate of some of the much-needed mammalian cells is slow. LLLT can enhance the proliferation rate of various cell lines. Literature review from 1923 to 2010. By investigating the outcome of LLLT on cell cultures, many articles report that it produces higher rates of ATP, RNA, and DNA synthesis in stem cells and other cell lines. Thus, LLLT improves the proliferation of the cells without causing any cytotoxic effects. Mainly, helium neon and gallium-aluminum-arsenide (Ga-Al-As) lasers are used for LLLT on cultured cells. The results of LLLT also vary according to the applied energy density and wavelengths to which the target cells are subjected. This review suggests that an energy density value of 0.5 to 4.0 J/cm2 and a visible spectrum ranging from 600 to 700 nm of LLLT are very helpful in enhancing the proliferation rate of various cell lines. With the appropriate use of LLLT, the proliferation rate of cultured cells, including stem cells, can be increased, which would be very useful in tissue engineering and regenerative medicine.
440 citations
TL;DR: In this paper, the authors presented methods of generating a neuronal cell from a differentiated non-neuronal cell by increasing the amount of miR-124 microRNA, a MYT1L transcription factor, and a BRN2 transcription factor.
TL;DR: Using rosiglitazone (2 μM) as an additional prodifferentiative agent, apparently complete differentiation of 3T3-L1 cells is achieved within 10 to 12 days that persisted for at least up to cell culture passage 10.
01 Jan 2012
TL;DR: It has to be kept in mind that cell lines do not behave identically with primary cells and should not be used to replace primary cells, and key control experiments using primary cells should always be performed.
Abstract: Cell lines are often used in place of primary cells to study biological processes. However, care must be taken when interpreting the results as cell lines do not always accurately replicate the primary cells. In this article, we will briefly talk about advantages and disadvantages of cell lines and then discuss results using the mouse Sertoli cell line, MSC-1, compared with primary mouse Sertoli cells. MSC-1 cells resemble Sertoli cells morphologically and possess several biochemical markers associated with Sertoli cells. Studies have demonstrated that the function and regulation of retinoic acid receptor α (RARα) is similar between MSC-1 and rat Sertoli cells. However, MSC-1 cells lack some of the immune privilege properties associated with primary Sertoli cells, including survival in animals with a fully functional immune system. Therefore, it has to be kept in mind that cell lines do not behave identically with primary cells and should not be used to replace primary cells. In order to strengthen the findings, key control experiments using primary cells should always be performed.
TL;DR: This study provides a method for generating patient-specific airway epithelial cells for disease modeling and in vitro drug testing and reports an in vitro directed differentiation protocol for generating functional CFTR-expressingAirway epithelia from human embryonic stem cells.
Abstract: Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene, which regulates chloride and water transport across all epithelia and affects multiple organs, including the lungs. Here we report an in vitro directed differentiation protocol for generating functional CFTR-expressing airway epithelia from human embryonic stem cells. Carefully timed treatment by exogenous growth factors that mimic endoderm developmental pathways in vivo followed by air-liquid interface culture results in maturation of patches of tight junction–coupled differentiated airway epithelial cells that demonstrate active CFTR transport function. As a proof of concept, treatment of CF patient induced pluripotent stem cell–derived epithelial cells with a small-molecule compound to correct for the common CF processing mutation resulted in enhanced plasma membrane localization of mature CFTR protein. Our study provides a method for generating patient-specific airway epithelial cells for disease modeling and in vitro drug testing.
TL;DR: The tissue specificity of both the global levels and locus-specific distribution of 5hmC in several human tissues and cell lines is investigated, finding that global 5mC content of normal human tissues is highly variable, does not correlate with global 5MC content, and decreases rapidly as cells from normal tissue adapt to cell culture.
Abstract: The discovery of substantial amounts of 5-hydroxymethylcytosine (5hmC), formed by the oxidation of 5-methylcytosine (5mC), in various mouse tissues and human embryonic stem (ES) cells has necessitated a reevaluation of our knowledge of 5mC/5hmC patterns and functions in mammalian cells. Here, we investigate the tissue specificity of both the global levels and locus-specific distribution of 5hmC in several human tissues and cell lines. We find that global 5hmC content of normal human tissues is highly variable, does not correlate with global 5mC content, and decreases rapidly as cells from normal tissue adapt to cell culture. Using tiling microarrays to map 5hmC levels in DNA from normal human tissues, we find that 5hmC patterns are tissue specific; unsupervised hierarchical clustering based solely on 5hmC patterns groups independent biological samples by tissue type. Moreover, in agreement with previous studies, we find 5hmC associated primarily, but not exclusively, with the body of transcribed genes, and that within these genes 5hmC levels are positively correlated with transcription levels. However, using quantitative 5hmC-qPCR, we find that the absolute levels of 5hmC for any given gene are primarily determined by tissue type, gene expression having a secondary influence on 5hmC levels. That is, a gene transcribed at a similar level in several different tissues may have vastly different levels of 5hmC (>20-fold) dependent on tissue type. Our findings highlight tissue type as a major modifier of 5hmC levels in expressed genes and emphasize the importance of using quantitative analyses in the study of 5hmC levels.
TL;DR: It is shown that soft fibrin gels promote the growth of colonies of tumorigenic cells from single cancer cells from mouse or human cancer cell lines, and that as few as ten fibrIn-cultured cells can lead to the formation of tumours in mice more efficiently than marker-selected cells.
Abstract: Conventional methods for the selection of tumorigenic cells from cancer cell lines rely on stem-cell markers. It is now shown that soft fibrin gels promote the growth of colonies of tumorigenic cells from single cancer cells from mouse or human cancer cell lines, and that as few as ten fibrin-cultured cells can lead to the formation of tumours in mice more efficiently than marker-selected cells.
TL;DR: SRSF 1 can promote breast cancer, and SRSF1 itself or its downstream effectors may be valuable targets for the development of therapeutics.
Abstract: The splicing-factor oncoprotein SRSF1 (also known as SF2/ASF or ASF/SF2) is upregulated in breast cancers. We investigated the ability of SRSF1 to transform human and mouse mammary epithelial cells in vivo and in vitro. SRSF1-overexpressing COMMA-1D cells formed tumors, following orthotopic transplantation to reconstitute the mammary gland. In three-dimensional (3D) culture, SRSF1-overexpressing MCF-10A cells formed larger acini than control cells, reflecting increased proliferation and delayed apoptosis during acinar morphogenesis. These effects required the first RNA-recognition motif and nuclear functions of SRSF1. SRSF1 overexpression promoted alternative splicing of BIM (also known as BCL2L11) and BIN1 to produce isoforms that lack pro-apoptotic functions and contribute to the phenotype. Finally, SRSF1 cooperated specifically with MYC to transform mammary epithelial cells, in part by potentiating eIF4E activation, and these cooperating oncogenes are significantly coexpressed in human breast tumors. Thus, SRSF1 can promote breast cancer, and SRSF1 itself or its downstream effectors may be valuable targets for the development of therapeutics.
TL;DR: It is shown that orthotopic transplantation of a CD44 variant isoform-expressing (CD44v(+)) subpopulation of 4T1 breast cancer cells, but not that of aCD 44v(-) subpopulation, in mice results in efficient lung metastasis accompanied by expansion of stem-like cancer cells.
Abstract: CD44 is a cell surface protein that is a marker for stem cell-like cancer cells and has a role in invasion and metastasis. Here, epithelial splicing regulatory protein 1 is shown to generate a CD44 variant protein that enhances metastasis in a mouse model and protects cells from oxidative stress.
TL;DR: The data indicate that platelets, by conferring an unsuspicious "pseudonormal" phenotype, may enable a molecular mimicry that allows metastasizing tumor cells to downregulate MHC class I, to escape T-cell-mediated immunity without inducing susceptibility to NK cell reactivity.
Abstract: Natural killer (NK) cells are cytotoxic lymphocytes that play an important role in tumor immunosurveillance, preferentially eliminating targets with low or absent expression of MHC class I and stress-induced expression of ligands for activating NK receptors. Platelets promote metastasis by protecting disseminating tumor cells from NK cell immunosurveillance, but the underlying mechanisms are not well understood. In this study, we show that tumor cells rapidly get coated in the presence of platelets in vitro, and circulating tumor cells of cancer patients display coexpression of platelet markers. Flow cytometry, immunofluorescent staining, confocal microscopy, and analyses on an ultrastructural level using immunoelectron microscopy revealed that such coating may cause transfer of MHC class I onto the tumor cell surface resulting in high-level expression of platelet-derived normal MHC class I. The resulting "phenotype of false pretenses" disrupts recognition of tumor cell missing self, thereby impairing cytotoxicity and IFN-γ production by NK cells. Thus, our data indicate that platelets, by conferring an unsuspicious "pseudonormal" phenotype, may enable a molecular mimicry that allows metastasizing tumor cells to downregulate MHC class I, to escape T-cell-mediated immunity without inducing susceptibility to NK cell reactivity.
TL;DR: It is found that P2X7 exhibits significant growth-promoting effects in vivo, and immunohistochemistry revealed strong P2x7 positivity in several human cancers.
Abstract: The P2X7 receptor is an ATP-gated ion channel known for its cytotoxic activity. However, recent evidence suggests a role for P2X7 in cell proliferation. Here, we found that P2X7 exhibits significant growth-promoting effects in vivo. Human embryonic kidney cells expressing P2X7 exhibited a more tumorigenic and anaplastic phenotype than control cells in vivo, and the growth rate and size of these tumors were significantly reduced by intratumoral injection of the P2X7 inhibitor-oxidized ATP. The accelerated growth of P2X7-expressing tumors was characterized by increased proliferation, reduced apoptosis, and a high level of activated transcription factor NFATc1. These tumors also showed a more developed vascular network than control tumors and secreted elevated amounts of VEGF. The growth and neoangiogenesis of P2X7-expressing tumors was blocked by intratumoral injection of the VEGF-blocking antibody Avastin (bevacizumab), pharmacologic P2X7 blockade, or P2X7 silencing in vivo. Immunohistochemistry revealed strong P2X7 positivity in several human cancers. Together, our findings provide direct evidence that P2X7 promotes tumor growth in vivo.
Garvan Institute of Medical Research1, University of New South Wales2, Necker-Enfants Malades Hospital3, Paris Descartes University4, Rockefeller University5, University of Manchester6, Hebrew University of Jerusalem7, Humboldt University of Berlin8, Uludağ University9, Tokyo Medical and Dental University10, National Defense Medical College11, Royal Perth Hospital12, University of Western Australia13, Royal Children's Hospital14, Children's Hospital at Westmead15, Canberra Hospital16, Australian National University17, Westmead Hospital18
TL;DR: CD4(+) T cells from patients deficient in IL-12Rβ1, TYK2, STAT1, and STAT3 are examined to further explore the pathways involved in human Tfh cell differentiation and suggest that defective T fh cell development and/or function contributes to the humoral defects observed in STAT3-deficient patients.
TL;DR: It is shown that expression of Oct4 gene or transmembrane delivery ofOct4 protein promoted dedifferentiation of melanoma cells to CSC-like cells, suggesting that CSC phenotype is dynamic and may be acquired through dedifferentiated cells.
Abstract: There is enormous interest to target cancer stem cells (CSCs) for clinical treatment because these cells are highly tumorigenic and resistant to chemotherapy. Oct4 is expressed by CSC-like cells in different types of cancer. However, function of Oct4 in tumor cells is unclear. In this study, we showed that expression of Oct4 gene or transmembrane delivery of Oct4 protein promoted dedifferentiation of melanoma cells to CSC-like cells. The dedifferentiated melanoma cells showed significantly decreased expression of melanocytic markers and acquired the ability to form tumor spheroids. They showed markedly increased resistance to chemotherapeutic agents and hypoxic injury. In the subcutaneous xenograft and tail vein injection assays, these cells had significantly increased tumorigenic capacity. The dedifferentiated melanoma cells acquired features associated with CSCs such as multipotent differentiation capacity and expression of melanoma CSC markers such as ABCB5 and CD271. Mechanistically, Oct4-induced dedifferentiation was associated with increased expression of endogenous Oct4, Nanog and Klf4, and global gene expression changes that enriched for transcription factors. RNAi-mediated knockdown of Oct4 in dedifferentiated cells led to diminished CSC phenotypes. Oct4 expression in melanoma was regulated by hypoxia and its expression was detected in a sub-population of melanoma cells in clinical samples. Our data indicate that Oct4 is a positive regulator of tumor dedifferentiation. The results suggest that CSC phenotype is dynamic and may be acquired through dedifferentiation. Oct4-mediated tumor cell dedifferentiation may have an important role during tumor progression.
TL;DR: Flow cytometry and western blot analysis showed that plasma treatment of ovarian cancer cells induced apoptosis, and nonequilibrium atmospheric pressure plasma could be a promising tool for therapy for ovarian cancers.
Abstract: Two independent ovarian cancer cell lines and fibroblast controls were treated with nonequilibrium atmospheric pressure plasma (NEAPP). Most ovarian cancer cells were detached from the culture dish by continuous plasma treatment to a single spot on the dish. Next, the plasma source was applied over the whole dish using a robot arm. In vitro cell proliferation assays showed that plasma treatments significantly decreased proliferation rates of ovarian cancer cells compared to fibroblast cells. Flow cytometry and western blot analysis showed that plasma treatment of ovarian cancer cells induced apoptosis. NEAPP could be a promising tool for therapy for ovarian cancers.
TL;DR: In this article, mesenchymal stem cells (MSCs) are recruited to the tumor stroma and, once present, are able to influence the phenotype of the carcinoma cells.
Abstract: Mesenchymal cells of the tumor-associated stroma are critical determinants of carcinoma cell behavior. We focus here on interactions of carcinoma cells with mesenchymal stem cells (MSCs), which are recruited to the tumor stroma and, once present, are able to influence the phenotype of the carcinoma cells. We find that carcinoma cell-derived interleukin-1 (IL-1) induces prostaglandin E2 (PGE2) secretion by MSCs. The resulting PGE2 operates in an autocrine manner, cooperating with ongoing paracrine IL-1 signaling, to induce expression of a group of cytokines by the MSCs. The PGE2 and cytokines then proceed to act in a paracrine fashion on the carcinoma cells to induce activation of β-catenin signaling and formation of cancer stem cells. These observations indicate that MSCs and derived cell types create a cancer stem-cell niche to enable tumor progression via release of PGE2 and cytokines.
TL;DR: It is suggested that immunosuppressive barriers erected by tumors greatly hamper the antitumor activity of human NK cells, thereby favoring tumor outgrowth and progression.
Abstract: Natural killer (NK) cells play a key role in tumor immune surveillance. However, adoptive immunotherapy protocols using NK cells have shown limited clinical efficacy to date, possibly due to tumor escape mechanisms that inhibit NK cell function. In this study, we analyzed the effect of coculturing melanoma cells and NK cells on their phenotype and function. We found that melanoma cells inhibited the expression of major NK receptors that trigger their immune function, including NKp30, NKp44, and NKG2D, with consequent impairment of NK cell-mediated cytolytic activity against various melanoma cell lines. This inhibitory effect was primarily mediated by indoleamine 2,3-dioxygenase (IDO) and prostaglandin E2 (PGE2). Together, our findings suggest that immunosuppressive barriers erected by tumors greatly hamper the antitumor activity of human NK cells, thereby favoring tumor outgrowth and progression.
TL;DR: This protocol to isolate PDGFR-α+Sca-1+ (PαS) MSCs using flow cytometry avoids cellular contamination that can complicate other methods and has augmented growth potential and robust tri-lineage differentiation compared with standard culture-selected M SCs.
Abstract: Platelet-derived growth factor receptor α (PDGFR-α) and stem cell antigen 1 (Sca-1) have recently been identified as selective markers of mouse mesenchymal stem cells (MSCs). PDGFR-α(+)Sca-1(+) (PαS) MSCs have augmented growth potential and robust tri-lineage differentiation compared with standard culture-selected MSCs. In addition, the selective isolation of PαS MSCs avoids cellular contamination that can complicate other methods. Here we describe in detail our protocol to isolate PαS MSCs using flow cytometry. In brief, the tibia and femora are isolated and crushed using a pestle and mortar. The crushed bones are then chopped and incubated for 1 h at 37 °C in 20 ml of DMEM containing 0.2% (wt/vol) collagenase. The cell suspension is filtered before red blood cell lysis and incubated with the following antibodies: allophycocyanin (APC)-conjugated PDGFR-α, FITC-conjugated Sca-1, phycoerythrin (PE)-conjugated CD45 and Ter119. Appropriate gates are constructed on a cell sorter to exclude dead cells and lineage (CD45(+)Ter-119(+))-positive cells. Approximately 10,000 PαS MSCs may then be isolated per mouse. The total protocol takes ~7 h to complete.
TL;DR: The role of microRNAs (miRNAs) in regulating autophagy of hepatocellular carcinoma (HCC) cells under hypoxic conditions was investigated in this article.
TL;DR: High-level expression of CSC-associated properties of MCF-7 cells cultured in 3D was further confirmed by high tumorigenicity in vivo, and 3D collagen scaffolds might provide a useful platform for anti-cancer therapeutics and CSC research.
TL;DR: Results suggest that secretory tumor-suppressive miRNAs can act as a death signal in a cell competitive process, providing a novel insight into a tumor initiation mechanism.
TL;DR: The results reveal that the rigidity of the substrate used to immobilize T cell stimulatory ligands is an important and previously unrecognized parameter influencing T cell activation, proliferation, and Th differentiation.
Abstract: Adoptive immunotherapy using cultured T cells holds promise for the treatment of cancer and infectious disease. Ligands immobilized on surfaces fabricated from hard materials such as polystyrene plastic are commonly employed for T cell culture. The mechanical properties of a culture surface can influence the adhesion, proliferation, and differentiation of stem cells and fibroblasts. We therefore explored the impact of culture substrate stiffness on the ex vivo activation and expansion of human T cells. We describe a simple system for the stimulation of the TCR/CD3 complex and the CD28 receptor using substrates with variable rigidity manufactured from poly(dimethylsiloxane), a biocompatible silicone elastomer. We show that softer (Young's Modulus [E] 2 MPa). Mixed peripheral blood T cells cultured on the stiffer substrates also demonstrate a trend (nonsignificant) toward a greater proportion of CD62L(neg), effector-differentiated CD4(+) and CD8(+) T cells. Naive CD4(+) T cells expanded on softer substrates yield an average 3-fold greater proportion of IFN-γ-producing Th1-like cells. These results reveal that the rigidity of the substrate used to immobilize T cell stimulatory ligands is an important and previously unrecognized parameter influencing T cell activation, proliferation, and Th differentiation. Substrate rigidity should therefore be a consideration in the development of T cell culture systems as well as when interpreting results of T cell activation based upon solid-phase immobilization of TCR/CD3 and CD28 ligands.