搜索结果: 'gmp t cell activator'

PURPOSE: The existence of tumor-initiating cells in breast cancer has profound implications for cancer therapy. In this study,we investigated the sensitivity of tumor-initiating cells isolated from human epidermal growth factor receptor type 2 (HER2)-overexpressing carcinoma cell lines to trastuzumab,a compound used for the targeted therapy of breast cancer. EXPERIMENTAL DESIGN: Spheres were analyzed by indirect immunofluorescence for HER2 cell surface expression and by real-time PCR for HER2 mRNA expression in the presence or absence of the Notch1 signaling inhibitor (GSI) or Notch1 small interfering RNA. Xenografts of HER2-overexpressing breast tumor cells were treated with trastuzumab or doxorubicin. The sphere-forming efficiency (SFE) and serial transplantability of tumors were assessed. RESULTS: In HER2-overexpressing carcinoma cell lines,cells with tumor-initiating cell properties presented increased HER2 levels compared with the bulk cell population without modification in HER2 gene amplification. HER2 levels were controlled by Notch1 signaling,as shown by the reduction of HER2 cell surface expression and lower SFE following gamma-secretase inhibition or Notch1 specific silencing. We also show that trastuzumab was able to effectively target tumor-initiating cells of HER2-positive carcinoma cell lines,as indicated by the significant decrease in SFE and the loss of serial transplantability,following treatment of HER2-overexpressing xenotransplants. CONCLUSIONS: Here,we provide evidence for the therapeutic efficacy of trastuzumab in debulking and in targeting tumor-initiating cells of HER2-overexpressing tumors. We also propose that Notch signaling regulates HER2 expression,thereby representing a critical survival pathway of tumor-initiating cells. View Publication

PI3K activity determines positive and negative selection of B cells,a key process for immune tolerance and B cell maturation. Activation of PI3K is balanced by phosphatase and tensin homolog (Pten),the PI3K's main antagonistic phosphatase. Yet,the extent of feedback regulation between PI3K activity and Pten expression during B cell development is unclear. Here,we show that PI3K control of this process is achieved post-transcriptionally by an axis composed of a transcription factor (c-Myc),a microRNA (miR17-92),and Pten. Enhancing activation of this axis through overexpression of miR17-92 reconstitutes the impaired PI3K activity for positive selection in CD19-deficient B cells and restores most of the B cell developmental impairments that are evident in CD19-deficient mice. Using a genetic approach of deletion and complementation,we show that the c-Myc/miR17-92/Pten axis critically controls PI3K activity and the sensitivity of immature B cells to negative selection imposed by activation-induced cell death. View Publication

Human pluripotent stem cell (hPSC) derived tissues often remain developmentally immature in vitro,and become more adult-like in their structure,cellular diversity and function following transplantation into immunocompromised mice. Previously we have demonstrated that hPSC-derived human lung organoids (HLOs) resembled human fetal lung tissue in vitro (Dye et al.,2015). Here we show that HLOs required a bioartificial microporous poly(lactide-co-glycolide) (PLG) scaffold niche for successful engraftment,long-term survival,and maturation of lung epithelium in vivo. Analysis of scaffold-grown transplanted tissue showed airway-like tissue with enhanced epithelial structure and organization compared to HLOs grown in vitro. By further comparing in vitro and in vivo grown HLOs with fetal and adult human lung tissue,we found that in vivo transplanted HLOs had improved cellular differentiation of secretory lineages that is reflective of differences between fetal and adult tissue,resulting in airway-like structures that were remarkably similar to the native adult human lung. View Publication

Bright/Arid3a has been characterized both as an activator of immunoglobulin heavy-chain transcription and as a proto-oncogene. Although Bright expression is highly B lineage stage restricted in adult mice,its expression in the earliest identifiable hematopoietic stem cell (HSC) population suggests that Bright might have additional functions. We showed that textgreater99% of Bright(-/-) embryos die at midgestation from failed hematopoiesis. Bright(-/-) embryonic day 12.5 (E12.5) fetal livers showed an increase in the expression of immature markers. Colony-forming assays indicated that the hematopoietic potential of Bright(-/-) mice is markedly reduced. Rare survivors of lethality,which were not compensated by the closely related paralogue Bright-derived protein (Bdp)/Arid3b,suffered HSC deficits in their bone marrow as well as B lineage-intrinsic developmental and functional deficiencies in their peripheries. These include a reduction in a natural antibody,B-1 responses to phosphocholine,and selective T-dependent impairment of IgG1 class switching. Our results place Bright/Arid3a on a select list of transcriptional regulators required to program both HSC and lineage-specific differentiation. View Publication

AIM: The generation and characterization of a human embryonic stem cell (hESC) line stably expressing red fluorescent mCherry protein.backslashnbackslashnMETHODS: Lentiviral transduction of a ubiquitously-expressed human EF-1α promoter driven mCherry transgene was performed in MEL2 hESC. Red fluore-scence was assessed by immunofluorescence and flow cytometry. Pluripotency of stably transduced hESC was determined by immunofluorescent pluripotency marker expression,flow cytometry,teratoma assays and embryoid body-based differentiation followed by reverse transcriptase-polymerase chain reaction. Quantification of cell motility and survival was performed with time lapse microscopy.backslashnbackslashnRESULTS: Constitutively fluorescently-labeled hESCs are useful tools for facile in vitro and in vivo tracking of survival,motility and cell spreading on various surfaces before and after differentiation. Here we describe the generation and characterization of a hESC line (MEL2) stably expressing red fluorescent protein,mCherry. This line was generated by random integration of a fluorescent protein-expressing cassette,driven by the ubiquitously-expressed human EF-1α promoter. Stably transfected MEL2-mCherry hESC were shown to express pluripotency markers in the nucleus (POU5F1/OCT4,NANOG and SOX2) and on the cell surface (SSEA4,TRA1-60 and TG30/CD9) and were shown to maintain a normal karyotype in long-term (for at least 35 passages) culture. MEL2-mCherry hESC further readily differentiated into representative cell types of the three germ layers in embryoid body and teratoma based assays and,importantly,maintained robust mCherry expression throughout differentiation. The cell line was next adapted to single-cell passaging,rendering it compatible with numerous bioengineering applications such as measurement of cell motility and cell spreading on various protein modified surfaces,quantification of cell attachment to nanoparticles and rapid estimation of cell survival.backslashnbackslashnCONCLUSION: The MEL2-mCherry hESC line conforms to the criteria of bona fide pluripotent stem cells and maintains red fluorescence throughout differentiation,making it a useful tool for bioengineering and in vivo tracking experiments. View Publication

We have established a novel cell line,designated as TF-1,from a patient with erythroleukemia,which showed complete growth dependency on granulocyte-macrophage colony-stimulating factor (GM-CSF) or on interleukin-3 (IL-3) and carried a homogeneous chromosomal abnormality (54X). Erythropoietin (EPO) also sustained the short-term growth of TF-1,but did not induce erythroid differentiation. These three hematopoietic growth factors acted on TF-1 synergistically. Transforming growth factor-beta and interferons inhibited the factor-dependent growth of TF-1 cells in a dose-dependent fashion,and monocyte-colony stimulating factor and interkeukin-1 enhanced the GM-CSF-dependent growth of TF-1. Ultrastructural studies revealed some very immature features in this cell line. Although TF-1 cells do not express glycophorin A or carbonyl anhydrase I,the morphological and cytochemical features,and the constitutive expression of globin genes,indicate the commitment of TF-1 to erythroid lineage. When induced to differentiate,TF-1 entered two different pathways. Specifically,hemin and delta-aminolevulinic acid induced hemoglobin synthesis,whereas TPA induced dramatic differentiation of TF-1 into macrophage-like cells. In summary,TF-1 is a cell line of immature erythroid origin that requires GM-CSF,IL-3,or EPO for its growth and that has the ability to undergo differentiation into either more mature erythroid cells or into macrophage-like cells. TF-1 is a useful tool for analyzing the human receptors for IL-3,GM-CSF,and EPO or the signal transduction of these hemopoietic growth factors. View Publication

Human pluripotent stem cells (hPSCs) require precise control of post-transcriptional RNA networks to maintain proliferation and survival. Using enhanced UV crosslinking and immunoprecipitation (eCLIP),we identify RNA targets of the IMP/IGF2BP family of RNA-binding proteins in hPSCs. At the broad region and binding site levels,IMP1 and IMP2 show reproducible binding to a large and overlapping set of 3' UTR-enriched targets. RNA Bind-N-seq applied to recombinant full-length IMP1 and IMP2 reveals CA-rich motifs that are enriched in eCLIP-defined binding sites. We observe that IMP1 loss in hPSCs recapitulates IMP1 phenotypes,including a reduction in cell adhesion and increase in cell death. For cell adhesion,we find IMP1 maintains levels of integrin mRNA specifically regulating RNA stability of ITGB5 in hPSCs. Additionally,we show that IMP1 can be linked to hPSC survival via direct target BCL2. Thus,transcriptome-wide binding profiles identify hPSC targets modulating well-characterized IMP1 roles. View Publication

Current EC differentiation protocols are inefficient,and the phenotypes of the differentiated ECs are only briefly stable,which significantly inhibits their utility for basic science research. Here,a remarkably more efficient hiPSC-EC differentiation protocol that incorporates a three-dimensional (3D) fibrin scaffold is presented. With this protocol,up to 45% of the differentiated hiPSCs assumed an EC phenotype,and after purification,greater than 95% of the cells displayed the EC phenotype (based on CD31 expression). The hiPSC-ECs continued to display EC characteristics for 4 weeks invitro. Gene and protein expression levels of CD31,CD144 and von Willebrand factor-8 (vWF-8) were significantly up-regulated in differentiated hiPSC-ECs. hiPSC-ECs also have biological function to up-take Dil-conjugated acetylated LDL (Dil-ac-LDL) and form tubular structures on Matrigel. Collectively,these data demonstrate that a 3D differentiation protocol can efficiently generate ECs from hiPSCs and,furthermore,the differentiated hiPSC-ECs are functional and can maintain EC fate up to 4 weeks invitro. ?? 2014 Elsevier Ltd. View Publication

BACKGROUND The success and limitations of current immunotherapies have pushed research toward the development of alternative approaches and the possibility to manipulate other cytotoxic immune cells such as natural killer (NK) cells. Here,we targeted an intracellular inhibiting protein 'cytokine inducible SH2-containing protein' (CISH) in NK cells to evaluate the impact on their functions and antitumor properties. METHODS To further understand CISH functions in NK cells,we developed a conditional Cish-deficient mouse model in NK cells (Cishfl/flNcr1Ki/+ ). NK cells cytokine expression,signaling and cytotoxicity has been evaluated in vitro. Using intravenous injection of B16F10 melanoma cell line and EO711 triple negative breast cancer cell line,metastasis evaluation was performed. Then,orthotopic implantation of breast tumors was performed and tumor growth was followed using bioluminescence. Infiltration and phenotype of NK cells in the tumor was evaluated. Finally,we targeted CISH in human NK-92 or primary NK cells,using a technology combining the CRISPR(i)-dCas9 tool with a new lentiviral pseudotype. We then tested human NK cells functions. RESULTS In Cishfl/flNcr1Ki/+ mice,we detected no developmental or homeostatic difference in NK cells. Global gene expression of Cishfl/flNcr1Ki/+ NK cells compared with Cish+/+Ncr1Ki/+ NK cells revealed upregulation of pathways and genes associated with NK cell cycling and activation. We show that CISH does not only regulate interleukin-15 (IL-15) signaling pathways but also natural cytotoxicity receptors (NCR) pathways,triggering CISH protein expression. Primed Cishfl/flNcr1Ki/+ NK cells display increased activation upon NCR stimulation. Cishfl/flNcr1Ki/+ NK cells display lower activation thresholds and Cishfl/flNcr1Ki/+ mice are more resistant to tumor metastasis and to primary breast cancer growth. CISH deletion favors NK cell accumulation to the primary tumor,optimizes NK cell killing properties and decreases TIGIT immune checkpoint receptor expression,limiting NK cell exhaustion. Finally,using CRISPRi,we then targeted CISH in human NK-92 or primary NK cells. In human NK cells,CISH deletion also favors NCR signaling and antitumor functions. CONCLUSION This study represents a crucial step in the mechanistic understanding and safety of Cish targeting to unleash NK cell antitumor function in solid tumors. Our results validate CISH as an emerging therapeutic target to enhance NK cell immunotherapy. View Publication

Stimulating erythropoiesis is essential in the treatment of various types of anemia. Sheng Xue Ning (SXN) is commonly used in China as an iron supplement to treat iron deficiency anemia,renal anemia,and anemia in pregnancy. This research reports a novel effect of SXN in enhancing the proliferation of hematopoietic stem/progenitor cell (HSPC) to promote erythropoiesis in the bone marrow,which is distinct from conventional iron supplements that primarily aid in the maturation of red blood cells. Employing a model of hematopoietic dysfunction induced by X-ray exposure,we evaluated the efficacy of SXN in restoring hematopoietic function. SXN significantly promoted the recovery of peripheral erythroid cells and enhanced the proliferation and differentiation of Lin − /c-KIT + /Sca-1 + HSPC in mice exposed to X-ray irradiation. Our results showed that SXN elevated the expression of stem cell factor (SCF) and activated the SCF/c-KIT/PI3K/AKT signaling pathway,facilitating the proliferation and differentiation of HSPC. In vitro,SXN markedly enhanced the proliferation of bone marrow nucleated cell (BMNC) and the colony-forming capacity of BFU-E,CFU-E,and CFU-GM,while also elevating the expression of proteins involved in the SCF/c-KIT/PI3K/AKT pathway in BMNC. Additionally,SXN enhanced the proliferation and differentiation of mesenchymal stem cell (MSC) and increased SCF secretion. In conclusion,SXN demonstrates the capacity to enhance erythropoiesis by upregulating SCF expression,thereby promoting HSPC proliferation and differentiation via the SCF/c-KIT/PI3K/AKT pathway. SXN may offer a new strategy for improving the activity of HSPC and promoting erythropoiesis in the treatment of hematopoiesis disorders. View Publication

Cancer stem cells (CSCs) play a key role in non-small cell lung cancer (NSCLC) chemoresistance and metastasis. In this study,we used two NSCLC cell lines to investigate the regulating effect of hypoxia in the induction and maintenance of CSC traits. Our study demonstrated hypoxia-induced stemness and chemoresistance at levels comparable to those in typical CSC sphere culture. Activation of the NF-κB pathway (by transfection of NF-κB-p65) plays a key role in NSCLC CSCs and chemoresistance. Disulfiram (DS),an anti-alcoholism drug,showed a strong in vitro anti-CSC effect. It blocked cancer cell sphere reformation and clonogenicity,synergistically enhanced the cytotoxicity of four anti-NSCLC drugs (doxorubicin,gemcitabine,oxaliplatin and paclitaxel) and reversed hypoxia-induced resistance. The effect of DS on CSCs is copper-dependent. A very short half-life in the bloodstream is the major limitation for the translation of DS into a cancer treatment. Our team previously developed a poly lactic-co-glycolic acid (PLGA) nanoparticle encapsulated DS (DS-PLGA) with a long half-life in the bloodstream. Intra venous injection of DS-PLGA in combination with the oral application of copper gluconate has strong anticancer efficacy in a metastatic NSCLC mouse model. Further study may be able to translate DS-PLGA into cancer applications. View Publication

Natural killer (NK) cell-based therapies represent a promising approach for acute myeloid leukemia (AML) relapse,yet their efficacy is hindered by immunosuppressive factors such as transforming growth factor beta (TGF-β) in the tumor microenvironment. This study investigated the effects of TGF-β on NK cell cytotoxicity and migration using 2D and 3D co-culture models that mimic the leukemic microenvironment. TGF-β production was evaluated in AML-derived leukemic cell lines and mesenchymal stromal cells (hTERT-MSCs) using ELISA. Bulk RNA sequencing (RNA-seq) was performed to analyze global gene expression changes in TGF-β-treated primary human NK cells. NK cell cytotoxicity and migration were assessed in 2D monolayer and 3D spheroid co-cultures containing hTERT-MSCs and leukemic cells using flow cytometry and confocal microscopy. Both leukemic cells and MSCs produced TGF-β,with increased levels observed in MSCs after co-culture with primary AML blasts. RNA sequencing revealed that TGF-β altered key gene pathways associated with NK cell cytotoxicity,adhesion,and migration,supporting its immunosuppressive role. In functional assays,TGF-β exposure significantly reduced NK cell-mediated cytotoxicity in a time-dependent manner and impaired NK cell infiltration into 3D spheroids,particularly in models incorporating MSCs. Additionally,MSCs themselves provided a protective environment for leukemic cells,further reducing NK cell effectiveness in 2D co-cultures. TGF-β suppresses both NK cell cytotoxicity and migration,limiting their ability to eliminate leukemic cells and infiltrate the bone marrow niche (BMN). These findings provide novel insights into TGF-β–mediated immune evasion mechanisms and provide important insights for the future design of NK-based immunotherapies and clinical trials. View Publication