搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

Retinoid-related molecules (RRM) are novel agents with tumor-selective cytotoxic/antiproliferative activity,a different mechanism of action from classic retinoids and no cross-resistance with other chemotherapeutics. ST1926 and CD437 are prototypic RRMs,with the former currently undergoing phase I clinical trials. We show here that ST1926,CD437,and active congeners cause DNA damage. Cellular and subcellular COMET assays,H2AX phosphorylation (gamma-H2AX),and scoring of chromosome aberrations indicate that active RRMs produce DNA double-strand breaks (DSB) and chromosomal lesions in NB4,an acute myeloid leukemia (AML) cell line characterized by high sensitivity to RRMs. There is a direct quantitative correlation between the levels of DSBs and the cytotoxic/antiproliferative effects induced by RRMs. NB4.437r blasts,which are selectively resistant to RRMs,do not show any sign of DNA damage after treatment with ST1926,CD437,and analogues. DNA damage is the major mechanism underlying the antileukemic activity of RRMs in NB4 and other AML cell lines. In accordance with the S-phase specificity of the cytotoxic and antiproliferative responses of AML cells to RRMs,increases in DSBs are maximal during the S phase of the cell cycle. Induction of DSBs precedes inhibition of DNA replication and is associated with rapid activation of ataxia telangectasia mutated,ataxia telangectasia RAD3-related,and DNA-dependent protein kinases with subsequent stimulation of the p38 mitogen-activated protein kinase. Inhibition of ataxia telangectasia mutated and DNA-dependent protein kinases reduces phosphorylation of H2AX. Cells defective for homologous recombination are particularly sensitive to ST1926,indicating that this process is important for the protection of cells from the RRM-dependent DNA damage and cytotoxicity. View Publication

The mTOR protein is a master regulator of cell growth and proliferation,and inhibitors of its kinase activity have the potential to become new class of anticancer drugs. Starting from quinoline 1,which was identified in a biochemical mTOR assay,we developed a tricyclic benzonaphthyridinone inhibitor 37 (Torin1),which inhibited phosphorylation of mTORC1 and mTORC2 substrates in cells at concentrations of 2 and 10 nM,respectively. Moreover,Torin1 exhibits 1000-fold selectivity for mTOR over PI3K (EC(50) = 1800 nM) and exhibits 100-fold binding selectivity relative to 450 other protein kinases. Torin1 was efficacious at a dose of 20 mg/kg in a U87MG xenograft model and demonstrated good pharmacodynamic inhibition of downstream effectors of mTOR in tumor and peripheral tissues. These results demonstrate that Torin1 is a useful probe of mTOR-dependent phenomena and that benzonaphthridinones represent a promising scaffold for the further development of mTOR-specific inhibitors with the potential for clinical utility. View Publication

BACKGROUND There is an unmet need for developing faithful animal models for preclinical evaluation of immunotherapy. The current approach to generate preclinical models for immunotherapy evaluation has been to transplant CD34+ cells from umbilical cord blood into immune-deficient mice followed by implantation of patient derived tumor cells. However,current models are associated with high tumor rejection rate secondary to the allograft vs. tumor response from human leukocyte antigen (HLA) mismatches. We herein report the first development of a novel,humanized patient-derived xenograft (PDX) model using autologous CD34+ cells from bone marrow aspirate obtained from a patient with metastatic clear cell renal cell carcinoma (mRCC) from whom a PDX had been developed. CASE DESCRIPTION This is a 68-year-old Caucasian man diagnosed with mRCC with metastasis to the liver in 2014. He was treated with sunitinib +/- AGS-003 and underwent a cytoreductive right nephrectomy,left adrenalectomy and partial liver resection. PDX was generated using resected nephrectomy specimen. After surgery,patient received multiple lines of standard of care therapy including sunitinib,axitinib,bevacizumab,everolimus and cabozantinib. While progressing on cabozantinib,he was treated with nivolumab. Seven years after initiation of nivolumab,and 4 years after stopping systemic therapy,he remains in complete remission. To generate autologous PDX model,bone marrow aspirate was performed and CD34+ hematopoietic stem/progenitor cells (HSPCs) were isolated and injected into 150 rad irradiated non-obese diabetic scid gamma null (NSG) mice. At 11 weeks post-transplant,the matched patient PDX was injected subcutaneously into the humanized mice and the mice were treated with nivolumab. CONCLUSIONS Our case represents successful therapy of nivolumab in mRCC. Furthermore,HPSCs obtained from a single bone marrow aspirate were able to reconstitute an immune system in the mice that allowed nivolumab to inhibit the tumor growth of PDX and recapitulated the durable remission observed in the patient with nivolumab. We observed the reconstitution of human T cells,B cells and natural killer (NK) cells and unlike the humanized mouse model using cord blood,our model system eliminates the tumor rejection from mis-matched HLA. Our autologous humanized renal cell carcinoma (RCC) PDX model provides an effective tool to study immunotherapy in a preclinical setting. View Publication

TOX is a DNA-binding factor required for development of CD4(+) T cells,natural killer T cells and regulatory T cells. Here we document that both natural killer (NK) cell development and lymphoid tissue organogenesis were also inhibited in the absence of TOX. We found that the development of lymphoid tissue-inducer cells,a rare subset of specialized cells that has an integral role in lymphoid tissue organogenesis,required TOX. Tox was upregulated considerably in immature NK cells in the bone marrow,consistent with the loss of mature NK cells in the absence of this nuclear protein. Thus,many cell lineages of the immune system share a TOX-dependent step for development. View Publication

The anti-proliferative activities of all twenty-five targeted kinase inhibitor drugs that are in clinical use were measured in two large assay panels: (1) a panel of proliferation assays of forty-four human cancer cell lines from diverse tumour tissue origins; and (2) a panel of more than 300 kinase enzyme activity assays. This study provides a head-on comparison of all kinase inhibitor drugs in use (status Nov. 2013),and for six of these drugs,the first kinome profiling data in the public domain. Correlation of drug activities with cancer gene mutations revealed novel drug sensitivity markers,suggesting that cancers dependent on mutant CTNNB1 will respond to trametinib and other MEK inhibitors,and cancers dependent on SMAD4 to small molecule EGFR inhibitor drugs. Comparison of cellular targeting efficacies reveals the most targeted inhibitors for EGFR,ABL1 and BRAF(V600E)-driven cell growth,and demonstrates that the best targeted agents combine high biochemical potency with good selectivity. For ABL1 inhibitors,we computationally deduce optimized kinase profiles for use in a next generation of drugs. Our study shows the power of combining biochemical and cellular profiling data in the evaluation of kinase inhibitor drug action. View Publication

The recently developed ability to differentiate primary adult stem cells and induced pluripotent stem cells (iPSCs) into cardiomyocytes is providing unprecedented opportunities to produce an unlimited supply of cardiomyocytes for use in patients with heart disease. Here,we examine the evidence for the preclinical use of such cells for successful heart regeneration. We also describe advances in the identification of new cardiac molecular and cellular targets to induce proliferation of cardiomyocytes for heart regeneration. Such new advances are paving the way for a new innovative drug development process for the treatment of heart disease. View Publication

Abelson kinase (c-abl) and platelet-derived growth factor (PDGF) are key players in the pathogenesis of systemic sclerosis (SSc). The aim of the present study was to evaluate the antifibrotic potential of dasatinib and nilotinib,2 novel inhibitors of c-abl and PDGF,which are well tolerated and have recently been approved. Dasatinib and nilotinib dose-dependently reduced the mRNA and protein levels of extracellular matrix proteins in human stimulated dermal fibroblasts from SSc patients (IC(50) of 0.5-2.0 nM for dasatinib and 0.8-2.5 nM for nilotinib). In a mouse model of bleomycin-induced dermal fibrosis,dasatinib and nilotinib potently reduced the dermal thickness,the number of myofibroblasts,and the collagen content of the skin in a dose-dependent manner at well-tolerated doses. These data indicate that dasatinib and nilotinib potently inhibit the synthesis of extracellular matrix in vitro and in vivo at biologically relevant concentrations. Thus,we provide the first evidence that dasatinib and nilotinib might be promising drugs for the treatment of patients with SSc. View Publication

Fibroblast growth factor receptor 1 (FGFR1) is overexpressed in multiple types of solid tumors,including head and neck squamous cell carcinoma (HNSCC). Being associated with poor prognosis,FGFR1 is a potential therapeutic target for aggressive tumors. T cell-based cancer immunotherapy has played a central role in novel cancer treatments. However,the potential of antitumor immunotherapy targeting FGFR1 has not been investigated. Here,we showed that FGFR-tyrosine kinase inhibitors (TKIs) augmented antitumor effects of immune checkpoint inhibitors in an HNSCC mouse model and upregulated tumoral MHC class I and MHC class II expression in vivo and in vitro. This upregulation was associated with the mitogen-activated protein kinase signaling pathway,which is a crucial pathway for cancer development through FGFR signaling. Moreover,we identified an FGFR1-derived peptide epitope (FGFR1305-319) that could elicit antigen-reactive and multiple HLA-restricted CD4+ T cell responses. These T cells showed direct cytotoxicity against tumor cells that expressed FGFR1. Notably,FGFR-TKIs augmented antitumor effects of FGFR1-reactive T cells against human HNSCC cells. These results indicate that the combination of FGFR-TKIs with immunotherapy,such as an FGFR1-targeting peptide vaccine or immune checkpoint inhibitor,could be a novel and robust immunologic approach for treating patients with FGFR1-expressing cancer cells. View Publication

Transition from the manual processes that are performed during the initial research and development (R&D) stage to automated processes for later and commercial stage cell therapy manufacturing can be challenging. It often requires significant effort,time,and costs – which hinders the therapy’s access to the clinic. To ease this transition,we have developed a novel and flexible manufacturing platform,Bioreactor with Expandable Culture Area (BECA),that aims to support both R&D and manufacturing to accelerate cell therapies from bench to bedside. This report introduces two models in this manufacturing platform: BECA-S for manual small-scale operation at R&D phase and BECA-Auto for functionally closed and automated scaled-out operation at manufacturing phase. We employed these two models to streamline transition of the T cell culture process from manual to automated and reported insignificant differences in the culture outcome between the two. Our work represents the first detailed development and demonstration of a standalone cell manufacturing platform that facilitates a seamless transition between manual and automated processing for autologous T cell therapy manufacturing. View Publication

In the context of pancreatic cancer,metastasis remains the most critical determinant of resectability,and hence survival. The objective of this study was to determine whether Hedgehog (Hh) signaling plays a role in pancreatic cancer invasion and metastasis because this is likely to have profound clinical implications. In pancreatic cancer cell lines,Hh inhibition with cyclopamine resulted in down-regulation of snail and up-regulation of E-cadherin,consistent with inhibition of epithelial-to-mesenchymal transition,and was mirrored by a striking reduction of in vitro invasive capacity (P textless 0.0001). Conversely,Gli1 overexpression in immortalized human pancreatic ductal epithelial cells led to a markedly invasive phenotype (P textless 0.0001) and near total down-regulation of E-cadherin. In an orthotopic xenograft model,cyclopamine profoundly inhibited metastatic spread; only one of seven cyclopamine-treated mice developed pulmonary micrometastases versus seven of seven mice with multiple macrometastases in control animals. Combination of gemcitabine and cyclopamine completely abrogated metastases while also significantly reducing the size of primary" tumors. Gli1 levels were up-regulated in tissue samples of metastatic human pancreatic cancer samples compared with matched primary tumors. Aldehyde dehydrogenase (ALDH) overexpression is characteristic for both hematopoietic progenitors and leukemic stem cells; cyclopamine preferentially reduced "ALDH-high" cells by approximately 3-fold (P = 0.048). We confirm pharmacologic Hh pathway inhibition as a valid therapeutic strategy for pancreatic cancer and show for the first time its particular efficacy against metastatic spread. By targeting specific cellular subpopulations likely involved in tumor initiation at metastatic sites View Publication

Recent methodological advances have improved the ease and efficiency of generating human induced pluripotent stem cells (hiPSCs),but this now typically results in a greater number of hiPSC clones being derived than can be wholly characterized. It is therefore imperative that methods are developed which facilitate rapid selection of hiPSC clones most suited for the downstream research aims. Here we describe a combination of procedures enabling the simultaneous screening of multiple clones to determine their genomic integrity as well as their cardiac differentiation potential within two weeks of the putative reprogrammed colonies initially appearing. By coupling splinkerette-PCR with Ion Torrent sequencing,we could ascertain the number and map the proviral integration sites in lentiviral-reprogrammed hiPSCs. In parallel,we developed an effective cardiac differentiation protocol that generated functional cardiomyocytes within 10 days without requiring line-specific optimization for any of the six independent human pluripotent stem cell lines tested. Finally,to demonstrate the scalable potential of these procedures,we picked 20 nascent iPSC clones and performed these independent assays concurrently. Before the clones required passaging,we were able to identify clones with a single integrated copy of the reprogramming vector and robust cardiac differentiation potential for further analysis. View Publication