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

IntroductionHumanization is typically adopted to reduce the immunogenicity of murine antibodies generated by hybridoma technology when used in humans.MethodsTwo different strategies of antibody humanization are popularly employed,including “complementarity determining region (CDR) grafting” and “framework (FR) shuffling” to humanize a murine antibody against human programmed death-1 (PD-1),XM PD1. In CDR-grafting humanization,the CDRs of XM PD-1,were grafted into the human FR regions with high homology to the murine FR counterparts,and back mutations of key residues were performed to retain the antigen-binding affinities. While in FR-shuffling humanization,a combinatorial library of the six murine CDRs in-frame of XM PD-1 was constructed to a pool of human germline FRs for high-throughput screening for the most favorable variants. We evaluated many aspects which were important during antibody development of the molecules obtained by the two methods,including antibody purity,thermal stability,binding efficacy,predicted humanness,and immunogenicity,along with T cell epitope prediction for the humanized antibodies.ResultsWhile the ideal molecule was not achieved through CDR grafting in this particular instance,FR-shuffling proved successful in identifying a suitable candidate. The study highlights FR-shuffling as an effective complementary approach that potentially increases the success rate of antibody humanization. It is particularly noted for its accessibility to those with a biological rather than a computational background. DiscussionThe insights from this comparison are intended to assist other researchers in selecting appropriate humanization strategies for drug development,contributing to broader application and understanding in the field. View Publication

Simple SummaryAcute myeloid leukemia (AML) is a challenging blood cancer to treat,with only about 24% of patients surviving for 5 years after diagnosis. A key challenge is that AML cells stick to normal cells in the bone marrow (BM),and these BM cells protect them from chemotherapy. The aim of this project is to find drugs that disrupt AML cell adherence to BM cells and release them into the blood,where chemotherapy will be more effective. To achieve this,we have created a model of adhesive BM and shown that it mimics the drug resistance seen clinically. We have used the model as a testing platform for drugs that disrupt AML cell adhesion. We have shown that the combined targeting of CD44 and FAK,using anti-CD44 and the clinical-grade FAK inhibitor defactinib,inhibits the adhesion of the most primitive AML cells that are associated with drug resistance and disease relapse. AbstractBackground/Objectives: Acute myeloid leukemia (AML) is an aggressive neoplasm. Although most patients respond to induction therapy,they commonly relapse due to recurrent disease in the bone marrow microenvironment (BMME). So,the disruption of the BMME,releasing tumor cells into the peripheral circulation,has therapeutic potential. Methods: Using both primary donor AML cells and cell lines,we developed an in vitro co-culture model of the AML BMME. We used this model to identify the most effective agent(s) to block AML cell adherence and reverse adhesion-mediated treatment resistance. Results: We identified that anti-CD44 treatment significantly increased the efficacy of cytarabine. However,some AML cells remained adhered,and transcriptional analysis identified focal adhesion kinase (FAK) signaling as a contributing factor; the adhered cells showed elevated FAK phosphorylation that was reduced by the FAK inhibitor,defactinib. Importantly,we demonstrated that anti-CD44 and defactinib were highly synergistic at diminishing the adhesion of the most primitive CD34high AML cells in primary autologous co-cultures. Conclusions: Taken together,we identified anti-CD44 and defactinib as a promising therapeutic combination to release AML cells from the chemoprotective AML BMME. As anti-CD44 is already available as a recombinant humanized monoclonal antibody,the combination of this agent with defactinib could be rapidly tested in AML clinical trials. View Publication

Co-stimulatory molecules are imperative for CD8+ T cells to eliminate target cell and maintain sustained cytotoxicity. Despite an advanced understanding of the co-stimulatory molecules deficiency that results in tumor escape,the tumor cell-intrinsic mechanisms that regulate co-stimulatory molecules remain enigmatic,and an in-depth dissection could facilitate the improvement of treatment options. To this end,in this study,we report that the deficiency of the critical costimulatory molecule CD58,mediated by the expression of ATF4 in tumor cells,impairs the formation of immunological synapses (IS) and leads to the deterioration of antitumor immune function of CD8+ T cells. Mechanistically,ATF4 transcriptionally upregulated dynamin 1 (DNM1) expression leading to DNM1-dependent endocytosis (DDE)-mediated degradation of CD58. Furthermore,administration of DDE inhibitor prochlorperazine or ATF4 knockdown effectively restored CD58 expression,boosting CD8+ T cell cytotoxicity and immunotherapy efficiency. Thus,our study reveals that ATF4 in tumor cells weakens CD58 expression to interfere with complete IS formation,and indicates potential approaches to improve the cytolytic function of CD8+ T cell in tumor immunotherapy.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-025-06245-4. View Publication

T cell dysfunction is a pivotal driving factor in autoimmune diseases,yet its underlying regulatory mechanisms remain incompletely understood. The role of long non-coding RNAs (lncRNAs) in immune regulation has gradually been recognized,although their functional mechanisms in T cells remain elusive. This study focuses on lncBADR (LncRNA Branched-chain Amino acids Degradation Regulator),elucidating its mechanism by which it regulates branched-chain amino acids (BCAAs) metabolism to influence T cell effector functions. Mice with specific knockout of lncBADR (T celllncBADR−/−) exhibited markedly ameliorated experimental autoimmune encephalomyelitis (EAE) symptoms. Mechanistic investigations revealed that lncBADR inhibits BCAAs degradation by binding to the enzymes Mccc1 and Pcca,leading to the accumulation of BCAAs within T-cells. This,in turn,activates the mTOR-Stat1 signaling pathway,promoting IFN-γ secretion and exacerbating EAE pathology. In contrast,knockout of lncBADR restored BCAAs degradation,significantly reducing IFN-γ secretion in T cells and suppressing their pathogenic functions. Further studies demonstrated that high-BCAAs feeding partially reversed the protective effects of lncBADR knockout,indicating that lncBADR plays a crucial role in autoimmune inflammation by regulating BCAAs metabolism. This study offers new insights into targeting lncBADR or modulating BCAAs metabolism as potential therapeutic strategies for autoimmune diseases. View Publication

Ferroptosis is a new form of nonapoptotic and iron-dependent type of cell death. Glutathione peroxidase-4 (GPX4) plays an essential role in anti-ferroptosis by reducing lipid peroxidation. Although acute myeloid leukemia (AML) cells,especially relapsed and refractory (R/R)-AML,present high GPX4 levels and enzyme activities,pharmacological inhibition of GPX4 alone has limited application in AML. Thus,whether inhibition of GPX4 combined with other therapeutic reagents has effective application in AML is largely unknown. Lipid reactive oxygen species (ROS),malondialdehyde (MDA),and glutathione (GSH) assays were used to assess ferroptosis in AML cells treated with the hypomethylating agent (HMA) decitabine (DAC),ferroptosis-inducer (FIN) RAS-selective lethal 3 (RSL3),or their combination. Combination index (CI) analysis was used to assess the synergistic activity of DAC + RSL3 against AML cells. Finally,we evaluated the synergistic activity of DAC + RSL3 in murine AML and a human R/R-AML-xenografted NSG model in vivo. We first assessed GPX4 expression and found that GPX4 levels were higher in AML cells,especially those with MLL rearrangements,than in NCs. Knockdown of GPX4 by shRNA and indirect inhibition of GPX4 enzyme activity by RSL3 robustly induced ferroptosis in AML cells. To reduce the dose of RSL3 and avoid side effects,low doses of DAC (0.5 µM) and RSL3 (0.05 µM) synergistically facilitate ferroptosis by inhibiting the AMP-activated protein kinase (AMPK)-SLC7A11-GPX4 axis. Knockdown of AMPK by shRNA enhanced ferroptosis,and overexpression of SLC7A11 and GPX4 rescued DAC + RSL3-induced anti-leukemogenesis. Mechanistically,DAC increased the expression of MAGEA6 by reducing MAGEA6 promoter hypermethylation. Overexpression of MAGEA6 induced the degradation of AMPK,suggesting that DAC inhibits the AMPK-SLC7A11-GPX4 axis by increasing MAGEA6 expression. In addition,DAC + RSL3 synergistically reduced leukemic burden and extended overall survival compared with either DAC or RSL3 treatment in the MLL-AF9-transformed murine model. Finally,DAC + RSL3 synergistically reduced viability in untreated and R/R-AML cells and extended overall survival in two R/R-AML-xenografted NSG mouse models. Our study first identify vulnerability to ferroptosis by regulating MAGEA6-AMPK-SLC7A11-GPX4 signaling pathway. Combined treatment with HMAs and FINs provides a potential therapeutic choice for AML patients,especially for R/R-AML. The online version contains supplementary material available at 10.1186/s40164-024-00489-4. View Publication

CRISPR-Cas9 genome editing often induces unintended,large genomic rearrangements,posing potential safety risks. However,there are no methods for mitigating these risks. Using long-read individual-molecule sequencing (IDMseq),we found the microhomology-mediated end joining (MMEJ) DNA repair pathway plays a predominant role in Cas9-induced large deletions (LDs). We targeted MMEJ-associated genes genetically and/or pharmacologically and analyzed Cas9-induced LDs at multiple gene loci using flow cytometry and long-read sequencing. Reducing POLQ levels or activity significantly decreases LDs,while depleting or overexpressing RPA increases or reduces LD frequency,respectively. Interestingly,small-molecule inhibition of POLQ and delivery of recombinant RPA proteins also dramatically promote homology-directed repair (HDR) at multiple disease-relevant gene loci in human pluripotent stem cells and hematopoietic progenitor cells. Our findings reveal the contrasting roles of RPA and POLQ in Cas9-induced LD and HDR,suggesting new strategies for safer and more precise genome editing. The online version contains supplementary material available at 10.1186/s12915-024-01896-z. View Publication

Epithelial-to-mesenchymal transition (EMT),cancer stem cells (CSCs),and colorectal cancer (CRC) therapy resistance are closely associated. Prior reports have demonstrated that sphingosine-1-phosphate (S1P) supports stem cells and maintains the CSC phenotype. We hypothesized that the EMT inducer SNAI1 drives S1P signaling to amplify CSC self-renewal capacity and chemoresistance. CRC cell lines with or without ectopic expression of SNAI1 were used to study the role of S1P signaling as mediators of cancer stemness and 5-fluorouracil (5FU) chemoresistance. The therapeutic ability of sphingosine kinase 2 (SPHK2) was assessed using siRNA and ABC294640,a SPHK2 inhibitor. CSCs were isolated from patient-derived xenografts (PDXs) and assessed for SPHK2 and SNAI1 expression. Ectopic SNAI1 expressing cell lines demonstrated elevated SPHK2 expression and increased SPHK2 promoter activity. SPHK2 inhibition with siRNA or ABC294640 ablated in vitro self-renewal and sensitized cells to 5FU. CSCs isolated from CRC PDXs express increased SPHK2 relative to the non-CSC population. Combination ABC294640/5FU therapy significantly inhibited tumor growth in mice and enhanced 5FU response in therapy-resistant CRC patient-derived tumor organoids (PDTOs). SNAI1/SPHK2 signaling mediates cancer stemness and 5FU resistance,implicating S1P as a therapeutic target for CRC. The S1P inhibitor ABC294640 holds potential as a therapeutic agent to target CSCs in therapy refractory CRC. View Publication

Micropapillary adenocarcinoma (MPC) is an aggressive histological subtype of lung adenocarcinoma (LUAD). MPC is composed of small clusters of cancer cells exhibiting inverted polarity. However,the mechanism underlying its formation is poorly understood. Here we show that hypoxia is involved in MPC formation. Hypoxia induced the formation of MPC-like structures (MLSs) in a three-dimensional culture system using A549 human LUAD cells,and HIF-1α was indispensable for MLS formation. RNA sequencing analysis demonstrated that A549 cells forming MLSs exhibited a gene expression signature similar to that of lung MPC. Moreover,MLS formation enhanced the resistance of A549 cells to natural killer cell cytotoxicity. Our findings suggest that hypoxia drives lung MPC formation through HIF-1α and that immune escape from natural killer cells might underlie the aggressiveness of MPC. View Publication

Functional cellular heterogeneity in tumours often underlies incomplete response to therapy and relapse. Previously,we demonstrated that the growth of the paediatric brain malignancy,sonic hedgehog subgroup medulloblastoma,is rooted in a dysregulated developmental hierarchy,the apex of which is defined by characteristically quiescent SOX2 + stem-like cells. Integrating gene expression and chromatin accessibility patterns in distinct cellular compartments,we identify the transcription factor Olig2 as regulating the stem cell fate transition from quiescence to activation,driving the generation of downstream neoplastic progenitors. Inactivation of Olig2 blocks stem cell activation and tumour output. Targeting this rare OLIG2-driven proliferative programme with a small molecule inhibitor,CT-179,dramatically attenuates early tumour formation and tumour regrowth post-therapy,and significantly increases median survival in vivo. We demonstrate that targeting transition from quiescence to proliferation at the level of the tumorigenic cell could be a pivotal medulloblastoma treatment strategy. Subject terms: Cancer stem cells,Mechanisms of disease,Cancer therapy View Publication

Cerebral organoids (COs) are valuable tools for studying the intricate interplay between glial cells and neurons in brain development and disease,including HIV-associated neuroinflammation. We developed a novel approach to generate microglia containing COs (CO-iMs) by co-culturing hematopoietic progenitors and inducing pluripotent stem cells. This approach allowed for the differentiation of microglia within the organoids concomitantly with the neuronal progenitors. Compared with conventional COs,CO-iMs were more efficient at generating CD45 + /CD11b + /Iba-1 + microglia and presented a physiologically relevant proportion of microglia (~ 7%). CO-iMs presented substantially increased expression of microglial homeostatic and sensome markers as well as markers for the complement cascade. CO-iMs are susceptible to HIV infection,resulting in a significant increase in several pro-inflammatory cytokines/chemokines,which are abrogated by the addition of antiretrovirals. Thus,CO-iM is a robust model for deciphering neuropathogenesis,neuroinflammation,and viral infections of brain cells in a 3D culture system. The online version contains supplementary material available at 10.1186/s12974-025-03353-2. View Publication

Biological mechanisms are inherently dynamic,requiring precise and rapid manipulations for effective characterization. Traditional genetic manipulations operate on long timescales,making them unsuitable for studying dynamic processes or characterizing essential genes,where chronic depletion can cause cell death. We compare five inducible protein degradation systems—dTAG,HaloPROTAC,IKZF3,and two auxin-inducible degrons (AID) using OsTIR1 and AtFB2—evaluating degradation efficiency,basal degradation,target recovery after ligand washout,and ligand impact. This analysis identifies OsTIR1-based AID 2.0 as the most robust system. However,AID 2.0’s higher degradation efficiency comes with target-specific basal degradation and slower recovery rates. To address these limitations,we employ base-editing-mediated mutagenesis followed by several rounds of functional selection and screening. This directed protein evolution generates several gain-of-function OsTIR1 variants,including S210A,that significantly enhance the overall degron efficiency. The resulting degron system,named AID 2.1,maintains effective target protein depletion with minimal basal degradation and faster recovery after ligand washout,enabling characterization and rescue experiments for essential genes. Our comparative assessment and directed evolution approach provide a reference dataset and improved degron technology for studying gene functions in dynamic biological contexts. Subject terms: Genetic engineering,CRISPR-Cas9 genome editing,Peptides View Publication