搜索结果: 'gmp t cell activator'

PurposeOsteosarcoma (OS),the most common primary bone malignancy in childhood poses a therapeutic challenge despite extensive research. Neutrophil extracellular traps (NETs) play a role in the tumor microenvironment (TME) in a variety of cancers,but their role in OS has not been characterized.Experimental DesignThis retrospective cohort study aimed to investigate immune cell infiltration and NETs formation in patients with OS and its association with chemotherapy response and overall survival using immunofluorescence of paraffin-embedded tissue samples.ResultsAs compared to the non-malignant bone tumor Osteoblastoma,OS samples were characterized by a higher proportion of neutrophils exhibiting NETs. High NETs formation on initial diagnostic biopsies,but not Neutrophil to Lymphocyte ratio,the number of tumor-infiltrating neutrophils,CD3+ T-cells or CD8+ T-cells,was associated with poor response to neoadjuvant chemotherapy. The NETs burden in diagnostic biopsies was also correlated with survival: patients with high NETs burden had a mean overall survival of 53.7 months,as compared with 71.5 months for patients with low NETs. Furthermore,metastatic sites exhibited elevated NETs formation compared to primary tumors,and sera from patients with OS induced NETs release in healthy neutrophils,while sera from healthy controls did not.ConclusionsThese data highlight the potential role of NETs in OS’s TME biology,and suggest that NETs released by tumor infiltrating neutrophils can serve as an independent prognostic factor for poor response to neoadjuvant therapy and overall survival in patients with OS. Such insights may inform the development of tailored treatment approaches in OS. View Publication

DEAD-box helicase 41 (DDX41) is implicated in germline (GL)-predisposed myeloid neoplasms,where pathogenic GL variants often lead to disease following the acquisition of a somatic variant in trans,most commonly p.R525H. However,the precise molecular mechanisms by which DDX41 variants contribute to the pathogenesis of myeloid neoplasms remain poorly understood,partly due to challenges in establishing cellular and animal models that faithfully recapitulate the human disease phenotype. This limitation highlights the necessity of directly analyzing primary human disease cells. In this case report,conducted to pursue this objective,we implemented single-cell RNA sequencing integrated with genotyping at the p.R525 locus in a myelodysplastic neoplasm (MDS) harboring both germline and somatic DDX41 variants,leveraging highly efficient Terminator-Assisted Solid-phase cDNA amplification and sequencing. We found that acquiring p.R525H induced G2/M cell cycle arrest selectively in colony-forming unit-erythroid cells,accompanied by R-loop accumulation,which impaired erythropoiesis through DNA damage. In hematopoietic stem and myeloid progenitor populations,gene expression profiles were largely similar between p.R525H-positive and -negative cells. However,ligand-receptor interaction and transcriptional regulation analyses suggested a non-cell-autonomous influence from p.R525H-expressing cells on GL variant-only cells. This interaction drove convergence toward a shared expression profile,highlighting an intricate interplay shaping the patient’s MDS phenotype. View Publication

Aging is associated with immune dysfunction,but long-term endurance training may confer protective effects on immune cell function. This study investigates how natural killer (NK) cell phenotypes,functional markers,and metabolism differ between endurance-trained and untrained older adults. Ex vivo expanded NK cells from endurance-trained (63.6 ± 2.1 years) and untrained (64.3 ± 3.3 years) males were exposed to adrenergic blockade (propranolol; 0–200 ng/mL) or mTOR inhibition (rapamycin; 10–100 ng/mL),both with or without PMA-induced inflammatory stimulation. Flow cytometry assessed NK subsets,activation (CD38,CD57,CD107a,NKG2D),senescence (KLRG1),and inhibitory markers (PD-1,LAG-3,TIM-3,NKG2A). Seahorse analysis measured metabolic parameters. Trained participants displayed healthier immune profiles (lower NLR,SII) and higher effector NK cells with lower cytotoxic subsets. Propranolol at 100 ng/mL blunted PMA-driven increases in CD57,CD107a,and NKG2D,while potentiating regulatory markers KLRG1,LAG-3,and PD-1 in the trained group,indicating stronger immunoregulation. With rapamycin,trained NK cells preserved NKG2D and CD107a at 10 ng/mL,maintaining cytotoxicity and degranulation. In contrast,at 100 ng/mL rapamycin plus PMA,trained NK cells shifted toward an effector phenotype with higher CD57 and CD107a,yet a blunted PMA-increased LAG-3 and TIM-3,suggesting resistance to exhaustion. PD-1 and KLRG1 remained elevated,reflecting balanced immune control. Mitochondrial analysis revealed that trained NK cells exhibited higher basal and maximal OCR,greater spare respiratory capacity,and OCR/ECAR ratio,reflecting superior metabolic fitness. These findings indicate that endurance-trained older adults have NK cells with greater functional adaptability,reduced senescence,and enhanced metabolism under inflammatory and pharmacological stress.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-06057-y. View Publication

BackgroundMacrophages play a crucial role in the development of early-onset preeclampsia (EOPE),which may be closely associated with an imbalance in macrophage M1/M2 polarization. Mesenchymal stem cell (MSC)-derived apoptotic vesicles (apoVs) have anti-inflammatory,tissue repair,and immunomodulatory functions. MSC-apoVs may ameliorate EOPE by regulating macrophage polarization,but the underlying mechanisms remain to be clarified.MethodsMacrophage infiltration and M1/M2 polarization were first analyzed in the placentas of PE patients and normal pregancies to identify macrophage alterations in EOPE placentas. MSC-apoVs were extracted and characterized. The effects of MSC-apoVs on macrophage polarization and trophoblasts invasion were validated in vivo and in vitro. miRNA transcriptomic sequencing of MSC-apoVs was conducted to identify key miRNAs involved in macrophage M2 polarization and to investigate upstream and downstream regulation factors,which were further validated in vivo and in vitro.ResultsThe proportion of M2 macrophages was significantly reduced in EOPE placentas. MSC-apoVs carrying high levels of miR-191-5p recruited macrophages,downregulated CDK6 protein expression,stabilized mitochondrial membrane potential (MMP),and promoted M2 polarization of macrophages. This enhanced the invasion of trophoblasts and improved EOPE pregnancy outcomes in mice,including reduced blood pressure,decreased urine protein,and improved embryo quality. Overexpression of miR-191-5p mimics in MSC-apoVs further alleviated EOPE-related symptoms,whereas inhibition of miR-191-5p reduced the therapeutic effect of MSC-apoVs. Further experiments confirmed that M2 macrophages polarized by MSC-apoVs promote trophoblasts invasion by secreting platelet-derived growth factor-AB (PDGF-AB),which binds to platelet-derived growth factor receptor-beta (PDGFR-β) on trophoblasts,directly activating the downstream PI3K-AKT-mTOR signaling pathway,thereby improving EOPE.ConclusionOur findings reveal the crucial role of M2 macrophages in the pathogenesis of EOPE. MSC-apoVs with high miR-191-5p recruit macrophages,downregulate CDK6,stabilize MMP,and promote M2 polarization,increasing PDGF-AB secretion,which enhances trophoblasts invasion and thereby treat EOPE. Therefore,MSC-apoVs therapy may serve as a promising strategy to improve the prognosis of EOPE.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04546-5. View Publication

Long-term imaging formats are ideal for capturing dynamic neuronal network formation in vitro,yet fluorescent techniques are often constrained by the impact of phototoxicity on cell survival. Here we present a live-imaging protocol that was optimised via quantitative analysis of 3 target culturing conditions on neuromorphological health: extracellular matrix (human- versus murine-derived laminin),culture media (Neurobasal™ versus Brainphys™ Imaging media),and seeding density (1 × 105 versus 2 × 105 cells/cm2). A cortical neuron reporter line was differentiated from human embryonic stem cells by transduction of Neurogenin-2 and green fluorescent protein,then fluorescently imaged in 8 different microenvironments daily for 33 days. Alongside viability analysis by PrestoBlue assay and gene quantification by digital polymerase chain reaction,an automated image analysis pipeline was developed to characterise network morphology and organisation over time. Brainphys™ Imaging medium was observed to support neuron viability,outgrowth,and self-organisation to a greater extent than Neurobasal™ medium with either laminin type,while the combination of Neurobasal™ medium and human laminin reduced cell survival. Further,a higher seeding density fostered somata clustering,but did not significantly extend viability compared to low density. These findings suggest a synergistic relationship between species-specific laminin and culture media in phototoxic environments,which is positively mediated by light-protective compounds found in Brainphys™ Imaging medium.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04591-0. View Publication

Inflammatory cytokines,particularly interferon-γ (IFN-γ),are markedly elevated in the peripheral blood of patients with immune checkpoint inhibitor-induced myocarditis (ICI-M). Endomyocardial biopsies from these patients also show GBP-associated inflammasome overexpression. While both factors are implicated in ICI-M pathophysiology,their interplay and cellular targets remain poorly characterized. Our aim was to elucidate how ICI-M-associated cytokines affect the viability and inflammatory responses of endothelial cells (ECs) and cardiomyocytes (CMs) using human induced pluripotent stem cell (hiPSC)-derived models. ECs and CMs were differentiated from the same hiPSC line derived from a healthy donor. Cells were exposed either to IFN-γ alone or to an inflammatory cytokine cocktail (CCL5,GZMB,IL-1β,IL-2,IL-6,IFN-γ,TNF-α). We assessed large-scale transcriptomic changes via microarray and evaluated inflammatory,apoptotic,and cell death pathways at cellular and molecular levels. hiPSC-ECs were highly sensitive to cytokine exposure,displaying significant mortality and marked transcriptomic changes in immunity- and inflammation-related pathways. In contrast,hiPSC-CM showed limited transcriptional changes and reduced susceptibility to cytokine-induced death. In both cell types,cytokine treatment upregulated key components of the inflammasome pathway,including regulators (GBP5,GBP6,P2X7,NLRC5),a core component (AIM2),and the effector GSDMD. Increased GBP5 expression and CASP-1 cleavage mirrored the findings found elsewhere in endomyocardial biopsies from ICI-M patients. This hiPSC-based model reveals a distinct cellular sensitivity to ICI-M-related inflammation,with endothelial cells showing heightened vulnerability. These results reposition endothelial dysfunction,rather than cardiomyocyte injury alone,as a central mechanism in ICI-induced myocarditis. Modulating endothelial inflammasome activation,particularly via AIM2 inhibition,could offer a novel strategy to mitigate cardiac toxicity while preserving antitumor efficacy. View Publication

Metastatic breast cancer remains largely incurable,and the mechanisms driving the transition from primary to metastatic breast cancer remain elusive. We analyzed the complex landscape of estrogen receptor (ER)-positive breast cancer primary and metastatic tumors using scRNA-seq data from twenty-three female patients with either primary or metastatic disease. By employing single-cell transcriptional profiling of unpaired patient samples,we sought to elucidate the genetic and molecular mechanisms underlying changes in the metastatic tumor ecosystem. We identified specific subtypes of stromal and immune cells critical to forming a pro-tumor microenvironment in metastatic lesions,including CCL2+ macrophages,exhausted cytotoxic T cells,and FOXP3+ regulatory T cells. Analysis of cell-cell communication highlights a marked decrease in tumor-immune cell interactions in metastatic tissues,likely contributing to an immunosuppressive microenvironment. In contrast,primary breast cancer samples displayed increased activation of the TNF-α signaling pathway via NF-kB,indicating a potential therapeutic target. Our study comprehensively characterizes the transcriptional landscape encompassing primary and metastatic breast cancer. View Publication

Accumulating evidence indicates that various oncogenic mutations interfere with normal myeloid differentiation of leukemogenic cells during the early process of acute myeloid leukemia (AML) development. Differentiation therapy is a therapeutic strategy capable of terminating leukemic expansion by reactivating the differentiation potential; however,the plasticity and instability of leukemia cells counteract the establishment of treatments aimed at irreversibly inducing and maintaining their differentiation states. On the basis of our previous observation that autophagy inhibitor treatment induces the accumulation of cytosolic DNA and activation of cytosolic DNA-sensor signaling selectively in leukemia cells,we herein examined the synergistic effect of cytosolic DNA-sensor signaling activation with conventional differentiation therapy on AML. The combined treatment succeeded in inducing irreversible differentiation in AML cell lines. Mechanistically,cytosolic DNA was sensed by absent in melanoma 2 (AIM2),a cytosolic DNA sensor. Activation of the AIM2 inflammasome resulted in the accumulation of p21 through the inhibition of its proteasomal degradation,thereby facilitating the myeloid differentiation. Importantly,the combined therapy dramatically reduced the total leukemia cell counts and proportion of blast cells in the spleens of AML mice. Collectively,these findings indicate that the autophagy inhibition-cytosolic DNA-sensor signaling axis can potentiate AML differentiation therapy. Clinical effects on AML therapy are closely associated with reactivating the normal myeloid differentiation potential in leukemia cells. This study shows that autophagosome formation inhibitors activate the cytosolic DNA-sensor signaling,thereby augmenting conventional differentiation therapy to induce irreversible differentiation and cell growth arrest in several types of AML cell lines. View Publication

Hepatocellular carcinoma (HCC) is widely recognized as a globally prevalent malignancy. Immunotherapy is a promising therapy for HCC patients. Increasing evidence suggests that lncRNAs are involved in HCC progression and immunotherapy. The study reveals the mechanistic role of long non‐coding RNA (lncRNA) FOXD1‐AS1 in regulating migration,invasion,circulating tumor cells (CTCs),epithelial‐mesenchymal transition (EMT),and immune escape in HCC in vitro. This study employed real‐time PCR (RT‐qPCR) to measure FOXD1‐AS1,miR‐615‐3p,and programmed death‐ligand 1 ( PD‐L1 ). The interactions of FOXD1‐AS1,miR‐615‐3p,and PD‐L1 were validated via dual‐luciferase reporter gene and ribonucleoprotein immunoprecipitation (RIP) assay. In vivo experimentation involves BALB/c mice and BALB/c nude mice to investigate the impact of HCC metastasis. The upregulation of lncRNA FOXD1‐AS1 in malignant tissues significantly correlates with poor prognosis. The investigation was implemented on the impact of lncRNA FOXD1‐AS1 on the migratory,invasive,and EMT of HCC cells. It has been observed that the lncRNA FOXD1‐AS1 significantly influences the generation and metastasis of M CTC in vivo analysis. In mechanistic analysis,lncRNA FOXD1‐AS1 enhanced immune escape in HCC via upregulation of PD‐L1,which acted as a ceRNA by sequestering miR‐615‐3p. Additionally,lncRNA FOXD1‐AS1 was found to modulate the EMT of CTCs through the activation of the PI3K/AKT pathway. This study presents compelling evidence supporting the role of lncRNA FOXD1‐AS1 as a miRNA sponge that sequesters miR‐655‐3p and protects PD‐L1 from suppression. View Publication

Myeloperoxidase (MPO) is found almost exclusively in granulocytes and immature myeloid cells. It plays a key role in the innate immune system,catalysing the formation of reactive oxygen species that are important in anti‐microbial action,but MPO also oxidatively transforms the topoisomerase II (TOP2) poison etoposide to chemical forms that have elevated DNA damaging properties. TOP2 poisons such as etoposide are widely used anti‐cancer drugs,but they are linked to cases of secondary acute myeloid leukaemias through a mechanism that involves DNA damage and presumably erroneous repair leading to leukaemogenic chromosome translocations. This leads to the possibility that myeloperoxidase inhibitors could reduce the rate of therapy‐related leukaemia by protecting haematopoietic cells from TOP2 poison‐mediated genotoxic damage while preserving the anti‐cancer efficacy of the treatment. We show here that myeloperoxidase inhibition reduces etoposide‐induced TOP2B‐DNA covalent complexes and resulting DNA double‐strand break formation in primary ex vivo expanded CD34 + progenitor cells and unfractionated bone marrow mononuclear cells. Since MPO inhibitors are currently being developed as anti‐inflammatory agents this raises the possibility that repurposing of these potential new drugs could provide a means of suppressing secondary acute myeloid leukaemias associated with therapies containing TOP2 poisons. View Publication

Prostate cancer (PCa) is one of the most prevalent cancers in men and is associated with high mortality and disability rates. β-hydroxybutyrate (BHB),a ketone body,has received increasing attention for its role in cancer. However,its role in PCa remains unclear. This study aimed to explore the mechanism and feasibility of BHB as a treatment alternative for PCa. Colony formation assay,flow cytometry,western blot assay,and transwell assays were performed to determine the effect of BHB on the proliferation and metastasis of PCa cells. Tumor sphere formation and aldehyde dehydrogenase assays were used to identify the impact of BHB or indoleacetamide-N-methyltransferase (INMT) on the stemness of PCa cells. N6-methyladenosine (m6A)–meRIP real-time reverse transcription polymerase chain reaction and dual luciferase assays were conducted to confirm INMT upregulation via the METTL3–m6A pathway. Co-IP assay was used to detect the epigenetic modification of INMT by BHB-mediated β-hydroxybutyrylation (kbhb) and screen enzymes that regulate INMT kbhb. Mouse xenograft experiments demonstrated the antitumor effects of BHB in vivo. BHB can inhibit the proliferation,migration,and invasion of PCa cells by suppressing their stemness. Mechanistically,INMT,whose expression is upregulated by the METTL3–m6A pathway,was demonstrated to be an oncogenic gene that promotes the stem-like characteristics of PCa cells. BHB can suppress the malignant phenotypes of PCa by kbhb of INMT,which in turn inhibits INMT expression. Our findings indicate a role of BHB in PCa metabolic therapy,thereby suggesting an epigenetic therapeutic strategy to target INMT in aggressive PCa. Not applicable. The online version contains supplementary material available at 10.1186/s12935-024-03277-6. View Publication

Tumor cells have the ability to invade and form small clusters that protrude into adjacent tissues,a phenomenon that is frequently observed at the periphery of a tumor as it expands into healthy tissues. The presence of these clusters is linked to poor prognosis and has proven challenging to treat using conventional therapies. We previously reported that p60AmotL2 expression is localized to invasive colon and breast cancer cells. In vitro,p60AmotL2 promotes epithelial cell invasion by negatively impacting E-cadherin/AmotL2-related mechanotransduction. Using epithelial cells transfected with inducible p60AmotL2,we employed a phenotypic drug screening approach to find compounds that specifically target invasive cells. The phenotypic screen was performed by treating cells for 72 h with a library of compounds with known antitumor activities in a dose-dependent manner. After assessing cell viability using CellTiter-Glo,drug sensitivity scores for each compound were calculated. Candidate hit compounds with a higher drug sensitivity score for p60AmotL2-expressing cells were then validated on lung and colon cell models,both in 2D and in 3D,and on colon cancer patient-derived organoids. Nascent RNA sequencing was performed after BET inhibition to analyse BET-dependent pathways in p60AmotL2-expressing cells. We identified 60 compounds that selectively targeted p60AmotL2-expressing cells. Intriguingly,these compounds were classified into two major categories: Epidermal Growth Factor Receptor (EGFR) inhibitors and Bromodomain and Extra-Terminal motif (BET) inhibitors. The latter consistently demonstrated antitumor activity in human cancer cell models,as well as in organoids derived from colon cancer patients. BET inhibition led to a shift towards the upregulation of pro-apoptotic pathways specifically in p60AmotL2-expressing cells. BET inhibitors specifically target p60AmotL2-expressing invasive cancer cells,likely by exploiting differences in chromatin accessibility,leading to cell death. Additionally,our findings support the use of this phenotypic strategy to discover novel compounds that can exploit vulnerabilities and specifically target invasive cancer cells. The online version contains supplementary material available at 10.1186/s13046-024-03031-w. View Publication