参考文献

Triple Negative Breast Cancers (TNBCs) are heterogeneous and aggressive tumors with a median overall survival of less than two years. Despite the availability of new drugs,the prognosis remains poor,implicating a more aggressive clinical course in the metastatic setting. This study investigated the effects of metronomic treatment (mCHT) with 5-fluorouracil (5-FU) plus vinorelbine (VNR) on spheroids derived from two different TNBC cell lines (BT-549 and MDA-MB-231) and a patient-derived primary cell line (MS-186). mCHT significantly reduced spheroid growth and altered spheroid architecture,with a pronounced effect in second-generation spheroids,enriched in self-renewing cancer stem cells (CSCs). Expression of CSC-related markers (CD44,CD133,NOTCH-1,and MYC) was more significantly altered—both at the mRNA and protein levels—by mCHT than by standard treatment (STD). In MS-186-derived spheroids,mCHT downregulated EZH2 and STAT3,key regulators of CSC maintenance,and reduced H3K27ac,suggesting a global epigenetic reprogramming. Unlike STD,which partially and transiently reduced stemness markers,mCHT achieved sustained suppression,indicating preferential targeting of therapy-resistant CSCs. These results indicate mCHT as a promising strategy for specifically aiming at the CSC-like compartment in TNBC,underscoring a therapeutic approach that reprograms key epigenetic networks and overcomes resistance to treatment. View Publication

Background/objectives: Interactions between colorectal tumours and their immune microenvironment critically influence disease progression and response to immunotherapy. However,most organoid systems fail to preserve the complex architecture and immune composition of the original tissue. Here,we applied the air-liquid interface (ALI) organoid model to paired tumour and perilesional colon tissues from colorectal cancer patients to evaluate its ability to retain immune and genetic features and to reproduce responses to chemotherapy and immune checkpoint blockade. Methods: Fresh human tumour and matched healthy colon tissues were processed to generate ALI organoids. Their histological organization,immune cell composition (including CD45+ subsets),and genomic profiles were compared with those of the parental tissues and with conventional Matrigel organoids,either alone or co-cultured with peripheral blood mononuclear cells (PBMCs). Organoids were exposed to 5-FU and nivolumab (anti-PD-1) to assess local immune modulation. Results: ALI organoids faithfully preserved the three-dimensional architecture,native immune infiltrates,and somatic mutational landscape of the source tissues. Importantly,upon PD-1 blockade with nivolumab,ALI organoids consistently exhibited a local expansion of regulatory T cells (Tregs),a phenomenon that could contribute to adaptive immune resistance. This response was not reproduced in PBMC-Matrigel co-culture systems,highlighting the importance of preserving endogenous tumour-immune interactions. Conclusions: Patient-derived ALI organoids represent a physiologically relevant platform that conserves key structural,immunological,and genomic hallmarks of colorectal cancer. By capturing clinically relevant immune remodeling events,such as Treg expansion following PD-1 blockade,this model provides a powerful tool for dissecting tumour-immune interactions. View Publication

Metabolic dysfunction–associated steatotic liver disease (MASLD) is reversible at early stages,making early identification critical. We previously demonstrated that patient-derived induced pluripotent stem cells (iPSCs) carrying MASLD-associated genetic risk variants exhibit greater oleate-induced intracellular lipid accumulation than those without these variants. This study aimed to develop an iPSC-based MASLD risk predictor using functional lipid accumulation assessments. We quantified oleate-induced lipid accumulation in iPSCs from three cohorts: (1) CIRM (22 cases,20 controls),(2) POST (18 cases,16 controls),and (3) UCSF (4 cases,8 controls). Data from the CIRM cohort was used to define an iPSC-based MASLD risk score,which was subsequently validated in the POST and UCSF cohorts. Lipid accumulation was consistently higher in MASLD iPSCs across cohorts. The risk score achieved 44% sensitivity/75% specificity in POST and 75%/100% in UCSF. These findings suggest that oleate-induced lipid accumulation in iPSCs may be a predictor of MASLD risk. Larger studies incorporating additional cellular phenotypes,clinical,and genetic data could enhance predictive accuracy for MASLD surveillance and prevention. View Publication

Super enhancers (SEs),characterized by clusters of enhancers,are instrumental in shaping cellular identity and function. Given this crucial involvement of SEs in cell lineage commitment,and considering the pivotal position of surface ectoderm in differentiating into a wide array of cell types,the study of these SEs holds immense promise for advancing cell-based therapeutic applications. In this study,we profiled the SE landscape in surface ectoderm cells derived from pluripotent stem cell differentiation. By leveraging 3D genomic data,we discerned active histone modifications and frequent chromatin interactions of SEs with target genes. Notably,perturbing specific SE using a CRISPR-dCas9-mediated approach resulted in decreased expression of the connected gene. Subsequently,we constructed a regulatory network of core transcription factors (TFs) operating on SEs and uncovered their control over the differentiation process by forming regulatory network with key TFs,such as TEAD1. Knocking down TEADs attenuated the differentiation process and target gene activation,whereas YAP-TEAD activation expedited the differentiation process by promoting the early establishment of SEs. Collectively,our findings shed light on the crucial role of SEs and identify YAP-TEAD as vital regulators controlling surface ectoderm commitment,thereby providing a novel insight into lineage commitment and stem cell-based epithelial regeneration. View Publication

Multiple myeloma (MM) is the second most common hematological malignancy,characterized by a clonal expansion of malignant plasma cells in bone marrow. Monoclonal gammopathy of undetermined significance (MGUS) is the premalignant condition of MM. The tumor microenvironment is thought to influence the progression from premalignant conditions. Myeloid‐derived suppressor cells (MDSCs) are a heterogenous group of different cellular subsets with myeloid origin,characterized by their ability to inhibit T‐cell responses. MDSC are thought to play an important immunoregulatory role in different diseases,and in many cancers their levels seem to correlate with a poor prognosis. There are three different subsets,the neutrophil‐like polymorphonuclear (PMN)‐MDSC,the monocyte‐like (M)‐MDSC,and the immature early (e)MDSC. In this study,we investigate the levels and functions of all MDSC subsets in the bone marrow of both MGUS and MM patients and compare it to blood MDSC. We found that MDSC levels are not increased in neither the blood nor bone marrow of MGUS or MM patients,and they lack strong T‐cell suppressive abilities. Blood PMN‐MDSC seems to have a small inhibitory effect,but mature neutrophils were more suppressive. Interestingly,eMDSC levels were decreased in the blood of MM patients. Our data indicate that MDSC are not key players in the pathogenesis of MM,but that mature neutrophils may be more important as they have a stronger immunoregulatory effect. View Publication

Ischemia-reperfusion injury (IRI) represents a major challenge in liver transplantation,driving acute dysfunction and contributing to long-term allograft rejection. This process triggers a robust inflammatory response,leading to hepatocyte damage,senescence,and impaired liver regeneration. While the underlying mechanisms remain incompletely understood,increasing evidence highlights macrophage-derived signaling as a pivotal driver of hepatocyte fate during IRI. Here,we identify iRhom2 as a key regulator of immune-mediated liver injury,orchestrating macrophage-driven inflammation and hepatocyte senescence. iRhom2 is known to modulate the secretion of multiple cytokines by macrophages,yet its specific contribution to IRI-driven hepatocyte senescence has not been fully elucidated. We reveal a significant upregulation of iRhom2 in IRI+ reperfused allografts,particularly in Kupffer cells and monocyte-derived macrophages. Functional characterization in iRhom2-deficient macrophages revealed reduced ER stress,preserved mitochondrial function,and attenuated apoptosis,indicating a protective role against IRI-induced cellular damage. Proteomic profiling further uncovers iRhom2-dependent secretion of inflammatory mediators,with HMGB1 emerging as a critical damage-associated molecular pattern (DAMP) molecule in this context. Notably,HMGB1 release occurs independently of TACE catalytic activity,suggesting an alternative unexplored regulatory mechanism. Furthermore,co-culture experiments confirm that macrophage-derived HMGB1 directly induces senescence of human induced pluripotent stem cell-derived hepatocytes (hiPSC-Heps) under in vitro IRI condition,driving the up-regulation of key senescence markers and disrupting cell cycle dynamics. Strikingly,HMGB1 neutralization enhances hepatocyte viability and mitigates senescence,underscoring its pathogenic role. Additionally,HMGB1 knockdown in macrophages protects hepatocytes,though p21 expression remains unaffected,hinting at additional senescence pathways. Our findings establish iRhom2 as a central orchestrator of macrophage-driven hepatocyte dysfunction in IRI and suggest that targeting the iRhom2-HMGB1 axis could represent a promising therapeutic strategy to improve post-transplant liver recovery and long-term graft survival. View Publication

Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic virus that causes Kaposi sarcoma,primary effusion lymphoma and multicentric Castleman disease. A vaccine that prevents KSHV infection or serves in the treatment of KSHV-related diseases represents a critical unmet need,however,the types of immune responses a vaccine should elicit have not been well defined. The gH/gL glycoprotein complex is an important target of KSHV-neutralizing antibodies,but the epitope specificities targeted by these antibodies remain unknown. Here,we isolated 12 gH/gL-specific monoclonal antibodies (mAbs) from KSHV-infected donors and performed structure/function analyses. These mAbs bind recombinant gH/gL with nanomolar affinities and epitope binning analyses revealed that the mAbs bind to 5 epitope clusters on gH/gL. Seven mAbs were able to neutralize KSHV infection of epithelial cell lines. Two potent neutralizing mAbs mapped to the EphA2 binding site as determined by inhibition of the receptor-ligand interaction and negative stain electron microscopy (nsEM) of the mAb/gH/gL complex. The epitopes of other neutralizing mAbs targeting novel sites of vulnerability were determined by a combination of cryogenic electron microscopy and nsEM. Together,these mAbs help to define the relevant epitope targets for KSHV vaccine design,have utility in understanding the role of antibodies in preventing KSHV infection,enable the development of immunotherapy approaches,and provide valuable tools to understand the molecular details of the KSHV entry process. Author summaryKSHV is an oncogenic virus that can cause cancer in infected individuals. The virus is most prevalent in sub-Saharan Africa and in men who have sex with men. It is possible this virus could be prevented with an effective vaccine,however,the immune response to this virus has not been well defined. gH/gL,a protein essential for viral fusion,plays an important role in infection and could be a possible vaccine target. To better understand the antibody response to this protein,we sought to isolate and characterize monoclonal antibodies that can bind gH/gL and neutralize viral infection. In this study,we isolate and characterize twelve monoclonal antibodies that could bind to five different regions of the gH/gL protein. Seven of these antibodies can neutralize infection,with two being able to block the gH/gL EphA2 receptor-ligand interaction. View Publication

Background: The pathology of Alzheimer’s Disease (AD) is characterized by aggregates of amyloid beta (Aβ) peptides and neurofibrillary tau tangles. Increased blood-brain barrier (BBB) permeability and reduced Aβ clearance,which signal neurovascular dysfunction,have also been proposed as early markers of AD. Despite intense scrutiny,the mechanisms of AD remain elusive and novel treatments that address core symptoms of dementia are limited. New alternative methods (NAMs) aim to develop in-vitro translational models that recapitulate human pathology more accurately than previous models and could contribute to the development of new therapies. Methods: Here,we developed a NAM model of the cortical neurovascular unit (NVU) using brain cells derived from human induced pluripotent stem cells (hiPSCs) from a patient with AD and a healthy individual. Differentiated neurons,astrocytes,pericytes,microglia,and brain-like microvascular endothelial cells were cultured in a microphysiological system to create a brain-chip model to evaluate NVU-related endpoints. Results: Compared to control,AD brain-chips had reduced claudin-5 and ZO-1 expression and increased paracellular permeability. AD brain-chips also had decreased activity of the efflux transporter P-glycoprotein (P-gp),but its expression was unchanged. In AD brain-chips,levels of Aβ42,total tau,and p-tau 181 were decreased in protein lysates from the brain channel,while levels of total tau and p-tau 181 were increased in protein lysates from the vascular channel. Finally,AD brain-chips had increased levels of the proinflammatory markers IL-6 and MCP-1 in effluent from both brain and vascular channels. Conclusion: In this brain-chip model,we showed Aβ-independent NVU dysfunction that was related to neuroinflammation and vascular tau accumulation. This study demonstrates the utility of the brain-chip model to evaluate changes in NVU functions induced by AD-like pathology and highlights donor-specific responses associated with the use of hiPSC-derived models. View Publication

Purpose: Current lung cancer organoid models often fail to replicate the complex tumor immune microenvironment,reducing their predictive value for immunotherapy and radiotherapy. Therefore,it is crucial to establish an optimized lung cancer organoid model which could recapitulate the tumor immune microenvironment,enabling more accurate evaluation of therapeutic responses. Methods: We developed an optimized air-liquid interface (ALI) culture method to generate patient-derived lung cancer organoids (ALI-LUOs) from 19 lung cancer samples. The tumor microenvironment,including immune and stromal components,was characterized using immunofluorescence,flow cytometry,and single-cell RNA sequencing. The organoids were further used to assess responses to αPD-1 therapy and radiotherapy. Results: The optimized method significantly improved organoid formation efficiency while preserving immune cell viability for up to 30 days. Immune and fibroblast populations were confirmed by immunofluorescence and flow cytometry. Single-cell RNA sequencing demonstrated that ALI-LUOs accurately replicate the tumor immune landscape. Key tumor immunity pathways such as cGAS-STING could be captured by ALI-LUOs. Importantly,ALI-LUOs modeled clinical responses to immune checkpoint inhibitors and radiotherapy with high fidelity. Conclusions: The ALI-LUOs,developed through an optimized culture method,faithfully capture the key characteristics of lung cancer,including its immunosuppressive tumor microenvironment. Our findings highlight this modified ALI-LUOs as a valuable preclinical platform for evaluating antitumor immunity and refining lung cancer treatments. View Publication

Background: Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy with poor overall survival (OS). Resistance to chemotherapeutic drugs such as idarubicin (IDA) remains a major cause of treatment failure. This study investigated the anti-leukemic activity of halofuginone (HF) a synthetic derivative of the natural compound from hydrangea Dichroa febrifuge and its potential to overcome IDA resistance in AML cells. Methods: Apoptosis,proliferation,cell cycle,and colony formation were assessed in AML cells treated with HF. RNA sequencing (RNA-seq) was performed to identify the potential molecular targets of HF. The anti-leukemic efficacy of HF was further assessed in NOD/SCID-IL2Rγ (NSG) mice xenografted with human relapsed/refractory (R/R) AML samples. Results: HF treatment significantly inhibited cell proliferation,reduced colony formation,and induced apoptosis in AML cells. By RNA-seq analysis,S100A8 and S100A9 (S100A8/A9) were identified as potential targets of HF,and HF treatment markedly suppressed their expression. Overexpression of S100A8/A9 abrogated the anti-leukemic effects of HF,indicating that S100A8/A9 are critical mediators of HF activity. Mechanistically,HF activated the amino acid starvation response (AAR),leading to phosphorylation of eukaryotic translation initiation factor 2 subunit alpha (p-eIF2α),subsequent downregulation of S100A8/A9,and elevation of cytoplasmic Ca2⁺ levels. Knockdown of eIF2α prevented HF-induced downregulation of S100A8/A9,confirming that HF regulates S100A8/A9 expression via the eIF2α pathway. Furthermore,HF treatment inhibited global protein synthesis,enhanced the cytotoxicity of chemotherapeutic drugs,and reversed IDA resistance by suppressing S100A8/A9 expression. Finally,HF inhibits leukemic infiltration and extended OS in MLL-AF9-transduced AML mice and enhanced IDA-induced anti-leukemic effects in R/R AML-xenografted NSG mice model. Conclusions: These findings reveal that HF exerts anti-leukemic effects by modulating the p-eIF2α–S100A8/A9–Ca2⁺ signaling axis in AML cells. HF represents a promising therapeutic candidate for AML,particularly for patients with IDA-resistant disease. View Publication