技术资料

Alzheimer’s disease (AD) is the leading cause of dementia globally. The accumulation of amyloid and tau proteins,neuronal cell death and neuroinflammation are seen with AD progression,resulting in memory and cognitive impairment. Microglia are crucial for AD progression as they engage with neural cells and protein aggregates to regulate amyloid pathology and neuroinflammation. Recent studies indicate that microglia contribute to the propagation of amyloid beta (Aβ) via their immunomodulatory functions including Aβ phagocytosis and inflammatory cytokine production. Three-dimensional cell culture techniques provide the opportunity to study pathophysiological changes in AD in human-derived samples that are difficult to recapitulate in animal models (e.g.,transgenic mice). However,these models often lack immune cells such as microglia,which play a critical role in AD pathophysiology. In this study,we developed a neuroimmune assembloid model by integrating cerebral organoids (COs) with induced microglia-like cells (iMGs) derived from human induced pluripotent stem cells from familial AD patient with PSEN2 mutation. After 120 days in culture,we found that iMGs were successfully integrated within the COs. Interestingly,our assembloids displayed histological,functional and transcriptional features of the pro-inflammatory environment seen in AD,including amyloid plaque-like and neurofibrillary tangle-like structures,reduced microglial phagocytic capability,and enhanced neuroinflammatory and apoptotic gene expression. In conclusion,our neuroimmune assembloid model effectively replicates the inflammatory phenotype and amyloid pathology seen in AD. The online version contains supplementary material available at 10.1186/s12974-025-03544-x. View Publication

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by the lack of ER,PR,and HER2 expression. Its aggressive behavior,high degree of tumor heterogeneity,and immunosuppressive tumor microenvironment (TME) are associated with poor clinical outcomes,rapid disease progression,and limited therapeutic options. Although chimeric antigen receptor (CAR)-engineered T cell therapy has shown certain promise,its applicability in TNBC is hindered by antigen escape,TME-mediated suppression,and the logistical constraints of autologous cell production. In this study,we employed hematopoietic stem and progenitor cell (HSPC) gene engineering and a feeder-free HSPC differentiation culture to generate allogeneic IL-15-enhanced,mesothelin-specific CAR-engineered invariant natural killer T ( Allo15 MCAR-NKT) cells. These cells demonstrated robust and multifaceted antitumor activity against TNBC,mediated by CAR- and NK receptor-dependent cytotoxicity,as well as selective targeting of CD1d + TME immunosuppressive cells through their TCR. In both orthotopic and metastatic TNBC xenograft models,Allo15 MCAR-NKT cells demonstrated potent antitumor activity,associated with robust effector and cytotoxic phenotypes,low exhaustion,and a favorable safety profile without inducing graft-versus-host disease. Together,these results support Allo15 MCAR-NKT cells as a next-generation,off-the-shelf immunotherapy with strong therapeutic potential for TNBC,particularly in the context of metastasis,immune evasion,and treatment resistance. The online version contains supplementary material available at 10.1186/s13045-025-01736-9. View Publication

Ovarian cancer remains the most lethal gynaecological malignancy,with tumour recurrence and chemoresistance posing significant therapeutic challenges. Emerging evidence suggests that cancer stem cells (CSCs),a rare subpopulation within tumours with self‐renewal and differentiation capacities,contribute to these hurdles. Therefore,elucidating the mechanisms that sustain CSCs is critical for improving treatment strategies. Mitophagy,a selective process for eliminating damaged mitochondria,plays a key role in maintaining cellular homeostasis,including CSC survival. Our study demonstrates that ovarian CSCs exhibit enhanced mitophagy,accompanied by elevated expression of the mitochondrial outer membrane receptors BNIP3 and BNIP3L. Knockdown of BNIP3 or BNIP3L significantly reduces mitophagy and impairs CSC self‐renewal,indicating that receptor‐mediated mitophagy is essential for CSC maintenance. Mechanistically,we identify that hyperactivated NF‐κB signalling drives the upregulation of BNIP3 and BNIP3L in ovarian CSCs. Inhibition of NF‐κB signalling,either via p65 knockdown or pharmacological inhibitors,effectively suppresses mitophagy. Furthermore,we demonstrate that elevated DNA‐PK expression contributes to the constitutive activation of NF‐κB signalling,thereby promoting mitophagy in ovarian CSCs. In summary,our findings establish that BNIP3/BNIP3L‐mediated mitophagy,driven by DNA‐PK‐dependent NF‐κB hyperactivation,is essential for CSC maintenance. Targeting the DNA‐PK/NF‐κB/BNIP3L‐BNIP3 axis to disrupt mitochondrial quality control in CSCs represents a promising therapeutic strategy to prevent ovarian cancer recurrence and metastasis. View Publication

Bovine viral diarrhea virus (BVDV) is an important viral disease of cattle that causes immune dysfunction. Macrophages are the key cells for the initiation of the innate immunity and play an important role in viral pathogenesis. In this in vitro study,we studied the effect of the supernatant of BVDV-infected macrophage on immune dysfunction. We infected bovine monocyte-derived macrophages (MDM) with high or low virulence strains of BVDV. The supernatant recovered from BVDV-infected MDM was used to examine the functional activity and surface marker expression of normal macrophages as well as lymphocyte apoptosis. Supernatants from the highly virulent 1373-infected MDM reduced phagocytosis,bactericidal activity and downregulated MHC II and CD14 expression of macrophages. Supernatants from 1373-infected MDM induced apoptosis in MDBK cells,lymphocytes or BL-3 cells. By protein electrophoresis,several protein bands were unique for high-virulence,1373-infected MDM supernatant. There was no significant difference in the apoptosis-related cytokine mRNA (IL-1beta,IL-6 and TNF-a) of infected MDM. These data suggest that BVDV has an indirect negative effect on macrophage functions that is strain-specific. Further studies are required to determine the identity and mechanism of action of these virulence factors present in the supernatant of the infected macrophages. View Publication

To date,the effects of endurance exercise training on lymphocyte physiology at the kinome level are largely unknown. Therefore,the present study used a highly sensitive peptide-based kinase activity profiling approach to investigate if the basal activity of tyrosine (Tyr) and serine/threonine (Ser/Thr) kinases of human lymphocytes is affected by the aerobic endurance training status. Results revealed that the activity of various tyrosine kinases of the FGFR family and ZAP70 was increased,whereas the activity of multiple Ser/Thr kinases such as IKK$\alpha$,CaMK4,PKA$\alpha$,PKC$\alpha$+$\delta$ (among others) was decreased in lymphocytes of endurance trained athletes (ET). Moreover,functional associations between several differentially regulated kinases in ET-derived lymphocytes were demonstrated by phylogenetic mapping and network analysis. Especially,Ser/Thr kinases of the AGC-kinase (protein kinase A,G,and C) family represent exercise-sensitive key components within the lymphocytes kinase network that may mediate the long-term effects of endurance training. Furthermore,KEGG (Kyoto Encyclopedia of Genes and Genomes) and Reactome pathway analysis indicate that Ras as well as intracellular signaling by second messengers were found to be enriched in the ET individuals. Overall,our data suggest that endurance exercise training improves the adaptive immune competence by modulating the activity of multiple protein kinases in human lymphocytes. View Publication

Tandemers 2MG,2MG3,3MG and 4MG are derivatives of the potent anti-HIV-1 microbicide candidate griffithsin (GRFT). We compared these compounds anti-HIV-1 activity to GRFT using the viruses CAP206.08 and CAAN5342.A2 that have decreased sensitivity to this lectin. The 2MG and 2MG3 tandemers had similar activity to GRFT against cell-free and cell-associated viruses,while 3MG and 4MG were significantly more potent. Furthermore,the restoration of the 234N or 295N glycan in these viruses,known to increase sensitivity to GRFT,also increased sensitivity to 2MG and 2MG3,and not to 3MG and 4MG. In addition,GRFT resistant viruses generated in-vitro were equally resistant to 2MG and 2MG3 while they had considerably low resistance to 3MG and 4MG. Lastly,all five compounds showed increased inhibitory activity in seminal and vaginal simulants although the effect was more pronounced in the former. These data support further studies of tandemers as potential microbicides. View Publication

Tumor complexity and intratumor heterogeneity contribute to subclonal diversity. Despite advances in next-generation sequencing (NGS) and bioinformatics,detecting rare mutations in primary tumors and metastases contributing to subclonal diversity is a challenge for precision genomics. Here,in order to identify rare mutations,we adapted a recently described epithelial reprograming assay for short-term propagation of epithelial cells from primary and metastatic tumors. Using this approach,we expanded minor clones and obtained epithelial cell-specific DNA/RNA for quantitative NGS analysis. Comparative Ampliseq Comprehensive Cancer Panel sequence analyses were performed on DNA from unprocessed breast tumor and tumor cells propagated from the same tumor. We identified previously uncharacterized mutations present only in the cultured tumor cells,a subset of which has been reported in brain metastatic but not primary breast tumors. In addition,whole-genome sequencing identified mutations enriched in liver metastases of various cancers,including Notch pathway mutations/chromosomal inversions in 5/5 liver metastases,irrespective of cancer types. Mutations/rearrangements in FHIT,involved in purine metabolism,were detected in 4/5 liver metastases,and the same four liver metastases shared mutations in 32 genes,including mutations of different HLA-DR family members affecting OX40 signaling pathway,which could impact the immune response to metastatic cells. Pathway analyses of all mutated genes in liver metastases showed aberrant tumor necrosis factor and transforming growth factor signaling in metastatic cells. Epigenetic regulators including KMT2C/MLL3 and ARID1B,which are mutated in {\textgreater}50{\%} of hepatocellular carcinomas,were also mutated in liver metastases. Thus,irrespective of cancer types,organ-specific metastases may share common genomic aberrations. Since recent studies show independent evolution of primary tumors and metastases and in most cases mutation burden is higher in metastases than primary tumors,the method described here may allow early detection of subclonal somatic alterations associated with metastatic progression and potentially identify therapeutically actionable,metastasis-specific genomic aberrations. View Publication

Prenatal cannabis exposure (PCE) influences human brain development,but it is challenging to model PCE using animals and current cell culture techniques. Here,we developed a one-stop microfluidic platform to assemble and culture human cerebral organoids from human embryonic stem cells (hESC) to investigate the effect of PCE on early human brain development. By incorporating perfusable culture chambers,air-liquid interface,and one-stop protocol,this microfluidic platform can simplify the fabrication procedure and produce a large number of organoids (169 organoids per 3.5 cm × 3.5 cm device area) without fusion,as compared with conventional fabrication methods. These one-stop microfluidic assembled cerebral organoids not only recapitulate early human brain structure,biology,and electrophysiology but also have minimal size variation and hypoxia. Under on-chip exposure to the psychoactive cannabinoid,$\Delta$-9-tetrahydrocannabinol (THC),cerebral organoids exhibited reduced neuronal maturation,downregulation of cannabinoid receptor type 1 (CB1) receptors,and impaired neurite outgrowth. Moreover,transient on-chip THC treatment also decreased spontaneous firing in these organoids. This one-stop microfluidic technique enables a simple,scalable,and repeatable organoid culture method that can be used not only for human brain organoids but also for many other human organoids including liver,kidney,retina,and tumor organoids. This technology could be widely used in modeling brain and other organ development,developmental disorders,developmental pharmacology and toxicology,and drug screening. View Publication

The recent advances,offered by cell therapy in the regenerative medicine field,offer a revolutionary potential for the development of innovative cures to restore compromised physiological functions or organs. Adult myogenic precursors,such as myoblasts or satellite cells,possess a marked regenerative capacity,but the exploitation of this potential still encounters significant challenges in clinical application,due to low rate of proliferation in vitro,as well as a reduced self-renewal capacity. In this scenario,induced pluripotent stem cells (iPSCs) can offer not only an inexhaustible source of cells for regenerative therapeutic approaches,but also a valuable alternative for in vitro modeling of patient-specific diseases. In this study we established a reliable protocol to induce the myogenic differentiation of iPSCs,generated from pericytes and fibroblasts,exploiting skeletal muscle-derived extracellular vesicles (EVs),in combination with chemically defined factors. This genetic integration-free approach generates functional skeletal myotubes maintaining the engraftment ability in vivo. Our results demonstrate evidence that EVs can act as biological shuttles" to deliver specific bioactive molecules for a successful transgene-free differentiation offering new opportunities for disease modeling and regenerative approaches." View Publication

Medulloblastoma (MB) is the most frequent malignant pediatric brain tumor,representing 20{\%} of newly diagnosed childhood central nervous system malignancies. Although advances in multimodal therapy yielded a 5-year survivorship of 80{\%},MB still accounts for the leading cause of childhood cancer mortality. In this work,we describe the epigenetic regulator BMI1 as a novel therapeutic target for the treatment of recurrent human Group 3 MB,a childhood brain tumor for which there is virtually no treatment option beyond palliation. Current clinical trials for recurrent MB patients based on genomic profiles of primary,treatment-naive tumors will provide limited clinical benefit since recurrent metastatic MBs are highly genetically divergent from their primary tumor. Using a small molecule inhibitor against BMI1,PTC-028,we were able to demonstrate complete ablation of self-renewal of MB stem cells in vitro. When administered to mice xenografted with patient tumors,we observed significant reduction in tumor burden in both local and metastatic compartments and subsequent increased survival,without neurotoxicity. Strikingly,serial in vivo re-transplantation assays demonstrated a marked reduction in tumor initiation ability of recurrent MB cells upon re-transplantation of PTC-028-treated cells into secondary recipient mouse brains. As Group 3 MB is often metastatic and uniformly fatal at recurrence,with no current or planned trials of targeted therapy,an efficacious targeted agent would be rapidly transitioned to clinical trials. View Publication

PURPOSE The outgrowth of antigen-negative variants is a significant challenge for adoptive therapy with T cells that target a single specificity. Chimeric antigen receptors (CAR) are typically designed with one or two scFvs that impart antigen specificity fused to activation and costimulation domains of T-cell signaling molecules. We designed and evaluated the function of CARs with up to three specificities for overcoming tumor escape using Designed Ankyrin Repeat Proteins (DARPins) rather than scFvs for tumor recognition. EXPERIMENTAL DESIGN A monospecific CAR was designed with a DARPin binder (E01) specific for EGFR and compared with a CAR designed using an anti-EGFR scFv. CAR constructs in which DARPins specific for EGFR,EpCAM,and HER2 were linked together in a single CAR were then designed and optimized to achieve multispecific tumor recognition. The efficacy of CAR-T cells bearing a multispecific DARPin CAR for treating tumors with heterogeneous antigen expression was evaluated in vivo. RESULTS The monospecific anti-EGFR E01 DARPin conferred potent tumor regression against EGFR+ targets that was comparable with an anti-EGFR scFv CAR. Linking three separate DARPins in tandem was feasible and in an optimized format generated a single tumor recognition domain that targeted a mixture of heterogeneous tumor cells,each expressing a single antigen,and displayed synergistic activity when tumor cells expressed more than one target antigen. CONCLUSIONS DARPins can serve as high-affinity recognition motifs for CAR design,and their robust architecture enables linking of multiple binders against different antigens to achieve functional synergy and reduce antigen escape. View Publication

BACKGROUND Lymphotoxin $\beta$ receptor (LT$\beta$R) plays important roles in the development of the immune system and immune response. At the cellular level,ligand-bound LT$\beta$R activates the pro-inflammatory NF-$\kappa$B pathway but the detailed mechanisms regulating its signaling remain unknown. Understanding them is of high importance since LT$\beta$R and its ligands are promising therapeutic targets. Here,we studied the consequences of perturbed cellular cholesterol content on LT$\beta$R-induced NF-$\kappa$B signaling. METHODS To modulate cholesterol availability and/or level in lung carcinoma A549 and H2228,and endothelial HUVEC cells different treatment regimens with filipin,methyl-$\beta$-cyclodextrin and simvastatin were applied. LT$\beta$R localization was studied by confocal microscopy. The activity of LT$\beta$R-induced NF-$\kappa$B pathway was assessed by measuring the levels of NF-$\kappa$B pathway inhibitor I$\kappa$B$\alpha$ and phosphorylation of RelA transcription factor by Western blotting. The NF-$\kappa$B transcriptional response,production of chemokines and adhesion molecules were examined by qRT-PCR,ELISA,and Western blotting,respectively. Adherence of different types of primary immune cells to epithelial A549 cells and endothelial HUVECs was measured fluorometrically. Interactions of LT$\beta$R with its protein partners were investigated by immunoprecipitation. RESULTS We showed that filipin-mediated sequestration of cholesterol or its depletion from the plasma membrane with methyl-$\beta$-cyclodextrin impaired LT$\beta$R internalization and potentiated LT$\beta$R-dependent activation of the canonical branch of the NF-$\kappa$B pathway. The latter was manifested by enhanced degradation of I$\kappa$B$\alpha$ inhibitor,elevated RelA phosphorylation,substantial increase in the expression of NF-$\kappa$B target genes encoding,among others,cytokines and adhesion molecules known to play important roles in immune response. It was followed by robust secretion of CXCL8 and upregulation of ICAM1,that favored the adhesion of immune cells (NK and T cells,neutrophils) to A549 cells and HUVECs. Mechanistically,we showed that cholesterol depletion stabilized interactions of ligand-stimulated LT$\beta$R with modified forms of TRAF2 and NEMO proteins. CONCLUSIONS Our results showed that the reduction of the plasma membrane content of cholesterol or its sequestration strongly potentiated signaling outcome initiated by LT$\beta$R. Thus,drugs modulating cholesterol levels could potentially improve efficacy of LT$\beta$R-based therapies. Video abstract. View Publication