技术资料

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

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

β-cell dysfunction and dedifferentiation towards an α-cell-like phenotype are hallmarks of type 2 diabetes. However,the cell subtypes involved in β-to-α-cell transition are unknown. Using single-cell and single-nucleus RNA-seq,RNA velocity,PAGA/cell trajectory inference,and gene commonality,we interrogated α-β-cell fate switching in human islets. We found five α-cell subclusters with distinct transcriptomes. PAGA analysis showed bifurcating cell trajectories in non-diabetic while unidirectional cell trajectories from β-to-α-cells in type 2 diabetes islets suggesting dedifferentiation towards α-cells. Ten genes comprised the common signature genes in trajectories towards α-cells. Among these,the α-cell gene SMOC1 was expressed in β-cells in type 2 diabetes. Enhanced SMOC1 expression in β-cells decreased insulin expression and secretion and increased β-cell dedifferentiation markers. Collectively,these studies reveal differences in α-β-cell trajectories in non-diabetes and type 2 diabetes human islets,identify signature genes for β-to-α-cell trajectories,and discover SMOC1 as an inducer of β-cell dysfunction and dedifferentiation. Subject terms: Cell signalling,Diabetes,Differentiation View Publication

The cell cycle (CC) underpins diverse cell processes like cell differentiation,cell expansion,and tumorigenesis but current single-cell (sc) strategies study CC as: coarse phases,rely on transcriptomic signatures,use imaging modalities limited to adherent cells,or lack high-throughput multiplexing. To solve this,we develop an expanded,Mass Cytometry (MC) approach with 48 CC-related molecules that deeply phenotypes the diversity of scCC states. Using Cytometry by Time of Flight,we quantify scCC states across suspension and adherent cell lines,and stimulated primary human T cells. Our approach captures the diversity of scCC states,including atypical CC states beyond canonical definitions. Pharmacologically-induced CC arrest reveals that perturbations exacerbate noncanonical states and induce previously unobserved states. Notably,primary cells escaping CC inhibition demonstrated aberrant CC states compared to untreated cells. Our approach enables deeper phenotyping of CC biology that generalizes to diverse cell systems with simultaneous multiplexing and integration with MC platforms. Subject terms: Assay systems,Proteomics,Cell biology,Immunology,Systems biology View Publication

Cancer drug resistance poses a significant challenge in oncology,often driven by intricate cross-talk among membrane-bound receptors that compromise mono-targeted therapies. We develop a dual membrane receptor degradation strategy leveraging Folate Receptor α (FRα) to address this issue. Folate Receptor α Targeting Chimeras-dual (FolTAC-dual) are engineered degraders designed to selectively and simultaneously degrade distinct receptor pairs: (1) EGFR/HER2 and (2) PD-L1/VISTA. Through modular optimization of modality configurations and geometries,we identify the “string” format as the most effective construct. Mechanistic studies demonstrate an ~85% increase in EGFR-binding affinity compared to the conventional knob-into-hole design,likely contributing to the improved efficiency of dual-target degradation. Proof-of-concept studies reveal that EGFR and HER2 FolTAC-dual effectively counteracts resistance in Trastuzumab/Lapatinib-resistant HER2-positive breast cancer models,while PD-L1 and VISTA FolTAC-dual rejuvenates immune responses in PD-L1 antibody-resistant syngeneic mouse models. These findings establish FolTAC-dual as a promising dual-degradation platform for clinical translation. Subject terms: Cancer immunotherapy,Targeted therapies,Protein design,Drug discovery and development View Publication

Extracellular vesicles (EVs) are nano-sized lipid vesicles released into the extracellular space. We investigated the role of mouse brain-derived EVs in α-synuclein (α-syn) degradation and pathology transmission. Using sucrose gradient isolation and biochemical characterization,we found that EVs harbor active proteases that cleave both monomeric α-syn and pre-formed fibrils (PFFs). Protease activity and inhibitor profiling identified cathepsins B and S as key enzymes mediating this cleavage. EV-mediated proteolysis reduced the seeding capacity of α-syn PFFs in vitro and in vivo,whereas protease inhibition enhanced aggregation. Proteomic analysis revealed a restricted protease repertoire within EV cargo. Our findings suggest that EVs regulate extracellular α-syn levels via proteolysis,thereby modulating its prion-like spreading potential. We suggest that EVs represent a novel post-translational mechanism to regulate the levels of extracellular α-syn and may thus affect the spreading of α-syn pathology. Targeting this proteolytic capacity may offer new therapeutic interventions for mitigating synucleinopathies. Subject terms: Biochemistry,Cell biology,Neuroscience,Pathogenesis View Publication

Background: Lipoprotein(a) [Lp(a)]-induced inflammation contributes to coronary artery spasm (CAS) by the contraction of vascular smooth muscle cells. However,the interaction between Lp(a) and soluble CD36 (sCD36)/interleukin (IL)-6/RAS Homolog Family Member A (RhoA)-GTP signaling pathway has not been evaluated. Methods: We investigated the relevance of Lp(a)/CD36 signaling in CAS patient monocyte-derived macrophages (PMDMs) and a human coronary artery smooth muscle cell (HCASMC) line using expression profile correlation analyses,molecular docking,RNA sequencing,flow cytometry,immunoblotting,and quantitative reverse transcription polymerase chain reaction. Results: Plasma Lp(a) and sCD36 levels in 41 CAS patients were significantly higher ( p = 0.001) and positively correlated (r 2 = 0.3145,p < 0.001),a trend not observed in 36 non-CAS controls. RNA sequencing indicated a significant co-overexpression of CD36 and RhoA in Lp(a)-treated CAS PMDMs and HCASMCs,of which the mRNA and protein expression of CD36 and RhoA were significantly enhanced ( p < 0.001) dose-dependently. Lp(a) rather than LDL preferentially induced CD80+ PMDM (M1) polarization. In HCASMCs,the CD36 knockdown using either short hairpin RNA or natural biflavonoid amentoflavone suppressed Lp(a)-upregulated protein expression of CD36,RhoA-GTP,IL-6,tumor necrosis factor (TNF)-α,nuclear factor (NF)-κB,and CD80; however,overexpressed CD36 increased their levels. Lp(a) decreased and amentoflavone increased the epigenetic expression of CD36 inhibitors,miR-335-5p,and miR-448,respectively. Reciprocally,an miRNA inhibitor or mimic could magnify or diminish Lp(a)-induced CD36,TNF-α,NF-κB and IL-6 expressions in HCASMCs,respectively. Conclusions: Elevated Lp(a) levels upregulate the CD36-dependent TNF-α/NF-κB/IL-6/RhoA-GTP signaling pathway in CAS PMDMs and HCASMCs,indicating that Lp(a)/CD36 inflammatory signaling,HCASMC activation,and macrophage M1 polarization mediate CAS development. View Publication