技术资料

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

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

Natural killer (NK) cells play a crucial role in immune surveillance by recognizing and eliminating tumor cells. However,tumors employ various mechanisms to evade NK cell-mediated immunity. NKp30 is a potent activating receptor on NK cells,but its function can be inhibited by specific ligands secreted by cancer cells. Here,we identified dipeptidase 1 (DPEP1) as a novel ligand for NKp30 in KM12C colon cancer cells,using co-immunoprecipitation,confocal microscopy,and flow cytometry. We examined how the DPEP1–NKp30 interaction affects NK cell activity and found that NK cytotoxicity increased in KM12C cells with DPEP1 knockdown but was significantly reduced in HCT116 cells overexpressing DPEP1. We further demonstrated that DPEP1 is secreted via extracellular vesicles and that its interaction with NKp30 suppressed the expression and secretion of perforin 1,granzyme B,CD107a,and interferon-γ in NK92 cells. In a xenograft mouse model treated with NK92 cells,tumors derived from HCT116/DPEP1 cells were significantly larger than those from HCT116/mock cells. Using peripheral blood-derived human NK cells,we confirmed that DPEP1 inhibited both cytotoxicity and granzyme B secretion. These findings suggest that disrupting the DPEP1–NKp30 interaction may enhance NK cell-mediated cytotoxicity and represent a novel therapeutic strategy for cancer immunotherapy. The online version contains supplementary material available at 10.1038/s41598-025-18475-z. View Publication

Cost-effective,practical,and reproducible culture of human pluripotent stem cells (hPSCs) is required for basic and translational research. Basal 8 (B8) has emerged as a cost-effective solution for weekend-free and chemically-defined hPSC culture. However,the requirement to home-produce some recombinant growth factors for B8 can hinder access and reproducibility. Moreover,we found the published B8 formulation suboptimal in widely-used normoxic hPSC culture. Lastly,the performance of B8 in functional applications such as genome editing or organoid differentiation required systematic evaluation. We formulated B8 with commercially available,growth factors and adjusted its composition to support normoxic culture of WTC11 human induced pluripotent stem cell line. We compared this formulation (B8+) with commercial Essential 8 (cE8) and a home-made,weekend-free E8 formulation (hE8). We measured pluripotency marker expression and cell cycle by flow cytometry,and investigated the transcriptional profiles by bulk and single-cell RNA sequencing. We further assessed genomic stability,genome editing efficiency,single-cell cloning,and differentiation in both monolayer and organoids. Finally,we validated key findings using male (H1) and female (H9) human embryonic stem cells. hE8 performed comparably to cE8 across most functional assays and cell lines. In contrast,cells in B8+ displayed higher NANOG expression and improved genome editing efficiency. At the same time,B8+ led to gene expression changes indicative of marked lineage priming,reflected in altered morphology and differential response to some differentiation protocols. Both weekend-free media resulted in a modest transcriptional shift towards a less metabolically active state,consistent with intermittent media starvation. Homemade weekend-free media can provide a cost-effective alternative to commercial formulations. hE8,integrating some features of B8 while resembling cE8,emerges as a robust and practical option with limited compromises. B8+,though advantageous in some contexts,warrants caution due to lineage priming effects that may impact differentiation outcomes. View Publication

Proliferating cell nuclear antigen (PCNA),a well-documented anticancer target,is critical for DNA synthesis,replication,and repair. AOH1996,a small-molecule PCNA inhibitor,is currently undergoing clinical trials for the treatment of advanced solid tumors. However,the therapeutic effect of AOH1996 on head and neck squamous cell carcinoma (HNSCC) remains unclear. The effects of AOH1996 on HNSCC biological behaviors and cancer stemness were tested in HNSCC cells and nude mice. The combination treatment of AOH1996 and anti-PD1 was performed in a 4-nitroquinoline N-oxide (4NQO)-induced HNSCC mouse model. RNA sequencing,Western Blotting,immunofluorescence staining,comet assays,and qRT‒PCR were conducted for mechanistic studies. Our results showed that AOH1996 effectively inhibited HNSCC proliferation and invasion both in vitro and in vivo. AOH1996 suppressed HNSCC stemness,development,and metastasis. Moreover,AOH1996 altered the tumor immune microenvironment into an inflamed state with increased CD8 + T-cell infiltration,rendering it a favorable partner for combination therapy with immune checkpoint inhibitors. Mechanistically,AOH1996 induced cellular DNA damage,suppressed cancer stemness through the upregulation of p-TBK1,and promoted the secretion of CD8 + T-cell-recruiting chemokines by stimulating IRF3-mediated transcription. Taken together,our results demonstrated that AOH1996 suppressed tumor growth,eliminated cancer stem cells (CSCs),and synergistically enhanced the efficacy of anti-PD1 immunotherapy in HNSCC. The online version contains supplementary material available at 10.1186/s13287-025-04607-9. 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