参考文献

Cell surface proteins offer significant cancer therapeutic potential attributable to their accessible membrane localization and central roles in cellular signaling,yet their promise remains largely untapped due to technical challenges inherent to profiling them. Here,we employ N-glycoproteomics to analyze 85 patient-derived xenografts (PDXs),constructing Glyco PDXplorer—an in vivo pan-cancer atlas of cancer-derived surface proteins. We develop a target discovery pipeline to prioritize proteins with favorable expression profiles for immunotherapeutic targeting and validate FAT2 as a squamous-cancer-enriched surface protein minimally detected in normal tissue. Functional studies reveal that FAT2 is essential for head and neck squamous cancer (HNSC) cell growth and adhesion through regulation of surface architecture and integrin-PI3K signaling. Chimeric antigen receptor (CAR)-T cells targeting FAT2 demonstrate anti-tumor activity. This work lays the foundation for developing FAT2-targeted therapies and represents a pivotal platform to inform therapeutic target discovery across cancers. Graphical abstract Highlights•Pan-cancer landscape of cancer-derived cell surface proteins detected in vivo•Discovery pipeline to prioritize proteins as immunotherapy target candidates•Validation of FAT2 as an SCC surface protein with minimal normal tissue expression•FAT2 CAR-T cells demonstrate anti-tumor activity in pre-clinical models Govindarajan et al. leverage N-glycoproteomics and PDX models to decode the in vivo cancer cell surfaceome and establish Glyco PDXplorer—a target discovery platform. The identification and validation of FAT2 as a previously undescribed,actionable antigen demonstrates the utility of Glyco PDXplorer for uncovering therapeutic vulnerabilities. View Publication

Ferroptosis,an iron-dependent,lipid peroxidation-driven programmed cell death,holds substantial promise for cancer therapy,yet its translational potential is hindered by widespread intrinsic resistance. While glutathione peroxidase 4 (GPX4) is a well-established ferroptosis suppressor,the epigenetic circuitry coordinating GPX4-related mechanisms remains elusive. Here,via genome-wide screening,we identify ten-eleven translocation 1 (TET1)—a key mediator of DNA 5-hydroxymethylation—as a master controller of cancer cell ferroptosis susceptibility. In acute myeloid leukemia (AML),TET1 enhances 5hmC deposition at the glutamate-cysteine ligase catalytic subunit (GCLC) promoter to activate glutathione/γ-glutamyl-peptide metabolism,fortifying GPX4-dependent defense. Concurrently,TET1 activates NFκB signaling to upregulate GTP cyclohydrolase-1 (GCH1),conferring GPX4-independent ferroptosis resistance. Critically,co-targeting TET1/GCLC/GCH1 with low-dose ferroptosis inducers exhibits potent therapeutic effects against both ferroptosis-sensitive and -resistant AML. Our work positions TET1 as a pivotal epigenetic hub governing ferroptosis surveillance,and provides a translatable strategy to overcome ferroptosis resistance in cancer,with AML as a paradigm. DNA demethylation enzyme TET1 is a known oncogene in leukemia. Here,the authors discover that TET1 is involved in GPX4-dependent and -independent ferroptosis in acute myeloid leukemia via the regulation of GSH synthesis enzyme GCLC and BH4 synthesis enzyme GCH1. View Publication

Baculovirus,an insect virus commonly used for recombinant protein expression in insect cells and gene delivery in mammalian systems,is often generated through bacmid-based engineering. To enable flexible and programmable bacmid engineering,we developed SHOT 2.0,an optimized CRISPR-associated transposon platform that mediates RNA-guided and customized bacmid editing in Escherichia coli. The edited bacmid can be transfected into insect cells to produce recombinant baculoviruses. SHOT 2.0 supported site-specific integration of large DNA cargos (at least 14 kb) into defined loci such as v-cath and ODVe56,with integration at ODVe56 markedly improving transgene stability during serial virus passaging. The system is fully compatible with the Bac-to-Bac® workflow,enabling dual-gene insertion into the bacmid and derived baculovirus. Leveraging this platform,we constructed an all-in-one baculovirus encoding the PE5max prime editor. This vector-mediated prime editing achieves efficiencies up to 85.6% in HEK293T cells and achieves robust prime editing in hard-to-transfect cell types,including iPSCs and liver cancer cells,with efficiencies up to 37.1%. These results demonstrate that SHOT 2.0 substantially expands the baculovirus engineering toolbox,providing a flexible platform for genome editing and future gene delivery. View Publication

Expansion microscopy (ExM) has enabled nanoscale imaging of tissues by physically enlarging biological samples in a swellable hydrogel. However,the increased sample size and water-based environment pose challenges for deep imaging using conventional inverted confocal microscopes,particularly due to the limited working distance of high-numerical-aperture (NA) water immersion objectives. Here,we introduce a practical imaging alternative that utilizes an inverted water-dipping objective and a refractive-index-matched optical path using fluorinated ethylene propylene (FEP) film. Through point spread function (PSF) measurements and simulations,we show that the FEP film introduces predominantly defocus-like wavefront profiles characteristic of high NA systems,which result in an easily correctable axial shift of the focal plane. To ensure stable immersion and refractive index continuity,we use an arrangement relying on an FEP film,Immersol W,water and a FEP-based imaging dish. This configuration achieves sub-micron lateral and axial resolution,supports large tile-scan acquisitions,and maintains image quality across depths exceeding 800 µm. We validate the system by imaging 4×-expanded U2OS cells and human cerebral organoids. Our approach provides a low-cost,plug-and-play solution for high-resolution volumetric imaging of expanded samples using standard inverted microscopes. View Publication

Cis-regulatory elements (CREs) are noncoding DNA regions regulating cell-type-specific gene expression programs by interacting with distal gene promoters. Here,we aim to decode the function and spatial organization of CRE-promoter interactions in human cardiomyocytes. We analyzed the epigenome and chromatin interactions of human male atrial,ventricular,and failing cardiomyocytes. Atrial and ventricular cardiomyocytes harbored chamber-specific CRE-promoter interactions modulating gene expression as confirmed by functional epigenetic silencing. These CRE-promoter interactions explain the distinct contribution of non-coding genetic variants to atrial and ventricular diseases,such as dilated cardiomyopathy and arrhythmias. We dissected the prototypic KCNJ2 locus,encoding a potassium channel associated with ventricular arrhythmia susceptibility. Functional epigenetic silencing confirmed that CREs,harboring QT-duration-associated genetic risk factors,modulate KCNJ2 gene expression levels,alter KCNJ2-dependent channel currents,and affect cardiomyocyte repolarization. The presented human CM-specific chromatin interaction analysis provides key insights into regulatory mechanisms and aids in interpreting genetic risk factors. Here the authors functionally test and resolve the spatial genome organization of cis-regulatory elements and genetic variants in atrial,ventricular,and failing human cardiomyocytes and linked them to heart disease traits,including QT syndrome. View Publication

Chronic neuroinflammation plays a key role in the progression of Alzheimer’s disease (AD),and the cytosolic calcium-dependent phospholipase A2 (cPLA2) enzyme is a critical mediator of inflammatory lipid signaling pathways. Here we investigate the therapeutic potential of novel cPLA2 inhibitors in modulating neuroinflammation in AD. By leveraging the giga-scale V-SYNTHES2 virtual screening in on-demand chemical space and conducting two rounds of optimization for potency and selectivity,we have identified BRI-50460,achieving an IC50 of 0.88 nM in cellular assays that measure cPLA2-mediated arachidonic acid release. In vivo studies revealed favorable brain-to-plasma ratios,highlighting the ability of BRI-50460 to penetrate the central nervous system,modulating neuroinflammatory pathways,and restoring lipid homeostasis. In astrocytes and neurons derived from human induced pluripotent stem cells,BRI-50460 mitigates the effects of amyloid beta 42 oligomers on cPLA2 activation,tau hyperphosphorylation,and synaptic loss. Our results support that small molecule inhibitors of cPLA2 can modulate the downstream inflammatory signaling,offering a promising therapeutic strategy for neurodegenerative diseases. View Publication

Interleukin-17 (IL-17) plays a central role in the pathogenesis of various autoimmune diseases. Soluble CD14 (sCD14),a marker of innate immune activation,is elevated in several inflammatory conditions. However,its influence on IL-17 production and the differentiation of Th17 cells remains poorly understood. We found that sCD14 enhances Th17-associated cytokine production and up-regulates critical transcription factors such as STAT3 and RORC. Notably,sCD14's effect on Th17 polarization was mediated indirectly through autologous sCD14-treated peripheral blood mononuclear cell (PBMC) supernatant (sCD14-PBMC-Sup). Additionally,we identified a distinct cytokine profile enriched for pro-inflammatory cytokines and chemokines in sCD14-treated T cells,further reinforcing the Th17-promoting role of sCD14. Interestingly,gamma-aminobutyric acid (GABA),a metabolite elevated in sCD14-treated monocytes,was identified as a potential contributor to Th17 polarization. GABA supplementation in T-cell cultures enhanced IL-17A secretion,indicating its role as a signaling molecule in T-cell differentiation. Our findings also revealed the expansion of innate lymphoid cell (ILC)2/3-like cells in T-cell cultures exposed to sCD14-PBMC-Sup and GABA,highlighting the potential role of monocytes in Th17-mediated immunity. Furthermore,while sCD14 promoted Th17 polarization,it simultaneously impaired T-cell activation and proliferation,suggesting an immunosuppressive effect mediated by soluble factors released from monocytes. These results underscore the dual role of sCD14 in modulating T-cell responses,promoting Th17 differentiation while suppressing T-cell effector functions. This study identifies a previously unrecognized role for sCD14 in promoting Th17 induction,highlighting its contribution to immune regulation and its potential as a therapeutic target in Th17-driven autoimmune conditions. View Publication

HIV-associated distal sensory polyneuropathy (HIV-DSP) remains prevalent even in the antiretroviral therapy (ART) era. Previously,we identified the upregulation of nociceptive ion channels transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1) in the dorsal root ganglia (DRG) of simian immunodeficiency virus (SIV)-infected ART-treated macaques. To investigate upstream mechanisms,we performed bulk RNA-seq and pathway analysis on DRGs from uninfected,SIV-infected,and SIV-infected/ART macaques. SIV infection drove strong activation of upstream regulators of interferon γ (IFNγ) and lipopolysaccharide (LPS). Although ART reduced overall IFNγ and LPS pathway activity,the IFNγ-inducible chemokines C-X-C motif chemokine ligand (CXCL)9 and CXCL10 remained significantly upregulated. To determine whether these chemokines influence TRPV1/TRPA1 expression,we treated induced pluripotent stem cell-derived peripheral sensory neurons (iPSC-PSNs) with CXCL9 and CXCL10,which induced a significant increase in TRPV1 but not TRPA1 expression. In parallel experiments,IFNγ but not LPS stimulated monocyte-derived macrophages (MDMs) to release CXCL9 and CXCL10. Conditioned media from IFNγ-treated MDMs modestly increased TRPV1 expression in iPSC-PSNs,and pharmacological inhibition of CXCR3,the receptor of CXCL9/10,did not reduce this effect. Together,these data indicate that persistent IFNγ-driven CXCL9/10 signaling may be one contributor to nociceptor sensitization underlying HIV-DSP,even in the presence of ART. View Publication

Human induced pluripotent stem cells (hiPSCs) offer significant potential for differentiation and research applications in cardiovascular diseases. When induced differentiated hiPSC-derived cardiomyocytes (hiPSC-CMs) are transplanted into the infarcted myocardial region,they exhibit extremely low survival rates and unsatisfactory therapeutic effects due to ischemia,hypoxia,and immune inflammation in the surrounding environment. To address this issue,we used Panax notoginseng saponin R1 (NGR1),which has demonstrated significant protective effects in prior research,to pretreat hiPSC-CMs before transplantation. Utilizing an in vitro H2O2 oxidative stress model and a nude mouse myocardial infarction (MI) model,we investigated the mechanism through which NGR1 pretreatment enhances the therapeutic efficacy of hiPSC-CM transplantation. The results revealed that the hiPSC-CMs expressed cTnT. NGR1 did not promote the proliferation of hiPSC-CMs but instead induced elevated levels of p-Akt protein in these cells. Compared to hiPSC-CM transplantation alone,transplantation of hiPSC-CMs pretreated with NGR1 exhibited higher ejection fraction (EF) and fractional shortening (FS) values,along with reduced infarct size and collagen deposition. Additionally,there were more HNA-positive cardiomyocytes in the cardiac tissue,fewer TUNEL-positive signals,and increased VWF-positive and Lyve1-positive signals. Furthermore,the gene expression levels of VEGFC,IGF-1,and SDF-1 were higher. Therefore,NGR1 pretreatment improves the survival of transplanted hiPSC-CMs in tissues,reduces myocardial apoptosis,enhances cardiac function,decreases infarct size and collagen deposition,promotes angiogenesis and lymphangiogenesis,and stimulates paracrine secretion. View Publication

The Mas-related G protein-coupled receptor (MRGPR) X2 is expressed on skin mast cells and can be stimulated by an unusually broad spectrum of ligands,including specific drugs and even endogenous peptides. MRGPRX2 activation can induce mast cell degranulation and consequently mediator release,leading to inflammatory and hypersensitivity reactions. In addition,MRGPRX2 mediates pain and itching sensations,leading to increased efforts to identify MRGPRX2 inhibitors,including plant-derived compounds. Components within oat extracts have been shown to mediate anti-inflammatory and itch-relieving properties,but a possible inhibitory effect on MRGPRX2 activation has not yet been investigated. We aimed to fill this gap and explored whether an oat kernel extract can modulate MRGPRX2 activation. For this purpose,we established a mast cell model with the human LAD2 cell line and used it to investigate the consequences of exposure to oat extract. While we did not observe any influence on cell viability,we analyzed the impact of oat extract on MRGPRX2-mediated mast cell activation and degranulation initiated by the three confirmed MRGPRX2 ligands c48/80,substance P,and cortistatin 14. Exposure to oat extract resulted in a significant reduction in mast cell degranulation for all three ligands,as assessed by the release of β-hexosaminidase,tryptase,cell surface expression of CD63 and CD107a,and phosphorylation of ERK. All results were confirmed with primary human mast cells. Thus,we demonstrated for the first time that oat extract leads to a significant reduction in MRGPRX2 activation,pointing to a previously unrecognized capacity of natural compounds to modulate this pathway. View Publication