技术资料

USP30,a ubiquitin‐specific protease,primarily characterized as a mitochondrial deubiquitinase regulating mitophagy,has not been previously reported to have nuclear functions. In this study,we demonstrate that USP30 is present in both mitochondrial and nuclear compartments. Nutrient deprivation triggers USP30 nuclear translocation via an N‐terminal nuclear localization signal (NLS),mediated through suppression of mTORC1‐dependent phosphorylation at serine 104,a modification constraining nuclear entry. Nuclear USP30 acts as a tumor suppressor by inhibiting cancer stemness and chemoresistance in triple‐negative breast cancer (TNBC) cells. Mechanistically,USP30 directly interacts with and deubiquitinates the transcription factor TCF/LEF1 at K379 and K382 residues,disrupting recruitment of CBP/P300 co‐activators to the β‐catenin/LEF1 complex. This abolishes β‐catenin/LEF1 transactivation and suppresses WNT signaling. Clinically,USP30 is downregulated in TNBC and cancer stem cells (CSCs),with notably reduced nuclear levels in cancer tissues. Overexpression of nuclear USP30 markedly reduces lung metastatic burden in TNBC mouse models. These findings uncover a novel role for nuclear USP30 in regulating cancer stemness and suggest that targeting the dynamic relocalization of USP30 from mitochondria to the nucleus could offer new therapeutic strategies for breast cancer metastasis. View Publication

Acute myeloid leukemia (AML) is primarily driven by leukemic stem cells (LSCs),the main cause of relapse and therapy resistance. Here,we discover that LSCs are predominantly small and mechanically soft. These mechanical properties enable their selective isolation using microfluidic chips. Single-cell RNA-sequencing of primary human AML bone marrow identifies enrichment of LSCs within the FSClow ALDH1A1+ subpopulation,which exhibits long-term stemness in functional assays. Notably,inhibiting ALDH1A1 in these cells promotes F-actin polymerization and increases cellular stiffness,reducing their stemness while enhancing their susceptibility to natural killer (NK) cell-mediated cytotoxicity. In AML patient-derived xenograft models,the combination of ALDH1A1 inhibition with NK cell therapy markedly suppresses leukemia progression. These findings suggest that targeting the mechanical properties of LSC offers a promising strategy to overcome AML treatment resistance,providing insights into stem cell mechanobiology and paving the way for combining targeted therapies with immunotherapy to improve clinical outcomes. Leukemic stem cells (LSCs) drive relapse and therapy resistance in acute myeloid leukemia (AML). Here,the authors show that increasing the stiffness of LSCs reduces their stemness and enhances their susceptibility to natural killer cell-mediated immunotherapy in AML. View Publication

Systemic sclerosis (SSc) is an autoimmune disease marked by fibrosis and extensive autoantibody production. Although B-cell depletion with rituximab (RTX) has shown clinical benefit,predictive biomarkers of response remain elusive. Here,we apply proteome-wide autoantibody screening using wet protein arrays covering 13,455 human antigens in serum samples from participants of the randomized trial of RTX. We identify a significant elevation in the total autoantibody levels in SSc compared to healthy controls,with greater reductions post-treatment observed in RTX high responders than in low responders. A stepwise selection highlights 88 clinically relevant autoantibodies,including those targeting G protein-coupled receptors. Among them,anti-C-C motif chemokine receptor 8 (CCR8) autoantibodies are functionally validated by cell-based assays using CCR8-overexpressing HEK293 cells. Furthermore,in a bleomycin-induced mouse model,anti-CCR8 antibody administration exacerbates dermal fibrosis and modifies immune cell infiltration. Although external validation with multiple comparison adjustment is further required,these findings reveal an autoantibody signature associated with therapeutic response and pathogenic potential in SSc,providing a foundation for precision immunotherapy and mechanistic insights into disease progression. B-cell depletion benefits systemic sclerosis,but predictive biomarkers remain limited. The authors here map autoantibody profiles using proteome-wide screening,identify C-C motif chemokine receptor 8-targeting autoantibodies with functional impact,suggesting novel pathophysiology and precision therapy targets. View Publication

Older adults have decreased vaccine efficacy,but the adjuvanted recombinant VZV-gE zoster vaccine (RZV) is highly efficacious. We investigated memory-like innate immune responses after RZV and after the zoster vaccine live (ZVL),which is much less efficacious. RZV increased NK,monocyte,and DC activation in response to in vitro VZV-gE stimulation for up to 5 years post-vaccination,while ZVL increased only DC responses to VZV for up to 90 days. In purified monocyte and NK cell cocultures,RZV recipients showed increased responses to VZV-gE,HCMV and HSV antigenic stimulation post-vaccination. ATAC-seq analysis of purified monocytes revealed decreased accessibility in areas of the TGFβ1 gene. scRNA-seq and immunoproteomics confirmed decreased TGFβ1 transcription and translation,respectively. Exogenous supplementation and inhibition of TGFβ1 modulated in vitro monocyte responses to VZV-gE. In conclusion,RZV generated homologous (VZV-gE) and heterologous (HCMV,HSV) trained immunity in monocytes through genomic repression of the regulatory cytokine TGFβ-1. Cytokine modulation may represent a novel mechanism of generating trained immunity in myeloid cells. Author summaryOlder adults have decreased vaccine efficacy,but the adjuvanted recombinant varicella zoster virus (VZV)-gE zoster vaccine (RZV; Shingrix™) is highly efficacious. We investigated memory-like innate immune responses after RZV and after the zoster vaccine live (ZVL; Zostavax™),which is much less efficacious than RZV. We found that RZV increased the functionality of several innate immune cell subsets against VZV-gE other herpesviruses. The increase in functionality was associated with decreased production of the inhibitory cytokine TGFβ1,which may have resulted from decreased ability to use the TGFβ1 gene as a template for the synthesis of its product. We concluded that RZV generated homologous (VZV-gE) and heterologous (other herpesviruses) memory-like responses in innate immune cell subsets through genomic repression of the regulatory cytokine TGFβ-1. View Publication

Osteosarcoma (OSA) is the most common primary bone tumor in children and adolescents,yet outcomes have remained largely unchanged for over 40 years. While chimeric antigen receptor (CAR) T cell therapy has shown success in blood cancers,it faces major limitations in solid tumors due to immune evasion,antigen loss,and immunosuppressive tumor microenvironments. Natural killer (NK) cells offer several advantages over T cells,including multiple killing mechanisms and lower risks of graft-versus-host disease,neurotoxicity,and cytokine release syndrome,making them promising candidates for off-the-shelf cell therapies. However,unmodified NK cells have shown limited efficacy in clinical settings due to poor engraftment,persistence,and tumor-mediated suppression. To overcome these barriers,we developed a cost-effective method to engineer CAR NK cells targeting CD70,a tumor antigen overexpressed in relapsed and metastatic OSA. We further enhanced these cells by incorporating soluble interleukin-15 (IL-15) and a dominant-negative TGF-β receptor,creating “armored” CAR NK cells. These engineered cells resist transforming growth factor β (TGF-β) suppression,secrete IL-15,and demonstrate improved cytotoxicity,persistence,and tumor homing in both in vitro and in vivo models. Our findings support CD70 CAR NK cells as a promising immunotherapeutic strategy for relapsed and metastatic OSA. Graphical abstract Engineered “armored” CAR NK cells targeting CD70 overcome immune suppression in osteosarcoma,enhancing persistence,tumor homing,and cytotoxicity. This study presents a promising off-the-shelf immunotherapy approach for relapsed and metastatic OSA,offering a potential advance where current treatments have stagnated for decades. View Publication

INTRODUCTION: Alzheimer's disease (AD) pathology is complex and involves mitochondrial dysfunction. There are emerging therapies targeting mitochondrial function in clinical trials for AD. This highlights the need for biomarkers that measure mitochondrial function. METHODS: We determined the utility of a novel blood‐based mitochondrial biomarker,the mitochondrial functional index (MFI),in the context of AD in a pilot study.RESULTS: In vitro and in vivo models of AD had a reduced MFI. MFI was lower in human AD subjects and APOE ????4 carriers. Receiver operating characteristic analysis showed MFI had a higher area under the curve than other plasma biomarkers. The MFI biomarker correlated with the Mini‐Mental State Examination (MMSE) and the Clinical Dementia Rating (CDR) scale. DISCUSSION: This study highlights the potential utility of MFI as a functional blood‐based mitochondrial biomarker to interrogate energy metabolism. Ongoing studies are examining the relationship of MFI with brain energy metabolism outcomes. Highlights: The MFI biomarker is reduced in cell and animal models of AD. The MFI biomarker is reduced in human AD subjects and APOE ε4 carriers. The MFI biomarker can discriminate between subjects with normal cognition and AD with better performance than other plasma biomarkers. The MFI biomarker correlates with cognitive scores. View Publication

BACKGROUND: The discovery of induced pluripotent stem cells revolutionized regenerative medicine,providing a source for generating induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs). AIM: To evaluate and compare five iMSC differentiation protocols,assessing their efficiency,phenotypic characteristics,and functional properties relative to primary mesenchymal stem cells (MSCs). METHODS: Five iMSC differentiation protocols were assessed: SB431542-based differentiation (iMSC1,iMSC3),an iMatrix-free method (iMSC2),growth factor supplementation (iMSC4),and embryoid body formation with retinoic acid (EB-iMSC). iMSC identity was confirmed according to the International Society for Cell & Gene Therapy 2006 criteria,requiring expression of surface markers (CD105,CD73,CD90) and absence of pluripotency markers. Functional assays were conducted to evaluate differentiation potential (osteogenic and adipogenic),proliferation,mitochondrial function,reactive oxygen species,senescence,and migration. RESULTS: All iMSC types expressed MSC markers and lacked pluripotency markers. EB-iMSC and iMSC2 showed enhanced osteogenesis (runt-related transcription factor 2; P ≤ 0.01 and P ≤ 0.0001,respectively),while adipogenic potential was reduced in iMSC2 (Adipsin; P ≤ 0.01) and EB-iMSC (Adipsin and peroxisome proliferator-activated receptor gamma; P ≤ 0.0001 and P ≤ 0.01,respectively). Proliferation was comparable or superior to bone marrow MSCs,except in iMSC1,with iMSC4 showing the highest rate (MTT assay; P values ranged from 0.01 to 0.001). Despite reduced mitochondrial health in iMSC3 and iMSC4 (P ≤ 0.001),reactive oxygen species levels were lower in all iMSCs (P values ranged from 0.001 to 0.0001),and senescence was significantly reduced in all iMSCs with the exception of iMSC1 (P values ranged from 0.01 to 0.0001). Migration was most reduced in iMSC4 (P ≤ 0.001 at 24 hours and P ≤ 0.0001 at 48 hours). CONCLUSION: While all protocols generated functional iMSCs,variations in differentiation,proliferation,and function emphasize the impact of protocol selection. These findings contribute to optimizing iMSC generation for research and clinical applications. 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

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

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

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

Cancer stem cells are critical for cancer initiation,development,and treatment resistance. Our understanding of these processes,and how they relate to glioblastoma heterogeneity,is limited. To overcome these limitations,we performed single-cell RNA sequencing on 53586 adult glioblastoma cells and 22637 normal human fetal brain cells,and compared the lineage hierarchy of the developing human brain to the transcriptome of cancer cells. We find a conserved neural tri-lineage cancer hierarchy centered around glial progenitor-like cells. We also find that this progenitor population contains the majority of the cancer's cycling cells,and,using RNA velocity,is often the originator of the other cell types. Finally,we show that this hierarchal map can be used to identify therapeutic targets specific to progenitor cancer stem cells. Our analyses show that normal brain development reconciles glioblastoma development,suggests a possible origin for glioblastoma hierarchy,and helps to identify cancer stem cell-specific targets. View Publication