参考文献

The efficacy of immunotherapy is often hindered by the suppression of immune responses via the tumor microenvironment (TME). The presence of cancer cells forces other proximal non-cancerous cells to support tumor growth and persistence. A clear example of this cancerous-to-non-cancerous communication is represented by the accumulation of myeloid-derived suppressor cells (MDSCs) within the TME. Several studies have convergently shown that the overexpression of DNA-methyl-transferase-1 (DNMT1) in these cells results in protection from necroptosis and enhanced accumulation in vivo. Conversely,targeting DNMT1 through hypo-methylating agents has shown promising therapeutic potential by not only reducing the levels of MDSCs but also enhancing cancer immunogenicity and the efficacy of immune checkpoint inhibitors (ICI). Methods: Murine 4T1 (triple-negative breast cancer (TNBC)) and CT26 (colon carcinoma) cell lines were cultured under standard conditions and used to generate tumor models in BALB/c mice. An oncolytic adenovirus expressing a DNMT1-targeting short hairpin RNA (OAd.shDNMT1) was engineered and validated for DNMT1 knockdown and genome-wide methylation reduction. Small extracellular vesicles (sEVs) were isolated from virus-infected cancer cells and characterized for RNA content and uptake by MDSCs. MDSC differentiation and suppressive function were assessed in vitro using flow cytometry and co-culture assays with murine splenocytes. In vivo,tumor-bearing mice received intratumoral OAd.shDNMT1,systemic decitabine,or immune checkpoint inhibitors (anti-Programmed cell Death protein-1),and tumor growth,immune infiltration,and systemic MDSC levels were evaluated. Results: In this study,we report that,by using virally infected TNBC murine cells as a source for shDNMT1-loaded sEVs,OAd.shDNMT1 successfully reduced MDSC levels in vitro and in vivo. Furthermore,the co-administration with ICI resulted in a significant tumor growth reduction in mice bearing poorly immunogenic TNBC 4T1 cells. Also,our treatment promoted antitumor immunity,prolonged survival,and complete tumor eradication in modestly immunogenic colon CT26 cancer cells. Conclusions: This multifaceted strategy,based on OV-mediated immune stimulation and reduction of MDSC levels via sEVs,may improve clinical outcomes and the success of immuno-based regimens for patients facing MDSC-rich and highly aggressive cancer subtypes. View Publication

Syngeneic mouse tumor models have shown that senescence influences the tumor immune response in multiple ways,including the induction of an immunosuppressive microenvironment or the promotion of immune cell recruitment. Yet,the impact of senescence on the tumor immune response in a humanized setting remains largely unexplored. MethodsTo address this question,we employed a combination cells co-culture models,tumor spheroids and mice bearing tumors immunogenic to human immune cells derived from the same donor. Results: We found that senescent fibroblasts exert a dual effect by enhancing the recruitment of immune cells into the tumor microenvironment while simultaneously promoting the apoptosis of T and NK cells. Mechanistically,we demonstrate that this apoptosis is primarily due to increased Fas ligand (FasL) expression on the surface of senescent fibroblasts. Increased FasL expression was observed on different human fibroblast cell lines in response to different senescence inducers with a particular robust effect in response to RAS-induced senescence. Deletion of FasL on fibroblasts was sufficient to prevent immune cell death and increase tumor cell killing in mice. Discussion: Our results identified the expression of FasL expression as a novel component of the senescent tumor microenvironment and highlight the importance of evaluating the impact of therapy-induced senescence in humanized models to understand and predict the outcome of cancer treatments. View Publication

Diffuse intrinsic pontine glioma (DIPG) is a fatal brainstem tumor desperately in need of better treatments. Chimeric antigen receptor (CAR) T cell therapies for DIPG have demonstrated clinical tolerability and bioactivity,but not universal benefit. A major obstacle is insufficient CAR T cell trafficking to the tumor. As our recent clinical trials have demonstrated locoregional elevation of CXCL10,a ligand of the chemokine receptor CXCR3,here we aim to leverage this CXCL10 upregulation to enhance cell trafficking by engineering our B7-H3-targeting CAR T cells to overexpress CXCR3 variants. We demonstrate that,compared to unmodified B7-H3 CAR T cells,CXCR3-A-modified CAR T cells migrate more efficiently toward CXCR3 ligands in vitro,and when delivered intracerebroventricularly in orthotopic DIPG mouse models,CXCR3-A-modified CAR T cells show enhanced trafficking into the tumor and improved therapeutic efficacy. Overall,our data support the potential for engineering CXCR3-A expression to enhance CAR T cell trafficking and efficacy against DIPG. CAR T cell therapies have been developed to treat paediatric diffuse intrinsic pontine glioma (DIPG),however,clinical efficacy remains limited. Here,the authors report that engineering B7-H3-targeting CAR T cells to express the chemokine receptor CXCR3-A enhances their trafficking and efficacy in DIPG preclinical models. View Publication

Aggregated α-synuclein (αSyn) is a pathological hallmark of Parkinson’s disease (PD),yet other protein aggregates,including tau,are commonly observed in PD brains. This suggests that PD is not solely a synucleinopathy but may involve multiple,coexisting proteinopathies. Mutations in LRRK2,particularly the G2019S (GS),are the most common cause of familial PD. LRRK2-PD has been associated with both αSyn and tau pathology; however the mechanistic links between LRRK2 dysfunction and protein aggregation remain incompletely defined. Here we opted to investigate whether LRRK2 contributes to αSyn and tau pathology through common molecular pathways or via distinct cellular mechanisms. Viral vector-mediated αSyn overexpression in GS LRRK2 knock-in mice led to enhanced dopaminergic neurodegeneration,increased phosphorylated αSyn levels,pronounced neuroinflammation,and accumulation of lysosomal proteins,suggesting impaired αSyn clearance and immune activation as key drivers. Human iPSC-derived dopaminergic neurons from GS LRRK2 PD patients mirrored these findings. In contrast viral vector-mediated overexpression of tau in GS LRRK2 knock-in mice promoted tau phosphorylation but did not significantly affect neuroinflammation,lysosomal markers,or neurodegeneration,indicating a primarily cell-autonomous mechanism. Our results reveal a mechanistic divergence in how GS LRRK2 impacts αSyn and tau pathologies,supporting the notion that LRRK2 kinase activity contributes to PD pathogenesis through different pathways,thereby highlighting its potential as a therapeutic target in both familial and sporadic PD. View Publication

Lipid regulation is crucial role in Alzheimer's disease (AD) pathogenesis. In AD,microglia show elevated sterol O‐acyltransferase 1/Acyl‐coenzymeA: Choleseterol Acyltransferase 1 (SOAT1) expression,encoding Acyl‐coenzymeA: Cholesterol Acyltransferase 1  (ACAT1),which produces cholesteryl esters (CEs) in lipid droplets. Inhibiting ACAT1 has been shown to reduce amyloid beta (Aβ) pathology,though the mechanism is unclear. Methods: We inhibited ACAT1 using avasimibe (AV) in wild‐type,triggering receptor expressed on myeloid cells 2 (TREM2) knockout (KO),and low‐density lipoprotein receptor related protein 1 (LRP1) KO mouse BV2 and human induced pluripotent stem cell‐derived microglia and measured the impact on Aβ uptake to determine the mechanism through which the inhibition of ACAT1 enhances Aβ uptake. Results: ACAT1 inhibition increased LRP1 levels and soluble TREM2 (sTREM2) release via enhanced TREM2 cleavage by ADAM metallopeptidase domain 10/17 (ADAM10/17). KO of TREM2 or blockade of sTREM2 release prevented AV‐enhanced Aβ uptake. This effect was rescued by recombinant sTREM2,but only when LRP1 was present. Discussion: ACAT1 inhibition promotes microglial Aβ uptake in a sTREM2‐ and LRP1‐dependent manner,offering insights into novel therapeutic strategies for AD. Highlights: Inhibition of ACAT1,the major enzyme that catalyzes cholesterol storage via esterification enhances microglia‐mediated Aβ uptake. Increased Aβ uptake is dependent on the presence of both TREM2 and LRP1. Inhibition of ACAT1 increases cleavage of TREM2 via ADAM10/17 to release sTREM2. Treatment of microglial cells with sTREM2 rescues Aβ uptake in TREM2 KO BV2 cells.Inhibition of ACAT1 promotes Aβ uptake through increased shedding of TREM2,which enhances Aβ uptake through a LRP1‐dependent mechanism. View Publication

Circulating tumor cell (CTC) detection in hepatocellular carcinoma (HCC) is limited not only by the rarity of CTCs but also by a heavy reliance on cell surface markers such as EpCAM,which are variably expressed or lost during tumor progression. Detecting intracellular markers,such as cytokeratin offers an important complementary and comprehensive strategy but remains technically limited in flow cytometry due to the need for fixation and permeabilization,which often lead to cell loss and surface epitope damage. In this study,we systematically evaluated the feasibility of using fixed samples for flow cytometry,using HepG2 cells,PBMCs,and CTCs from patients with HCC. Our results demonstrate that fixation enabled intracellular staining without compromising cell surface marker detection,even after short-term storage at 4 °C and long-term storage at -80 °C. Fixed samples,particularly fixed unfrozen,exhibited comparable staining performance to fresh samples with only a 7–10% reduction in cell recovery. Clinical validation in HCC patients confirmed successful CTC detection,and tumor-specific CTNNB1 mutations were identified in CTC-derived DNA but not in matched plasma cfDNA. These findings support fixed CTC sample workflows as a reliable and practical approach for CTC analysis in HCC. View Publication

The maintenance of a basal immunoinflammatory signature in hematopoietic stem/progenitor cells (HSPCs) constitutes a fundamental regulatory axis governing hematopoietic competence and immune effector generation. While epigenetic repressors constrain this inflammatory phenotype,the molecular amplifiers that preserve this critical state remain undefined. Through integrated single-cell transcriptomic/epigenomic profiling and functional interrogation,we identify Setd2-mediated H3K36me3 as an indispensable epigenetic amplifier sustaining baseline inflammation in murine HSPCs. Setd2 ablation specifically eliminated interferon (IFN)-enriched HSPC subpopulations and attenuated inflammatory signaling cascades. Functionally,Setd2-deficient HSPCs exhibited impaired IFNγ responsiveness,compromised B-lymphopoiesis,and diminished reconstitution capacity due to Lin−c-Kit+Sca1high cell depletion. Paradoxically,Setd2 loss conferred resistance to IFNγ-induced HSPCs exhaustion,which may contribute to the maintenance of Setd2-deficient HSPCs in our myelodysplastic syndrome (MDS) model under the inflammatory milieu. Mechanistically,Setd2 sustained chromatin accessibility and enhancer (H3K27ac) activity at inflammatory gene loci. This work delineates a critical link between Setd2-mediated chromatin regulation,baseline inflammation,HSPC function,and immune competence,providing insights into inflammatory dysregulation in hematopoietic malignancies like MDS. View Publication

In the arms race between a pathogen and the host,the defense mechanisms of the host cell,including the ubiquitin system,are often counteracted by bacteria. Simkania negevensis (Sne),an obligate intracellular Chlamydia-like bacterium connected with respiratory diseases,possesses numerous deubiquitinases,but not much is known about its other ubiquitin-modifying enzymes. Sne infects a wide range of hosts,developing inside a tubular vacuole in close contact with the host endoplasmic reticulum (ER) and mitochondria. Our study describes an uncharacterized Sne ubiquitin E3 RING-ligase (SNE_A12920 or SneRING),which primarily generates K63- and K11-linked ubiquitin chains and preferentially interacts with UbcH5b and UBE2T E2 enzymes. SneRING is expressed upon infection of various human cell lines,as well as amoebae. We show that a portion of the expressed SneRING co-localizes with mitochondria and ER and that the SneRING interactome includes mitochondrial and ER proteins involved in organelle morphology and stress response. Our work offers an initial characterization of a bacterial RING ligase potentially involved in the host cell remodeling to accommodate the unique intracellular lifestyle of Sne. Author summaryUbiquitination is a protein modification system that regulates protein degradation,localization,or interactions. As such,ubiquitination has many important functions in cell signalling,and its dysregulation can lead to cancer and neurodegenerative diseases. Bacteria that live and develop inside human or other eukaryotic cells,such as Chlamydia,often modulate the ubiquitination system to ensure their own survival. Simkania negevensis is a Chlamydia-like bacterium connected to respiratory diseases in humans. We have discovered a novel enzyme expressed by these bacteria that can ubiquitinate other proteins and thus potentially modify host cell processes that would otherwise hinder infection. In this work,we explore the function of this enzyme and determine its possible cellular localization,as well as some of the proteins it interacts with. Our study provides new insights into how bacterial pathogens adapt to and manipulate host cells using one of the major cell function regulatory systems. View Publication

BackgroundCancer stem cells (CSCs) are considered the ‘seeds’ of recurrence after chemotherapy,but eliminating CSCs remains notoriously challenging. This study aims to examine whether cell cycle checkpoint kinase 1 (CHK1) blockade can abrogate the stemness of ovarian cancer (OC) cells,making them easier targets of anti-tumor immunity. Methods: Prexasertib was used to block CHK1 in OC cell lines and xenografts,and its cytotoxicity was assessed in vitro and in vivo. In vitro tumor-sphere formation assays and stemness markers were used to evaluate cell stemness. PD-L1 expressions were examined via qRT-PCR,Western blot,flow cytometry,and immunohistochemistry. Prexasertib in combination with anti-PD-L1 antibody Atezolizumab was tested in immune-proficient mice bearing OC xenografts in terms of effects on tumor growth,tumor cell stemness,and tumor infiltrating lymphocytes via tumor volume monitoring,immunohistochemistry,and flow cytometry. Results: Prexasertib effectively inhibited CHK1 phosphorylation,exhibited significant anti-tumor effects in vitro and in vivo,accompanied by decreased OC cell stemness. CHK1 was highly expressed in tumor spheres versus tumor cells cultured in 2D system,and Prexasertib treatment suppressed sphere formation and reduced the ALDH+ cell fraction. Unexpectedly,Prexasertib upregulated PD-L1 expression in tumor cells. In vivo,combining Prexasertib with Atezolizumab led to more remarkable remission of tumors,when compared with Prexasertib or Atezolizumab alone. Meanwhile,the tumor-infiltrating CD8+ T cells significantly increased in the combination group,while exhausted T cells decreased; the treatments did not affect CD4+ cell infiltration. Conclusions: Dual targeting of CHK1 and PD-L1 may improve OC treatment by simultaneously suppressing stemness and enhancing anti-tumor immunity. View Publication

Since the COVID-19 pandemic,there has been a documented rise in the incidence of neurological manifestations among individuals complicated with encephalitis or myelitis. The spectrum of neurological symptoms associated with HCoVs infections is expanding. However,the infection characteristics and pathogenesis of seasonal HCoVs to the central nervous system remain obscure. No pharmacological agents have demonstrated the capacity to specifically and efficaciously mitigate the neurological symptoms induced by HCoVs infections to date. Methods: We developed human cerebral organoids (HCOs) derived from human induced pluripotent stem cells and established a blood–brain barrier (BBB) HCOs co-culture model. We subjected these models to seasonal human coronavirus (HCoV) infections to investigate the viral characteristics within the central nervous system (CNS). Utilizing RNA sequencing,we conducted a preliminary exploration of the mechanisms underlying virus-induced inflammatory responses in the CNS. Furthermore,we assessed the efficacy of antiviral and anti-inflammatory drugs using the HCO model. Results: Our results showed that among seasonal coronaviruses,HCoV-OC43 replicates efficiently within the organoids,primarily targeting neurons and astrocytes,and disrupts the barrier function of the BBB. RNA sequencing analysis revealed that HCoV-OC43 infection triggers an inflammatory response through the TNF and NF-κB signaling pathways,leading to cell death,impaired neuronal function,and disrupted interneuron signaling. Interestingly,Bardoxolone methyl (CDDO-Me) demonstrated antiviral effects comparable to remdesivir,reducing both inflammation and cell death. Conclusions: Conclusively,HCOs infected with HCoV-OC43 offer valuable insights into the pathogenesis of HCoVs in central nervous system (CNS),and might serve as a tool for developing novel therapeutic strategies for HCoVs infections,including COVID-19,especially on exploring treatment candidates.Graphical abstract View Publication