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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

Antibody–drug conjugates (ADCs) represent a promising strategy in cancer therapy,enabling the targeted delivery of cytotoxic agents to tumor cells. In this study,we developed and characterized novel ADCs combining the anti-EGFR monoclonal therapeutic antibody Cetuximab (Cet) with two aminobisphosphonates (N-BPs) analogues of zoledronic acid (ZA): DC310 and the aminothiazole DC315. These conjugates aim to enhance antitumor efficacy of Cet in colorectal cancer (CRC) by both directly inhibiting tumor cell growth and activating Vδ2 T lymphocytes. We optimized the drug-antibody ratio (DAR),achieving significantly higher DARs compared to previously reported Cet-ZA conjugate,particularly with Cet-DC315 (DAR ≈ 23). Both ADCs retained selective EGFR binding in CRC cell lines and patient-derived organoids (PDO). Functionally,Cet-DC315 markedly inhibited proliferation of EGFR⁺ CRC cell lines in conventional cultures and 3D spheroids. Furthermore,Cet-DC-315 uniquely induced expansion and cytotoxic activation of Vδ2 T cells in co-cultures with CRC cell lines,PDO,and primary tumor samples. These findings suggest that ADCs incorporating novel N-BPs such as DC315 represent a potent approach for dual antitumor targeting through direct cytostatic effects and immune activation,offering a potential therapeutic advantage in the treatment of EGFR+ colorectal cancer. View Publication

Parechovirus ahumpari 3 (HPeV-3) is among the main agents causing severe neonatal neurological infections such as encephalitis and meningitis. However,the underlying molecular mechanisms and changes to the host cellular landscape leading to neurological disease has been understudied. Through quantitative proteomic analysis of HPeV-3 infected neural organoids,we identified unique metabolic changes following HPeV-3 infection that indicate immunometabolic dysregulation. Protein and pathway analyses showed significant alterations in neurotransmission and potentially,neuronal excitotoxicity. Elevated levels of extracellular glutamate,lactate dehydrogenase (LDH),and neurofilament light (NfL) confirmed glutamate excitotoxicity to be a key mechanism contributing to neuronal toxicity in HPeV-3 infection and can lead to apoptosis induced by caspase signaling. These insights are pivotal in delineating the metabolic landscape following severe HPeV-3 CNS infection and may identify potential host targets for therapeutic interventions. View Publication

Regulatory T cells are a class of T lymphocytes which respond to activation signals by expanding their cell numbers,and whose culturing and expansion are of significant clinical interest. Cellular activation states are used to inform process control decisions such as restimulation and can be probed with experimental measurements of cell surface markers. However,these measurements are expensive,time-consuming,and invasive,and an urgent need exists for devising a non-invasive method for activation state monitoring that could be deployed on-line. Raman spectroscopy is a label-free and information-rich optical method that,when coupled to data analytical methods,can ameliorate these experimental issues. In this work,we quantitatively estimated experimental measurements of regulatory T cell activation markers with high accuracy. We simulated a clinical manufacturing setting by building an L1-regularized least-squares model with spectroscopic data from six regulatory T cell donors. Then,we validated the constructed model by accurately estimating different experimental measurements of biomarker values from two external donors,unseen by the model. We have devised a robust program to effectively estimate the activation state of regulatory T cells. We anticipate our method to be used with on-line Raman probes integrated into cell manufacturing devices for label-free monitoring of these processes. View Publication

USH2A mutations are the leading cause of autosomal recessive retinitis pigmentosa (RP),a progressive blinding disease marked by photoreceptor degeneration. Animal models fail to recapitulate the features of USH2A RP seen in humans,and its earliest pathogenic events remain unknown. Here,we established a human model of USH2A RP using retinal organoids derived from patient induced pluripotent stem cells and CRISPR-Cas9-engineered isogenic-USH2A−/− induced pluripotent stem cells. Methods: We assessed organoids for cellular,molecular,and morphological defects using serial live imaging and whole organoid and fixed section analyses. Results: Both patient-derived and isogenic-USH2A−/− organoids showed preferential rod photoreceptor loss followed by widespread degeneration,consistent with the clinical phenotype. Additionally,isogenic-USH2A−/− organoids showed early defects in proliferation and structure. Conclusions: Our findings suggest that molecular changes precede overt photoreceptor loss in USH2A RP,and pathogenesis may begin before clinical symptoms emerge. By defining early and late disease features,we provide new insight on the developmental origins of USH2A RP to guide therapeutic strategies. View Publication