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Mutations causing Parkinson’s disease (PD) give diverse pathological phenotypes whose cellular correlates remain to be determined. Those with PRKN mutations have significantly earlier selective vulnerability of dopamine neurons,those with SNCA mutations have increased alpha-synuclein deposition,while those with LRRK2 mutations have additional deposition of tau. Yet all three mutation types are implicated in mitochondrial and/or lysosomal dysfunction. To compare cellular dysfunctions associated with these different pathological phenotypes,an unbiased high-content imaging platform was developed to assess both lysosomal and mitochondrial dysfunction,along with alpha-synuclein and tau protein deposition using induced pluripotent stem cell (iPSC) derived cortical and ventral midbrain neurons. Different PD mutations caused cell type specific dysfunctions,likely to impact on both selective neuronal vulnerability and the pathologies observed in PD. Comparison of dopamine neurons identified that both lysosomal and mitochondrial dysfunction were predominant with PRKN lof mutations,whereas SNCA A53T and LRRK2 R1441G mutations had increased tau deposition. In contrast,cortical neurons with SNCA and LRRK2 mutations both had mitochondrial and autophagy impairments without protein deposition,with LRRK2 cells additionally showing decreased glucocerebrosidase activity and increased alpha-synuclein phosphorylation. View Publication

SummaryDiffuse midline glioma (DMG) is a devastating pediatric brain tumor. The oncolytic adenovirus Delta-24-RGD has shown promising efficacy and safety in DMG patients but is not yet curative. Thus,we hypothesized that activating dendritic cells (DCs) through the CD40 costimulatory receptor could increase antigen presentation and enhance the anti-tumor effect of the virus,resulting in long-term responses. This study shows that the intratumoral co-administration of Delta-24-RGD and a CD40 agonistic antibody is well tolerated and induces long-term anti-tumor immunity,including complete responses (up to 40%) in DMG preclinical models. Mechanistic studies revealed that this therapy increased tumor-proliferating T lymphocytes and proinflammatory myeloid cells,including mature DCs with superior tumor antigen uptake capacity. Moreover,the lack of cross-presenting DCs and the prevention of DC recruitment into the tumor abolish the Delta-24-RGD+anti-CD40 anti-DMG effect. This approach shows potential for combining virotherapy with activating antigen-presenting cells in these challenging tumors. Graphical abstract Highlights•CD40 agonism boosts Delta-24-RGD efficacy in pediatric DMG models•cDC1s are essential for the long-lasting anti-tumor effect of Delta-24-RGD and anti-CD40•Microglia and tumor macrophages contribute to the cDC1 recruitment following the therapy Labiano et al. show that co-administration of the Delta-24-RGD virus and a CD40 agonistic antibody is safe,yielding complete responses in DMG-bearing mice. The combination relies on type 1 conventional dendritic cells (cDC1s),which remodel the tumor microenvironment into a proinflammatory landscape,fostering long-term anti-tumor immunity against this challenging tumor. View Publication

Tumor-associated macrophages (TAMs) are the most abundant non-cancerous cell type in glioblastoma (GBM) and heavily influence GBM biology,contributing to tumor progression,therapeutic resistance,immune evasion,and neovascularization. Current in vitro models that utilize IL-4/IL-13 stimulation fail to capture the transcriptional and functional heterogeneity of TAMs observed in vivo. In this study,we utilize a serum-free indirect co-culture model with patient-derived xenolines to polarize primary human macrophages and characterize their molecular and functional phenotypes. We demonstrate that xenoline-polarized macrophages diverge from classical M1/M2 states and instead adopt transcriptional signatures reflective of TAM subsets identified from patients. Notably,macrophages polarized with the radiation-therapy selected xenoline,JX14P-RT,exhibited gene expression patterns enriched for interferon response and hypoxia,mirroring recurrent GBM samples. In contrast,JX14P TAMs showed enrichment in phagocytic gene sets. Functional validation of these phenotypes revealed discrepancies between the transcriptionally predicted and observed phenotypes,emphasizing the importance of integrating phenotypic validation in sequencing studies. Altogether,our findings establish xenoline-polarized macrophages as a useful alternative to traditional models that can be used to study immune-interactions in vitro.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40478-025-02057-1. View Publication

Human Papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) remains a challenging malignancy,with radiotherapy,alone or combined with immune checkpoint inhibitors,often failing to achieve durable disease control. Here,by conducting longitudinal multi-omic analyses of pre- and post-radiation biopsies from patients receiving a pre-operative hypofractionated radiation regimen,we uncover that radiation rapidly depletes a subpopulation of tumor-infiltrating lymphocytes (TIL),characterized by a proliferative,cytotoxic,and tissue-resident gene signature (TProlif_Tox). We provide multi-dimensional evidence for tumor antigen-specificity of TProlif_Tox clonotypes and show that post-radiation tumors are instead repopulated by regulatory and non-specific clones. Finally,TIL depletion correlates with radiorecurrent disease after conventional radiation,emphasizing the potential impact of radiation-induced TIL loss regardless of fractionation. Thus,this study provides key insights into radiotherapy-induced alterations in the immune microenvironment that drive immunologic radioresistance and proposes restoring tumor antigen-specific T cell clonotypes as a strategy to improve radioimmunotherapy responses in HNSCC. Combining radiation therapy (RT) with immunotherapy has had limited therapeutic benefits in the treatment of head and neck cancer (HNSCC). Here,the authors present a multi-omics analysis of patient biopsies pre- and post- hypofractionated RT and uncover RT-mediated depletion of tumor-reactive CD8+ T cells as the underlying cause of immunologic radioresistance in HNSCC. View Publication

Hepatocellular carcinoma (HCC) is a highly heterogeneous and immunosuppressive malignancy that frequently exhibits poor responses to immunotherapy,primarily due to immune evasion mediated by myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment. Despite increasing interest in MDSC-targeted strategies,effective approaches to selectively modulate MDSC function and enhance immunotherapeutic efficacy remain limited. Recent advances in nanotechnology have led to the development of nanodrug delivery systems,particularly for small interfering RNA (siRNA),offering advantages such as protection from degradation and improved delivery specificity. However,traditional liposomal carriers often suffer from low selectivity and widespread biodistribution,increasing the risk of off-target effects. In this study,we designed a pH-responsive lipid nanoparticle (Lip@si-YTHDF2) for the targeted delivery of siRNA against YTHDF2. This approach aimed to suppress MDSC function,inhibit CSC-mediated immune escape,and enhance immunotherapy in HCC. Bioinformatic analyses of GEO and TCGA-LIHC datasets revealed elevated YTHDF2 expression in HCC and its association with poor prognosis. Functional studies in a conditional YTHDF2-knockout mouse model demonstrated that YTHDF2 regulates MDSC activity and promotes tumor progression by stabilizing MYC mRNA through N6-methyladenosine (m6A) modification. Our findings demonstrated that Lip@si-YTHDF2 effectively downregulated MYC expression,diminished the immunosuppressive phenotype of MDSCs,restored T cell-mediated anti-tumor immunity,and significantly inhibited tumor growth in combination with PD-1 checkpoint blockade. This study not only elucidates a novel YTHDF2/m6A/MYC axis in immune evasion but also provides a clinically relevant siRNA delivery platform with promising therapeutic implications for improving HCC immunotherapy.Graphical Abstract Supplementary InformationThe online version contains supplementary material available at 10.1186/s12951-025-03538-0. View Publication

AbstractColorectal cancer is the second leading cause of cancer-related death in the United States and high-risk individuals face a notably higher likelihood of developing colorectal cancer based on their genetic background. Hence,there is a compelling need for innovative chemopreventive treatments aimed at minimizing colorectal cancer tumorigenesis. Exportin 1 (XPO1; also referred to as CRM1) plays a pivotal role in transporting proteins from the nucleus to the cytoplasm. Various cancers overexpress XPO1,including colorectal cancer,and selective inhibitors of nuclear export compounds,such as eltanexor (KPT-8602),have been developed to target XPO1. Eltanexor demonstrates fewer adverse effects than its precursors and is currently under evaluation in phase I/II clinical trials. This research evaluates eltanexor as a chemopreventive agent for colorectal cancer. Our findings indicate that eltanexor treatment inhibits expression of the common chemoprevention target in colorectal cancer,COX-2. This occurs by eltanexor-dependent reduction of Wnt/β-catenin signaling. Furthermore,XPO1 inhibition leads to forkhead transcription factor O subfamily member 3a nuclear retention,which can modulate β-catenin/TCF transcriptional activity. The in vivo oral treatment of eltanexor to Apcmin/+ mice (a mouse model for familial adenomatosis polyposis) was well tolerated and reduced tumor burden by approximately threefold,along with decreased tumor size. Drug sensitivity assays using organoids from Apcmin/+ mice tumors showed increased sensitivity to eltanexor compared with wild-type organoids. Collectively,these findings highlight XPO1 as a potent target for colorectal cancer chemoprevention.Significance:In this study,we show the XPO1 inhibitor eltanexor acts as an effective colorectal cancer chemopreventive agent both in vivo and in vitro. This occurs by reducing COX-2 expression by modulating the Wnt/β-catenin signaling pathway. Collectively,these findings highlight XPO1 as a potent target for colorectal cancer chemoprevention. View Publication

Electronic cigarettes (e-cigs) have been introduced as a safer alternative to traditional combustible cigarettes and have been growing in popularity. E-cig e-liquids all contain the carrier compounds,vegetable glycerin (VG),propylene glycol (PG),and nicotine,together with different flavors,but the effects of inhalation of these compounds on the airway are not well understood. This study investigates the effects of e-cig exposure on primary human airway epithelial cells grown in air–liquid interface (ALI) cultures,specifically focusing on mucociliary clearance,the lung’s primary host defense mechanism whereby pathogens and particles trapped by mucus are cleared by unidirectional beating by ciliated cells. We developed a microcontroller-based exposure system to reproducibly examine cellular and molecular changes in ALI cultures from e-cig exposure. Here we show heterogeneous,donor-dependent effects of different e-cig flavors on airway epithelial cells. Examining the effects of the unflavored carrier compounds common to all e-cigs,we found that ALI airway cultures exposed to PG:VG (30:70 ratio) with 5% nicotine unflavored e-cigs show a reduction in ciliary beat frequency. Moreover,using transmission electron microscopy,we identified defects in ciliary ultrastructure induced by unflavored e-cigs. Phosphoproteomic analysis uncovered changes in phosphorylation of proteins involved in cadherin and actin binding and the Rho GTPase signaling pathway,which are all involved in cytoskeletal remodeling that may influence ciliary structure and function. Altogether,our findings suggest that exposure to all e-cigs reduces mucociliary clearance.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12931-025-03302-w. View Publication

IntroductionCoronavirus disease 2019 (COVID-19),caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2),has been a major health emergency since its emergence in late 2019. Endothelial dysfunction is a hallmark of COVID-19,leading to severe illness,i.e. coagulopathy,multi-organ failure. FX06,a fibrin-derived peptide naturally occurring in the human body,formerly known as Bβ15-42,is a promising therapeutic candidate for endothelial complications like capillary leakage in COVID-19 and other forms of acute respiratory disorders. The aim of this project is to investigate whether FX06 can attenuate COVID-19 cytokine-triggered inflammatory processes in vitro.MethodsTo mimic the inflammatory status of COVID-19,a human pulmonary microvascular endothelial cell line (ECs) – HULEC-5a,was treated with a cytokine cocktail comprised of ten different cytokines or chemokines at concentrations found in serum profiles of COVID-19 patients with severe illness,further referred to as the severe cytokine cocktail. ECs were treated with the severe cytokine cocktail for 24 h,in the absence or presence of FX06 for 2 h.ResultsThe severe cytokine cocktail enhanced peripheral blood mononuclear cell (PBMC)-endothelial adhesion and monolayer transmigration. This deleterious effect was significantly reduced by FX06. FX06 was also shown to mitigate the cytotoxic activity of allogeneic CD8+ T cells,which increased upon cytokine treatment. FX06 restored continuous vascular endothelial (VE)-cadherin/CD144 distribution on the EC surface and reversed morphological changes mediated by the severe cytokine cocktail,such as the elongation of F-actin stress fibers. FX06 reduced capillary-like structure formation of the severe cytokine cocktail treated-ECs,indicating FX06 down-regulated the pro-inflammatory angiogenic activity caused by the severe cytokine cocktail. Additionally,FX06 might assist in maintaining the normal barrier function of ECs by altering the surface expression of Syndecan-1 (SDC1/CD138). Proteomics and phosphoproteomics analyses demonstrated that FX06 in the presence of the severe cytokine cocktail inactivated RhoGTPase,which was confirmed by western blotting that FX06 attenuated RhoA,a member of RhoGTPase,enhanced by the severe cytokine cocktail and down-regulated the expression of the phosphorylated downstream protein,ROCK1.ConclusionOverall,FX06 shows promising potential in normalizing ECs and reducing vascular leakage to protect the endothelium against the proinflammatory effect of COVID-19-triggered cytokines. View Publication

Glucose transporter 4 (GLUT4) expression on white adipocytes is critical for facilitating cellular uptake of blood glucose,failure of which promotes hyperglycemia. Matrix metalloproteinases (MMPs) play a crucial role in remodeling the white adipose tissue (WAT) during obesity. MMPs have multiple protein substrates,and surprisingly,it is unknown if they can directly target GLUT4 on the adipocyte surface and impair glucose uptake. We identified MMP2 as the highly active gelatinase,a class of MMP,in the gonadal WAT of high-fat diet-induced obese mice. In vitro,metabolic studies in 3T3-L1 adipocytes revealed MMP2 attenuated glucose uptake and glycolysis,which were recovered by an MMP2 inhibitor. In silico structural Analysis using AlphaFold identified a putative MMP2 cleavage site on the extracellular domain of GLUT4. Further,in a substrate competition assay,a peptide mimicking the MMP2 cleavage site on GLUT4 attenuated the cleavage of an MMP substrate by MMP2. Altogether,our results suggest a novel mechanism of impaired glucose utilization by adipocytes,which may contribute to hyperglycemia during obesity.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-06252-x. View Publication

SummaryPrimary human cells cultured in long-term expandable 3D organoid format have great promise as potential regenerative cellular therapies,but their immunogenicity has not yet been fully characterized. In this study,we use in vitro co-cultures and in vivo humanized mouse experimental models to examine autologous and allogeneic immune response to human primary cholangiocyte organoids (PCOs) as treatment for bile duct disorders. Our data demonstrate that PCOs upregulate the expression of human leukocyte antigen (HLA)-I and HLA-II in inflammatory conditions. The allogeneic immune response to PCOs is driven by both HLA-I and HLA-II and is substantially ameliorated by donor-recipient HLA matching. While allogeneic cells display evolving stages of immune rejection in vivo,autologous PCOs induce a low-level immune infiltration into the graft site possibly influenced by acquired mutations in culture,cell viability,and culture matrix. Our findings have important implications for the design and clinical translation of autologous and allogeneic organoid cellular therapies. Graphical abstract Highlights•Human primary cholangiocyte organoids (PCOs) upregulate HLA-I/II in inflammatory conditions•Allogeneic immune response is substantially ameliorated by donor-recipient HLA matching•Autologous PCOs induce a low-level immune infiltration into the graft site•Humanized mice distinguish early/vigorous and late/resolved stages of allogeneic response Petrus-Reurer et al. use in vitro co-cultures and in vivo humanized mice to examine autologous and allogeneic immune response to human primary cholangiocyte organoids (PCOs). The authors show that autologous cells induce a low-level immune infiltration into the graft and allogeneic cells display HLA-driven evolving stages of immune rejection. View Publication

Neuropsychiatric disorders remain difficult to treat due to complex and poorly understood mechanisms. NeuroPainting is a high-content morphological profiling assay based on Cell Painting and optimized for human stem cell–derived neural cell types,including neurons,progenitors,and astrocytes. The assay quantifies over 4000 features of cell structure and organelle organization,generating a dataset suitable for phenotypic screening in neural models. Here,we show that,in studies of the 22q11.2 deletion—a strong genetic risk factor for schizophrenia—we observe cell-type-specific effects,particularly in astrocytes,including mitochondrial disruption,altered endoplasmic reticulum organization,and cytoskeletal changes. Transcriptomic analysis shows reduced expression of cell adhesion genes in deletion astrocytes,consistent with post-mortem brain data. Integration of RNA and morphology data suggests a link between adhesion gene dysregulation and mitochondrial abnormalities. These results illustrate how combining image-based profiling with gene expression analysis can reveal cellular mechanisms associated with genetic risk in neuropsychiatric disease. Here,authors use NeuroPainting,a high-content imaging assay,to reveal cell-type-specific effects of 22q11.2 deletion in neural cells,linking adhesion gene dysregulation to mitochondrial and structural abnormalities,especially in astrocytes. View Publication

IntroductionThe main cause of death for patients with non-Hodgkin lymphoma (NHL) remains therapy resistant relapses. Chemoresistance is commonly associated with apoptosis defects and upregulated autophagy. Therefore,novel therapeutic options that do not rely on apoptosis and target autophagy would be of interest to treat NHL. An agent that may fulfill these requirements is the glycan-binding protein Galectin-9 (Gal-9).MethodsA panel of B cell lymphoma NHL cell lines,including diffuse large B cell lymphoma (DLBCL),mantle cell lymphoma (MCL),Burkitt’s lymphoma (BL),and (chemoresistant) follicular lymphoma (FL),were treated with Gal-9 after which cell counts and cell viability were determined. Basal mRNA and protein expression levels were respectively determined by RTqPCR and western blot. The impact of Gal-9 treatment on the autophagy pathway was determined using lysotracker,Cyto-ID and western blot (targeting LAMP2,p62,LC3B-I/LC3B-II).ResultsTreatment with Gal-9 reduced total cell counts and cell viability of various DLBCL,MCL,BL and FL cell lines. Gal-9-induced cell death was associated with the inhibition of autophagy,as demonstrated by the accumulation of LC3B-II and p62. In addition,Gal-9-sensitive cells expressed lower basal protein levels of LC3B-I as compared to cells that responded less to this lectin. Furthermore,Gal-9 was cytotoxic for chemoresistant Sc-1 cells (Sc-1-RES),which were even more sensitive toward Gal-9 treatment than the parental cells (Sc-1-PAR).ConclusionGal-9 is a potent inducer of B cell lymphoma cell dead by inhibiting the proper execution of autophagy. View Publication