技术资料

Gestational transfer of brain‐reactive antibodies is a risk factor for neurodevelopmental disorders. Contactin‐associated protein‐like 2 (CASPR2) is a known target for pathogenic maternal autoantibodies which have been proposed to interfere with fetal neurodevelopment. However,the impact of CASPR2 antibodies on human brain development remains largely unknown. Here,to better understand the neurophysiological changes that occur in the presence of these pathogenic autoantibodies,we cultured unguided human neural organoids for a period of 6‐months in media containing anti‐CASPR2 antibodies. We then performed neurophysiological characterization via whole‐cell patch‐clamp and calcium imaging in acute organoid slices. Our results reveal that CASPR2 antibody exposure increased spontaneous synaptic activity,enhanced the maximal frequency of action potential firing and of spontaneous network activity. These findings are consistent with a state of neuronal hyperexcitability,a phenotype which is observed in several models of neurodevelopmental disorders. Mechanistically,the alterations observed in action potential waveform are in accordance with a role for CASPR2 in the regulation of voltage‐gated potassium channels and a pathological role for CASPR2 autoantibodies in driving neuronal hyperexcitability. Maternal antibodies targeting CASPR2 are a known risk factor for neurodevelopmental disorders,yet their impact on early human brain development remains unclear. We modeled this exposure using human neural organoids treated with patient‐derived CASPR2 antibodies up to the age of 6 months. Our study reveals that these antibodies drive neurons into a state of pathological hyperexcitability by specifically impairing action potential repolarization and enhancing excitatory synaptic transmission. These findings provide novel mechanistic evidence linking maternal autoimmunity to the excitation/inhibition imbalance characteristic of autism,highlighting a potential biological origin for antibody‐mediated neurodevelopmental conditions. View Publication

Three-dimensional (3D) culture systems that recapitulate the tumor microenvironment are essential for studying cancer cell behavior,drug response,and cell–matrix interactions. Here,we present a detailed protocol for generating 3D spheroid cultures from murine breast cancer cells using methacrylated gelatin (GelMA)-based bioink and a CELLINK BioX bioprinter. This method enables precise deposition of spheroid-laden GelMA droplets into low-attachment plates,facilitating high-throughput and reproducible 3D culture formation. The protocol includes steps for spheroid formation,GelMA preparation,bioprinting,and post-printing analysis using a customized CellProfiler pipeline. The analysis pipeline takes advantage of the functionality of CellProfiler and ImageJ software (version 2.14.0) packages to create a versatile and accessible analysis tool. This approach provides a robust and adaptable platform for in vitro cancer research,including studies of metastasis,drug resistance,cancer cell lipid metabolism,and TME-associated hypoxia. View Publication

Epstein-Barr virus (EBV) causes infectious mononucleosis and contributes to neurodegenerative disorders and malignancies,particularly in immune-compromised hosts. Transplant patients face high risk of post-transplant lymphoproliferative disease,a life-threatening EBV-driven lymphoma. There are no EBV-specific vaccines or treatments; however,neutralizing antibodies against EBV glycoproteins may offer utility as therapeutic agents. EBV entry into B cells involves gp350,which binds complement receptors,and gp42,which engages HLA class II to trigger fusion. Most existing monoclonal antibodies (mAbs) against these antigens are non-human,limiting clinical use. Using a transgenic mouse model,we generate two gp350 and eight gp42 genetically human neutralizing mAbs that block receptor binding. Structural analyses reveal extended sites of vulnerability relevant to vaccine development. Delivery of a gp42 mAb protects humanized mice from EBV challenge,while a gp350 mAb provides partial protection. These mAbs highlight the utility of transgenic mice to produce therapeutic mAbs for preventing EBV-driven disease. Graphical abstract Highlights•Transgenic mice were used to make genetically human EBV mAbs against gp350 and gp42•mAbs potently neutralize EBV infection by blocking receptor-ligand interactions•mAbs prevent EBV infection following EBV challenge in humanized mice Epstein-Barr virus (EBV) can cause serious illness,including cancer,especially in immunocompromised patients. There are no EBV-specific treatments. Chhan et al. leverage a transgenic mouse model to develop human monoclonal antibodies that block EBV entry. These antibodies prevent EBV infection in a murine challenge model offering hope for new therapies. View Publication

The immunomodulatory function of the gastric microbiota in cancer is poorly understood,partly due to the stomach’s acidic environment and limited microbial colonization. Here,by analyzing 68 paired human gastric cancer (GC) samples,we identify Ligilactobacillus salivarius as a commensal bacterium depleted in tumors but enriched in immune checkpoint blockade (ICB) responders. Oral administration of L. salivarius enhances anti-PD-1 efficacy in multiple GC mouse models by promoting pro-inflammatory macrophage activation. Mechanistically,bacterial extracellular vesicles (bEVs) derived from L. salivarius deliver 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase (2,3-BdpM) to tumors,where it activates formyl peptide receptor 1 (FPR1) on macrophages,triggering mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) signaling. Moreover,2,3-BdpM augments the cytotoxic activity of chimeric antigen receptor (CAR)-Claudin18.2+ macrophages in an FPR1-dependent manner. These findings describe a microbial-macrophage axis that enhances GC immunotherapy and highlights the translational potential of orally deliverable microbial adjuvants. Graphical abstract Highlights•Intratumoral reduction of L. salivarius correlates with GC immunotherapy efficacy•bEVs derived from L. salivarius enhance immunotherapy efficacy in GC mouse models•2,3-BdpM in bEV triggers pro-inflammatory macrophage remodeling via FPR1•The cytotoxicity of CAR-Claudin18.2+ macrophages was amplified with 2,3-BdpM alone Yu et al. identify Ligilactobacillus salivarius as a gastric commensal enriched in immunotherapy responders. Oral administration enhances anti-PD-1 efficacy by delivering 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase (2,3-BdpM) via bacterial extracellular vesicles (bEVs) to activate pro-inflammatory macrophages through formyl peptide receptor 1 (FPR1),revealing a microbial-macrophage axis that potentiates gastric cancer immunotherapy. View Publication

Metastatic melanoma is an aggressive malignancy with limited long-term treatment success due to therapeutic resistance and immune evasion. The transient receptor potential melastatin 8 (TRPM8) ion channel is overexpressed in melanoma but its role as therapeutic target remains unexplored. We investigated the anti-tumor effects of novel TRPM8 modulators in metastatic melanoma cells using viability assays,apoptosis markers,mitochondrial function analyses,reactive oxygen species (ROS) measurements and gene silencing. Their functional impact was further assessed in 3D melanoma organoids,clonogenic survival assays,and natural killer (NK) cell co-culture systems. TRPM8 is significantly overexpressed in metastatic melanoma,as compared with the normal counterparts. Its pharmacological inhibition with novel modulators selectively induces calcium-independent mitochondrial apoptosis characterized by ROS accumulation,mitochondrial membrane depolarization,cytochrome c release,and caspase-3 activation. This process involves activation of the ATM/p53 pathway and upregulation of pro-apoptotic proteins. Additionally,TRPM8 modulators increase expression of the NK cell-activating ligand ULBP1,enhancing melanoma susceptibility to NK-mediated cytotoxicity. Our study identifies TRPM8 as a promising biomarker in melanoma. Its targeting triggers mitochondrial cell death and simultaneously boosts NK cell recognition via ULBP1/NKG2D engagement. TRPM8 targeting in combination with immunotherapy might be,hence,further explored in clinical setting of advanced melanoma. View Publication