�����ƽ��

Bioresorbable nanopatterned scaffolds functionalized with polydopamine (PDA) and graphene oxide (GO) have been shown to promote the differentiation of murine neural stem cells (mNSCs) toward neural and glial lineages. Herein, we aim to evaluate the compatibility of these scaffolds for the culture and differentiation of both human embryonic (hESCs) and induced pluripotent (hiPSCs) stem cells. Our results indicate that PDA and GO scaffolds support the topographic alignment of hESCs and hiPSCs cultures, while preserving their pluripotency characteristics. Upon differentiation, PDA and GO scaffolds guide cell specification toward the neuroectoderm germ layer and the neural crest. This promotes enhanced differentiation into both neural and supportive glial cells of the central nervous system (CNS), as well as Schwann cells of the peripheral nervous system (PNS). Moreover, nanopatterned scaffolds also support the differentiation of hESCs and hiPSCs toward endothelial precursors. These findings establish a novel culture platform that enables combined differentiation pathways, potentially relevant for applications in personalized medicine and regenerative cell therapy.

Bone marrow fibrosis is the most extensive matrix remodeling of the microenvironment and can include de novo formation of bone (osteosclerosis). Spatiotemporal information on the contribution of distinct bone marrow niche populations to this process is incomplete. We demonstrate that fibrosis‐inducing hematopoietic cells cause profibrotic reprogramming of perivascular CXCL12‐abundant reticular (CAR) progenitor cells, resulting in loss of their hematopoiesis‐support and upregulation of osteogenic and pro‐apoptotic programs. In turn, peritrabecular osteolineage cells (OLCs) are activated in an injury‐specific, Wnt‐dependent manner, comparable to skeletal repair. OLCs fuel bone marrow fibrosis through their expansion and skewed differentiation, resulting in osteosclerosis and expansion of Ly6a+ fibroblasts. NCAM1 expression marks peritrabecular OLCs and their expansion into the central marrow is specific for fibrosis in mice and patients. Peritrabecular stromal β‐catenin expression is linked to fibrosis in patients, and inhibition of Wnt signaling reduces bone marrow fibrosis and osteosclerosis, possibly being a clinically relevant therapeutic target.

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.

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.

Myocardial infarction (MI) is a leading cause of death globally. Following MI, the heart undergoes remodeling leading to heart failure. The Hippo pathway is a major regulator of cell growth and survival in cardiomyocytes. Here, we show that serotonin receptor 2B (5-HT2B) regulates the Hippo pathway in cardiomyocytes and modulates heart remodeling following MI. 5-HT2B expression significantly enhanced the Hippo pathway effector Yes-associated protein (YAP) activity resulting in increased cardiomyocyte proliferation and decreased apoptosis. However, transgenic mice overexpressing 5-HT2B in cardiomyocytes had a lower survival rate post-MI. Conversely, modified mRNA (modRNA)-mediated transient 5-HT2B expression in the heart was sufficient to inhibit post-MI remodeling. Pharmacological screening of serotonergic compounds identified SB204741 as a modulator of the Hippo/YAP pathway in cardiomyocytes. SB204741 has been shown to protect the heart from adverse remodeling post-MI. Our findings identify 5-HT2B as a regulator of the Hippo pathway that can be targeted to improve cardiac phenotype following MI. Highlights•Serotonin receptor 2B (5HT2B) regulates the Hippo pathway and activates YAP in cardiomyocytes•5HT2B promotes cardiomyocyte proliferation and survival in vitro•Transgenic overexpression of 5HT2B exacerbates hypertrophic remodeling following MI•modRNA delivery of exogenous 5HT2B was partially protective against MI-induced remodeling Biochemical mechanism; Human metabolism; Molecular network

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.

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.

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.

Chimeric antigen receptor (CAR)-T cell therapy targeting antigens shared with normal T cells requires genetic modifications to prevent fratricide. This phase 1 trial evaluates autologous CD5-targeting CAR-T cells with CD5 gene deletion (CT125A) in seven patients with relapsed/refractory CD5+ hematologic malignancies. The overall response rate is 85.7%, including four complete responses. All patients experience cytokine release syndrome (six grade 1–2, one grade 3), and two patients develop immune effector cell-associated neurotoxicity syndrome. The most common grade ≥3 adverse events are cytopenia and infection, with unique observations of rash and autoimmune-related events. Post-infusion immunophenotyping shows persistent depletion of CD5+ T cells and CD19+ B cells, with reduced CD4/CD8 ratios. The human CD5 knockin murine model reveals skin lesions without significant vital organ involvement. These findings demonstrate CT125A’s therapeutic potential in CD5+ malignancies while highlighting the need for safety optimization. The trial has been registered at ClinicalTrials.gov (NCT04767308). Graphical abstract Highlights•CT125A achieves 85.7% response rate in relapsed/refractory CD5+ malignancies•CD5 gene deletion prevents fratricide and enhances CAR-T cell persistence•Prolonged CD5+ T cell aplasia associates with infections and autoimmune events•Mouse model reveals on-target, off-tumor effects primarily affecting skin tissue Cheng et al. report a phase 1 trial of autologous CD5-targeting CAR-T cells with CD5 gene deletion (CT125A) in seven patients with relapsed/refractory CD5+ malignancies. CT125A achieves an 85.7% response rate but causes prolonged immunosuppression, infections, and autoimmune events, highlighting the need for safety optimization strategies.

Directed differentiation of human induced pluripotent stem cells (iPSCs) into anterior foregut endoderm (AFE) and lung progenitors (LPs) has wide-ranging implications for lung developmental biology, disease modeling, and regenerative medicine. We expand on a previously developed mathematical modeling framework and apply it to the directed differentiation of AFE into LPs. A model-based approach guides experimental design, followed by a multistage model inference process: maximum likelihood estimation based on in vitro data and identifiability analyses to eliminate unidentifiable candidates, thereby guiding model selection. To the authors’ knowledge, this is the first mathematical model of the population dynamics of directed differentiation of AFE into LPs. The model suggests that the overall dynamics are primarily driven by AFE proliferation and differentiation into LPs. In silico experiments predict that daily media change nearly doubles LP yields compared to cultures without media replenishment. Moreover, the model suggests that higher split ratios on day 10 enhance yield per input cell, a measure of differentiation efficiency, by 26%. This work provides a blueprint for refining iPSC-based lung lineage differentiation protocols by combining empirical data and mathematical modeling.

Cell surface proteins offer significant cancer therapeutic potential attributable to their accessible membrane localization and central roles in cellular signaling, yet their promise remains largely untapped due to technical challenges inherent to profiling them. Here, we employ N-glycoproteomics to analyze 85 patient-derived xenografts (PDXs), constructing Glyco PDXplorer—an in vivo pan-cancer atlas of cancer-derived surface proteins. We develop a target discovery pipeline to prioritize proteins with favorable expression profiles for immunotherapeutic targeting and validate FAT2 as a squamous-cancer-enriched surface protein minimally detected in normal tissue. Functional studies reveal that FAT2 is essential for head and neck squamous cancer (HNSC) cell growth and adhesion through regulation of surface architecture and integrin-PI3K signaling. Chimeric antigen receptor (CAR)-T cells targeting FAT2 demonstrate anti-tumor activity. This work lays the foundation for developing FAT2-targeted therapies and represents a pivotal platform to inform therapeutic target discovery across cancers. Graphical abstract Highlights•Pan-cancer landscape of cancer-derived cell surface proteins detected in vivo•Discovery pipeline to prioritize proteins as immunotherapy target candidates•Validation of FAT2 as an SCC surface protein with minimal normal tissue expression•FAT2 CAR-T cells demonstrate anti-tumor activity in pre-clinical models Govindarajan et al. leverage N-glycoproteomics and PDX models to decode the in vivo cancer cell surfaceome and establish Glyco PDXplorer—a target discovery platform. The identification and validation of FAT2 as a previously undescribed, actionable antigen demonstrates the utility of Glyco PDXplorer for uncovering therapeutic vulnerabilities.

Ferroptosis, an iron-dependent, lipid peroxidation-driven programmed cell death, holds substantial promise for cancer therapy, yet its translational potential is hindered by widespread intrinsic resistance. While glutathione peroxidase 4 (GPX4) is a well-established ferroptosis suppressor, the epigenetic circuitry coordinating GPX4-related mechanisms remains elusive. Here, via genome-wide screening, we identify ten-eleven translocation 1 (TET1)—a key mediator of DNA 5-hydroxymethylation—as a master controller of cancer cell ferroptosis susceptibility. In acute myeloid leukemia (AML), TET1 enhances 5hmC deposition at the glutamate-cysteine ligase catalytic subunit (GCLC) promoter to activate glutathione/γ-glutamyl-peptide metabolism, fortifying GPX4-dependent defense. Concurrently, TET1 activates NFκB signaling to upregulate GTP cyclohydrolase-1 (GCH1), conferring GPX4-independent ferroptosis resistance. Critically, co-targeting TET1/GCLC/GCH1 with low-dose ferroptosis inducers exhibits potent therapeutic effects against both ferroptosis-sensitive and -resistant AML. Our work positions TET1 as a pivotal epigenetic hub governing ferroptosis surveillance, and provides a translatable strategy to overcome ferroptosis resistance in cancer, with AML as a paradigm. DNA demethylation enzyme TET1 is a known oncogene in leukemia. Here, the authors discover that TET1 is involved in GPX4-dependent and -independent ferroptosis in acute myeloid leukemia via the regulation of GSH synthesis enzyme GCLC and BH4 synthesis enzyme GCH1.