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Chimeric antigen receptor (CAR) T cells have become an established immunotherapy with promising results for the treatment of hematological malignancies. However, modulation of the targeted antigen’s surface level in cancer cells affects the quality and safety of CAR-T cell therapy. Here we present an engineered yeast-based antigen system for simulation of cancer cells with precise regulation of surface-antigen densities, providing a tool for controlled activation of CAR T cells and systematic assessment of antigen density effects. This Synthetic Cellular Advanced Signal Adapter (SCASA) system uses G protein-coupled receptor signaling to control cancer antigen densities on the yeast surface and provides a customizable platform allowing selectable signal inputs and modular pathway engineering for precise output fine-tuning. In relation to CD19+ cancers, we demonstrate synthetic cellular communication between CD19-displaying yeast and human CAR T cells as well as applications in high-throughput characterization of different CAR designs. We show that yeast is an alternative to conventional technologies (e.g. microbeads) and can provide higher activation control of clinically derived CAR T cells in vitro, relative to cancer cells. In summary, we present a customizable yeast-based platform for high-throughput characterization of CAR-T cell functionality and show potential applications within therapeutic T cells in clinical settings. Chimeric antigen receptor T (CAR-T) cell therapy uses engineered donor T cells to recognize and eliminate cancer cells through cognate antigen-dependent activation. Here authors develop an alternative to bead-based or cancer-cell-induced CAR-T cell activation by presenting the antigen on the surface of engineered yeast cells, which allows precise regulation of antigen density.

Changes in the immune microenvironment are frequent in cancers occurring in adult patients, yet our understanding of the pediatric cancer immune microenvironment and its clinical relevance is limited. We investigate the immune microenvironment in pediatric T cell acute lymphoblastic leukemia (T-ALL), using single-cell CITE-seq and immune repertoire analyses. We identify a T-ALL subgroup characterized by a remodeled immune microenvironment, which is associated with adverse clinical outcome in minimal residual disease low patients. This adverse immune landscape is dominated by the presence of a population of non-malignant CD4-CD8-TCRαβ T cells that interact with CXCL16 expressing non-classical monocytes. Leukemia cell intrinsic transcriptional rewiring in these patients is associated with activation of Rap1 signaling. Inhibiting Rap1 signaling results in increased sensitivity to the BCL2/BCL-XL inhibitor navitoclax. Our study provides insights into the immune microenvironment of pediatric hematologic malignancies, forming the basis for identifying potential (immuno) therapeutic targets and risk stratification for treatment. Understanding of the immune microenvironment in pediatric acute T cell lymphoblastic leukemia is limited. By analyzing single-cell transcriptome, surface protein expression and immune repertoire data, the authors here identify non-malignant CD4-CD8- TCRαβ T cells that are present in a subset of patients with Rap1 signaling in leukemia cells and are associated with adverse clinical outcome in patients with low minimal residual disease.

ABSTRACTCD36 is a transmembrane glycoprotein that facilitates the uptake of fatty acids and oxidized low‐density lipoproteins. CD36 is overexpressed in various types of cancer and CD36+ cancer cells display enhanced malignancy, including resistance to therapy and elevated stemness and metastatic ability. Thus, CD36 is a therapeutic target in cancer. This study aimed to discover new CD36 antagonists through virtual screening. We identified a druggable pocket in CD36 that is functionally relevant and serves as the binding site for reported antagonists. Consensus molecular docking of a chemical library containing over 25,000 drug‐like compounds identified 15 computational hits with structural diversity. Experimental evaluation of seven compounds revealed that compounds 8, 13, and 14 are novel inhibitors of CD36‐mediated palmitate and/or oxLDL uptake in cellular models. Compound 14 reduced the clonogenicity of HepG2 cells without impacting cell viability, showing that it modulates CD36‐triggered phenotypes associated with disease progression. Analysis of molecular interactions of compounds 8, 13, or 14 with CD36 by molecular dynamics simulations showed that the identified antagonists had stable binding and favorable binding energy, leading to distinct conformational states of the protein. These results support the use of the antagonists identified here as lead compounds for developing CD36‐targeted therapies. Virtual screening identified drug‐like molecules as potential CD36 antagonists; some of them inhibited CD36 function in relevant cellular models. These antagonists could drive the development of CD36‐targeted therapies.

AbstractINTRODUCTIONRecent studies have identified important species‐dependent differences in the response of microglia to β‐amyloid (Aβ) pathology. Yet, whether human microglia also interact differently with the pathognomonic combination of amyloid and tau pathologies that occur in Alzheimer's disease (AD) remains unclear.METHODSWe generated a xenotolerant mouse model of AD that develops both plaque and tangle pathologies, transplanted stem cell‐derived microglial progenitors and examined the interactions between human microglia and AD pathologies with scRNA sequencing, immunohistochemistry, and in vitro modeling.RESULTSThe combined amyloid and tau pathologies induced robust type‐I interferon and proinflammatory cytokine responses, as well as an increased adoption of a distinct “rod” morphology in human microglia. The rod morphology could be induced with type‐I interferon treatment in vitro.DISCUSSIONWe provide new insights into human microglial responses to combined AD pathologies and a novel platform to investigate and manipulate human microglia in vivo.Highlights Amyloid pathology promotes the rapid development of neurofibrillary tangles and neuronal loss in a novel chimeric model of AD.Combined Alzheimer's disease pathologies lead to an expansion of disease‐associated microglia (DAM) and exacerbate Interferon‐responsive and cytokine/chemokine‐enriched states in xenotransplanted human microglia.The combination of amyloid and tau promotes the development of a distinctive rod microglial phenotype that closely correlates with tau pathology and neurodegeneration.Rod morphology and transcriptional changes can be modeled in vitro by treatment of induced pluripotent stem cells (iPSC) ‐microglia with type‐I interferons.

The choroid plexus-cerebrospinal fluid (ChP-CSF) interface regulates a microenvironment supporting neural stem cell growth, strongly affected by hypoxia through ChP function. From human induced pluripotent stem cells (hiPSCs), here we established and validated in vitro ChP organoid secreting CSF-like fluid (iCSF) and exposed them to low oxygen atmosphere for 24 h. Transcriptomic indicated major data on morphological and functional alterations in the ChP cells and shotgun proteomics revealed significant changes in proteins involved in energy metabolism and mitochondrial function. We found that H2AZ and ITM2B, involved in neurogenesis and neurite growth, were the key proteins downregulated in hypoxic iCSF and ChP organoids, respectively. Positive correlation analysis between hypoxia-induced mRNA expression of the neuronal progenitor biomarkers SOX2 and PAX6. Mature neuron MAP2 and H2AZ also confirmed impairment of neurogenesis. The results from this study suggest that ChP-CSF interface opens new opportunities to characterize hypoxic brain pathophysiology and discover novel biomarkers.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12987-025-00731-z.

The limited clinical translation of preclinical anticancer drug efficacy underscores the urgent need for advanced models that faithfully replicate tumor pathophysiology. While three-dimensional (3D) tumor cultures improve the fidelity of microenvironmental modeling, most existing systems lack vascularization, which is a critical element influencing tumor progression and treatment resistance. In this study, a vascularized 3D lung cancer model was established by co-seeding decellularized lung scaffold with human embryonic stem cell-derived endothelial cells, pericytes, and A549 adenocarcinoma cells. This tri-culture system successfully formed a hierarchical vascular network and recapitulated key features of the tumor microenvironment, including hypoxia-driven lysyl oxidase (LOX) overexpression, and integrin-mediated fibronectin-rich desmoplastic niches accumulation. Compared to traditional cultures, this 3D bioengineered platform demonstrated excellent cell compatibility and architectural complexity, supporting enhanced cell migration and MUC5AC hypersecretion. Importantly, cancer cells cultured in this 3D vascularized system exhibited reduced chemosensitivity relative to monolayer cultures. Moreover, patient-derived lung cancer organoids were integrated into the pre-vascularized 3D compartment for individualized drug response testing. Mechanically, hypoxia-activated HIF-1α/LOX signaling promoted ITGA5/FN1-dependent extracellular matrix remodeling and contributed to a chemoprotective niche. This vascularized 3D lung cancer model offers a physiologically relevant and translationally valuable platform for investigating non-small cell lung cancer progression and optimizing patient-specific drug screening.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12935-025-04064-7.

The rapid increase in global meat demand has placed significant pressure on traditional meat production. This has led to the emergence of the cultured meat industry as a promising alternative offering sustainable and ethical solutions. This study explored the efficacy of chick embryo extract (CEE) as a potential economic supplement. Specifically, we aimed to identify the optimal culture conditions for chicken muscle satellite cells (CMSCs) using CEE and evaluate their effects on cell proliferation and differentiation. The CEE+horse serum (HS) condition was found to be the most effective for CMSC proliferation, with a significant increase in the expression of myogenic transcription factors. CMSCs cultured in 30% CEE + 3% HS-containing differentiation medium (CEE + 3%HS-DM) showed higher expression of myogenic differentiation markers and formed thicker myotubes compared to the CMSCs cultured in the 5% fetal bovine serum (FBS)-containing differentiation medium (5% FBS-DM). Notably, CMSCs differentiated in CEE + 3%HS-DM displayed consistently aligned myotubes in the predominant direction. In contrast, myotubes in the 5% FBS-DM showed less directional preference. To investigate 3D myofiber formation on plant-based scaffolds, CMSCs were cultured on a decellularized green onion scaffold. CMSCs successfully attached and differentiated into myofibers, with significantly enhanced attachment and differentiation observed in CMSCs cultured in CEE + 3%HS-DM compared to those in 5% FBS-DM. These results demonstrate that CEE + 3%HS-DM is suitable for CMSC differentiation and 3D myotube formation, providing a promising culture system for sustainable cultured meat production.

Adipocyte deposition is believed to be a primary characteristic of age-related thymic involution, but the underlying cellular and molecular mechanisms remain unknown. We show here that thymic mesenchymal stromal cells (tMSCs) have a higher tendency to differentiate into adipocytes and melanocortin-2 receptor accessory protein (MRAP) is a potential driver of tMSCs adipogenesis. Furthermore, we discover that thymosin-α1 promotes MRAP expression in tMSCs through FoxO1 signaling pathway. Additionally, the proportion of tMSCs increase in older mice compared to young mice. Importantly, MRAP is also necessary for human thymic MSCs to differentiate into adipocytes when exposed to thymosin-α1. Single-cell RNA-seq analysis of human thymus revealed an accumulation of tMSCs and adipocytes during aging, indicating a strong potential for adipogenic differentiation in age-related thymic involution. Thus, we have revealed MRAP as a key factor in promoting thymic MSCs adipogenesis triggered by thymosin-α1 and FoxO1 pathway, which may serve as potential target to hinder adiposity in age-related thymic involution. Adipocyte deposition is believed to be a primary characteristic of age-related thymic involution. Here, the authors show that MRAP is a key factor in promoting thymic MSCs adipogenesis triggered by thymosin-α1 and FoxO1 pathway, which provide a new mechanism for age-related thymic involution

ABSTRACTThe emergence of SARS‐CoV‐2 posed a major global public health threat, necessitating urgent development of therapeutics. Despite vaccine availability, continuous emergence of viral variants with enhanced transmissibility and immune escape capabilities, and consequential impacts on health services, requires effective antiviral therapeutics. Drug repurposing offers an expeditious strategy to identify therapeutics with established safety profiles. We implemented a comprehensive three‐tiered validation approach, screening 2,570 compounds against SARS‐CoV‐2 in vitro, followed by ex vivo validation in well‐differentiated primary human bronchial epithelial cell (WD‐PBEC) cultures, and rigorous in vivo assessment. This methodical progression identified Azatadine‐Dimaleate, a H1‐receptor antagonist, as an exceptional candidate with consistent efficacy across all systems. Azatadine‐Dimaleate demonstrated potent antiviral activity‐ EC50: 4.0 µM (95% CI: 3.2–4.8 µM), reducing viral replication by ~5,000‐fold at 25 µM in epithelial cultures and lowering peak viral titers in WD‐PBECs by 1.4 log10, and 2.33 log10 at 48 and 96 hpi, respectively, compared to controls. There was also a concomitant reduction in expression of interferons and pro‐inflammatory genes, including IL‐6. Combination with Remdesivir synergistically enhanced antiviral activity, reducing the EC50 of both drugs by > 60%. In the K18‐hACE2 transgenic mouse model, Azatadine‐Dimaleate significantly reduced weight loss (4% vs. 12%, p ≤ 0.05), decreased viral loads, and halved viral antigen expression in lung tissues. Unlike many candidates that faltered in complex models, Azatadine‐Dimaleate maintained efficacy across all platforms. These findings support its clinical evaluation, alone or in combination with Remdesivir, as a versatile therapeutic with strong potential to address current and emerging SARS‐CoV‐2 variants. SummaryMulti‐tiered validation proves Azatadine‐Dimaleate potently inhibits SARS‐CoV‐2 with synergistic Remdesivir enhancement, offering immediate clinical potential.

AbstractBackgroundWhile CD19-targeting chimeric antigen receptor T-cell (CAR-T) therapies have shown efficacy in B-cell hematological malignancies, treatment of antigen-negative or relapsed tumors remains challenging. The recombinant malaria protein VAR2CSA (rVAR2) binds specifically to placental-like chondroitin sulfate A (pl-CSA), which is expressed on many cancer types, enabling its use in various tumor-targeting platforms. However, rVAR2-based CAR-T strategies are limited by poor protein thermostability, short serum half-life, and safety concerns related to irreversible CAR activation. To address these limitations, we developed a navigable CAR-T cell system that uses an rVAR2-based navigator to direct CAR-T cells to pl-CSA+ tumors.MethodsWe overexpressed and purified rVAR2 and three catenation proteins in Escherichia coli, then produced and epitope-mapped the anti-rVAR2 monoclonal antibody 5H4VAR2omab following murine immunization. Using its single-chain fragment variable, we constructed a third-generation CAR-T with CD28 and 4-1BB co-stimulatory domains. An interlocking rVAR2 catenane, AXVB, was identified via orthogonal screening and employed as a navigator for directing CAR-T cells to pl-CSA+ tumors. The antitumor efficacy of this navigable CAR-T cell system was evaluated in vitro and in hematologic cancer cell line-derived xenograft models.ResultsWe developed an orthogonal CAR-T cell system that employs a navigator protein (rVAR2 or AXVB) to target pl-CSA+ tumors. Through a reversible switch mechanism, this system enables indirect tumor recognition by CAR-T cells, thereby improving the safety profile of conventional CAR-T therapies. Compared with the rVAR2 monomer, the AXVB multimer exhibited enhanced thermostability and target affinity. Both in vitro and in vivo, AXVB-(navigation)-CAR T cells potently eliminated CD19-positive and CD19-negative pl-CSA+ tumor cells and significantly prolonged survival in tumor-bearing mice.ConclusionsProtein catenation improved the thermostability and functional activity of rVAR2. In a murine model of hematologic malignancy, CAR-T cells utilizing the rVAR2 catenane (AXVB) as a targeting module exhibited superior antitumor activity.

AbstractTissue engineering for cardiovascular implants has largely utilized primary human cells to generate human tissue-engineered matrices (hTEMs). However, due to donor-to-donor variability and limited passage numbers, a more robust alternative to primary cells would be beneficial. To overcome these limitations, we have defined a new differentiation protocol for human-induced pluripotent stem cells (hiPSCs) into isogeneic cardiac fibroblast-like cells (iCFs) using animal sera-free and chemically defined methods. Morphology, extracellular matrix (ECM) deposition, and global transcriptomics revealed similarity between iCFs and primary human cardiac fibroblasts. Additionally, by overexpressing specific ECM and ECM-related proteins through gene-editing approaches, the ECM composition can be modulated as a building block to create “designer” next-generation hTEMs. Proteomics of gene-edited iCF-derived hTEMs demonstrated an increase in proteins involved in collagen and elastic fiber assembly. Furthermore, analysis of gene-edited iCF-derived hTEM mechanical functionality through biaxial mechanical testing exhibited increased collagen function, attributed to increased crosslinking and maturation. In sum, we have combined hiPSC technology with genome engineering to lay the foundation for next-generation tissue engineering applications by generating a novel cell source, gene-edited iCFs, that are able to modulate the composition as well as the functional mechanics of hTEMs. Graphical abstract Graphical Abstract Depicts the building blocks of next-generation human tissue-engineered matrices (hTEMs). Human-induced pluripotent stem cells (hiPSCs) as the starting cell source were gene edited, using the TALEN-based genome engineering approach, to induce a stable upregulation of genes involved in the biosynthesis and regulation of extracellular matrix (ECM) proteins (ie, lysyl oxidase [LOX], lysyl oxidase like 1 [LOXL1], elastin [ELN], and fibulin 5 [FBLN5]). These isogeneic hiPSCs were then differentiated into isogeneic cardiac fibroblast-like cells (iCFs) and used as building blocks to manufacture hTEMs for cardiovascular applications with modulated ECM composition and tissue mechanical properties.

Over the past decade, non-viral DNA template delivery has been used with engineered nucleases to target single-stranded DNA sequences in hematopoietic stem and progenitor cells. While effective for gene therapy, this method is limited to short DNA donor templates, restricting its applications to gene corrections. To expand its scope, we developed an editing process using kilobase-long circular single-stranded DNA donor templates and TALEN technology. Our results show that the CssDNA editing process achieves high gene insertion frequency in HSPCs. Compared to AAV-edited HSPCs, CssDNA-edited HSPCs show a higher propensity to engraft and maintain gene edits in a female NCG murine model. This positive outcome is partly due to higher levels of primitive edited HSPCs, a more quiescent metabolic state, and elevated expression of bone marrow niche adhesion markers. Our findings highlight the strong potential of CssDNA as a universal, scalable and efficient non-viral DNA template for gene therapy applications. Letort et al. demonstrate that Circular single-stranded DNA enables efficient and precise gene insertion in hematopoietic stem cells, outperforming other DNA delivery formats and supporting progress toward scalable next-generation gene therapies.