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BACKGROUND: Current antibody-drug conjugates (ADCs) face limitations due to a lack of tumor-selective targets, inefficient internalization, and challenges in reaching tumors in challenging sites, ultimately limiting their therapeutic efficacy. We developed and characterized V66-exatecan, a novel ADC composed of V66, a humanized antibody with high affinity for extracellular DNA (exDNA), conjugated to exatecan via a cleavable linker. This ADC employs a dual-targeting mechanism based on exDNA and ENT2 transporter expression to enhance nuclear drug delivery and tumor specificity. This study evaluates its anti-tumor activity, mechanism of action, ability to treat challenging tumors, and safety profile. METHODS: To validate tumor selectivity, V66 or a control antibody were conjugated to a fluorescent tag and injected intravenously into tumor-bearing mice; biodistribution analysis demonstrated selective accumulation in tumors and nuclear localization within tumor cells. V66 was then conjugated to exatecan via a cleavable linker. In vitro assays across diverse cancer cell lines assessed cytotoxicity, DNA damage response (DDR) activation, and TOP1 degradation. In vivo efficacy was evaluated in xenograft models of triple-negative breast cancer (TNBC) and BRCA1/2-deficient tumors, including intracranial medulloblastoma. These models were used to assess tumor growth inhibition, survival benefit, and blood-brain barrier (BBB) permeability. Toxicity was assessed through a dose-escalation study, with analysis of hematologic parameters, histopathology of major organs, and liver and kidney function tests (ALT, AST, BUN, total protein) following short- and long-term treatment. RESULTS: V66-exatecan demonstrated potent anti-tumor activity in multiple cancer cell lines but not on healthy mouse primary fibroblasts, with EC50 values in the low nanomolar range. It induced robust DDR signaling, TOP1 degradation, and bystander killing effects. BRCA1/2-deficient models exhibited enhanced penetration and sensitivity, with up to 17-fold lower EC50 compared to BRCA-proficient controls. In vivo, V66-exatecan significantly inhibited tumor growth and extended survival in both TNBC and BRCA-mutant CNS tumors, including complete regressions and prolonged median survival in BRCA2-deficient models. Toxicology studies revealed no significant hematologic, renal, hepatic, or bone marrow toxicity, even at high or repeated doses. CONCLUSIONS: V66-exatecan represents a next-generation of ADCs that overcomes key limitations of traditional platforms by exploiting exDNA-driven tumor selectivity and ENT2-mediated nuclear delivery. It demonstrates broad therapeutic efficacy and a favorable safety profile, supporting its potential for treating DDR-deficient and hard-to-reach tumors. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12943-025-02462-z.

Chemically-defined (CD) media offer critical advantages for in vitro systems, requiring consistency, tunability, and component transparency, particularly in applications such as bioassays, drug testing, and translational research. While serum-containing media remain widely used, reliable protocols for adapting cells to CD medium, especially for sensitive adherent cell types, are underreported. This study presents a streamlined protocol for adapting human umbilical vein endothelial cells (HUVECs) to a custom CD medium, with a focus on optimizing weaning strategies and identifying surface coatings that support robust attachment and viability. The protocol evaluates gradual and stepwise adaptation approaches to minimize cellular stress, while incorporating defined extracellular matrix proteins to promote adherence under serum-free conditions. Among tested coatings, fibronectin substantially improved cell attachment and viability during CD medium adaptation, outperforming laminin and collagen IV. To support quantifiable and reproducible tracking of cell growth, we applied a trainable AI-based image analysis method for confluence assessment throughout the adaptation process. This work provides a reproducible, modular framework for transitioning HUVECs, and potentially other human cell lines, to CD medium, while preserving cell health and experimental utility across multiple passages. The methodology may also facilitate the design of quantitative and ethically aligned bioassays, accelerating progress in standardized cell culture practices.

In vitro evaluation of novel therapeutic approaches often fails to reliably predict efficacy and toxicity, especially when recapitulating conditions involving recirculating cells. Current testing strategies are often based on static co-culturing of cells in suspension and 3D tissue models, where cell sedimentation on the target tissue can occur. The observed effects may then mostly be a consequence of sedimentation and of the corresponding forced cell-tissue interactions. The realization of continuous medium flow helps to better recapitulate physiological conditions and cell-tissue interactions. To tackle current limitations of perfused organ-on-chip approaches, we developed a microfluidic chip and operation concept, which prevents undesired sedimentation and accumulation of suspended cells during multiple days by relying on gravity-driven perfusion. Our platform, which we termed “human immune flow (hiFlow) chip”, enables to co-culture cells in suspension with up to 7 preformed microtissue models. Here, we present the design principle and operation of the platform, and we validate its performance by culturing cells and microtissues of a variety of different origins. Cells and tissues could be monitored on chip via high-resolution microscopy, while cell suspensions and microtissues could be easily retrieved for off-chip analysis. Our results demonstrate that primary immune cells and a range of different spheroid models of healthy and diseased tissues can be maintained for over 6 days on chip. As proof-of-concept cell-tissue interaction assay, we used an antibody treatment against diffuse midline glioma, a highly aggressive pediatric tumor. We are confident that our platform will help to increase the prediction power of in vitro preclinical testing of novel therapeutics that rely on the interaction of circulating cells with organ tissues.

ObjectiveThere is currently no long-term treatment for the repair of damaged cartilage and osteoarthritis (OA). Induced pluripotent stem cells (iPSCs) are an ideal cell source for screening platforms due to their ability to self-renew and differentiate to cell types that would otherwise require invasive surgeries to obtain, such as chondrocytes and mesenchymal stromal cells (MSCs). Here, we developed an iPSC-based screening platform and tested previously described pro-chondrogenic small molecule compounds, to determine their potential to identify hits.DesigniPSC derived chondroprogenitors (iCPs) and neural crest cell (NCC) derived MSCs (iNCC-MSCs) were generated, and their chondrogenic potential was confirmed. The iPSC derived cells and a primary bone marrow derived MSC (BM-MSC) line were cultured as pellets and treated with different concentrations of small molecule compounds, in the presence of chondrogenic inducing growth factors, over 14 days at 2 ​% O2. Glycosaminoglycan (GAG) synthesis was quantified by a 1,9- dimethylmethylene blue (DMMB) assay.ResultsAfter 14 days of chondrogenesis, forskolin, baicalin and sesamin enhanced GAG synthesis in the iCPs, and forskolin enhanced GAG synthesis in the iNCC-MSCs, while no small molecule compounds enhanced GAG synthesis in the BM-MSCs.ConclusionOur findings further demonstrate how the small molecules pro-chondrogenic effects are dependent on the screening platform conditions, including the cell type, molecule concentration, 3D culture, hypoxia, and the inclusion of additional growth factors. The iPSC-based screening platform developed has the potential to identify disease modifying OA drugs (DMOADs) in novel compound screening libraries.

Glucocorticoids (GC) are useful adjunctive host directed therapies (HDT) for sub-types of tuberculosis (TB). Macrophages play a central role in controlling Mycobacterium tuberculosis (Mtb) infection, relying on glycolytic reprogramming to support an effective host defence, yet the influence of GC on these important phagocytes is poorly understood. Here, we examined the impact of dexamethasone on metabolic and functional responses of primary human airway macrophages (AM) from bronchoalveolar lavage fluid and monocyte-derived macrophages (MDM). We found that dexamethasone significantly reduced basal and compensatory glycolysis in both AM and MDM, and decreased expression of the glycolytic enzyme PFKFB3. Oxidative metabolism was lower in dexamethasone AM but not MDM, indicating different specific metabolic sensitivity of macrophages. Dexamethasone also inhibited the glycolytic response to Mtb and reduced secretion of IL-1β, TNF, IL-6, IL-8, and IL-10. Dexamethasone-treated macrophages showed enhanced survival following Mtb infection and these cells had a significant reduction in bacterial burden. This antimicrobial effect was impaired when macrophages were pre-treated with bafilomycin A1, implicating that phagosomal acidification may at least in part mediate dexamethasone-induced bacterial control. Collectively, these findings demonstrate that dexamethasone reprograms human macrophage metabolism toward a less glycolytic state while preserving their ability to limit Mtb growth. These results may provide a basis for the clinical benefit of GC in some TB presentations and support the development of targeting GC therapies to macrophages, thereby mitigating inflammation without compromising host antimicrobial defence.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-20188-2.

Gene regulatory mechanisms that underlie tissue regeneration have been largely studied at the level of transcription. Here, proximity labeling methods identify increased presence of the RNA helicase and P-body marker Ddx61 in adult zebrafish cardiomyocytes induced to divide by injury or mitogens. Ddx61 molecules form complex condensates in cardiomyocytes during cardiogenic settings in zebrafish, developing mice, and cultured human cells. ddx61 mutations disrupt cardiomyocyte proliferation and heart regeneration indices in adult zebrafish, and DDX6 knockdown reduces proliferation of cultured human cardiomyocytes. During heart regeneration, Ddx61 associates with and is required to restrain expression of mRNA encoding Chordin, a secreted BMP inhibitor that impedes regeneration if present at high levels. Our experiments indicate that mRNA sorting by context-dependent condensates can impact tissue regenerative capacity. Pronobis et al. show that Ddx61 localizes to P-bodies and regulates heart muscle proliferation during cardiac regeneration in zebrafish. Ddx61 is required is required to restrain expression of Chordin, a secreted BMP inhibitor that impedes regeneration if present at high levels.

IntroductionThe role of prolactin (PRL) in breast cancer and its role within the context of the tumor microenvironment are not well understood. In our previous study, we demonstrated a cross-talk between the ataxia telangiectasia-mutated (ATM) DNA damage response pathway and the PRL-Janus-kinase-2 (JAK2)-signal transducer and activator of transcription-5 (STAT5)-heat shock protein-90 (HSP90) pathway. Here we investigated the role of PRL in tumor initiation and the effect of DNA damage.MethodsWe used an in vivo model to assess the ability of breast cancer cells to initiate orthotopic xenograft tumor formation after DNA damage. Breast cancer cells engineered to secrete human PRL were treated with the DNA damaging agent doxorubicin and injected into the mammary fat pad of immune-deficient severe combined immunodeficiency disease (SCID) mice.ResultsDoxorubicin and PRL combination increased the tumor latency, although PRL secretion alone did not change the tumor latency compared to the controls. Depletion of glycolipid asialo ganglioside-GM1-positive immune cells using anti-asialo GM1 antibody resulted in faster tumor formation only in the PRL-secreting breast cancer cells that were pre-treated with doxorubicin. Additionally, doxorubicin plus the PRL treatment of breast cancer cells was shown in vitro to attract cytotoxic NK cells compared to the controls, and this was dependent on the PRLR.DiscussionThese results demonstrate that combined breast cancer cell DNA damage and PRL exposure results in the anti-tumor cell activity of asialo-GM1-positive immune cells.

Human intestinal organoids (HIOs) are a model system for studying human intestinal epithelium. Utilizing HIOs for high-throughput studies remains inefficient, as analyzing their cellular composition and responses to varying experimental conditions requires extensive time and labor. We describe a 96-well plate-based automated pipeline for rapidly imaging and quantifying fluorescent labeling in HIOs using a high-throughput confocal microscope and image analysis software. The pipeline was leveraged to quantify varying levels of cell proliferation among donor HIO lines in response to microbial products. Cytoplasmic fluorescence via antibody labeling was also quantified with the pipeline, enabling measurement of the prevalence of specific cell types in HIOs. This platform offers a novel approach to efficiently and rapidly image and quantify fluorescent staining and immunolabeling in HIOs and has many potential applications, including drug screening, toxicity testing, intestinal barrier integrity and transport studies, microbiome and host-pathogen interaction studies, and lineage tracking.

BackgroundSchlafen family member 11 (SLFN11) has been implicated in cancer biology and immune modulation, but its expression patterns, prognostic value, and role in tumor immunity in melanoma remain incompletely defined.MethodsThrough multi-omics analyses of public databases (The Human Protein Atlas, TIMER2, BEST) and functional validation, we characterized SLFN11 in melanoma. Functional assays were conducted in SLFN11-overexpressing melanoma cells to evaluate effects on M0 macrophage polarization, recruitment of macrophages and CD8⁺ T cells, and CD8⁺ T cell cytotoxic activity.ResultsSLFN11 mRNA levels are reduced in skin cutaneous melanoma (SKCM) compared to normal skin, yet higher in metastatic lesions than in primary tumors. High SLFN11 expression correlates with favorable overall and progression-free survival across multiple independent melanoma cohorts, with consistent prognostic value across clinical subgroups (tumor stages, nodal/metastatic status). Multivariable Cox regression analysis, adjusting for factors like gender, age, and pathologic T/N/M stages, confirmed SLFN11 expression as an independent predictor of favorable overall survival. SLFN11 expression associates with enhanced infiltration of immune cells along with co-expression of immune checkpoint molecules. Furthermore, SLFN11 expression is associated with favorable prognosis in immunotherapy-treated patients. Functional assays show that SLFN11-overexpressing melanoma cells promote M0 macrophage polarization toward an M1 phenotype, enhance recruitment of macrophages and CD8⁺ T cells, and slightly increase CD8⁺ T cell cytotoxic activity.ConclusionsThese findings provide evidence that SLFN11 is associated with immune microenvironment changes in melanoma, correlates with favorable prognosis, and may be linked to immunotherapy response, supporting its potential as a candidate biomarker and therapeutic target for further investigation.

Preclinical studies provided convincing evidence that umbilical cord derived mesenchymal stem cells (UC-MSC) prevent lung injury and promote lung regeneration. We hypothesized that cyclic mechanical stretch (CMS) and hyperoxia (HOX) during mechanical ventilation account for their limited therapeutic efficacy within the clinics. UC-MSC cultures were subjected to CMS and HOX and evaluated for proliferation, cell viability and further functional properties. Reversibility of the phenotype changes was evaluated after recovery in room air following these exposures. CMS and HOX compromised cell viability and proliferation, altered phenotypic characteristics, particularly PDGFRα expression, and induced cellular senescence in UC-MSC. Effects were most pronounced for CMS plus HOX. The alterations of UC-MSC were mediated by p21 accumulation. As inhibition of p21 aggravated cell death of UC-MSC, the results indicated a cell defense mechanism to ensure survival. This assumption was underpinned by the principal reversibility of the phenotype alterations and regrowth after removal of CMS and HOX. But prolonged strongest exposures resulted in definite phenotype changes. CMS and HOX have comparable effects on UC-MSC as described for lung resident MSC. Our results explain their timely limited presence in the diseased lung after therapeutic application. Future research should therefore focus on their repetitive application.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-22330-6.

The glia limitans ensheathes the entire central nervous system (CNS) parenchyma towards the outer surfaces and the perivascular spaces and is formed by a subset of astrocytes strategically localized at these outer parenchymal borders. Barrier properties of the glia limitans during health and neuroinflammation are incompletely understood. By developing an aquaporin-4 (Aqp4)-mRuby3 knock-in reporter mouse that allows for in vivo imaging of the superficial and perivascular glia limitans, we here show that the glia limitans forms a barrier for soluble mediators, beads and immune cells. Combining the Aqp4-mRuby3 reporter strain with additional reporter alleles for vascular, leptomeningeal or myeloid cells ensures precise localization of immune cells to CNS border zones versus the CNS parenchyma allowing to assign functional roles in CNS immune surveillance versus neuropathology. Availability of the Aqp4-mRuby3 reporter mouse will further advance our understanding of the active role of the glia limitans in CNS immune privilege. The glia limitans is formed by astrocytes at CNS borders and acts as a barrier to molecules and immune cells. Here, using an Aqp4-mRuby3 knock-in mouse, the authors visualize this barrier and its role in CNS immune surveillance and neuroinflammation.

ABSTRACTSevere COVID‐19 is an immunological disorder characterized by a hyper‐inflammatory reaction of the immune system. SARS‐CoV‐2 anti‐spike antibodies of the IgG isotype are known to strongly contribute to this hyperinflammation by overactivation of alveolar macrophages. However, while the pathogenic function of IgG has been extensively studied, very little is known about the function of IgA, the most abundant immunoglobulin isotype in the airways. Although IgA is generally considered noninflammatory, in this study, we show that anti‐spike IgA induces pronounced proinflammatory responses. We demonstrate that stimulation of macrophages with anti‐spike IgA immune complexes in combination with a viral stimulus amplifies proinflammatory cytokine production. This IgA‐induced inflammation is particularly driven by IgA2, the IgA subclass that is increased in the plasma of severely ill COVID‐19 patients. We identified that IgA2‐induced inflammation is predominantly dependent on FcαRI‐Syk signaling. Mechanistically, IgA2‐induced inflammation is linked to enhanced glycolysis and altered mitochondrial function, indicating subclass‐specific immunometabolic reprogramming. Taken together, these data indicate a pathogenic role for IgA2 in severe COVID‐19 and highlight its signaling cascades and metabolic pathways as potential druggable targets to counteract hyperinflammation in severe coronavirus infections, such as COVID‐19, SARS, MERS, and potential future outbreaks. Anti‐SARS‐CoV‐2 spike IgA drives inflammation in human macrophages through FcαRI‐Syk signaling. IgA2 immune complexes amplify cytokine production and alter glycolysis and mitochondrial function more potently than IgA1, indicating subclass‐specific immunometabolic reprogramming. These findings identify IgA2 as a pathogenic factor in severe COVID‐19 and highlight pathways with potential therapeutic relevance.