Journal of Extracellular Vesicles

Lipophilic dyes are widely used to track extracellular vesicles (EVs), yet their labeling efficiency toward bona fide small EVs (sEVs) remains poorly defined. Here, we critically reassess this efficiency using a serum-free HEK293F system that generates endogenously fluorescent protein-tagged sEVs (sEVs-FPT) as an unambiguous positive reference, thereby minimizing interference from co-isolated, dye-labelable non-vesicular extracellular particles (NVEPs). Two orthogonal methods, nanoflow cytometry and fluorescence microscopy, were employed for cross-validation. We found that PKH26, PKH67, and DiD labeled <0.5% of sEVs-FPT, regardless of vesicle heterogeneity. In vivo tracking confirmed that dye-derived signals were far weaker than FPT signals and strikingly failed to colocalize with them. Preliminary mechanistic evidence indicates that this failure is due to an inability of sEVs to actively internalize dye aggregates. Our findings raise serious concerns about the validity of lipophilic dye-based EV tracking and call for a critical reevaluation of the relevant literature.

Extracellular vesicles (EVs) derived from activated dendritic cells (DCs) are promising cell-free mediators capable of shaping CD8+ T-cell responses. However, their early molecular and functional effects on CD8+ T cells remain incompletely characterized, and whether engineering activated DC-derived EVs with immunomodulatory cargo can fine-tune these responses remains largely unexplored. Here, we investigated whether curcumin loading into EVs derived from CpG-activated and peptide-pulsed DC2.4 cells (EV-ACT) modulates early activation of primary CD8+ T cells. EVs were isolated by ultrafiltration coupled with size-exclusion chromatography (UF-SEC) and characterized physicochemically and molecularly. Exploratory proteomic profiling identified an activation-associated EV protein signature enriched in antigen-processing and immune-related pathways. Curcumin loading achieved an encapsulation efficiency of 16.4% while preserving EV properties, and spectral confocal fluorescence microscopy revealed heterogeneous fluorescence emission patterns consistent with distinct EV-associated curcumin microenvironments. Following rapid cellular association, EV-ACT promoted early CD8+ T-cell activation, inducing an effector-like phenotype characterized by increased CD69 expression, TNF- and Granzyme B production, and reduced Bcl-2 levels without compromising cell viability. Unlike free curcumin, EV-mediated curcumin delivery selectively reinforced these immunostimulatory responses by significantly increasing CD69 expression and STAT3 phosphorylation, sustaining early activation-associated functional and molecular reprogramming of primary CD8+ T cells.

Harnessing the immunomodulatory capacity of commensal bacteria is an emerging avenue in cancer therapy. Bacterial extracellular vesicles (BEVs) provide a non-replicating, nanoscale alternative to live microbes with the potential for safer systemic delivery. Here, we investigated BEVs from a novel Gram-positive strain of Bifidobacterium pseudocatenulatum (Bif-210). Intravenous administration of Bif-210 BEVs reduced B16-F10 melanoma growth in C57BL/6J mice. Mechanistically, BEVs increased tumour-infiltrating Ly6G+ granulocytes in vivo, increased CD11b+Ly6G+ and ICAM-1+Ly6G+ bone marrow populations, and induced production of the neutrophil-attracting chemokines KC/CXCL1 (mouse) and IL-8 (human). Although Ly6G+ depletion independently inhibited tumour growth, it did not combine additively with BEVs, supporting a model in which Bif-210 BEVs alter Ly6G+ granulocyte function rather than simply expanding a conventional pro-tumour granulocyte pool. BEVs activated TLR2, did not activate TLR4, and upregulated TLR2 on Ly6G+ cells, while proxy assays provided no evidence of NETosis-associated activation. Repeated intravenous BEV administration produced no overt toxicity by tissue histology, body temperature, or body weight monitoring. These findings position B. pseudocatenulatum BEVs as a promising systemic immunotherapy that recruits and re-educates granulocytes via a TLR2-centred pathway to restrain melanoma progression. HIGHLIGHTSO_LIIntravenous Bif-210 BEVs reduce established B16-F10 melanoma growth in mice. C_LIO_LIBif-210 BEVs selectively increase tumour-associated Ly6G+ granulocytes. C_LIO_LIBEV treatment and Ly6G depletion are non-additive, linking BEV activity to granulocyte biology. C_LIO_LIBif-210 BEVs expand Ly6G+ bone marrow populations and induce granulocyte-recruiting chemokines. C_LIO_LIBif-210 BEVs engage TLR2 and enhance granulocyte fitness without NETosis-associated activation. C_LI

Background. Myocardial infarction (MI) triggers splenic immune cell trafficking to the heart. Vehicles that carry these signals and mediate this crosstalk are unknown. Hypothesis: We hypothesize that extracellular vesicles (EVs) released post-MI mediate splenic immune trafficking to the heart. Methods. Mice were treated daily with an EV biogenesis inhibitor (GW4869) or vehicle. Splenic/cardiac immune cells were assessed at 3d while survival, cardiac function, hypertrophy, and fibrosis were evaluated at 8w post-MI. Plasma EVs from 1d MI mice or from the hearts that underwent MI/sham in a Langendorff system induced splenic immune trafficking to the heart within 3d and systolic dysfunction at 8w in naive mice. Results. GW4869 i) inhibited splenic regression, ii) increased splenic retention of neutrophils, monocytes, dendritic cells (DCs), and CD4+ T-cells, iii) decreased cardiac gene expression of pro-inflammatory cytokines/chemokines, and iv) decreased trafficking of immune cells to the hearts at 3d post-MI, and iii) improved systolic function and attenuated hypertrophy at 8w post-MI. MI EVs accumulated in the spleen and promoted egress of matured splenic immune cells upon administration to naive mice. Cardiac pro-inflammatory cytokines/chemokines expression and CCR2+MHC-IIhi infiltrating macrophages, CD11c+ DCs, and CD4+ and CD4+TNF+ T-cell levels were also increased in naive mice at 3d post-injection. Importantly, transfer of MI EVs for 2 days induced systolic dysfunction, cellular hypertrophy, and fibrosis in naive mice at 8 w post-injection. DCs process MI EVs for T-cells activation. Conclusions: EVs mobilize splenic immune cells to the heart post-MI and their inhibition can subdue inflammatory tissue-damage to promote healing post-MI.

BackgroundReliable tracking of extracellular vesicles (EVs), key biological nanocarriers in nanomedicine, remains a major technical challenge due to the limitations of conventional lipophilic dyes, including aggregation, micelle formation, and nonspecific background signals that compromise biodistribution analyses. MethodsHere, we present a fluorogenic labeling strategy based on Aco-600, a water-soluble probe exhibiting a "light-on" activation in hydrophobic environments. Medium/large EVs (m/lEVs) derived from murine BV2 microglial cells were labeled and intranasally administered to adult C57BL/6 mice. EV biodistribution and brain uptake were quantitatively assessed by ex vivo fluorescence imaging on brain cryosections at multiple time points (5-1440 min), focusing on the cortex and hippocampus. ResultsAco-600 labeling enabled high signal-to-noise detection with minimal background and no evidence of dye aggregation artifacts. Quantitative analysis revealed a consistent spatiotemporal distribution profile across brain regions, with peak signal intensity at 60 minutes post-administration, followed by progressive clearance. This approach provided reproducible and sensitive tracking of EV biodistribution following a clinically relevant intranasal delivery route. ConclusionsOur findings establish fluorogenic labeling as a robust and artifact-minimizing strategy for in vivo EV tracking. This method enhances the accuracy of biodistribution studies and supports the development of EV-based nanomedicine platforms, particularly for central nervous system delivery applications.

Dendritic cells (DCs) are an important type of antigen presentation cell that regulate immunity by initiating antigen-specific immunity and tolerance through T cell activation. The interaction between DCs and T cells can be mediated through direct cell-cell contact or via the release of extracellular vesicles (EVs) from DCs that harbour antigen presentation machineries. Although small EVs (<200 nm in diameter) such as exosomes released by DCs have been shown to regulate immunity, whether other EV subtypes, in particular those that are released by dying DCs due to homeostatic turnover or following infection, can modulate immune responses is not defined. In this study, we demonstrated that DCs undergoing apoptosis can generate a subclass of large EVs ([~]1,000-5,000 nm in diameter) known as apoptotic bodies (ApoBDs) via distinct morphological steps. Mechanistically, ApoBD formation by apoptotic DCs is regulated by Rho-associated kinase 1 and T-type calcium channels. Functionally, DC-derived ApoBDs were found to mediate direct antigen presentation. These data demonstrate a novel function of ApoBDs and highlight the ability of apoptotic materials derived from dying DCs to continue mediating intercellular communication and regulating immune responses.

Although micro- and nanoplastics have been detected in human atherosclerotic plaques, their mechanistic contribution to disease pathogenesis remains poorly defined. Most experimental studies have used microplastics (particles > 1 micrometer) in non-atherosclerotic animal models or the ApoE KO; mouse, relying on short-term exposure or single-pathway analyses, whereas the chronic cardiovascular effects of nanoplastics (< 100 nm) remain exceedingly scarce, despite their higher biological reactivity and greater tissue penetrance. To address this gap, this study employs a multi-omics approach to investigate the chronic (12-week) oral exposure to 80 nm polystyrene nanoplastics in LDLr KO; mice. We uniquely integrate aortic plaque quantification, hepatic transcriptomics with global alternative splicing profiling, gut microbiome 16S sequencing, and liver untargeted metabolomics to construct a unified host-microbiome-metabolite network. Nanoplastics exposure significantly exacerbates aortic lipid deposition, suppresses hepatic detoxification and anti-atherogenic lipid pathways primarily through transcriptional and post-transcriptional level changes driven by alternative splicing events (e.g., intron retention and isoform switching), and induces gut dysbiosis marked by a reduction in SCFA-producing commensals and enrichment of pro-atherogenic pathobionts-perturbations that correlate with specific hepatic functional modules. Metabolomic changes, including decreased levels of the glutathione precursor gamma-glutamylcysteine and the choline-derived metabolite neurine, implicate oxidative stress and TMAO-related pathways. Cross-species validation using human atherosclerotic transcriptomic and metagenomic datasets supports the clinical translatability. By integrating multi-level biological responses, this work establishes nanoplastics as an environmental cardiovascular risk factor and uncovers novel regulatory mechanisms involving splicing-associated transcriptional reprogramming and gut-liver crosstalk, offering potential early-warning biomarkers and therapeutic targets for nanoplastics-associated cardiovascular disease.

miRNA-based transcriptomic analysis of extracellular vesicles (EVs) provide a promising strategy for identifying non-invasive biomarkers and understanding complex pathological mechanisms. Recently, however, urinary extracellular vesicles (uEVs) have emerged as a valuable window into molecular alterations. Despite the high morbidity and mortality associated with alcohol use disorder (AUD), the molecular mechanisms underlying its sex-specific differences remain poorly understood. To address this, we characterize for the first time the uEV miRNome in AUD, revealing its sexually dimorphic profile. We employed uEVs from actively drinking AUD patients of both sexes who did not have advanced liver disease, alongside matched controls. Deep sequencing revealed 14 differentially expressed miRNAs in females (e.g., hsa-miR-197-3p, hsa-miR-19b-3p, hsa-miR-505-3p, hsa-miR-625-5p, and hsa-miR-27a-5p) and 6 in males (e.g., hsa-miR-1290, hsa-miR-1246, hsa-miR-450a-5p, and miR-590-5p). Notably, whereas hsa-miR-4787-5p was consistently overexpressed in uEVs from both sexes, it was absent in plasma-derived EVs, highlighting the specificity of the urinary compartment. Remarkably, the miRNA signatures we uncovered reflect the multiorgan impact of AUD. For instance, hsa-miR-1290 and hsa-miR-197-3p point to alcohol-related liver injury and systemic inflammation, whereas hsa-miR-19b-3p and hsa-miR-1246 signal neuroinflammation and neuronal stress. A subset, including hsa-miR-1290, hsa-miR-1246, and hsa-miR-27a-5p, has been implicated in cancer contexts. Collectively, these findings support the uEV miRNome as a promising sex-informed molecular signature of AUD with biomarker and mechanistic relevance.

PurposeExtracellular vesicles (EVs) are increasingly explored as natural vehicles for drug delivery and gene therapy approaches. However, reproducible yield and scalability of EV production still pose major challenges in the clinical translation of EV-based therapies. In this study, we sought to quantify and characterize EVs released by suspension-cultured HEK293 cells (Expi293F cells) grown in shaker flasks or small-scale bioreactors, to investigate how the culturing environment affects EV production yield. MethodsExpi293F cells were cultivated (N=3) in either shaker flasks or a bioreactor system, and total cell density, viability, and size were monitored. Supernatants were drawn daily post-cell seeding and were analyzed for EV quantity, size, morphology, and CD63 expression. ResultsNo significant differences were observed in terms of total cell density, viability, and cell size between both cultivation settings. However, cultivation of Expi293F cells in the bioreactor environment significantly increased EV yield by 3-fold compared to shaker flask cultivation (p < 0.01). Other parameters such as average nanoparticle size, EV morphology, and CD63 expression remained comparable between both cultivation methods. ConclusionThese results demonstrate that Expi293F-derived EV yield can be increased by culturing cells in a scalable bioreactor system. These findings pave the way towards the production of therapeutic-based EVs in a scalable and reproducible manner suitable for future (pre-)clinical applications.

Extracellular vesicles (EVs) are mediators of intercellular communication, yet their impact on Chinese Hamster Ovary (CHO) cell physiology and bioprocess performance remains poorly understood. Here, we investigated whether small EVs (sEVs) and large EVs (LgEVs) that accumulate during fed-batch and perfusion cultures modulate CHO cell growth, metabolism, apoptosis, and monoclonal antibody (mAb) production. EVs isolated from early- and late-stage cultures were added to fresh CHO cultures grown with or without glutamine supplementation. Only LgEVs had a significant impact. Late-stage LgEVs markedly altered CHO-cell behavior, reducing cell proliferation, increasing apoptosis under glutamine-limited conditions, and substantially enhancing mAb productivity in a dose-dependent manner. Glutamine supplementation largely alleviated the growth-inhibitory and pro-apoptotic effects of LgEVs while preserving their positive impact on productivity, suggesting that glutamine decouples EV-mediated stress from productivity enhancement. Metabolic analyses revealed increased glucose consumption, a glutamine-dependent shift between glycine and alanine overflow metabolism, and remodeling of amino-acid utilization. Metabolic flux analysis further demonstrated enhanced glycolytic overflow and increased reliance on amino acid-supported anaplerosis. Conversely, selective removal of LgEVs from perfusion medium significantly improved cell expansion without reducing antibody production, supporting an inhibitory role for late-stage LgEVs. These LgEVs were enriched in let-7 family miRNAs and miR-21, consistent with RNAseq analyses demonstrating stress-associated enrichment of these miRNAs in CHO EVs and with functional studies showing that let-7a and miR-21reduce CHO-cell growth. Together, these observations suggest that selective miRNA loading contributes to the growth, metabolic, and productivity phenotypes elicited by late-stage LgEVs. Our findings identify LgEVs as endogenous regulators of CHO-cell physiology and potential targets for optimizing high-density fed-batch and perfusion biomanufacturing processes. HighlightsO_LIEndogenous late-stage Large Extracellular Vesicles (LgEVs) reduce CHO cell growth but boost specific mAb productivity. C_LIO_LIGlutamine supplementation rescues LgEV-mediated growth inhibition and apoptosis. C_LIO_LIMetabolic Flux Analysis (MFA) based on the dynamic behavior of amino acid and other metabolite and substrate concentrations reveals the pyruvate node as a metabolic bottleneck and the associated lactate overflow metabolism as resulting from LgEV exposure. C_LIO_LIStress-associated let-7 and miR-21 microRNAs are highly enriched on a per-EV basis in late-stage LgEVs. C_LIO_LISelective removal of LgEVs improves perfusion cell growth without impacting antibody titer. C_LI

Lipid-processing microglia are among the microglial states most strongly associated with Alzheimers disease (AD) pathology, yet whether this association reproduces across independent cohorts, what transcriptional programs define the state, and which upstream signals and small molecules can modulate it remain unsolved. We address these questions through a cross-cohort analysis of one such substate (MG4) by integrating differential expression, transcription factor activity inference, gene set enrichment, and cell-cell communication across five independent single-nucleus RNA sequencing cohorts (ntotal = 140 donors), with paired single-nucleus ATAC sequencing in one multi-omic cohort for epigenomic corroboration. A held-out cohort (n = 150 donors) supported donor-level regression of MG4 proportion on ligand expression, and two spatial transcriptomics datasets (ntotal = 30 donors) related ligand expression to MG4 identity in neighboring spots. MG4 was reproducibly enriched in AD across all five cohorts (pooled log2 fold change = 0.90, p = 3.0 x 10-4). Expression-based inference and motif accessibility jointly nominated MITF and BACH1 as regulators of a program led by V-ATPase-driven lysosomal acidification and cholesterol efflux, a lysosomal-biogenesis signature distinct from the catabolic DAM and lipid-storage LDAM programs, with AD-specific upregulation of energy metabolism. FGF1 and TGFB2 were the most supported candidate upstream ligands, each significant in donor-level regression with further spatial evidence. Computational drug repurposing nominated ten blood-brain barrier-penetrant compounds as perturbational probes. Together, these results advance a described disease-associated microglial state into a reproducible, mechanistically framed regulatory model, providing candidate regulators, upstream ligands, and pharmacological probes for functional validation.

Glioblastoma is characterized by spatial heterogeneity, with tumor-core and invasive-edge regions differing in cellular composition, transcriptional state, and metabolic context. Spatial transcriptomics has improved understanding of glioblastoma tissue organization. However, cellular transcriptional programs and metabolic interpretation remain poorly resolved. Here, single-cell matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) with spatial transcriptomics were integrated on the same tissue sections to map lipid and transcriptomic organization across matched tumor-core and invasive-region samples from 10 glioblastoma patients. Common region-dependent cellular organization along the core - versus invasive regions were identified after accounting for patient specific signatures. Tumor cores were enriched with astrocyte-like malignant and immune cell types, whereas invasive regions showed increased contribution from oligodendrocyte (progenitor cells). Despite these compositional differences, tumor and invasive cells had a shared transcriptional state space, indicating that regional identity is shaped by altered spatial organization of shared cell states. Transcriptional programs associated with proliferation, hypoxia, extracellular matrix remodeling, tumor associated macrophages and microglia (TAMs) phagocytic activity, T-cell infiltration, and lipid synthesis were spatially structured and differed between tumor and invasive compartments. MALDI-MSI revealed broad lipidomic remodeling across these regions. Tumor regions were enriched in membrane and storage lipid classes, whereas invasive regions showed relative enrichment of lipid species associated with membrane turnover. Integrating lipid and transcriptomic layers revealed spatial lipid-gene program coupling, with tumor cores showing stronger and more coherent coupling than invasive regions. TAM phagocytic activity co-localized with cholesteryl ester abundance, while lipid synthesis coupled to phosphatidylcholine-rich astrocyte-like tumor niches. Exploratory analysis indicated that MGMT promoter methylation may be associated with this altered lipid-transcriptional coupling, particularly in TAM-associated lipid-handling programs. Together, these findings imply that spatial coordination between lipid states and transcriptional programs is a key feature of glioblastoma metabolic heterogeneity.

Dysregulated gut homing of leukocytes drives chronic inflammation in Crohns disease (CD). We employed phage display selection campaigns with libraries of stabilized, constrained peptides against endogenous conformation states of the cannabinoid receptor CB2R on human T cells, to discover novel receptor antagonists with potential to inhibit gut homing. Cluster and frequency analysis of 50,000 enriched sequences resulted in expression and functional characterisation of 10 protein candidates using assays of glucose uptake, ERK phosphorylation (pERK) and beta-arrestin recruitment. Each candidate antagonised CB2R activity with recorded IC50 values of between 5-10 nM. Cannabinoid receptor nanodisc binding experiments and SPR confirmed CB2R selectivity. SLKC_09 with an IC50 of 5.4 nM, was studied in a mouse model of chronic ileitis where it significantly inhibited gut homing of CD4+ & CD8+ naive, effector and memory cell types. Our findings highlight an alternative route to therapeutic inhibition of leukocyte trafficking in CD with a biologic inhibitor of CB2R.

Viruses make long (>40 bp) double stranded RNA (LdsRNA) during replication, which stimulates the innate antiviral immune response. In vertebrates, LdsRNA can induce the type I interferon response (IFN) or the antiviral RNA interference response (dsRNAi) to limit viral replication. Extracellular vesicles (EVs) have previously been shown to carry a variety of nucleic acids for intercellular signaling, and insects and plants have been shown to package LdsRNA in EVs as part of their antiviral immune response. We hypothesized that a similar phenomenon occurs in vertebrates in which EVs traffic LdsRNA between cells during viral infection to induce an antiviral response in naive cells. In this study we showed that both vesicular stomatitus virus (VSV)-derived and in vitro transcribed (ivt)-LdsRNA can be packaged into EVs. LdsRNA was detectable by immunoblots in EVs extracted by both differential ultracentrifugation from ivt-LdsRNA treated U937 and ExoQuick-TCTM precipitation from VSV-infected U937 cells (dsRNA-EVs) but not uninfected controls (control EVs). Isolated EVs were roughly 100 nm in diameter and were able to protect LdsRNA from degradation by RNase III. LdsRNA delivery by dsRNA-EVs was visualized in HEL-299 cells via immunocytochemistry (ICC). The LdsRNA-EVs protected against infection from HCoV-229E, while control EVs did not. Together these results indicate EVs can package and deliver long dsRNA to provide antiviral protection in naive vertebrate cells. Author SummaryWhen viruses infect cells, they produce double-stranded RNA, a molecule that alerts the body to the presence of infection and triggers antiviral defenses. Previous studies have shown that cells can release small membrane-bound packages called extracellular vesicles, which carry biological messages to other cells. However, it was not known whether antiviral double-stranded RNA could be transported in these vesicles and shared with neighboring cells. In this study, we investigated whether human cells package double-stranded RNA into extracellular vesicles and whether this cargo helps protect other cells from viral infection. We found that both synthetic and virus-derived double-stranded RNA were incorporated into extracellular vesicles and shielded from degradation. These vesicles successfully delivered double-stranded RNA to untreated cells, substantially protecting these cells from infection with a human coronavirus. Our findings suggest that cells can communicate antiviral warnings to neighboring cells by packaging double-stranded RNA into extracellular vesicles. This work reveals a previously unrecognized way that antiviral protection may spread through tissues during infection. By extending antiviral signals beyond directly infected cells, extracellular vesicles may help coordinate a broader host defense response. Understanding this natural communication system could also inform the development of new RNA-based antiviral therapies.

Although microplastics and nanoplastics (MP/NP) are pervasive environmental contaminants, our understanding of cellular toxicity remains incomplete, as adverse effects are often attributed to long-term intracellular accumulation, while the spatiotemporal onset of cellular damage remains poorly defined. Here, we employ chemical-bond-selective stimulated Raman scattering (SRS) microscopy and cell models that decouple continuous exposure from intracellular retention to directly visualize clinically derived MP/NP-cell interactions. Cellular stress occurs primarily during MP/NP exposure, accompanied by alterations in lipid droplet (LD) composition. In contrast, following extracellular removal, intracellularly retained MP/NP become largely inert, with recovery of lipid metabolism and cellular functions. Lipidomics identifies arachidonic acid (AA) as a key dysregulated metabolite, and SRS imaging further reveals transient, spatially confined AA enrichment in MP/NP-proximal LDs during uptake. Importantly, phospholipid coating of MP/NP attenuates LD alterations and cytotoxicity while preserving particle internalization, establishing uptake-driven metabolic stress, rather than long-term intracellular retention, as primary source of MP/NP-induced damage.

Existing interfaces modulate user experience through visual, auditory, and haptic channels, but direct physiological modulation, which programmatically alters a user's internal state, remains largely underexplored. We present a wearable sonophoresis patch that uses low-frequency acoustic stimulation to deliver psychoactive substances transdermally, and evaluate its potential for programmable physiological modulation in HCI. We tested this in a double-blinded study (N=26) delivering 100 mg caffeine versus sham control, recording physiological signals during rest and a sustained attention task (SART). The planned comparison for heart rate standard deviation during rest was significant (HR-SD p=0.025, d=1.48), with the caffeine group showing suppressed HR~SD consistent with sympathetic activation. Mean heart rate at rest was not significant (p=0.365), but exploratory analyses during the cognitive task revealed significant cardiovascular divergence: heart rate (p=0.003) and heart rate standard deviation (p=0.027) both moved in directions consistent with systemic caffeine delivery, with effects emerging within minutes of device activation and a sustained group effect across all task rounds (p<0.001). These results provide indirect evidence that wearable sonophoresis can deliver substances to modulate user physiology, opening the design space for on-skin chemical interfaces that adapt delivery in real time to change the user's physiological state on demand.

Cellular senescence is characterized by stable cell cycle arrest and the senescence-associated secretory phenotype (SASP), which drives tissue remodeling and inflammation. Underlying SASP with its increased secretion of cytokines and other secreted proteins is a massive reorganization of the secretory pathway. While transcriptional regulation of senescence has been extensively studied, the contribution of post-translational modifications (PTM) to secretory pathway regulation remains poorly understood. Here, we combined quantitative proteomics with multi-layered PTM profiling of phosphorylation, ubiquitination and acetylation to investigate how intracellular trafficking and secretion are regulated in senescence. Using doxorubicin-induced senescence as the primary model, we identified extensive proteome remodeling, with pronounced changes in ER-Golgi-associated pathways and secretory machinery. Acetylation emerged as the most prominently regulated PTM, particularly affecting proteins involved in vesicle trafficking and ER proteostasis. Comparable proteome and PTM remodeling were also observed in replicative senescence, indicating that these changes are not restricted to a single senescence model. Functional analyses revealed activation signatures of the acetyltransferases p300/CBP, linking global acetylation changes to enzymatic activity. Pharmacological inhibition of p300/CBP using A485 selectively modulated senescence-associated features without reversing growth arrest, consistent with a senomorphic-like effect during senescence establishment. Secretome profiling further demonstrated changes in the composition of secreted factors, consistent with modulation of the senescence-associated secretory phenotype. Together, these findings indicate that acetylation-dependent regulation of the secretory pathway shapes the senescence-associated secretome, revealing a mechanistic link between post-translational regulation, intracellular trafficking, and extracellular signaling in senescence.

Acute ischemic stroke (AIS) induces profound systemic immune alterations that contribute to infection susceptibility. Here, we identify lipocalin-2 (LCN-2) as a rapidly induced and conserved regulator of stroke-associated immunosuppression. Using 3-MACE-Seq, cytokine profiling, and immunofluorescence in C57BL/6J mice subjected to transient middle cerebral artery occlusion (tMCAO), we show that LCN-2 is strongly upregulated in splenic red pulp macrophages (RPMs) within 24 hours and again 7 days post-tMCAO. LCN-2-expressing RPMs form immunological synapses with CD3+ T cells, thereby impacting T cell trafficking. Recombinant LCN-2 directly reprogrammed T cells and monocytes toward hyporesponsive, tolerogenic phenotypes by suppressing inflammatory cytokines, impairing chemotaxis, enhancing phagocytosis, and uncoupling oxidative burst. Human spleens likewise displayed LCN-2-expressing CD68+ RPMs, and LCN-2 preconditioning of monocytes reproduced reduced HLA-DR, CD80, CD206, and ROS with increased uptake of E. coli bioparticles. These findings identify LCN-2 signaling as a central orchestrator of stroke-induced peripheral immunoreprogramming and a potential therapeutic target to mitigate post-stroke immunodepression. SummaryAcute ischemic stroke induces LCN-2 in splenic red pulp macrophages, which reprogram T cells and monocytes toward tolerogenic, hyporesponsive states. Mouse and human data identify LCN-2 as a driver of peripheral immunodepression and a potential target to reduce infection risk. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/733904v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@149bd46org.highwire.dtl.DTLVardef@2974aaorg.highwire.dtl.DTLVardef@1aa7a52org.highwire.dtl.DTLVardef@144dbe5_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

Motor Neurone Disease (MND) is characterised by progressive degeneration of upper and lower motor neurons, accompanied by cytoplasmic mislocalisation and hyperphosphorylation of TDP-43, hallmarks that implicate failure of endolysosomal proteostasis. The Endosomal Sorting Complexes Required for Transport (ESCRT) pathway governs multivesicular body (MVB) formation, lysosomal cargo delivery, and autophagosome closure, yet its expression profile in human MND tissue and mechanistic contribution to disease pathology have not been established. Here, we report subunit-specific dysregulation of ESCRT proteins in postmortem motor cortex and spinal cord from MND patients: CHMP2B (ESCRT-III) is significantly upregulated in both regions, whilst TSG101 (ESCRT-I) and VPS37A (ESCRT-I) are significantly downregulated in motor cortex, indicating a region-specific remodelling of the ESCRT network. In a tunicamycin-induced ER stress model using NSC-34 motor neuron-like cells and primary cortical neurons, TSG101 overexpression reduced total and phosphorylated TDP-43, suppressed mTOR signalling, and restored autophagic flux, whereas TSG101 knockdown exacerbated TDP-43 accumulation and cytoplasmic mislocalisation. CHMP2B modulation selectively regulated TDP-43 phosphorylation without altering total TDP-43 levels, consistent with a casein kinase 1-dependent mechanism operating independently of bulk autophagy. Both TSG101 and VPS4a were required to maintain neuronal CD9 tetraspanin localisation to early endosomes; their depletion redirected CD9 to late endosomal and lysosomal compartments under ER stress. Extracellular vesicle characterisation revealed a functional divergence: TSG101 is required for general exosome biogenesis, whereas VPS4a ATPase activity specifically mediates loading of pathological TDP-43 cargo into EVs. Dynamic light scattering confirmed that ER stress and ESCRT modulation produce distinct, condition-specific alterations in EV size and polydispersity. These findings establish ESCRT dysfunction as a multifaceted contributor to MND pathogenesis and identify TSG101, CHMP2B, and VPS4a as mechanistically distinct therapeutic targets warranting preclinical validation.

High-risk neuroblastoma patients face poor survival despite intensive treatment. Drug testing using patient-derived models can support therapy prioritization for precision medicine and drug development. Models incorporating tumour microenvironmental components, such as co-cultures and short-term cultured patient samples containing substantial non-malignant cell fractions, could better recapitulate microenvironment-dependent drug responses. However, conventional viability assays measure the combined signal from all viable cells in a well and therefore cannot determine tumour-specific drug responses. Here, we establish a microscopy-based readout to quantify cell-type-specific viability in two complementary settings: neuroblastoma-PBMC co-cultures and freshly dissociated patient tumour samples. In the co-cultures, PBMCs were pre-labelled with a cell-tracking dye, and Calcein staining was used to independently quantify the viability of tumour cells and PBMCs in the same well. The Calcein-based viability readout correlated strongly with conventional CellTiter-Glo measurements and was compatible with automated high-throughput drug screening. The imaging workflow enabled identification of compounds with differential efficacy in co-culture versus monoculture and distinguished tumour-specific effects from PBMC toxicity. The microscopy-based viability readout was further adapted to short-term cultured patient samples. Neuroblastoma tumour cells were distinguished from the non-malignant cells using a combination of tumour-specific surface markers NCAM, L1CAM and B7H3. This enabled determination of tumour fractions and measurement of tumour-specific drug responses. Tumour fractions varied substantially between patient samples, highlighting the importance of tumour-specific viability measurements. Together, the microscopy-based viability readout for co-cultures and patient samples enables scalable assessment of tumour-specific drug responses.