Journal of Extracellular Biology

Matrix-bound nanovesicles (MBVs) are a type of small extracellular vesicle (EV) embedded in the extracellular matrix (ECM) throughout the body. MBVs have been previously isolated from various tissues and in vitro-cultured cell sheets, demonstrating remarkable attributes in regenerative medicine. However, differences between MBVs and conditioned culture medium-derived EVs (liquid-EVs) have yet to be characterized, and the field currently lacks specific protein markers that can identify MBVs from other EV subtypes. Here, we isolate MBVs and liquid-EVs from bone marrow mesenchymal stem cell (MSC) sheets and define differences in size, protein, and zeta potential between these EVs. We show that there is a correlation between cell-driven ECM deposition and MBV and liquid-EV production. We also find that MBVs are smaller, contain less protein per particle, and possess lower zeta potential than liquid-EVs. Interestingly, MBVs also comprise a distinct tetraspanin profile compared to liquid-EVs, with MBVs containing more CD63 and little to no CD81. Finally, we define that CD63, LAMP1, Alix, ITG{beta}1, and GRP94 and their abundance, may be markers specifically used to identify MBVs from liquid-EVs. Our study paves the way for the characteristic differentiation between MBVs from liquid-EVs, elucidates their differences in biogenesis, and reveals a potential connection between EV and ECM production.

Extracellular vesicles (EVs) are promising nanocarriers for therapeutic delivery; however, the factors governing EV uptake by recipient cells remain incompletely understood. In this study, we investigated whether EV internalization is primarily influenced by donor-cell origin or recipient-cell phenotype. Fluorescently labeled EVs derived from HEK293T, or SKBR-3 cells were incubated with a range of human epithelial, immune, and murine cancer cell lines at different doses and time points. HEK293T-derived EVs showed highly variable uptake across recipient cells, with hepatocellular carcinoma cell lines Huh7 and HepG2 exhibiting the highest internalization, while parental HEK293T cells showed the lowest. THP-1 immune cells also demonstrated strong uptake, whereas Jurkat cells showed moderate uptake. In murine melanoma models, Yummer cells internalized more EVs than B16F10 cells. Importantly, similar uptake trends were observed using SKBR-3-derived EVs, where Huh7 and HepG2 again displayed the highest uptake despite originating from a different donor cell source. EV internalization increased with dose and incubation time until saturation at higher concentrations. Together, these results demonstrate that EV uptake is predominantly determined by recipient-cell characteristics rather than EV source. These findings provide important mechanistic insight for the development of EV-based therapeutics and suggest that optimizing recipient-cell targeting is essential for efficient vesicle-mediated delivery. Graphical abstractEV uptake is determined by cell membrane properties rather than by the source of the EVs. The image was created by Biorender. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/726167v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@f5c1cborg.highwire.dtl.DTLVardef@860962org.highwire.dtl.DTLVardef@1d20239org.highwire.dtl.DTLVardef@9003af_HPS_FORMAT_FIGEXP M_FIG C_FIG

Extracellular vesicles (EVs) are versatile therapeutic candidates due to biological roles in intercellular communication and amenability to bioengineering. Compared with lipid nanoparticles (LNPs), native or surface-modified EVs may have favorable immunogenicity and biodistribution profiles. However, when administered intravenously (IV), EVs are rapidly cleared and accumulate mostly in the liver and spleen. With the goal of modifying EV biodistribution, we engineered EVs to display the human endogenous retrovirus (HERV) envelope glycoprotein Syncytin-1, an SLC1A5-binding fusogenic viral protein essential for syncytiotrophoblast formation in pregnancy. Here, we comprehensively characterize engineered Syncytin-1+ EVs, examine their interactions with cells in vitro, and assay biodistribution, immunogenicity, and pharmacokinetics ex vivo and in vivo in non-human primates. IV-administered Syncytin-1+ EVs are well tolerated, persist in the blood stream, and have altered organ biodistribution compared with unmodified EVs, suggesting therapeutic potential of Syncytin-1+ EVs at specific sites.

Antibody-based therapeutics have revolutionized disease treatment, and recent advances in messenger RNA (mRNA) technologies have opened new opportunities for their intracellular production. In particular, in vitro-transcribed mRNA encapsulated in lipid nanoparticles (LNPs) enables targeted delivery to specific cells, where it can enable the synthesis of therapeutic antibodies with prolonged half-lives in a cost-effective manner. Despite rapidly growing experimental data, a modeling framework that integrates mRNA delivery, intracellular expression kinetics, and whole-body antibody disposition remains unavailable. To address this gap, we extended a Physiologically Based Pharmacokinetic model with a novel multiscale layer describing mRNA trafficking, cellular uptake, translation, and degradation. The integrated model was calibrated and validated using five datasets of mRNA-based cancer therapeutics, demonstrating strong predictive performance for the biodistribution of mRNA-encoded antibodies. The newly introduced mRNA layer, while minimally parameterized, effectively represents complex intracellular and systemic processes, enabling quantitative investigation of antibody biodistribution, optimization of dose scheduling, and providing an initial framework for future exploration of how LNP-mRNA formulation influences delivery and pharmacokinetics.

The PRESERVE trial (NCT04972097) is a prospective, single-arm pivotal IDE study evaluating focal irreversible electroporation (IRE) using the NanoKnife System for intermediate-risk prostate cancer. Men with Gleason Grade Group 2-3 disease underwent focal IRE and were followed for durability of oncologic control and safety. At 24 months, 68 patients completed follow-up with no new treatment failures identified. PSA levels were below baseline in 97% of patients, and one clinically triggered biopsy was negative for cancer. No new device- or procedure-related adverse events occurred beyond 12 months. These findings demonstrate durable efficacy and sustained safety of focal IRE.

Aims Active surveillance (AS) allows selected men with localized prostate cancer to defer curative therapy and reduce treatment morbidity. Conversion from AS to treatment is commonly triggered by Gleason grade group (GGG) upgrading on confirmatory biopsy. We developed and validated a digital pathology artificial intelligence (DPAI) biomarker to predict GGG upgrading in AS-eligible patients. Materials & Methods The DPAI model was trained using histopathology image features from diagnostic biopsies of 998 patients and validated in an independent cohort of 296 patients meeting criteria for AS. Logistic regression estimated the probability of confirmatory-biopsy GGG increase, and feature selection identified the most predictive variables. Results AI-GUR (Artificial Intelligence-Gleason Upgrade Risk) predicted GGG reclassification at confirmatory biopsy (OR 1.60; p=0.0003), and provided information beyond conventional stratification (risk group, CAPRA) and cribriform morphology (all p<0.01). Predicted risks were similar across time from diagnosis (~10-15% to ~85% at 1, 1.5, or 2 years; p for time=0.50), consistent with initial biopsy mischaracterization rather than time-dependent progression. Conclusions AI-GUR provides individualized estimates of confirmatory-biopsy GGG upgrading for AS candidates. Using DPAI may improve shared decision-making by complementing standard clinicopathologic tools and molecular testing using the same biopsy specimen, while informing the likelihood of grade upgrade at confirmation.

While engineered nanomaterials offer unprecedented precision in targeting tumor cells, their efficacy is often limited by rapid clearance from the bloodstream via the mononuclear phagocyte system (MPS). To overcome this limitation, a promising strategy known as MPS-cytoblockade has been developed. This approach involves administering antibodies against host erythrocytes. The resulting saturation of the MPS with erythrocyte clearance creates a critical window, allowing subsequently administered nanoparticles to evade immune surveillance and circulate for a significantly extended period. However, MPS-cytoblockade induces a transient reduction in hematocrit, which can lead to adverse effects. Here, we demonstrate that approaches to restore hematocrit, specifically through the administration of donor erythrocyte suspension or the hormone erythropoietin, effectively prevent this drop while maintaining the efficacy of the MPS-cytoblockade. Notably, these interventions do not compromise the prolonged circulation time of the nanoparticles or alter their biodistribution, preserving high accumulation in tumors. Our findings establish a viable strategy to mitigate a key side effect of MPS-cytoblockade, thereby enhancing its therapeutic potential and safety profile.

Manufacturers are adopting propellants for use in pressurized metered-dose inhalers (pMDIs) that have lower global warming potentials (GWPs) than the propellants traditionally used in pMDIs. Hydrofluoroalkane (HFA)-134a has been used as the propellant in the pMDI used to deliver the fixed-dose triple combination of budesonide, glycopyrrolate and formoterol fumarate (BGF); following successful clinical evaluation, the BGF pMDI is now being transitioned to the next generation propellant hydrofluoroolefin (HFO)-1234ze(E), which has near-zero GWP. We describe formulation development efforts that led to selection of HFO-1234ze(E) over another propellant, HFA-152a, for reformulation. Propellant-specific studies evaluated active pharmaceutical ingredient (API) stability and aerodynamic particle size distribution (aPSD). Those analyses have been complemented by in silico regional lung deposition modeling conducted after the clinical evaluation of the reformulated BGF pMDI. HFO-1234ze(E) supported favorable stability and aPSD characteristics for BGF pMDI reformulation, compared with HFA-152a, and modeling predicted regional deposition consistent with therapeutic intent. Given that each pMDI is a unique combination of APIs, device, propellant, and excipients, propellant substitution requires product-specific evidence and regulatory approval, and typically takes several years. Targeted analyses, such as those described here, helped to identify the most suitable candidate propellant for successful substitution in the BGF pMDI. HighlightsO_LIFormulation development efforts that led to evaluation of a budesonide-glycopyrrolate-formoterol fumarate pressurized metered-dose inhaler (BGF pMDI) reformulated with the next generation propellant HFO-1234ze(E) in a clinical trial program are described; the suitability of another propellant, HFA-152a, was also assessed C_LIO_LIOver 6 months under accelerated storage conditions (40{degrees}C/75% relative humidity [RH]), the HFA-152a formulation approached and, in one replicate, fell below the 90% of formulation label claim threshold of evaluation, whereas the original HFA-134a product and the HFO-1234ze(E) formulation remained above that threshold C_LIO_LIOver 6 months under accelerated storage conditions (40{degrees}C/75% RH) and 18 months under long-term stability storage conditions (25{degrees}C/60% RH), the fine particle mass and fine particle fraction for all active pharmaceutical ingredients (APIs) showed that the HFO-1234ze(E) formulation tracked more closely than the HFA-152a formulation to the original HFA-134a product C_LIO_LILater in silico modeling, conducted after clinical testing, predicted a trend for greater deposition of APIs in early airway generations with HFA-152a, whereas HFO-1234ze(E) was predicted to more closely match HFA-134a, indicating a greater likelihood of achieving equivalence to the original HFA-134a product with HFO-1234ze(E) than with HFA-152a C_LIO_LIBased on these analyses and other formulation development efforts, HFO-1234ze(E) was identified as the most suitable propellant for reformulation of the BGF pMDI; for HFA-152a, analyses raised concerns about storage stability, and differences in aerosol characteristics that can impact API deposition in the lungs and, in turn, efficacy C_LI

TRPA1 is a polymodal ion channel receptor known for its role in nociception. TRPA1 can be activated by local mechanical perturbations in the surrounding plasma membrane (PM) by molecules that insert in the lipid bilayer. Here, we tested whether TRPA1 function can be modulated by lipid nanoparticles (LNPs) while interacting with the target cell plasma membrane. We found that LNP induce irregular Ca2+ transients in heterologous and native TRPA1-expressing cells, which may reflect stochastic LNP-PM interactions. By using different cell types and applying selective and non-selective TRPA1 inhibitors, we revealed that the cytosolic [Ca2+] is elevated transients arise as a result through multiple mechanisms: TRPA1-dependent Ca2+ influx, TRPA1-independent Ca2+ influx, and Ca2+ mobilization from the endoplasmic reticulum. Our results describe a novel, non-canonical TRPA1 activation mechanism by LNPs, that may be relevant in the context of the development of cancer and nasal vaccines.

Class B1 G-protein-coupled receptors (GPCRs), such as the calcitonin gene-related peptide (CGRP) receptor and parathyroid hormone 1 (PTH1) receptor, require native lipid interactions to maintain signalling-competent conformations. However, conventional detergents disrupt these environments. Amphipathic copolymers offer a detergent-free alternative, yet the field still lacks a clear understanding of which polymer architectures best preserve active-state GPCR pharmacology, limiting their broader translational utility. Here, we examine how distinct copolymer chemistries influence the functional integrity of class B1 GPCRs by comparing SMA 2000, DIBMA-12, and the electroneutral sulfo-DIBMA. Using NanoLuciferase bioluminescence resonance energy transfer (NanoBRET) ligand-binding, competition, and mini-G-protein recruitment assays on nanodisc-encapsulated receptors, we show that all three copolymers maintain high-affinity extracellular ligand binding but differ markedly in their ability to preserve intracellular signalling. Despite lower receptor extraction efficiency, only sulfo-DIBMA support mini-Gs engagement at the CGRP receptor and enable G-protein-dependent allosteric modulation at the PTH1 receptor, including conserved ligand affinity and prolonged residence time. These data reveal that polymer charge and backbone chemistry, rather than extraction yield, determine whether native-like nanodiscs retain the conformational landscape required for active-state signalling. Controlling non-specific ligand binding to the copolymer is a key requirement for a successful assay. Our findings identify sulfo-DIBMALP as a particularly superior environment for preserving native signalling behaviour in class B1 GPCRs, highlighting copolymer chemistry as an important determinant in detergent-free membrane protein studies. HIGHLIGHTSO_LISulfo-DIBMA encapsulated nanodiscs preserve active-state conformation of human calcitonin gene-related peptide receptor and parathyroid hormone 1 receptor. C_LIO_LIAll three copolymers (SMA 2000, DIBMA-12 and sulfo-DIBMA) preserve extracellular ligand binding but only sulfo-DIBMA preserves intracellular functional competence, including mini-Gs recruitment and G-protein-dependent allosteric modulation. C_LIO_LICopolymer chemistry, particularly the electroneutral, aliphatic nature of sulfo-DIBMA, may influence the preservation of signalling-competent states in two class B1 GPCRs by minimising charge-driven perturbations during solubilisation. C_LIO_LISulfo-DIBMALP provides a novel platform for studying dynamic membrane proteins with potential to provide mechanistic insights and facilitate drug discovery programmes in the future. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/724797v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@12db163org.highwire.dtl.DTLVardef@d8efb3org.highwire.dtl.DTLVardef@610dbaorg.highwire.dtl.DTLVardef@1cc3ce4_HPS_FORMAT_FIGEXP M_FIG C_FIG

De novo vessel formation (vasculogenesis) in vitro is a key step in tissue engineering to preserve tissue viability for long-term assays and testing therapeutic agents. However, in vitro vasculogenesis is often unreliable due to differences in vascular-supporting cells, including endothelial cells and stromal cells such as smooth muscle cells (SMCs) and fibroblasts. Here, we developed a robust co-culture system of HUVECs and SMCs to generate stable vascular networks capable of maintaining tissue viability over extended periods. Given that SMC plasticity is a major limitation in supporting endothelial network formation, we systematically evaluated the effects of passage number, confluency, and freezing on primary SMC function. To overcome this limitation, we generated immortalized supportive SMCs, which preserved their vasculogenic gene program and functional capacity even at high passage. In addition, we identified and validated key genes associated with endothelial support, including CD248, C3, and FBLN1, all essential for vasculogenesis. Immortalized SMCs consistently maintained expression of these genes and supported robust vessel formation under variable culture conditions. Collectively, this study demonstrates that immortalized SMCs provide a stable, reproducible platform for endothelial-SMC co-cultures, enabling long-term vascularized tumor models suitable for functional studies and therapeutic screening.

Extracellular vesicles (EVs) carry microRNAs (miRNAs) that mediate intercellular communication and have strong potential as disease biomarkers, yet the roles of miRNA isoforms (isomiRs) in EVs remain poorly understood. Here, we analyzed 96 human EV and corresponding source samples from nine public datasets. We found that EV samples consistently contained substantially higher proportions of isomiR reads than their corresponding source samples, indicating widespread isomiR enrichment in EVs. Although individual isomiRs showed limited reproducibility across biological replicates and limited sharing between EVs and their corresponding source samples, the parent miRNAs that generated these isomiRs remained highly reproducible across replicates and strongly shared between EV-source pairs. Despite extensive isomiR diversification, EV-source pairs retained highly correlated miRNA expression profiles. Using integrated miRNA- and isomiR-related features, we further developed a random forest model that successfully associated EV samples with their corresponding source samples, with improved performance when isomiR information was included. Together, our results demonstrate that EVs are enriched for biologically meaningful isomiRs while preserving source-associated miRNA landscapes, highlighting the importance of incorporating isomiRs into future EV studies.

IntroductionImpaired motile cilia function contributes to many respiratory disorders, but therapies targeting this cellular defect are currently lacking. Personalized airway epithelial models combined with quantitative, complementary ciliary assays can pave the way for the development of such therapies. However, existing airway epithelial cultures often show variable ciliogenesis, and ciliary function is frequently assessed using a single assay that does not capture the phenotypic heterogeneity of ciliary dysfunction. Here, we established a personalized, multi-assay in vitro platform using human nasal epithelial cells (HNECs) to assess ciliary function and therapeutic response, using primary ciliary dyskinesia (PCD) as a model disease. MethodsHNECs from 8 healthy individuals and 13 individuals with PCD carrying distinct disease-associated variants were obtained by nasal brushing. Cells were differentiated under optimized conditions, including {gamma}-secretase/Notch and BMP pathway inhibitors and a low liquid-liquid interface, to generate highly ciliated 2D epithelial cultures. Ciliary function was assessed using ciliary beat frequency, bead transport, and apical-out nasal organoid rotation assays. Therapeutic rescue was assessed in HNECs harboring DNAI1 alterations using DNAI1 mRNA-loaded lipid nanoparticles. ResultsOptimized differentiation yielded reproducibly multiciliated HNEC cultures. The multi-assay platform distinguished healthy from PCD-derived HNECs and revealed individual- and genotype-specific patterns of ciliary dysfunction not captured by a single assay. Basolateral administration of DNAI1 mRNA-loaded lipid nanoparticles resulted in partial, dose-dependent recovery of ciliary function in DNAI1-deficient HNECs. ConclusionThis study establishes a standardized, individual-specific multi-assay nasal epithelial platform for functional phenotyping of motile cilia and preclinical evaluation of emerging therapies, with demonstrated utility in PCD.

This work aimed to establish a translationally viable, xeno-free, serum-free platform and protocol for the isolation and expansion of human salivary stem/progenitor cells (hS/PCs) suitable for regulatory qualification and future FDA-approved first-in-human autologous regenerative therapy trials for the treatment of hyposalivation disorders. Parotid gland specimens from non-cancerous regions/tissues were collected from consented surgical patients. Primary hS/PCs were isolated from tissue specimens, cultured in animal-component-free conditions, expanded to produce millions of cells, then enriched for CD44+ stem/progenitor cells by magnetic cell sorting. Normal epithelial purity was assessed using cytokeratins 5/14. Anti-CD133/PROM1 (cancer marker) and anti- fibroblast (clone TE-7) antibodies were used to demonstrate a lack of contaminating cells. Phenotype validation was performed by flow cytometry and immunocytochemistry on both CD44+ sorted and unsorted populations. Senescence-associated beta-galactosidase (SA-{beta}-gal) assays were performed across serial passages (P1-P6). Pluripotency was demonstrated by culture under conditions supporting lineage-specific differentiation. Primary hS/PCs demonstrated consistent expansion and epithelial morphology under serum-free conditions. CD44 expression remained high (>95%) throughout expansion, with negligible detection of CD133 or fibroblast markers, confirming epithelial purity and absence of tumorigenic or stromal contamination. Immunocytochemistry corroborated these expression profiles. SA-{beta}-gal staining revealed only a minor, passage-dependent increase (5-16%) in senescent cells from multiple donors, indicating retention of proliferative potential. Our defined, animal-free culture system supports stable expansion of pure low passage hS/PCs under conditions compatible with good manufacturing practice (GMP).

In the terminal segment of the seminiferous tubules, SOX17 expression in the rete testis (RT) epithelium plays a crucial role in the formation of the Sertoli valve (SV), as revealed by phenotypic analyses of RT-specific Sox17 conditional knockout (cKO) mouse testes. In these RT-specific Sox17 cKO testes, SV disruption leads to the backflow of RT fluid into the seminiferous tubules, resulting in defective spermiogenesis and male infertility. Although valve deformation in the Sox17 cKO testes is likely caused indirectly by impaired downstream actions of Sox17 in the RT, the mechanisms by which SOX17 in RT influences SV formation in the seminiferous tubules remain unclear. To address this, we generated a novel AMH-Sox17 transgenic (Tg) mouse line carrying a human AMH promoter-driven Sox17 cDNA cassette. We analyzed the phenotypes of the Sertoli valve and spermatogenesis in AMH-Sox17 Tg mice, as well as in RT-specific Sox17 cKO; AMH-Sox17 Tg double mutant mice. Ectopic SOX17 (SOX17+) expression in Sertoli cells resulted in excessive Sertoli valve structures with acetylated tubulin bundles in the terminal segment of the AMH-Sox17 Tg testes, along with enhanced WNT4/RSPO1 signaling, suggesting the enhanced valve formation of ectopic SOX17+ Sertoli cells by themselves. Moreover, the AMH-Sox17 Tg could partially rescue the SV deformation and infertility in RT-specific Sox17 cKO mice, leading to proper SV formation, normal spermiogenesis and a partial recovery of male fertility in AMH-Sox17 Tg; RT-specific Sox17 cKO double mutant mice. These findings genetically demonstrate that ectopic SOX17+ Sertoli cells can compensate for SOX17 paracrine signaling in the RT, underscoring a key shared downstream pathway between RT and SV. Summary statementThe paracrine actions downstream of ectopic SOX17 expression in the Sertoli cells not only promote the valve formation, but also partially rescue the defective spermiogenesis of the rete testis-specific Sox17-null mice.

Single-cell RNA sequencing (scRNA-seq) effectively captures the differences in transcriptomic landscape of cell types and cell states between benign and cancer tissues. Pooling publicly available datasets distributed across independent studies enables increased sample representation and cross-study comparisons. Here we present a harmonized scRNA-seq atlas of the human prostate constructed by integrating 17 available studies, comprising 163 samples from 106 donors. The dataset contains benign tissue, primary tumors, and metastatic disease profiles. Raw sequencing FASTQ data files were uniformly reprocessed to minimize technical variability. Study metadata were curated and standardized using a unified schema capturing donor identity, tissue site, disease context, and histologic grade. Post quality control, the integrated dataset contains 754,000 high-quality cells. Harmonized cell type annotations were generated using a pseudobulk correlation framework informed by multiple reference resources. The workflow identified 17 distinct cell types representing epithelial, mesenchymal, and immune compartments of the prostate. The processed expression matrices, standardized metadata, and analysis workflows are publicly available to support reproducible analysis and enable exploration of heterogeneity across prostate disease states.

Whole-tissue spatial proteomics level provides critical insights into region-specific biological regulations but remains challenging. Previously, we introduced the Micro-scaffold Assisted Spatial Proteomics (MASP) concept for whole-tissue mapping. However, this prototype required substantial development in spatial resolution, practicality, and throughput for practical application. Here we present a next-generation MASP technique (hex-MASP) featuring i) a new design of hexagonal-micro-wells fabricated with optimized Projection Micro-Stereolithography (P{micro}SL) 3D-printing, achieving high spatial resolution, sampling robustness and mechanical strength for reproducibly compartmentalizing even tough tissues; ii) enhanced throughput/effectiveness in sample preparation and LC-MS analysis with high quantitative quality. Applied to mouse brain, hex-MASP for the first time achieved in-depth, whole-tissue mapping for >6,000 proteins in mouse brains, with high spatial accuracy and excellent data quality. The substantially improved resolution revealed critical regional details across the entire brain, that were not previously captured, enabling precise depiction of protein distribution heterogeneity. This technique enabled the discovery of many unreported regionally-enriched proteins across brain structures. We further applied hex-MASP to investigate the intra-brain distribution of intracerebroventricularly-dosed antibody therapeutics and related proteins, which to our knowledge, enabled whole tissue mapping of protein drugs for the first time and revealed novel mechanistic insights into antibody distribution and localized treatment effects. Hex-MASP represent a robust, scalable platform for whole-tissue spatial proteomics.

Background: Immune-oncology has revolutionized cancer treatment, but some patients fail to benefit due to primary resistance and tumour-immune evasion. Extracellular vesicles (EVs) are secreted by both tumour and immune cells and mediate communication between cancer cells and the immune system. Our study used proteomic profiling of circulating EVs collected from NSCLC patients treated with immune checkpoint inhibitors (ICI) to identify predictive biomarkers of response as well as immune evasion mechanisms related to treatment resistance. Methods: EVs were isolated from plasma collected prior to ICI treatment using peptide-affinity purification and high-throughput proteomics was performed using Proximal Extension Assay. Differentially expressed EV proteins between durable (DR) and non-durable responders (NDR) were identified and evaluated using Cox proportional hazards regression, survival analysis, sex-stratified analysis, as well as pathway and network analysis. Results: Proteomics analysis identified 116 differentially expressed EV proteins between DR and NDR. NDR was characterized by enrichment of inflammatory, angiogenic, and immune-suppressive EV proteins, such as IL1RL1, TFRC, IL6ST, galectins, TNF superfamily death receptors, chemokines, and PCSK9. Pathway analysis revealed enrichment of angiogenesis, chemotaxis, ECM remodeling, and neutrophil degranulation associated with poor progression-free survival (PFS). In contrast, DR to ICI treatment was associated with EV proteins related to T- and B-cell activation and adaptive immunity. Sex-related differences in abundance and association with PFS was observed for certain EV proteins, including IL1RL1 and TFRC. A six protein EV model (IL1RL1, TFRC, ERI1, CCN5, IGFBPL1, and TNFRSF13C) demonstrated good prognostic performance for identifying NDR (AUC = 0.907) and stratified patients into three discrete risk groups. Conclusions: High-plex EV proteomics revealed biologically coherent tumour-immune signaling programs that are associated with ICI treatment resistance. Profiling circulating EVs may improve our understanding of EV-mediated immune evasion mechanisms and identify protein signatures that reflect the tumour immune microenvironment and predict response to immune checkpoint blockade.

Coronavirus disease 2019 (COVID-19) shows highly variable clinical outcomes that are not fully explained by age or comorbidities, underscoring the importance of host molecular responses in determining disease severity. Proteomic and multi-omics studies have linked severe COVID-19 to profound dysregulation of immune, inflammatory, and coagulation pathways, and have shown that circulating protein signatures can predict clinical trajectories. Tocilizumab (TCZ), a monoclonal antibody targeting the interleukin-6 receptor (IL-6R), is an established therapy for IL-6-driven inflammatory diseases and can normalize aberrant molecular profiles. Here, we applied longitudinal serum proteomics to patients with severe SARS-CoV-2 pneumonia treated with TCZ to further characterize how IL-6R blockade reshapes the systemic inflammatory milieu. After TCZ administration, several clinical and inflammatory markers, including C-reactive protein (CRP), CCL5 and CXCL10, decreased. Proteomic profiling revealed that TCZ exerts a sustained effect on the serum proteome, with the most pronounced changes emerging 7 days after treatment. These changes were associated with a broad reconfiguration of the proteomic profile toward a pattern resembling a healthy physiological state, characterized by the restoration of key protein abundances to levels comparable to those observed under homeostatic conditions. Collectively, our findings support that TCZ treatment contributes to the normalization of the inflammatory state in severe COVID-19 and represents a viable therapeutic option for managing the acute inflammatory phase of the disease, while also highlighting additional pathways and biomarkers involved in this recovery process.

BackgroundPer- and polyfluoroalkyl substances (PFAS), particularly perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), are persistent organic pollutants ubiquitous in the environment. Epidemiological evidence has closely linked them to an elevated risk of prostate cancer (PCa). However, the precise molecular mechanisms by which combined PFOS/PFOA exposure promotes prostate cancer and their dynamic effects on the tumor microenvironment remain unclear. MethodsThis study constructed a multi-module analytical framework integrating network pharmacology and computational biology: (1) Through ADMET toxicity prediction, multi-database target collection (three-way Venn analysis), panoramic GO/KEGG enrichment, focused androgen receptor (AR) axis analysis, GWAS genetic association validation, protein-protein interaction (PPI) network construction, machine learning-based independent screening, and a relaxed intersection strategy, we systematically identified PFOS/PFOA-prostate cancer core targets. (2) Subsequently, a PFAS-PTS score weighted purely by Cox coefficients was employed to drive gene set variation analysis (GSVA)-based pathway enrichment, tumor microenvironment (TME) deconvolution, ordinary differential equation (ODE)-based kinetic modeling, and drug intervention prediction. ResultsTarget collection identified 100 shared PFOS/PFOA-prostate cancer targets, from which 18 core targets were determined after multi-module screening. These targets were significantly enriched in the AR signaling axis, the PI3K-AKT pathway, and cell cycle regulation. Molecular docking confirmed strong binding affinities of PFOS/PFOA with AR (-9.49/-8.56 kcal/mol), AKT1 (-7.56/-6.93 kcal/mol), and PTEN (-6.36/-6.08 kcal/mol). GSVA revealed that the G2M checkpoint and E2F target gene pathways were significantly upregulated in the high-risk group (padj < 0.001), whereas the androgen response pathway was downregulated (padj = 4.8e-4). TME deconvolution (GSE141445, NNLS) revealed a significantly increased proportion of tumor cells (PCa) (p = 2.4e-4) and markedly reduced CD8+ T cell infiltration (p = 5.7e-4) in the high-risk group, indicating immunosuppressive microenvironment remodeling. ODE-based kinetic modeling confirmed that PFAS promoted tumor cell proliferation and suppressed immune surveillance in a dose-dependent manner. Drug intervention simulation demonstrated that the combination of enzalutamide and Alpelisib achieved optimal tumor cell inhibition (33.9% predicted by the ODE model). ConclusionPFOS/PFOA promote prostate cancer progression primarily through multi-target synergy involving AR axis disruption, PI3K-AKT pathway activation, and cell cycle dysregulation, while reshaping an immunosuppressive tumor microenvironment. The integrative computational framework established in this study provides systematic computational evidence for risk assessment and therapeutic intervention in PFAS-associated prostate cancer.