TL;DR
This chapter illustrates how EV-focused proteomics enables the systematic dissection of microenvironment-driven oncogenic signaling and provides a translational framework for discovering actionable stromal targets in CLL and other Lyn-dependent malignancies.
Pancreatic ductal adenocarcinoma remains a formidable clinical challenge, with a very poor survival rate. It is highly metastatic and quickly exhibits chemoresistance, both of which are mediated by interactions of the tumor cells with their microenvironment. Extracellular vesicles are key mediators by which tumor cells interact with their microenvironment. Here, we utilized a data-independent acquisition proteomics workflow to characterize the protein cargo of extracellular vesicles isolated from patient-derived primary cells and cell lines of varying aggressiveness. Mapping 1432 unique proteins with high reproducibility, we identified protein signatures that robustly discriminated between short-term and long-term survival phenotypes. While the data set was highly enriched for established exosomal markers, differential expression and PLS-DA modeling revealed that the most significant indicators of short survival (high aggressiveness) were ribosomal proteins. STRING analysis confirmed these as highly connected interactome hubs. These findings suggest that the differential loading of translational machinery into the extracellular space─likely via microvesicles or coisolated nonvesicular particles─contributes to poor prognosis, offering new insights into the systemic secretome of aggressive tumors.
Integrated multi-omic profiling of human tumor-infiltrating T cells reveals cell-intrinsic regulators of T cell dysfunction that are missed by transcriptomic analyses alone, highlighting an additional layer of regulatory control.
Extracellular vesicles (EVs) are key mediators of intercellular communication that transfer functional molecules reflective of their parent cells and are increasingly explored as cell-free therapeutic agents. Immunoregulatory cell types, including regulatory T cells (Tregs), produce EVs with potential anti-inflammatory properties. While thymus-derived Tregs (thyTregs) exhibit high stability and potent suppressive activity, their capacity to generate functional EVs remains unknown. EVs were isolated by size exclusion chromatography or by immunocapture from ex vivo–expanded human thyTregs and characterized by electron microscopy, nanoparticle tracking analysis, and Western blotting. Proteomic and miRNA transcriptomic analyses were performed to define EV cargo. Functional activity was assessed by co-culturing EVs with activated allogeneic peripheral blood lymphocytes (PBLs) and evaluating proliferation, activation markers, and cytokine production. Tetraspanin-positive ThyTreg-derived EVs exhibited typical vesicular morphology, a mean size of ~ 200 nm, and expression of canonical EV markers (CD9, TSG101). Proteomic profiling identified 314 proteins enriched in immune-related pathways, including CD25, CTLA-4, TGF-β1, ICOS, and granzyme B, reflecting the parental Treg phenotype. Transcriptomic analysis revealed a distinct miRNA signature dominated by let-7 family members and miR-148a-3p, with predicted targets involved in TGF-β/BMP and MAPK signaling. Functionally, thyTreg-EVs suppressed CD4⁺ and CD8⁺ T cell proliferation in a dose-dependent but heterogeneous manner and, in some donors, induced responder cell death. No significant changes in activation marker expression or cytokine production were observed. Human thyTregs release EVs with immunosuppressive properties and a defined molecular cargo. These findings identify thyTreg-derived EVs as a novel, cell-free platform with potential for immunomodulatory therapies.
Tumor hypoxia is a hallmark of head and neck squamous cell carcinoma (HNSCC) and promotes tumor prgression. Tumor-derived small extracellular vesicles (sEVs) are mediators of hypoxia-driven signaling and contribute to immune suppression within the tumor microenvironment. ADAM17, a membrane-associated sheddase regulating inflammatory and immune pathways, is sensitive to membrane lipid composition, particularly phosphatidylserine (PS). We investigated how hypoxia-induced lipidomic remodeling of tumor-derived sEVs affects ADAM17 activity and CD8+ T-cell apoptosis. HNSCC cells (PCI-30) and normal keratinocytes (HaCaT) were cultured under normoxia (21% O₂) or hypoxia (1% O₂). sEVs were isolated by size-exclusion chromatography and characterized by nanoparticle tracking analysis, transmission electron microscopy, immunoblotting, and high-resolution untargeted lipidomics. Surface PS and ADAM17 expression were assessed by bead-based flow cytometry, and ADAM17 activity was measured using fluorogenic assays. Human CD8+ T cells were incubated with hypoxic or normoxic tumor-derived sEVs. PS-mediated interactions were blocked using Annexin V, and CD8+ T-cell apoptosis was quantified by flow cytometry. sEVs (125—135 nm) expressed Alix, CD63 and CD9 but not Grp94. Hypoxia selectively enriched PS species with mono-, polyunsaturated and very long acyl chains, reducing surface-exposed PS. Despite decreased PS exposure, hypoxic TEX displayed significantly increased ADAM17 expression and enzymatic activity, resulting in an elevated ADAM17/PS ratio. Functionally, hypoxic TEX induced higher levels of CD8+ T-cell apoptosis compared with normoxic TEX. Blockade of PS with annexin V significanlty reduced ADAM17 activity and attenuated TEX-induced CD8+ T-cell apoptosis. Hypoxia alters phosphatidylserine organization in tumor-derived sEVs, enhancing ADAM17 activation, promoting PS-dependent CD8+ T-cell apoptosis, and facilitating immune evasion in HNSCC. n/a Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Thyroid cancer (TC) is one of the most common endocrine malignancies and ranks among the ten most frequently diagnosed cancers worldwide, highlighting the need for improved diagnostic and monitoring strategies. Extracellular vesicles (EVs) are nano-sized, membrane-enclosed particles released by nearly all cell types that carry selectively sorted bioactive cargo capable of influencing the behavior and fate of recipient cells. Although their molecular composition is shaped by their cells of origin, cargo loading is a regulated process that contributes to EV-mediated intercellular communication and cell-specific targeting. In TC, EVs have emerged as important mediators of tumor progression and microenvironment modulation. Recent advances underscore the diagnostic and prognostic value of EVs as non-invasive biomarkers, particularly through the detection of EV-associated non-coding RNAs, proteins, lipids, and other molecular signatures. However, methodological variability and limited clinical validation remain key barriers to clinical translation. Further preclinical and clinical studies are needed to establish the role of EVs in liquid biopsy and personalized targeted therapy for TC. This review provides a comprehensive and systematic overview of EVs in TC, with particular emphasis on methodological factors influencing EV research, including sample-specific isolation and characterization strategies. Based on a systematic literature survey, this review integrates current knowledge on EV-associated cargo, biological functions, biomarker and therapeutic potential while critically evaluating methodological challenges and outlining future directions for clinical translation.
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