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In Asian populations with non-small-cell lung cancer (NSCLC), EGFR mutations are highly prevalent, occurring in roughly half of these patients. Studies have revealed that individuals with EGFR mutation typically fare worse with immunotherapy. In patients who received EGFR tyrosine kinase inhibitor (TKI) treatment followed by anti-PD-1 therapy, poor results were observed. The underlying mechanism remains unclear. We used high-resolution flow cytometry and ELISA to detect the circulating level of small extracellular vesicle (sEV) PD-L1 in NSCLC individuals with EGFR mutations before and after receiving TKIs. The secretion amount of sEV PD-L1 of lung cancer cell lines with EGFR mutations under TKI treatment or not were detected using high-resolution flow cytometry and Western blotting. The results revealed that patients harboring EGFR mutations exhibit increased levels of sEV PD-L1 in circulation, which inversely correlated with the presence of CD8 + T cells in tumor tissues. Furthermore, tumor cells carrying EGFR mutations secrete a higher quantity of PD-L1-positive sEVs. TKI treatment appeared to amplify the levels of PD-L1-positive sEVs in the bloodstream. Mutation-induced and TKI-induced sEVs substantially impaired the functionality of CD8 + T cells. Importantly, our findings indicated that EGFR mutations and TKI therapies promote secretion of PD-L1-positive sEVs via distinct molecular mechanisms, namely the HRS and ALIX pathways, respectively. In conclusion, the increased secretion of PD-L1-positive sEVs, prompted by genetic alterations and TKI administration, may contribute to the limited efficacy of immunotherapy observed in EGFR -mutant patients and patients who have received TKI treatment.

Small extracellular vesicles (sEVs) residing at tumor tissues are valuable specimens for biopsy. Tumor heterogeneity is common across all cancer types, but the heterogeneity of tumor tissue-derived sEVs (Ti-sEVs) is undefined. This study aims to discover the spatial distributions of Ti-sEVs in oral squamous cell carcinoma (OSCC) tissues and explore how these vesicle distributions affect the patients' prognosis. Multi-regional sampling enabled us to uncover that Ti-sEVs' accumulation at peritumoral sites correlates with a higher disease-free survival rate, and conversely, sparse peritumoral Ti-sEVs tend to forecast a higher risk of relapse. Of those relapsed patients, Ti-sEVs strongly bind to extracellular matrix and subsequently degrade it for allowing themselves enter the bloodstream rather than staying in situ. In advanced OSCC patients, the quantity and spatial distribution of Ti-sEVs prior to anti-PD-1 treatment, as well as the temporal variance of Ti-sEVs before and after immunotherapy, strongly map the clinical response and can help to distinguish the patients with shrinking tumors from those with growing tumors. Our work elucidates the correlation of spatiotemporal features of Ti-sEVs with patients' therapeutic outcomes and exhibit the potential for using Ti-sEVs as a predictor to forecast prognosis and screen the responders to anti-PD-1 therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Only a minority of cancer patients benefit from immune checkpoint blockade therapy. Sophisticated cross-talk among different immune checkpoint pathways as well as interaction pattern of immune checkpoint molecules carried on circulating small extracellular vesicles (sEV) might contribute to the low response rate. Here we demonstrate that PD-1 and CD80 carried on immunocyte-derived sEVs (I-sEV) induce an adaptive redistribution of PD-L1 in tumour cells. The resulting decreased cell membrane PD-L1 expression and increased sEV PD-L1 secretion into the circulation contribute to systemic immunosuppression. PD-1/CD80 + I-sEVs also induce downregulation of adhesion- and antigen presentation-related molecules on tumour cells and impaired immune cell infiltration, thereby converting tumours to an immunologically cold phenotype. Moreover, synchronous analysis of multiple checkpoint molecules, including PD-1, CD80 and PD-L1, on circulating sEVs distinguishes clinical responders from those patients who poorly respond to anti-PD-1 treatment. Altogether, our study shows that sEVs carry multiple inhibitory immune checkpoints proteins, which form a potentially targetable adaptive loop to suppress antitumour immunity., (© 2024. The Author(s).)

Objective: Small extracellular vesicle (sEV)-mediated intercellular communication is increasingly the key for the understanding of venous malformations (VMs). This study aims to clarify the detailed changes of sEVs in VMs., Subjects and Methods: Fifteen VM patients without treatment history and twelve healthy donors were enrolled in the study. sEVs were isolated from both fresh lesions and cell supernatant, and were examined by western blotting, nanoparticle tracking analysis and transmission electron microscopy. Western blot analysis, immunohistochemistry and immunofluorescence were adopted to screening candidate regulator of sEV size. Specific inhibitors and siRNA were employed to validate the role of dysregulated p-AKT/vacuolar protein sorting-associated protein 4B (VPS4B) signaling on the size of sEVs in endothelial cells., Results: The size of sEVs derived from both VM lesion tissues and cell model was significantly increased. VPS4B, whose expression level was mostly significantly downregulated in VM endothelial cells, was responsible for the size change of sEVs. Targeting abnormal AKT activation corrected the size change of sEVs by recovering the expression level of VPS4B., Conclusion: Downregulated VPS4B in endothelial cells, resulted from abnormally activated AKT signaling, contributed to the increased size of sEVs in VMs., (© 2023 Wiley Periodicals LLC.)

Immune checkpoint molecule PD-1, expressed on the cell surface, impairs antigen-driven activation of T cells and thus plays a critical role in tumorigenesis, progression, and the poor prognosis of oral squamous cell carcinoma (OSCC). In addition, increasing evidence indicates that PD-1 carried on small extracellular vesicles (sEVs) also mediates tumor immunity, although their contributions to OSCC are yet unclear. Here, we investigated the biological functions of sEV PD-1 in patients with OSCC. The cell cycle, proliferation, apoptosis, migration, and invasion of CAL27 cell lines treated with or without sEV PD-1 were examined in vitro . We performed mass spectrometry to investigate the underlying biological process, combined with an immunohistochemical study of SCC7-bearing mice models and OSCC patient samples. In vitro data demonstrated that sEV PD-1 induced senescence and subsequent epithelial-mesenchymal transition (EMT) in CAL27 cells by ligating with tumor cell surface PD-L1 and activating the p38 mitogen-activated protein kinase (MAPK) pathway. Comprehensive immunohistochemical analysis of the xenograft mice models and OSCC patient samples revealed a very close correlation between the level of circulating sEV PD-1 and lymph node metastasis. These results demonstrate that circulating sEV PD-1 triggers senescence-initiated EMT in a PD-L1-p38 MAPK-dependent manner, contributing to tumor metastasis. It also suggests that the inhibition of sEV PD-1 may be a promising therapeutic target for the treatment of OSCC.

Extracellular vesicles (EVs) are cell-derived membrane-enclosed structures that deliver biomolecules for intercellular communication. Developing visualization methods to elucidate the spatiotemporal dynamics of EVs' behaviors will facilitate their understanding and translation. With a quantum dot (QD) labeling strategy, a single particle tracking (SPT) platform is proposed here for dissecting the dynamic behaviors of EVs. The interplays between tumor cell-derived small EVs (T-sEVs) and endothelial cells (ECs) are specifically investigated based on this platform. It is revealed that, following a clathrin-mediated endocytosis by ECs, T-sEVs are transported to the perinuclear region in a typical three-stage pattern. Importantly, T-sEVs frequently interact with and finally enter lysosomes, followed by quick release of their carried miRNAs. This study, for the first time, reports the entire process and detailed dynamics of T-sEV transportation and cargo-release in ECs, leading to better understanding of their proangiogenic functions. Additionally, the QD-based SPT technique will help uncover more secrets of sEV-mediated cell-cell communication., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

PD-L1 localized to immunosuppressive small extracellular vesicles (sEV PD-L1) contributes to tumor progression and is associated with resistance to immune-checkpoint blockade (ICB) therapy. Here, by establishing a screening strategy with a combination of tissue microarray (TMA), IHC staining, and measurement of circulating sEV PD-L1, we found that the endosomal sorting complex required for transport (ESCRT) member protein hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) was the key regulator of circulating sEV PD-L1 in head and neck squamous cell carcinoma (HNSCC) patients. Increased HRS expression was found in tumor tissues and positively correlated with elevated circulating sEV PD-L1 in patients with HNSCC. The expression of HRS was also negatively correlated to the infiltration of CD8+ T cells. Knockdown of HRS markedly reduced PD-L1 expression in HNSCC cell-derived sEVs, and these sEVs from HRS knockdown cells showed decreased immunosuppressive effects on CD8+ T cells. Knockout of HRS inhibited tumor growth in immunocompetent mice together with PD-1 blockade. Moreover, a higher HRS expression was associated with a lower response rate to anti-PD-1 therapy in patients with HNSCC. In summary, our study reveals HRS, the core component of ESCRT-0, regulates sEV PD-L1 secretion, and is associated with the response to ICB therapy in patients with HNSCC, suggesting HRS is a promising target to improve cancer immunotherapy., (©2022 American Association for Cancer Research.)

Venous malformations (VMs), featuring localized dilated veins, are the most common developmental vascular anomalies. Aberrantly organized perivascular extracellular matrix (ECM) is one of the prominent pathological hallmarks of VMs, accounting for vascular dysfunction. Although previous studies have revealed various proteins involved in ECM remodeling, the detailed pattern and molecular mechanisms underlying the endothelium-ECM interplay have not been fully elucidated. Our previous studies revealed drastically elevated extracellular vesicle (EV) secretion in VM lesions. Here, we identified increased EV-carried MMP14 in lesion fluids of VMs and culture medium of TIE2-L914F mutant endothelial cells (ECs), along with stronger ECM degradation. Knockdown of RAB27A, a required regulator for vesicle docking and fusion, led to decreased secretion of EV-carried MMP14 in vitro. Histochemical analysis further demonstrated a highly positive correlation between RAB27A in the endothelium and MMP14 in the perivascular environment. Therefore, our results proved that RAB27A-regulated secretion of EV-MMP14, as a new pattern of endothelium-ECM interplay, contributed to the development of VMs by promoting ECM degradation., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Circulating small extracellular vesicles (sEVs) are naturally occurring nanosized membrane vesicles that convey bioactive molecules between cells. Conventionally, to evaluate their behaviors in vivo, circulating sEVs have to be isolated from the bloodstream, then labeled with imaging materials in vitro, and finally injected back into the circulation of animals for subsequent detection. The tedious isolation-labeling-reinfusion procedures might have an undesirable influence on the natural properties of circulating sEVs, thereby changing their behaviors and the detected kinetics in vivo. Herein, we proposed an in situ biotinylation strategy to directly label circulating sEVs with intravenously injected DSPE-PEG-Biotin, aiming to evaluate the in vivo kinetics of circulating sEVs more biofriendly and accurately. Such an analysis strategy is free of isolation-labeling-reinfusion procedures and has no unfavorable influence on the natural behaviors of sEVs. The results showed that the lifetime of generic circulating sEVs in mice was around 3 days. Furthermore, we, for the first time, revealed the distinct in vivo kinetics of circulating sEV subpopulations with different cell sources, among which erythrocyte-derived sEVs showed the longest lifespan. Moreover, compared with circulating sEVs in situ or used as autograft, circulating sEVs used as allograft had the shortest lifetime. In addition, the in situ biotinylation strategy also provides a way for the enrichment of biotinylated circulating sEVs. In summary, this study provides a novel strategy for in situ labeling of circulating sEVs, which would facilitate the accurate characterization of their kinetics in vivo, thereby accelerating their future application as biomarkers and theranositic vectors.