Anti-PD-1 therapy, effective in patients with various advanced tumors, still encounters the challenge of insensitivity in most patients. Here, we demonstrate that PD-L1 on tumor cell-derived extracellular vesicles (TEVs) is critical for anti-PD-1 therapy resistance. Reducing endogenous and transferring exogenous TEVs abrogates and induces anti-PD-1 therapy resistance, respectively. Notably, PD-L1 is sorted onto TEVs via the endosomal sorting complex required for transport after ubiquitination by UBE4A and gradually upregulated on TEVs with tumor progression. During progression, increased MFGE8 from tumor cells promotes self αv integrin signaling activation, enabling themselves to upregulate UBE4A, thereby increasing PD-L1 on TEVs and enhancing their immunosuppressive abilities. Translationally, anti-MFGE8-neutralizing antibodies effectively downregulate UBE4A and TEV PD-L1, thereby negating anti-PD-1 therapy resistance. Furthermore, serum MFGE8 and PD-L1+ EV levels of tumor patients correlate positively, and high levels of both indicate poor prognosis after anti-PD-1 therapy. Thus, MFGE8 is a promising target for overcoming resistance and predicting responsiveness to anti-PD-1 therapy.
Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.

Assessing therapeutic response in glioblastoma (GBM) is a major factor limiting the clinical development of new and effective therapies. The intracranial location limits serial biopsies, and only provides an intermittent view of the tumor molecular profile from the initial resection. Liquid biopsy techniques, specifically small extracellular vesicle (sEV) analysis, have the potential to overcome these limitations by providing a window into the brain using peripheral blood. To address the need for monitoring tumor evolution and therapeutic resistance, we developed a GBM biomarker panel (ATP1B2, EAAT2, CD24, CD44, CD133 and EGFR) for multiplexed profiling of sEVs using an advanced GBM Extracellular vesicle Monitoring Phenotypic Analyzer Chip (GEMPAC). We successfully tracked patient response to treatment by monitoring changes in glioma stem cell markers on circulating sEVs. We propose that these results provide a strong rationale for using GBM sEVs as a serial monitoring tool in the future clinical management of GBM patients.

Autophagy dysfunction has been closely related with pathogenesis of many neurodegenerative diseases and therefore represents a potential therapeutic target. Extracellular vesicles (EVs) may act as potent anti-inflammatory agents and also modulators of autophagy in target cells. However, the molecular mechanisms by which EVs modulate autophagy flux in human microglia remain largely unexplored. In the present study, we investigated the effects of EVs derived from human oral mucosa stem cells on the autophagy in human microglia. We demonstrate that EVs promoted autophagy and autophagic flux in human microglia and that this process was dependent on the integrity of lipid rafts. Lipopolysaccharide (LPS) also activated autophagy, but combined treatment with EVs and LPS suppressed autophagy response, indicating interference between these signaling pathways. Blockage of Toll-like receptor 4 (TLR4) with anti-TLR4 antibody suppressed EV-induced autophagy. Furthermore, inhibition of the EV-associated heat shock protein (HSP70) chaperone which is one of the endogenous ligands of the TLR4 also suppressed EV-induced lipid raft formation and autophagy. Pre-treatment of microglia with a selective inhibitor of αvβ3/αvβ5 integrins cilengitide inhibited EV-induced autophagy. Finally, blockage of purinergic P2X4 receptor (P2X4R) with selective inhibitor 5-BDBD also suppressed EV-induced autophagy. In conclusion, we demonstrate that EVs activate autophagy in human microglia through interaction with HSP70/TLR4, αVβ3/αVβ5, and P2X4R signaling pathways and that these effects depend on the integrity of lipid rafts. Our findings could be used to develop new therapeutic strategies targeting disease-associated microglia.
© 2024 Federation of European Biochemical Societies.

Despite significant therapeutic advances, lung cancer remains the leading cause of cancer-related death worldwide1. Non-small cell lung cancer (NSCLC) patients have a very poor overall five-year survival rate of only 10-20%. Currently, TNM staging is the gold standard for predicting overall survival and selecting optimal initial treatment options for NSCLC patients, including those with curable stages of disease. However, many patients with locoregionally-confined NSCLC relapse and die despite curative-intent interventions, indicating a need for intensified, individualised therapies. Epithelial-to-mesenchymal transition (EMT), the phenotypic depolarisation of epithelial cells to elongated, mesenchymal cells, is associated with metastatic and treatment-refractive cancer. We demonstrate here that EMT-induced protein changes in small extracellular vesicles are detectable in NSCLC patients and have prognostic significance. Overall, this work describes a novel prognostic biomarker signature that identifies potentially-curable NSCLC patients at risk of developing metastatic NSCLC, thereby enabling implementation of personalised treatment decisions.
© 2023. The Author(s).

Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stability in bodily fluids. As a result, the field of sEV research has expanded exponentially. Nevertheless, there is a lack of standardisation in methods for sEV isolation from cells grown in serum-containing media. The majority of researchers use serum-containing media for sEV harvest and employ ultracentrifugation as the primary isolation method. Ultracentrifugation is inefficient as it is devoid of the capacity to isolate high sEV yields without contamination of non-sEV materials or disruption of sEV integrity. We comprehensively evaluated a protocol using tangential flow filtration and size exclusion chromatography to isolate sEVs from a variety of human and murine cancer cell lines, including HeLa, MDA-MB-231, EO771 and B16F10. We directly compared the performance of traditional ultracentrifugation and tangential flow filtration methods, that had undergone further purification by size exclusion chromatography, in their capacity to separate sEVs, and rigorously characterised sEV properties using multiple quantification devices, protein analyses and both image and nano-flow cytometry. Ultracentrifugation and tangential flow filtration both enrich consistent sEV populations, with similar size distributions of particles ranging up to 200 nm. However, tangential flow filtration exceeds ultracentrifugation in isolating significantly higher yields of sEVs, making it more suitable for large-scale research applications. Our results demonstrate that tangential flow filtration is a reliable and robust sEV isolation approach that surpasses ultracentrifugation in yield, reproducibility, time, costs and scalability. These advantages allow for implementation in comprehensive research applications and downstream investigations.
© 2022 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.