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Roseburia intestinalis-derived extracellular vesicles ameliorate colitis by modulating intestinal barrier, microbiome, and inflammatory responses 萝芙木肠源性细胞外囊泡通过调节肠道屏障、微生物群和炎症反应改善结肠炎。
IF 15.5 1区 医学 Q1 CELL BIOLOGY Pub Date : 2024-08-21 DOI: 10.1002/jev2.12487
Hwa Seung Han, Soonjae Hwang, Seung Young Choi, Emmanuel Hitayezu, Mabwi A. Humphrey, Altai Enkhbayar, Dae-Geun Song, Myungsuk Kim, Jong-Sung Park, Young-Tae Park, Jin-Soo Park, Kwang Hyun Cha, Ki Young Choi

Inflammatory bowel disease (IBD) is a chronic disorder characterized by recurrent gastrointestinal inflammation, lacking a precise aetiology and definitive cure. The gut microbiome is vital in preventing and treating IBD due to its various physiological functions. In the interplay between the gut microbiome and human health, extracellular vesicles secreted by gut bacteria (BEVs) are key mediators. Herein, we explore the role of Roseburia intestinalis (R)-derived EVs (R-EVs) as potent anti-inflammatory mediators in treating dextran sulfate sodium-induced colitis. R was selected as an optimal BEV producer for IBD treatment through ANCOM analysis. R-EVs with a 76 nm diameter were isolated from R using a tangential flow filtration system. Orally administered R-EVs effectively accumulated in inflamed colonic tissues and increased the abundance of Bifidobacterium on microbial changes, inhibiting colonic inflammation and prompting intestinal recovery. Due to the presence of Ile-Pro-Ile in the vesicular structure, R-EVs reduced the DPP4 activity in inflamed colonic tissue and increased the active GLP-1, thereby downregulating the NFκB and STAT3 via the PI3K pathway. Our results shed light on the impact of BEVs on intestinal recovery and gut microbiome alteration in treating IBD.

炎症性肠病(IBD)是一种以反复发作的胃肠道炎症为特征的慢性疾病,缺乏确切的病因和明确的治疗方法。肠道微生物组具有多种生理功能,对预防和治疗 IBD 至关重要。在肠道微生物组与人类健康的相互作用中,肠道细菌分泌的细胞外囊泡是关键的介质。在本文中,我们探讨了肠道罗斯布氏菌(R)衍生的EVs(R-EVs)作为强效抗炎介质在治疗葡聚糖硫酸钠诱导的结肠炎中的作用。通过 ANCOM 分析,R 被选为治疗 IBD 的最佳 BEV 生产者。利用切向流过滤系统从 R 中分离出了直径为 76 nm 的 R-EV。口服 R-EVs 能有效积聚在发炎的结肠组织中,并增加微生物变化中双歧杆菌的丰度,从而抑制结肠炎症,促进肠道恢复。由于囊泡结构中存在Ile-Pro-Ile,R-EVs降低了炎症结肠组织中DPP4的活性,增加了活性GLP-1,从而通过PI3K途径下调了NFκB和STAT3。我们的研究结果阐明了 BEVs 在治疗 IBD 过程中对肠道恢复和肠道微生物组改变的影响。
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引用次数: 0
Extracellular vesicles carry transcriptional ‘dark matter’ revealing tissue-specific information 细胞外囊泡携带的转录 "暗物质 "揭示了组织特异性信息。
IF 15.5 1区 医学 Q1 CELL BIOLOGY Pub Date : 2024-08-15 DOI: 10.1002/jev2.12481
Navneet Dogra, Tzu-Yi Chen, Edgar Gonzalez-Kozlova, Rebecca Miceli, Carlos Cordon-Cardo, Ashutosh K. Tewari, Bojan Losic, Gustavo Stolovitzky

From eukaryotes to prokaryotes, all cells secrete extracellular vesicles (EVs) as part of their regular homeostasis, intercellular communication, and cargo disposal. Accumulating evidence suggests that small EVs carry functional small RNAs, potentially serving as extracellular messengers and liquid-biopsy markers. Yet, the complete transcriptomic landscape of EV-associated small RNAs during disease progression is poorly delineated due to critical limitations including the protocols used for sequencing, suboptimal alignment of short reads (20–50 nt), and uncharacterized genome annotations—often denoted as the ‘dark matter’ of the genome. In this study, we investigate the EV-associated small unannotated RNAs that arise from endogenous genes and are part of the genomic ‘dark matter’, which may play a key emerging role in regulating gene expression and translational mechanisms. To address this, we created a distinct small RNAseq dataset from human prostate cancer & benign tissues, and EVs derived from blood (pre- & post-prostatectomy), urine, and human prostate carcinoma epithelial cell line. We then developed an unsupervised data-based bioinformatic pipeline that recognizes biologically relevant transcriptional signals irrespective of their genomic annotation. Using this approach, we discovered distinct EV-RNA expression patterns emerging from the un-annotated genomic regions (UGRs) of the transcriptomes associated with tissue-specific phenotypes. We have named these novel EV-associated small RNAs as ‘EV-UGRsʼ or “EV-dark matter”. Here, we demonstrate that EV-UGR gene expressions are downregulated by ∼100 fold (FDR < 0.05) in the circulating serum EVs from aggressive prostate cancer subjects. Remarkably, these EV-UGRs expression signatures were regained (upregulated) after radical prostatectomy in the same follow-up patients. Finally, we developed a stem-loop RT-qPCR assay that validated prostate cancer-specific EV-UGRs for selective fluid-based diagnostics. Overall, using an unsupervised data driven approach, we investigate the ‘dark matter’ of EV-transcriptome and demonstrate that EV-UGRs carry tissue-specific Information that significantly alters pre- and post-prostatectomy in the prostate cancer patients. Although further validation in randomized clinical trials is required, this new class of EV-RNAs hold promise in liquid-biopsy by avoiding highly invasive biopsy procedures in prostate cancer.

从真核生物到原核生物,所有细胞都分泌胞外囊泡 (EV),作为其正常平衡、细胞间通讯和货物处理的一部分。越来越多的证据表明,小EV携带功能性小RNA,有可能成为细胞外信使和液体活检标志物。然而,由于测序协议、短读数(20-50 nt)的次优比对以及未定性的基因组注释(通常被称为基因组的 "暗物质")等关键限制因素,在疾病进展过程中,EV相关小RNA的完整转录组图谱还没有得到很好的描述。在这项研究中,我们调查了与 EV 相关的未注释的小 RNA,它们来自内源基因,是基因组 "暗物质 "的一部分,可能在调控基因表达和翻译机制方面发挥着新出现的关键作用。为了解决这个问题,我们从人类前列腺癌和良性组织以及从血液(前列腺切除术前后)、尿液和人类前列腺癌上皮细胞系中提取的 EVs 中创建了一个独特的小 RNAseq 数据集。然后,我们开发了一种基于无监督数据的生物信息学管道,它能识别与生物相关的转录信号,而不管其基因组注释如何。利用这种方法,我们发现了来自转录组中未注释基因组区域(UGR)的与组织特异性表型相关的独特 EV-RNA 表达模式。我们将这些新型 EV 相关小 RNA 命名为 "EV-UGRs "或 "EV-暗物质"。在这里,我们证明了 EV-UGR 基因表达下调了 ∼100 倍(FDR
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引用次数: 0
Biopotency and surrogate assays to validate the immunomodulatory potency of extracellular vesicles derived from mesenchymal stem/stromal cells for the treatment of experimental autoimmune uveitis 验证间充质干细胞/基质细胞衍生的细胞外囊泡治疗实验性自身免疫性葡萄膜炎的免疫调节效力的生物效力和替代测定。
IF 15.5 1区 医学 Q1 CELL BIOLOGY Pub Date : 2024-08-14 DOI: 10.1002/jev2.12497
Gagandeep Kaur, Eun-Hye Bae, Yu Zhang, Nicole Ciacciofera, Kyung Min Jung, Heather Barreda, Carol Paleti, Joo Youn Oh, Ryang Hwa Lee

Extracellular vesicles (EVs) derived from mesenchymal stem/stromal cells (MSCs) have been recognized as promising cytotherapeutics due to their demonstrated immunomodulatory effects in various preclinical models. The immunomodulatory capabilities of EVs stem from the proteins and genetic materials they carry from parent cells, but the cargo contents of EVs are significantly influenced by MSC tissues and donors, cellular age and culture conditions, resulting in functional variations. However, there are no surrogate assays available to validate the immunomodulatory potency of MSC-EVs before in vivo administration. In previous work, we discovered that microcarrier culture conditions enhance the immunomodulatory function of MSC-EVs, as well as the levels of immunosuppressive molecules such as TGF-β1 and let-7b in MSC-EVs. Building on these findings, we investigated whether TGF-β1 levels in MSC-EVs could serve as a surrogate biomarker for predicting their potency in vivo. Our studies revealed a strong correlation between TGF-β1 and let-7b levels in MSC-EVs, as well as their capacity to suppress IFN-γ secretion in stimulated splenocytes, establishing biopotency and surrogate assays for MSC-EVs. Subsequently, we validated MSC-EVs generated from monolayer cultures (ML-EVs) or microcarrier cultures (MC-EVs) using murine models of experimental autoimmune uveoretinitis (EAU) and additional in vitro assays reflecting the Mode of Action of MSC-EVs in vivo. Our findings demonstrated that MC-EVs carrying high levels of TGF-β1 exhibited greater efficacy than ML-EVs in halting disease progression in mice with EAU as well as inducing apoptosis and inhibiting the chemotaxis of retina-reactive T cells. Additionally, MSC-EVs suppressed the MAPK/ERK pathway in activated T cells, with treatment using TGF-β1 or let-7b showing similar effects on the MAPK/ERK pathway. Collectively, our data suggest that MSC-EVs directly inhibit the infiltration of retina-reactive T cells toward the eyes, thereby halting the disease progression in EAU mice, and their immunomodulatory potency in vivo can be predicted by their TGF-β1 levels.

间充质干细胞/基质细胞(间充质干细胞)产生的胞外囊泡(EVs)在各种临床前模型中显示出免疫调节作用,因此被认为是有前景的细胞治疗药物。EVs的免疫调节能力源于它们从母细胞携带的蛋白质和遗传物质,但EVs的货物含量受间叶干细胞组织和供体、细胞年龄和培养条件的显著影响,从而导致功能上的差异。然而,目前还没有替代检测方法可以在体内给药前验证间充质干细胞-EVs的免疫调节效力。在之前的工作中,我们发现微载体培养条件能增强间充质干细胞-EVs的免疫调节功能,同时也能提高间充质干细胞-EVs中TGF-β1和let-7b等免疫抑制分子的水平。在这些发现的基础上,我们研究了间充质干细胞-EVs中的TGF-β1水平是否可以作为预测其体内效力的替代生物标志物。我们的研究揭示了间充质干细胞-EVs中TGF-β1和let-7b水平之间的强相关性,以及它们抑制刺激脾细胞分泌IFN-γ的能力,从而建立了间充质干细胞-EVs的生物有效性和替代检测方法。随后,我们利用实验性自身免疫性葡萄膜视网膜炎(EAU)小鼠模型和其他反映间充质干细胞-EV在体内作用模式的体外试验验证了由单层培养物(ML-EV)或微载体培养物(MC-EV)产生的间充质干细胞-EV。我们的研究结果表明,与ML-EV相比,携带高水平TGF-β1的MC-EV在阻止EAU小鼠疾病进展、诱导细胞凋亡和抑制视网膜反应性T细胞趋化方面表现出更强的功效。此外,间充质干细胞-EVs还能抑制活化T细胞中的MAPK/ERK通路,使用TGF-β1或let-7b处理也能对MAPK/ERK通路产生类似的影响。总之,我们的数据表明间充质干细胞-EV直接抑制了视网膜反应性T细胞向眼部的浸润,从而阻止了EAU小鼠的疾病进展,而且它们在体内的免疫调节效力可以通过其TGF-β1水平来预测。
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引用次数: 0
Comparison of EV characterization by commercial high-sensitivity flow cytometers and a custom single-molecule flow cytometer 比较商用高灵敏度流式细胞仪和定制单分子流式细胞仪对 EV 的表征。
IF 15.5 1区 医学 Q1 CELL BIOLOGY Pub Date : 2024-08-14 DOI: 10.1002/jev2.12498
James Kim, Shihan Xu, Seung-Ryoung Jung, Alya Nguyen, Yuanhua Cheng, Mengxia Zhao, Bryant S. Fujimoto, Wyatt Nelson, Perry Schiro, Jeffrey L. Franklin, James N. Higginbotham, Robert J. Coffey, Min Shi, Lucia N. Vojtech, Florian Hladik, Muneesh Tewari, John Tigges, Ionita Ghiran, Tijana Jovanovic-Talisman, Louise C. Laurent, Saumya Das, Olesia Gololobova, Kenneth W. Witwer, Tuoye Xu, Al Charest, Kendall Van Keuren Jensen, Robert L. Raffai, Jennifer C. Jones, Joshua A. Welsh, John P. Nolan, Daniel T. Chiu

High-sensitivity flow cytometers have been developed for multi-parameter characterization of single extracellular vesicles (EVs), but performance varies among instruments and calibration methods. Here we compare the characterization of identical (split) EV samples derived from human colorectal cancer (DiFi) cells by three high-sensitivity flow cytometers, two commercial instruments, CytoFLEX/CellStream, and a custom single-molecule flow cytometer (SMFC). DiFi EVs were stained with the membrane dye di-8-ANEPPS and with PE-conjugated anti-EGFR or anti-tetraspanin (CD9/CD63/CD81) antibodies for estimation of EV size and surface protein copy numbers. The limits of detection (LODs) for immunofluorescence and vesicle size based on calibration using cross-calibrated, hard-dyed beads were ∼10 PE/∼80 nm EV diameter for CytoFLEX and ∼10 PEs/∼67 nm for CellStream. For the SMFC, the LOD for immunofluorescence was 1 PE and ≤ 35 nm for size. The population of EVs detected by each system (di-8-ANEPPS+/PE+ particles) differed widely depending on the LOD of the system; for example, CellStream/CytoFLEX detected only 5.7% and 1.5% of the tetraspanin-labelled EVs detected by SMFC, respectively, and median EV diameter and antibody copy numbers were much larger for CellStream/CytoFLEX than for SMFC as measured and validated using super-resolution/single-molecule TIRF microscopy. To obtain a dataset representing a common EV population analysed by all three platforms, we filtered out SMFC and CellStream measurements for EVs below the CytoFLEX LODs as determined by bead calibration (10 PE/80 nm). The inter-platform agreement using this filtered dataset was significantly better than for the unfiltered dataset, but even better concordance between results was obtained by applying higher cutoffs (21 PE/120 nm) determined by threshold analysis using the SMFC data. The results demonstrate the impact of specifying LODs to define the EV population analysed on inter-instrument reproducibility in EV flow cytometry studies, and the utility of threshold analysis of SMFC data for providing semi-quantitative LOD values for other flow cytometers.

高灵敏度流式细胞仪是为单个细胞外囊泡 (EV) 的多参数表征而开发的,但不同仪器和校准方法的性能各不相同。在这里,我们比较了三种高灵敏度流式细胞仪(两种商用仪器 CytoFLEX/CellStream 和一种定制的单分子流式细胞仪 (SMFC))对来自人类结直肠癌(DiFi)细胞的相同(分裂)EV 样品的表征。DiFi EV用膜染料di-8-ANEPPS和PE结合的抗EGFR或抗tetraspanin(CD9/CD63/CD81)抗体染色,以估计EV大小和表面蛋白拷贝数。免疫荧光和囊泡大小的检测限(LODs)是使用交叉校准的硬染色珠校准的,CytoFLEX 的检测限为 ∼10 PE/∼80 nm EV 直径,CellStream 的检测限为 ∼10 PEs/∼67 nm。对于 SMFC,免疫荧光的 LOD 为 1 PE,尺寸≤ 35 nm。每种系统(di-8-ANEPPS+/PE+颗粒)检测到的EV数量因系统的LOD不同而有很大差异;例如,CellStream/CytoFLEX检测到的四聚乙二醇标记EV分别只有SMFC的5.7%和1.5%,而且CellStream/CytoFLEX的中位EV直径和抗体拷贝数远大于SMFC,这是用超分辨/单分子TIRF显微镜测量和验证的。为了获得代表所有三种平台分析的共同 EV 群体的数据集,我们过滤掉了 SMFC 和 CellStream 测量的低于 CytoFLEX LODs 的 EV,这些 LODs 是通过珠子校准(10 PE/80 nm)确定的。使用该过滤数据集的平台间一致性明显优于未过滤的数据集,但通过使用 SMFC 数据的阈值分析确定更高的临界值(21 PE/120 nm),结果间的一致性甚至更好。这些结果表明了指定 LOD 来定义所分析的 EV 群体对 EV 流式细胞仪研究中仪器间重现性的影响,以及 SMFC 数据的阈值分析为其他流式细胞仪提供半定量 LOD 值的实用性。
{"title":"Comparison of EV characterization by commercial high-sensitivity flow cytometers and a custom single-molecule flow cytometer","authors":"James Kim,&nbsp;Shihan Xu,&nbsp;Seung-Ryoung Jung,&nbsp;Alya Nguyen,&nbsp;Yuanhua Cheng,&nbsp;Mengxia Zhao,&nbsp;Bryant S. Fujimoto,&nbsp;Wyatt Nelson,&nbsp;Perry Schiro,&nbsp;Jeffrey L. Franklin,&nbsp;James N. Higginbotham,&nbsp;Robert J. Coffey,&nbsp;Min Shi,&nbsp;Lucia N. Vojtech,&nbsp;Florian Hladik,&nbsp;Muneesh Tewari,&nbsp;John Tigges,&nbsp;Ionita Ghiran,&nbsp;Tijana Jovanovic-Talisman,&nbsp;Louise C. Laurent,&nbsp;Saumya Das,&nbsp;Olesia Gololobova,&nbsp;Kenneth W. Witwer,&nbsp;Tuoye Xu,&nbsp;Al Charest,&nbsp;Kendall Van Keuren Jensen,&nbsp;Robert L. Raffai,&nbsp;Jennifer C. Jones,&nbsp;Joshua A. Welsh,&nbsp;John P. Nolan,&nbsp;Daniel T. Chiu","doi":"10.1002/jev2.12498","DOIUrl":"10.1002/jev2.12498","url":null,"abstract":"<p>High-sensitivity flow cytometers have been developed for multi-parameter characterization of single extracellular vesicles (EVs), but performance varies among instruments and calibration methods. Here we compare the characterization of identical (split) EV samples derived from human colorectal cancer (DiFi) cells by three high-sensitivity flow cytometers, two commercial instruments, CytoFLEX/CellStream, and a custom single-molecule flow cytometer (SMFC). DiFi EVs were stained with the membrane dye di-8-ANEPPS and with PE-conjugated anti-EGFR or anti-tetraspanin (CD9/CD63/CD81) antibodies for estimation of EV size and surface protein copy numbers. The limits of detection (LODs) for immunofluorescence and vesicle size based on calibration using cross-calibrated, hard-dyed beads were ∼10 PE/∼80 nm EV diameter for CytoFLEX and ∼10 PEs/∼67 nm for CellStream. For the SMFC, the LOD for immunofluorescence was 1 PE and ≤ 35 nm for size. The population of EVs detected by each system (di-8-ANEPPS<sup>+</sup>/PE<sup>+</sup> particles) differed widely depending on the LOD of the system; for example, CellStream/CytoFLEX detected only 5.7% and 1.5% of the tetraspanin-labelled EVs detected by SMFC, respectively, and median EV diameter and antibody copy numbers were much larger for CellStream/CytoFLEX than for SMFC as measured and validated using super-resolution/single-molecule TIRF microscopy. To obtain a dataset representing a common EV population analysed by all three platforms, we filtered out SMFC and CellStream measurements for EVs below the CytoFLEX LODs as determined by bead calibration (10 PE/80 nm). The inter-platform agreement using this filtered dataset was significantly better than for the unfiltered dataset, but even better concordance between results was obtained by applying higher cutoffs (21 PE/120 nm) determined by threshold analysis using the SMFC data. The results demonstrate the impact of specifying LODs to define the EV population analysed on inter-instrument reproducibility in EV flow cytometry studies, and the utility of threshold analysis of SMFC data for providing semi-quantitative LOD values for other flow cytometers.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":"13 8","pages":""},"PeriodicalIF":15.5,"publicationDate":"2024-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.12498","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141975818","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Potential of extracellular vesicles in the pathogenesis, diagnosis and therapy for parasitic diseases 细胞外囊泡在寄生虫病的发病机制、诊断和治疗中的潜力。
IF 15.5 1区 医学 Q1 CELL BIOLOGY Pub Date : 2024-08-08 DOI: 10.1002/jev2.12496
Ana Acacia Sá Pinheiro, Ana Claudia Torrecilhas, Bruno Solano de Freitas Souza, Fernanda Ferreira Cruz, Herbert Leonel de Matos Guedes, Tadeu Diniz Ramos, Miqueias Lopes-Pacheco, Celso Caruso-Neves, Patricia R. M. Rocco

Parasitic diseases have a significant impact on human and animal health, representing a major hazard to the public and causing economic and health damage worldwide. Extracellular vesicles (EVs) have long been recognized as diagnostic and therapeutic tools but are now also known to be implicated in the natural history of parasitic diseases and host immune response modulation. Studies have shown that EVs play a role in parasitic disease development by interacting with parasites and communicating with other types of cells. This review highlights the most recent research on EVs and their role in several aspects of parasite-host interactions in five key parasitic diseases: Chagas disease, malaria, toxoplasmosis, leishmaniasis and helminthiases. We also discuss the potential use of EVs as diagnostic tools or treatment options for these infectious diseases.

寄生虫病对人类和动物的健康有着重大影响,是对公众的一大危害,并在全球范围内造成经济和健康损失。细胞外囊泡(EVs)一直被认为是诊断和治疗工具,但现在也被认为与寄生虫病的自然史和宿主免疫反应调节有关。研究表明,EVs 通过与寄生虫的相互作用以及与其他类型细胞的交流,在寄生虫病的发展过程中发挥作用。本综述重点介绍了有关 EVs 的最新研究及其在五种主要寄生虫病中寄生虫与宿主相互作用的几个方面所起的作用:南美锥虫病、疟疾、弓形虫病、利什曼病和蠕虫病。我们还讨论了将 EVs 用作这些传染病的诊断工具或治疗方案的可能性。
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引用次数: 0
Extracellular vesicles miR-31-5p promotes pancreatic cancer chemoresistance via regulating LATS2-Hippo pathway and promoting SPARC secretion from pancreatic stellate cells 细胞外囊泡miR-31-5p通过调节LATS2-Hippo通路和促进胰腺星状细胞分泌SPARC促进胰腺癌化疗抗性
IF 15.5 1区 医学 Q1 CELL BIOLOGY Pub Date : 2024-08-06 DOI: 10.1002/jev2.12488
Cheng Qin, Bangbo Zhao, Yuanyang Wang, Zeru Li, Tianyu Li, Yutong Zhao, Weibin Wang, Yupei Zhao

Pancreatic cancer remains one of the most lethal malignant diseases. Gemcitabine-based chemotherapy is still one of the first-line systemic treatments, but chemoresistance occurs in the majority of patients. Recently, accumulated evidence has demonstrated the role of the tumour microenvironment in promoting chemoresistance. In the tumour microenvironment, pancreatic stellate cells (PSCs) are among the main cellular components, and extracellular vesicles (EVs) are common mediators of cell‒cell communication. In this study, we showed that SP1-transcribed miR-31-5p not only targeted LATS2 in pancreatic cancer cells but also regulated the Hippo pathway in PSCs through EV transfer. Consequently, PSCs synthesized and secreted protein acidic and rich in cysteins (SPARC), which was preferentially expressed in stromal cells, stimulating Extracellular Signal regulated kinase (ERK) signalling in pancreatic cancer cells. Therefore, pancreatic cancer cell survival and chemoresistance were improved due to both the intrinsic Hippo pathway regulated by miR-31-5p and external SPARC-induced ERK signalling. In mouse models, miR-31-5p overexpression in pancreatic cancer cells promoted the chemoresistance of coinjected xenografts. In a tissue microarray, pancreatic cancer patients with higher miR-31-5p expression had shorter overall survival. Therefore, miR-31-5p regulates the Hippo pathway in multiple cell types within the tumour microenvironment via EVs, ultimately contributing to the chemoresistance of pancreatic cancer cells.

胰腺癌仍然是最致命的恶性疾病之一。以吉西他滨为基础的化疗仍是一线系统治疗方法之一,但大多数患者会出现化疗耐药性。近来,不断积累的证据证明了肿瘤微环境在促进化疗耐药方面的作用。在肿瘤微环境中,胰腺星状细胞(PSCs)是主要的细胞成分之一,而细胞外囊泡(EVs)是细胞与细胞间通讯的常见介质。本研究表明,SP1转录的miR-31-5p不仅能靶向胰腺癌细胞中的LATS2,还能通过EV转移调控PSCs中的Hippo通路。因此,胰腺干细胞合成并分泌了酸性富含半胱氨酸的蛋白质(SPARC),这种蛋白质优先在基质细胞中表达,从而刺激了胰腺癌细胞的细胞外信号调节激酶(ERK)信号传导。因此,miR-31-5p调控的内在Hippo通路和SPARC诱导的外部ERK信号都能改善胰腺癌细胞的生存和化疗耐受性。在小鼠模型中,miR-31-5p 在胰腺癌细胞中的过表达促进了共注射异种移植的化疗抗性。在组织芯片中,miR-31-5p 表达较高的胰腺癌患者总生存期较短。因此,miR-31-5p通过EVs调节肿瘤微环境中多种细胞类型的Hippo通路,最终导致胰腺癌细胞的化疗抗性。
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引用次数: 0
A Predictive Model for Initial Platinum-Based Chemotherapy Efficacy in Patients with Postoperative Epithelial Ovarian Cancer Using Tissue-Derived Small Extracellular Vesicles 利用组织来源的细胞外小泡建立卵巢上皮癌术后患者初始铂类化疗疗效预测模型
IF 15.5 1区 医学 Q1 CELL BIOLOGY Pub Date : 2024-08-06 DOI: 10.1002/jev2.12486
Shizhen Shen, Conghui Wang, Jiaxin Gu, Feifei Song, Xiaodong Wu, Fangfang Qian, Xiaojing Chen, Lingfang Wang, Qiaohua Peng, Ziyu Xing, Lingkai Gu, Fenfen Wang, Xiaodong Cheng

Epithelial ovarian cancer (EOC) is an often-fatal malignancy marked by the development of resistance to platinum-based chemotherapy. Thus, accurate prediction of platinum drug efficacy is crucial for strategically selecting postoperative interventions to mitigate the risks associated with suboptimal therapeutic outcomes and adverse effects. Tissue-derived extracellular vesicles (tsEVs), in contrast to their plasma counterparts, have emerged as a powerful tool for examining distinctive attributes of EOC tissues. In this study, 4D data-independent acquisition (DIA) proteomic sequencing was performed on tsEVs obtained from 58 platinum-sensitive and 30 platinum-resistant patients with EOC. The analysis revealed a notable enrichment of differentially expressed proteins that were predominantly associated with immune-related pathways. Moreover, pivotal immune-related proteins (IRPs) were identified by LASSO regression. These factors, combined with clinical parameters selected through univariate logistic regression, were used for the construction of a model employing multivariate logistic regression. This model integrated three tsEV IRPs, CCR1, IGHV_35 and CD72, with one clinical parameter, the presence of postoperative residual lesions. Thus, this model could predict the efficacy of initial platinum-based chemotherapy in patients with EOC post-surgery, providing prognostic insights even before the initiation of chemotherapy.

上皮性卵巢癌(EOC)是一种经常致命的恶性肿瘤,其特点是对铂类化疗产生耐药性。因此,准确预测铂类药物的疗效对于战略性地选择术后干预措施以降低与次优治疗效果和不良反应相关的风险至关重要。组织来源的细胞外囊泡(tsEVs)与血浆来源的细胞外囊泡不同,是研究 EOC 组织独特属性的有力工具。在这项研究中,对从58名铂敏感和30名铂耐药的EOC患者体内获得的tsEV进行了4D数据独立采集(DIA)蛋白质组测序。分析结果表明,差异表达的蛋白质明显富集,这些蛋白质主要与免疫相关通路有关。此外,还通过 LASSO 回归确定了关键的免疫相关蛋白(IRPs)。这些因素与通过单变量逻辑回归筛选出的临床参数相结合,用于构建多变量逻辑回归模型。该模型将三个 tsEV IRPs(CCR1、IGHV_35 和 CD72)与一个临床参数(是否存在术后残留病灶)整合在一起。因此,该模型可以预测EOC患者术后初始铂类化疗的疗效,甚至在开始化疗之前就能提供预后信息。
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引用次数: 0
Expression of the αVβ3 integrin affects prostate cancer sEV cargo and density and promotes sEV pro-tumorigenic activity in vivo through a GPI-anchored receptor, NgR2 αVβ3整合素的表达会影响前列腺癌sEV的载货量和密度,并通过GPI锚定受体NgR2促进体内sEV的促肿瘤活性。
IF 15.5 1区 医学 Q1 CELL BIOLOGY Pub Date : 2024-08-06 DOI: 10.1002/jev2.12482
Cecilia E. Verrillo, Fabio Quaglia, Christopher D. Shields, Stephen Lin, Andrew V. Kossenkov, Hsin-Yao Tang, David Speicher, Nicole M. Naranjo, Anna Testa, William K. Kelly, Qin Liu, Benjamin Leiby, Luca Musante, Khalid Sossey-Alaoui, Navneet Dogra, Tzu-Yi Chen, Dario C. Altieri, Lucia R. Languino

It is known that small extracellular vesicles (sEVs) are released from cancer cells and contribute to cancer progression via crosstalk with recipient cells. We have previously reported that sEVs expressing the αVβ3 integrin, a protein upregulated in aggressive neuroendocrine prostate cancer (NEPrCa), contribute to neuroendocrine differentiation (NED) in recipient cells. Here, we examine the impact of αVβ3 expression on sEV protein content, density and function. sEVs used in this study were isolated by iodixanol density gradients and characterized by nanoparticle tracking analysis, immunoblotting and single vesicle analysis. Our proteomic profile of sEVs containing αVβ3 shows downregulation of typical effectors involved in apoptosis and necrosis and an upregulation of tumour cell survival factors compared to control sEVs. We also show that the expression of αVβ3 in sEVs causes a distinct reposition of EV markers (Alix, CD81, CD9) to a low-density sEV subpopulation. This low-density reposition is independent of extracellular matrix (ECM) protein interactions with sEVs. This sEV subset contains αVβ3 and an αVβ3 downstream effector, NgR2, a novel marker for NEPrCa. We show that sEVs containing αVβ3 are loaded with higher amounts of NgR2 as compared to sEVs that do not express αVβ3. Mechanistically, we demonstrate that sEVs containing NgR2 do not affect the sEV marker profile, but when injected in vivo intratumorally, they promote tumour growth and induce NED. We show that sEVs expressing NgR2 increase the activation of focal adhesion kinase (FAK), a known promoter of cancer cell proliferation, in recipient cells. We also show that NgR2 mimics the effect of sEVs containing αVβ3 since it displays increased growth of NgR2 transfectants in vivo, as compared to control cells. Overall, our results describe the changes that occur in cargo, density and functions of cancer cell-derived sEVs containing the αVβ3 integrin and its effector, NgR2, without affecting the sEV tetraspanin profiles.

众所周知,小细胞外囊泡(sEVs)从癌细胞中释放出来,并通过与受体细胞的串联促进癌症的发展。我们以前曾报道过,表达αVβ3整合素(一种在侵袭性神经内分泌前列腺癌(NEPrCa)中上调的蛋白质)的sEVs有助于受体细胞的神经内分泌分化(NED)。本研究采用碘克沙醇密度梯度分离 sEV,并通过纳米颗粒追踪分析、免疫印迹和单囊分析对其进行表征。与对照组相比,我们对含有αVβ3的sEVs进行的蛋白质组学分析表明,参与细胞凋亡和坏死的典型效应因子下调,而肿瘤细胞存活因子上调。我们还发现,在 sEVs 中表达 αVβ3 会导致 EV 标记(Alix、CD81、CD9)明显重新定位到低密度 sEV 亚群。这种低密度重新定位与细胞外基质(ECM)蛋白与 sEV 的相互作用无关。这种 sEV 亚群包含 αVβ3 和 αVβ3 下游效应物 NgR2,NgR2 是 NEPrCa 的新型标记物。我们发现,与不表达αVβ3的sEV相比,含有αVβ3的sEV负载了更多的NgR2。从机理上讲,我们证明了含有 NgR2 的 sEVs 不会影响 sEV 的标记特征,但当它们在体内肿瘤内注射时,会促进肿瘤生长并诱导 NED。我们的研究表明,表达 NgR2 的 sEV 会增加受体细胞中焦点粘附激酶(FAK)的活化,FAK 是一种已知的癌细胞增殖促进因子。我们还表明,NgR2 可模拟含有 αVβ3 的 sEVs 的效应,因为与对照细胞相比,NgR2 转染细胞在体内的生长速度加快。总之,我们的研究结果描述了含有αVβ3整合素及其效应物NgR2的癌细胞衍生sEV在货物、密度和功能方面发生的变化,而不影响sEV的四聚体概况。
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引用次数: 0
Mesenchymal stromal/stem cell tissue source and in vitro expansion impact extracellular vesicle protein and miRNA compositions as well as angiogenic and immunomodulatory capacities 间充质基质/干细胞组织来源和体外扩增会影响细胞外囊泡蛋白和 miRNA 成分以及血管生成和免疫调节能力。
IF 15.5 1区 医学 Q1 CELL BIOLOGY Pub Date : 2024-08-02 DOI: 10.1002/jev2.12472
Yuan Liu, Li Sun, Yan Li, Christina Holmes

Recently, therapies utilizing extracellular vesicles (EVs) derived from mesenchymal stromal/stem cells (MSCs) have begun to show promise in clinical trials. However, EV therapeutic potential varies with MSC tissue source and in vitro expansion through passaging. To find the optimal MSC source for clinically translatable EV-derived therapies, this study aims to compare the angiogenic and immunomodulatory potentials and the protein and miRNA cargo compositions of EVs isolated from the two most common clinical sources of adult MSCs, bone marrow and adipose tissue, across different passage numbers. Primary bone marrow-derived MSCs (BMSCs) and adipose-derived MSCs (ASCs) were isolated from adult female Lewis rats and expanded in vitro to the indicated passage numbers (P2, P4, and P8). EVs were isolated from the culture medium of P2, P4, and P8 BMSCs and ASCs and characterized for EV size, number, surface markers, protein content, and morphology. EVs isolated from different tissue sources showed different EV yields per cell, EV sizes, and protein yield per EV. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of proteomics data and miRNA seq data identified key proteins and pathways associated with differences between BMSC-EVs and ASC-EVs, as well as differences due to passage number. In vitro tube formation assays employing human umbilical vein endothelial cells suggested that both tissue source and passage number had significant effects on the angiogenic capacity of EVs. With or without lipopolysaccharide (LPS) stimulation, EVs more significantly impacted expression of M2-macrophage genes (IL-10, Arg1, TGFβ) than M1-macrophage genes (IL-6, NOS2, TNFα). By correlating the proteomics analyses with the miRNA seq analysis and differences observed in our in vitro immunomodulatory, angiogenic, and proliferation assays, this study highlights the trade-offs that may be necessary in selecting the optimal MSC source for development of clinical EV therapies.

最近,利用间充质基质/干细胞(MSCs)产生的细胞外囊泡(EVs)进行治疗的方法开始在临床试验中显示出前景。然而,EV 的治疗潜力因间叶干细胞组织来源和体外扩增传代而异。为了找到可转化为临床EV衍生疗法的最佳间充质干细胞来源,本研究旨在比较从临床上最常见的两种成人间充质干细胞来源(骨髓和脂肪组织)分离出来的EV在不同通过数下的血管生成和免疫调节潜能以及蛋白质和miRNA载体组成。从成年雌性 Lewis 大鼠体内分离出原发性骨髓间充质干细胞(BMSCs)和脂肪间充质干细胞(ASCs),并在体外扩增至指定的培养倍数(P2、P4 和 P8)。从 P2、P4 和 P8 BMSCs 和 ASCs 的培养液中分离出 EVs,并对 EVs 的大小、数量、表面标记、蛋白质含量和形态进行表征。从不同组织来源分离的EV显示出不同的单位细胞EV产量、EV大小和单位EV蛋白产量。通过对蛋白质组学数据和 miRNA seq 数据进行基因本体论(GO)和京都基因组百科全书(KEGG)通路分析,确定了与 BMSC-EVs 和 ASC-EVs 之间的差异有关的关键蛋白质和通路,以及因通过数而产生的差异。利用人体脐静脉内皮细胞进行的体外血管形成试验表明,组织来源和通过数对EVs的血管生成能力都有显著影响。无论是否有脂多糖(LPS)刺激,EVs 对 M2-巨噬细胞基因(IL-10、Arg1、TGFβ)表达的影响比 M1-巨噬细胞基因(IL-6、NOS2、TNFα)更明显。通过将蛋白质组学分析与 miRNA 序列分析以及在体外免疫调节、血管生成和增殖试验中观察到的差异联系起来,本研究强调了在为开发临床 EV 疗法选择最佳间充质干细胞来源时可能需要进行的权衡。
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引用次数: 0
Normothermic liver perfusion derived extracellular vesicles have concentration-dependent immunoregulatory properties 常温肝脏灌注衍生的细胞外囊泡具有浓度依赖性免疫调节特性。
IF 15.5 1区 医学 Q1 CELL BIOLOGY Pub Date : 2024-07-25 DOI: 10.1002/jev2.12485
Heather Jennings, Stacey McMorrow, Peter Chlebeck, Grace Heise, Mia Levitsky, Bret Verhoven, John A. Kink, Kristin Weinstein, Seungpyo Hong, David P. Al-Adra

Extracellular vesicles (EVs) are major contributors to immunological responses following solid organ transplantation. Donor derived EVs are best known for their role in transplant rejection through transferring donor major histocompatibility complex proteins to recipient antigen presenting cells, a phenomenon known as ‛cross-decoration’. In contrast, donor liver-derived EVs are associated with organ tolerance in small animal models. Therefore, the cellular source of EVs and their cargo could influence their downstream immunological effects. To investigate the immunological effects of EVs released by the liver in a physiological and transplant-relevant model, we isolated EVs being produced during normothermic ex vivo liver perfusion (NEVLP), a novel method of liver storage prior to transplantation. We found EVs were produced by the liver during NEVLP, and these EVs contained multiple anti-inflammatory miRNA species. In terms of function, liver-derived EVs were able to cross-decorate allogeneic cells and suppress the immune response in allogeneic mixed lymphocyte reactions in a concentration-dependent fashion. In terms of cytokine response, the addition of 1 × 109 EVs to the mixed lymphocyte reactions significantly decreased the production of the inflammatory cytokines TNF-α, IL-10 and IFN-γ. In conclusion, we determined physiologically produced liver-derived EVs are immunologically regulatory, which has implications for their role and potential modification in solid organ transplantation.

细胞外囊泡(EVs)是实体器官移植后免疫反应的主要因素。供体衍生的EVs最著名的作用是通过将供体主要组织相容性复合体蛋白转移到受体抗原呈递细胞而导致移植排斥反应,这种现象被称为 "交叉修饰"。与此相反,在小动物模型中,供体肝脏来源的EV与器官耐受有关。因此,EVs 及其载体的细胞来源可能会影响其下游免疫效应。为了研究肝脏在生理学和移植相关模型中释放的EVs的免疫效应,我们分离了在常温体外肝脏灌注(NEVLP)过程中产生的EVs,这是一种在移植前储存肝脏的新方法。我们发现肝脏在NEVLP过程中产生了EVs,这些EVs含有多种抗炎miRNA。在功能方面,肝脏衍生的EVs能够交叉修饰异体细胞,并以浓度依赖的方式抑制异体混合淋巴细胞反应中的免疫反应。就细胞因子反应而言,在混合淋巴细胞反应中加入 1 × 109 EVs 能显著减少炎性细胞因子 TNF-α、IL-10 和 IFN-γ 的产生。总之,我们确定了生理产生的肝源性 EVs 具有免疫调节作用,这对它们在实体器官移植中的作用和可能的改变具有影响。
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引用次数: 0
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Journal of Extracellular Vesicles
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