Hyo Kyeong Kim, Yujeong Choi, Kyoung Hwa Kim, Yeongju Byun, Tae Hee Kim, Jae Hwan Kim, Shung Hyun An, DaeHo Bae, Myeong Kwan Choi, Minyoung Lee, Gwansuk Kang, Jihwa Chung, Seok-Hyun Kim, Kihwan Kwon
Extracellular vesicles (EVs) have emerged as a potential delivery vehicle for nucleic-acid-based therapeutics, but challenges related to their large-scale production and cargo-loading efficiency have limited their therapeutic potential. To address these issues, we developed a novel “shock wave extracellular vesicles engineering technology” (SWEET) as a non-genetic, scalable manufacturing strategy that uses shock waves (SWs) to encapsulate siRNAs in EVs. Here, we describe the use of the SWEET platform to load large quantities of KRASG12C-targeting siRNA into small bovine-milk-derived EVs (sBMEVs), with high efficiency. The siRNA-loaded sBMEVs effectively silenced oncogenic KRASG12C expression in cancer cells; they inhibited tumour growth when administered intravenously in a non-small cell lung cancer xenograft mouse model. Our study demonstrates the potential for the SWEET platform to serve as a novel method that allows large-scale production of cargo-loaded EVs for use in a wide range of therapeutic applications.
{"title":"Scalable production of siRNA-encapsulated extracellular vesicles for the inhibition of KRAS-mutant cancer using acoustic shock waves","authors":"Hyo Kyeong Kim, Yujeong Choi, Kyoung Hwa Kim, Yeongju Byun, Tae Hee Kim, Jae Hwan Kim, Shung Hyun An, DaeHo Bae, Myeong Kwan Choi, Minyoung Lee, Gwansuk Kang, Jihwa Chung, Seok-Hyun Kim, Kihwan Kwon","doi":"10.1002/jev2.12508","DOIUrl":"https://doi.org/10.1002/jev2.12508","url":null,"abstract":"<p>Extracellular vesicles (EVs) have emerged as a potential delivery vehicle for nucleic-acid-based therapeutics, but challenges related to their large-scale production and cargo-loading efficiency have limited their therapeutic potential. To address these issues, we developed a novel “shock wave extracellular vesicles engineering technology” (SWEET) as a non-genetic, scalable manufacturing strategy that uses shock waves (SWs) to encapsulate siRNAs in EVs. Here, we describe the use of the SWEET platform to load large quantities of KRAS<sup>G12C</sup>-targeting siRNA into small bovine-milk-derived EVs (sBMEVs), with high efficiency. The siRNA-loaded sBMEVs effectively silenced oncogenic KRAS<sup>G12C</sup> expression in cancer cells; they inhibited tumour growth when administered intravenously in a non-small cell lung cancer xenograft mouse model. Our study demonstrates the potential for the SWEET platform to serve as a novel method that allows large-scale production of cargo-loaded EVs for use in a wide range of therapeutic applications.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":null,"pages":null},"PeriodicalIF":15.5,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.12508","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142324670","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}
Jannik Hjortshøj Larsen, Iben Skov Jensen, Per Svenningsen
Extracellular vesicles (EVs) contain cell-derived lipids, proteins and RNAs; however, determining the tissue- and cell-type-specific EV abundances in body fluids remains a significant hurdle for our understanding of EV biology. While tissue- and cell-type-specific EV abundances can be estimated by matching the EV's transcriptome to a tissue's/cell type's expression signature using deconvolutional methods, a comparative assessment of deconvolution methods' performance on EV transcriptome data is currently lacking. We benchmarked 11 deconvolution methods using data from four cell lines and their EVs, in silico mixtures, 118 human plasma and 88 urine EVs. We identified deconvolution methods that estimated cell type-specific abundances of pure and in silico mixed cell line-derived EV samples with high accuracy. Using data from two urine EV cohorts with different EV isolation procedures, four deconvolution methods produced highly similar results. The three methods were also concordant in their tissue- and cell-type-specific plasma EV abundance estimates. We identified driving factors for deconvolution accuracy and highlighted the importance of implementing biological knowledge in creating the tissue/cell type signature. Overall, our analyses demonstrate that the deconvolution algorithms DWLS and CIBERSORTx produce highly similar and accurate estimates of tissue- and cell-type-specific EV abundances in biological fluids.
细胞外囊泡(EV)含有细胞衍生的脂质、蛋白质和 RNA;然而,确定体液中组织和细胞类型特异的 EV 丰度仍然是我们了解 EV 生物学的一个重大障碍。虽然组织和细胞类型特异性的 EV 丰度可以通过使用去卷积方法将 EV 的转录组与组织/细胞类型的表达特征相匹配来估算,但目前还缺乏对去卷积方法在 EV 转录组数据上的性能的比较评估。我们使用来自四种细胞系及其 EV、硅学混合物、118 人血浆和 88 尿液 EV 的数据,对 11 种去卷积方法进行了基准测试。我们确定了一些去卷积方法,这些方法能高精度地估算纯细胞系和硅学混合细胞系衍生 EV 样本的细胞类型特异性丰度。使用来自两个尿液EV队列的数据和不同的EV分离程序,四种解卷积方法得出了高度相似的结果。这三种方法对组织和细胞类型特异性血浆 EV 丰度的估计也是一致的。我们确定了去卷积准确性的驱动因素,并强调了在创建组织/细胞类型特征时应用生物学知识的重要性。总之,我们的分析表明,解卷积算法 DWLS 和 CIBERSORTx 对生物液体中组织和细胞类型特异性 EV 丰度的估计高度相似且准确。
{"title":"Benchmarking transcriptome deconvolution methods for estimating tissue- and cell-type-specific extracellular vesicle abundances","authors":"Jannik Hjortshøj Larsen, Iben Skov Jensen, Per Svenningsen","doi":"10.1002/jev2.12511","DOIUrl":"https://doi.org/10.1002/jev2.12511","url":null,"abstract":"<p>Extracellular vesicles (EVs) contain cell-derived lipids, proteins and RNAs; however, determining the tissue- and cell-type-specific EV abundances in body fluids remains a significant hurdle for our understanding of EV biology. While tissue- and cell-type-specific EV abundances can be estimated by matching the EV's transcriptome to a tissue's/cell type's expression signature using deconvolutional methods, a comparative assessment of deconvolution methods' performance on EV transcriptome data is currently lacking. We benchmarked 11 deconvolution methods using data from four cell lines and their EVs, in silico mixtures, 118 human plasma and 88 urine EVs. We identified deconvolution methods that estimated cell type-specific abundances of pure and in silico mixed cell line-derived EV samples with high accuracy. Using data from two urine EV cohorts with different EV isolation procedures, four deconvolution methods produced highly similar results. The three methods were also concordant in their tissue- and cell-type-specific plasma EV abundance estimates. We identified driving factors for deconvolution accuracy and highlighted the importance of implementing biological knowledge in creating the tissue/cell type signature. Overall, our analyses demonstrate that the deconvolution algorithms DWLS and CIBERSORTx produce highly similar and accurate estimates of tissue- and cell-type-specific EV abundances in biological fluids.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":null,"pages":null},"PeriodicalIF":15.5,"publicationDate":"2024-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.12511","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142320706","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}
In melanoma, carcinoma-associated fibroblasts (CAFs) are important cellular components in the tumour microenvironment due to their potential to promote tumour growth and metastatic spread of malignant cells. Melanoma cells have the ability to affect non-tumour cells in the microenvironment by releasing extracellular vesicles (EVs). The mechanisms responsible for reprogramming normal dermal fibroblasts (NHDFs) into CAFs remain incompletely understood. However, it is likely thought to be mediated by melanoma-specific miRNAs, which are transported by EVs derived from melanoma cells. Therefore, we wondered if one of the most enriched miRNAs in EVs secreted by melanoma cells, miR-92b-3p, is involved in the conversion of normal fibroblasts into CAFs. We observed that melanoma cell-derived EVs indeed delivered miR-92b-3p into NHDFs and that its accumulation correlated with CAF formation, as demonstrated by enhanced expression of CAF marker genes and increased proliferation and migration. Overexpression of miR-92b-3p in NHDFs revealed similar results, while EVs deficient of miR-92b-3p did not induce a CAF phenotype. As a target we identified PTEN, whose repression led to increased expression of CAF markers. We thus provide a novel pathway of intercellular communication by which melanoma cells control the transformation of CAFs by virtue of EV-transported miRNAs.
{"title":"Extracellular vesicles derived from melanoma cells induce carcinoma-associated fibroblasts via miR-92b-3p mediated downregulation of PTEN","authors":"Stefanie Kewitz-Hempel, Nicola Windisch, Gerd Hause, Lutz Müller, Cord Sunderkötter, Dennis Gerloff","doi":"10.1002/jev2.12509","DOIUrl":"10.1002/jev2.12509","url":null,"abstract":"<p>In melanoma, carcinoma-associated fibroblasts (CAFs) are important cellular components in the tumour microenvironment due to their potential to promote tumour growth and metastatic spread of malignant cells. Melanoma cells have the ability to affect non-tumour cells in the microenvironment by releasing extracellular vesicles (EVs). The mechanisms responsible for reprogramming normal dermal fibroblasts (NHDFs) into CAFs remain incompletely understood. However, it is likely thought to be mediated by melanoma-specific miRNAs, which are transported by EVs derived from melanoma cells. Therefore, we wondered if one of the most enriched miRNAs in EVs secreted by melanoma cells, miR-92b-3p, is involved in the conversion of normal fibroblasts into CAFs. We observed that melanoma cell-derived EVs indeed delivered miR-92b-3p into NHDFs and that its accumulation correlated with CAF formation, as demonstrated by enhanced expression of CAF marker genes and increased proliferation and migration. Overexpression of miR-92b-3p in NHDFs revealed similar results, while EVs deficient of miR-92b-3p did not induce a CAF phenotype. As a target we identified PTEN, whose repression led to increased expression of CAF markers. We thus provide a novel pathway of intercellular communication by which melanoma cells control the transformation of CAFs by virtue of EV-transported miRNAs.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":null,"pages":null},"PeriodicalIF":15.5,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.12509","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142307876","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}
Yee Huang, Jiaying Sun, Xuemei Cui, Xuefeng Li, Zizhe Hu, Quanan Ji, Guolian Bao, Yan Liu
With the coming of the post-antibiotic era, there is an increasingly urgent need for safe and efficient antibacterial vaccines. Bacterial outer membrane vesicles (OMVs) have received increased attention recently as a potential subunit vaccine. OMVs are non-replicative and contain the principle immunogenic bacterial antigen, which circumvents the safety concerns of live-attenuated vaccines. Here, we developed a novel nano-vaccine by coating OMVs onto PEGylated nano-Rehmannia glutinosa polysaccharide (pRL) in a structure consisting of concentric circles, resulting in a more stable vaccine with improved immunogenicity. The immunological function of the pRL-OMV formulation was evaluated in vivo and in vitro, and the underlying mechanism was studied though transcriptomic analysis. The pRL-OMV formulation significantly increased dendritic cell (DC) proliferation and cytokine secretion. Efficient phagocytosis of the formulation by DCs was accompanied by DC maturation. Further, the formulation demonstrated superior lymph node targeting, contributing to a potent mixed cellular response and bacterial-specific antibody response against Bordetella bronchiseptica infection. Specifically, transcriptomic analysis revealed that the immune protection function correlated with T-cell receptor signalling and Th1/Th2/Th17 differentiation, among other markers of enhanced immunological activity. These findings have implications for the future application of OMV-coated nano-carriers in antimicrobial immunotherapy.
{"title":"Enhancing protective immunity against bacterial infection via coating nano-Rehmannia glutinosa polysaccharide with outer membrane vesicles","authors":"Yee Huang, Jiaying Sun, Xuemei Cui, Xuefeng Li, Zizhe Hu, Quanan Ji, Guolian Bao, Yan Liu","doi":"10.1002/jev2.12514","DOIUrl":"10.1002/jev2.12514","url":null,"abstract":"<p>With the coming of the post-antibiotic era, there is an increasingly urgent need for safe and efficient antibacterial vaccines. Bacterial outer membrane vesicles (OMVs) have received increased attention recently as a potential subunit vaccine. OMVs are non-replicative and contain the principle immunogenic bacterial antigen, which circumvents the safety concerns of live-attenuated vaccines. Here, we developed a novel nano-vaccine by coating OMVs onto PEGylated nano-<i>Rehmannia glutinosa</i> polysaccharide (pRL) in a structure consisting of concentric circles, resulting in a more stable vaccine with improved immunogenicity. The immunological function of the pRL-OMV formulation was evaluated in vivo and in vitro, and the underlying mechanism was studied though transcriptomic analysis. The pRL-OMV formulation significantly increased dendritic cell (DC) proliferation and cytokine secretion. Efficient phagocytosis of the formulation by DCs was accompanied by DC maturation. Further, the formulation demonstrated superior lymph node targeting, contributing to a potent mixed cellular response and bacterial-specific antibody response against <i>Bordetella bronchiseptica</i> infection. Specifically, transcriptomic analysis revealed that the immune protection function correlated with T-cell receptor signalling and Th1/Th2/Th17 differentiation, among other markers of enhanced immunological activity. These findings have implications for the future application of OMV-coated nano-carriers in antimicrobial immunotherapy.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":null,"pages":null},"PeriodicalIF":15.5,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.12514","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142307875","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}
Hannah J. O'Toole, Neona M. Lowe, Vishalakshi Arun, Anna V. Kolesov, Tina L. Palmieri, Nam K. Tran, Randy P. Carney
Sepsis following burn trauma is a global complication with high mortality, with ∼60% of burn patient deaths resulting from infectious complications. Diagnosing sepsis is complicated by confounding clinical manifestations of the burn injury, and current biomarkers lack the sensitivity and specificity required for prompt treatment. There is a strong rationale to assess circulating extracellular vesicles (EVs) from patient liquid biopsy as sepsis biomarkers due to their release by pathogens from bacterial biofilms and roles in the subsequent immune response. This study applies Raman spectroscopy to patient plasma-derived EVs for rapid, sensitive, and specific detection of sepsis in burn patients, achieving 97.5% sensitivity and 90.0% specificity. Furthermore, spectral differences between septic and non-septic burn patient EVs could be traced to specific glycoconjugates of bacterial strains associated with sepsis morbidity. This work illustrates the potential application of EVs as biomarkers in clinical burn trauma care and establishes Raman analysis as a fast, label-free method to specifically identify features of bacterial EVs relevant to infection amongst the host background.
{"title":"Plasma-derived extracellular vesicles (EVs) as biomarkers of sepsis in burn patients via label-free Raman spectroscopy","authors":"Hannah J. O'Toole, Neona M. Lowe, Vishalakshi Arun, Anna V. Kolesov, Tina L. Palmieri, Nam K. Tran, Randy P. Carney","doi":"10.1002/jev2.12506","DOIUrl":"https://doi.org/10.1002/jev2.12506","url":null,"abstract":"<p>Sepsis following burn trauma is a global complication with high mortality, with ∼60% of burn patient deaths resulting from infectious complications. Diagnosing sepsis is complicated by confounding clinical manifestations of the burn injury, and current biomarkers lack the sensitivity and specificity required for prompt treatment. There is a strong rationale to assess circulating extracellular vesicles (EVs) from patient liquid biopsy as sepsis biomarkers due to their release by pathogens from bacterial biofilms and roles in the subsequent immune response. This study applies Raman spectroscopy to patient plasma-derived EVs for rapid, sensitive, and specific detection of sepsis in burn patients, achieving 97.5% sensitivity and 90.0% specificity. Furthermore, spectral differences between septic and non-septic burn patient EVs could be traced to specific glycoconjugates of bacterial strains associated with sepsis morbidity. This work illustrates the potential application of EVs as biomarkers in clinical burn trauma care and establishes Raman analysis as a fast, label-free method to specifically identify features of bacterial EVs relevant to infection amongst the host background.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":null,"pages":null},"PeriodicalIF":15.5,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.12506","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142273133","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}
Jun Ho Lee, Hyungtaek Jeon, Jan Lötvall, Byong Seung Cho
Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have shown anti-inflammatory potential in multiple inflammatory diseases. In the March 2022 issue of the Journal of Extracellular Vesicles, it was shown that EVs from human MSCs can suppress severe acute respiratory distress syndrome, coronavirus 2 (SARS-CoV-2) replication and can mitigate the production and release of infectious virions. We therefore hypothesized that MSC-EVs have an anti-viral effect in SARS-CoV-2 infection in vivo. We extended this question to ask whether also other respiratory viral infections could be treated by MSC-EVs. Adipose stem cell-derived EVs (ASC-EVs) were isolated using tangential flow filtration from conditioned media obtained from a multi-flask cell culture system. The effects of the ASC-EVs were tested in Vero E6 cells in vitro. ASC-EVs were also given i.v. to SARS-CoV-2 infected Syrian Hamsters, and H1N1 influenza virus infected mice. The ASC-EVs attenuated SARS-CoV-2 virus replication in Vero E6 cells and reduced body weight and signs of lung injury in infected Syrian hamsters. Furthermore, ASC-EVs increased the survival rate of influenza A-infected mice and attenuated signs of lung injury. In summary, this study suggests that ASC-EVs can have beneficial therapeutic effects in models of virus-infection-associated acute lung injury and may potentially be developed to treat lung injury in humans.
间充质干细胞(MSC)衍生的细胞外囊泡(EVs)已在多种炎症疾病中显示出抗炎潜力。2022年3月出版的《细胞外囊泡杂志》(Journal of Extracellular Vesicles)显示,来自人类间充质干细胞的细胞外囊泡能抑制严重急性呼吸窘迫综合征冠状病毒2(SARS-CoV-2)的复制,并能减轻传染性病毒的产生和释放。因此,我们假设间充质干细胞-EVs 在体内感染 SARS-CoV-2 时具有抗病毒作用。我们将这一问题延伸至间叶干细胞-EV是否也能治疗其他呼吸道病毒感染。我们使用切向流过滤法从多层细胞培养系统获得的条件培养基中分离出了脂肪干细胞衍生的EVs(ASC-EVs)。在体外 Vero E6 细胞中测试了 ASC-EVs 的作用。此外,还为感染了 SARS-CoV-2 的叙利亚仓鼠和感染了 H1N1 流感病毒的小鼠静脉注射了 ASC-EV。ASC-EVs 可减轻 SARS-CoV-2 病毒在 Vero E6 细胞中的复制,减轻受感染叙利亚仓鼠的体重和肺损伤症状。此外,ASC-EVs 还能提高甲型流感感染小鼠的存活率,减轻肺损伤症状。总之,这项研究表明,ASC-EVs 可在病毒感染相关急性肺损伤模型中产生有益的治疗效果,并有可能开发用于治疗人类肺损伤。
{"title":"Therapeutic potential of mesenchymal stem cell-derived extracellular vesicles in SARS-CoV-2 and H1N1 influenza-induced acute lung injury","authors":"Jun Ho Lee, Hyungtaek Jeon, Jan Lötvall, Byong Seung Cho","doi":"10.1002/jev2.12495","DOIUrl":"https://doi.org/10.1002/jev2.12495","url":null,"abstract":"<p>Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have shown anti-inflammatory potential in multiple inflammatory diseases. In the March 2022 issue of the <i>Journal of Extracellular Vesicles</i>, it was shown that EVs from human MSCs can suppress severe acute respiratory distress syndrome, coronavirus 2 (SARS-CoV-2) replication and can mitigate the production and release of infectious virions. We therefore hypothesized that MSC-EVs have an anti-viral effect in SARS-CoV-2 infection in vivo. We extended this question to ask whether also other respiratory viral infections could be treated by MSC-EVs. Adipose stem cell-derived EVs (ASC-EVs) were isolated using tangential flow filtration from conditioned media obtained from a multi-flask cell culture system. The effects of the ASC-EVs were tested in Vero E6 cells in vitro. ASC-EVs were also given i.v. to SARS-CoV-2 infected Syrian Hamsters, and H1N1 influenza virus infected mice. The ASC-EVs attenuated SARS-CoV-2 virus replication in Vero E6 cells and reduced body weight and signs of lung injury in infected Syrian hamsters. Furthermore, ASC-EVs increased the survival rate of influenza A-infected mice and attenuated signs of lung injury. In summary, this study suggests that ASC-EVs can have beneficial therapeutic effects in models of virus-infection-associated acute lung injury and may potentially be developed to treat lung injury in humans.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":null,"pages":null},"PeriodicalIF":15.5,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.12495","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142165630","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}
Paolo Bergese, Marcella Chiari, Alessandro Gori, Benedetta Bussolati, Pietro Parisse, the EVIta Board
<p></p><p>It is with profound sadness and sorrow that we announce the unexpected passing of our cherished friend and colleague, Marina Cretich. Marina, a renowned scientist in the field of bioanalytical chemistry, passed away on June 29th 2024, leaving behind a heritage of invaluable contributions to science. Her career was marked by a relentless pursuit of knowledge and an unwavering dedication to advancing scientific understanding.</p><p>Marina graduated in Biological Sciences with a specialization in Molecular Biology from the University of Milano in 1998 with Prof. Piergiorgio Righetti. She then joined the National Research Council of Italy (CNR), where she served as a Researcher at the Institute of Chemistry for Molecular Recognition in Milan, becoming a key figure in the Analytical Microsystem laboratory guided by Dr. Marcella Chiari. Thanks to the interdisciplinarity of her approach, she made significant strides in the progress of advanced methods and materials for bio-molecular recognition, together with high-sensitive and selective biosensors, providing new tools for detecting biomolecules with unprecedented accuracy and efficiency. Her strong focus on integrating bioanalytical techniques, microfluidics and detection tools, enabled more rapid and precise analyses, enhancing the capabilities of lab-on-a-chip devices, towards point-of-care diagnostics.</p><p>These studies paved the road to her approach to extracellular vesicles, addressing the challenges posed by their separation and analysis. Specifically, Marina was working on affinity-capturing protocols from complex samples for EV isolation and on microarray platforms for their molecular characterization (Daaboul et al., <span>2016</span>). Her interest in the field was raised by participating in the EU project INDEX, coordinated by Dr Marcella Chiari, and became true love for this fascinating and challenging area. This led Marina to establish her own research team and to find the Extracellular Vesicle Lab at SCITEC-CNR. Soon after, she was the Coordinator of the EU project MARVEL, a multi-partner project at the intersection of chemistry and technology, biology and translational medicine, which was centred around the use of membrane-sensing peptides (MSP) as enabling tools for the multiscale EV isolation (Gori et al., <span>2020</span>, Gori et al, <span>2024</span>). She was a true pioneer in this field and she consolidated a leading expertise in the area of ultrasensitive EV analysis, aiming to fill existing gaps in the clinical translation of EVs in diagnostics. Her contribution is witnessed by remarkable scientific contributions (Frigerio et al., <span>2022</span>; Musicò et al., <span>2024</span>) and, despite very recent, the concepts and technologies that she developed set the basis for an ever-increasing number of preclinical and clinical collaborations encompassing EV analysis in the fields of neurodegeneration, cancer, and heart diseases.</p><p>In recent years, she also dedic
{"title":"Extracellular vesicle analytical science loses a touch of creativity and kindness","authors":"Paolo Bergese, Marcella Chiari, Alessandro Gori, Benedetta Bussolati, Pietro Parisse, the EVIta Board","doi":"10.1002/jev2.12504","DOIUrl":"https://doi.org/10.1002/jev2.12504","url":null,"abstract":"<p></p><p>It is with profound sadness and sorrow that we announce the unexpected passing of our cherished friend and colleague, Marina Cretich. Marina, a renowned scientist in the field of bioanalytical chemistry, passed away on June 29th 2024, leaving behind a heritage of invaluable contributions to science. Her career was marked by a relentless pursuit of knowledge and an unwavering dedication to advancing scientific understanding.</p><p>Marina graduated in Biological Sciences with a specialization in Molecular Biology from the University of Milano in 1998 with Prof. Piergiorgio Righetti. She then joined the National Research Council of Italy (CNR), where she served as a Researcher at the Institute of Chemistry for Molecular Recognition in Milan, becoming a key figure in the Analytical Microsystem laboratory guided by Dr. Marcella Chiari. Thanks to the interdisciplinarity of her approach, she made significant strides in the progress of advanced methods and materials for bio-molecular recognition, together with high-sensitive and selective biosensors, providing new tools for detecting biomolecules with unprecedented accuracy and efficiency. Her strong focus on integrating bioanalytical techniques, microfluidics and detection tools, enabled more rapid and precise analyses, enhancing the capabilities of lab-on-a-chip devices, towards point-of-care diagnostics.</p><p>These studies paved the road to her approach to extracellular vesicles, addressing the challenges posed by their separation and analysis. Specifically, Marina was working on affinity-capturing protocols from complex samples for EV isolation and on microarray platforms for their molecular characterization (Daaboul et al., <span>2016</span>). Her interest in the field was raised by participating in the EU project INDEX, coordinated by Dr Marcella Chiari, and became true love for this fascinating and challenging area. This led Marina to establish her own research team and to find the Extracellular Vesicle Lab at SCITEC-CNR. Soon after, she was the Coordinator of the EU project MARVEL, a multi-partner project at the intersection of chemistry and technology, biology and translational medicine, which was centred around the use of membrane-sensing peptides (MSP) as enabling tools for the multiscale EV isolation (Gori et al., <span>2020</span>, Gori et al, <span>2024</span>). She was a true pioneer in this field and she consolidated a leading expertise in the area of ultrasensitive EV analysis, aiming to fill existing gaps in the clinical translation of EVs in diagnostics. Her contribution is witnessed by remarkable scientific contributions (Frigerio et al., <span>2022</span>; Musicò et al., <span>2024</span>) and, despite very recent, the concepts and technologies that she developed set the basis for an ever-increasing number of preclinical and clinical collaborations encompassing EV analysis in the fields of neurodegeneration, cancer, and heart diseases.</p><p>In recent years, she also dedic","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":null,"pages":null},"PeriodicalIF":15.5,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.12504","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142165745","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}
Stephan P. Ebenberger, Fatih Cakar, Yi-Chi Chen, Katharina Pressler, Leo Eberl, Stefan Schild
Vibrio cholerae, a facultative human pathogen and causative agent of the severe diarrheal disease cholera, transits between the human intestinal tract and aquatic reservoirs. Like other bacterial species, V. cholerae continuously releases bacterial extracellular vesicles (BEVs) from its surface, which have been recently characterised for their role during in vivo colonisation. However, between epidemic outbreaks, V. cholerae persists in the biofilm mode for extended periods in aquatic reservoirs, which enhances environmental fitness and host transition. In this study, we investigated the effect of V. cholerae BEVs on biofilm formation, a critical feature for ex vivo survival. In contrast to BEVs from planktonic cultures, our results show that physiological concentrations of BEVs from dynamic biofilm cultures facilitate V. cholerae biofilm formation, which could be linked to a proteinaceous factor. Comparative proteomic analyses of planktonic- and biofilm-derived BEVs identified a previously uncharacterised outer membrane protein as an abundant component of dynamic biofilm-derived BEVs, which was found to be responsible for the BEV-dependent enhancement of biofilm production. Consequently, this protein was named outer membrane-associated biofilm facilitating protein A (ObfA). Comprehensive molecular studies unravelled ObfA as a negative modulator of HapR activity. HapR is a key transcriptional regulator of the V. cholerae quorum sensing (QS) cascade acting as a potent repressor of biofilm formation and virulence. Consistently, obfA mutants not only exhibited reduced biofilm production but also reduced colonisation fitness. Surprisingly, our results demonstrate that ObfA does not affect HapR through the canonical QS system but via the Csr-cascade altering the expression of the small regulatory RNAs CsrC and CsrD. In summary, this study elucidates a novel intraspecies BEV-based communication in V. cholerae that influences biofilm formation and colonisation fitness via a new regulatory pathway involving HapR, Csr-cascade and the BEV-associated protein ObfA.
{"title":"The activity of the quorum sensing regulator HapR is modulated by the bacterial extracellular vesicle (BEV)-associated protein ObfA of Vibrio cholerae","authors":"Stephan P. Ebenberger, Fatih Cakar, Yi-Chi Chen, Katharina Pressler, Leo Eberl, Stefan Schild","doi":"10.1002/jev2.12507","DOIUrl":"https://doi.org/10.1002/jev2.12507","url":null,"abstract":"<p><i>Vibrio cholerae</i>, a facultative human pathogen and causative agent of the severe diarrheal disease cholera, transits between the human intestinal tract and aquatic reservoirs. Like other bacterial species, <i>V. cholerae</i> continuously releases bacterial extracellular vesicles (BEVs) from its surface, which have been recently characterised for their role during in vivo colonisation. However, between epidemic outbreaks, <i>V. cholerae</i> persists in the biofilm mode for extended periods in aquatic reservoirs, which enhances environmental fitness and host transition. In this study, we investigated the effect of <i>V. cholerae</i> BEVs on biofilm formation, a critical feature for ex vivo survival. In contrast to BEVs from planktonic cultures, our results show that physiological concentrations of BEVs from dynamic biofilm cultures facilitate <i>V. cholerae</i> biofilm formation, which could be linked to a proteinaceous factor. Comparative proteomic analyses of planktonic- and biofilm-derived BEVs identified a previously uncharacterised outer membrane protein as an abundant component of dynamic biofilm-derived BEVs, which was found to be responsible for the BEV-dependent enhancement of biofilm production. Consequently, this protein was named <b>o</b>uter membrane-associated <b>b</b>iofilm <b>f</b>acilitating protein <b>A</b> (ObfA). Comprehensive molecular studies unravelled ObfA as a negative modulator of HapR activity. HapR is a key transcriptional regulator of the <i>V. cholerae</i> quorum sensing (QS) cascade acting as a potent repressor of biofilm formation and virulence. Consistently, <i>obfA</i> mutants not only exhibited reduced biofilm production but also reduced colonisation fitness. Surprisingly, our results demonstrate that ObfA does not affect HapR through the canonical QS system but via the Csr-cascade altering the expression of the small regulatory RNAs CsrC and CsrD. In summary, this study elucidates a novel intraspecies BEV-based communication in <i>V. cholerae</i> that influences biofilm formation and colonisation fitness via a new regulatory pathway involving HapR, Csr-cascade and the BEV-associated protein ObfA.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":null,"pages":null},"PeriodicalIF":15.5,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.12507","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142165748","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}
Reactive oxygen species (ROS)-induced oxidative DNA damages have been considered the main cause of mutations in genes, which are highly related to carcinogenesis and tumour progression. Extracellular vesicles play an important role in cancer metastasis. However, the precise role of DNA oxidative damage in extracellular vesicles (EVs)-mediated cancer cell migration and invasion remains unclear. Here, we reveal that ROS-mediated DNA oxidative damage signalling promotes tumour metastasis through increasing EVs release. Mechanistically, 8-oxoguanine DNA glycosylase (OGG1) recognises and binds to its substrate 8-oxo-7,8-dihydroguanine (8-oxoG), recruiting NF-κB to the synaptotagmin 7 (SYT7) promoter and thereby triggering SYT7 transcription. The upregulation of SYT7 expression leads to increased release of E-cadherin-loaded EVs, which depletes intracellular E-cadherin, thereby inducing epithelial-mesenchymal transition (EMT). Notably, Th5487, the inhibitor of DNA binding activity of OGG1, blocks the recognition and transmission of oxidative signals, alleviates SYT7 expression and suppresses EVs release, thereby preventing tumour progression in vitro and in vivo. Collectively, our study illuminates the significance of 8-oxoG/OGG1/SYT7 axis-driven EVs release in oxidative stress-induced tumour metastasis. These findings provide a deeper understanding of the molecular basis of cancer progression and offer potential avenues for therapeutic intervention.
活性氧(ROS)诱导的 DNA 氧化损伤被认为是基因突变的主要原因,而基因突变与癌变和肿瘤进展高度相关。细胞外囊泡在癌症转移中发挥着重要作用。然而,DNA氧化损伤在细胞外囊泡介导的癌细胞迁移和侵袭中的确切作用仍不清楚。在这里,我们揭示了 ROS 介导的 DNA 氧化损伤信号通过增加 EVs 释放促进肿瘤转移。从机理上讲,8-氧鸟嘌呤 DNA 糖基化酶(OGG1)识别并与其底物 8-氧代-7,8-二氢鸟嘌呤(8-oxoG)结合,将 NF-κB 募集到突触柄蛋白 7(SYT7)启动子上,从而触发 SYT7 的转录。SYT7 表达的上调导致 E-cadherin 负载的 EVs 释放增加,从而消耗细胞内的 E-cadherin,从而诱导上皮-间质转化(EMT)。值得注意的是,OGG1 DNA结合活性抑制剂Th5487能阻断氧化信号的识别和传递,减轻SYT7的表达并抑制EVs的释放,从而阻止肿瘤在体外和体内的进展。总之,我们的研究阐明了 8-oxoG/OGG1/SYT7 轴驱动的 EVs 释放在氧化应激诱导的肿瘤转移中的重要作用。这些发现加深了人们对癌症进展分子基础的理解,并为治疗干预提供了潜在途径。
{"title":"The 8-oxoguanine DNA glycosylase-synaptotagmin 7 pathway increases extracellular vesicle release and promotes tumour metastasis during oxidative stress","authors":"Ying Ma, Jiarong Guo, Haipeng Rao, Jingyu Xin, Xinyi Song, Rui Liu, Shan Shao, Jiajia Hou, Liyu Kong, Zhigang Hu, Lingfeng He, Feiyan Pan, Zhigang Guo","doi":"10.1002/jev2.12505","DOIUrl":"10.1002/jev2.12505","url":null,"abstract":"<p>Reactive oxygen species (ROS)-induced oxidative DNA damages have been considered the main cause of mutations in genes, which are highly related to carcinogenesis and tumour progression. Extracellular vesicles play an important role in cancer metastasis. However, the precise role of DNA oxidative damage in extracellular vesicles (EVs)-mediated cancer cell migration and invasion remains unclear. Here, we reveal that ROS-mediated DNA oxidative damage signalling promotes tumour metastasis through increasing EVs release. Mechanistically, 8-oxoguanine DNA glycosylase (OGG1) recognises and binds to its substrate 8-oxo-7,8-dihydroguanine (8-oxoG), recruiting NF-κB to the synaptotagmin 7 (SYT7) promoter and thereby triggering SYT7 transcription. The upregulation of SYT7 expression leads to increased release of E-cadherin-loaded EVs, which depletes intracellular E-cadherin, thereby inducing epithelial-mesenchymal transition (EMT). Notably, Th5487, the inhibitor of DNA binding activity of OGG1, blocks the recognition and transmission of oxidative signals, alleviates SYT7 expression and suppresses EVs release, thereby preventing tumour progression in vitro and in vivo. Collectively, our study illuminates the significance of 8-oxoG/OGG1/SYT7 axis-driven EVs release in oxidative stress-induced tumour metastasis. These findings provide a deeper understanding of the molecular basis of cancer progression and offer potential avenues for therapeutic intervention.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":null,"pages":null},"PeriodicalIF":15.5,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11375530/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142132933","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}
The translation of discoveries on extracellular vesicle (EV) based cancer biomarkers to personalised precision oncology requires the development of robust, sensitive and specific assays that are amenable to adoption in the clinical laboratory. Whilst a variety of elegant approaches for EV liquid biopsy have been developed, most of them remain as research prototypes due to the requirement of a high level of microfabrication and/or sophisticated instruments. Hence, this study is set to develop a simple DNA aptamer-enabled and fluorescence polarisation-based homogenous assay that eliminates the need to separate unbound detection ligands from the bound species for EV detection. High specificity is achieved by immobilising EVs with one set of antibodies and subsequently detecting them with a DNA aptamer targeting a distinct EV biomarker. This two-pronged strategy ensures the removal of most, if not all, non-EV substances in the input biofluids, including soluble proteins, protein aggregates or non-vesicular particles, prior to quantifying biomarker-positive EVs. A limit of detection of 5.0 × 106 EVs/mL was achieved with a linear quantification range of 5.0 × 108 to 2.0 × 1010 EVs/mL. Facilitated by a multiple parametric analysis strategy, this aptamer-guided fluorescence polarisation assay was capable of distinguishing EVs from three different types of solid cancer cells based on quantitative differences in the levels of the same sets of biomarkers on EVs. Given the simplicity of the method and its ease of implementation in automated clinical biochemistry analysers, this assay could be exploited for future EV-based continuous and real-time monitoring of the emergence of new macro- or micro-metastasis, cancer progression as well as the response to treatment throughout different stages of cancer management in the clinic.
要将基于细胞外囊泡(EV)的癌症生物标记物的发现转化为个性化的精准肿瘤学,就必须开发出稳健、灵敏和特异的检测方法,以便在临床实验室中应用。虽然目前已开发出多种优雅的 EV 液体活检方法,但由于对微细加工和/或精密仪器的要求较高,大多数方法仍停留在研究原型阶段。因此,本研究将开发一种简单的 DNA 配体和基于荧光偏振的均质检测方法,无需将未结合的检测配体与结合的物种分开,即可进行 EV 检测。先用一组抗体固定 EV,然后再用针对不同 EV 生物标记物的 DNA 类似物检测它们,从而实现高特异性。这种双管齐下的策略可确保在量化生物标记物阳性 EV 之前,去除输入生物流体中的大部分(如果不是全部)非 EV 物质,包括可溶性蛋白质、蛋白质聚集体或非囊泡颗粒。检测限为 5.0 × 106 EVs/mL,线性定量范围为 5.0 × 108 至 2.0 × 1010 EVs/mL。在多参数分析策略的帮助下,这种由适配体引导的荧光极化测定能够根据EVs上同一组生物标记物水平的定量差异,区分来自三种不同类型实体癌细胞的EVs。鉴于该方法简单且易于在自动临床生化分析仪中实施,该测定可用于未来基于EV的连续和实时监测,以监测新的大转移或微转移的出现、癌症的进展以及在临床癌症治疗的不同阶段对治疗的反应。
{"title":"An aptamer-guided fluorescence polarisation platform for extracellular vesicle liquid biopsy","authors":"Cuong Viet Pham, Rocky Chowdhury, Shweta Patel, Satendra Kumar Jaysawal, Yingchu Hou, Huo Xu, Lee Jia, Yu-mei Zhang, Xiaowei Wang, Wei Duan, Dongxi Xiang","doi":"10.1002/jev2.12502","DOIUrl":"10.1002/jev2.12502","url":null,"abstract":"<p>The translation of discoveries on extracellular vesicle (EV) based cancer biomarkers to personalised precision oncology requires the development of robust, sensitive and specific assays that are amenable to adoption in the clinical laboratory. Whilst a variety of elegant approaches for EV liquid biopsy have been developed, most of them remain as research prototypes due to the requirement of a high level of microfabrication and/or sophisticated instruments. Hence, this study is set to develop a simple DNA aptamer-enabled and fluorescence polarisation-based homogenous assay that eliminates the need to separate unbound detection ligands from the bound species for EV detection. High specificity is achieved by immobilising EVs with one set of antibodies and subsequently detecting them with a DNA aptamer targeting a distinct EV biomarker. This two-pronged strategy ensures the removal of most, if not all, non-EV substances in the input biofluids, including soluble proteins, protein aggregates or non-vesicular particles, prior to quantifying biomarker-positive EVs. A limit of detection of 5.0 × 10<sup>6</sup> EVs/mL was achieved with a linear quantification range of 5.0 × 10<sup>8</sup> to 2.0 × 10<sup>10</sup> EVs/mL. Facilitated by a multiple parametric analysis strategy, this aptamer-guided fluorescence polarisation assay was capable of distinguishing EVs from three different types of solid cancer cells based on quantitative differences in the levels of the same sets of biomarkers on EVs. Given the simplicity of the method and its ease of implementation in automated clinical biochemistry analysers, this assay could be exploited for future EV-based continuous and real-time monitoring of the emergence of new macro- or micro-metastasis, cancer progression as well as the response to treatment throughout different stages of cancer management in the clinic.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":null,"pages":null},"PeriodicalIF":15.5,"publicationDate":"2024-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.12502","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142107993","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}