Megan I. Mitchell, Iddo Z. Ben-Dov, Kenny Ye, Christina Liu, Miao Shi, Ali Sadoughi, Chirag Shah, Taha Siddiqui, Aham Okorozo, Martin Gutierrez, Rashmi Unawane, Lisa Biamonte, Kaushal Parihk, Simon Spivack, Olivier Loudig
{"title":"呼出的气体冷凝物中含有细胞外囊泡 (EV),这些囊泡携带肺组织来源的 miRNA,可选择性地对其进行纯化和分析","authors":"Megan I. Mitchell, Iddo Z. Ben-Dov, Kenny Ye, Christina Liu, Miao Shi, Ali Sadoughi, Chirag Shah, Taha Siddiqui, Aham Okorozo, Martin Gutierrez, Rashmi Unawane, Lisa Biamonte, Kaushal Parihk, Simon Spivack, Olivier Loudig","doi":"10.1002/jev2.12440","DOIUrl":null,"url":null,"abstract":"<p>Lung diseases, including lung cancer, are rising causes of global mortality. Despite novel imaging technologies and the development of biomarker assays, the detection of lung cancer remains a significant challenge. However, the lung communicates directly with the external environment and releases aerosolized droplets during normal tidal respiration, which can be collected, stored and analzsed as exhaled breath condensate (EBC). A few studies have suggested that EBC contains extracellular vesicles (EVs) whose microRNA (miRNA) cargos may be useful for evaluating different lung conditions, but the cellular origin of these EVs remains unknown. In this study, we used nanoparticle tracking, transmission electron microscopy, Western blot analyses and super resolution nanoimaging (ONi) to detect and validate the identity of exhaled EVs (exh-EVs). Using our customizable antibody-purification assay, EV-CATCHER, we initially determined that exh-EVs can be selectively enriched from EBC using antibodies against three tetraspanins (CD9, CD63 and CD81). Using ONi we also revealed that some exh-EVs harbour lung-specific proteins expressed in bronchiolar Clara cells (Clara Cell Secretory Protein [CCSP]) and Alveolar Type II cells (Surfactant protein C [SFTPC]). When conducting miRNA next generation sequencing (NGS) of airway samples collected at five different anatomic levels (i.e., mouth rinse, mouth wash, bronchial brush, bronchoalveolar lavage [BAL] and EBC) from 18 subjects, we determined that miRNA profiles of exh-EVs clustered closely to those of BAL EVs but not to those of other airway samples. When comparing the miRNA profiles of EVs purified from matched BAL and EBC samples with our three tetraspanins EV-CATCHER assay, we captured significant miRNA expression differences associated with smoking, asthma and lung tumor status of our subjects, which were also reproducibly detected in EVs selectively purified with our anti-CCSP/SFTPC EV-CATCHER assay from the same samples, but that confirmed their lung tissue origin. Our findings underscore that enriching exh-EV subpopulations from EBC allows non-invasive sampling of EVs produced by lung tissues.</p>","PeriodicalId":15811,"journal":{"name":"Journal of Extracellular Vesicles","volume":null,"pages":null},"PeriodicalIF":15.5000,"publicationDate":"2024-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.12440","citationCount":"0","resultStr":"{\"title\":\"Exhaled breath condensate contains extracellular vesicles (EVs) that carry miRNA cargos of lung tissue origin that can be selectively purified and analyzed\",\"authors\":\"Megan I. Mitchell, Iddo Z. Ben-Dov, Kenny Ye, Christina Liu, Miao Shi, Ali Sadoughi, Chirag Shah, Taha Siddiqui, Aham Okorozo, Martin Gutierrez, Rashmi Unawane, Lisa Biamonte, Kaushal Parihk, Simon Spivack, Olivier Loudig\",\"doi\":\"10.1002/jev2.12440\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>Lung diseases, including lung cancer, are rising causes of global mortality. Despite novel imaging technologies and the development of biomarker assays, the detection of lung cancer remains a significant challenge. However, the lung communicates directly with the external environment and releases aerosolized droplets during normal tidal respiration, which can be collected, stored and analzsed as exhaled breath condensate (EBC). A few studies have suggested that EBC contains extracellular vesicles (EVs) whose microRNA (miRNA) cargos may be useful for evaluating different lung conditions, but the cellular origin of these EVs remains unknown. In this study, we used nanoparticle tracking, transmission electron microscopy, Western blot analyses and super resolution nanoimaging (ONi) to detect and validate the identity of exhaled EVs (exh-EVs). Using our customizable antibody-purification assay, EV-CATCHER, we initially determined that exh-EVs can be selectively enriched from EBC using antibodies against three tetraspanins (CD9, CD63 and CD81). Using ONi we also revealed that some exh-EVs harbour lung-specific proteins expressed in bronchiolar Clara cells (Clara Cell Secretory Protein [CCSP]) and Alveolar Type II cells (Surfactant protein C [SFTPC]). When conducting miRNA next generation sequencing (NGS) of airway samples collected at five different anatomic levels (i.e., mouth rinse, mouth wash, bronchial brush, bronchoalveolar lavage [BAL] and EBC) from 18 subjects, we determined that miRNA profiles of exh-EVs clustered closely to those of BAL EVs but not to those of other airway samples. When comparing the miRNA profiles of EVs purified from matched BAL and EBC samples with our three tetraspanins EV-CATCHER assay, we captured significant miRNA expression differences associated with smoking, asthma and lung tumor status of our subjects, which were also reproducibly detected in EVs selectively purified with our anti-CCSP/SFTPC EV-CATCHER assay from the same samples, but that confirmed their lung tissue origin. Our findings underscore that enriching exh-EV subpopulations from EBC allows non-invasive sampling of EVs produced by lung tissues.</p>\",\"PeriodicalId\":15811,\"journal\":{\"name\":\"Journal of Extracellular Vesicles\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":15.5000,\"publicationDate\":\"2024-04-25\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://onlinelibrary.wiley.com/doi/epdf/10.1002/jev2.12440\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of Extracellular Vesicles\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1002/jev2.12440\",\"RegionNum\":1,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"CELL BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Extracellular Vesicles","FirstCategoryId":"3","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/jev2.12440","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"CELL BIOLOGY","Score":null,"Total":0}
引用次数: 0
摘要
包括肺癌在内的肺部疾病是全球死亡率不断上升的原因。尽管采用了新型成像技术并开发了生物标志物检测方法,但肺癌的检测仍然是一项重大挑战。然而,肺与外界环境直接沟通,在正常潮式呼吸过程中会释放出气溶胶状液滴,这些液滴可作为呼出气体冷凝物(EBC)进行收集、储存和分析。一些研究表明,EBC 中含有细胞外囊泡(EVs),其携带的 microRNA(miRNA)可能有助于评估不同的肺部状况,但这些 EVs 的细胞来源仍然未知。在这项研究中,我们利用纳米粒子追踪、透射电子显微镜、Western 印迹分析和超分辨率纳米成像(ONi)来检测和验证呼出的 EVs(呼出-EVs)的身份。利用我们可定制的抗体纯化检测方法 EV-CATCHER,我们初步确定了利用针对三种四泛蛋白(CD9、CD63 和 CD81)的抗体可以从 EBC 中选择性地富集呼出的 EV。我们还利用 ONi 发现,一些外显子-EV 含有在支气管克拉细胞(克拉细胞分泌蛋白 [CCSP])和肺泡 II 型细胞(表面活性蛋白 C [SFTPC])中表达的肺特异性蛋白。在对从 18 名受试者的五个不同解剖层面(即漱口液、口腔清洗液、支气管刷、支气管肺泡灌洗液 [BAL] 和 EBC)采集的气道样本进行 miRNA 下一代测序(NGS)时,我们发现呼出物-EVs 的 miRNA 图谱与 BAL EVs 的 miRNA 图谱密切相关,但与其他气道样本的 miRNA 图谱不同。用我们的三种四泛素 EV-CATCHER 检测法比较从匹配的 BAL 和 EBC 样本中纯化的 EV 的 miRNA 图谱时,我们捕捉到了与受试者的吸烟、哮喘和肺部肿瘤状态有关的显著 miRNA 表达差异,这些差异在用我们的抗CCSP/SFTPC EV-CATCHER 检测法从相同样本中选择性纯化的 EV 中也可重复检测到,但这证实了它们的肺组织来源。我们的研究结果表明,从 EBC 中富集外源性 EV 亚群可对肺组织产生的 EV 进行非侵入性采样。
Exhaled breath condensate contains extracellular vesicles (EVs) that carry miRNA cargos of lung tissue origin that can be selectively purified and analyzed
Lung diseases, including lung cancer, are rising causes of global mortality. Despite novel imaging technologies and the development of biomarker assays, the detection of lung cancer remains a significant challenge. However, the lung communicates directly with the external environment and releases aerosolized droplets during normal tidal respiration, which can be collected, stored and analzsed as exhaled breath condensate (EBC). A few studies have suggested that EBC contains extracellular vesicles (EVs) whose microRNA (miRNA) cargos may be useful for evaluating different lung conditions, but the cellular origin of these EVs remains unknown. In this study, we used nanoparticle tracking, transmission electron microscopy, Western blot analyses and super resolution nanoimaging (ONi) to detect and validate the identity of exhaled EVs (exh-EVs). Using our customizable antibody-purification assay, EV-CATCHER, we initially determined that exh-EVs can be selectively enriched from EBC using antibodies against three tetraspanins (CD9, CD63 and CD81). Using ONi we also revealed that some exh-EVs harbour lung-specific proteins expressed in bronchiolar Clara cells (Clara Cell Secretory Protein [CCSP]) and Alveolar Type II cells (Surfactant protein C [SFTPC]). When conducting miRNA next generation sequencing (NGS) of airway samples collected at five different anatomic levels (i.e., mouth rinse, mouth wash, bronchial brush, bronchoalveolar lavage [BAL] and EBC) from 18 subjects, we determined that miRNA profiles of exh-EVs clustered closely to those of BAL EVs but not to those of other airway samples. When comparing the miRNA profiles of EVs purified from matched BAL and EBC samples with our three tetraspanins EV-CATCHER assay, we captured significant miRNA expression differences associated with smoking, asthma and lung tumor status of our subjects, which were also reproducibly detected in EVs selectively purified with our anti-CCSP/SFTPC EV-CATCHER assay from the same samples, but that confirmed their lung tissue origin. Our findings underscore that enriching exh-EV subpopulations from EBC allows non-invasive sampling of EVs produced by lung tissues.
期刊介绍:
The Journal of Extracellular Vesicles is an open access research publication that focuses on extracellular vesicles, including microvesicles, exosomes, ectosomes, and apoptotic bodies. It serves as the official journal of the International Society for Extracellular Vesicles and aims to facilitate the exchange of data, ideas, and information pertaining to the chemistry, biology, and applications of extracellular vesicles. The journal covers various aspects such as the cellular and molecular mechanisms of extracellular vesicles biogenesis, technological advancements in their isolation, quantification, and characterization, the role and function of extracellular vesicles in biology, stem cell-derived extracellular vesicles and their biology, as well as the application of extracellular vesicles for pharmacological, immunological, or genetic therapies.
The Journal of Extracellular Vesicles is widely recognized and indexed by numerous services, including Biological Abstracts, BIOSIS Previews, Chemical Abstracts Service (CAS), Current Contents/Life Sciences, Directory of Open Access Journals (DOAJ), Journal Citation Reports/Science Edition, Google Scholar, ProQuest Natural Science Collection, ProQuest SciTech Collection, SciTech Premium Collection, PubMed Central/PubMed, Science Citation Index Expanded, ScienceOpen, and Scopus.