肺部疾病,包括肺癌,是全球死亡率上升的原因。尽管新的成像技术和生物标志物检测的发展,肺癌的检测仍然是一个重大挑战。然而,在正常的潮气呼吸过程中,肺直接与外界环境连通并释放雾化液滴,可以收集,储存并作为呼出气冷凝液(EBC)进行分析。一些研究表明,EBC含有细胞外囊泡(EV),其microRNA(miRNA)货物可能有助于评估不同的肺部状况。但是这些电动汽车的细胞起源仍然未知。在这项研究中,我们使用了纳米粒子跟踪,透射电子显微镜,蛋白质印迹分析和超分辨率纳米成像(ONi),以检测和验证呼出电动汽车(exh-EV)的身份。使用我们可定制的抗体纯化测定法,EV-CATCHER,我们最初确定,使用抗三种四跨膜蛋白(CD9,CD63和CD81)的抗体,可以从EBC中选择性富集exh-EV.使用ONi,我们还发现一些exh-EV含有在细支气管克拉拉细胞(克拉拉细胞分泌蛋白[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。当在五个不同的解剖水平(即,漱口水,漱口水,支气管刷,支气管肺泡灌洗[BAL]和EBC)来自18名受试者,我们确定exh-EV的miRNA谱与BALEV的谱紧密聚类,但不与其他气道样本的谱聚类.当将从匹配的BAL和EBC样品中纯化的EV的miRNA谱与我们的三种四跨膜蛋白EV-CATCHER测定进行比较时,我们捕获了与吸烟相关的显著miRNA表达差异,我们受试者的哮喘和肺部肿瘤状态,在使用我们的抗CCSP/SFTPCEV-CATCHER测定法从相同样品中选择性纯化的EV中也可重复检测到,但这证实了它们的肺组织起源.我们的发现强调,从EBC中富集exh-EV亚群可以对肺组织产生的EV进行非侵入性采样。
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.