It is a well-known phenomenon that cancer cells release key biological information such as DNA, RNA or proteins into body fluids (e.g., blood, urine or saliva). The analysis of these molecules—often encapsulated within nanovesicules called exosomes—is highly attractive, because it could replace current surgical biopsies, which are painful, costly and potentially risky for patients. For example, current strategies in lung cancer diagnosis involve genetic analyses from tumour tissues to detect the presence of underlying DNA mutations, known to alter the phosphorylation status and function of proteins. This information is used to direct therapy, as aberrantly phosphorylated proteins are the main targets of current drugs such as Tyrosine Kinase Inhibitors (TKIs). An alternative and less invasive strategy would be the remote analysis of these phospho-proteins by isolating them from cancer-derived exosomes. This would allow evaluating not only their phosphorylation status at diagnosis, but also the timely restoration of protein phosphorylation levels during therapy with TKIs. Yet, this proteomic approach remains vastly unexplored. Herein, we demonstrate that key lung cancer phosphoproteins, such as EGFR and ERK, are expressed in lung cancer exosomes and we outline a new exosomal proteomic-based approach for their fast and convenient detection. This approach, which could complement current genetic analysis for lung cancer detection, easily detects the phosphorylation status of lung cancer exosomal proteins within minutes after their extraction, bringing hope of circumventing the need for tissue biopsy and costly and cumbersome DNA sequencing techniques. It exploits the fact that phosphorylation induces protein conformational changes, which in turn alter protein's ability to effectively interact with bare gold surfaces. This leads to phosphorylated and non-phosphorylated protein isoforms displaying different gold-adsorption profiles. Using single-use and inexpensive, gold (Au) screen-printed electrodes (SPEs), we demonstrate the successful detection of aberrantly phosphorylated EGFR and ERK protein isoforms derived from lung cancer cell exosomes with a sensitivity down to 15 ng μL⁻¹ in samples with up to 90% excess of their non-phosphorylated (wild-type) forms. We further show the applicability of this strategy for monitoring the action of Tyrosine Kinase Inhibitors over time. We believe that this non-invasive technique will open up new avenues for facilitating cancer diagnosis and time-point monitoring of therapeutic responses.

中文翻译：

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基于外泌体和界面生物传感的策略，用于远程监测肺癌细胞中异常磷酸化的蛋白质†

一个众所周知的现象是癌细胞将诸如DNA，RNA或蛋白质之类的关键生物学信息释放到体液中（例如，血液，尿液或唾液）。这些分子（通常封装在称为囊泡的纳米囊泡中）的分子分析具有很高的吸引力，因为它可以代替当前的外科活检组织，因为活检组织是痛苦的，昂贵的，并且可能给患者带来风险。例如，当前的肺癌诊断策略涉及从肿瘤组织进行遗传分析，以检测潜在的DNA突变的存在，已知该突变会改变蛋白质的磷酸化状态和功能。该信息用于直接治疗，因为异常磷酸化的蛋白质是当前药物（例如酪氨酸激酶抑制剂（TKIs））的主要靶标。另一种侵入性较小的策略是通过从癌症衍生的外来体中分离这些磷酸蛋白来进行远程分析。这样一来，不仅可以在诊断时评估其磷酸化状态，而且还可以在使用TKI进行治疗时及时恢复蛋白质的磷酸化水平。但是，这种蛋白质组学方法仍未开发。本文中，我们证明了关键的肺癌磷蛋白（例如EGFR和ERK）在肺癌外泌体中表达，并且我们概述了一种基于外泌体蛋白质组学的新方法，可快速，方便地进行检测。这种方法可以补充当前用于肺癌检测的遗传分析方法，可以在提取后几分钟内轻松检测出肺癌外泌体蛋白的磷酸化状态，从而为避免组织活检和昂贵而繁琐的DNA测序技术带来了希望。它利用了一个事实，即磷酸化会诱导蛋白质构象变化，进而改变蛋白质与裸金表面有效相互作用的能力。这导致磷酸化和非磷酸化的蛋白质同工型显示出不同的金吸附曲线。使用一次性且价格便宜的金（Au）丝网印刷电极（SPE），我们证明了成功检测出源自肺癌细胞外泌体的异常磷酸化的EGFR和ERK蛋白同工型，其灵敏度低至15 ngμL样品中非磷酸化（野生型）形式最多90％的样品中的^-1。我们进一步显示了该策略在一段时间内监测酪氨酸激酶抑制剂作用的适用性。我们相信，这种非侵入性技术将为促进癌症诊断和治疗反应的时间点监测开辟新途径。