Exosome-Protein-microRNA-OneStop (Exo-PROS) biosensor: a new liquid biopsy for cancer screening and early detection

项目总结 目前的癌症筛查测试，如肺癌的低剂量计算机断层扫描(LDCT)和 结肠镜检查在结直肠癌诊断中存在敏感性低、假阳性率高、肿瘤范围小等问题。 信息，不舒服或侵入性的程序和高昂的成本。检测循环的液体活组织检查 生物标志物，允许顺序监测癌症的发展，补充医学成像和风险因素 数据提供了对癌症的更全面的描述，因此它显示了作为体外实验的巨大前景 癌症诊断学。外体蛋白和外体microRNAs是很有希望的癌症生物标记物 液体活组织检查，在诊断许多癌症方面显示出非常高的敏感性和特异性。不幸的是 目前的检测技术，如ELISA、LC-MS、qRT-PCR、微阵列和下一代测序， 繁琐、昂贵和耗时，这限制了它们作为筛查测试的临床应用。此外， 许多方法无法区分肿瘤来源的外切体和非肿瘤来源的外切体，因此受到了 检测灵敏度和特异度较差。为了应对这些挑战，我们开发了一种表面 基于等离子体共振(SPR)的分子信标辅助液体活检装置，名为Exosome Protein MicroRNA OneStop(Exo-Pros)生物传感器。这种生物传感器可以捕获肿瘤衍生的外切体 癌症过度表达的蛋白质。它还可以提供表面蛋白质和内部蛋白质的一站式定量 在4小时内在单个设备上捕获的外切体的囊泡状microRNA。有效地分离了 肿瘤来源的外切体-来自干扰性外切体的外切体(如正常细胞来源的外切体)和 结合胞外体蛋白和microRNA生物标志物可显著提高诊断准确率。 我们已经成功地使用Exo-Pros生物传感器测量了外体EGFR和EGFR+外体miR- 21在细胞培养上清液和肺癌患者血清标本中。该生物传感器具有较高的灵敏度和较高的灵敏度。 在区分肿瘤和正常对照方面的特异性优于ELISA和qRT-PCR。在这项研究中，我们建议 以(1)推动Exo-pros生物传感器的发展，提高其传感性能，实现 多重生物标志物检测；(2)展示了其在肺癌筛查和早期检测中的临床应用价值， 并探讨胞外蛋白和microRNA联合检测对肿瘤的诊断价值。 尚未接受调查。我们将评估Exo-pros生物传感器的检测灵敏度和特异性。 非小细胞肺癌患者与健康对照及良性孤立性吸烟者的鉴别 肺结节是肺癌的高危人群。我们将展示Exo-One的卓越传感性能。 PROS生物传感器的检测优于酶联免疫吸附试验和定量RT-PCR检测。我们希望将Exo-Pros生物传感器开发成 用于癌症筛查和早期检测的高精度、简单、快速和成本效益高的液体活组织检查。

PROJECT SUMMARY Current cancer screening tests, such as low dose computed tomography (LDCT) for lung cancer and colonoscopy for colorectal cancer, are challenged by low sensitivity, high false positive rate, limited tumor information, uncomfortable or invasive procedures and high cost. Liquid biopsy that detects circulating biomarkers, allows sequential monitoring of cancer development, complements medical imaging and risk factor data to provide a more comprehensive portrait of cancer, and thus it has shown great promise as the in vitro diagnostics for cancer. Exosomal proteins and exosomal microRNAs are promising cancer biomarkers for liquid biopsy, and have shown very high sensitivity and specificity in diagnosing many cancers. Unfortunately current detection technologies, such as ELISA, LC-MS, qRT-PCR, microarray and next generation sequencing, are tedious, expensive and time consuming, which limits their clinical utilities as screening tests. Moreover, many methods cannot distinguish tumor-derived exosomes from their non-tumor counterparts, and thus suffer from poor detection sensitivity and specificity. To address these challenges, we have developed a surface plasmon resonance (SPR)-based, molecular beacon-assisted liquid biopsy device, named Exosome Protein microRNA OneStop (Exo-PROS) biosensor. This biosensor can capture tumor-derived exosomes using cancer-overexpressed proteins. It can also provide one-stop quantification of surface proteins and intra- vesicular microRNAs of captured exosomes on a single device within 4 hours. The effective separation of tumor-derived-exosomes from interfering exosomes (such as normal-cell-derived exosomes) and the combination of exosomal protein and microRNA biomarkers may significantly increase the diagnostic accuracy. We have successfully used the Exo-PROS biosensor to measure exosomal EGFR and EGFR+ exosomal miR- 21 in cell culture medium and lung cancer patient serum samples. The biosensor showed higher sensitivity and specificity than ELISA and qRT-PCR in distinguishing cancer from normal controls. In this study, we propose to (1) advance the development of the Exo-PROS biosensor to improve its sensing performance and to realize multiplexed biomarker detection; (2) demonstrate its clinical utility in lung cancer screening and early detection, and explore the diagnostic value of exosomal protein and microRNA combined biomarkers in cancer, which has not yet been investigated. We will evaluate the detection sensitivity and specificity of Exo-PROS biosensor in distinguishing nonsmall cell lung cancer patients from healthy controls and smokers with benign solitary pulmonary nodule at high risk for lung cancer. We will demonstrate the superior sensing performance of Exo- PROS biosensor over ELISA and qRT-PCR assays. We expect to develop the Exo-PROS biosensor into a highly accurate, simple, fast, and cost-effective liquid biopsy test for cancer screening and early detection.