Renal Cell Carcinoma Surveillance by Immuno-Lipoplex Nanoparticle Platform

项目摘要 肾癌是一种常见且致命的恶性肿瘤，有超过73，000例新发病例和14，000例死亡 预计2019年在美国发生。肾细胞癌（RCC）是最常见的类型， 占所有肾癌的90%以上。手术切除是局部肾癌的治疗选择， 然而，由于多种合并症，RCC患者通常被认为不适合手术。立体定向 体部放射治疗（SBRT），其特征在于在几个部分中递送高剂量的电离辐射， 能克服肾细胞癌的放射抵抗性，治疗效率高。因此，SBRT目前 被认为是管理不可手术患者的替代治疗选择。治疗后监测， 建立在这样的信念上，即检测无症状的疾病复发提供了最佳的机会， 有益的干预，是RCC护理的一个组成部分。然而，通过常规方法检测RCC生物标志物， 方法需要大量的样品和繁琐的工作。没有分子生物标志物得到可靠验证 或融入日常临床实践，合理指导肾癌SBRT后的治疗。因此，识别 辐射后潜在的RCC治疗后生物标志物，样品制备最少，灵敏度高 将是无价的最近，“液体活检”依赖于检测循环肿瘤细胞（CTC），循环肿瘤细胞（CTCs）， 转录物、RNA（miRNA/mRNA）和肿瘤相关基因已被研究作为潜在的诊断 和各种癌症的预后生物标志物。细胞外囊泡（EV）如外泌体很难与细胞内的其他物质结合。 检测和检测EV的所有当前方法已被证明是繁琐、昂贵和耗时的。的 这项R21提案的主要目的是开发和评估一种新开发的免疫栓系脂复合物 用于临床前分子诊断的纳米颗粒（ILN）技术系统。ILN被设计为提供一个简单的， 非侵入性，低成本的方法，用于肾细胞癌的监测后SBRT。为了实现这一目标，我们开发了 一种生物芯片，其内部含有含有特定分子信标（MB）的纳米颗粒， 表面上抗体可以捕获具有特异性RCC表面受体的EV，然后MB可以结合到具有特异性RCC表面受体的EV。 靶向EV RNA以产生可通过全内反射荧光记录荧光 显微镜（TIRF）。这种新的生物芯片可以捕获RCC肿瘤分泌的均匀EV亚群 细胞，并在单个步骤中测量EV RNA和膜蛋白靶标。如果成功实施， 这种诊断和监测工具，对于癌症患者的监测可能具有很大的变革性。

Project Summary Kidney cancer is a common and deadly malignancy, with more than 73,000 new cases and 14,000 deaths estimated to occur in 2019 in the United States. Renal cell carcinoma (RCC) is the most prevalent type, accounting for more than 90% of all kidney cancers. Surgical resection is the treatment choice for localized RCC, however, patients with RCC are often deemed unfit for surgery because of multiple comorbidities. Stereotactic body radiotherapy (SBRT), characterized by the delivery of high doses of ionizing radiation in a few fractions, can overcome the radioresistance of RCC and achieve high therapeutic efficiency. Therefore, SBRT is currently considered as an alternative treatment option to manage non-operable patients. Post-treatment surveillance, established on the belief that detecting asymptomatic disease recurrence offers an optimal opportunity for beneficial intervention, is an integral part of RCC care. However, detecting RCC biomarkers by conventional methods requires large sample volumes and tedious work. No molecular biomarkers have been reliably validated or integrated into daily clinical practice to rationally guide kidney cancer therapy after SBRT. Thus, identifying potential RCC post-treatment biomarkers after irradiation with minimum sample preparation and high sensitivity would be invaluable. Recently, ‘liquid biopsy’ relying on detecting circulating tumor cells (CTCs), circulating transcripts, RNAs (miRNAs/mRNAs), and tumor-related genes has been investigated as a potential diagnostic and prognostic biomarker for various cancers. Extracellular vesicles (EVs) such as exosomes are difficult to detect and all current approaches to detect EVs have proven to be tedious, expensive, and time consuming. The primary objective of this R21 proposal is to develop and evaluate a newly developed immune-tethered lipoplex nanoparticle (ILN) technology system for preclinical molecular diagnosis. ILNs are designed to provide a simple, non-invasive, and low-cost method for RCC surveillance after SBRT. To achieve this goal, we have developed a biochip with nanoparticles containing specific molecular beacons (MBs) inside and RCC-specific antibody on the surface. The antibodies can capture EVs with specific RCC surface receptors, and then MBs can bind to the target EV RNAs to produce fluorescence that can be recorded by total internal reflection fluorescence microscope (TIRF). This new biochip allows capturing homogeneous EV subgroups secreted from RCC tumor cells, and measuring both EV RNA and membrane protein targets in a single step. If successfully implemented, this diagnostic and surveillance tool, could be highly transformative for the surveillance of patients with cancer.