Click Chemistry-Mediated Microfluidic Sorting for HCC CTCs

项目摘要/摘要 此R21提案专门响应RFA-CA-18-002-创新的分子和细胞分析 基础和临床癌症研究技术。长期目标是开发和验证一部小说 点击化学介导的细胞分选法(即点击芯片)用于计数和定量分子 肝细胞癌循环肿瘤细胞(CTCs)的特征我们设想Click 芯片可以与多标记分选策略无缝结合，以从 采集血液样本，以加快检测和鉴定肝细胞癌CTC。 CTC被认为是肿瘤的液体活检，允许对肿瘤进行非侵入性和系统性的采样。 疾病。CTC可以在疾病过程中重复恢复和分析，提供潜在的见解 控制疾病发展的分子机制，同时避免了大量侵入性 活组织检查。肝细胞癌是全球癌症相关死亡的第二大常见原因，迫切需要预测预后。 生物标志物。目前的临床病理和放射学分期系统以及血清生物标记物(如甲胎蛋白) 适于手术治疗的早期患者和晚期患者之间的区别很差 接受化疗。我们在加州大学洛杉矶分校的联合研究团队最近开发了一种多标记捕获 鸡尾酒，允许检测所有疾病阶段的肝细胞癌CTC。开发新的液体活检 诊断学能够同时进行肝细胞癌CTC计数和分子分析，具有很大的前景 显著增强当前分期标准实现疾病纵向监测的能力 进展和治疗反应。 认识到与传统抗体介导的CTC分选相关的局限性，我们的团队 目的开发一种基于点击化学介导的细胞捕获的新型CTC检测方法。与之形成鲜明对比的是 抗体介导的CTC分选方法，一对高活性的点击化学基序(即四嗪，TZ，和 将反式环辛烯(TCO)分别接枝到细胞捕获底物和CTCs上。当TCO嫁接时 CTCs流经集成设备，即点击反应(CTCs上的TCO和衬底上的TZ之间) 导致CTCs的不可逆固定化，大大提高了灵敏度和特异度。此外，通过 将二硫键结合到将TZ连接到底物上的表面连接物中，CTCs被捕获在 底物可在暴露于温和的二硫化物裂解剂(即， 二硫代丁胺)，可实现有效的四氯化碳提纯。点击芯片的创新包括：i)增加了 用点击化学代替抗体介导的捕获来浓缩CTC的敏感性和特异性，以及 Ii)由于二硫键裂解驱动的CTC释放机制，高效提纯CTC。这项建议 将通过具体目标1实施：进行用于肝细胞癌CTC的Click芯片的探索性开发，以及 具体目标2：使用肝细胞癌血液样本进行Click芯片的初步临床验证。

PROJECT SUMMARY/ABSTRACT This R21 proposal specifically responds to RFA-CA-18-002 – Innovative Molecular and Cellular Analysis Technologies for Basic and Clinical Cancer Research. The long-term goal is to develop and validate a novel click chemistry-mediated cell sorting method (i.e., Click Chips) for enumeration and quantitative molecular characterization of circulating tumor cells (CTCs) in hepatocellular carcinoma (HCC). We envision that Click Chips can be seamlessly coupled with a multi-marker sorting strategy to capture and purify HCC CTCs from blood samples to expedite the detection and characterization of HCC CTCs. CTCs are regarded as a liquid biopsy of tumors, allowing non-invasive and systemic sampling of the disease. CTCs can be recovered and analyzed repeatedly over the disease course, providing potential insights into the molecular mechanisms governing disease progression, while averting the need for numerous invasive biopsy. HCC, the 2nd most common cause of cancer-related deaths worldwide, is in dire need of prognostic biomarkers. Current clinicopathologic and radiographic staging systems, and serum biomarkers (e.g., AFP) poorly discriminate between early-stage patients amenable to surgical therapy and advanced-stage patients receiving chemotherapy. Our joint research team at UCLA has recently developed a multi-marker capture cocktail that allows for detection of HCC CTCs across all disease stages. Developing new liquid biopsy diagnostics, capable of conducting both HCC CTC enumeration and molecular analysis, holds great promise to significantly augment the ability of current staging criteria to realize longitudinal monitoring of disease progression and treatment responses. Recognizing the limitations associated with the conventional antibody-mediated CTC sorting, our team aims to develop a new class of CTC assays based on click chemistry-mediated cell capture. In contrast to antibody-mediated CTC sorting methods, a pair of highly reactive click chemistry motifs (i.e., tetrazine, Tz, and trans-cyclooctene, TCO) were grafted onto cell-capture substrates and CTCs, respectively. When TCO-grafted CTCs flow through the integrated device, a click reaction (between TCO on CTCs and Tz on the substrate) leads to irreversible immobilization of CTCs with dramatically improved sensitivity and specificity. Further, by incorporating a disulfide bond into the surface linker that tethers Tz onto the substrate, the CTCs captured on the substrate can be released/recovered upon exposure to a mild disulfide cleavage agent (i.e., dithiobutylamine), allowing for effective CTC purification. The innovation of Click Chips includes i) increased sensitivity and specificity of CTC enrichment by replacing antibody-mediated capture with click chemistry, and ii) highly effective CTC purification due to the disulfide-cleavage driven CTC release mechanism. The proposal will be implemented via Specific Aim 1: to conduct exploratory development of Click Chips for HCC CTCs, and Specific Aim 2: to conduct initial clinical validation of Click Chips using HCC blood samples.