Click Chemistry-Mediated Microfluidic Sorting for HCC CTCs

点击化学介导的 HCC CTC 微流控分选

• 批准号: 9905498
• 负责人: Vatche Agopian
• 金额: $ 16.97万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-02 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9905498
• 关键词: AFP gene; Affect; Affinity; Antibodies; Biological Assay; Biological Markers; Biometry; Biopsy; Blood; Blood Substitutes; Blood specimen; Cancer Etiology; Cell Separation; Cells; Cellular Assay; Cessation of life; Chemistry; Clinical; Clinical Trials; Colorectal Cancer; Coupled; Custom; Cyclooctenes; Cytology; Detection; Development; Devices; Diagnosis; Diagnostic; Diagnostic radiologic examination; Diels Alder reaction; Disease; Disease Progression; Disulfides; Electrons; Exposure to; FABP1 gene; GPC3 gene; Goals; Immobilization; In Vitro; Joints; Liver; Malignant neoplasm of prostate; Mediating; Messenger RNA; Methods; Microfabrication; Microfluidic Microchips; Microfluidics; Modification; Molecular; Molecular Analysis; Monitor; Motivation; NanoVelcro; Nanostructures; Nanotechnology; Neoplasm Circulating Cells; Oncology; Operative Surgical Procedures; Patients; Performance; Population; Postoperative Period; Primary carcinoma of the liver cells; Procedures; Prognostic Marker; Public Health; RBP4 gene; Reaction; Recurrence; Research; Research Personnel; Sampling; Sensitivity and Specificity; Series; Serum; Solid Neoplasm; Sorting - Cell Movement; Staging; Staging System; Surface; TACSTD1 gene; Technology; Testing; Tumor Biology; Validation; anticancer research; base; chemotherapy; design; disulfide bond; hepatocellular carcinoma cell line; improved; innovation; insight; liquid biopsy; malignant breast neoplasm; molecular pathology; nano; nanomaterials; novel; predicting response; prognostic significance; treatment response; tumor

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.

项目总结/摘要 本R21提案专门响应RFA-CA-18-002 -创新分子和细胞分析 基础和临床癌症研究技术。长期目标是开发和验证一种新的 点击化学介导的细胞分选方法（即，点击芯片）进行计数和定量分子 肝细胞癌（HCC）中循环肿瘤细胞（CTC）的表征。我们设想， 芯片可以与多标志物分选策略无缝耦合，以从HCC细胞中捕获和纯化HCC CTC。 血液样本，以加快HCC CTC的检测和表征。 CTC被认为是肿瘤的液体活检，允许对肿瘤进行非侵入性和全身性取样。 疾病CTC可以在疾病过程中反复回收和分析，提供潜在的见解 进入控制疾病进展的分子机制，同时避免了对许多侵入性 活检HCC是世界范围内癌症相关死亡的第二大常见原因，迫切需要预后评估。 生物标志物。目前的临床病理学和放射学分期系统，以及血清生物标志物（例如，法新社） 难以区分适合手术治疗的早期患者和晚期患者 接受化疗我们在加州大学洛杉矶分校的联合研究小组最近开发了一种多标记捕获 混合物，允许在所有疾病阶段检测HCC CTC。开发新的液体活检 诊断，能够进行HCC CTC计数和分子分析，具有很大的希望， 显著增强了当前分期标准实现疾病纵向监测的能力 进展和治疗反应。 认识到与常规抗体介导的CTC分选相关的局限性，我们的团队 旨在开发一类新的基于点击化学介导的细胞捕获的CTC测定。相比 抗体介导的CTC分选方法，一对高反应性点击化学基序（即，四嗪，Tz，和 反式-环辛烯，TCO）分别接枝到细胞捕获基底和CTC上。当TCO接枝时 CTCs流过集成器件，发生点击反应（CTCs上的TCO和衬底上的Tz之间） 导致CTC的不可逆固定，具有显著提高的灵敏度和特异性。此外，由 将二硫键掺入将Tz拴系到基底上的表面接头中， 底物可以在暴露于温和的二硫化物裂解剂时释放/回收（即， 二硫代丁胺），从而允许有效的CTC纯化。点击芯片的创新包括i）增加 - 通过用点击化学代替抗体介导的捕获来富集CTC的灵敏度和特异性，以及 ii）由于二硫键裂解驱动的CTC释放机制，高度有效的CTC纯化。该提案 将通过具体目标1实施：探索性开发用于HCC CTC的点击芯片，以及 具体目标2：使用HCC血液样本对点击芯片进行初步临床验证。