Thermoresponsive NanoVelcro CTC Purification System for Prostate Cancer Profiling

 描述（由申请人提供）：本U 01提案的长期目标是开发热响应（TR）-NanoVelcro循环肿瘤细胞（CTC）纯化系统，该系统可进行数字编程，以实现回收前列腺癌（PC）患者血液中活CTC的最佳性能，允许与各种下游功能和分子测定无缝耦合。Dr. Tseng（PI/UCLA）和Dr.波萨达斯（co-PI/Cedars Sinai Medical Center）将组建一个跨学科研究团队，以实施拟议的研究活动。 CTC被认为是肿瘤的液体活检，允许非侵入性的、重复的和非侵入性的。 疾病的系统采样。尽管检测和计数CTC在转移性PC中具有预后意义，但可以想象，对CTC进行分子和功能表征将揭示对驱动致死性PC的致病机制的前所未有的洞察。为了获得CTC衍生的分子特征和功能读数，重要的是开发改进的方法，其不仅可以以高灵敏度检测/计数CTC，而且还可以以最小的白色血细胞污染和对CTC活力的可忽略的破坏来回收CTC。 基于使用聚合物刷接枝的纳米基底收集的活CTC的温度依赖性纯化的初步数据，我们的工作假设是所提出的TR-NanoVelcro CTC纯化系统的性能可以i）通过合理调节表面化学、混合物捕获剂、流速和加热/冷却循环来优化，以及ii）使用人工和前列腺癌细胞来验证。 病人的血液样本 通过TR-NanoVelcro CTC纯化系统纯化的PC CTC将具有足够的活力和纯度，为i）短期体外培养，ii）长期体外维持和iii）作为患者来源的异种移植物的体内致瘤模型铺平了道路。离体扩增的CTC将提供足够的高质量gDNA和mRNA，其可以通过下一代测序（NGS）表征，用于从头鉴定PC中的关键分子事件。使用生物信息学方法，我们将使用从多个PC患者新鲜分离的CTC，通过NGS组装PC特异性基因组/转录组学面板进行交叉验证。我们设想TR-NanoVelcro系统将能够从PC患者中即时纯化活性CTC，为进行各种下游分子和功能测定铺平道路，这些测定可以显著有助于理解PC进展，实施个性化治疗和开发新疗法。

 DESCRIPTION (provided by applicant): The long-term goal of this U01 proposal is to develop Thermoresponsive (TR)-NanoVelcro circulating tumor cell (CTC) purification system that can be digitally programmed to achieve optimal performance for recovering viable CTCs in prostate cancer (PC) patients' blood, allowing seamless coupling with various downstream functional and molecular assays. Dr. Tseng (PI/UCLA) and Dr. Posadas (co-PI/Cedars Sinai Medical Center) will bring together an interdisciplinary research team to implement the proposed research activities. CTCs are regarded as a liquid biopsy of tumors, allowing non-invasive, repetitive, and systemic sampling of the disease. Although detecting and enumerating CTCs is of prognostic significance in metastatic PC, it is conceivable that performing molecular and functional characterization on CTCs will reveal unprecedented insight into the pathogenic mechanisms driving lethal PC. In order to obtain CTC-derived molecular signatures and functional readouts, it is important to develop improved methodologies that can not only detect/enumerate CTCs with high sensitivity, but also recover CTCs with minimum contamination by white blood cells and negligible disruption to CTCs' viability. Our working hypothesis, based on preliminary data gathered of temperature-dependent purification of viable CTCs using polymer brush-grafted nanosubstrates, is that the performance of the proposed TR- NanoVelcro CTC purification system can be i) optimized by rationally modulating surface chemistry, cocktail capture agents, flow rates, and heating/cooling cycles, and ii) validated using both artificial and prostate cancer patient blood samples. PC CTCs purified by TR-NanoVelcro CTC purification system will be of sufficient viability and purity, paving the way for i) short-term in vitro culture, ii) long-term i vitro maintenance, and iii) in vivo tumorigenic models as patient-derived xenografts. The ex vivo expanded CTCs will provide sufficient high-quality gDNA and mRNA that can be characterized by next-generation sequencing (NGS) for de novo identification of key molecular events in PC. Using bioinformatic approaches, we will assemble PC-specific genomic/transcriptomic panels for cross-validation by NGS using CTCs freshly isolated from multiple PC patients. We envision that TR-NanoVelcro system will enable instant purification of viable CTCs from PC patients, paving the way for performing a variety of downstream molecular and functional assays that can significantly contribute to understanding PC progression, implementation of personalized treatment, and development of new therapeutics.