Thermoresponsive NanoVelcro CTC Purification System for Prostate Cancer Profiling

 描述(申请人提供)：这项U01提案的长期目标是开发温度响应型(TR)-纳米级尼龙搭扣循环肿瘤细胞(CTC)纯化系统，该系统可以进行数字编程，以实现回收前列腺癌(PC)患者血液中活的CTC的最佳性能，从而能够与各种下游功能和分子分析无缝耦合。曾博士(PI/加州大学洛杉矶分校)和Posadas博士(PI/Cedars Sinai医学中心联合)将召集一个跨学科研究团队来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、重复性和 对疾病进行系统抽样。虽然检测和计数CTCs在转移性PC中具有预后意义，但可以想象，对CTCs进行分子和功能表征将揭示致死性PC的致病机制。为了获得CTC衍生的分子标记和功能读数，重要的是开发改进的方法，不仅可以高灵敏度地检测/计数CTCs，而且可以最大限度地减少对白细胞的污染和对CTCs活性的干扰来恢复CTCs。我们的工作假设是，基于使用聚合物刷子接枝的纳米基质对活性CTC进行依赖于温度的纯化的初步数据，提出的tr-NanoVeloVelcro CTC纯化系统的性能可以：i)通过合理地调节表面化学、鸡尾酒捕获剂、流速和加热/冷却循环来优化；ii)使用人工和前列腺癌进行验证 病人的血样。经TR-NanoVelcro CTC纯化系统纯化的PC CTCs将具有足够的活性和纯度，为以下方面铺平道路：1)短期的体外培养，2)长期的体外维持，3)体内致瘤模型作为患者来源的异种移植。体外扩增的CTCs将提供足够高质量的gDNA和mRNA，可以通过下一代测序(NGS)来表征PC中的关键分子事件的从头识别。利用生物信息学方法，我们将组装PC特异的基因组/转录模板，以供NGS使用从多个PC患者新鲜分离的CTC进行交叉验证。我们预计，TR-NanoVelcro系统将能够即时纯化PC患者的活性CTCs，为进行各种下游分子和功能分析铺平道路，这些分析可以显著有助于了解PC的进展、实施个性化治疗和开发新的治疗方法。

 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.