Molecular and Functional Analysis of Single Circulating Melanoma Cells

描述（申请人提供）：本研究提案的长期目标是i）通过将NanoVelcro芯片与激光显微切割（LMD）技术相结合，开发单细胞分离技术，以实现从血液中高效计数和特异性分离活/无增殖性循环黑色素瘤细胞（CMC），和ii）证明对分离的单一CMC进行分子和功能分析的可行性。 与UCLA黑色素瘤团队合作，我们将验证所提出的单CMC分子测定的临床实用性，用于动态监测接受BRAF抑制剂（BRAFi）治疗的黑色素瘤患者的疾病进展、治疗结果和耐药性。 我们在加州大学洛杉矶分校的团队已经证明了一种高效，廉价的循环肿瘤细胞（CTC）检测方法，能够富集，识别和分离从不同实体瘤患者收集的全血样本中的CTC。 首先，我们开创了“NanoVelcro”细胞亲和基底的独特概念，通过该概念，捕获剂（抗体或适体）涂覆的纳米结构化表面被用于在固定装置设置中捕获CTC。 第二，通过将NanoVelcro基底与可以产生垂直流动的覆盖微流体组件整合，由于CTC和基底之间的碰撞增强，已经实现了进一步提高的CTC捕获效率（>85%）。 使用人工和患者CTC样本进行的并行分析验证研究表明，NanoVelcro CTC Assay的灵敏度优于CellSearchTM。 CTC和CMC是从原发肿瘤或转移部位脱离并在外周血中循环的癌细胞。 CTC/CMC计数已在转移性实体瘤患者中建立了临床实用性，其中CTC/CMC数量成为患者对化疗的反应、无病/总生存期的独立且准确的预测因子。可以想象，微创血液诊断技术可以重复表征CTC/CMC，在干预实际上可以发挥作用的关键窗口期间提供对肿瘤生物学的洞察。 目前，FDA批准的CellSearchTM检测试剂盒成本高昂，且在不污染周围白色血细胞的情况下捕获CTCs/CMC的效率低下，因此CTCs/CMC的诊断价值没有得到充分利用。 在本文中，我们将探索新的NanoVelcro测定和LMD技术的组合使用，用于从BRAFi治疗过程中从黑素瘤患者收集的血液样品中分离活的/无增殖性的单个CMC。 然后，我们将对分离的CMC进行分子和功能分析。 我们设想CMC数的变化和所得的基于CMC的分子特征可用于更好地研究和监测BRAFi治疗期间抗性机制的演变，并指导下一代基于激酶受体的黑素瘤治疗的开发。

DESCRIPTION (provided by applicant): The long-term objective of this research proposal is to i) develop a single-cell isolation technology by coupling a NanoVelcro Chip with Laser MicroDissection (LMD) techniques to enable highly efficient enumeration and specific isolation of viable/preservative-free circulating melanoma cells (CMCs) from blood, and ii) to demonstrate the feasibility of performing molecular and functional analyses of the isolated single CMCs. In collaboration with the UCLA melanoma team, we will validate the clinical utility of the proposed single-CMC molecular assays for dynamically monitoring disease progression, treatment outcomes and drug resistance in melanoma patients treated with BRAF inhibiters (BRAFi). Our team at UCLA has demonstrated a highly efficient, inexpensive circulating tumor cell (CTC) assay capable of enriching, identifying and isolating CTCs in whole-blood samples collected from patients with different solid tumors. First, we pioneered a unique concept of "NanoVelcro" cell-affinity substrates, by which capture agent (antibodies or aptamers) -coated nanostructured surfaces were utilized to immobilize CTCs in a stationary device setting. Second, by integrating the NanoVelcro substrate with an overlaid microfluidic component that can generate vertical flows, further improved CTC capture efficiency (>85%) has been achieved as a result of the enhanced collisions between CTCs and the substrate. Side-by-side analytical validation studies using both artificial and patient CTC samples suggested that the sensitivity of NanoVelcro CTC Assay outperformed that of CellSearchTM. CTCs and CMCs are cancer cells that break away from either the primary tumor or metastatic sites and circulate in the peripheral blood. Enumeration of CTCs/CMCs has established clinical utility in patients with metastatic solids tumors, in whom the CTC/CMCs number becomes an independent and accurate predictor for a patient's response to chemotherapy, disease free/overall survival. It is conceivable that a minimally invasive blood-based diagnostic technique could allow repeated characterization of CTCs/CMCs, providing insight into tumor biology during the critical window where intervention could actually make the difference. Currently, FDA- cleared CellSearchTM Assay is costly and inefficient in capturing CTCs/CMCs without contamination of surrounding white blood cells, thus the diagnostic values of CTCs/CMCs are not fully utilized. Herein, we will explore the combined use of new NanoVelcro Assay and LMD technique for isolating viable/preservative-free single CMCs from blood samples collected from melanoma patients over the course of BRAFi treatment. We will then subject the isolated CMCs for molecular and functional analysis. We envision the variation of CMC number and resulting CMC-based molecular signatures can be used to better investigate and monitor evolution of resistance mechanisms during BRAFi treatment, and to guide development of next- generation kinase inhibitor-based melanoma treatments.