Molecular and Functional Analysis of Single Circulating Melanoma Cells

描述(由申请人提供)：本研究建议的长期目标是：i)通过将纳米尼龙搭扣芯片与激光显微解剖(LMD)技术相结合，开发一种单细胞分离技术，以实现从血液中高效计数和特定分离活的/无防腐剂的循环黑色素瘤细胞(CMC)，以及ii)论证对分离的单个CMC进行分子和功能分析的可行性。在与加州大学洛杉矶分校黑色素瘤团队的合作下，我们将验证拟议的单一CMC分子分析在动态监测使用BRAF抑制剂(BRAF)治疗的黑色素瘤患者的疾病进展、治疗结果和耐药性方面的临床实用性。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)分析方法，该方法能够从不同实体肿瘤患者的全血样本中浓缩、鉴定和分离CTC。首先，我们开创了“纳米尼龙搭扣”细胞亲和性底物的独特概念，通过这种底物，捕捉剂(抗体或适配子)覆盖的纳米结构表面被用来在固定设备中固定CTCs。其次，通过将纳米尼龙搭扣衬底与可产生垂直流动的覆盖微流控组件集成在一起，由于CTCs与衬底之间的碰撞增强，CTC捕获效率进一步提高(&gt；85%)。使用人工CTC样本和患者CTC样本的并列分析验证研究表明，NanoVelcro CTC检测的灵敏度优于CellSearchTM。CTCs和CMCs是从原发肿瘤或转移部位分离出来并在外周血液中循环的癌细胞。CTC/CMCs计数已在转移性实体肿瘤患者中建立了临床实用价值，其中CTC/CMCs数量成为独立而准确的预测患者对化疗的反应、无病生存/总生存期的指标。可以想象，一种基于血液的微创诊断技术可以重复表征CTCs/CMCs，在干预实际上可能产生影响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试验在捕获CTCs/CMCs而不污染周围白细胞方面成本较高且效率较低，因此CTCs/CMCs的诊断价值没有得到充分利用。在这里，我们将探索结合使用新的纳米尼龙搭扣技术和LMD技术，从BRAFi治疗过程中收集的黑色素瘤患者的血液样本中分离出活的/不含防腐剂的单个巨噬细胞。然后，我们将对分离的CMC进行分子和功能分析。我们预计CMC数量的变化和由此产生的基于CMC的分子签名可以用来更好地研究和监测BRAFi治疗过程中耐药机制的演变，并指导下一代基于激酶抑制剂的黑色素瘤治疗的发展。

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.