Molecular and Functional Analysis of Single Circulating Melanoma Cells

描述（由申请人提供）：本研究计划的长期目标是i)通过将NanoVelcro芯片与激光显微解剖（LMD）技术相结合，开发单细胞分离技术，以实现血液中活的/无防腐剂的循环黑色素瘤细胞（cmc）的高效枚举和特异性分离，ii)证明对分离的单个cmc进行分子和功能分析的可行性。通过与加州大学洛杉矶分校黑色素瘤研究小组的合作，我们将验证拟议的单cmc分子检测的临床应用，以动态监测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。我们加州大学洛杉矶分校的团队已经展示了一种高效、廉价的循环肿瘤细胞（CTC）检测方法，能够富集、识别和分离来自不同实体肿瘤患者的全血样本中的循环肿瘤细胞。首先，我们开创了“NanoVelcro”细胞亲和底物的独特概念，利用捕获剂（抗体或适配体）涂覆的纳米结构表面将ctc固定在固定设备设置中。其次，通过将NanoVelcro衬底与可产生垂直流动的覆盖微流体组件集成，由于CTC与衬底之间的碰撞增强，进一步提高了CTC捕获效率（>85%）。使用人工CTC和患者CTC样品进行的对比分析验证研究表明，NanoVelcro CTC Assay的灵敏度优于CellSearchTM。ctc和cmc是脱离原发肿瘤或转移部位并在外周血中循环的癌细胞。CTC/CMCs计数在转移性实体瘤患者中具有临床应用价值，CTC/CMCs数量成为患者对化疗反应、无病生存/总生存的独立准确预测指标。可以想象，一种基于血液的微创诊断技术可以重复表征ctc / cmc，在关键窗口期间提供对肿瘤生物学的深入了解，而干预实际上可以发挥作用。目前，FDA批准的CellSearchTM检测方法在不污染周围白细胞的情况下捕获ctc / cmc成本高且效率低，因此ctc / cmc的诊断价值没有得到充分利用。在此，我们将探索结合使用新的NanoVelcro Assay和LMD技术，从黑色素瘤患者在BRAFi治疗过程中采集的血液样本中分离出有活力/无防腐剂的单个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的变化数量和产生CMC-based分子特征可以用来更好的调查和监测在BRAFi治疗耐药机制的进化,来指导发展下一代激酶inhibitor-based黑色素瘤的治疗方法。

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.