Microfluidic Western Blotting for Targeted Proteomic Analysis of Single Circulating Tumor Cells

 描述（由申请人提供）：最近的研究强调了癌症诊断的乏善可陈的性能，包括前列腺癌中前列腺特异性抗原（PSA）的测量。PSA水平作为一种微创血液检查常规测量可能不准确地反映疾病。为了提高我们识别和治疗前列腺癌的能力，需要疾病状态，治疗反应和前景的新指标。前列腺循环肿瘤细胞（CTC）为了解癌症提供了潜在的强大的新生物标志物。单CTC分析的动力源于在实体瘤微环境中观察到的细胞异质性。CTC被认为引发转移，占实体瘤死亡的约90%。先前的研究将CTC计数与患者结局联系起来。CTC分析可以提供对治疗的见解。虽然单细胞的分子分析在过去十年中取得了巨大的进步，揭示了肿瘤细胞亚群的遗传多样性，但对癌症进展和变异性的全面了解需要直接评估蛋白质介导的信号传导的工具。查询CTC以获得蛋白质水平信息的主要限制源于血液中发现的CTC数量少得惊人（即，每十亿正常血细胞1 CTC）。出于必要，对CTC群体进行蛋白质测定，忽略细胞之间的固有变异性。然而，我们知道细胞间的变异性可能对理解转移的起始和最终阶段以及确定有希望的药物靶点和阻止转移的候选者至关重要。与我们在斯坦福大学的临床合作者一起，我们将显著推进和扩展我们最近推出的单细胞微流控蛋白质印迹阵列，以量化通过MagSweeper从前列腺癌患者收集的单个CTC中的蛋白质表达。我们的工作将使单一CTCs的同类蛋白质印迹成为可能。为此，我们将：（1）引入针对低起始数目的CTC（10-100个细胞）优化的单细胞scWestern阵列，同时引入高灵敏度读数，（2）应用单CTC Western印迹来分析具有增加程度的假设转移潜能的一组细胞类型中靶蛋白的差异表达，和（3）将流体流动控制引入到靶蛋白的差异表达中。 用于原位细胞表面抗原染色的单CTC Western阵列，以允许与细胞内蛋白质介导的信号传导相关。总之，我们的工作将通过允许纳入CTC水平的蛋白质数据来推进癌症研究。展望未来，由于我们的IMAT成功而获得的新知识将通过研究对新型癌症疗法的敏感性/获得性耐药性的有前景的CTC生物标志物以及确定阻止癌症转移的潜在治疗靶点来显著推进临床实践。蛋白质水平表征 CTCs对于实现真正的个性化医疗非常重要。

 DESCRIPTION (provided by applicant): Recent studies underscore the lackluster performance of cancer diagnostics, including measurement of prostate specific antigen (PSA) in prostate cancer. PSA levels measured routinely as a minimally invasive blood test may inaccurately reflect disease. To advance our capacity to identify & treat prostate cancer, new indicators of disease state, therapeutic response and outlook are needed. Prostate circulating tumor cells (CTCs) offer potentially powerful new biomarkers for understanding cancer. The impetus for single-CTC analysis stems from the observed cellular heterogeneity in solid tumor microenvironments. CTCs are believed to initiate metastasis, accounting for approximately 90% of deaths from solid tumors. Previous studies link CTC enumeration to patient outcome. CTC analysis may provide insight into therapies. While molecular analyses of single-cells has advanced tremendously over the last decade, revealing genetic diversity in tumor cell subpopulations, a complete understanding of cancer progression and variability requires tools to assess protein-mediated signaling directly. A major limitation on querying CTCs for protein- level information stems from the staggeringly low number of CTCs found in blood (i.e., 1 CTC per billion normal blood cells). Out of necessity, protein assays are performed on CTC populations, ignoring the inherent variability between cells. Yet, we know that cell-to-cell variability may hold information crucial to understanding the initiation and final stages of metastasis, as well as in identifying promising drug targets and candidates for stemming metastasis. In conjunction with our clinical collaborators at Stanford, we will significantly advance and expand on our recently introduced single-cell microfluidic Western blot array to quantify protein expression in single CTCs collected via MagSweeper from prostate cancer patients. Our work will enable first-in-kind Western blotting of single CTCs. To do this, we will: (1) introduce a single-cell scWestern array optimized for low starting numbers of CTCs (10-100 cells), in tandem with introducing high sensitivity readouts, (2) apply single CTC Western blotting to analyses of differential expression of targeted proteins in a suite of cell types with increasing degree of hypothesized metastatic potential, and (3) introduce fluid flow control to the single-CTC Western array for in situ cell surface antigen staining to allow correlation with intracellular protein-mediated signaling. Taken together, our work will advance cancer research by allowing for inclusion of CTC-level protein data. Looking forward, the new knowledge gained as a result of our IMAT success would notably advance clinical practice by enabling study of promising CTC biomarkers of sensitivity/acquired resistance to novel cancer therapies and in identifying potential therapeutic targets to halt cancer metastasis. Protein-level characterization of CTCs is important to realizing truly personalized medicine.