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平淡无奇的表现，包括前列腺癌的前列腺特异性抗原(PSA)的测量。作为微创血液检测的常规PSA水平可能不能准确反映疾病。为了提高我们识别和治疗前列腺癌的能力，需要新的疾病状态、治疗反应和前景指标。前列腺循环肿瘤细胞(CTCs)为了解癌症提供了潜在的强大的新生物标志物。单一CTC分析的推动力来自观察到的实体肿瘤微环境中细胞的异质性。CTC被认为是引发转移的原因，约占实体肿瘤死亡的90%。以前的研究将CTC计数与患者预后联系起来。CTC分析可能为治疗提供洞察力。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，但要完全了解癌症的进展和可变性，需要直接评估蛋白质介导的信号传递的工具。查询CTC以获取蛋白质水平信息的一个主要限制源于血液中发现的CTC数量惊人地少(即每10亿个正常血细胞中有1个CTC)。出于需要，蛋白质分析是在CTC群体上进行的，忽略了细胞之间的固有变异性。然而，我们知道，细胞间的可变性可能包含对理解转移的开始和最后阶段至关重要的信息，以及在确定有希望的药物靶点和阻止转移的候选药物方面。与我们在斯坦福大学的临床合作者一起，我们将在我们最近推出的单细胞微流控Western印迹阵列上取得重大进展并进行扩展，以量化通过MagSweeper从前列腺癌患者身上收集的单个CTC中的蛋白质表达。我们的工作将使单一CTC的蛋白质印迹成为第一例。为此，我们将：(1)引入一种针对低起始CTC数量(10-100个细胞)进行优化的单细胞scWestern阵列，同时引入高灵敏度读数，(2)应用单个CTC Western blotting分析一组假设转移潜能增加的细胞类型中靶蛋白的差异表达，以及(3)将流体流动控制引入 用于细胞表面抗原原位染色的单CTC Western阵列，以允许与细胞内蛋白介导的信号相关。总而言之，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，我们的IMAT成功所获得的新知识将极大地推动临床实践，使人们能够研究对新的癌症疗法敏感/获得性耐药的有前景的CTC生物标记物，并确定阻止癌症转移的潜在治疗靶点。蛋白质水平的表征 对于实现真正的个性化医疗来说，CTC是非常重要的。

 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.