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

用于单个循环肿瘤细胞的靶向蛋白质组分析的微流控蛋白质印迹

• 批准号: 9085245
• 负责人: Amy Elizabeth Herr
• 金额: $ 22.27万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-09 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9085245
• 关键词: Accounting; Address; Advanced Malignant Neoplasm; Biochemical; Biological Assay; Biological Markers; Blood; Blood Cells; Blood Tests; Cancer Diagnostics; Cancer Patient; Cardiovascular system; Cell Line; Cells; Cessation of life; Clinical; Complex; Cytolysis; DU145; Data; Development; Disease; Doctor of Medicine; Dose; Drug Targeting; Epithelial Cells; Exhibits; Flow Cytometry; Fostering; Generations; Genetic Variation; Genomics; Goals; Gold; Health; Heterogeneity; Human; Image; In Situ; Intracellular Signaling Proteins; Knowledge; LNCaP; Lead; Letters; Link; Liquid substance; Malignant Neoplasms; Malignant neoplasm of prostate; Measurement; Measures; Mediating; Messenger RNA; Methodology; Microfluidics; Modification; Molecular; Neoplasm Circulating Cells; Neoplasm Metastasis; Noise; PC3 cell line; PIM1 gene; Patient-Focused Outcomes; Patients; Pattern; Performance; Play; Population; Primary Neoplasm; Prostate; Prostate specific antigen measurement; Prostate-Specific Antigen; Protein Analysis; Proteins; Proteomics; Reproducibility; Research; Resistance; Resolution; Role; Serum; Signal Transduction; Solid; Solid Neoplasm; Specificity; Staging; Staining method; Stains; Surface; Surface Antigens; System; TACSTD1 gene; TimeLine; Treatment Protocols; Validation; Western Blotting; Work; analytical tool; anticancer research; base; cancer therapy; cell type; clinical application; clinical practice; clinically relevant; combinatorial; design; differential expression; drug candidate; epithelial to mesenchymal transition; fluid flow; hepsin; innovation; insight; minimally invasive; neoplastic cell; new technology; novel; personalized medicine; protein expression; single cell proteins; stem; success; technology development; therapeutic target; tool; treatment response; tumor microenvironment; tumor progression

 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.

 描述（由适用提供）：最近的研究强调了癌症诊断的表现不足，包括在前列腺癌中对前列腺特异性抗原（PSA）的测量。定期测量的PSA水平作为微创血液检查可能不准确地反映出疾病。为了提高我们识别和治疗前列腺癌的能力，需要新的疾病状态指标，治疗反应和前景。前列腺循环肿瘤细胞（CTC）为理解癌症提供了潜在的强大新生物标志物。从实体瘤微环境中观察到的细胞异质性的单CCTC分析植物的动力。据信CTC启动转移，约占实体瘤死亡的90％。先前的研究将CTC枚举与患者结局联系起来。 CTC分析可以提供有关疗法的见解。虽然在过去的十年中，单细胞的分子分析已大大提高，揭示了肿瘤细胞亚群中的遗传多样性，但对癌症进展和可变性的完全了解需要直接评估蛋白质介导的信号传导的工具。从血液中发现的CTC数量惊人的CTC中查询CTC的主要局限性（即每十亿个正常血细胞）。在必要的情况下，对CTC种群进行了蛋白质测定，忽略了细胞之间的继承变异性。但是，我们知道细胞间的可变性可能对了解转移的主动性和最终阶段以及确定有希望的药物靶标和候选者的促进转移的阶段至关重要。与我们在斯坦福大学的临床合作者一起，我们将在最近引入的单细胞微流体蛋白质印迹阵列上大大提高和扩展，以量化从前列腺癌患者通过Magsweeper收集的单个CTC中的蛋白质表达。我们的工作将实现单个CTC的第一印迹。为此，我们将：（1）引入一个针对较低的CTC（10-100个单元）优化的单细胞scwestern阵列，并与引入高灵敏度读数一起引入高灵敏度读数，（2）将单个CTC Western印迹在靶向蛋白质的差异化蛋白质的分析中，以增加流动性的流动型和均具有高素质量的细胞类型的靶向蛋白质，并（3. 用于原位细胞表面抗原染色的单-CTC西部阵列，以允许与细胞内蛋白质介导的信号传导相关。综上所述，我们的工作将通过允许包含CTC级蛋白数据来提高癌症研究。展望未来，由于我们的IMAT成功而获得的新知识将通过研究有希望的CTC生物标志物的敏感性/对新型癌症疗法的耐药性以及鉴定潜在的治疗靶点以阻止癌症转移的潜在耐药性，这将显着提高临床实践。蛋白质水平的特征 CTC的重要性对于实现真正的个性化医学很重要。