Accelerating Cancer Research with Single Cell Arrays

利用单细胞阵列加速癌症研究

• 批准号: 7434648
• 负责人: Heinz-Ulrich Guenter Weier
• 金额: $ 23.67万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7434648
• 关键词: Address; Affect; Benign; Biological Assay; Breast; Buffers; Cancer Patient; Cancer cell line; Cell Cycle Stage; Cell Nucleus; Cells; Chromatin; Chromosome Band; Chromosome Banding; Chromosome Painting; Chromosome abnormality; Classification; Clinical; Clinical Research; Color; Cytogenetic Analysis; Cytogenetics; DNA; DNA Sequence Rearrangement; Detection; Development; Diagnosis; Disease; Feasibility Studies; Fine-needle biopsy; Fluorescent in Situ Hybridization; G-Banding; Gene Amplification; Genomics; Glass; Harvest; Heterogeneity; Human; Humidity; In Situ; Individual; Interphase; Interphase Cell; Karyotype; Lateral; Lead; Lesion; Malignant Neoplasms; Malignant neoplasm of thyroid; Metaphase; Methods; Microscopic; Neoplasm Metastasis; Neoplasms; Normal Cell; Nucleic Acids; Numbers; Patients; Pattern; Phenotype; Premalignant; Proliferating; Protocols documentation; Public Health; Range; Reaction; Research; Residual Tumors; Resolution; Sampling; Slide; Spectral Karyotyping; Staging; Stretching; Supporting Cell; Syndrome; Techniques; Technology; Temperature; Testing; Thyroid carcinoma; Tissue Sample; Tissues; anticancer research; base; cell fixation; cost; design; disease phenotype; innovation; neoplastic; neoplastic cell; response; size; technology development; tool; tumor; tumor progression

DESCRIPTION (provided by applicant): This proposal addresses the sensitive detection of chromosomal changes such as small translocations, rearrangements or genomic imbalances in apparently normal individuals, benign neoplasia, premalignant lesions, and cancer. Current techniques for full karyotype analysis of individual cells require metaphase cells, and cells in interphase or non-viable cells cannot be analyzed. Many cells that can be obtained from human tumors are not in metaphase. The objective of the proposed research is the development of technologies to support the cytogenetic analysis of small amounts of fresh, fixed or archival tissues regardless of the cells' proliferative stage. A highly sensitive, fluorescence in situ hybridization (FISH)-based technology platform termed Single Cell Arrays (SCAs) will allow the detection of small rearrangements in interphase and metaphase cells by combining the high-resolution DNA in situ analysis with sensitivity in the kb range. This will be achieved by immobilizing cell nuclei on glass slides and controlled stretching of chromatin in specially designed micro-chambers followed by cytogenetic analysis using FISH. The Specific Aims of this R21 feasibility study are 1. Demonstrate the feasibility that interphase cell nuclei can be immobilized in a defined pattern and reproducibly extended for subsequent cytogenetic analysis. We will demonstrate the feasibility of preparing SCAs comprised of individual cell nuclei arranged in a defined pattern inside microscopic reaction chambers and elongated/stretched by a constant force. Importantly, the extent of chromatin stretching will be controlled by cell fixation and adjusting environmental parameters such as buffer, chamber temperature, and humidity, and the force applied to pull the chromatin. 2. Develop an optimized assay for the sensitive, high-resolution cytogenetic analysis of SCAs. We will develop a protocol for a FISH-based multi-locus cytogenetic analysis of SCAs. The assay is expected to provide near kilobase sensitivity for the detection of single copy nucleic acids with a resolution in the order of 10-20 kb, while minimizing the overall loss of DNA. The assay will be tested by analyzing SCAs prepared from different breast or thyroid cancer cell lines. SCAs will become powerful tools in basic and applied/clinical research, where chromosomal changes often affect a cell's phenotype and the fate of its progeny. In clinical practice, for example, such a sensitive assay may support cell classifications, thereby benefiting patients with de novo translocations or premalignant lesions as well as cancer patients. Furthermore, SCAs will allow the analysis of very small samples, regardless of their integrity or cell cycle stage. This will open new avenues for the analysis of small samples like those obtained by fine needle biopsies as well as the analysis of circulating or exfoliated tumor cells. Public health relevance statement: At present, no technology exists to prepare small samples of non-proliferating cells and screen them for karyotypic abnormalities. Highly sensitive, FISH-based assays termed Single Cell Arrays (SCAs) will provide a platform technology with which one can develop a multitude of tests tailored to specific diseases and cell or tissue samples. Due to their versatility, SCAs may become powerful tools in basic and clinical research, thereby benefiting patients with de novo translocations or premalignant lesions as well as cancer patients.

描述（由申请人提供）：该提案涉及染色体变化的敏感检测，例如显然正常个体，良性肿瘤，预调子病变和癌症的小易位，重排或基因组失衡。目前的单个细胞全核型分析的技术需要中期细胞，并且不能分析相间或不可生存的细胞中的细胞。许多可以从人类肿瘤中获得的细胞不在中期。拟议的研究的目的是发展技术，以支持对少量新鲜，固定或档案组织的细胞遗传学分析，而不论细胞的增殖阶段如何。称为单细胞阵列（SCAS）的高度敏感的荧光原位杂交（FISH）的技术平台将通过将高分辨率DNA原位分析与KB范围的敏感性结合，从而允许检测相间和中期细胞中的小重排。这将通过将细胞核固定在载玻片上，并在特殊设计的微室中受控的染色质伸展，然后使用FISH进行细胞遗传学分析。这项R21可行性研究的具体目的是1。我们将证明制备由单个细胞核组成的SCA的可行性，该细胞核以微观反应室内定义的模式排列，并通过恒定力伸长/拉伸。重要的是，染色质拉伸的程度将由细胞固定和调节环境参数（例如缓冲液，腔室温度和湿度）以及施加的力来控制。 2。为SCAS的敏感，高分辨率的细胞遗传学分析开发优化的测定。我们将为SCAS的基于鱼类的多洛克斯细胞遗传学分析开发一种方案。预计该测定将提供接近千倍酶灵敏度，以检测单拷贝核酸的分辨率为10-20 kb，同时最大程度地减少了DNA的总体损失。该测定将通过分析从不同乳腺或甲状腺癌细胞系制备的SCA进行测试。 SCAS将成为基本和应用/临床研究的强大工具，在这些工具中，染色体变化通常会影响细胞的表型及其后代的命运。例如，在临床实践中，这种敏感的测定可能支持细胞分类，从而使从头易位或预易病变的患者以及癌症患者受益。此外，SCAS将允许对非常小的样品进行分析，无论其完整性或细胞周期阶段如何。这将为分析小样品（例如通过细针活检获得的小样本）以及循环或去角质肿瘤细胞的分析提供新的途径。 公共卫生相关性声明：目前，尚无技术来制备小型细胞样本并筛选它们的核型异常。高度敏感的，基于鱼类的单细胞阵列（SCA）将提供一种平台技术，可以通过该技术开发针对特定疾病，细胞或组织样本量身定制的多种测试。由于它们的多功能性，SCAS可能会成为基础和临床研究中的强大工具，从而使从头易位或预先抗衰老病变以及癌症患者的患者受益。