Accelerating Cancer Research with Single Cell Arrays

利用单细胞阵列加速癌症研究

基本信息

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
项目类别：
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

项目摘要

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）：该提案解决了对染色体变化的敏感检测，例如表面正常个体、良性肿瘤、癌前病变和癌症中的小易位、重排或基因组失衡。目前对单个细胞进行全面核型分析的技术需要中期细胞，而无法分析间期细胞或非存活细胞。从人类肿瘤中获得的许多细胞并不处于中期。拟议研究的目标是开发技术来支持少量新鲜、固定或存档组织的细胞遗传学分析，无论细胞的增殖阶段如何。一种称为单细胞阵列 (SCA) 的高灵敏度、基于荧光原位杂交 (FISH) 的技术平台将通过将高分辨率 DNA 原位分析与 kb 范围内的灵敏度相结合，可以检测间期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专门设计的微室中控制染色质拉伸，然后使用 FISH 进行细胞遗传学分析来实现。该 R21 可行性研究的具体目标是 1. 证明间期细胞核可以以定义的模式固定并可重复地扩展以用于后续细胞遗传学分析的可行性。我们将演示制备由单个细胞核组成的 SCA 的可行性，这些细胞核在微观反应室内以定义的模式排列，并通过恒定的力拉长/拉伸。重要的是，染色质拉伸的程度将通过细胞固定和调整环境参数（例如缓冲液、室温度和湿度以及拉伸染色质所施加的力）来控制。 2. 开发一种优化的分析方法，用于 SCA 的灵敏、高分辨率细胞遗传学分析。我们将开发一个基于 FISH 的 SCA 多位点细胞遗传学分析方案。该测定预计可为单拷贝核酸检测提供近千碱基的灵敏度，分辨率约为 10-20 kb，同时最大限度地减少 DNA 的总体损失。该测定将通过分析从不同乳腺癌或甲状腺癌细胞系制备的 SCA 进行测试。 SCA 将成为基础和应用/临床研究中的强大工具，其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，这种灵敏的测定可以支持细胞分类，从而使患有新生易位或癌前病变的患者以及癌症患者受益。此外，SCAs 可以分析非常小的样品，无论其完整性或细胞周期阶段如何。这将为分析小样本（例如通过细针活检获得的样本）以及分析循环或脱落肿瘤细胞开辟新途径。公共卫生相关声明：目前，尚无技术可以制备非增殖细胞的小样本并筛选其核型异常。称为单细胞阵列 (SCA) 的高灵敏度、基于 FISH 的检测将提供一种平台技术，人们可以利用该技术开发针对特定疾病和细胞或组织样本的多种测试。由于其多功能性，SCAs 可能成为基础和临床研究的强大工具，从而使患有新生易位或癌前病变的患者以及癌症患者受益。