Accelerating Cancer Research with Single Cell Arrays

描述(由申请人提供)：这项建议涉及对染色体变化的敏感检测，例如明显正常的个体中的小易位、重排或基因组不平衡、良性肿瘤、癌前病变和癌症。目前对单个细胞进行完全核型分析的技术需要中期细胞，而间期或非活细胞中的细胞无法分析。许多可以从人类肿瘤中获得的细胞并不处于中期。拟议研究的目标是开发支持对少量新鲜、固定或归档组织进行细胞遗传学分析的技术，而不考虑细胞的增殖阶段。一种基于荧光原位杂交(FISH)的高灵敏度技术平台，称为单细胞阵列(SCA)，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微小重排。这将通过将细胞核固定在玻片上并在特殊设计的微室中控制染色质的拉伸来实现，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标是：1.证明间期细胞核可以固定在特定的模式下并可重复扩展用于后续细胞遗传学分析的可行性。我们将论证制备SCA的可行性，该SCA由单个细胞核在微观反应室内以特定的模式排列，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节环境参数(如缓冲液、腔室温度和湿度)以及拉动染色质的力来控制。2.建立灵敏、高分辨率的SCA细胞遗传学分析方法。我们将开发一种基于FISH的SCA多位点细胞遗传学分析方法。该方法有望为单拷贝核酸的检测提供近千碱基的灵敏度，分辨率约为10-20kb，同时将DNA的总损失降至最低。该检测将通过分析从不同的乳腺或甲状腺癌细胞株制备的SCA来进行测试。SCAS将成为基础和应用/临床研究的有力工具，在这些研究中，染色体的变化往往会影响细胞的表型和后代的命运。例如，在临床实践中，这种灵敏的检测方法可以支持细胞分类，从而使新发易位或癌前病变患者以及癌症患者受益。此外，SCA将允许对非常小的样本进行分析，而不考虑其完整性或细胞周期阶段。这将为分析小样本开辟新的途径，如通过细针活检获得的样本，以及循环或脱落的肿瘤细胞的分析。 与公共卫生相关的声明：目前还没有技术来制备小样本的非增殖细胞并对其进行核型异常筛查。被称为单细胞阵列(SCA)的高灵敏度、基于FISH的分析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，SCA可能成为基础和临床研究的有力工具，从而使新生易位或癌前病变患者以及癌症患者受益。

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.