Accelerating Cancer Research with Single Cell Arrays

描述（由申请人提供）：本提案涉及染色体变化的敏感检测，如明显正常个体的小易位，重排或基因组失衡，良性肿瘤，癌前病变和癌症。目前单个细胞的全核型分析技术需要中期细胞，而间期细胞或无活力细胞不能分析。从人类肿瘤中获得的许多细胞并不处于中期。拟议研究的目的是开发技术，以支持少量新鲜，固定或档案组织的细胞遗传学分析，而不管细胞的增殖阶段。一种高度敏感的、基于荧光原位杂交（FISH）的技术平台，称为单细胞阵列（SCAs），通过结合高分辨率DNA原位分析和kb范围内的灵敏度，可以检测间期和中期细胞中的小重排。这将通过在玻璃载玻片上固定细胞核和在特殊设计的微室中控制染色质的拉伸，然后使用FISH进行细胞遗传学分析来实现。这项R21可行性研究的具体目的是：证明间期细胞核可以固定在一个确定的模式和可重复扩展的后续细胞遗传学分析的可行性。我们将展示制备由单个细胞核组成的SCAs的可行性，这些细胞核在微观反应室内以确定的模式排列，并通过恒定的力拉长/拉伸。重要的是，染色质拉伸的程度将由细胞固定和调整环境参数（如缓冲液、室温度和湿度）以及用于拉染色质的力来控制。2. 开发一种优化的检测方法，用于敏感、高分辨率的细胞遗传学分析。我们将开发一种基于fish的SCAs多位点细胞遗传学分析方案。该分析预计将为单拷贝核酸的检测提供近千碱基的灵敏度，分辨率为10-20 kb，同时最大限度地减少DNA的总体损失。该试验将通过分析从不同的乳腺癌或甲状腺癌细胞系制备的sca来测试。SCAs将成为基础和应用/临床研究的有力工具，在这些研究中，染色体变化经常影响细胞的表型和后代的命运。例如，在临床实践中，这种敏感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，SCAs将允许分析非常小的样品，而不考虑其完整性或细胞周期阶段。这将为分析小样本（如细针活检获得的样本）以及分析循环或脱落的肿瘤细胞开辟新的途径。

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.