Accelerating Cancer Research with Single Cell Arrays

描述（由申请人提供）：本申请是根据NIH通知no - od -09-058提交的，标题为“NIH宣布竞争性修订申请的恢复法案资金可用性”。目前的全核型分析技术依赖于中期扩散，而间期细胞不能分析。由于从肿瘤中获得的细胞很少处于中期，我们正在进行的研究重点是开发间期细胞的细胞遗传学分析技术。这项名为“利用单细胞阵列（SCAs）加速癌症研究”的亲本资助旨在对从乳腺癌或甲状腺癌细胞系中分离出来的少量细胞中的染色体变化进行敏感检测，例如小易位或基因组失衡。母项目将证明间期细胞核可以在固体支持上可重复延长的可行性，并开发延长染色质纤维的细胞遗传学分析方法。此竞争性修订应用程序将通过分析在可能产生部分DNA降解的条件下处理的一系列相关样品来定义SCA技术的工作限制。另一方面，我们的初步研究表明，通过将探针与DNA转录本（即存在于这些细胞中的RNA）杂交，对经历最小降解的标本的分析变得复杂。因此，本研究的第二个目标是开发通过RNA消化或阻断来提高信噪比的技术。本应用程序的两个具体目的是：1。通过分析经过DNA降解的样本来定义SCA技术的局限性。大多数档案样本将遭受固定和储存。为了研究SCA技术在癌症研究界感兴趣的样本中应用的广泛程度，我们将研究由乙酸、甲醇固定淋巴细胞、冷冻胎盘组织以及福尔马林固定石蜡包埋组织块组成的档案样本中DNA切口或丢失和蛋白质交联的影响。2. 通过从染色质中去除RNA，增加信噪比，从而提高检测灵敏度。利用从哺乳动物细胞培养中获得的乳腺癌和甲状腺癌细胞的扩展染色质制备，我们将制定酶消化和去除干扰检测或阻断DNA探针与残留RNA杂交的RNA的方案，同时最大限度地减少DNA损失。SCAs将成为基础和应用/临床研究的有力工具，在这些研究中，这种敏感的检测方法可以支持细胞分类，从而使新生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，而不管它们的活力或细胞周期阶段。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 application is submitted in response to NIH Notice Number NOT-OD-09-058 entitled 'NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications'. Current techniques for full karyotype analysis rely on metaphase spreads, and cells in interphase cannot be analyzed. Since cells obtained from tumors are rarely in metaphase, our ongoing research focuses on the development of technologies for the cytogenetic analysis of interphase cells. The parent grant entitled 'Accelerating cancer research with single cell arrays (SCAs)' addresses the sensitive detection of chromosomal changes such as small translocations or genomic imbalances in small numbers of cells isolated from breast or thyroid cancer cell lines. The parent project will demonstrate the feasibility that interphase cell nuclei can be extended reproducibly on a solid support and develop methods for the cytogenetic analysis of extended chromatin fibers. This Competing Revision Application will define the working limits of the SCA technology by analyzing a range of relevant samples that has been processed under conditions that are likely to produce partial DNA degradation. On the other hand, our preliminary studies have demonstrated that the analysis of specimens which have undergone minimal degradation is complicated by hybridization of probes to DNA transcripts, i.e., RNA present in these cells. Thus, a second objective of the proposed research is the development of techniques to increase signal-to-noise ratios through RNA digestion or blocking. The two Specific Aims of the present application are 1. Define the limits of the SCA technology by analyzing samples that have undergone DNA degradation. Most archival samples will have suffered from fixation and storage. To investigate how widely the SCA technology can be applied to samples of interest to the cancer research community, we will study the effects of DNA nicks or loss and protein cross-links in archival samples comprised of acetic acid: methanol fixed lymphocytes, frozen placental tissues as well formalin-fixed paraffin-embedded tissue blocks. 2. Increase signal-to-noise ratios and thus the detection sensitivity by removal of RNA from chromatin. Using extended chromatin preparations from breast and thyroid cancer cells harvested from mammalian cell cultures, we will develop protocols to enzymatically digest and remove RNAs that interfere with the assay or to block hybridization of DNA probes to residual RNA, while minimizing DNA loss. SCAs will become powerful tools in basic and applied/clinical research, where 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 viability 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.