Accelerating Cancer Research with Single Cell Arrays

描述（由申请人提供）：本申请是为了回应NIH通知编号NOT-OD-09-058，标题为“NIH宣布恢复法案资金可用于竞争性修订申请”。目前的全核型分析技术依赖于中期扩散，间期细胞不能进行分析。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集中在间期细胞的细胞遗传学分析技术的发展上。题为“用单细胞阵列（SCA）加速癌症研究”的母基金解决了对染色体变化的敏感检测，例如从乳腺癌或甲状腺癌细胞系中分离的少量细胞中的小易位或基因组不平衡。母项目将证明间期细胞核在固体支持物上可重复延伸的可行性，并开发用于延伸染色质纤维的细胞遗传学分析的方法。 本竞争修订申请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本来定义SCA技术的工作限值。另一方面，我们的初步研究表明，经过最小降解的样品的分析由于探针与DNA转录物的杂交而变得复杂，即，RNA存在于这些细胞中。因此，拟议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。 本申请的两个具体目的是1.通过分析已发生DNA降解的样品来确定SCA技术的限度。 大多数档案样本将受到固定和储存的影响。为了研究SCA技术在癌症研究界感兴趣的样本中的应用范围，我们将研究档案样本中DNA缺口或缺失和蛋白质交联的影响，这些样本包括乙酸：甲醇固定的淋巴细胞，冷冻胎盘组织以及福尔马林固定的石蜡包埋组织块。2.通过去除染色质中的RNA提高信噪比，从而提高检测灵敏度。 使用从哺乳动物细胞培养物中收获的乳腺癌和甲状腺癌细胞的延长染色质制备物，我们将开发协议，以酶促消化和去除干扰测定的RNA或阻断DNA探针与残留RNA的杂交，同时最大限度地减少DNA损失。 SCA将成为基础和应用/临床研究中的有力工具，在这些研究中，这种敏感的测定可以支持细胞分类，从而使患有新发易位或癌前病变的患者以及癌症患者受益。此外，SCA将允许分析非常小的样本，无论其活力或细胞周期阶段如何。这将为分析小样本（如通过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.