Accelerating Cancer Research with Single Cell Arrays

描述(由申请人提供)：本申请是针对NIH通知编号NOT-OD-09-058提交的，标题为“NIH宣布恢复法案资金可用于竞争性修订申请”。目前的完全核型分析技术依赖于中期分裂，而间期的细胞无法分析。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开发间期细胞的细胞遗传学分析技术。这项名为“用单细胞阵列(SCA)加速癌症研究”的母基金致力于灵敏地检测从乳腺或甲状腺癌细胞系分离出的少量细胞中的染色体变化，如微小易位或基因组不平衡。母项目将证明间期细胞核可以在固体载体上重复延伸的可行性，并为延伸的染色质纤维的细胞遗传学分析开发方法。这一竞争性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技术的工作极限。另一方面，我们的初步研究表明，由于探针与DNA转录本(即存在于这些细胞中的RNA)的杂交，对经历了最小降解的样本的分析变得复杂。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.通过分析经历了DNA降解的样品来确定SCA技术的限制。大多数档案样本都会受到固定和储存的影响。为了研究SCA技术能在多大程度上应用于癌症研究界感兴趣的样本，我们将研究DNA缺口或丢失以及由醋酸组成的档案样本中的蛋白质交联的影响：甲醇固定的淋巴细胞、冰冻的胎盘组织以及福尔马林固定的石蜡包埋组织块。2.通过从染色质中去除RNA来提高信噪比，从而提高检测灵敏度。使用从哺乳动物细胞培养中收获的乳腺和甲状腺癌细胞的延伸染色质制剂，我们将制定方案，以酶法消化和移除干扰分析的RNA，或阻止DNA探针与残留RNA的杂交，同时将DNA损失降至最低。SCAS将成为基础和应用/临床研究的有力工具，在这些研究中，这种敏感的分析可能支持细胞分类，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，而不考虑其活性或细胞周期阶段。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。

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.