Laser Capture For Macromolecular Analysis Of Normal Development And Pathology

激光捕获用于正常发育和病理学的大分子分析

• 批准号: 8351097
• 负责人: Robert F Bonner
• 金额: $ 16.93万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8351097
• 关键词: Algorithms; Animal Model; Antigens; Area; Award; Biological Assay; Biological Markers; Biopsy; Blood; Brain; Cancer Histology; Cell Nucleus; Cells; Clinical; Clinical Pathology; Collaborations; Coloboma; Color; Complex; Computer Simulation; Cytology; Cytology Histology; Data; Data Set; Defect; Development; Diagnostic; Disease; Disease Marker; Disease Progression; Dose; Embryonic Development; Evaluation; Evolution; Film; Formalin; Gene Expression; Gene Expression Microarray Analysis; Genes; Genetic; Genomics; Goals; Heating; Human; Image; Imagery; Immunohistochemistry; Lasers; Lead; Legal patent; Light; Macromolecular Complexes; Malignant neoplasm of prostate; Measures; Messenger RNA; Methods; Microbond; Microdissection; Microscope; Microscopic; Mitochondria; Molecular; Molecular Analysis; Molecular Biology; Molecular Classification of Tumors; Molecular Diagnosis; Molecular Medicine; Morphology; National Institute of Biomedical Imaging and Bioengineering; Neurons; Nuclear Envelope; Operative Surgical Procedures; Oranges; Organelles; Paraffin Embedding; Pathologist; Pathology; Pathway interactions; Patient Selection; Phenotype; Physics; Physiologic pulse; Play; Polymers; Population; Process; Proteins; Proteomics; Research; Resolution; Retina; Retinal; Role; Sampling; Scanning; Scanning Electron Microscopy; Site; Slide; Specimen; Speed; Staging; Staining method; Stains; Stem cells; Subcellular structure; Surface; System; Techniques; Technology; Therapeutic; Thick; Time; Tissues; Tolonium chloride; United States National Institutes of Health; Visual; Xenon; absorption; animal tissue; base; brain cell; cancer cell; cell type; cost; design; dosimetry; genome-wide; human tissue; improved; instrument; interest; knowledge base; laser capture microdissection; macromolecule; meetings; melting; millisecond; new technology; novel; nuclear transfer; programs; prototype; receptor; research study; spatial relationship; time use; tool; transcription factor

Integrative molecular biology requires understanding interactions of large numbers of pathways. Similarly, molecular medicine increasingly relies on complex macromolecular diagnostics to guide therapeutic choices. However, genome-wide protein and mRNA copy number distributions in each cell type generally have highly skewed Pareto distributions in which the vast majority of genes have low expression levels. Thus contamination by more highly expressing cell phenotypes is particularly problematic in omic analyses of complex tissues without first isolating specific cell populations. Laser capture microdissection (LCM) of tissues provides a robust method to separate of specific cell populations from complex tissues and thus allow evaluation of thousands of regulated transcription factors, cell regulators, and receptors that are expressed at low copy number. For example, in a recent collaboration with the NEI, we adapted LCM method to isolate localized (3D) cells at the site of retinal topological closure and performed microarray analysis of gene expression at 8 time points of embryonic development. This enabled us to identify low copy number transcriptions factors that when blocked lead to loss of closure in animal models. These transcription factors and approximately 200 other temporally and spatially covariant genes appear likely to play a role in coloboma, an inborn developmental defect of the retina occurring in humans. We are developing novel mathematical approaches to identify better the specific networks of genes that drive such local tissue development within such datasets. The LCM techniques that we invented sixteen years ago are now widely used in molecular analysis of genetics and gene expression changes within target cells within complex tissues. This microscope-based microdissection, which uniquely allows high-resolution imaging of the captured material before submission for downstream multiplex molecular analysis, has a proven role in research studies. However, the requirement for visual targeting has limited its application in proteomic research where an impractical number of target decisions are required for analysis of less abundant proteins. Similarly microdissection use in clinical diagnostics has been limited by subjectivity, low through-put and cost associated with targeting of specific cells in a specialized LCM microscope. Recently, in collaboration with NCI and CIT, we invented and patented an automatic target-directed microtransfer technique based on specific staining of cells that does not require user visualization or microscopic targeting. This technique combines our physical understanding of light-activated thermoplastic microbonding with standard histochemical staining techniques that provide high absorptive contrast for innumerable specific targets within tissues. For example, standard immunohistochemistry creates absorption specific to the presence of a specific antigen or protein. We irradiate the entire immunostained tissue section, but only the specifically stained targets are heated and bond to the thermoplastic polymer on its surface. The light dose needed is much less than needed in commercial laser microdissection. This reduces the thermal transients in the tissue and film and improves spatial resolution to better than 1 micron while dramatically increasing the rate at which our laser system can scan the whole section. Consequently the new method is particularly well suited to isolate highly dispersed, specific cell populations (e.g., specific neurons within a brain section) or specific organelles (e.g., nuclei of invasive cancer cells). The spatial relationships (morphology) among the specific cells in the tissue are preserved on the transfer film. This year we have demonstrated for the first time the use of a Xenon flashlamp to simultaneously capture all specifically stained targets within a large region of interest with one or two 0.5 msec white light pulses. The robustness and precision of this approach relies on new transfer films that we have developed with Nicole Morgan (NIBIB) which have an ultrathin (1 micron) thermoplastic polymer coating on a thermally stable, clear polymer film. The broad spectrum flashtube allows spectral filtering to be optimized for the specific stain colors. For example, we have used the standard histochemical stain, toluidine blue, with orange filtered flash to capture cancer cell nuclei from a high grade prostate cancer histology slide. In collaboration with Drs Markey, Lippincott-Schwartz, and Morgan under an NIH Directors Challenge Award, we applied our technology to proteomics studies of subcellular organelles. We are optimizing this approach for specifically stained neuronal nuclei within formalin-fixed, paraffin-embedded brain sections. We hope to integrate our ability to microtransfer specific organelles with a variety of omic multiplex molecular analyses and thereby increase our sensitivity to molecular species associated with specific subcellular structures. For a number of years clinical molecular diagnostics have focused on discovery of concise sets of pathology specific biomakers that might be quantitatively assayed from a routinely accessible clinical sample (e.g., blood, biopsy or surgical sample). Microdissection currently has an important role in such discovery, by isolating phenotypically pure pathological samples. The utility of microdissection in clinical diagnostics will require integration with current clinical pathology methods while efficiently providing increased accuracy by analyzing many stage-specific disease markers within such purified phenotypic targets compared to the complex variable mixture in the original clinical specimen. For this to be practical, microdissection of clinical samples (e.g., formalin-fixed paraffin-embedded tissue sections and cytology specimens) must be reduced to a simpler, lower-cost, robust method. To meet this need, we are developing simple, robust approaches for integration of our thermoplastic microtransfer methods of microdissection with downstream macromolecular analysis for clinically practical multiplex molecular diagnostics. Given the potential of such integration to more reliably measure larger numbers of interacting biomolecules with specific pathophysiological cells, we foresee an evolution of molecular diagnosis from one based on the qualitative or quantitative analysis of a few abundant, specific biomarker macromolecules to one in which special knowledge-based clustering algorithms characterize disease state with high-dimensional molecular data from microdissected samples. In summary, we are developing new technologies that integrate microdissection with macromolecular analysis of histology and cytology samples that would allow the study of many less abundantly expressed genes in determining normal function and pathological changes. Our goal is to have our new technology commercialized and integrated into better molecular diagnostics to guide selection of patient appropriate clinical therapies.

综合分子生物学需要了解大量途径的相互作用。 同样，分子医学越来越依赖复杂的大分子诊断来指导治疗选择。 然而，每种细胞类型中的全基因组蛋白质和 mRNA 拷贝数分布通常具有高度倾斜的帕累托分布，其中绝大多数基因具有低表达水平。因此，在不首先分离特定细胞群的情况下，在复杂组织的组学分析中，更高表达的细胞表型的污染尤其成问题。组织的激光捕获显微切割 (LCM) 提供了一种从复杂组织中分离特定细胞群的可靠方法，从而可以评估数千种低拷贝数表达的受调节转录因子、细胞调节因子和受体。例如，在最近与 NEI 的合作中，我们采用 LCM 方法分离视网膜拓扑闭合部位的局部 (3D) 细胞，并对胚胎发育的 8 个时间点的基因表达进行微阵列分析。 这使我们能够识别低拷贝数转录因子，这些转录因子在动物模型中被阻断时会导致封闭性丧失。这些转录因子和大约 200 个其他时间和空间共变基因似乎可能在缺损（一种人类视网膜先天性发育缺陷）中发挥作用。我们正在开发新的数学方法，以更好地识别在此类数据集中驱动局部组织发育的特定基因网络。 我们十六年前发明的 LCM 技术现已广泛应用于复杂组织内靶细胞内的遗传学和基因表达变化的分子分析。这种基于显微镜的显微切割独特地允许在提交下游多重分子分析之前对捕获的材料进行高分辨率成像，在研究中具有已被证明的作用。然而，视觉靶向的要求限制了其在蛋白质组学研究中的应用，在蛋白质组学研究中，分析丰度较低的蛋白质需要不切实际的目标决策数量。同样，显微切割在临床诊断中的使用也受到主观性、低通量和与在专门的 LCM 显微镜中靶向特定细胞相关的成本的限制。 最近，我们与 NCI 和 CIT 合作，发明了一种基于细胞特定染色的自动靶向微转移技术并获得了专利，该技术不需要用户可视化或显微靶向。该技术将我们对光激活热塑性微粘合的物理理解与标准组织化学染色技术相结合，为组织内无数特定目标提供高吸收对比度。 例如，标准免疫组织化学产生针对特定抗原或蛋白质的存在的特异性吸收。我们照射整个免疫染色的组织切片，但只有特定染色的目标被加热并与其表面的热塑性聚合物结合。所需的光剂量远低于商业激光显微切割所需的光剂量。这减少了组织和薄膜中的热瞬变，并将空间分辨率提高到优于 1 微米，同时显着提高了我们的激光系统扫描整个切片的速率。 因此，新方法特别适合分离高度分散的特定细胞群（例如，大脑部分内的特定神经元）或特定细胞器（例如，侵袭性癌细胞的细胞核）。组织中特定细胞之间的空间关系（形态）保留在转移膜上。 今年，我们首次展示了使用氙气闪光灯通过一两个 0.5 毫秒白光脉冲同时捕获大范围感兴趣区域内的所有特定染色目标。这种方法的稳健性和精确性依赖于我们与 Nicole Morgan (NIBIB) 合作开发的新型转印膜，该膜在热稳定的透明聚合物膜上具有超薄（1 微米）热塑性聚合物涂层。广谱闪光管可以针对特定染色颜色优化光谱过滤。 例如，我们使用标准组织化学染色剂甲苯胺蓝和橙色过滤闪光从高级前列腺癌组织学载玻片中捕获癌细胞核。 在 NIH 董事挑战奖下，我们与 Markey、Lippincott-Schwartz 和 Morgan 博士合作，将我们的技术应用于亚细胞器的蛋白质组学研究。 我们正在优化这种方法，用于福尔马林固定、石蜡包埋的脑切片中特定染色的神经元核。我们希望将我们微转移特定细胞器的能力与各种组学多重分子分析相结合，从而提高我们对与特定亚细胞结构相关的分子种类的敏感性。 多年来，临床分子诊断一直致力于发现简明的病理学特异性生物标记物，这些生物标记物可以从常规可获取的临床样本（例如血液、活检或手术样本）中进行定量分析。 显微解剖目前通过分离表型纯病理样本在此类发现中发挥着重要作用。显微切割在临床诊断中的应用将需要与当前的临床病理学方法相结合，同时通过分析这种纯化表型目标中的许多阶段特异性疾病标记物（与原始临床标本中的复杂变量混合物相比），有效地提供更高的准确性。 为了使其实用化，必须将临床样本（例如福尔马林固定石蜡包埋的组织切片和细胞学标本）的显微切割简化为更简单、成本更低、稳健的方法。为了满足这一需求，我们正在开发简单、稳健的方法，将我们的显微切割热塑性微转移方法与下游大分子分析相结合，以实现临床实用的多重分子诊断。鉴于这种集成有可能更可靠地测量大量与特定病理生理细胞相互作用的生物分子，我们预见分子诊断将从基于对一些丰富的特定生物标志物大分子的定性或定量分析的诊断演变为基于特殊知识的聚类算法利用来自显微解剖样本的高维分子数据来表征疾病状态的诊断。 总之，我们正在开发将显微切割与组织学和细胞学样本的大分子分析相结合的新技术，这将允许研究许多表达量较低的基因，以确定正常功能和病理变化。 我们的目标是将我们的新技术商业化并整合到更好的分子诊断中，以指导选择适合患者的临床疗法。