Direct Tumor Glycan Profiling on Tissue Microarrays

 描述（由申请人提供）：组织微阵列技术在癌症基因组时代不断发展，在验证与新兴遗传突变表型和转录谱研究相关的蛋白质表达方面发挥着核心作用。TMA的主要用途一直是用于特定癌症类型中差异表达蛋白质的免疫组织化学验证，特别是应用于生物标志物、诊断、预后、预防和治疗研究目的。用于DNA或RNA谱分析和蛋白质组学分析的TMA的直接分析也是主要的研究用途。用TMA进行实验的主要优点是可获得来自多个个体样品的组织病理学和临床相关组织切片，提供通量、统计相关性和多种分子靶标的多重分析的要素。本申请的目的是扩展TMA作为研究癌症发展和进展中糖基化的工具的用途。据充分记载，恶性转化和癌症进展导致细胞表面和分泌的糖蛋白的糖基化模式的根本变化。目前很难以全局方式评估组织中的这些糖基化变化，因为只有少数对一种糖型或广泛亲和力凝集素具有特异性的碳水化合物抗原抗体可用。为了解决这个问题，我们的实验室最近开发了一种新的方法，使用MALDI成像质谱法直接在组织上分析N-连接聚糖。根据组织的不同，目前可以通过该方法同时检测30-40种N-聚糖种类，与其组织病理学表达和组织分布直接相关。该方法已适用于福尔马林固定的组织，并与定制的TMA和高分辨率MALDI-FTICR质谱仪相结合，将针对多种组织类型优化以TMA格式分析多种N-聚糖的方法。我们预计优化的方法适用于任何TMA，并实现这一目标，提出了三个具体目标：目标1。使用8种癌症组织类型的混合TMA优化N-连接聚糖的MALDI成像质谱分析。目标2.使用载玻片上化学衍生法增加检出聚糖的总数，并改善对更高质量聚糖的检测。目标3.确认聚糖结构，按癌组织对其检测进行分类，并定义检测试剂盒的分析检测参数。我们的方法将是变革性的，因为与给定癌症类型相关的所有可检测的聚糖变化都可以同时以TMA格式进行分析。在病理学家的指导下，该方法的潜在影响是其用于任何TMA癌症类型的预防，进展，预后和生物标志物研究。

 DESCRIPTION (provided by applicant): Tissue microarray technology continues to evolve in the cancer genomic era, and has a central role in validating the expression of proteins associated with emerging genetic mutation phenotypes and transcriptional profiling studies. Primary uses of TMAs have always been for immunohistochemistry validation of differentially expressed proteins in a particular cancer type, particularly as applied to biomarker, diagnostic, prognostic, prevention and therapeutic research purposes. Direct analysis of TMAs for DNA or RNA profiling and proteomic analyses are also major research uses. The main advantages of experiments performed with TMAs are the availability of histopathology and clinically relevant tissue sections from multiple individual samples, providing elements of throughput, statistical relevance and multiplexed analysis of diverse molecular targets. The goal of this application is to extend use of TMAs as a tool for the study of glycosylation in cancer development and progression. It is well documented that malignant transformation and cancer progression result in fundamental changes in the glycosylation patterns of cell surface and secreted glycoproteins. It is currently difficult to assess these glycosylation changes in tissues in a global manner, as only a few carbohydrate antigen antibodies specific to one glycoform or broad affinity lectins are available. To address this, our lab has recently developed a novel method to profile N-linked glycans directly on tissue using MALDI-imaging mass spectrometry. Depending on the tissue, currently 30-40 N-glycan species can be simultaneously detected by this method, linked directly with their histopathology expression and tissue distribution. This approach has been adapted to formalin-fixed tissues, and in combination with a custom TMA and high-resolution MALDI-FTICR mass spectrometer, a method to profile multiple N-glycans in a TMA format will be optimized for multiple tissue types. We anticipate optimizing the approach for applicability to any TMA, and to accomplish this, three Specific Aims are proposed: Aim 1. Optimize MALDI imaging mass spectrometry analysis of N-linked glycans using a mixed TMA of 8 cancer tissue types. Aim 2. Increase the total number of detected glycans and improve detection of higher mass glycans using on-slide chemical derivatization. Aim 3. Confirm glycan structures, categorize their detection per cancer tissue and define analytical detection parameters of the assay. Our approach will be transformative in that all detectable glycan changes associated with a given cancer type can be profiled simultaneously in a TMA format. As guided by a pathologist, the potential impact of the approach is its use with any TMA cancer type for prevention, progression, prognosis, and biomarker studies.