Highly Multiplexed Nanoscale Mass Spectrometric Imaging of Cancer Tissues

总结/摘要 在过去的三十年里，质谱技术在蛋白质组学领域发挥了主导作用。的组合 创新的基于MS的技术为蛋白质组学发现提供了强大的工具， 鉴定复杂样品中蛋白质生物标志物，定量蛋白质表达的变化并表征蛋白质- 蛋白质相互作用质谱的第二个重要进展是引入了质谱成像 (MSI)其将MS扩展到空间维度，允许绘制生物分子的分布，包括 蛋白质、核酸、代谢物甚至是复杂组织中的小药物化合物。第二阶段的目标是 一个项目是进一步发展MSI的能力，以执行高度多路复用成像，甚至对亚细胞 纳米级的生物样品中的靶生物分子。这种能力将为系统提供一个主要工具， 生物学家和癌症研究人员，他们需要复杂的关键分子分布的详细知识， 在细胞、亚细胞和分子水平上对组织的影响。它也将为病理学家提供一个强大的新工具 分析肿瘤组织样本，以最终获得改善的治疗和患者结果。期间 第一阶段，我们已经成功地证明了：i）能够同时成像的MSI潜在的数百个 来自福尔马林固定石蜡包埋（FFPE）的小鼠脑薄切片的靶向生物标志物， 专利的改进的光可裂解质量标签（iPC-MT），其被掺入抗体中，或 寡核苷酸探针。相比之下，常规的基于光学显微镜的免疫组织化学（IHC）和 荧光原位杂交（FISH）方法只能对少数靶向生物分子成像;和ii）设计 新的iPC-MT探针，与新的扩展显微镜（ExM）方法完全兼容， 纳米级亚细胞MSI分辨率。在第二阶段，我们将通过应用iPC-MT-probe 该技术用于分析存档的乳腺癌FFPE标本，以最终改善癌症 组织病理学用于常规临床实践。作为模型系统，使用10个iPC-MT-抗体探针和10个iPC-MT-抗体探针。 将开发针对特定乳腺癌肿瘤抗原和miRNA生物标志物的寡核苷酸探针 最初使用AmberGen专有的珠阵列质谱法测试潜在的交叉反应性 技术（Bead-AMS™）。然后使用这些探针通过MSI分析乳腺癌组织切片， 与从常规的基于荧光的IHC和FISH方法获得的结果相比。为了获得 增加空间分辨率，与膨胀的丙烯酸酯凝胶交联或递送至膨胀的丙烯酸酯凝胶的iPC-MT探针 将使用包含扩展的FFPE切片的材料。将使用专门的低- 温度，红外激光为基础的MALDI-MSI仪器。我们将继续推动这项工作， 与MS和ExM领域的领先专家合作，包括Cathy Costello博士（BU，William Fairfield 沃伦杰出教授，BU生物医学质谱中心主任），艾德博伊登（麻省理工学院，Y。 伊娃谭神经技术教授）和贾森阿姆斯登（杜克大学，助理研究教授）。

Summary/Abstract Mass spectrometry (MS) has played a leading role in the past three decades in the field of proteomics. A combination of innovative MS-based techniques has provided powerful tools for proteomic discovery including the ability to identify protein biomarkers in a complex sample, quantify changes in protein expression and characterize protein- protein interactions. A second important advance in MS has been the introduction of mass spectrometric imaging (MSI) which extends MS to the spatial dimension, allowing mapping of the distribution of biomolecules including proteins, nucleic acids, metabolites and even small drug compounds in complex tissues. The goal of this Phase II project is to further develop the ability of MSI to perform highly multiplexed imaging, even on the subcellular nanoscale, of targeted biomolecules in biospecimens. Such a capability would provide a major tool for systems biologists and cancer researchers, who require a detailed knowledge of the distribution of key molecules in complex tissues at the cellular, subcellular and molecular levels. It would also provide pathologists with a powerful new tool to analyze tumor tissue specimens in order to ultimately obtain improved therapy and patient outcomes. During Phase I we have successfully demonstrated: i) the ability to simultaneously image by MSI potentially hundreds of targeted biomarkers from formalin fixed paraffin embedded (FFPE) thin sections from mouse brain using proprietary improved photocleavable mass-tags (iPC-MTs) which are incorporated into antibodies or oligonucleotide probes. In contrast, conventional light microscopy-based immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) methods can image only a few targeted biomolecules; and ii) designed new iPC-MT-probes which are fully compatible with the new method of expansion microscopy (ExM) to obtain nanoscale subcellular MSI resolution. During Phase II, we will build on this progress by applying iPC-MT-probe technology to the analysis of archived breast cancer FFPE specimens in order to ultimately achieve improved cancer histopathology for routine clinical practice. As a model system, 10 iPC-MT-antibody probes and 10 iPC-MT- oligonucleotide probes targeted at specific breast cancer tumor antigens and miRNA biomarkers will be developed and initially tested for potential cross-reactivity using AmberGen's proprietary Bead-Array Mass Spectrometry technology (Bead-AMS™). Breast cancer tissue slices will then be analyzed by MSI using these probes and compared to results obtained from conventional fluorescence based IHC and FISH methods. In order to obtain increased spatial resolution, iPC-MT-probes that cross-link with or are delivered to expanded acrylate gels containing expanded FFPE slices will be used. MSI of the expanded gels will be achieved using specialized low- temperature, infrared laser-based MALDI-MSI instrumentation. This work will be facilitated by our continued collaboration with leading experts in the MS and ExM fields including Drs. Cathy Costello (BU, William Fairfield Warren Distinguished Professor, Director of BU Center for Biomedical Mass Spectrometry), Ed Boyden (MIT, Y. Eva Tan Professor of Neurotechnology) and Jason Amsden (Duke University, Assistant Research Professor).