Imaging the native 3D architecture of pancreatic and breast tumor patient tissue at single-cell resolution

项目摘要 癌症仍然是美国成年人死亡的第二大原因，然而， 癌细胞的生长是如何开始的，以及转移和耐药性等关键性转变是如何发生的。 并没有得到很好的理解。在实体瘤的多重分析方面已经取得了重大进展， 使用单细胞测序和空间分子定位技术。然而，尽管独特的 尽管这些方法提供了对肿瘤异质性的深入了解，但它们仅限于二维（2D）薄 这些分析提供的关于肿瘤的天然三维结构的信息很少。串行 重建可以提供一些三维背景，但是这样的方法在重建方面都是低效的。 样品通过量，并且对组织结构具有固有的破坏性。我们提出了一个跨学科的， 定量表征人实体瘤组织的天然三维结构的方法 来自三阴性乳腺癌（TNBC）和胰腺导管腺癌（PDAC）患者， 结合了最先进的、成熟的组织清除、免疫荧光和原位技术， 杂交标记和高分辨率光片荧光显微术。分析这些组织体积 将能够阐明不同肿瘤、免疫和基质细胞的空间相互作用组， 肿瘤异质性以及它们与肿瘤微环境组分的相互作用。我们的具体 目的是（1）成像肿瘤，免疫和基质细胞的三维空间分布， 血管系统、血管系统和细胞外基质在来自人胰腺和胰腺癌的天然实体瘤组织中的表达 使用组织清除、免疫荧光、原位杂交和3D 光片显微镜，和（2）开发一个计算管道，以建立三维空间地图的 人胰腺和乳腺完整实体瘤组织中蛋白质表达和RNA转录本定位 恶性肿瘤。 本研究的创新之处在于采用了跨学科的研究策略， 边缘组织清除，多重标记和高分辨率光片显微镜，以更好地了解 实体瘤的天然三维结构。具体而言，通过分类和量化先前 实体瘤组织的未知三维特征，我们将能够将实体瘤组织的空间组织与实体瘤组织的三维特征相关联。 肿瘤与取自同一标本的基因组和蛋白质组数据。这一建议的意义在于， 人类肿瘤组织的成功三维表征将使关键的见解， 肿瘤内和肿瘤间的异质性，从而促进细胞-细胞和细胞-微环境的鉴定 相互作用，可以作为潜在的治疗目标，从而提供新的治疗途径，以减少 降低患者死亡率，提高生活质量。

PROJECT SUMMARY Cancer remains the second-leading cause of adult death in the United States, yet the mechanisms by which cancerous growths are initiated and how crucial transitions such as metastasis and therapeutic resistance occur are not well understood. Significant progress has been made in the multiplexed analysis of solid tumors through the use of single-cell sequencing and spatio-molecular mapping techniques. However, despite the unique insights into tumor heterogeneity these methods have afforded, they are limited to two-dimensional (2D) thin analyses which provide little information on the native three-dimensional architecture of the tumor. Serial reconstruction can provide some three-dimensional context, but such approaches are both inefficient in terms of sample throughput and are inherently destructive to the tissue architecture. We propose a cross-disciplinary, approach to quantitatively characterize the native three-dimensional architecture of human solid tumor tissue from triple negative breast cancer (TNBC) and pancreatic ductal adenocarcinoma (PDAC) patients using a combination of the state-of-the art, yet mature technologies of tissue clearing, immunofluorescence and in situ hybridization labeling, and high-resolution lightsheet fluorescence microscopy. Analysis of these tissue volumes will enable the elucidation of the spatial interactome of different tumor, immune and stromal cells that give rise to tumor heterogeneity as well as their interactions with components of the tumor microenvironment. Our Specific Aims are (1) to image the three-dimensional spatial distribution of tumor, immune and stromal cells in relation to vasculature, lymphatics and the extracellular matrix in native solid tumor tissue from human pancreatic and breast malignancies using a combination of tissue clearing, immunofluorescence, in situ hybridization and 3D lightsheet microscopy, and (2) to develop a computational pipeline to build three-dimensional spatial maps of protein expression and RNA transcript localization in intact solid tumor tissue from human pancreatic and breast malignancies. The innovation of the proposed work lies in our cross-disciplinary strategy of combining the cutting- edge tissue clearing, multiplexed labelling and high-resolution lightsheet microscopy to better understand the native three-dimensional architecture of solid tumors. Specifically, by classifying and quantitating previously unknown three-dimensional features of solid tumor tissue we will be able to relate the spatial organization of the tumor to genomic and proteomic data taken from the same specimen. The significance of this proposal is that successful three-dimensional characterization of human tumor tissues will enable key insights to be derived on both intra- and inter-tumor heterogeneity thus facilitating the identification of cell-cell and cell-microenvironment interactions that could serve as potential therapeutic targets thus providing new routes to treatments to decrease patient mortality and improve quality of life.