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

项目总结 癌症仍然是美国成年人死亡的第二大原因，但 癌细胞的生长是如何启动的，以及转移和治疗耐药等关键转变是如何发生的 都没有被很好地理解。在实体肿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测绘技术。然而，尽管独一无二 这些方法提供了对肿瘤异质性的见解，它们仅限于二维(2D)薄层 分析提供的关于肿瘤的天然三维结构的信息很少。连载 重建可以提供一些三维背景，但这种方法在以下方面都是低效的 样本吞吐量和对组织结构的固有破坏。我们提出了一个跨学科的， 定量表征人体实体瘤组织天然三维构筑的方法 来自三阴性乳腺癌(TNBC)和胰腺导管腺癌(PDAC)患者的 结合最先进、但成熟的组织清除、免疫荧光和原位技术 杂交标记和高分辨率光片荧光显微镜。对这些组织体积的分析 将有助于阐明不同肿瘤细胞、免疫细胞和基质细胞之间的空间相互作用 肿瘤的异质性以及它们与肿瘤微环境成分的相互作用。我们的特定 目的是(1)成像肿瘤、免疫和间质细胞的三维空间分布 人胰腺癌实体瘤组织中血管、淋巴管和细胞外基质的表达 应用组织清除、免疫荧光、原位杂交和3D相结合的乳腺恶性肿瘤 以及(2)开发一种计算管道来建立三维空间地图 人胰腺和乳腺完整实体瘤组织中蛋白质表达及RNA转录本定位 恶性肿瘤。 建议工作的创新之处在于我们的跨学科战略，将切割- 边缘组织清除，多路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。具体地说，通过分类和量化以前的 实体肿瘤组织的未知三维特征我们将能够联系到 肿瘤的基因组和蛋白质组数据取自同一标本。这项提议的意义在于 成功的人体肿瘤组织的三维特征将使关键的洞察力能够在 肿瘤内和肿瘤间的异质性，从而有助于识别细胞-细胞和细胞-微环境 可作为潜在治疗靶点的相互作用，从而提供了治疗减少 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.