Highly multiplexed tissue imaging with high-plex in situ signal amplification

项目摘要 生物分子的身份和空间背景（例如，蛋白质和RNA）是癌细胞中的关键成分 他们的病理学。因此，可以同时映射图像的可扩展的和多路复用的成像平台是可行的。 蛋白质和RNA景观组织将成为深入分析和绘制癌细胞类型的重要工具 在其空间背景下。为了达到这一目的，已经做出了广泛的努力， 细胞水平和分子水平。然而，这些方法通常缺乏高的多重性或遭受低的复杂性。 灵敏度随着目标丰度的降低而降低。此外，用于蛋白质共成像的高度多重方法 而RNA在整个组织水平上仍然落后。我们在这里的目标是通过开发一个 用于细胞的高度多重、快速、可扩展的蛋白质组和转录组作图的多功能成像平台 线和哺乳动物组织样品与高倍信号放大。我们建议（目标1）开发一个简单的， 高度可控的聚合酶介导的迭代原位DNA延伸（伊势）和连接。我们将 展示伊势在组织和细胞样本中蛋白质和RNA靶标成像上的应用， 高信噪比和信号特异性。我们还将（目标2）验证伊势成像在细胞中的可扩展性， 组织样本我们将优化该技术，以实现50至100重蛋白质和RNA的空间定位 哺乳动物组织样本中的靶点。最后，我们将（目标3）在FFPE和厚组织中验证伊势成像， 正常和癌性样本类型。我们将在临床上共同检测RNA和蛋白质肿瘤生物标志物， 最后，我们将整合伊势成像方法与现有的组织清除方法 （iDISCO、EQUITY等）以实现从细胞水平的高通量和高度复用的组织成像， 在分子水平上对正常脑组织和癌性脑组织进行检测。我们将建立一个50-plex成像平台， 数百微米至毫米厚的哺乳动物组织标本，以揭示前所未有的细节， 组织.拟议的工作将提供一个全面的成像工具集，包括低成本，简单的设计， 用于蛋白质和RNA分子多重成像的正交DNA探针，以及可扩展的信号 本发明提供了用于在不同类型的组织中进行多重荧光成像的放大方法。我们设想， 技术将被广泛使用，并无缝地纳入管道，工作流程和协调 临床研究人员、病理学家以及更广泛的生物成像社区的使命框架， 促进癌症研究和临床实践。

Project Summary The identity and spatial context of biomolecules (e.g., protein and RNA) in cancer cells are crucial components of their pathology. Therefore, a scalable and multiplexed imaging platform that can simultaneously map the protein and RNA landscapes tissue would be a vital tool towards deeply profiling and mapping cancer cell types in their spatial context. Extensive efforts have been made toward this end to reveal unprecedented details at both cellular level and molecular level. However, these methods generally lack high multiplexity or suffer low sensitivity as the target abundance decreases. In addition, highly multiplexed methods for co-imaging of protein and RNA at the whole tissue level still lag behind. We here aim to address these limitations by developing a versatile imaging platform for highly multiplexed, rapid, scalable proteomic and transcriptomic mapping of cell line and mammalian tissue samples with high-plex signal amplification. We propose to (Aim 1) develop a simple, highly controllable polymerase mediated iterative in situ DNA extension (ISE) and concatenation. We will demonstrate the application of ISE on the imaging of protein and RNA targets in both tissue and cell sample with high signal-to-noise and signal specificity. We will also (Aim 2) validate the scalability of ISE imaging in cell and tissue samples. We will optimize the technology to achieve spatial mapping of 50 to 100-plex protein and RNA targets in mammalian tissue samples. Finally, we will (Aim 3) validate ISE imaging in both FFPE and thick tissue, both normal and cancerous sample types. We will co-detect both RNA and protein tumor biomarkers in clinical FFPE samples and finally, we will integrate the ISE imaging methods with existing tissue clearing methods (iDISCO, CLARITY, etc.) to enable high-throughput and highly multiplexed tissue imaging from cellular level to molecular level for both normal and cancerous brain tissue. We will establish a platform for 50-plex imaging in hundreds micrometer to millimeter thick mammalian tissue specimens to unveil unprecedented detail in the tissue. The proposed work will deliver a comprehensive imaging toolset including a low-cost, simple design of orthogonal DNA probes for multiplexing imaging on protein and RNA molecules, and a scalable signal amplification method for multiplexed fluorescence imaging in different types of tissues. We envision that our technologies will be widely accessible and seamlessly incorporated into the pipelines, workflows, and coordinate frameworks of the mission for clinical researchers, pathologists, as well as the wider bio-imaging community to facilitate cancer research and clinical practice.