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

项目摘要 癌细胞中生物分子(如蛋白质和RNA)的特性和空间背景是至关重要的组成部分 他们的病态。因此，一个可扩展和多路复用的成像平台可以同时映射 蛋白质和RNA景观组织将是深入分析和绘制癌细胞类型的重要工具 在他们的空间背景下。为此已经做出了广泛的努力，以揭示前所未有的细节 无论是细胞水平还是分子水平。然而，这些方法通常缺乏高度的多解性或遭受较低的影响 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元化的方法 而整个组织水平的RNA仍然落后。我们在这里的目标是通过开发一个 多功能成像平台，用于细胞的高度多元化、快速、可扩展的蛋白质组学和转录图谱绘制 克隆和哺乳动物组织样本进行高复合信号放大。我们建议(目标1)开发一种简单、 高度可控的聚合酶介导的迭代原位DNA延伸(ISE)和串联。我们会 演示ISE在组织和细胞样本中蛋白质和RNA靶标成像中的应用 高信噪比和信号特异性。我们还将(目标2)验证ISE映像在细胞和 组织样本。我们将优化技术，实现50到100个蛋白质和RNA的空间映射 哺乳动物组织样本中的靶标。最后，我们将(目标3)验证FFPE和厚组织中的ISE成像， 包括正常样本和癌变样本。我们将在临床上同时检测RNA和蛋白质肿瘤生物标记物 最后，我们将把ISE成像方法与现有的组织清除方法相结合 (iDISCO、Clarity等)实现高通量和高度多路复用的组织成像 正常脑组织和癌变脑组织的分子水平。我们将在中国建立50路成像平台 数百微米到毫米厚的哺乳动物组织标本将在 组织。拟议的工作将提供全面的成像工具集，包括低成本、简单的设计 用于蛋白质和RNA分子的多路成像的正交DNA探针和可缩放的信号 不同类型组织中多重荧光成像的放大方法。我们设想我们的 技术将被广泛访问，并无缝整合到管道、工作流和协调中 临床研究人员、病理学家以及更广泛的生物成像社区的任务框架 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.