Advancing technologies for the collection and analysis of high dimensional immunoprofiles and tumor images

摘要-摘要 该提案的总体目标是使用高度复用、高分辨率的成像技术 组织和肿瘤来深入表征不同实体瘤的微环境 由哈佛大学系统药理学实验室 NCI 资助的合作者进行研究 (LSP)、达纳法伯/哈佛大学癌症中心和其他四个 NCI 癌症中心。这个的PI 研究专家 Jerry Lin 博士提议，发明了基于组织的循环免疫荧光 (CyCIF) 于 2018 年推出，并使其成为领先的公共领域（免许可）执行方法 高强度组织分析。他将该方法应用于选定的高分辨率 3D 成像 视野（使用解卷积显微镜约 103 个细胞）以及整个细胞的快速分析 幻灯片（约 106 个单元格）的分辨率较低。开发的 CyCIF 可以收集 20-60 个蛋白质的数据 来自单个样本的标记使识别细胞类型和状态成为可能 保存的组织环境。它还可以对像免疫突触这样小的结构进行成像 允许在功能水平上分析细胞间的相互作用。林博士的工作直接支持 由 18 个 NCI 资助的实验室进行的研究以及他发布的 CyCIF 协议被使用 多个其他实验室独立于 HMS 团队工作。林博士也表演了自己的 作为人类肿瘤图谱网络跨网络项目的一部分的研究，该项目构思并 现在领先。这些活动在过去三年内出版了 20 份合作出版物 多年来，其中包括林博士作为第一作者或共同第一作者的多篇高影响力期刊。 作为 R50 提案的一部分，林博士将从事三项主要活动。首先，他会 继续与研究小组合作，获取之前不同阶段的CyCIF数据 以及治疗后。这将使识别分子和形态特征成为可能 与疾病的发生、进展和治疗反应有关。第二，他会 继续改进CyCIF方法并将其与其他空间方法集成 人类和小鼠肿瘤的询问（例如转录谱分析和成像质量 光谱测定）。林博士还将继续领导 HTAN TNP，旨在比较空间 跨技术和性能站点的分析方法。这项研究已经导致 对充分供电的空间轮廓以及相对优点的意外见解 全幻灯片 2D 和 3D 成像。第三，他将继续开发和验证新方法 组织成像，特别是那些适用于数字病理学工作流程的组织成像 临床试验和患者诊断的设置。这建立在 CyCIF 已证实的收集能力的基础上 定期从甲醛固定石蜡包埋 (FFPE) 标本中获取高质量数据 获取用于患者诊断和分期（包括核心活检和细针抽吸）。 这种协作和原创研究与技术开发的结合是 预计将对转化癌症生物学的新兴领域产生重大影响，并且完全 与基于实验室的 R50 研究专家的预期角色一致。

SUMMARY-ABSTRACT The overall goal of this proposal is to use highly multiplexed, high-resolution imaging of tissues and tumors to deeply characterize the microenvironments of diverse solid tumors being studied by NCI-funded collaborators at the Harvard Laboratory of Systems Pharmacology (LSP), Dana Farber/Harvard Cancer Center and four other NCI cancer centers. The PI of this proposal, Research Specialist Jerry Lin PhD, invented tissue-based cyclic immunofluorescence (CyCIF) in 2018 and has made it the leading public domain (license-free) method for performing high-plex tissue analysis. He has adapted the method to high resolution 3D imaging of selected fields of view (~103 cells using deconvolution microscopy) as well as rapid analysis of whole slides (~106 cells) at lower resolution. As developed CyCIF can collect data from 20-60 protein markers from a single specimen making it possible to identify cell types and states in a preserved tissue environment. It can also image structures as small as immune synapses allowing cell-cell interactions to be analyzed at a functional level. Dr. Lin’s work directly supports research by 18 NCI-funded laboratories and the CyCIF protocols he has published are used by multiple other labs working independent of the HMS team. Dr. Lin also performs his own research as part of a Human Tumor Atlas Network Trans Network Project that conceived and now leads. These activities have resulted in 20 collaborative publications over the past three years including several in high impact journals with Dr. Lin as first or co-first author. As part of this R50 proposal Dr Lin will engage in three primary activities. First, he will continue to collaborate with research groups to acquire CyCIF data at different stages before and after treatment. This is will make it possible to identify molecular and morphological features associated with disease initiation, progression, and therapeutic response. Second, he will continue to improve the CyCIF method and integrate it with other methods for spatial interrogation of human and murine tumors (e.g. transcript profiling and imaging mass spectrometry). Dr. Lin will also continue to lead an HTAN TNP designed to compare spatial profiling methods across technologies and performance sites. This research has already led to unexpected insights into adequately powering spatial profiles as well as the relative merits of whole-slide 2D and 3D imaging. Third, he will continue to develop and validate new approaches to tissue imaging, particularly those that are applicable to digital pathology workflows in the setting of clinical trials and patient diagnosis. This builds on the proven ability of CyCIF to collect high quality data from the formaldehyde-fixed paraffin embedded (FFPE) specimens routinely acquired for patient diagnosis and staging (including core biopsies and fine needle aspirates). This combination of collaborative and original research and technology development is expected to have a high impact on an emerging area of translational cancer biology and is fully consistent with the roles expected of a laboratory-based R50 Research Specialist.