Intravital 2-photon microscopy enabling 6D single cell RNA seq in immunocompetent glioblastoma xenografts

免疫活性胶质母细胞瘤的活体双光子显微镜实现6D单细胞RNA序列 异种移植物 已使用各种体外和体外方法来研究哺乳动物细胞破译信号。 途径和验证新的药物靶点。然而，从自然环境中分离的细胞的属性是 不可避免地改变了。组织学只提供关于分子和细胞过程的静态信息。医疗 CT、PET和MRI等成像范例不能在显微镜下动态显示细胞 在活体动物身上的分辨率。双光子(2P)成像和标记工具的最新进展带来了一种 活体成像的新时代，它不仅能够在高空间和高分辨率下对细胞进行纵向可视化 时间分辨率，但也通过各种指标阐明细胞的功能。胶质母细胞瘤 (GBM)是成人中最具侵袭性和致命性的原发脑瘤。有一种迫切需要的高 分辨率活体功能成像工具询问基底细胞瘤的异质性，这是关键 基底膜复发和耐药的决定因素。这项提议旨在解决这一紧迫需求 通过建立具有分子图谱能力的体内功能单细胞示踪平台。使用 在2P成像和标记工具方面拥有丰富的研究经验，PI开发了一种细胞定位 用于对活体动物进行精确的、纵向功能的单细胞跟踪的系统(CPS)。在这项提案中，我们 将长期功能的单细胞跟踪平台与单细胞RNA测序(ScRNA Seq)相结合。 与传统的scRNA序列相比，增加了两个维度：时间(T)和细胞功能(F)， 构成6DscRNA序列(x，y，z，颜色，t和f)。6d scrna序列将被用来描绘分子。 在2p成像过程中收集到的GSCs的细胞行为特征。如果成功，这个6D序列 平台将显著推进单细胞测序领域，并提供一种使能技术， 有望更深入地了解脑癌或其他疾病的发病机制和治疗方法。 我们的平台还允许在体内全面评估肿瘤对化疗作为一种药物的反应 提供活体筛查平台，为GBM及以上疾病建立个体化靶向治疗。

Intravital 2-photon microscopy enabling 6D single cell RNA seq in immunocompetent glioblastoma xenografts A variety of in vitro and ex vivo methods have been used to study mammalian cells deciphering signaling pathways and validating new drug targets. However, properties of cell isolated from native environment are unavoidably altered. Histology provides only static information on molecular and cellular processes. Medical imaging paradigms such as CT, PET and MRI do not allow dynamic visualization of cells at microscopic resolution in living animals. Recent progress in two-photon (2p) imaging and labeling tools has ushered in a new era of intravital imaging, which not only enables the longitudinal visualization of cells at high spatial and temporal resolution, but also sheds light on the function of cells through a variety of indicators. Glioblastoma (GBM) is the most aggressive and lethal primary brain tumor in adults. There is a pressing need of high resolution intravital functional imaging tools to interrogate GBM tumor heterogeneity, which is the key determinant of GBM recurrence and resistance to therapy. This proposal aims to address this pressing need by establishing an in vivo functional single cell tracking platform with the capacity of molecular profiling. With extensive research experience in 2p imaging and labeling tools, the PI has developed a cell positioning system (CPS) for precise, longitudinal functional single cell tracking in live animals. In this proposal, we combine long-term functional single cell tracking platform with single cell RNA sequencing (scRNA seq). Compared with conventional scRNA seq, two more dimensions are added: time (t) and cell function (f), constituting the 6D scRNA seq (x, y, z, color, t, and f). 6D scRNA seq will be used to delineate the molecular features of GSCs whose cellular behaviors have been collected during 2p imaging. If successful, this 6D seq platform will significantly advance the field of single-cell sequencing and provide an enabling technology that promises a more in-depth understanding of the pathogenesis and therapy of brain cancers or other diseases. Our platform also allows comprehensive in vivo evaluation of tumor responses to chemotherapy as a drug in vivo screening platform toward establishing individualized targeted therapy for GBM and beyond.