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

活体双光子显微镜在免疫活性胶质母细胞瘤中实现6D单细胞RNA测序 异种移植 各种体外和离体方法已被用于研究哺乳动物细胞破译信号 途径和验证新药靶点。然而，从天然环境中分离的细胞的性质是不稳定的。 不可避免地改变了。组织学仅提供分子和细胞过程的静态信息。医疗 诸如CT、PET和MRI的成像范例不允许在显微镜下对细胞进行动态可视化 在活体动物中的分辨率。双光子（2p）成像和标记工具的最新进展迎来了一个新的时代。 活体成像的新时代，它不仅能够在高空间和高分辨率下纵向观察细胞， 时间分辨率，而且还通过各种指标揭示了细胞的功能。胶质瘤 (GBM)是成人中最具侵袭性和致命性的原发性脑肿瘤。迫切需要高 分辨率活体功能成像工具来询问GBM肿瘤异质性，这是关键 GBM复发和治疗抗性的决定因素。这项建议旨在解决这一迫切需要 通过建立具有分子谱分析能力的体内功能性单细胞追踪平台。与 在2p成像和标记工具方面拥有丰富的研究经验，PI开发了细胞定位 CPS系统（CPS）用于在活体动物中进行精确的纵向功能性单细胞跟踪。在本提案中，我们 联合收割机将长期功能性单细胞跟踪平台与单细胞RNA测序（scRNA seq）相结合。 与传统的scRNA seq相比，增加了两个维度：时间（t）和细胞功能（f）， 构成6D scRNA seq（x，y，z，color，t，and f）。6D scRNA seq将用于描绘分子 GSC的细胞行为已在2p成像期间收集。如果成功，这个6D序列 该平台将大大推进单细胞测序领域，并提供一种使能技术， 有望对脑癌或其他疾病的发病机制和治疗有更深入的了解。 我们的平台还允许在体内全面评估肿瘤对化疗作为药物的反应， 体内筛选平台，以建立GBM和超越的个体化靶向治疗。

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.