Mapping Single Extracellular Vesicles to Parent Cells for Immunotherapy Monitoring

这项建议旨在开发一种超高灵敏度的平台，可以映射细胞和细胞外 单颗粒水平的囊泡(EV)分子信息，并将其应用于描述肿瘤免疫 免疫治疗监测的微环境(时间)。这项技术可以解决 生物系统的异质性，并有可能发现强大的生物标志物 准确地描述身体状态，以进行疾病诊断。这项提议假定 单细胞-单EV图谱将为细胞间通讯提供新的分子信息 以更好地了解病理发展和疾病诊断。这样做的目的是 建议包括三个方面：i)开发单一EV蛋白测序技术以解决EV 异质性和发现罕见的EV亚型作为疾病生物标志物，II)超快循环 多路活细胞荧光成像监测细胞表型变化和 识别多种免疫细胞类型，以及iii)单细胞-单EV映射以发现新的 有关囊泡分泌的细胞间通讯的分子信息。这项技术将 允许在免疫治疗过程中重复采样和监测时间，并提供 指导以获得尽可能最佳的患者结果。 凭借在微流体学、分子生物学和机器学习方面的专业知识，Ko Jina(这一领域的PI Proposal)开发了新的微米和纳米技术，用于液体活检，可以提取 来自血液生物标记物(例如循环肿瘤细胞)的多维分子数据。 她将自己的专业知识扩展到化学、液滴微流体学和转化医学，以 开发新的平台，这些平台将作为该提案的基础工作。她最近 开发了一)使用液滴数字聚合酶链式反应的超高灵敏度单电动汽车分析技术 用于多路细胞荧光成像的超快循环。通过这项工作，吉娜锻造了 马萨诸塞州总医院和哈佛大学内外的研究合作 威斯研究所和哈佛大学医学院。在这些成就的基础上， 这项工作将由世界液滴微流体学和物理学专家组成的团队共同完成 (Weitz)、分子成像和诊断学(Weissleder)、免疫学(Pittet)、外显体生物学 和神经遗传学(Breakefield)，以及神经外科和肿瘤学(Chiocca)，谁将提供完整的 对这项工作的支持。与这些导师合作将使她能够解决临床上的挑战 问题，并进一步发展她作为独立调查员的职业生涯，具有发展的能力 下一代医疗诊断。

This proposal aims to develop an ultra-high sensitive platform that can map cell and extracellular vesicle (EV) molecular information at the single particle level, and apply it to profile tumor immune microenvironment (TIME) for immunotherapy monitoring. This technology can resolve heterogeneity of biological systems and has the potential to discover robust biomarkers that can accurately profile status of the body for disease diagnostics. This proposal hypothesizes that single cell-single EV mapping will provide new molecular information on cell-cell communication for better understanding of pathological development and disease diagnosis. The goals of this proposal are threefold: i) development of single EV protein sequencing technology to resolve EV heterogeneity and discover rare EV subtypes for disease biomarkers, ii) ultra-fast cycling for multiplexed live cell fluorescence imaging to monitor the changes of cellular phenotypes and identify multiple immune cell types, and iii) single cell-single EV mapping to discover new molecular information on cell-cell communication through vesicle secretion. This technology will allow repeat sampling and monitoring of TIME during the course of immunotherapy and provide guidance to achieve the best possible patient outcomes. With the expertise in microfluidics, molecular biology, and machine learning, Jina Ko (the PI of this proposal) has developed new micro- and nano-technologies for liquid biopsy that can extract multidimensional molecular data from blood-based biomarkers (e.g. circulating tumor cells, EV). She has extended her expertise to chemistry, droplet microfluidics, and translational medicine to develop novel platforms that will serve as a fundamental work to this proposal. She has recently developed i) ultra-high sensitive single EV profiling technology using droplet digital PCR and ii) ultra-fast cycling for multiplexed cellular fluorescence imaging. Through this work, Jina has forged research collaborations within and outside the Massachusetts General Hospital and Harvard Medical School at the Wyss Institute and Harvard University. Building upon these achievements, this work will be executed with a team of world experts in droplet microfluidics and physics (Weitz), molecular imaging and diagnostics (Weissleder), immunology (Pittet), exosome biology and neurogenetics (Breakefield), and neurosurgery and oncology (Chiocca) who will provide a full support on this work. Working with these mentors will allow her to tackle clinically challenging problems and further develop her career as an independent investigator with the ability to develop next generation medical diagnostics.