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.