Single-Molecule Imaging of Biological Trauma:Cytokine-Based Intracellular Communication

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PROJECT ABSTRACT Research Abstract: The detection of unlabeled cytokines in real-time and from single cells could provide a robust platform for understanding the ‘molecular language of biological trauma and disease’. However, tools to visualize cytokines at the cellular level, particularly in their secreted form, are lacking. We have developed a generic nanomaterial-based near-infrared fluorescent sensor and accompanying microscopy platform which produces a unique intensity and wavelength shift in the presence of a specific target molecule (Zhang*, Landry* et al. Nature Nanotechnology 2013; Landry et al. Sensors 2015; Landry et al. Nature Nanotechnology 2017). In this 5-year proposal, I (i) will develop synthetic sensors for VEGF, IL-6, and IL-8 cytokines, (ii) validate their use to monitor constitutive cytokine secretion from macrophage and epithelial cells, and (iii) directly visualize the spatio-temporal profiles of intercellular cytokine-based synergies. Direct cellular measurement of secreted cytokines will inform how cytokine secretion profiles from single or few individual cells are stimulated by chemokines and cytokines, which forms the basis of the cytokine secretion profiles currently used in biomarker- based diagnostics. The research we propose herein has – to the best of our knowledge – only been explored theoretically (Thurley et al. POLS Comp. Bio. 2015). Landry Laboratory Research Program: I am a single-molecule biophysicist by training, having developed several instruments capable of detecting piconewton-scale forces (Landry et al. Biophys. J. 2009), and nanometer-scale fluorescence localization (Landry et al. Nucl. Ac. Res. 2012) for my doctoral work. In transitioning to my postdoctoral position, my goal was to leverage my expertise in single-molecule spectroscopy and molecular biophysics to design purely synthetic molecular recognition tools. My scientific training in as a postdoctoral fellow in Chemical Engineering at MIT focused on merging these two previously disparate areas of science: optical microscopy and nanosensor development, yielding a platform for the optical detection of any generic molecular analyte. I began my faculty appointment at UC Berkeley in June 2016, with a research portfolio motivated by translating the technical strengths of my lab in microscopy (O’Donnel et al. Adv. Funct. Mater 2017), sensor development (Beyene et al. ACS Chem Neruo 2017 & Luo et al. ACS Sensors 2017), and molecular recognition (Li et al. RSC Chemical Science 2017) to addressing the need to develop methods to detect cytokine efflux from immune cells. In the first two years of my research plan, my group will synthesize and characterize nanomaterial-based sensors for cytokines in vitro. The remaining three years of the R35 award will implement the use of cytokine sensors to measure constitutive (year 3), induced (year 4), and intracellular (year 5) cytokine signaling from cultured cell samples. My long-term research goals focus on the application of nanosensors for cytokines, chemokines, and other important biomarkers in environments such as multicellular tumor spheroids and live tumor biopsies, in which biomarker detection has traditionally been difficult. Landry ABSTRACT AB-1