Mauro Ferrari (1) describes cancer as a multi-scale transport problem inside cells and notes that, "the physics of transport within the cytoplasm, such as trafficking to different subcellular locations...requires much deeper understanding." Towards this end, new techniques are needed to study mass transport in single cells in order to 1- answer fundamental questions about the effects of cancer on transport within the cytoplasm, and 2- design novel therapeutic agents to attack altered mass transport mechanisms within cancer cells. In order to address the physics of transport within the cytoplasm, I propose to fully develop a novel approach that enables the analysis of mass transport through the cytoplasm linked to the trafficking of specifically tagged proteins. Our live cell interferometry system already tracks single cell mass changes in real time for hundreds of cells simultaneously with sub-cellular resolution. I will add simultaneous high-resolution fluorescence capabilities to link mass transport to the transport of specific cellular components. This will establish time as an essential 4th dimension for mass transport studies beyond traditional static snap-shot based approaches. I will apply this new approach to studies of mass transport and linked protein translocation in HeLa, HEK293T, and A549 human cell lines first as a model for system optimization and to establish utility. In the project second phase, I will use the live cell interferometer to study mass transport in a mouse primary model of human acute myeloid leukemia (AML) to provide translational relevance for this approach in cancer. This will include silencing hairpin RNA (shRNA) knockdown and eGFP-tagged studies of the Wiskott-Aldrich Syndrome proteins WASP and N-WASP, which are known components of the cellular transport machinery that traffic widely and are involved in regulation of the actin cytoskeleton network inside the cytoplasm and nucleus, respectively. My goal is to understand how cytoskeleton regulatory components affect, and are affected by, the transport of mass within cells under native and altered environmental conditions, far beyond standard studies that employ non-localized actin or microtubule dissociating agents. Most importantly, I will be immersed in a focused, comprehensive mentored training program with an emphasis on cancer cell biology, technology development, and applications. This program will include coursework and seminars in cancer and cell biology and formal mentoring, by individuals and a mentoring committee, from leading experts in cancer/cell biology and engineering disciplines in optics/microscopy, electronics and fluidics. These rigorous training components complement my extensive classroom background in molecular biology and engineering acquired during my Ph.D. studies at Stanford. Together with a focused, interdisciplinary research project, this structured training and mentored program will help propel me into an early career independent investigator position in cancer cell biology with powerful applications in targeted technology development for fundamental investigations in translational medicine.
Cancer involves disruptions of transport and signaling pathways at all levels, from the whole organism to a single cell (1-5), with new approaches required to study mass transport in single cells in order to 1- answer fundamental questions about the effects of cancer on transport within the cytoplasm and 2- design novel therapeutic strategies which attack altered mass transport mechanisms within cancer cells to improve the retention of nanoparticle or large-macromolecule-based treatments (1). In this K25 project I propose to develop a new capability that enables the tracking of cytoplasmic mass distribution in relation to specific protein transport within individual cells in a population of hundreds of cells simultaneously. I also propose to characterize mass transport and disruptions to mass transport in leukemia cells from a mouse model of human acute myeloid leukemia (AML) in order to support a broader goal of developing novel therapeutic agents or targeting mechanisms for existing agents.
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