Phenotypic sorting of cancer cells to study the role and control of cell stiffness in the in vivo metastatic cascade

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PROJECT SUMMARY/ABSTRACT A majority of cancer-related deaths are the result of the metastatic spread of cancer cells from their primary tumor location to distant sites in the body. Cell mechanical properties, including stiffness, are related to the migratory and metastatic potential of tumor cells. However, previous studies are limited by observing cell mechanics as an effect rather than as a potential driving force of metastasis. If a causal link between cell stiffness and metastatic potential in vivo can be established, direct modulation of cell mechanics could constitute a therapeutic strategy to slow or stop the metastatic spread of cancer cells. The long term goal of this research is to connect cell behaviors and mechanical properties studied in vitro with in vivo metastatic phenotypes to identify therapeutic control points of cell mechanotype and metastasis. A microfluidic stiffness-based cell sorting device will be used to generate stiff and soft cell subpopulations to compare the effect of cell stiffness on the various stages of metastasis that occur in an orthotopic breast cancer mouse model. Preliminary experiments have established our ability to successfully sort cells based on several biophysical properties. Additionally, the Reinhart-King lab is uniquely positioned to compare in vitro and in vivo cell behaviors to understand metastasis. This project will explore the question of the causal link between cell stiffness and cell metastatic potential by 1) determining the heritability of cancer cell mechanotypes through microfluidic stiffness-based cell sorting, 2) investigating the role of cell stiffness on multiple stages of in vivo metastasis, and 3) exploring the genetic and epigenetic control points of cell stiffness for abatement of metastatic spread. First, the established microfluidic device will be used to repeatedly sort four breast cancer cell lines into mechanical subpopulations, tracking whether cells remain stiff or soft after cell division and passaging. The sorted populations will then be injected into the mammary fat pad of a mouse to form an orthotopic breast cancer model monitoring the effect of cell stiffness on metastatic tumor formation as well as studying the effect of stiffness of in vivo and in vitro models of each step of the metastatic cascade. Finally, multi-omics analyses will be used to understand the underlying molecular mechanisms that result in each mechanical subpopulation. The result of the proposed study will show, for the first time, the causal relationship between cell mechanical properties and their ability to successfully traverse each step in the metastatic cascade in vivo. The applicant’s long-term career goal is to become a leading researcher and expert in the field of metastatic cancer biology. The fellowship will help the applicant augment her previous expertise in microfluidics and atomic force microscopy with training in in vivo models of cancer and metastasis with an expert in cancer mechanobiology, Dr. Cynthia Reinhart-King, skills that will be invaluable to her as an independent researcher studying metastasis in a clinically relevant model.