Sorting and characterization of mechanically heterogeneous cell populations based on cellular contractility - Project Summary Cellular contractility plays a critical role in both development and disease. Recent evidence suggests that even within a cell population derived from the same source, vast heterogeneity exists in terms of cellular contractility. Dysregulation of spatiotemporally organized cellular contractility often results in developmental defects. In invasive diseases like cancer which are often highly contractile, the existence of a weakly contractile subpopulation is receiving increasing attention. Adherent cells are known for their ability to sense and dynamically adapt to their local microenvironment. Hence, the heterogeneity in mechanical phenotype may be a result of genetic heterogeneity or cellular plasticity and mechanical adaptation. Mechanomedicines or mechano- based therapies that target specific physical cellular and tissue interactions, including abnormal cellular contractility, in diseases like cancer, fibrosis, and cardiovascular disease, as well as aging, are emerging and hold great potential. While mechanical heterogeneity and plasticity are known to contribute to resistance to therapies that target a specific molecular pathway, it is not clear whether a change in mechanical phenotype predicts disease outcome or if mechanical adaption happens as a result of disease progression. This project proposes to phenotypically sort adherent cells into subpopulations with distinct contractile phenotypes and use these sorted subpopulations to test the hypothesis that the initial contractile phenotype and heterogeneity determine the disease outcome against the alternative hypothesis that mechanical adaptation to the local microenvironment and phenotypical switching contribute to disease progression regardless of the initial mechanical heterogeneity. Cancer metastasis will be used as the main biological model for hypothesis testing. In Aim 1, the engineering approach for cell sorting based on cellular contractility will be optimized. Fluorescence- activated cell sorting (FACS) will be coupled with an engineered high throughput cell contractility screening platform, automated microscopy, and photoactivation and fluorescent labeling of cells for cell separation. In Aim 2, the sorted contractile subpopulations will be used to test the main biological hypothesis in vitro and in vivo against the alternative hypothesis. Engineered systems mimicking the environmental conditions in cancer progression will be designed to characterize the migration, proliferation, survival, and metabolism of the subpopulations, as well as their mechanical adaptation. The metastatic potential of these subpopulations and their mechanical adaptations at various stages along the metastatic cascade will be examined in a mouse tumor model. The innovative aspect of this proposal is the concept to sort by cellular contractility with the goal of uncovering the role of initial mechanical phenotype in the progression of diseases like cancer and in development. This project will use the novel engineered cell sorting approach to dissect the respective roles of mechanical heterogeneity and adaptability in disease progression, thus laying the foundation for future work to identify the key molecular pathways to precisely target for the development of mechanomedicine.
