INTRACELLULAR FORCES AND NUCLEAR MECHANOBIOLOGY DURING CONFINED CELL MIGRATION IN 3-DIMENSIONS

232 pagesSupplemental file(s) description: None.Cell migration is a required step in many biological and pathological processes. During wound healing, embryogenesis, and immune surveillance, cells migrate to sites where they can repair, develop, and protect tissues, which are all essential for maintaining healthy physiology. In contrast, during cancer metastasis, individual cancer cells escape from the primary tumor and migrate into blood and lymphatic vessels, where they spread through the body to form secondary tumors. This process of cancer metastasis is responsible for 90% of cancer related deaths. Despite its centrality to these essential phenomena, there is still much that is unknown about how cells migrate in vivo. The nucleus limits the rate at which cells can migrate through tissues, as it is the largest and stiffest organelle in the cell. As cells move through narrow spaces in the dense matrix of endothelial cell layers and extracellular fibers that make up biological tissues, they must apply considerable intracellular force to squeeze the nucleus through these spaces. It is still unclear how exactly cells apply sufficient force to complete this deformation of the nucleus through constrictions. Here, we established a microfluidic platform for studying nuclear deformation through precisely defined constrictions. Using this platform, we demonstrate evidence of a novel mechanism by which cells push their nucleus through constrictions through contraction of the rear cortex. We subsequently demonstrate that contraction of the rear cortex induces nuclear blebbing in cancer cells through pressure-driven nuclear influx. This rear cortex-contraction mechanism is significant as it represents a previously unrecognized mechanism which enables confined migration, opening future avenues of study which can inform the treatment of metastatic cancer. We also used this microfluidic platform to evaluate the viability of engineered enucleated mesenchymal stromal cells (MSCs) as a targeted therapeutic delivery system. This work revealed that enucleated cells showed improved invasive ability in comparison to their nucleus-containing counterparts. These findings are significant in that they both support the use of these enucleated MSCs as a targeted therapeutic delivery platform, and that they revealed that cells do not require a nucleus to migrate through 3D constrictions. Later, we assessed the impact of nuclear size restoring compounds on the invasion of prostate cancer cells. This work identified compounds which reversed nuclear size defects commonly observed in cancer cells, and reduced migration in 2D wound healing assays and Boyden chambers, but not through microfluidic constrictions. While ample evidence has shown that the deformability of the nucleus limits its ability to squeeze through constrictions, changes in nuclear size have not shown a consistent relationship with cell’s invasive ability. These findings are significant as they provide evidence that a variety of compounds may present viable treatments for reducing the metastatic capacity of some cancer cell lines. However, it is unclear whether the principal mechanism which reduces migration is directly related to the observed changes in nuclear size. This thesis expands the existing knowledge on the role of the nucleus in confined migration, and the impact of intracellular force generation on nuclear movement