Field of Biochemistry, Molecular and Cell Biology Ph.D. Thesis Defense Seminar – Richa Agrawal, Lammerding Lab. 

Friday, April 26, 2024, 1:00 PM – 125 Weill Hall. 

“Design and Use of a Microfluidic Device to Understand the Consequences of Persistent Confined Migration”.

In vivo, only a small fraction of cells are successfully able to form the complete metastatic cascade, including leaving the primary tumor, travelling through the blood stream, extravasating into a distal tissue, and colonizing a secondary site. During the metastatic cascade, cells must tolerate numerous mechanical stresses, such as compressive stress in the primary tumor, migration through the dense origin and distal tissues including through collagen tracks, shear stress in the circulatory system, and migration through confined spaces as small as 1-2 µm in the basement membrane lining organs and blood vessels. In the last two decades, it has become clear that this confined migration (CM) requires severe nuclear deformation, and can result in nuclear rupture, replication stress, and DNA-damage. To date, the immediate and long-term consequences of persistent confined migration are still becoming clear, but have been limited by the availability of existing methods to study individual cell fates during migration through multiple pores to determine the balance of “selection” vs. “adaptation” within the population, as well as incompatibility with harvest of successful cells for other downstream characterization without expansion. Here, I describe the development and use of a microfluidic device and protocol to overcome these limitations which is capable of tracking individual cell fates to quantify the fraction which apoptose during transit, but which is also compatible with collecting cells after migration for functional and sequencing-based studies. As proof of principle, we subjected MDA-MB-231 breast cancer cells to varying densities of confinement and performed bulk mRNA sequencing. We demonstrate that cells are “selected”, as a small fraction of cells apoptose during transit. The remaining portion of cells which survive confinement only transiently alter their transcriptional signature and display indicators of cellular stress such as increased baseline autophagic flux and p62 accumulation, indicating that MDA-MB-231 cells primarily withstand the associated stresses of persistent confined migration.

Publications:

1. Agrawal,A. Varlet, S. Henretta, M. Elpers, J. Morival, J. Lammerding, Accrued stress during confined migration leads to divergent cell fates in MDA-MB-231 cells. (in preparation).
2. Agrawal, A. Windsor, J. Lammerding, Assembly and use of a microfluidic device to study confined migration. Methods Mol. Biol. (2022).
3. C.R. Hsia, Jawuanna McCallister, Ovais Hasan, Julius Judd, Seoyeon Lee,  R. Agrawal, C. Chang, C. Feschotte, P. Soloway, J. Lammerding, Confined migration induces heterochromatin formation and alters chromatin accessibility. iScience. (2022).