Contact Guidance During Breast Cancer Cell Dissemination

Abstract

Cancer invasion and metastasis are major life-threatening events in patients with cancer, yet the mechanisms by which cancer cells invade neighboring tissues remain poorly understood. It has been demonstrated that physical features of the dysregulated tumor extracellular matrix (ECM), such as fiber alignment, influence invasion phenotypes. Cells can polarize and migrate along aligned architectures, such as collagen fibers, through a process called contact guidance. In this dissertation, we investigate the mechanisms by which collectively migrating cancer cells sense and respond to fiber alignment.

This dissertation examines how collectively migrating breast cancer cells sense fibrous topographies. Chapter 2 details an adaptable collective cell migration assay we developed to study the effects of synthetic, electrospun fibers on collective migration behaviors. First, we described how to fabricate electrospun fiber mats using dextran vinyl sulfone (DexVS). Next, we describe a collective migration assay using cancer cell spheroids and DexVS fiber matrices. To quantify the migration phenotypes, we outlined the MATLAB code we developed to track cell migration over time. To demonstrate the capabilities of our system, we analyzed the collective migration behavior of an invasive breast cancer cell line on fibers oriented parallel to each other or randomly distributed. We explained how to calculate several migration parameters including cell speed, distance traveled, and directionality. In addition, we calculated the dispersal of individual cells from the migrating sheet and compared the migration phenotypes between individual cells and collectively migrating cells. Lastly, we report how to determine protein localization and expression after migration on fiber mats.

In chapter 3, we investigated the role of RhoA GTPase, a key regulator of the cytoskeleton and actomyosin contractility, during collective contact guidance. Our studies demonstrate that RhoA is crucial in the control of contact guidance in collectively migrating breast cancer cells by regulating cell-ECM and cell-cell adhesions. Using the methods outlined in Chapter 2, we found that the loss of RhoA resulted in decreased contact guidance and fractured cell-cell junctions in collectively migrating cells. We identified ROCK, a well-characterized RhoA effector, as being partially responsible for these effects. Notably, we determined that RhoA was dispensable for contact guidance in single cell migration. Additionally, we found that the loss of RhoA resulted in decreased focal adhesion lifetime during collective migration. Lastly, we discovered that RhoA was crucial for proper formation of both adherens junctions and desmosomes, two types of epithelial cell junctions.

Together, this dissertation provides a novel understanding of how cancer cells sense biophysical cues during invasive migration and moreover, uncovers a novel role for RhoA GTPase signaling during collective contact guidance.