Cancer metastasis, the spread of cancer from the primary site to distant regions in the body, is the major cause of cancer mortality, accounting for almost 90% of cancer related deaths. During metastasis, cancer cells from the primary tumor initially probe the surrounding fibrous tumor microenvironment (TME) prior to detaching and subsequently migrating towards the blood vessels for further dissemination. It has widely been acknowledged that the biophysical cues provided by the fibrous TME greatly facilitate the metastatic cascade. Consequently, there has been a tremendous wealth of work devoted towards elucidating different modes of cancer cell migration. However, our knowledge of how cancer cells at the primary tumor site initially sense their fibrous surroundings prior to making the decision to detach and migrate remains in infancy. In part, this is due to the lack of a fibrous in vitro platform that allows for precise, repeatable manipulation of fiber characteristics. In this study, we use the non-electrospinning, Spinneret based Tunable Engineered Parameters (STEP) technique to manufacture suspended nanofiber networks with exquisite control on fiber dimensions and network architecture and use these networks to investigate how single cancer cells biophysically sense fibers mimicking in vivo dimensions. Using high spatiotemporal resolution imaging (63x magnification/1-second imaging interval), we report for the first time, that cancer cells sense individual fibers by coiling (i.e. wrapping around the fiber axis) at the tip of a cell protrusion. We find that coiling dynamics are mediated by both the fiber curvature and the metastatic capacity of the cancer cells with less aggressive cancer cells showing diminished coiling. Based on these results, we explore the possibility of using coiling in conjunction with other key biophysical metrics such as cell migration dynamics and forces exerted in the development of a genetic marker independent, biophysical predictive tool for disease progression. Finally, we identify the membrane curvature sensing Insulin Receptor tyrosine kinase Substrate protein of 53 kDa (IRSp53) as a key regulator of protrusive activity with IRSp53 knockout (KO) cells exhibiting significantly slower protrusion dynamics and diminished coil width compared to their wild-type (WT) counterparts. We demonstrate that the hindered protrusive activity ultimately translates to impaired contractility, alteration in the nucleus shape and slower migration dynamics, thus highlighting the unique role of IRSp53 as a signal transducer – linking the protrusive activity at the cell membrane to changes in cytoskeletal contractility. Overall, these findings offer novel perspectives to our understanding of how cancer cells biophysically sense their fibrous surroundings. The results from this study could ultimately pave the way for elucidating the precise fiber configurations that either facilitate or hinder cancer cell invasion, allowing for the development of new therapeutics in the long term that could inhibit the metastatic cascade at a relatively nascent stage and yield a more promising prognosis in the perennial fight against cancer.
Doctor of Philosophy
Cancer is a leading cause of death worldwide. Almost ninety percent of cancer related deaths arise from the spreading of cancer cells from the primary tumor site to secondary sites in the body – a processed termed as metastasis. The environment surrounding a tumor (tumor microenvironment) is highly fibrous in nature and can assist in the metastatic process by providing biophysical cues to the cells at the tumor boundary. These cells sense the presence of the surrounding fibers by extending "arms" termed as protrusions, and then eventually detach from the primary tumor and start migrating through the fibrous microenvironment. While numerous studies have investigated the various modes of cell migration in fibrous environments, there is very little information regarding how cancer cells use protrusions to initially sense the fibers prior to detaching. In this study, we used the Spinneret based Tunable Engineered Parameters (STEP) technique to manufacture suspended nanofiber networks with robust control on fiber diameter and network architecture and use these networks to systematically investigate how single cancer cells biophysically sense fibers that mimic in vivo dimensions. We discovered that cancer cells sense individual fibers by "wrapping-around" the axis of the fiber at the tip of the protrusion – a phenomenon we refer to as coiling. We found both the fiber diameter as well as the invasive capacity of cells can influence the coiling mechanics. Based on these results, we explored the use of coiling in conjunction with other key biophysical metrics such as the cell migration speed and how much force a cell can exert to develop a biophysical predictor for cancer cell aggressiveness. Finally, given that cells sense the fiber curvature by coiling, we explored the role of a key curvature sensing protein Insulin Receptor tyrosine kinase Substrate protein of 53 kDa (IRSp53) in mediating coiling activity and found that knocking out (KO) IRSp53 results in reduced coiling and slower protrusions compared to wild-type (WT) cells. Furthermore, IRSp53 KO cells showed impaired contractility which led to an alteration in the nucleus shape and slower migration dynamics thus highlighting the role of IRSp53 in linking changes at the cell membrane to the underlying cell cytoskeleton. The results from this study could ultimately help us understand what type of fiber conditions around a primary tumor would either help or delay the emergence of the tumor boundary cells and thus allow for the development of therapeutics that could significantly slow down the metastatic process at a relatively early stage.