Austin, Robert HamiltonHosny, Noreen2025-08-012025-08-012025-04-22https://theses-dissertations.princeton.edu/handle/88435/dsp01z890rx69fWhile metastasis accounts for the majority of cancer-related deaths, the mechanisms by which rare subpopulations of cancer cells complete the metastatic cascade remain poorly understood. Emerging evidence implicates polyaneuploid cancer cells (PACCs), a subpopulation that arises via endoreplication in response to environmental stressors such as hypoxia, as potential drivers of metastasis due to their enhanced survival and regenerative capabilities. However, the physical and molecular means by which PACCs acquire heightened metastatic potential remain unclear. Here, we used a membrane-based hypoxia culture system combined with live-cell imaging to first investigate how hypoxia-induced PACCs in prostate cancer gain motility and directional migration – two key features that predict a cell's ability to invade the surrounding tissue. Spatial mapping of oxygen levels across the system revealed the formation of gradients that simulate those found in the tumor microenvironment, allowing us to assess cell behavior under physiologically relevant conditions. To then validate that our system effectively induced the PACC state, we performed FACS-based DNA content analysis post-hypoxia, which revealed a nearly threefold increase in the proportion of PACCs (defined as cells with >4N genomic content). PC3-GFP prostate cancer cells were then tracked over 16 hours of self-induced hypoxia, and single-cell trajectories were quantified using a custom Cellpose/TrackMate pipeline. We then quantified directionality along the oxygen gradient to assess aerotaxis, a behavior that may enable cells to migrate toward the oxygen-rich bloodstream and facilitate intravasation during metastasis. We found that PACCs displayed significantly increased net displacement, total distance traveled, and directional migration toward higher oxygen concentrations relative to non-PACC and normoxic cells. These results suggest that PACCs may have a unique ability to complete the early, critical steps of invasion and intravasation in the metastatic cascade. To identify molecular drivers of these behaviors, we next investigated two key regulators of cancer cell motility under hypoxia: HIF-1α, which promotes transcriptional programs for survival and invasion, and RhoA, which controls actin cytoskeletal remodeling. Although prior studies suggest a regulatory relationship between these proteins to drive invasion, the directionality of this interaction appears to be cell type-specific. We therefore examined this relationship specifically in prostate cancer and assessed whether this pathway is particularly upregulated in PACCs. Single-cell tracking under hypoxia, followed by fixed-cell immunofluorescence microscopy, revealed that PACCs exhibit increased motility and oxygen-directed migration in a HIF-1α- and RhoA-dependent manner. Additionally, RhoA expression was found to be HIF-1α- dependent. Based on these findings, we propose a potential mechanism (HIF-1α → RhoA → motility/aerotaxis) that enables PACCs to escape hypoxic tumor cores, invade the surrounding tissue, and enter oxygen-rich vasculature. Altogether, these results suggest that PACCs are a uniquely invasive subpopulation and offer new insight into the molecular basis of prostate cancer metastasis, with implications for the development of targeted anti-metastatic therapies.en-USPrinceton University Senior Theses