Motility as a Proxy for Metastasis: Hypoxia-induced Aerotaxis and RhoA-ROCK1 Activation in Ovarian Cancer

Abstract

Although metastasis accounts for approximately two-thirds of cancer-associated mortality, only a small subset of tumor cells acquires the traits necessary to complete the metastatic cascade. Oxygen deprivation within the tumor microenvironment imposes strong selective pressure, giving rise to morphologically and functionally distinct subpopulations with differential capacity for stress tolerance and migration. Among these, polyaneuploid cancer cells (PACCs) are a stress-adapted subpopulation that emerges via endoreduplication under hypoxia and has been implicated in driving metastatic behavior, though the mechanisms underlying their enhanced migratory and invasive capacity remain unclear. A prior study in prostate cancer demonstrated that hypoxia-induced PACCs exhibit increased motility and aerotaxis through HIF-1α-dependent RhoA activation linking cytoskeletal signaling to stress-adapted metastatic behavior. Whether this framework extends to other cancer types remains unclear, particularly given differences in metastatic dissemination routes. Prostate cancer metastasizes via hematogenous dissemination, with oxygen gradients along the vasculature orienting directed migration, whereas ovarian cancer metastasizes via transcoelomic dissemination, with tumor cells dispersing through peritoneal fluid to seed abdominal surfaces. These differences suggest that the selective advantage of aerotaxis, and the underlying HIF-1α- RhoA axis, may not be conserved across cancer types. Here, we used a membrane-based culture system with live-cell fluorescence imaging to investigate how self-generated hypoxia shapes PACC emergence and migration in ovarian cancer, recapitulating the O2 gradients of poorly perfused tumor regions in vivo. FACS-based DNA content analysis confirmed hypoxia-induced enrichment of ≥4N cells, validating our system. SKOv3-GFP cells were tracked over 21 hours using a custom Cellpose-SAM/TrackMate pipeline, and directionality along the oxygen gradient was quantified to assess aerotaxis, a migratory behavior that may enable ovarian cancer cells to navigate peritoneal oxygen gradients to locate and implant in oxygen-rich metastatic sites. Under extreme self-generated hypoxia, PACCs displayed significantly increased net displacement, directional migration, and aerotaxis relative to non-PACC hypoxic and normoxic cells. Subsequent immunofluorescence microscopy identified upregulation of the RhoA-ROCK1 signaling axis in PACCs, linking actomyosin- driven cytoskeletal remodeling to their enhanced motility and directed migration, and suggesting a mechanism by which PACCs escape hypoxic tumor cores. Together, these findings establish a context-dependent role for RhoA-ROCK1 signaling in hypoxia-induced PACC motility in ovarian cancer, positioning PACCs as a hypoxia-adapted subpopulation with implications for targeted anti-metastatic therapeutic development.