Abstract Metastasis accounts for the majority of cancer-related deaths, yet only a rare subset of tumor cells can successfully complete this process. Among these, polyaneuploid cancer cells (PACCs), which arise via endoreplication in response to stressors such as hypoxia, have been implicated as stress-resistant drivers of metastasis. In prostate cancer, the presence of PACCs within primary tumors correlates with poor prognosis and reduced metastasis-free survival, and these cells are consistently detected in metastatic lesions from patients. However, identifying PACCs and reproducibly modeling their formation in vitro remain major challenges to understanding their metastatic potential. Here, we build upon our validated in vitro model of tumor hypoxia to develop a reliable system for PACC induction and detection within prostate cancer. This membrane-based culture system permits the self-generated development of hypoxia as prostate cancer cells consume a limited amount of oxygen, while a phosphorescent oxygen-sensing film enables real-time, spatially resolved mapping of oxygen distribution across the culture. Under these conditions, heterogeneous populations of PACC and non-PACC cells emerge along an oxygen gradient, with cells at the core of the system experiencing the most severe hypoxia, thus effectively recapitulating the tumor microenvironment. This approach represents a marked departure from traditional PACC models, which rely on high doses of chemotherapy or hypoxia-mimetic agents to artificially induce their formation. To validate PACC induction, we performed flow cytometric DNA content analysis following 16 hours of hypoxia and observed a nearly threefold increase in the proportion of cells exceeding 4N genomic content. These results suggest that the tumor model promotes the emergence of prostate cancer-derived PACCs. To enable live detection of PACCs without DNA-binding dyes that may interfere with replication and cause phototoxicity, we established a morphology-based criterion. Cells exceeding 1500 µm2 in area and demonstrating ≥3-fold growth over 16 hours corresponded to the polyaneuploid population identified by conventional nuclear area analysis following DNA dye staining. Preliminary single-cell tracking using this classification showed that PACCs exhibit greater net and total displacements than non-PACCs under hypoxia, which are behaviors consistent with successful invasion during metastasis. Altogether, this system provides a physiologically-relevant model for inducing and identifying prostate cancer-derived PACCs in vitro. Coupled with the robust criteria for real-time PACC detection, this model establishes a foundation for future work to investigate the mechanisms by which PACCs may promote metastatic progression in prostate cancer. Citation Format: Noreen Hosny, Shengkai Li, Sarah Amend, Robert Gatenby, Kenneth J. Pienta, Joel Brown, Junle Qu, Robert H. Austin. In vitro tumor hypoxia model enables induction and real-time detection of polyaneuploid cancer cells (PACCs) in prostate cancer abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Prostate Cancer Research and Treatment; 2026 Jan 20-22; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86 (2Suppl): Abstract nr B030.
Hosny et al. (Tue,) studied this question.