Protocol to model tumor hypoxia in vitro using real-time phosphorescence-based sensing of O2 gradients generated by metastatic cancer cells
Tumor hypoxia plays a critical role in cancer progression and therapeutic resistance. Here, we present a protocol for the self-generation of hypoxia by metastatic cancer cells using phosphorescence-based O<sub>2</sub> sensing. We describe steps for phosphorescent film calibration, system assembly, and real-time imaging of hypoxia development. We then detail approaches to spatially map resulting O<sub>2</sub> gradients. This protocol supports applications for studying key behaviors linked to metastatic progression, including motility and aerotaxis, under physiologically relevant hypoxic conditions. For complete details on the use and execution of this protocol, please refer to Hosny et al.<sup>1</sup>.
Authors
- Sarah R. Amend (ORCID: https://orcid.org/0000-0002-5606-1262)
- Trung V. Phan (ORCID: https://orcid.org/0000-0001-7891-8119)
- Robert A. Gatenby (ORCID: https://orcid.org/0000-0002-1621-1510)
- Junle Qu (ORCID: https://orcid.org/0000-0001-7833-4711)
- Yihua Zhao (ORCID: https://orcid.org/0000-0003-4471-2379)
- Yusha Sun (ORCID: https://orcid.org/0000-0003-4835-3000)
- Emma U. Hammarlund (ORCID: https://orcid.org/0000-0001-7625-4793)
- George B. Butler (ORCID: https://orcid.org/0000-0002-6207-6225)
- Robert H. Austin (ORCID: https://orcid.org/0000-0002-4269-6793)
- Joel S. Brown (ORCID: https://orcid.org/0000-0002-0958-9494)
- Kenneth J. Pienta (ORCID: https://orcid.org/0000-0002-4138-2186)
- Shengkai Li (ORCID: https://orcid.org/0000-0003-1867-6802)
- Kimberly Shen
- Noreen Hosny (ORCID: https://orcid.org/0009-0001-1804-252X)
- Stephano Boyer-Paulet
Institutions
- Scripps College (US)
- Shenzhen University (CN)
- Lund University (SE)
- Princeton University (US)
- Moffitt Cancer Center (US)
- Sidney Kimmel Comprehensive Cancer Center (US)
Publication Details
- Journal
- STAR Protocols
- Published
- 2025-10-28
- DOI
- https://doi.org/10.1016/j.xpro.2025.104163
- Primary Topic
- Cancer, Hypoxia, and Metabolism
- Type
- article
- Field-Weighted Citation Impact
- 0.00