Physiological media and oxygen conditions promote transcriptomic, proteomic, and functional changes in breast cancer cells

Cell culture is a foundational tool for research investigating cell physiology. However, it is not without drawbacks. Standard cell culture conditions may not fully recapitulate the in vivo microenvironment of individual tissues, limiting the physiological relevance of experimental findings. For example, traditional media such as DMEM contain supraphysiological levels of key metabolites such as glucose, while completely lacking others. Further, standard cell culture incubators expose cells to near-atmospheric oxygen levels (~18% O 2 ) that significantly exceed those experienced by most cells in vivo (2-9% O 2 ). However, these two factors, media composition and oxygen levels, are virtually always investigated separately. Here, we investigated the impact of both media composition and oxygen levels on the physiology of breast cancer cells. We performed a series of transcriptomic, proteomic, and functional analyses of MCF7 cells grown in 18% O 2 or 5% O 2 (physioxia) and in DMEM or Plasmax, a physiological medium modeled after human plasma metabolome. RNA-seq data demonstrated both O 2 - and media-dependent trends in gene expression, particularly involving pathways associated with cell cycle regulation, metabolism, and redox homeostasis. While the overlap between differentially expressed genes and proteins was low, pathways related to proliferation and energy metabolism were also enriched in the proteomic analysis. Follow-up functional assays identified differences in proliferation, migration, metabolic activity, glucose uptake, and reactive oxygen species production associated with different culture conditions. Generally, physiological culture conditions promoted cell proliferation, migration, and metabolic activity in MCF7 cells. Our results indicate that both O 2 and media have robust and widespread effects on cancer cell biology and highlight the importance of implementing physiologically relevant conditions in research. Research funded by a Natural Science and Engineering Council of Canada Discovery Grant to JAS. JW is supported by an Ontario Graduate Scholarship. RA is supported by MITACS. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.