Physiological Oxygen and Media Conditions Influence Breast Cancer Hallmarks in Vitro

Standard cell culture conditions often fail to replicate the in vivo microenvironment, limiting the reproducibility and physiological relevance of experimental findings. For example, commonly used media such as Dulbecco’s Modified Eagle Medium (DMEM) contain supraphysiological levels of key metabolites like glucose while lacking others entirely. Additionally, standard cell culture incubators maintain near-atmospheric oxygen levels (~18% O2), far exceeding the physiological oxygen levels (2–9% O2) typical of most tissues. Despite their significance, these factors are often studied independently. The main objective of this thesis was to investigate the effects of both media and O2 on breast cancer cells in culture, particularly focusing on cancer hallmarks at the levels of gene expression, metabolism, and proliferation. To that end, I cultured MCF7 cells in 18% O2 or 5% O2 and in DMEM or Plasmax, a recently developed medium with metabolite levels designed to mimic human blood plasma. Using RNA-sequencing and proteomics, I identified widespread changes in gene expression driven by O2 and media at the mRNA and protein levels, with a strong enrichment of genes associated with proliferation, migration, and glucose metabolism. These molecular changes translated into functional outcomes, with physiologically relevant conditions generally enhancing cell migration, glucose uptake, and metabolic activity. Additionally, I developed and optimized a novel protocol for performing Seahorse metabolic flux analysis under hypoxic conditions, enabling more accurate assessment of metabolism in low-O₂ environments. Collectively, our results demonstrate that both oxygen levels and media composition profoundly influence cancer cell behavior, underscoring the necessity of implementing physiologically relevant culture conditions in in vitro cancer research.

Name thesis:degree_name

M.Sc. Biological Sciences

Level thesis:degree_level

Master

Discipline thesis:degree_discipline

Faculty of Mathematics and Science

Department dc:contributor.department

Department of Biological Sciences

Grantor dc:publisher

Brock University

Year dc:date.issued

2026

Author dc:creator

• Wiebe, Jacob

Advisor dc:contributor.advisor

• Stuart, Jeff

• NATURAL SCIENCES::Biology::Cell and molecular biology::Cell biology
• NATURAL SCIENCES::Biology::Cell and molecular biology::Molecular biology
• NATURAL SCIENCES::Biology::Other biology

Language dc:language.iso

eng

Handle dc:identifier.uri

https://hdl.handle.net/10464/20005

OAI identifier oai:identifier

oai:brocku.scholaris.ca:10464/20005