Understanding natural isotopic variations in cultured cancer cells

Rationale

Natural variations in the abundance of the stable isotopes of nitrogen (δ¹⁵N) and carbon (δ¹³C) offer valuable insights into metabolic fluxes. In the wake of strong interest in cancer metabolism, recent research has revealed δ¹⁵N and δ¹³C variations in cancerous compared to non-cancerous tissues and cell lines. However, our understanding of natural isotopic variations in cultured mammalian cells, particularly in relation to metabolism, remains limited. This study aims to start addressing this gap using metabolic modulations in cells cultured under controlled conditions.

Methods

Prostate cancer cells (PC3) were cultured in different conditions and their δ¹⁵N and δ¹³C were measured using isotope ratio mass spectrometry. Isotopic variations during successive cell culture passages were assessed and two widely used cell culture media (RPMI and DMEM) were compared. Metabolism was modulated through glutamine deprivation and hypoxia.

Results

Successive cell culture passages generally resulted in reproducible δ¹⁵N and δ¹³C values. The impact of culture medium composition on δ¹⁵N and δ¹³C of the cells highlights the importance of maintaining a consistent medium composition across conditions whenever possible. Glutamine deprivation and hypoxia induced a lower δ¹³C in bulk cell samples, with only the former affecting δ¹⁵N. Gaps between theory and experiments were bridged and the lessons learned throughout the process are provided.

Conclusions

Exposing cultured cancer cells to hypoxia allowed us to further investigate the relation between metabolic modulations and natural isotopic variations, while mitigating the confounding impact of changing culture medium composition. This study highlights the potential of natural δ¹³C variations for studying substrate fluxes and nutrient allocation in reproducible culture conditions. Considering cell yield and culture medium composition is pivotal to the success of this approach.