Geomedical application of copper isotope ratios: change of δ65Cu in xenograft model of human cancers

In geosciences, high precision isotope ratio determination provides essential information about processes in geological systems. Novel ambitions evolve closer to biological applications. Copper is an essential metal for human body taking part of several cellular processes (e.g. respiratory chain, enzyme function, iron metabolism, elimination of reactive oxygen species, cell signalling pathways etc). However, the disorder of copper homeostasis causes serious diseases like Wilson disease (Cu accumulation in liver caused by genetical disorder) and it could also promote tumour growth by supporting angiogenesis and metastasis formation [Denoyer et al., 2015]. Despite numerous experiments, focusing on copper concentration determination in different tumour tissues (e.g. breast, lung cancer, etc.) hoping to assist in tumour diagnosis, the results are not convincing enough. However, previous studies on hepatocellular cancer and oral squamous cell carcinoma showed that tumour tissue appears to be relatively enriched in ⁶⁵Cu compared to normal tissue whereas the δ⁶⁵Cu in blood of tumorous patient decreased according to data obtained from control population [Balter et al., 2015, Lobo et al., 2017]. Our main aim is to elaborate a method to understand better the change in ⁶³Cu/⁶⁵Cu stable isotope ratio during tumour growth. In this approach, we present our first results on copper isotope ratio determination in a xenograft mouse model. Our model was established in SCID (severe combined immunodeficiency disease) mice by injecting human cancer cells (1x10⁷ cells) subcutaneously. After the tumour reached approximately 2-3 cm diameter, the tumour mass was cut it in small, equal pieces and transplanted further into 10 mice increasing the experimental set-up homogeneity. All the animals were sacrificed by cardiac puncture under deep terminal anaesthesia within four weeks. Tumour and organs were removed by ceramic knife then were frozen with liquid nitrogen and stored at -80°C. We measured the copper concentration and δ⁶⁵Cu in the tumour tissue, blood, liver, kidney and brain. A clean laboratory ambience was chosen to perform the sample preparation processes decreasing the environmental contamination. Separation of copper from other biologically essential element (Na, Mg, Fe, Zn) interfering the copper isotope measurement is a serious condition of the preparation [Lauwens et al., 2017]. Effects of sodium (²³Na⁴⁰Ar⁺) and magnesium (²⁵Mg⁴⁰Ar⁺) on copper isotope ratio were solved by choosing not the peak center but the interference-free plateau. Our measurements have been carried out on a Thermo Neptune PLUS multicollector mass spectrometer equipped with 9 moveable Faraday detectors, 3 amplifiers with a resistance of 10¹³ Ohm, and 6 amplifiers with a resistance of 10¹¹ Ohm, in wet plasma conditions. The mass spectrometric measurement of the copper isotope ratio is doped either with Ni or Ga reference material which have a well-known isotope ratios.

 

References:

Balter V. et al. PNAS 2015; 112: 982−985.

Denoyer D. et al. Metallomics 2015; 7: 1459−76.

Lauwens S. et al. J. Anal. At. Spectrom. 2017; 32: 597−608.

Lobo L. et al. Talanta 2017; 165: 92−97.