C and 15 N natural isotope abundance reflects breast cancer cell metabolism OPEN

Breast cancer is the most common cancer in women worldwide. Despite the information provided by anatomopathological assessment and molecular markers (such as receptor expression ER, PR, HER2), breast cancer therapies and prognostics depend on the metabolic properties of tumor cells. However, metabolomics have not provided a robust and congruent biomarker yet, likely because individual metabolite contents are insufficient to encapsulate all of the alterations in metabolic fluxes. Here, we took advantage of natural 13 C and 15 N isotope abundance to show there are isotopic differences between healthy and cancer biopsy tissues or between healthy and malignant cultured cell lines. Isotope mass balance further suggests that these differences are mostly related to lipid metabolism, anaplerosis and urea cycle, three pathways known to be impacted in malignant cells. Our results demonstrate that the isotope signature is a good descriptor of metabolism since it integrates modifications in C partitioning and N excretion altogether. Our present study is thus a starting point to possible clinical applications such as patient screening and biopsy characterization in every cancer that is associated with metabolic changes. Medical applications of stable isotopes are now widespread, like the well-known 13 C-urea breath assay for ulcer detection 1. This takes advantage of 13 C-labelling and thus usually neglects differences in reaction rates between isotopic forms, because the isotopic signal used for diagnosing is far above small natural variations in 13 C. By contrast, the use of isotopes at natural abundance exploits such subtle differences (referred to as isotope effects) to identify bottlenecks in metabolic pathways (rate-limiting steps) or the contribution of multiple elemental sources (mass balance), without the need to introduce expensive isotope tracers into the patient. Isotope effects in metabolism are mostly caused by enzymatic reactions that preferentially consume substrates containing either the light or the heavy isotope (isotopologues) and therefore, the natural isotope abundance in metabolites depends on metabolic fluxes and source substrates 2. For example, the natural 13 C abundance in respired CO 2 has been used to trace diet composition and substrate changes during exercise 3,4. In cancer biology, the use of natural variations in Cu and S stable isotopes in hepatocellular carcinoma has been attempted recently 5. But to our knowledge, no study has looked at alterations of natural isotope abundance in breast cancer. Due to changes in primary C and N metabolism such as increased glycolysis, glutaminolysis and nucleotide synthesis 6 , important changes in 13 C and 15 N natural abundance can be anticipated. To address this question, we examined the isotopic signature of intact breast cancer biopsies (mostly from invasive ductal carcinoma, IDC) and cultured breast cancer cell lines (Supplementary Tables S1 and S2) using elemental analysis coupled to isotope ratio mass spectrometry (EA-IRMS). This technique has been recently shown to be applicable to the biochemical analysis of cancerous cell lines 7 .