Metabolic stress induces GD2⁺ cancer stem cell-like phenotype in triple-negative breast cancer

Background: Metabolic stress resulting from nutrient deficiency is one of the hallmarks of a growing tumour. Here, we tested the hypothesis that metabolic stress induces breast cancer stem-like cell (BCSC) phenotype in triple-negative breast cancer (TNBC). Methods: Flow cytometry for GD2 expression, mass spectrometry and Ingenuity Pathway Analysis for metabolomics, bioinformatics, in vitro tumorigenesis and in vivo models were used. Results: Serum/glucose deprivation not only increased stress markers but also enhanced GD2⁺ BCSC phenotype and function in TNBC cells. Global metabolomics profiling identified upregulation of glutathione biosynthesis in GD2^high cells, suggesting a role of glutamine in the BCSC phenotype. Cueing from the upregulation of the glutamine transporters in primary breast tumours, inhibition of glutamine uptake using small-molecule inhibitor V9302 reduced GD2⁺ cells by 70–80% and BCSC characteristics in TNBC cells. Mechanistic studies revealed inhibition of the mTOR pathway and induction of ferroptosis by V9302 in TNBC cells. Finally, inhibition of glutamine uptake significantly reduced in vivo tumour growth in a TNBC patient-derived xenograft model using NSG (non-obese diabetic/severe combined immunodeficiency with a complete null allele of the IL-2 receptor common gamma chain) mice. Conclusion: Here, we show metabolic stress results in GD2⁺ BCSC phenotype in TNBC and glutamine contributes to GD2⁺ phenotype, and targeting the glutamine transporters could complement conventional chemotherapy in TNBC.