Fig 2: Expression of genes related to hypoxia (Endothelial PAS Domain Protein 1, EPAS1), cellular quiescence and extracellular matrix remodelling (Quiescin Sulfhydryl Oxidase 1, QSOX1) is elevated in tumor-initiating cells (TICs)Expression of the EPAS1 gene at the mRNA level in breast non-malignant cell line MCF10A, in TICs derived from breast cancer cell lines MCF-7, BT-474, T-47D and ZR-75-30 as well as from prostate cancer cell lines DU-145 and LNCaP has been determined (A) together with protein levels in the MCF-7 cell line (CTRL) and MCF-7 derived spheres (SPH) (B). Similarly, the expression of the QSOX1 gene at the mRNA (C) level as well as protein level (D) in TICs is documented. Experiments were performed at least in triplicate, standard error is SEM, p-values lower than 0.05 are denoted with a star and were calculated by the GenEx software using the unpaired t-test and plotted with GraphPad prism software. Number sign denotes statistical significance involving Dun-Bonferroni correction. The protein expression was quantified by the image J software from 2 to 5 independent samples, standard error is SEM, p-values lower than 0.05 are denoted with a star and were calculated and plotted in GraphPad prism, using the unpaired t-test.

Fig 3: Putative scheme depicting changes in iron metabolism of tumor-initiating cells (TICs)TICs show higher levels of labile iron pool (LIP) and subsequently higher reactive oxygen species (ROS) which in connection with lower Fes cluster biogenesis (lower expression of ABCB10 and GLRX5) leading to accumulation of iron in mitochondria and possibly affecting genome stability and increasing plasticity of these cells. Alterations in the FeS cluster assembly lead to reduced activity of ACO1/IRP1 as well as mitochondrial respiratory complex I. The IRP binding activity of ACO1 is elevated resulting in higher TFRC and lower ferritin levels. Furthermore, TICs show an increase in the protein level of of EPAS1/ HIF2A connected with higher iron uptake (CYBRD1, TFRC) as well as with the extracellular matrix remodelling and redox balance equilibrium via the expression of QSOX1. On the other hand, reduced glutathione (GSH) is lower in TICs and these cells seem to be in a more pro-oxidative state and contain lower protein levels of IREB2/IRP2 likely reflecting increased LIP.