Fig. 5

Fumarate accumulation increases ROS levels via forming succinic glutathione in hypoxic breast cancer TRCs. a Levels of α-ketoglutarate, succinate, fumarate and malate in hypoxic or normoxic breast TRCs were analyzed by LC-MS. b A metabolic model describing the flow of [U5]-¹³C-glutamine into the TCA cycle. c MCF-7 cells were seeded in 90 Pa soft 3D fibrin gels for 4 days under normoxic or hypoxic conditions, and then incubated with ¹³C-glutamine for another 24 h, LC-MS were performed for m + 5 isoform of α-KG and m + 4 isoforms of succinate, fumarate, and malate. d MCF-7 cells were seeded in 90 Pa soft 3D fibrin gels and treated with different doses of MOG, SAD, DMF, and malate for 5 days in hypoxia, ROS levels were determined by flow cytometry. e GSF levels of normoxic or hypoxic breast TRCs were analyzed by LC-MS as described in methods. f, g GSH/GSSG, NADPH/NADP+ratio from normoxic and hypoxic breast cancer TRCs were analyzed by assay kits as described in methods. h, i MCF-7 cells were seeded in soft 3D fibrin gels and treated with different doses of ethyl-GSH for 5 days in hypoxia, NADPH/NADP + ratio, ROS levels (h) and colony size (i) were analyzed. Scale bar, 20 μm. j, k MCF-7 cells were transfected with IDH3G siRNA and cultured in 90 Pa soft 3D fibrin gels under hypoxic condition for 5 days, the NADPH/NADP + ratio (j) and GSF (k) were analyzed. Data shown are representative of three independent experiments and error bars represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. NS, no significance