Establishment of cell model of breast cancer-associated fibroblasts’ energy metabolism reprogramming induced by hypoxia

Abstract:

Objective：To establish the cell model of energy metabolism reprogramming（EMR） of breast cancer-associated fibroblasts（CAF） induced by hypoxia and to lay a firm foundation for subsequently exploring its molecular mechanism and biological functions. Methods：Based on the O2 concentration，paired immortalized normal fibroblasts（NF） and CAF derived from the same breast cancer patient were divided into hypoxic group and normoxic group. NF/CAF were treated with different O2 concentrations for 0，1，3，6，12 and 24 h. The glycolysis activity of NF and CAF was tested by the glucose consumption assay and the lactate generation assay；the oxidative phosphorylation activity of NF and CAF was detected by the mitochondrial activity assay；the protein expression levels of MCT4，COX Ⅳ and HIF1α were determined by Western blot；the data were analyzed with the SPSS 17.0 software. Results：There was dose-effect and time-effect between hypoxic levels and the EMR degree of CAF. 1%[O2] for 24 h was optimum for inducing the EMR of CAF. Hypoxic treatment increased the glycolysis activity，suppressed the oxidative phosphorylation levels，in－creased the protein expression of MCT4 and HIF1α 2 times or 3.9 times respectively，and decreased the protein expression of COX Ⅳ by 10% in CAF compared with NF. Conclusion：Hypoxic treatment（1%[O2] for 24 h） maked CAF have a typical EMR pheno－type. It is successfully established that the cell model of the EMR of breast cancer-derived CAF induced by hypoxia（1%[O2] for 24 h）.