Fig. 4.

Aberrant Pfkfb3 expression drives emergence from metastatic dormancy and increased BCSC frequency. a D2.OR and D2.A1 cells were incubated in the absence or presence of the Pfkfb3 inhibitor, 3PO (0–500 μM), and differences in cell viability were quantified by CellTiter-Glo Assay. Data are the mean (±SD; n = 3). **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. This experiment was independently repeated for a total of three experiments, all with similar results. b Pfkfb3 overexpression drives D2.OR organoid growth in 3D cultures, while Pfkfb3-deficiency suppresses the growth of D2.A1 organoids in 3D culture, c, d Parental and Pfkfb3-deficient D2.A1 cells (1 × 10⁶ cells/injection) were inoculated into the lateral tail veins of BALB/c mice (n = 5). Pulmonary tumor growth was monitored by BLI and normalized to Day 0 readings. IHC images (×20 magnification) show loss of Pfkfb3 knockdown in tumors excised on Day 72. e Parental and Pfkfb3-overexpressing D2.OR cells or f parental and Pfkfb3-deficient D2.A1 cells were monitored for mammosphere forming ability by ELDA (n = 8). Stem cell frequency was evaluated using the ELDA: Extreme Limiting Dilution Analysis (http://bioinf.wehi.edu.au/software/elda/). These experiments were independently repeated for a total of three experiments, all with similar results. g Evaluating CD49f/CD24 expression levels by flow cytometry in parental and Pfkfb3-overexpressing D2.OR cells and in parental and Pfkfb3-deficient D2.A1 cells