Fig. 3.
The mechanism responsible for the increased stemness by MDSC. a Effects of MDSC on the induction of CSC in vitro. HM-1 cells (3 × 105 cells /well) were cultured with MDSC or splenocytes (excluding MDSC) (3 × 104 cells /well) in the presence of 0.1% FBS for 18 h in 6-well dishes. The mouse EpCAM+ CD45− cells were gated using flow cytometry and then the percentages of ALDH-high cells were assessed using the Aldefluor assay (n = 5). Bars, SD. **p < 0.01, according to two-sided Student’s t test. b Production of PGE2 by MDSC. MDSC that had been isolated from spleens of mice bearing HM-1-GCSF-derived tumors were cultured in serum-free medium. Splenocytes (excluding MDSC) were also used for comparison. The PGE2 concentrations in the culture media were measured by the Prostaglandin E2 Express ELISA Kit (n = 3). Bars, SD. **p < 0.01, according to two-sided Student’s t test. c PGE2 expression in HM-1-Control cell- or HM-1-GCSF cell-derived tumors. Scale bar, 50 μm. d Expression levels of EP2 and EP4 receptors in HM-1 cells. Ep2 and Ep4 receptors and Gapdh mRNA levels in HM-1 cells assessed by RT-PCR. e Effects of PGE2 on the induction of CSC in vitro. HM-1 cells were treated with PGE2 (50 ng/mL) with or without EP4 antagonist (200 nM) in the presence of 0.1% FBS for 18 h. The frequencies of ALDH-high HM-1 cells were assessed using an Aldefluor assay (n = 5) Bars, SD. **p < 0.01, according to two-sided Student’s t test. f Effect of PGE2-inhibition on the MDSC-mediated CSC induction. HM-1 cells (3 × 105 cells /well) and MDSC (3 × 104 cells /well) were co-cultured in 6-well dishes and treated either with celecoxib (20 μm) or EP4 antagonist (200 nM) in the presence of 0.1% FBS for 18 h in vitro. The mouse EpCAM+ CD45− cells were gated using flow cytometry and then the percentages of ALDH-high cells were assessed using the Aldefluor assay (n = 5). Bars, SD. **p < 0.01, according to two-sided Student’s t test. g Serum PGE2 concentrations in ovarian cancer patients with or without leukocytosis were measured using the Prostaglandin E2 Express ELISA Kit. *p < 0.05, according to Welch’s t test