Fig. 4

Estrogen treatment increases LDN cell death and reduces bone marrow neutrophil production in tumour-bearing Inv mice. A-C, At Inv D2, OVX mice were inoculated with 4 T1-Luc2 cells at the 9th abdominal mammary gland. At 48 h prior to sample collection, mice were treated with either E2V or Ctrl. Subsequently, at 24 h before collection, mice were administered with BrdU. Mice were euthanized and tumour harvested at day 14 post-tumour inoculation. A, 48 h estrogen treatment did not affect the percentage of newly generated circulating LDN and HDN in the blood; (i) Percentage of BrdU⁺PI⁺ cells out of total circulating LDN (CD45⁺CD11b⁺Ly6G⁺), (ii) Percentage of BrdU⁺PI⁺ cells out of total circulating HDN (CD45⁺CD11b⁺Ly6G⁺) via flow cytometry analysis, Inv + Ctrl n = 3, Inv + E2V n = 4. B, 48 h estrogen treatment reduces the amount of newly generated neutrophils in the bone marrow (BM); Percentage of BrdU⁺PI⁺ cells out of total BM neutrophils (CD45⁺CD11b⁺Ly6G⁺) via flow cytometry analysis, Inv + Ctrl n = 3, Inv + E2V n = 4. C, 48 h estrogen treatment increases circulating LDN cell death; (i) Percentage of dead cells (Annexin V⁺ Dead stain⁺) out of total circulating LDN (CD45⁺CD11b⁺Ly6G⁺), (ii) Percentage of early apoptotic cells (Annexin V⁺ Dead stain⁻) out of total circulating LDN via flow cytometry analysis. Inv + Ctrl n = 3, Inv + E2V n = 4. D-E, Experiment is conducted according to the description in Fig. 1. D, Estrogen treatment led to a reduction in splenic neutrophils in tumour-bearing Null and Inv mice; Percentage of splenic neutrophils (CD45⁺CD11b⁺Ly6G⁺) out of viable CD45⁺ population via flow cytometry analysis, Null+Ctrl (N.T) n = 3, Null+Ctrl n = 5, Null+E2V n = 5, Inv + Ctrl n = 5, Inv + E2V n = 6. E, Estrogen treatment did not result in any significant changes in tumour neutrophils; Percentage of neutrophils (CD45⁺CD11b⁺Ly6G⁺) out of viable CD45⁺ population in tumour via flow cytometry analysis, Null+Ctrl n = 5, Null+E2V n = 5, Inv + Ctrl n = 5, Inv + E2V n = 6. Data represented as mean ± SEM. Statistical significances determined using two-tailed unpaired student’s T-test in (A-C); one-way ANOVA followed by post-hoc tukey test in (D and E)