Estrogen Markedly Reduces Circulating Low-density Neutrophils and Enhances Pro-tumoral Gene Expression in Neutrophil of Tumour-bearing Mice

Background: Neutrophils are important for immune surveillance of tumour cells. Neutrophils may also be epigenetically programmed in the tumour microenvironment to promote tumour progression. In addition to the commonly known high-density neutrophils (HDN) based on their separation on density gradient, recent studies have reported the presence of high levels of low-density neutrophils (LDN) in tumour-bearing mice and cancer patients. We reported previously that estrogen promotes the growth of estrogen receptor α-negative mammary tumours in mice undergoing mammary involution through stimulating pro-tumoral activities of neutrophils in the mammary tissue. Methods: : Female BALB/cAnNTac mice at 7-8 weeks old were mated and bilateral ovariectomy performed two days post-partum. At 24h post-weaning, mice were treated with either E2V, or vehicle control once every 2 days. On 48h post-weaning, mice were inoculated subcutaneously with 4T1-Luc2 cells into the 9 th abdominal mammary gland. Age-matched nulliparous female was treated similarly. For 48h E2V treatment, mice were treated with one dose of E2V on day 12 post-tumour inoculation. Animals were euthanized on day 14 post-tumour inoculation for analysis. Results: : Estrogen treatment for 2-weeks reduces the number of blood LDN by more than 10-fold in tumour-bearing nulliparous and involuting mice, whilst it had no effect on blood HDN. The effect on tumour-bearing mice is associated with reduced number of mitotic neutrophils in the bone marrow and increased apoptosis in blood neutrophils. Since estrogen enhanced tumour growth in involuting mice, but not in nulliparous mice, we assessed the effect of estrogen on the gene expression associated with pro-tumoral activities of neutrophils. Whilst 48h treatment with estrogen had no effect, 2-weeks treatment significantly increased the expression of Arg1 , Il1b and Tgfb1 in both HDN and LDN of involuting mice. In contrast, estrogen increased the expression of Arg1 and Ccl5 in HDN and LDN of nulliparous mice. Conclusions: : Prolonged estrogenic stimulation in tumour-bearing mice markedly hampered tumour-associated increase of LDN plausibly by inhibiting their output from the bone marrow and by shortening their life span. Estrogen also alters the gene expression in neutrophils that is not seen in tumour-free mice. The results imply that estrogen may significantly influence the tumour-modulating activity of blood neutrophils.