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. Female BALB/cAnNTac mice at 7–8 weeks old were mated and bilateral ovariectomy was performed 2 days post-partum. At 24 h after forced-weaning of pups to induce mammary involution, post-partum female mice were injected with either E2V, or vehicle control on alternative days for 2-weeks. On 48 h post-weaning, treated female mice were inoculated subcutaneously with 4 T1-Luc2 cells into the 9th abdominal mammary gland. Age-matched nulliparous female was treated similarly. Animals were euthanized on day 14 post-tumour inoculation for analysis. To evaluate the short-term effect of estrogen, post-partum females were treated with only one dose of E2V on day 12 post-tumour inoculation. 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 significant 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 48 h 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. 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.

中文翻译：

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雌激素显着降低循环低密度中性粒细胞并增强荷瘤小鼠中性粒细胞中促肿瘤基因的表达

中性粒细胞对肿瘤细胞的免疫监视很重要。中性粒细胞也可能在肿瘤微环境中进行表观遗传编程以促进肿瘤进展。除了众所周知的基于密度梯度分离的高密度中性粒细胞 (HDN) 之外，最近的研究还报道了荷瘤小鼠和癌症患者中存在高水平的低密度中性粒细胞 (LDN)。我们之前报道过，雌激素通过刺激乳腺组织中中性粒细胞的促肿瘤活性，促进了经历乳腺复旧的小鼠中雌激素受体 α 阴性乳腺肿瘤的生长。将 7-8 周龄的雌性 BALB/cAnNTac 小鼠交配，并在产后 2 天进行双侧卵巢切除术。在幼仔强制断奶后 24 h 诱导乳腺复旧，产后雌性小鼠在 2 周内每隔几天注射 E2V 或载体对照。在断奶后 48 小时，将 4 个 T1-Luc2 细胞皮下接种到经处理的雌性小鼠第 9 腹部乳腺中。年龄匹配的未生育女性也接受了类似的治疗。在肿瘤接种后第 14 天对动物实施安乐死以进行分析。为了评估雌激素的短期作用，产后女性在肿瘤接种后第 12 天仅接受一剂 E2V 治疗。雌激素治疗 2 周后，荷瘤未产和退化小鼠的血液 LDN 数量减少了 10 倍以上，而对血液 HDN 没有显着影响。对荷瘤小鼠的影响与骨髓中有丝分裂中性粒细胞数量减少和血液中性粒细胞凋亡增加有关。由于雌激素增强了退化小鼠的肿瘤生长，而不是未产小鼠，我们评估了雌激素对与中性粒细胞促肿瘤活性相关的基因表达的影响。虽然用雌激素处理 48 小时没有效果，但 2 周的处理显着增加了退化小鼠 HDN 和 LDN 中 Arg1、Il1b 和 Tgfb1 的表达。相比之下，雌激素增加了未经产小鼠 HDN 和 LDN 中 Arg1 和 Ccl5 的表达。荷瘤小鼠体内的长期雌激素刺激明显阻碍了 LDN 与肿瘤相关的增加，这似乎是通过抑制它们从骨髓的输出和缩短它们的寿命来实现的。雌激素还会改变中性粒细胞中的基因表达，这在无肿瘤小鼠中是看不到的。