Abstract 3181: Nicotine promotes pre-metastatic niche formation in hormone receptor negative breast cancer through paracrine signaling in the tumor microenvironment

Hormone receptor negative breast cancer is responsible for a majority of the mortalities through aggressive metastatic spread, which accounts for a poor patient survival. Increasing epidemiological evidence indicates significant risk of pulmonary metastasis among smokers in breast cancer. However, little is known whether smoking or cessation affects clinical outcomes in breast cancer with pulmonary metastasis particularly in patients with hormone receptor negative status. To address this question, we employed spontaneous and experimental pre-metastasis in syngeneic mice model with or without i.p. injection of nicotine in line with human blood cotinine level and measured metastatic progression. We found that nicotine promoted the pulmonary metastasis burden by 100-fold in vivo. Immune profiling of the pulmonary metastatic lesions showed predominant infiltration of neutrophils compared to primary tumors. Notably, mice pre-exposed to nicotine showed a significantly increased number of neutrophils in the lungs even before injection of cancer cells (pre-metastatic lung). Remarkably, neutrophil depletion during metastatic colonization caused a decrease of spontaneous metastasis. Furthermore, infiltrated neutrophils in the pulmonary metastasis lesions under nicotine treatment selectively overexpressed N2-polarization phenotype in a STAT3-dependent manner. We then examined secretory factor(s) from nicotine-treated primary neutrophils using a qPCR array system. We found that the expression of LCN2 was significantly augmented in the conditioned medium of nicotine treated primary neutrophils. Next, we investigated functional contribution of neutrophil-secreted factors on tumor metastasis. MCF10CA cells were cultured with the conditioned medium (CM) of primary neutrophils that were treated with or without nicotine. Importantly, mesenchymal-like MCF10CA cells displayed epithelial phenotype with increase in the expression of epithelial markers when they were treated with the CM. These results strongly implies that nicotine-induced polarization of N2-neutrophils plays a critical role in regulating cancer cell plasticity. In addition, levels of LCN2 were significantly higher in the serum and urine of cancer patients and cancer-free healthy subjects who were smokers compared to non-smokers. Collectively, our results suggest a novel pro-metastatic role of nicotine-induced N2-neutrophils via secretion of LCN2 that facilitate metastatic colonization of hormone receptor negati