Abstract 801: Uptake of exogenous neutrophil elastase by breast cancer cells: A novel link between innate immunity, inflammation and breast cancer immunity

Abstract

Abstract Introduction: High level of neutrophil elastase (NE) in tumor tissue from breast cancer (BrCa) patients is associated with poor metastasis-free survival. Modest endogenous expression of NE in some BrCa cell lines has also been observed. However, because the major source of NE is from azurophil granules of activated neutrophils, we hypothesized that breast cancer cells may take up soluble NE released by abundant inflammatory cells in the tumor microenvironment. Methods: Endogenous NE expression was evaluated in MCF7, HER18, MDA-MB-231 (231) and MDA-MB-453 BrCa cell lines by western blot analysis and RT-PCR. Uptake of soluble NE was quantified by flow cytometry by intracellular staining of NE-pulsed BrCa and compared to non-specific uptake of OVA. Dose and time course experiments were performed. Confocal immunoflourescence microscopy was used to confirm uptake and demonstrate subcellular localization of NE. BrCa cells were also co-incubated with activated neutrophils to determine if they were able to take up cell-associated NE. Results: Western blot analysis of BrCa cell lysates showed no NE protein expression in all 4 cell lines. Furthermore, RT-PCR with NE-specific primers demonstrated no NE mRNA expression in the 4 BrCa cell lines. However, BrCa cells pulsed with 5 to 25 μg/ml of soluble NE took up NE in a time and dose dependent manner, consistent with a receptor-mediated mechanism. Maximal intracellular uptake of NE by 231 cells occurred by 24 hours when NE accumulated at comparable levels to that of HL60 leukemia cells that express high levels of NE (987 vs 1300 MFI, p=ns). Uptake was enzyme-independent because it was not affected by alpha-1-antitrypsin, a protease inhibitor. Confocal microscopy demonstrated that NE accumulates in membrane bound compartments by 20 minutes after uptake, whereas there was no significant uptake of OVA by BrCa cells. Similarly, after co-incubation of 231 cells with activated neutrophils, significant uptake of NE was detected (p<.001). Conclusions: These data show that BrCa cells do not express NE but the cells take up extracellular NE by a receptor-mediated enzyme-independent mechanism. This establishes a novel link between NE, a naturally secreted mediator of inflammation and cell growth, and BrCa. Because neutrophils are the major source of NE and chronic inflammation by tumor-infiltrating neutrophils is associated with positive tumor growth, this suggests NE could mediate growth control of BrCa. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 801. doi:10.1158/1538-7445.AM2011-801