Effect of Extracellular Histones on Cancer Progression and Immune Cell Survival
Date
2021
Authors
Yan, Claudia
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Abstract
Neutrophil extracellular traps (NETs), DNA containing structures released from neutrophils and other immune cells as an ancient, antimicrobial defence mechanism, are believed to promote cancer growth and metastasis. Some studies have shown that tumours induce neutrophil infiltration and NET formation that aid metastasis. Other studies have shown that histones, released from NETs by DNases, cause immunosuppression. Hence, in order to resolve these differences in this Thesis the role of NETs in the B16 melanoma and 4T1 mammary adenocarcinoma cancer models in mice were studied, and the commonly accepted views for the role of NETs in tumour growth and metastasis questioned. Finally, the role of cytotoxic histones in immunosuppression was considered along with the contrasting activity of immune cells.
In Chapter 3, the role of NETs in tumour growth and metastasis was investigated using a range of mouse models. Inhibiting NET formation, using a mouse model that was genetically modified to be deficient in neutrophil elastase, showed no effect on tumour growth and metastasis. In addition, inhibiting NET activity via neutralising histone cytotoxicity, using a heparin-like polyanionic compound, mCBS, led to no change in tumour growth or metastasis. Activation of TLR4 to promote NET formation, in addition to stimulating the tumour microenvironment, also resulted in no change in tumour growth. These results suggest that NETs may not promote tumour growth and metastasis, despite being actively induced. In Chapter 4, the cytotoxicity of histones, released from NETs by DNases, for immune cells was investigated. Histones were found to be cytotoxic for a range of immune cells, with T cells and dendritic cells becoming more sensitive to histone killing when activated. On the other hand, red pulp macrophages (RPMOs) and eosinophils from the peritoneal cavity (PEX eos) were very resistant to histone cytotoxicity, which potentially provides a mechanism for the clearance of histones from the circulation due to the high phagocytic and endocytic activity of these cells. Conversely, Chapter 4 shows that histones are likely to be immunosuppressive due
to the susceptibility of most immune cells to histone-mediated cytotoxicity. Chapter 5 investigates the role of endocytosis and the presence of histone-neutralising proteins on the cell surface as possible mechanisms for RPMO and PEX eos resistance to histone cytotoxicity. To determine if endocytosis of histones provides RPMOs and PEX eos with resistance to histone killing, a range of chemical inhibitors were used to inhibit endocytosis. Overall, the results in this Chapter suggest that clathrin-mediated endocytosis could be the mechanism that allows uptake of histones, with degradation occurring in lysosomes, providing RPMOs and PEX eos with resistance to histone cytotoxicity. In conclusion, this Thesis contains data that suggests that, although tumours induce NET production, NET release does not necessarily aid tumour cells in evading anti-tumour immune responses. This conclusion may result from the varied response of immune cells to histone cytotoxicity, with RPMOs and PEX eos showing resistance to histone cytotoxicity, likely as a result of clathrin-mediated endocytosis of histones. This Thesis also proposes that macrophage and eosinophil endocytosis of histones and NETs results in the lack of detection of NETs in primary tumour samples. This suggests that in studies where histones and NETs are detected in tumours, macrophage and eosinophil mediated endocytosis of histones and NETs may have reached a state of exhaustion.
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