Characterisation of Heparanase in Natural Killer Cell Function

Submission note: This thesis is submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, School of Molecular Sciences, College of Science, Health and Engineering, La Trobe University, Victoria, Australia.

Heparanase is the only mammalian enzyme capable of cleaving heparan sulfate, a complex glycosaminoglycan that comprises an important component of the extracellular matrix and cell surfaces. By cleaving heparan sulfate heparanase mediates several cellular processes, including cytokine release, cell migration and cell signalling, as well as pathological processes including metastasis and angiogenesis. These cellular functions of heparanase have been defined in a number of immune cell types, but the role of the enzyme in natural killer (NK) cells has not been investigated. NK cells are innate lymphoid cells capable of inducing cell death in virally infected and malignantly transformed cells. In this thesis, conditional NK cell-heparanase knock out mice (Hpsefl/fl NKp46-iCre) and constitutive heparanase knock out mice (Hpse-/- ) have been used to explore the function of heparanase in NK cells during tumour surveillance. NK cell-heparanase was found to be critical for NK cell migration into tumours, and consequent tumour surveillance in B16F10 and RM-1 experimental metastasis models, and in the EO771.LMB spontaneous metastasis model. It was also found that in Hpsefl/fl NKp-46-iCre mice, immunotherapies targeting the programmed cell death protein (PD)-1 and cytotoxic T-lymphocyte-associated antigen (CTLA)-4 axes were less effective than in Hpsefl/fl NKp46-WT mice. Furthermore, using constitutive Hpse-/- mice, heparanase was shown to be important for NK cell activation in vivo in response to polyinosinic–polycytidylic acid (poly(I:C)). Following poly(I:C) stimulation, there was a reduction in expression of CD69 and NKG2D on Hpse-/- NK cells, and lower ex vivo cytotoxicity of Hpse-/- NK cells against EO771.LMB cells when compared to NK cells from wild type mice. In response to poly(I:C), murine NK cells are activated by dendritic cells (DCs) in vivo. Data presented here suggest that heparanase is important for DC activation and DC-NK cell crosstalk. Heparanase was also found to be important for NK cell production of monocyte chemoattractant protein-1 (MCP-1). Interestingly, the constitutive deletion of heparanase in mice does not affect tumour surveillance of B16F10 or EO771.LMB metastasis. In addition, poly(I:C)-mediated immune activation during EO771.LMB experimental metastasis is not dependent on heparanase. However, the absence of heparanase did impact tumour-mediated NK cell activation. Taken together, the results presented in this thesis have important implications for the use of heparanase inhibitors in cancer therapy. This thesis also lays the foundation for future work into exploring the role of heparanase during tumour surveillance and potential applications in immunotherapies.