Studying the responses of macrophages and microglia to brain tumour development in the zebrafish

Macrophages are important immune cells and primary phagocytes of the immune system. Microglia are the resident macrophages of the brain. In contrast to their physiological functions to recognise and eliminate pathogenic threats, microglia and macrophages have been found to promote brain tumour growth. It has since been shown that microglia and macrophages can infiltrate the brain tumour mass, with their density correlating directly to worsened prognosis through various tumour promoting activities. However, their involvement during the initial onset and development of brain tumours remains elusive. In this thesis, the research focussed on identifying two main aspects; (1) the impact of the initial steps of neoplastic transformation on the microglia and macrophage population, as well as (2) the impact of microglia and macrophages on early pre-neoplastic cells within the brain parenchyma. Oncogene overexpression is one of the classic methods to induce and study tumour development. To induce pre-neoplastic transformation and tumour growth within the brain, the human AKT1 oncogene was overexpressed in neural cells of zebrafish (Danio rerio) larvae. AKT1 overexpression led to evidence of pre-neoplastic development such as morphological transformation and enhanced proliferation. Importantly, within one month of AKT1 overexpression in neural cells, solid tumours were detected that showed neural and glial characteristics. Based on this model, macrophage and microglia responses to AKT1 expressing cells during the initial steps of neoplastic transformation were analysed. Interestingly, using wholemount immunohistochemistry, it was discovered that the microglia population was significantly increased within 4 days of AKT1 overexpression in the brain. This increase was a result of peripheral macrophages infiltrating into the brain parenchyma and their differentiation into microglial cells over time. To identify the mechanism involved, this study focussed on Sdf1b-Cxcr4b signalling. Gene expression analysis demonstrated that AKT1 cells had significantly upregulated levels of Sdf1b. Using a combination of the zebrafish Cxcr4b mutant line and pharmacological interventions, Cxcr4b activation on macrophages was found to be directly responsible for the recruitment of macrophages into the brain and the increased microglia population following oncogene activation. In addition to the increased population, microglia were significantly activated within the brain parenchyma of AKT1 overexpressing larvae. Interestingly, in vivo imaging showed dynamic interactions between the microglia/macrophages and the pre-neoplastic cells. Of note, phagocytosis and clearance of AKT1 positive cells by the microglia and macrophages were not observed. To address the impact of the immune cells on pre-neoplastic development, the microglia and macrophage populations were depleted. This was carried out through the use of various zebrafish mutant lines either devoid of microglia through development or incapable of macrophage Cxcr4 activation. In addition, treatments with specific inhibitors and antagonists were also carried out to pharmacologically deplete the microglia and macrophage populations. Intriguingly, quantification of AKT1 cellular proliferation upon macrophage and microglia depletion revealed that proliferation rates of the oncogenic AKT1 cells were significantly reduced. This therefore provided evidence that microglia and macrophages are required for promoting pre-neoplastic development. To understand the functional role of the cellular interactions between microglia and the pre-neoplastic cells and to identify the underlying mechanism involved, this study investigated the role of Ca2+-ATP signalling. Utilizing a transgenic zebrafish line that ubiquitously expresses the GCaMP6f calcium indicator, AKT1 expressing cells were found to express significantly increased intracellular levels of Ca2+. As increased Ca2+ levels were shown to lead to downstream ATP release and subsequent activation of microglia via microglial P2RY12 receptors, the role of Ca2+-ATP signalling involved in microglial interactions toward pre-neoplastic cells was examined. Preliminary evidence obtained through interfering with ATP release as well as inhibition of P2RY12 receptor activation on microglia suggest a role for Ca2+-ATP-P2RY12 signalling in mediating cell-to-cell interactions between the microglia and pre-neoplastic cells. Furthermore, proliferation rates of AKT1 expressing cells were significantly reduced upon inhibition of P2RY12 signalling, revealing the role of microglial interactions in stimulating proliferation of the pre-neoplastic cells. Taken together, results from this thesis demonstrate for the first time that microglia and macrophages act to promote tumour growth from the onset and earliest stages of brain tumour development. As similar mechanisms may occur during tumour recurrence, understanding how microglia and macrophages respond during tumour initiation may provide insights into the strategies to develop future cancer therapies