5 research outputs found
Interleukin-1β stimulates stromal-derived factor-1α expression in human subacromial bursa
Visfatin induces stromal cell-derived factor-1 expression by β1 integrin signaling in colorectal cancer cells
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