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    Interactions between MCF-7 and MDA-MB-231 breast cancer cell lines and neutrophils : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Anatomy and Physiology at Massey University, Albany, New Zealand
    (Massey University, 2019) Holland, Sherina
    The tumour micro-environment (TME) has an essential role in tumour development and progression. Immune cells recruited to the site of the tumour secrete soluble factors such as proteinases, growth factors, survival factors and angiogenic factors into the TME. The secretion of these factors is up-regulated via inflammatory mediators secreted by tumour cells, resulting in a pro-malignant cycle between the cancer and immune cells. A greater understanding of the molecular mechanisms underpinning these interactions is required, as this will assist towards identifying potential new drug targets for cancer and ultimately, will aid in the long-term development of targeted and effective treatments for breast cancer and MBC. The activities of certain immune cells, such as tumour associated macrophages, have been reasonably well characterised in cancer, however, until recently, less was known regarding the role of neutrophils in tumour progression. The goal of the research described in this thesis was to determine whether soluble factors secreted by breast cancer cells might alter the phenotype or lifespan of neutrophils. The latter may allow neutrophils sufficient time to participate in activities within the TME that may either help or hinder tumour progression, while soluble factors released by the neutrophils might influence the invasiveness of breast cancer cells. To investigate whether soluble factors released by breast cancer cells could delay neutrophil apoptosis, neutrophils were cultured in conditioned medium (CM) prepared from highly metastatic MDA-MB-231 or poorly metastatic MCF-7 cells. Flow cytometry experiments showed a delay in apoptosis for neutrophils cultured in MDA-MB-231 CM, but not MCF-7 CM. Quantitative RT-PCR was used to measure neutrophil mRNA expression of pro- versus anti-apoptosis peptides; neutrophils incubated in MDA-MB-231 CM, but not MCF-7 CM, demonstrated a significantly higher expression of the anti-apoptosis peptide BCL2 (A1) and significantly lower expression of the pro-apoptosis peptide BAK compared to control. Western blots showed extensive caspase-8 activation for neutrophils cultured in MCF-7 CM, consistent with apoptosis, whilst neutrophils cultured in MDA-MB-231 CM showed little activation of caspase-8, indicating low levels of apoptosis. The soluble factor contained within the MDA-MB-231 CM, responsible for the delay in neutrophil apoptosis was found to be heat stable and have a molecular weight of between 10-100kDA. Prostaglandin E2 (PGE2) was identified as a potential candidate molecule, as it is a heat stable lipid, and when bound to plasma proteins, fits the molecular weight criteria. In addition, neutrophils cultured with 10μM native or heat treated PGE2 demonstrated a delay in apoptosis, however, this was to a lesser extent compared to neutrophils cultured in MDA-MB-231 CM. Cycoloxygenase-2 (COX-2), the enzyme responsible for PGE2 synthesis, was shown to be expressed in MDA-MB-231 cells but not MCF-7 cells, which is in agreement with the results demonstrating a delay in apoptosis for neutrophils cultured in MDA-MB-231 CM but not MCF-7 CM. Freshly isolated human neutrophils, obtained from the peripheral blood of healthy volunteers, cultured in MDA-MB-231 or MCF-7 CM for 7hrs were not polarised toward a pro or anti-tumour phenotype, as determined via the expression of ICAM-1 and MMP-9. Finally, to investigate whether neutrophils could influence the process of EMT and alter the migration of breast cancer cells, neutrophils were indirectly cultured, via transwell plates, with MDA-MB-231 or MCF-7 cells. Neutrophils were not found to enhance the migration of the cancer cells, as determined via a wound scratch assay. Likewise, neutrophils were not shown to influence the process of EMT in the cancer cells, as determined by changes to cell morphology or the expression of EMT markers.
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    The chemiluminescence of ovine neutrophils : a thesis presented in partial fulfilment of the requirement for the degree of Master of Veterinary Science at Massey University
    (Massey University, 1984) Eagleson, Jane
    The development, structure and function of polymorphonuclear leucocytes (PMN) is reviewed and methods for determining neutrophil competence are discussed. A technique, based on differential centrifugation and red blood cell lysis, is described for isolating neutrophils of 80 to 90% purity from ovine blood. A standardised, luminol-enhanced chemiluminescence (CL) assay was developed for ovine neutrophils using latex beads as the phagocytic stimulus and some conditions influencing the level of CL generated are described. Normal sheep of similar age, housed under identical conditions and bled at approximately the same time on different days produced CL responses ranging from 386 to 3084 millivolts (mV). Animals sampled once daily over 5 days showed large fluctuations in CL values both between and within individuals. Furthermore, sheep bled at 4 hourly and 6 hourly intervals for 48 and 96 hrs respectively produced CL responses in a single individual with a range of 618 to 2946 mV. There was no evidence of periodicity in CL activity over the time periods examined. Since peak CL responses showed such large variations between individuals, integrated CL values were also measured. Variations between and within individuals similar to those recorded by peak CL were seen in these results. To examine the possible role of genetic differences in neutrophil function on the variability of CL, pairs of bovine monozygous twins were sampled. There was no correlation in CL response between genetically identical animals with the CL values from pairs of animals differing by as much as 2943 mV. The effect of cortisol on PMN CL was assessed. Synthetic corticosteroid in vivo and in vitro did not increase the peak CL response from isolated neutrophils. Profiles produced by recording CL against time were examined. Some cell isolates produced single peaked profiles while others gave a double peaked response. Single and double peaked profiles were recorded from the same donor at different times during a 24 hr period. Storage of the cells for prolonged periods sometimes resulted in an increase in the magnitude of the first peak possibly indicating an increase in the amount of more readily available myeloperoxidase (MPO). Prominent first peaks were still displayed after the cells were washed and resuspended in fresh media suggesting that the more readily available MPO was cell attached rather than truely extracellular. Neutrophils from ceroid lipfuscinosis-affected sheep produced peak CL responses and CL profiles similar to those given by normal sheep. These results did not confirm the postulated myeloperoxidase deficiency of this condition. It is concluded that ovine neutrophil CL is subject to large variations which cannot be controlled by standardising the cell isolation and CL analysis techniques. The assay is therefore unsuitable as a measure of neutrophil function where single samples are examined. Where there are consistant differences between individuals over a number of days, then CL may be of use when considered in conjunction with other tests of PMN function.