Pathology - Doctoral Theses

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    Characterisation of the effect of electroporation and electrochemotherapy on cancer cells and immune cells in the tumour microenvironment
    (University College Cork, 2022-10) Bendix, Maura; Brint, Elizabeth K.; Houston, Aileen M.; Amu, Sylvie; Forde, Patrick; Health Research Board; Breakthrough Cancer Research
    Lung cancer is the leading cause of cancer-related death worldwide, with the lung cancer incidence rate expected to rise further. Despite recently developed novel therapy options, 5-year survival rates for lung cancer patients remains below 20% generally and below 5% for late-stage diagnosis, thus additional therapy options are still needed. Electrochemotherapy (ECT), the application of an electric pulse to deliver chemotherapy drugs into cells, could be a new treatment option for lung cancer patients. ECT is a locally very effective treatment, with local tumour reduction of up to 85%, while the systemic effects are more varied. For clinical application ECT treatment modalities have been standardized since 2006, after the ESOPE study, which optimized ECT parameters to 8 pulses at 1000V/cm with 100µs pulse length at 1Hz frequency and either bleomycin or cisplatin as the drug of choice. To evaluate whether ECT could be a potential treatment option for lung cancer patients’ ECT parameters, the needed electric field strength and the needed drug and drug concentration, were optimized for in vitro lung cancer research. In our study, we initially developed a standard operating protocol (SOP) to determine the optimal electric field strength for a given cancer cell line in vitro, while keeping the other ESOPE parameters constant. The developed SOP combined short-, medium-, and long-term assays to fully visualize the impact treatment, at a given field strength, has on the tested cancer cell line. This evaluation showed that human lung cancer cell lines (A549, H460 and SK-MES 1) and the human pancreatic Pan02 cell line have an optimal electric field strength of 800V/cm, the melanoma A375 (human) and B16F10 (murine) cell lines as well as the murine pancreatic Mia-PACA2 cell line have an optimal electric field strength of 700V/cm, while the murine Lewis Lung carcinoma (LLC) cell line has an optimal electric field strength of 1300V/cm. In addition, our study findings demonstrate that cisplatin at 11µM would be the drug of choice when using ECT for lung cancer treatments. In recent years while the importance of the immune system in lung cancer development and treatment results has become increasingly clear, little is known about how ECT treatments impact immune cells. Therefore, the impact of ECT on murine T cells, dendritic cells (DCs) and macrophages was evaluated in vitro. Our data indicates that while T cells are able to tolerate electric field strengths of up to 1400V/cm, DCs and macrophages are significantly negatively impacted by electric field strengths exceeding 800V/cm. Further investigation on the impact of ECT on dendritic cells demonstrated that DCs die via necrosis following ECT treatment, while ECT at electric field strength exceeding 1000V/cm leads to DC maturation and activation of the surviving cells. In addition, DCs remain partially functional following ECT treatment in a stimuli and treatment dependent manner with distinctively different sets of genes upregulated 4-hours post treatment at 800V/cm compared to treatment at 1000V/cm and 1300V/cm. Taken together, our data indicates that it is worthwhile to further investigate ECT as a potential therapy option for lung cancer patients, while more attention needs to be paid to the impact ECT has on immune cells in order to maximize treatment results.
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    Characterisation of the role of Fas in intestinal inflammation and cancer
    (University College Cork, 2015) Fernandes, Philana; Houston, Aileen M.; Brint, Elizabeth K.; Science Foundation Ireland
    Background: The role of Fas (CD95) and its ligand, Fas ligand (FasL/CD95L), is poorly understood in the intestine. Whilst Fas is best studies in terms of its function in apoptosis, recent studies suggest that Fas ligation may mediate additional, non-apoptotic functions such as inflammation. Toll like Receptors (TLRs) play an important role in mediating inflammation and homeostasis in the intestine. Recent studies have shown that a level of crosstalk exists between the Fas and TLR signalling pathways but this has not yet been investigated in the intestine. Aim: The aim of this study was to evaluate potential cross-talk between TLRs and Fas/FasL system in intestinal cancer cells. Results: Treatment with TLR4 and TLR5 ligands, but not ligands for TLR2 and TLR9 increased the expression of Fas and FasL in intestinal cancer cells in vitro. Consistent with this, expression of Fas and FasL was reduced in the distal colon tissue from germ-free (GF), TLR4 and TLR5 knock-out (KO) mice but was unchanged in TLR2KO tissue, suggesting that intestinal cancer cells display a degree of specificity in their ability to upregulate Fas and FasL expression in response to TLR ligation. Expression of both Fas and FasL was significantly reduced in TRIF KO tissue, indicating that signalling via TRIF by TLR4 and TLR5 agonists may be responsible for the induction of Fas and FasL expression in intestinal cancer cells. In addition, modulating Fas signalling using agonistic anti-Fas augmented TLR4 and TLR5-mediated tumour necrosis factor alpha (TNFα) and interleukin 8 (IL)-8 production by intestinal cancer cells, suggesting crosstalk occurs between these receptors in these cells. Furthermore, suppression of Fas in intestinal cancer cells reduced the ability of the intestinal pathogens, Salmonella typhimurium and Listeria monocytogenes to induce the expression of IL-8, suggesting that Fas signalling may play a role in intestinal host defence against pathogens. Inflammation is known to be important in colon tumourigenesis and Fas signalling on intestinal cancer cells has been shown to result in the production of inflammatory mediators. Fas-mediated signalling may therefore play a role in colon cancer development. Suppression of tumour-derived Fas by 85% led to a reduction in the tumour volume and changes in tumour infiltrating macrophages and neutrophils. TLR4 signalling has been shown to play a role in colon cancer via the recruitment and activation of alternatively activated immune cells. Given the crosstalk seen between Fas and TLR4 signalling in intestinal cancer cells in vitro, suppressing Fas signalling may enhance the efficacy of TLR4 antagonism in vivo. TLR4 antagonism resulted in smaller tumours with fewer infiltrating neutrophils. Whilst Fas downregulation did not significantly augment the ability of TLR4 antagonism to reduce the final tumour volume, Fas suppression may augment the anti-tumour effects of TLR4 antagonism as neutrophil infiltration was further reduced upon combinatorial treatment. Conclusion: Together, this study demonstrates evidence of a new role for Fas in the intestinal immune response and that manipulating Fas signalling has potential anti-tumour benefit.