Cancer Research @ UCC - Doctoral Theses
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Item 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 ResearchLung 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.Item Investigating the utility of blood borne oncological biomarkers in solid tumours: glioma and melanoma(University College Cork, 2021-07-15) Ita, Michael Itak; Lim, Chris; Wang, Jianghuai; Redmond, Henry Paul; University College CorkBloodborne molecular biomarkers are increasingly emerging as significant non-invasive adjuncts to current methods of disease status evaluation in cancer patients. Investigations into the potential utility of these circulating biomarkers as analytic test measures complementing radiological imaging have been occasioned by the invasive nature of malignant tumour tissue biopsy, the need for serial evaluation of tumour burden during therapy, and the need for prognostication. In a series of five studies (four clinical studies and one pre-clinical study), this research work explored the potential utility of plasma cell-free DNA, circulating tumour DNA, cell-free messenger RNA, and bloodborne tumour related proteins as disease biomarkers in patients with glioma and metastatic melanoma. The in vivo research work employed an animal model to study micro RNA mediated epigenetic regulatory mechanisms implicated in therapeutic resistance which is prevalent in melanoma brain metastasis. Specifically, this research work sought to determine whether somatic mutations identified in the plasma samples of patients with glioma were identical or representative of the somatic mutations in synchronously obtained glioma tumour tissue samples. It further sought to determine whether significant differences exist in the plasma transcriptomic profile of glioma patients relative to differences in their tumour characteristics, and also whether any observed differences were representative of synchronously obtained glioma samples and the human cancer genome atlas (TCGA) glioma derived RNA profile. Moreover, this research work explored the relationship between plasma cell-free DNA (cfDNA), serum lactate dehydrogenase (LDH), plasma vascular endothelial growth factor (VEGF), programmed death ligand-1 (PD-L1), interferon-gamma (IFN-γ), and tumour burden in advanced melanoma patients. Furthermore, it sought to examine whether important differences exist in the plasma transcriptomic profile of advanced melanoma patients with a high disease burden compared to patients with a low disease burden or therapeutic response and whether the plasma transcriptomic profile of advanced melanoma patients was representative of TCGA melanoma tumour tissue-derived RNA profile. The methods employed in this research work include; the purification and quantification of circulating cell-free DNA and total RNA from the plasma samples of glioma and metastatic melanoma patients, somatic mutation profiling using DNA derived from FFPE glioma tumour tissue curls and plasma circulating cell-free DNA by amplification refractory mutation system (ARMS®) PCR, pathway-focused gene expression analysis using complementary DNA synthesized from the plasma circulating cell-free messenger ribonucleic acid (ccfmRNA) samples of patients with glioma and advanced melanoma, the extraction and quantification of tumour-related proteins such as LDH, VEGF, PD-L1, and IFN-γ from patients with advanced melanoma by the enzyme-linked immunosorbent assay technique (ELISA), in vivo malignant brain tumour model development, bioluminescence imaging study, immunohistochemistry and microscopy, evaluation of protein expression by flow cytometry, and genomic profiling of total cellular micro RNA using RT- PCR. This research work was able to establish that the detection of plasma circulating tumour DNA originating from the glioma tumour tissues of affected patients is feasible, albeit with a low tumour to plasma mutation concordance. It identified significant differential expression of genes involved in cancer inflammation and immunity crosstalk among patients with different glioma grades, and a positive correlation between the transcriptomic profile of these genes in plasma and tumour samples, and with TCGA glioma derived RNA. Moreover, this research work identified that the incorporation of the quantitative measures of cfDNA, LDH, VEGF and PD-L1 in a suitable multiple regression analysis model was capable of predicting changes in tumour burden in patients with advanced melanoma. Furthermore, it identified and characterized the plasma transcriptomic profile associated with therapeutic response in advanced melanoma patients during immunotherapy. This research work also characterized in a limited way, the tissue and blood markers of therapeutic response and resistance in an in vivo model of melanoma brain metastasis.Item Malnutrition & altered body composition in oncology: prevalence, aetiology, consequences & potential therapies(University College Cork, 2020-08-10) Sullivan, Erin S.; Ryan, Aoife; Power, DerekMalnutrition is common across all cancer sites and stages and its aetiology is multifactorial and complex. It is associated with poorer quality of life, increased morbidity and mortality and is often considered an inevitable consequence of cancer and its treatments. However, we lack efficacious treatments for cancer-related malnutrition. The aim of this thesis was to describe the epidemiology of malnutrition in cancer, examine the causes and consequences of the condition and explore potential treatment strategies. This thesis begins by estimating that across Ireland and the UK, 34% of cancer patients (128,892) are affected by clinically significant weight loss annually and there are 133,707 annual cases of cancer-related sarcopenia (35% patients affected). This thesis shows using computed tomography scans (the gold standard in body composition analysis) that abnormalities of body composition, including loss of fat without loss of muscle, are predictive of poor survival in advanced cancer. Furthermore, cachexia (a syndrome of disease-related appetite loss and wasting) was shown to be more prevalent in those with inflammation and poor performance status and the obesity paradox in colorectal cancer was confirmed (obesity is a risk factor for the disease, but is associated with improved survival). The nutritional experience of patients with cancer is described, namely that nutrition is a high priority for patients, who experience many dietary issues throughout their journey, but that information available to patients is lacking and referral to dietitians is very inconsistent. Finally, a placebo controlled trial of 2 novel dairy-derived, ghrelinergic peptides showed that one of the peptides investigated increased protein intake in healthy males by 23 g per day. Prompt identification of patients with cancer-related malnutrition must be optimised and development of an effective, evidence-based treatment strategy is of the utmost importance as it stands to improve longevity and quality of life for cancer survivors.Item Evaluation of the role of autophagy in ovarian cancer chemoresistance(University College Cork, 2020) Quinn, Jennifer; McKenna, Sharon L.; O'Donovan, Tracey; Breakthrough Cancer ResearchOvarian cancer (OC) is the seventh most commonly diagnosed cancer in women worldwide. In Ireland, OC was the fourth most common cancer for women between 2015 and 2017 (1). A major challenge in the clinical management of OC is the high rate of disease recurrence. Approximately 80% of women who exhibit an excellent response to first line therapy, will present again with recurrent disease. A panel of ovarian cancer cell lines were evaluated for their response to two clinically relevant chemotherapies, paclitaxel and carboplatin. Apoptosis was assessed via morphological analysis and quantitation of active caspase-3. Apoptosis was significantly induced in response to treatment in the ovarian cancer cell lines, with the highest induction evident in the most drug sensitive cells. When these cell lines were re-challenged with a second round of paclitaxel treatment – apoptosis levels were significantly reduced and clonogenic recovery enhanced. This provides a novel insight into the mechanisms by which ovarian cancer cells may gain resistance to treatment. Autophagy is a highly conserved catabolic process, that enables cells to cope with stressful conditions. We have demonstrated, using flow cytometry and western blot analysis of LC3, that three ovarian cancer cell lines, that induced only limited apoptosis to chemotherapy, showed significant elevation of autophagy. In these cell lines, co-treatment with the autophagy modulators chloroquine and lithium impeded clonogenic recovery following treatment. siRNA knockdown of the autophagy genes, BECN1 and ATG5, significantly reduced the recovery of OC cells that undergo autophagy following treatment with paclitaxel. In addition, cell cycle analysis demonstrated that OC cells can regain a ‘normal/parental’ DNA profile 7-days after drug treatment, which was impeded when autophagy was inhibited with chloroquine or when key autophagy genes BECN1 and ATG5 were knocked down. Confocal analysis showed nuclear material colocalised with autophagic vesicles, suggesting that nucleophagy may play a role in the recovery of the parental DNA content. The potential contribution of non-canonical autophagy was also evaluated using the inhibitor Brefeldin A. Autophagosome accumulation was reduced following treatment with Brefeldin A and following siRNA knockdown of BECN1 with the non-canonical regulator Rab9, which significantly reduced the recovery of OC cells following paclitaxel treatment. These data demonstrate the importance of autophagy in OC cell recovery following drug treatment and suggests that more than one form of autophagy may be involved. It is also notable that only three of the five cell lines tested demonstrated elevated autophagy – and these are the cells that respond to chloroquine. This may be important for clinical trials that are currently incorporating hydroxychloroquine without any guidance on the contribution of autophagy to the tumour phenotype. It is likely that biomarkers will be needed to guide the administration of autophagy inhibitors in the future. Here, we have made potentially clinically relevant observations. We have highlighted reduced apoptosis competency following drug re-challenge and shown that autophagy modulators are effective in a subgroup of cell lines with high autophagy levels. We have provided a novel insight into a role for canonical and non-canonical autophagy in OC chemoresistance, and demonstrated for the first time a potential role for nucleophagy in OC cell recovery. It is hoped that autophagy inhibitors, along with appropriate biomarkers, may be incorporated into treatment strategies to overcome treatment resistance and improve OC patient outcomes in the future. 1. National Cancer Registry IrelandItem Development of methods for the microbiome analysis of formalin fixed paraffin embedded tissue specimens(University College Cork, 2019-04-30) Flores, Yensi; Tangney, Mark; Irish Research CouncilFor Synthetic Biology to reach its potential, it necessitates foundational knowledge of the organisms that can be engineered. The remarkable influence our microbiome has on our health status has made it a focus of attention for engineering possibilities aiming at its modulation. As the field of the human microbiome expands, it necessitates access to high-quality nucleic acid samples which are truly representative of the community of bacteria under study. Formalin-fixed, paraffin-embedded (FFPE) samples represent the most comprehensive collections of patient materials in hospital pathology archives. However, for this sample to become reliably accessible for microbiome studies, the effects of FFPE processing on bacteria must be considered. Any sample processing method should be based upon specific study aims, target organisms and sample types. It is only through a holistic understanding of FFPE-induced changes to the bacterial cellular structure and its DNA content, that a reliable method can be developed It is hypothesised here that with a sample-prep workflow considering the effects of FFPE on bacterial cells, their DNA content and the overall contamination introduced, a reliable and reproducible analysis of the microbiome of FFPE samples could be achieved. As such, the overall aim of this thesis was to characterise FFPE induced changes to the bacterial cell walls/membranes and their DNA content, and with this information, to propose strategies for purifying and repairing DNA suitable for microbiome analysis, while also characterising the common contaminants found in samples processed in this manner. To achieve this, an appropriate FFPE bacterial study model was first developed. With this in place, a thorough characterisation of the state of bacterial FFPE DNA was performed and strategies to reduce this damage assessed. Finally, to develop an appropriate method for bacterial DNA extraction from FFPE samples (unavailable at the time of writing), the state of the bacterial cell wall/membrane was assessed and strategies for a uniform bacterial lysis and host depletion evaluated. Chapter 2 Describes methods for creating a mock bacterial FFPE block (Protoblock) that serves as a standard for FFPE samples. The Protoblock is a cell matrix which can be populated with cell types and numbers as desired, so as to resemble those of the FFPE tissue specimens. Its accuracy for representing bacterial load and cell architecture was validated by microscopy. With this model, the performance of the human gold-standard FFPE kit for microbiome analysis of FFPE samples was evaluated and found unsuitable for microbiome research. Additionally, the Protoblock permitted the characterisation of bacterial FFPE DNA, where it was found to be highly fragmented, a poor PCR template and featured significant sequence alterations. Finally, this model also permitted the characterisation of contaminants originating from the FFPE process, the most common being Xanthomonadaceae, Pseudomonadaceae and Clostridiaceae. Chapter 3 Makes a thorough investigation of the state of bacterial FFPE DNA in terms of PCR readability, formalin crosslinking, and the presence of sequence artefacts. Here, bacterial FFPE DNA was found to be highly fragmented, with a significant inverse correlation between fragment size and PCR recovery and a log-fold reduction between the recovery of 200 bp and 500 bp fragments. It was also evident that 95-97% of DNA present in these samples was crosslinked and that the most evident sequence artefacts were those derived from oxidative damage. Two strategies to reduce this damage were investigated. (1) An optimised decrosslinking procedure (10 oC lower than current methods) significantly reduced sequence artefacts. (2) The in vitro reconstitution of the Base Excision Repair pathway targeting oxidative DNA damage, using FPG and Endo VIII DNA glycosylases. Samples treated with both strategies showed a 3X increase in fragment length and a significant reduction in sequence chimeras and SNPs, leading to a significant improvement in sequencing readability. Chapter 4 Investigates the state of the bacterial cell wall/envelope and mammalian membrane to assess the state of their permeabilisation in FFPE samples. In this chapter, mammalian and Gram-negative bacterial cells were found to be impermeable to molecules with dimensions of 3-5 nm. A host depletion strategy was devised using a combination of Saponin and DNAse (Benzonase). It was also found that FFPE bacterial cells require a lysis strategy, and the use of a mix of bacterial-lytic enzymes was found to provide a uniform cross-taxa bacterial lysis. The collection of methods developed were tested by 16S rRNA gene sequence analysis of protoblocks, murine FFPE faeces and human breast tumour samples. The collection of methods provided an overall increase in recovery of 16S PCR amplicons, a higher uniformity in bacterial lysis, and a higher bacterial to host DNA ratio in high biomass models. However, these improvements were obscured for low biomass samples, where contaminants dominated the sequencing reads. It is concluded from this work that to unlock the potential of FFPE specimens for the microbiome field, a full dedicated workflow, comprising not only sample-prep, but also QC, 16S PCR and 16S sequencing, needs to be in place. This workflow should be directed by a robust QC system. In addition, a database for known FFPE derived common contaminants is essential to inform future strategies for the biological removal of contaminants from these samples.