Surgery - Doctoral Theses

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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 Cork
Bloodborne 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.
Macrophage polarisation: the impact of M1 versus M2 polarisation on host innate immune responses to bacterial infection
(University College Cork, 2015-12) Foley, Niamh M.; Redmond, Henry Paul; Wang, Jianghuai
Background and Aim: Infection is a global burden causing millions of deaths per annum worldwide. In the US sepsis is the tenth leading cause of death with the mortality associated with severe sepsis estimated at 30-50%. Innate immunity is a generic response mediated by the host to protect it from bacterial infection. The recognition of foreign microbes leads to activation of pattern recognition receptors and recruitment of macrophages. In acute bacterial infections, activated macrophages polarised to M1 or M2 states play a major role in the host cytokine response which drives the immune response until the host has overcome the invading microbial pathogen. The aim of this study was to 1) to characterise the cytokine profile of M1 and M2 polarised macrophages 2) to investigate the changes in the cytokine profile of polarised macrophages in response to bacterial stimulation 3) to examine the role of the MAPK and NFκB signalling pathways in the response of naïve and polarised macrophages to bacterial infection. Results: (i) polarisation of macrophages to an M1 state resulted in a higher secretion of pro-inflammatory cytokines (IL-6, IL12p70 and TNF-.). (ii) following bacterial stimulation M1 polarised macrophages had reduced pro-inflammatory cytokine release. (iii) M1 polarised macrophages have reduced MAPK and NFκB signalling as detected by western blot analysis. Conclusion: Following bacterial stimulation M1 polarised macrophages had reduced pro-inflammatory cytokine release which may in part be due to reduced MAPK and NFκB signalling. This data suggests that M1 polarisation states may play important roles in an endotoxin tolerant phenomenon in acute bacterial sepsis.
Investigation of orally administrable bacteria for cancer therapy
(University College Cork, 2021-01-06) Hogan, Glenn; Tangney, Mark
Diminished progress in improving prognoses and treatment outcomes of several cancers, using conventional drugs, is being countered by investigations into a range of new, advanced formulations, of which bacteria-based anticancer therapies are one. Bacteria-based treatments hold an especially intriguing place within this group of disruptive medical technologies for the well-documented ability of bacteria to selectively colonise tumours following IV administration and replicate therein. Based on this property, bacteria have been studied both clinically and preclinically to evaluate their potential to promote tumour regression and confine noxious effects to the malignant tissue. Some of these studies have attempted to engineer the bacteria genetically to reduce their virulence and/or accentuate their capability to exert tumour-selective damage, but efforts to date have failed to penetrate clinical barriers and produce a safe, effective, and marketable bacteria-based product for cancer therapy. This thesis examines ways in which bacteria that are truly optimised for cancer treatment might be acquired and/or developed, using data derived from patient (Chapters 2, 3, and 4) and preclinical studies (Chapters 3, 4, and 5). The thesis can be divided into three main hypotheses: i) if naturally occurring bacteria can be identified in human tumour tissues, then these may be better tumour-targeting agents than the laboratory and probiotic strains that are routinely used in tumour-targeting studies (Chapters 2 and 3); ii) if bacteria that are administered orally to cancer patients can successfully colonise their tumours, then these may be safer and equally effective alternatives to intravenously administered microbes (Chapter 4); and iii) if bacteria are continually recycled through tumours via IV administration, then this may result in strains with improved tumour selectivity over their parental counterparts (Chapter 5). A prevailing concept throughout this work is that exposure to tumours, either via purposeful human intervention or natural colonisation, and the resultant evolutionary effects could adapt bacteria to the intratumoural environment more effectively than what genetic engineering would allow. This thesis contributes considerably to several bodies of knowledge, including the human tumour microbiota and the feasibility of administrable bacteria as vehicles for tumour-selective transmission of therapeutics. Chapters 2 and 3 use sequencing and culture techniques to develop an accurate and comprehensive picture of the human breast tumour microbiota. A culture-based assay was able to define the microbial diversity and community structure of breast tumours, when placed in the context of refined bioinformatic analyses. Despite a lack of evidence for viable, endogenous, tumour-selective bacteria, breast tumours contained a characteristic microbiological signature, as determined by deep sequencing, that warrants further exploration in varying tumour types to establish the diagnostic potential of this finding. Chapter 4 reports clinical data in relation to the translocation of probiotic bacteria from the GIT to distal tumours, using both FFPE specimens and fresh tissues. Results of this study, and corresponding animal experiments, shed light on the viability, or lack thereof, of this route of administration for prospective bacterial tumour-targeting studies in the clinic. Lastly, Chapter 5 is an account of the effects of tumour-to-tumour cycling on probiotic bacterial strains. The data in this chapter indicate that recycling bacteria through murine tumours can alter intratumoural growth dynamics and metabolic capacity, which was reflected in enhanced tumour persistence and biofilm formation. This work emphasises tumour recycling as a method by which bacteria can be adapted to the complex, heterogenous environment of cancerous tissue in a relatively simplistic way, which may have implications for the success of this treatment modality as it gains more clinical traction.
In silico-aided design, build and test of synthetic proteins
(University College Cork, 2019-12-20) Yallapragada, V. V. B.; Tangney, Mark
Since the discovery of proteins in 1838, the field of protein engineering and our understanding of proteins have improved exponentially. Synthetic proteins have found applications in various biomedical, food and material-based settings. This rise in synthetic proteins was complemented with the parallel expansion in the availability of in silico tools for protein modelling. The complexity in the composition and design of synthetic proteins requires careful in silico validation to screen for potential pitfalls in the design. In silico tools for protein modelling and design have been used extensively to computationally validate the structure and functioning of the synthetic proteins prior to wet-lab testing. In this thesis, the workflow of design-model-build-test of synthetic proteins with novel applications in imaging is described. The in silico-aided design, screening and the in vitro testing of synthetic proteins targeting S. aureus surface antigen Clumping factor A are discussed in Chapter 2. In this chapter, a suitable candidate worthy of examining in a future in vivo setting was identified. During the in silico-aided screening, the complexity of data obtained from various in silico tools posed new challenges. This was termed as ‘the in silico myriad problem’. In Chapter 3, a mathematical strategy (Function2Form bridge) was tested to address the in silico myriad problem, by combining the scores of different design parameters pertaining to the synthetic protein being analysed into a single easily interpreted output describing overall performance. The strategy comprises 1. A mathematical strategy combining data from a myriad of in silico tools into an Overall Performance-score (a singular score informing on a user-defined overall performance); 2. The F2F-Plot, a graphical means of informing the wet-lab biologist holistically on designed construct suitability in the context of multiple parameters, highlighting scope for improvement. F2F bridge was implemented during the design process of all the synthetic proteins in Chapter 4 and Chapter 5. The synthetic protein design strategy used in Chapter 2 was implemented to design synthetic proteins targeting cancer cells, and to assess their potential as in vivo imaging agents in Chapter 4. For both MUC1 and ClfA targeted proteins, in vivo luminescence imaging studies involving systemic intravenous administration of proteins, validated synthetic protein specific accumulation at target cell locations within mice as evidenced by localised luminescence. Dose response studies indicated that luminescence output was both target cell and administered protein quantity related. In Chapter 5, a self-assembling protein ‘cage’ was designed, built and tested in vitro. An accompanying novel fluorescence-based protein-protein interaction reporting strategy was introduced, involving incorporation of cysteine residues at the interaction interface of monomeric proteins of the self-assembling protein cage. In silico tools were used to ensure the conformational and functional stability. FlAsH EDT2 (fluorescin arsenical hairpin binder-ethanedithiol) mediated fluorescence was used to confirm the self-assembly. This demonstrates the level of accuracy and detail that can be incorporated into synthetic protein design using in silico tools. In Chapter 6, the scope of introducing miniaturised optical devices to aid biological experimentation was explored. A novel handheld device for monitoring continuous bacterial growth, with prospects of measuring biofluorescence was developed. The device was tested using different bacterial strains and showed accuracy levels similar to a standard benchtop spectrophotometer. This thesis demonstrates the use of computational methods and various in silico tools for protein design. Modern day biomedical science demands novel concepts with deployable technology to assist their translation into user-based settings. In this thesis, various interdisciplinary concepts have been applied to deliver on a holistic end-goal.
The characterization of macrophages in melanoma and the effect of electroporation on melanoma conditioned macrophages
(University College Cork, 2019) Tremble, Liam Friel; Forde, Patrick; Heffron, Cynthia; Breakthrough Cancer Research
Approximately 11,000 people are diagnosed with skin cancer in Ireland every year and approximately 1,000 of these present with malignant melanoma. Due to lifestyle changes and an increase in UV exposure, incidences are expected to continue to rise despite the presence of health campaigns. Immune cells, called macrophages, have been documented to represent up to 50% of the tumour mass in some melanomas. However, we have only a nascent understanding of the role of these cells in tumour biology and treatment responses. One treatment offered to advanced melanoma patients is electrochemotherapy, which has exceptionally high complete local response rates of up to 80%, and is under investigation with the replacement of chemotherapy with non-cytotoxic drugs such as calcium. Calcium electroporation has shown the ability to induce comparable response rates and is under increased scrutiny due to the presence of a case report in which a systemic anti-melanoma response was seen following treatment. While the effect of electroporation, and increasingly, calcium electroporation on tumour cells has been well documented. The effect of these treatments on bystander cells in the treatment area, such as tumour-associated macrophages, has not been investigated. Here we present clinical findings of the presence of distinct macrophage populations recruited to melanoma tissue. Using their inflammatory phenotype, effect on gene expression within the tumour, and correlation with survival outcomes we give compelling evidence that melanomas contain distinct populations of both active, and relatively inactive macrophages, which can vary depending on the pathological features of the tumour, such as Breslow depth and BRAF mutational status. Given the presence of an inflammatory population of macrophages in the tumour, we sought to develop an in vitro model in which we could examine the effect of electroporation on melanoma conditioned macrophages. Using an adapted model of bone marrow-derived monocyte development followed by melanoma conditioning, we were able to generate an immunologically active model of melanoma conditioned monocytes, which upregulated M2-associated surface receptors, similar to the predominant population of intratumoural macrophages. These conditioned cells showed no major increase in inducible nitric oxide synthase or arginase expression, as was seen clinically, but were able to affect T cell proliferation and polarization, indicating an influential immunological phenotype. In chapter 5, we investigate the effect of electroporation on these cells, and show that, similarly to tumour cells, their membranes do become reversibly electroporated. Using the parameters investigated in chapter 4 we show that calcium electroporation does impact their phenotype and functionality, and critically, influences their ability to subsequently activate and polarize T cells. Our data indicate that electroporation does not deplete intratumoural macrophages or inhibit their ability to drive cytotoxic T cell responses, suggesting that, from a macrophage perspective, calcium electroporation may be complementary to other immunogenic treatments.

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