New bioimaging techniques have recently been proposed to visualise the colocation or interaction of several proteins within individual cells, displaying the heterogeneity of neighbouring cells within the same tissue specimen. Such techniques could hold the key to understanding complex biological systems such as the protein interactions involved in cancer. However, there is a need for new algorithmic approaches that analyse the large amounts of multi-tag bioimage data from cancerous and normal tissue specimens in order to begin to infer protein networks and unravel the cellular heterogeneity at a molecular level. In the firrst part of the thesis, we propose an approach to analyses cell phenotypes in normal and cancerous colon tissue imaged using the robotically controlled Toponome Imaging System (TIS) microscope. It involves segmenting the DAPI labelled image into cells and determining the cell phenotypes according to their protein-protein dependence profile. These were analysed using two new measures, Difference in Sums of Weighted cO-dependence/Anti-co-dependence profiles (DiSWOP and DiSWAP) for overall co-expression and anti-co-expression, respectively. This approach enables one to easily identify protein pairs which have significantly higher/lower co-dependence levels in cancerous tissue samples when compared to normal colon tissue. The proposed approach could identify potentially functional protein complexes active in cancer progression and cell differentiation. Due to the lack of ground truth data for bioimages, the objective evaluation of the methods developed for its analysis can be very challenging. To that end, in the second part of the thesis we propose a model of the healthy and cancerous colonic crypt microenvironments. Our model is designed to generate realistic synthetic fluorescence and histology image data with parameters that allow control over differentiation grade of cancer, crypt morphology, cellularity, cell overlap ratio, image resolution, and objective level. The model learns some of its parameters from real histology image data stained with standard Hematoxylin and Eosin (H&E) dyes in order to generate realistic chromatin texture, nuclei morphology, and crypt architecture. To the best of our knowledge, ours is the first model to simulate image data at subcellular level for healthy and cancerous colon tissue, where the cells are organised to mimic the microenvironment of tissue in situ rather than dispersed cells in a cultured environment. The simulated data could be used to validate techniques such as image restoration, cell segmentation, cell phenotyping, crypt segmentation, and differentiation grading, only to name a few. In addition, developing a detailed model of the tumour microenvironment can aid the understanding of the underpinning laws of tumour heterogeneity. In the third part of the thesis, we extend the model to include detailed models of protein expression to generate synthetic multi-tag fluorescence data. As a first step, we have developed models for various cell organelles that have been learned from real immunofluorescence data. We then develop models for five proteins associated with microsatellite instability, namely MLH1, PMS2, MSH2, MSH6 and p53. The protein models include subcellular location, which cells express the protein and under what conditions.

Although haemopoietic stem cells (HSCs) represent one of the best-defined stem cell systems, the pathways regulating HSC development and maintenance are not fully understood. HSCs reside in the hypoxic niche and maintain intracellular hypoxia. Hypoxia and hypoxia signalling pathways are thought to play a vital role in HSC maintenance. Hypoxia inducible factors (Hifs) are evolutionarily conserved and are the key regulators of hypoxia. Hifs consist of an unstable, oxygen-dependent α-subunit and an oxygen-independent stable β-subunit. The two main isoforms of Hif-α, namely Hif-1α and Hif-2α, are critical for the response to hypoxia. Hif-mediated pathways have been extensively studied and have been shown to regulate metabolic adaptation and to influence various cellular mechanisms, including cell growth, survival, differentiation and apoptosis, erythropoiesis and angiogenesis. Hif-1α has been shown to be essential for maintenance of HSC functions under stressful conditions of serial transplantation and aging, but the role of Hif-2α and the interplay between Hif-1α and Hif-2α in regulating HSC functions and their niche is not known. Hence, in this study, I have investigated the role of Hif-α in HSC functions. Furthermore, published evidence suggested that leukaemic stem cells (LSC) share the hypoxic properties with HSCs. Cited2, a hypoxia-inducible Hif-1α and Hif-2α target gene, is critical for embryonic and adult haemopoiesis and possesses oncogenic properties. I have investigated the role of Cited2 in AML generation. The results demonstrate that Hif-2α is not essential for maintenance of HSC functions in a cell-autonomous manner under steady state and stressful conditions of serial transplantation and aging. It is also evident that HSCs lacking Hif-2α together with Hif-1α successfully maintain normal haemopoiesis. However, the data in this thesis show that Hif-2α is essential for non-cell-autonomous maintenance of HSC functions, particularly in males and current work also indicate that a previously unappreciated complex interplay between Hif-1α- and Hif-2α-dependent signalling is required for adult HSC maintenance in a non-cell-autonomous manner. Additionally, the data demonstrate that haemopoietic stem and progenitor cells (HSPCs) lacking Cited2 display reduced transformation potential and failure to generate transplantable AML in vivo. Overexpression of Mcl-1 (an anti-apoptotic gene), in Cited2Δ/Δ cells bypassed their defective transformation potential forming transformed colonies in vitro. Hence, the data in this thesis provide evidence that Cited2 is essential for leukaemic transformation at least in part via Mcl-1 regulation.

This thesis is concerned with the design of half-sandwich iridium(III) complexes of the type [(η5-Cp*)Ir(2-(Rˈ-phenyl)pyridine-R)X]0/+ (Cp* = pentamethylcyclopentadienyl), X = Cl- or pyridine derivatives) as anticancer agents, with particular focus on the effects that functionality in the chelating and monodentate ligands has on their chemical and biological properties. A set of phenyliminopyridyl (ImPy) complexes of the type [(η5-Cpx)Ir(ImPy)Cl]PF6 (Cpx = Cp*, tetramethyl(phenyl)-cyclopentadienyl (CpxPh) or tetramethyl(biphenyl)- cyclopentadienyl (CpxBiPh) was also synthesised and their solution chemistry and potential applications as antibacterial and anticancer agents investigated. Electron donating (-CH3, -OH, -CH2OH and –OCH3) or electron withdrawing (-F, -CF3 -CHO and –NO2) groups were introduced to various positions on the 2-PhPy ligand, giving rise to seventeen complexes [(η5-Cp*)Ir(2-(Rˈ- phenyl)pyridine-R)Cl]. Three X-ray crystal structures were determined, showing the expected pseudo-octahedral configuration. The functional groups have a profound effect on the resulting anticancer activities against a range of cell lines. Some complexes showed activity against A2780 human ovarian cancer cells comparable to cisplatin, and similar activity to [(η5-CpxPh)Ir(2-phenyl)pyridine)Cl]. The complexes all show similar extents of hydrolysis. The complexes preferentially bind to the model nucleobase 9-EtG over 9-MeA, and show the ability to catalytically oxidise the coenzyme NADH to NAD+. Contrasting anticancer activities were found for structural isomers. The hydrophobicity is related to substituent type and position on the ligand. The more hydrophobic complexes accumulated in A2780 cells to a greater extent. The extent of accumulation appeared to correlate with the potency of the complexes. The most potent complex [(η5-Cp*)Ir(2-(2ˈ-methylphenyl)pyridine)Cl] 13 (IC50 = 1.18 μM against A2780 cells) was modified via replacement of the chlorido monodentate ligand with pyridine derivatives (18 = pyridine (Py), 19 = 4- dimethylaminopyridine (Py-NMe2), 20 = 4-trifluoromethylpyridine (Py-CF3). The X-ray crystal structure was determined for 19. These pyridine complexes show less monodentate ligand release in aqueous solution and less binding to 9-EtG than parent complex 13. Complexes 13 and 19 show mild catalytic activity towards the oxidation of NADH to NAD+. Reactivity towards glutathione (GSH) decreased only in the case of 19 when compared with the parent complex 13. The reaction products include [(η5-Cp*)Ir(2-(2ˈ-methylphenyl)pyridine)(S-O)G] (sulfenate) and [(η5- Cp*)Ir(2-(2ˈ-methylphenyl)pyridine)(S-O2)G] (sulfinate) which are reliant on the presence of O2. The antiproliferative activity against A2780 cells increased upon enhancement of stability at the monodentate site (19 > 18 > 20 ~ 13) when activity was measured with no cell recovery time, with 19 exhibiting nanomolar activity (IC50 = 650 nM). All of the complexes induced high levels of total reactive oxygen species (ROS). Apoptotic cell death after 24 h recovery time was only observed for the pyridyl complexes. The ability to functionalise 2-PhPy complexes via Schiff base formation was examined using the aldehyde-containing complex [(η5-Cp*)Ir(2-phenyl-5- pyridinecarboxaldehyde)Cl] 6, and new conjugates were synthesised by conjugation to primary amines. Reactions with lysine-containing peptides were analysed by ultrahigh resolution mass spectrometry (UHR-MS) techniques which showed formation of new iridium-peptide conjugates via Schiff base formation with the free amino group of lysine. The imine bond in a complex bearing the fluorescent dansyl moiety (for fluorescence microscopy) was reduced using the hydride source Et3SiH, which appears to involve the metal centre through proposed formation of an Ir-H species. Eighteen [(η5-Cpx)Ir(ImPy)Cl]PF6 complexes with various ImPy ligands were synthesised and characterised, and six X-ray crystal structures were reported. They were found to exhibit more complex aqueous chemistry than the 2-PhPy complexes, but exhibit minimal hydrolysis at biologically-relevant concentrations of chloride, remaining predominantly as the chlorido species. The hydrophobicity of the complexes increased upon extension of the Cpx capping ligand: CpxBiPh > CpxPh > Cp*. The complexes show minimal activity against Gram-negative E. coli but CpxBiPh complexes show good activity (MIC = 8 – 15 μM) against Gram-positive S. aureus bacteria. The antibacterial activity is generally dependent on the hydrophobicity and extension of the Cpx capping ligand. Disruption of the bacterial cell membrane appears to be involved in the mechanism of action. The antiproliferative activity against A2780 cells follows the trend CpxBiPh > CpxPh > Cp*, where the complex [(η5-CpBiPh)Ir(ImPy-NMe2)Cl]PF6 31 exhibits an IC50 value against A2780 cells of 640 nM.

Normal human keratinocytes possess a finite replicative lifespan whereas most advanced squamous cells carcinomas are immortal. The mechanisms, whose abrogation's can be considered necessary to achieve immortality, include those involving the negative cell cycle regulators p53, p16INK4A, and the telomere repair reverse-transcriptase enzyme, telomerase. Other specific chromosomes have also been demonstrated to carry functions whose loss is necessary for the development of immortality, through immortal phenotype reversion upon reintroduction into an immortal cell line. We demonstrate here the phenotypic reversion of immortal HNSCC-derived keratinocyte cell lines to a mortal growth-arrest upon reintroduction of a resistance marker tagged wild-type human chromosome 4. We further demonstrate that this phenotypic reversion occurs only in cell lines, which display LOH on 4q (BICR6 and BICR31), and not in those with intact endogenous chromosome 4 copies (BICR3 and BICR19), and that it is chromosome 4-specific, chromosomes 6, 11, and 15 having no effect on proliferative lifespan following introduction into BICR6 by MMCT. Through XMMCT-based truncated chromosomal fragment generation the functional complementation was localised to 4cen-q23, whilst fine mapping in segregants arising from the MMCT experiments identified an approximate 1.5Mb locus containing a minimal number of candidate genes. Through biological assay we have further determined the growth-arrest to have characteristics of crisis. This was determined through low BrdU incorporation balanced with high levels of apoptosis to statistically significant levels (less than 0.05). We found no evidence for involvement of telomeric attrition in the observed phenotype, through insufficient phenotypic lag (3-10 MPD) and growth-arrest in the presence of ectopic hTERT expression, suggesting the operation of an alternative mechanism. This suggests the presence of gene(s) at 4cen-q23 whose loss is advantageous to the development of immortality in advanced tumours including HNSCC.

Using existing software and six novel scripts, we developed a pipeline for variant calling using exome re-sequencing data of germline blood deoxyribonucleic acid (DNA) samples. We observed >99% (6,723/6,739) concordance between calls from our pipeline and previous genotyping. We identified >93% of single nucleotide variants (SNVs) and >94% of insertion/deletions called by a commercial partner using the same sequencing reads. Using a subset of genes, we showed that around half of predicted pathogenic variants could be validated in vitro. Together, these data showed that our pipeline was reliable for variant calling. Next generation sequencing (NGS) of DNA from formalin-fixed, paraffin-embedded (FFPE) tumours is technically challenging. We sought to determine the sensitivity of NGS to detect known somatic hotspot mutations (n=25) in 19 FFPE advanced colorectal cancers and to optimise it for the identification of novel somatic variants. Analysis using Illumina’s MiSeq software identified 100% of somatic mutations with 93% specificity, whereas the Genome Analysis Tool Kit’s (GATK) HaplotypeCaller identified 80-92% of somatic mutations with 100% specificity. We investigated the background mutator profile and found that normal FFPE DNA had 3-fold more SNVs than matched blood DNA, with an excess of FFPE-associated C:G>T:A substitutions (27.1 vs. 5.3%). Uracil DNA glycosylase treatment reduced this excess. Only ~5% of variants were consistently called in replicate runs and were likely to be genuine somatic variants. We detected potential candidates for genetic biomarkers of cetuximab-resistance in colorectal cancers that were previously shown to be wild type for KRAS, NRAS, BRAF and PIK3CA. We found that 7/21 (33%) of the CRCs analysed harboured mutations at either codon 12 or 61, whileother CRCs carried truncating KRAS mutations. NRAS also carried a codon 12 mutation in 1 CRC, and PIK3CA possessed mutations at codons 542 and 545. PTEN was found to harbour 5 truncating mutations at codons 71, 140, 155, 178 and 189, potentially leading to loss of function.

This study explores the day-to-day application of digital radiography (DR) within the X-ray environment. This study presents the voices of the radiographers' untold views, attitudes and experiences of DR through the process of observing, listening, retelling and interpreting junior and senior radiographers' responses. There were three stages to this ethnographic study. Firstly, exploring 'what radiographers did' environment by observing clinical practices. This provided 'first-hand' experience of action-in-process. Secondly, 22 semi-structured interviews were undertaken, directed by emerging themes and informal discussions from the clinical observations. Semi-structured interviews provided an understanding of the experiences, behaviours and attitudes of radiographers providing a deeper understanding of the relationship between practice and context. Thirdly, X-ray experiments were undertaken contributing to 'what had been seen and said by participants'. This data was later triangulated to support the research objectives outlined in this PhD research. Observation and interview data were analysed using thematic analysis and grouped into four overarching categories; learning, radiographer challenges, ionising radiation and patient care delivery. X-ray experimental data was inputted into SPSS and later coded. The qualitative data had numerous codes, which generated themes and could be linked in order to generate theoretical descriptions. Multiple-linear regression analysis and Pearson's Correlation provide statistically significant values (p < 0.001) for the experimental models contributing to 'what had been seen and said' by radiographers in the clinical environment. This thesis provides new insights into general radiographic practices using advancing technology. The conclusions that can be drawn from the empirical data is that advancing technology has impacted the day-to-day practices of diagnostic radiographers. Complex phenomena include; current knowledge and understanding, the practice of keeping doses 'as low as reasonably practicable' and impact on patient care delivery. These insights suggest that healthcare and academic environments may require additional support in the aim of delivering optimum patient care.

Introduction: Glioblastoma (GBM) is characterised by local recurrence following surgery, radiotherapy and chemotherapy. GBM has a poor prognosis and novel approaches are required. Recently, a hierarchical organisation of tumour cells in GBM has been proposed. This hypothesis suggests only a subset of cancer cells, termed ‘cancer stem-like cells’ (CSCs) drive tumour growth and possess properties of self renewal and unlimited proliferative capacity. CSCs have been described as radioresistant, implicating CSCs as a determinant of tumour recurrence following therapy. Therefore improved patient outcomes could potentially be achieved by targeting GBM CSCs. Nevertheless, reports of GBM CSC radioresistance have been conflicting, with some authors demonstrating CSC radiosensitivity. Furthermore, investigations of GBM CSC radioresponse have lacked robust radiobiological quantification and this aspect of the CSC phenotype remains controversial. Aims: To investigate the radioresponse of GBM CSCs in comparison to non CSCs, characterise the DNA damage response (DDR) in GBM CSCs to radiation and investigate effects of inhibition of DNA damage response (DDR) in GBM CSCs. Methods: Primary GBM cells were cultured in CSC enriching conditions and differentiating (‘tumour bulk’) conditions. The radioresponse of CSC and tumour bulk cultures derived from single parental tumours were thus compared by clonogenic survival assay. DDR was analysed in CSC and tumour bulk cells via Western blotting for DDR phosphoproteins and flow cytometric quantification of mitotic cells. DNA double strand break (DSB) repair was quantified by analysis of gamma H2AX foci. CSCs and tumour bulk response to irradiation in combination with inhibition of key DDR elements (ataxia telangiectasia mutated, (ATM); ataxia telangiectasia and Rad3 related, (ATR); and poly (ADP-ribose) polymerase, (PARP) by small molecule inhibitor agents was characterised. Results: CSC cultures were tumourigenic or recapitulated pathological features of parental tumours in orthotopic mouse models, whereas differentiated tumour bulk cultures did not. CSC cultures exhibited upregulation of putative CSC markers relative to tumour bulk. CSC cultures were radioresistant, demonstrated upregulated DDR and more efficient activation of the G2/M checkpoint compared to tumour bulk. CSC cultures repaired DNA DSBs more efficiently at 24 hours following irradiation. Inhibition of ATM in CSCs led to abrogation of the G2/M checkpoint response, reduced efficiency of DNA DSB repair and potent radiosensitisation. Inhibition of PARP in CSCs produced an increase in unresolved DNA DSBs in GBM CSCs at 24 hours post irradiation in G2 phase cells and modest levels of radiosensitisation. Inhibition of ATR in CSCs abrogated the G2/M checkpoint in CSCs efficiently and was associated with modest radiosensitisation. Dual ATR and PARP inhibition provided highly potent radiosensitisation of GBM CSCs. Conclusions: GBM CSCs were shown to be radioresistant relative to tumour bulk cells due to upregulated DDR, in support of the hypothesis that CSCs contribute to local recurrence, implying a need for CSC targeted therapies. The inhibition of G2/M checkpoint activation and DNA DSB repair via ATM inhibition or combined ATR/PARP inhibition potently radiosensitised GBM CSCs suggesting targeting both checkpoint and DNA DSB repair is important for optimal radiosensitisation of GBM CSCs. This study has demonstrated that DDR is a potential therapeutic target for radiosensitisation of GBM CSCs.

Lung cancer is the commonest cancer in Scotland and survival rates for patients in Scotland appear lower than in many other European countries. Although this variation in survival is usually interpreted as evidence of variation in facilities, access to care and clinical practice it is possible that the increased comorbidity and poor performance status of the Scottish population may contribute to the observed disparities in treatment and outcomes, although this has never been proven. The overall aim of the Thesis was to examine the impact of comorbidity in lung cancer, to attempt to quantify the extent and severity of comorbidity and to explore its relationship with treatment and survival. Between 2005 and 2008 all newly diagnosed lung cancer patients coming through the Multi-Disciplinary Teams (MDTs) in four Scottish Centres were included in the study. Patient demographics, World Health Organization/Eastern Cooperative Oncology Group performance status (PS), clinic-pathological features, stage, comorbidity, markers of systemic inflammation and proposed primary treatment modality were all recorded. Information on date of death was obtained via survival analysis undertaken by the Information Service Division (ISD) of NHS Scotland. Death records were complete until 1 June 2011, which served as the censor date for those alive. Chapter 4 examines the variations in demographics and baseline characteristics seen between the centres and reveals significant differences between the centres such as deprivation, stage at presentation, PS and treatments offered. Chapter 5 explores the relationship between comorbidity and the patient cohort. It shows that comorbidity can be quantified using a scoring index (the Scottish Comorbidity Scoring System (SCSS)) and that increasing comorbidity is associated with treatment centre and socio-economic status, with the most deprived patients having increased levels of co-morbidity. It also demonstrates that comorbidity appears to have an impact on treatment offered. Chapter 6 examines the relationship between systemic inflammation (utilizing the well established modified Glasgow Prognostic Score (mGPS)) and outcome in the patient cohort. It confirms previous work supporting the use of the mGPS in predicting lung cancer survival and also shows how it might be used to provide more objective risk stratification in patients diagnosed with lung cancer. Chapter 7 explores the relationship between a novel comorbidity scoring system (SCSS) and the already established Charlson Comorbidity Index (CCI) and the modified Glasgow Prognostic Score (mGPS). This study aimed to determine which of these factors provided the most accurate information on survival. The novel comorbidity scoring system, the SCSS compares very favourably with the more established CCI. In addition this study demonstrates clear differences between patients having potentially radically treatable disease (NSCLC stage I – IIIa) and disease which would generally be considered incurable (NSCLC IIIb/IV and SCLC). Chapter 8 examines the reasons for the clinician decision-making process and if these reasons do indeed mirror the individual patient’s demographics, fitness and stage. In the majority of patients, both in the early and advanced stage at presentation, the treatment decision appears to be appropriate given the recorded fitness, PS and comorbidity. However in a small but significant number of patients there did appear to be discrepancies between the clinician’s reasons for sub-optimal therapy and the recorded objective assessment of the patient in question. The work presented in this thesis has demonstrated the significant extent of comorbidity in lung cancer and the important role it appears to play (along with systemic inflammation) in determining treatment choice and survival.

Dynamic alterations in the actin cytoskeleton, under the regulation of the Rho/ROCK pathway, permit cell motility, cell-to-cell and cell-to-matrix adhesion, and have also been shown to participate in apoptosis and cell proliferation. These facets of cellular behaviour all have the capacity to become dysregulated in cancer; components of the Rho/ROCK pathway are known to play varying roles in these processes, both within primary tumours and within the tumour microenvironment. The LIM kinases are phosphorylated and activated by ROCK, leading to inactivation of cofilin and subsequent stabilisation of actin filaments. In addition, LIM kinase 2 serves as a p53 target and is upregulated in response to DNA damage. In some solid tumours (e.g. breast and prostate), LIM kinase levels are elevated. However, we found that LIM kinase 2 expression is downregulated in colon cancer, with a progressive reduction noted with advancing tumour stage. I found that LIMK2 expression in colon cancer is under epigenetic regulation, with hypermethylation of the promoters leading to transcriptional silencing; this implicates LIMK2 as a tumour suppressor gene in this context. This has potential translational implications as loss of LIMK2 could be utilised as a biomarker to stratify patients in the future. Elevated mechanical tension within the tumour microenvironment is known to be an adverse prognostic indicator due to its association with desmoplasia. ROCK activation has previously been shown to increase epidermal tissue stiffness and thickness, but little was known about the mechanisms by which this occurs. I found that ROCK activation leads to the deposition of extracellular matrix components, with a presumed consequent further increase in stromal stiffness. This indicates that a positive feedback cycle is established in the tumour microenvironment, maintaining a fibrotic stromal reaction that permits tumour progression. These results highlight the disparate roles that the actin cytoskeleton and constituents of the Rho/ROCK pathway play in tumour initiation and propagation, indicating the need for further research.

Scientific background. The inherent resistance of breast cancer stem cells (CSCs) to existing therapies has largely hampered effective treatments for advanced breast cancer. My research aimed at establishing novel immunotherapy approaches efficiently targeting CSCs by harnessing human γδ T cells as non-MHC-restricted killer cells and simultaneously as APCs to induce tumour-specific CD8+ T cell responses. Approach. An experimental model allowing reliable distinction of CSCs and non-CSCs was set up to study their interaction with γδ T cells and CD8+ T cells. FluM1 and CMVpp65 viral epitopes were used as surrogates for yet-to-be-discovered CSC-associated antigens. Results. Stable sublines with characteristics of CSCs and non-CSCs were generated from ras-transformed human mammary epithelial (HMLER) cells as confirmed by their (i) distinct expression profiles of CD24, CD44 and GD2, (ii) mesenchymal- and epitheliallike characteristics, (iii) differential growth patterns in mammosphere culture and (iv) distinct tumourigenicity, self-renewal and differentiation in NSG mice. The resistance of both CSCs and non-CSCs to γδ T cells could be overcome by inhibition of FPPS through pretreatment with zoledronate or FPPS-targeting shRNA, resulting in increased cytotoxicity and APC function of γδ T cells. CSCs presenting FluM1 or CMVpp65 exhibited stronger resistance to antigen-specific CD8+ T cells as compared to their non-CSC counterparts. Of note, pretreatment of Flu M1- or CMVpp65-presenting CSCs with γδ T cell conditioned supernatant significantly increased surface expression of MHC class I and ICAM-1 by both CSCs and non-CSCs as well as their susceptibility to CD8+ T cellmediated killing. Moreover, using the humanised anti-GD2 monoclonal antibody,Hu14.18K322A, a specific direction of γδ T cell responses against CSCs could be achieved. In addition to their direct cytotoxicity and ability to modulate the susceptibility of CSCs and non-CSCs to CD8+ T cell-mediated killing, γδ T cells concomitantly functioned as APCs to initiate de novo tumour-specific cytotoxic CD8+ T cell responses. Conclusions. My findings identify a powerful synergism between MHC-restricted and non-MHC-restricted T cells in the eradication of both CSCs and non-CSCs, thus establishing a powerful positive feedback loop for the eradication of residual cancer cells survived from killing by γδ T cells. My research suggests that novel immunotherapies may benefit from a two-pronged approach combining γδ T cell and CD8+ T cell targeting strategies that triggers effective innate-like and tumour-specific adaptive responses.