Saudi Cultural Missions Theses & Dissertations
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Item Restricted Exploring the role of STING type-I Interferon response as a therapeutic in myeloid leukaemia(University of Liverpool, 2024-10) Alsufyani, Abdullah Meshal A; Dahal, Lekh; Woolley, JohnMyeloid leukaemia is a type of blood cancer that originates from the development of abnormal myeloid cells in the bone marrow and classified into either acute or chronic myeloid leukaemia (AML/CML). Acute Myeloid Leukaemia (AML) poses a unique challenge in haematological oncology due to its aggressive nature and significant genetic diversity. Unlike Chronic Myeloid Leukaemia (CML), which is primarily driven by the BCR-ABL1 fusion gene and can be effectively managed with tyrosine kinase inhibitors (TKIs). Intensive chemotherapy is the front-line treatment for AML, and not all patients fit or can tolerate it owing to its toxicity. Targeted therapies customised to the molecular and cytogenetic profiles of each patient have been administered for AML treatment and improved the management of AML. Nevertheless, their efficacy is often limited, since some individuals may acquire resistance over time. Thus, AML patients have the worse overall survival (OS) compared to CML patients. Type-I IFN signalling through STING pathway appears to negatively impact the development of leukaemia, in line with its downstream functions, such as increase in rate of apoptosis, increase in AML immunogenicity, and decrease in cell proliferation. STING is found highly expressed in AML, making it an appealing therapeutic target. Its activation has the potential to overcome the immunosuppressive bone marrow microenvironment, where conventional immune responses are less effective, and could stimulate a robust immune response against AML. This highlights the promise of STING-based therapies in a disease where conventional treatments often fall short, offering a new strategy to address the challenges posed by AML's genetic complexity. The role of STING gene in the outcome of AML were studied through various approaches including bioinformatics, cellular and molecular biology and immunoassay techniques. Bioinformatics databases were used to analyse and evaluate STING expression in AML and how this affects the OS and patients’ prognosis. The efficacy of STING agonist (CDA) in human AML and CML cell lines was assessed by western blotting to study the functioning of STING signalling pathway in AML and CML, and RT-qPCR to evaluate the production of type-I IFN and its ability to induce of innate immune response in those cells. Flow cytometry was used to assess apoptosis and cell proliferation. Application of Immune assay in a co-culture system enabled us to study tumour microenvironment in vitro by assessing immune cells response against AML/CML upon STING activation in the co-culture through performing T-cell proliferation, NK cell lysis of tumour cells (ADCC assay), macrophage phagocytosis (ADCP assay) and Dendritic cells efferocytosis assay. Finally, Studying the synergetic effect of STING agonist in combination with hypomethylating agent such as 5’Azacytidine and how these combinations enhance tumour anti-immunity. Publicly available database was used for bioinformatic analysis and showed that STING gene is highly expressed and associated with myeloid lineage while MAVS gene which encodes Mitochondrial Antiviral Signalling Protein (MAVS) was associated with lymphoid lineage in HSCs. Significantly, elevated STING expression was positively correlated with increased overall survival in AML dataset (OHSU, TCGA). An In vitro study demonstrated that STING agonist was able to activate STING pathway in AML and CML cell lines within 1 hour, leading to IFN-β upregulation. I demonstrated that STING pathway activation inhibited proliferation and induced apoptosis. These physiological changes upon STING activation were accompanied by alterations in the AML and CML immunophenotype within 24 hours such as (HLA A,B,C and CD86). Furthermore, the In vitro study of the tumour microenvironment through employing immune cells and AML/CML cells in a co-culture system showed that treating the co-culture with STING agonist restores CD8 T-cell proliferation comparing to suppressed CD8 T-cells in untreated co-culture. STING agonist also enhanced macrophage and dendritic cells phagocytosis in the co-culture to the target cells when measured by flow cytometry and upregulated the activator FcγR and reduced the inhibitory one in macrophage as well as enhanced antigen presenting markers on DC. Significantly, transcriptomic analysis revealed that the high state of immune response induced by a hypomethylating agent treatment 5-Azacytidine (5'Aza), through "viral mimicry induction," enhanced the sensitivity of an AML cell line that was less responsive to STING activation when treated with the STING agonist alone, making the cell line more responsive to combination therapy. Taken together, these results pointed to the synergetic effect of STING agonist in combination with chemotherapy such as 5’Aza and these combinations enhanced tumour anti immunity.20 0Item Restricted Assessment of Immunotherapy-Related Adverse Events Among Patients with Cancer in Saudi Arabia: A Mixed Methods Study(Queensland university of techonology, 2024) Alquzi, Fatimah; Bradford, NatalieThis thesis contributes significantly to cancer care by focusing on patients undergoing immunotherapy in Saudi Arabia. it is the first study to explore the challenges these patients face, offering valuable insight into their experience. the research translated and validated an immunotherapy assessment tool and assessed its real world use by evaluating its acceptability, feasibility, and clinical utility. this study deepens the understanding of immune related adverse events and provide critical perspectives for clinicians, patients, and researcher. Ultimately, it contributes to improving the management of adverse events in cancer immunotherapy. Background: Cancer is a rapidly growing healthcare system challenge, with more than 19.3 million incidences recorded globally in 2020; this is projected to surpass 29.5 million by 2040. The emergence of immunotherapy has positively changed many advanced cancer outcomes and improved survival rates. Despite immunotherapy being an efficacious and reliable treatment option for cancer, its success is tempered by associated immune-related adverse events (irAEs). Limited understanding of proactive irAE assessment further complicates this issue. Patient Reported Outcome (PRO) measures are standardised tools used to systematically collect data from patients about their health status, symptoms, functioning, and well-being, and are increasingly advocated for use in routine clinical care. However, their use in clinical care, and more specifically to assess irAEs and symptoms, is an understudied area. Objectives: To investigate the feasibility, acceptability, and usefulness of systematic assessment of irAEs from the perspectives of patients with cancer living in Saudi Arabia. Methods: The doctorate project involved five inter-related studies, underpinned by the theory of Symptoms Experience in Time (SET), a conceptual framework aimed at understanding the temporal dynamics of symptom manifestation and progression. In Study 1, a systematic literature review was completed. A comprehensive search strategy was employed to identify relevant literature on symptom assessment and management strategies related to irAEs in cancer patients. The review identified a PRO tool developed specifically for irAEs to assess 20 symptom and six interference items. In Study 2, the PRO tool identified for irAEs assessment from the review underwent robust translation from English into Arabic using the Brislin translation method. This involved forward and backward translations, consensus committees with health experts and linguists, and iterative adjustments to ensure linguistic accuracy and cultural appropriateness. Study 3 was an observational study undertaken in Saudi Arabia to evaluate the psychometric properties of the tool among Saudi Arabian cancer patients undergoing immunotherapy. The assessment involved mixed methods, including cognitive interviewing to assess item clarity and relevance, content validity and test–retest reliability analysis, and a post-assessment feedback survey to assess acceptability. Study 4 was a prospective observational cohort study over four weeks that investigated the feasibility, acceptability, and usefulness of the translated tool to assess irAEs and symptom experiences of patients undergoing immunotherapy for cancer in Saudi Arabia. Finally, in Study 5, a subset of participants from Study 4 participated in in-depth semi-structured interviews, which were analysed using qualitative content analysis. Results: Study 1: The systematic literature review identified a suitable PRO tool for assessing diverse irAEs among cancer patients undergoing immunotherapy. The review also identified gaps in evidence, including the unclear frequency of symptom assessment and the role of non-pharmacological approaches for managing mild irAEs. In Study 2, the PRO tool was translated, creating the Arabic version of the tool: the Arabic Immunotherapy Symptom Assessment Inventory (AISAI). Study 3 evaluated the psychometric properties of the AISAI, which demonstrated content validity, reliability, linguistic precision, and cultural relevance. The Cronbach’s alpha coefficients for the internal consistency were calculated as 0.90 for the 20 symptom items and 0.88 for the interference scale, indicating satisfactory reliability. The test–retest reliability, assessed through intraclass correlation coefficients (ICC), showed excellent agreement between Time 1 and Time 2, with ICC values above 0.90. In Study 4, a real-world, 4-week evaluation involving a cohort of 69 patients undergoing cancer immunotherapy treatment was completed. The feasibility and acceptability of administering the AISAI were high, with 97.1% affirming its acceptability and applicability. The five most prevalent and severe symptoms, based on mean scores, were: numbness or tingling; pain; rash or skin change; interference with general activity; and impaired walking. Over the 4- week immunotherapy period, there was a notable increase in the severity of symptoms, with statistically significant changes observed. The number of participants with moderate (score 4–6) and severe (score 6–10) symptoms increased, indicating a worsening pattern over time. In Study 5, a qualitative evaluation affirmed that the AISAI was perceived as a valuable tool for early recognition and assessment of irAEs, with a preference for routine usage. The evaluation also highlighted knowledge gaps, emphasising the need for educational interventions to enhance comprehension and management capabilities among patients. Conclusion: This thesis represents a novel contribution to the field, particularly in the context of cancer patients undergoing immunotherapy treatment in Saudi Arabia. It introduces an innovative approach by incorporating PROs in Saudi Arabia. This marks a significant advancement in the assessment practices related to irAEs. Notably, it is the first study to explore the experience of irAEs in Saudi Arabia using PRO, providing a comprehensive understanding of the challenges faced by these patients. Moreover, this research pioneers the investigation of the use of the AISAI in real-world settings for cancer patients undergoing immunotherapy. It assesses the tool’s acceptability, feasibility, and usefulness, shedding light on its practical implications in the clinical setting. The research contributes a valid and comprehensive tool, the AISAI, designed specifically for assessing irAEs in Arabic cancer patients. This research makes a valuable contribution to clinicians, patients, and researchers, enhancing the overall understanding and approach to assessment of adverse events in the context of cancer immunotherapy.22 0Item Restricted A portrait of gamma delta T cell exhaustion: Chronic stimulation models, RNA transcriptomics and CRISPR screens.(University College London, 2024) Alturki, Meshael; Anderson, JohnT cell exhaustion has been identified as a major challenge for T cell therapeutics. In conventional T cells (ab T cells), the study of exhaustion has led to insights into its aetiology, and potential therapeutic approaches to overcome it. Our research focuses on a novel area of study-T cell exhaustion in human γδ T cells, particularly the Vγ9Vδ2 subtype, which is the most prevalent in peripheral blood. These cells hold therapeutic promise due to their broad cytotoxic characteristics and ability to bridge innate and adaptive immunity. However, the understanding of how exhaustion can affect the behaviour and functionality of these cells is yet to be established. Our study involved in vitro chronic stimulation protocols using combinations of zoledronic acid (ZOL) and OKT3 (anti-CD3 monoclonal antibody) or Daudi target cells, both stimulating via the Vγ9Vδ2 T cell receptor. The findings from our ZOL model, which showed a notable decrease in absolute T-cell numbers and significant upregulation of genes associated with T cell exhaustion, could have significant implications for understanding and potentially overcoming T cell exhaustion in Vγ9Vδ2 T cells. Bulk-RNA sequencing of Vγ9Vδ2 T cells identified distinct signatures for the multiple stimulated samples, and a list of highly differentiated genes was selected for further investigation by a pooled CRISPR screen. We optimised and tested a suitable protocol for a CRISPR screen in Vδ2 T cells. Two gRNA hits were enriched in the ZOL and multiple stimulated samples (targets for SLC37A3 and GORASP2 genes). In conclusion, a simple in vitro assay was developed to study hypofunctionaity in Vδ2 T cells, allowing studies to evaluate RNA profiles and regulatory mechanisms of exhaustion induction in this cell type. Finally, selected genes were screened in a CRISPR-based pooled screen and hit gRNAs from stimulated Vδ2 T cells were identified.27 0Item Restricted TIM3 is a context-dependent coregulator of cytotoxic T cell function(Saudi Digital Library, 2023-12-05) Alamir, Hanin; Wuelfing, ChristophCytotoxic T lymphocytes (CTLs) are essential effectors in the antiviral and antitumour immune response and attractive targets in cancer immunotherapy. Although CTLs can directly recognise and kill tumour cells, CTLs become suppressed in the tumour microenvironment. This project investigated the inhibitory receptor T cell immunoglobulin and mucin domain 3 (TIM3). TIM3 is expressed on T cells after chronic antigen exposure and marks the most exhausted tumour infiltrating CTLs in multiple solid tumours. However, it is unclear whether TIM3 directly regulates CTL function. In addition, despite its predominantly inhibitory role in vivo, TIM3 can promote cellular activation in T and non-T cells, and the roles of putative ligands in TIM3 function are disputed. Therefore, we aimed to determine the effect of TIM3 on direct CTL antitumour function and how the TIM3 ligands Galectin9 (GAL9) and CEACAM1 regulate its function. We employed three-dimensional (3D) tumour spheroids that effectively induce CTL suppression similar to the in vivo tumour microenvironment in comparison to conventional two-dimensional (2D) tumour cell culture. In the 3D spheroid model, TIM3 significantly inhibited CTL cytotoxicity and cytoskeletal polarisation as a key mechanism of effective cytolysis in murine and human CTLs. In contrast, in the 2D tumour model, TIM3 stimulated CTL cytotoxicity, cytoskeletal polarisation, and secretion of the immune-stimulatory cytokine interferon γ (IFNγ). Expression of GAL9 and CEACAM1 in trans on tumour cells further suppressed the CTL killing ability in the 3D spheroid model and enhanced costimulatory function in 2D. CEACAM1 in cis neutralised TIM3 functions in both 3D and 2D. We suggest that TIM3 functions as a context-dependent coregulatory receptor, as supported by the engagement of its ligands GAL9 and CEACAM1. In a largely stimulatory signalling context of a CTL, TIM3 functions as a costimulator, and in a more inhibitory context, TIM3 functions as a coinhibitor.8 0Item Restricted Remodeling the Lung Tumor Microenvironment with Locally Administered Nano-Immunochemotherapies for Osteosarcoma Lung Metastases(Saudi Digital Library, 2021-12) Sunbul, Fatemah; Da Rocha, SandroOsteosarcoma (OS) is the most common type of primary bone cancer in all ages1 and metastasizes exclusively to the lungs2. The majority of patients present with micrometastases at the time OS is first diagnosed. When lung metastasis (OSLM) develops, and gross lesions are detected, the curability of disease and the overall survival rate diminish drastically (<20%)2,3. OSLM is thus the leading cause of death in those patients1,4. The current standard of care with chemotherapy has failed to improve patients' therapeutic outcomes with OSLM2,3. The interplay between tumor cells and immune infiltrates in the tumor microenvironment (TME) is a critical determinant of metastasis growth. Systemically administered gemcitabine (GMT) chemotherapy is currently being used in the clinic to treat patients with OSLM, alone or in combination with other chemotherapies, but the response rates are very low. Early studies leading to current trials with GMT administered via pulmonary route have shown promise in decreasing tumor burden – but unfortunately not eradicating the tumor. New therapeutic strategies are needed to tackle this unmet need in clinical oncology. We propose the combination of orally inhaled GMT chemotherapy with colony-stimulating factor-1 receptor inhibitors (CSF-1Ris), macrophage immunotherapy, for the treatment of OSLM also via the pulmonary route. The benefits of CSF-1Ris have been recently shown in manipulating TME away from tolerance (but not for OSLM yet) and is now in the clinic 5. Immunotherapies for tumorassociated macrophages (TAMs) are particularly important in OSLM as immune checkpoint inhibitors (ICIs) have yielded very low response rates in such "cold tumors". Moreover, TAMs are the most abundant infiltrates in OSLM tumors. Our particular interest is PLX-3397 (PLX, or Pexidertinib), which has been recently approved by the FDA. As with systemic chemotherapy, potential limitations of CSF-1Ris are the poor biodistribution to the lungs upon systemic administration and the high off-target toxicity (mainly hepatotoxicity)6. Thus, there is an opportunity to develop novel combination immunochemotherapy and local lung delivery strategies to improve the therapeutic potential and decrease off-target toxicity of currently available therapeutics. In this work, we develop a reproducible, robust in vivo model of OSLM that enables screening of various therapeutics via local administration to the lungs and their influence on tumor growth in vivo and ex vivo. We also investigated the effect of gender as a variable in this model, given that tumor growth and treatment outcomes have been shown to be impacted by gender in the clinic. We evaluated the tolerability and efficacy of the local lung delivery of PLX TAM immunotherapy alone and in combination with GMT chemotherapy, and their effect in remodeling the TME to prevent the development of micrometastases to large lesions. We studied the impact of those therapies at the TME level using a combination of flow cytometry, immunofluorescence, and H&E staining, to assess levels of expression of upregulated Fas/FasL in both OS cells and infiltrating lymphocytes, and also the abundance of classically vs. alternatively activated TAMs with the CSF-1Ri treatment. We correlate those results with tumor burden and survival. As lung tissue retention and clearance limit this combination therapy's potential, we started our investigations of the effect of nanoformulation as a potential strategy to enhance drug efficacy by improving the lung pharmacokinetic profile of the treatments. We started with a translatable, lipid-based platform for the development of GMT nanoformulations that can be in the future delivered in nebulizer form; initial studies in the lung retention of GMT and optimization of the lipid formulation are presented. Our studies are clinically significant as they have the potential to introduce new translatable approaches to treat OSLM. They are scientifically significant and innovative, as for the first time, we are elucidating the tolerability and effect of TAM immunotherapy locally delivered to the lungs in combination with chemotherapy in the TME of an immunocompetent OSLM model.23 0Item Restricted Targeting oral epithelial dysplasia with antigen-specific immunity(Abdulaziz Alboushi, 2022-12-01) Alboushi, Abdulaziz; Savelyeva, NataliaThis project aimed to establish a novel approach that these two antigens may become potential targets in OED and/or biomarkers for malignant transformation. With the broad expression of MAGED4B and FJX1 antigens in head and neck cancer, these two antigens would be potential targets in OED. MAGED4B and FJX1 antigens are expressed in OED and HNSCC with immune response against these antigens. MAGED4B and FJX1 antigens expression is prospective to be high in progressed OED. Also, the more the grading the more the expression and in the same case the immune response.14 0