Gregor Reid

Neuroblastoma (NB), a complex pediatric cancer primarily affecting the adrenal medulla and paraspinal sympathetic ganglia, presents significant clinical challenges due to its occurrence in young children and low incidence rate. These factors complicate clinical trial recruitment, emphasizing the need for accurate pre-clinical models. My thesis aimed to enhance pre-clinical mouse models of NB to expedite the identification and assessment of new treatments for this disease.The first focus of my work was to refine the widely-used TH-MYCN transgenic mouse model. I have enabled early detection of NB progression through luciferase expression in developing tumours by employing a triple-transgenic Cre-lox approach. This modification facilitates non-invasive monitoring of tumour growth and is further leveraged to demonstrate the effectiveness of NB cell transplant experiments for establishing tumours at various organ sites. This new model overcomes previous limitations of TH-MYCN mice, including the limited metastatic spread and lack of GD2 expression on tumour-derived cell lines.Building on this approach, I next established a panel of human NB cell lines engineered for bioluminescence-based detection in xenografts. This was achieved through lentiviral transduction, allowing for the stable co-expression of luciferase and green fluorescent protein in eleven human NB cell lines. These cell line xenografts were characterized for tumour localization, growth rates, and mouse survival. This work revealed significant consistency and diversity among the different cell lines that could inform the design of future pre-clinical studies.To expand on the success of the luciferase-tagged mouse and human NB models, I lastly explored dual-colour in vivo imaging techniques. Challenges with spectral overlap were addressed through innovative approaches, combining fluorescence and bioluminescent imaging to enhance signal separation and sensitivity. This approach could contribute to immune therapy development by enabling simultaneous detection of effector and target cells.My thesis establishes novel, complementary mouse NB models that harness luciferase expression to provide real-time, non-invasive tracking of tumour growth and treatment response. These developments hold significant potential to deepen our understanding of NB biology, immune system interactions, and therapeutic susceptibility, potentially guiding future research and development of treatment approaches for NB.

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Remarkable clinical successes have been achieved with targeted immunotherapies directed at a surface antigen of leukemia blasts in patients with relapse or chemotherapy-refractory B-ALL. This single antigen-targeted approach, however, is highly prone to tumor immune escape. Development of resistance to therapy, commonly caused by the emergence of target-negative escape variants, remains a major drawback of CD19-directed therapies for B-ALL. The efficacy of strategies that direct T cell-mediated cytotoxicity towards leukemic cells bearing non-immunogenic antigens is limited, with a lack of evidence that these interventions establish immunological memory. In this study, I use the Eμ-ret mouse model to better understand the limitations of current single antigen-targeted immunotherapies and to identify immune responses required for the achievement and, more importantly, maintenance of remission in childhood B-ALL. My results uncovered the ability of target-directed therapy to elicit epitope spreading, enabling the generation of a secondary immune response against additional non-targeted leukemia-associated antigens that contributes to sustaining durable remission. Importantly, these results also suggest that such diversification of protective immune response is limited in an immunological setting where immune tolerance towards leukemia-associated antigens is established early in the course of leukemia progression. Furthermore, I have shown the ability of TLR agonist-mediated immune modulation to target leukemia cells in bone marrow, and induce durable immune control of primary B-ALL cells. Finally, I have demonstrated that NKT cells as a population is capable of influencing disease progression in the Eμ-ret mouse by playing a role in immunoediting.Overall, these findings support that the generation of immune response with a broad specificity for range of leukemia-associated antigens contributes to the maintenance of remission. Furthermore, my results suggest that overcoming immune tolerance established against leukemia-associated antigens may be critical for maximizing the therapeutic benefits of immunotherapies for childhood B-ALL. Collectively, the therapeutic impact of innate immune modulation presented here in the context of B-ALL may contribute to the eradication of MRD, and thus reduce the risk of relaspse in MRD-positive patients.

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The immune system has been proposed to have an impact on the etiology of B-cell ALL; however, a mechanistic explanation of this influence remains elusive. Epidemiological studies have uncovered a paradox, such that surrogates of infection exposure have consistently been associated with reduced ALL risk, while documented infections have a more variable influence, with both positive and negative risk associations being uncovered. Despite these contradictory findings, timing of infection exposure has been identified as a critical variable. A mechanistic explanation for the variable influences of infection or the importance of timing remain poorly defined. In this study, I use the Eµ-RET and E2A-PBX1 mouse models to assess how the basal and stimulated immune system is capable of influencing disease progression. My results indicate that in the absence of infection, resting IFN-γ is capable of influencing disease progression in the Eμ-RET mouse. By modulating SOCS-1 expression levels, IFN-γ restricted the IL-7-driven proliferation of leukemia-initiating cells (LICs) and caused a significant delay in disease progression. Furthermore, TLR ligand-mediated immune modulation inhibited disease progression by inducing the depletion of LICs through a mechanism that involved the direct activity of type-1 and type-2 interferon. Importantly, the relevance of this mechanism in humans was validated through the use of both human immune-effector and leukemia cells. Finally, I demonstrate that quantitative and qualitative differences in neonatal immune responses, in particular the increased capacity for IL-17A production by γδ T-cells, confer significant infection-induced protection from ALL progression in neonatal mice.Mechanistically these findings represent several firsts in the investigation of how the immune system influences B-cell ALL progression. They identify both type-1 and type-2 IFN and IL-17A as important inhibitory factors, and demonstrate the significant impact of immune modulation during the pre-leukemic phase on subsequent disease risk and progression. Furthermore, the results presented here provide a mechanistic explanation for the importance of timing in the association between infection exposure and ALL risk. Collectively, my results indicate that in addition to the educational influence suggested by the “delayed infection” hypothesis, early-life infection exposure may also have an active inhibitory impact on the progression of B-cell ALL.

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