Quantitative mapping of boron drug uptake in a preclinical cancer model to inform boron neutron capture therapy (BNCT)

The work presented in this thesis was undertaken to address critical challenges in accurately visualising and quantifying the distribution of boron-containing drugs for boron neutron capture therapy (BNCT). Current imaging techniques and their associated limitations were reviewed, and research gaps were identified to provide a rationale for the methodological approaches adopted in this study. The factors influencing the uptake of boronophenylalanine (BPA) in cancer cells were systematically investigated. Variables relating to cell lines, including morphology, doubling time, cell status, and LAT1 expression, were assessed alongside parameters associated with BPA concentration, incubation time, and conjugates. The impact of the cellular microenvironment, specifically hypoxia and amino acid deprivation, was also explored. NanoSIMS was applied to investigate the intracellular and intratumoural distribution of BPA in CRC models. Subcellular mapping in monolayer HT29 and HCT116 cells revealed differences in 10B localisation, with HCT116 showing higher nuclear accumulation. To extend this to more complex systems, NanoSIMS was used on CRC spheroid sections, with consecutive sections immunostained for Ki67 and HIF1α to define proliferative and hypoxic zones. Although no significant differences were detected between spheroid edge and core, BPA-treated spheroids displayed significantly higher boron levels compared to controls. Validation was achieved using bulk inductively coupled plasma-mass spectrometry (ICP-MS), confirming higher uptake in HCT116 than HT29 cells, and confocal microscopy with DAHMI, supporting the subcellular distribution observed. These findings demonstrate the feasibility of NanoSIMS for boron drug imaging in CRC, while also highlighting its limitations for larger samples due to restricted FOV and long acquisition times. Optimisation of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was undertaken for the spatial mapping of boron within three-dimensional CRC spheroids. Protocols for embedding, cryosection thickness, and freeze-drying were refined to ensure morphological preservation and consistent sectioning. Bulk ICP-MS was employed to validate LA-ICP-MS data, confirming reproducibility across experimental conditions. A correlative imaging platform was subsequently developed by combining LA-ICP-MS with immunohistochemistry on consecutive spheroid sections. Quantitative boron mapping was performed in parallel with immunostaining for proteins of interest (LAT1, GLUT1, Ki67, HIF1α, and cleaved caspase-3), enabling investigation of spatial relationships between boron uptake and cellular phenotype. Live-cell flow cytometry using DAHMI was further employed to validate boron localisation and correlations with protein expression. This multi-modal approach provided novel insights into the intra-spheroid microdistribution of BPA and its association with biological markers relevant to BNCT efficacy. The findings of this thesis contribute to the advancement of preclinical imaging approaches for BNCT drug evaluation. By establishing optimised workflows for both subcellular and spheroid-level imaging, the work lays a foundation for the future development and assessment of novel boron-based therapeutics.