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Title: Multimodal imaging of CXCR4 expression in mouse tumour models
Author: Braga, Marta
ISNI:       0000 0004 7658 193X
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2019
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CXCR4, the C-X-C chemokine receptor 4, has been gaining interest as a therapeutic target since its overexpression has been identified in more than 20 solid tumours and correlated with tumour progression and therapy resistance. Several CXCR4 antagonist molecules are under investigation, including AMD3100, a small molecule CXCR4 inhibitor, which is already undergoing clinical trials for the treatment of several tumour types. With the emerging role of this receptor as a therapeutic agent, there is a demand for imaging agents capable of sensitive and fast detection of CXCR4. This work aimed at the development of a microbubble (MB) for molecular ultrasound (US) imaging of CXCR4; in order to do so, a second objective was the development of a CXCR4-specific positron emission tomography (PET) tracer for characterisation of the biological models prior to US imaging. Lastly, a US-dedicated contrast agent was radiolabelled to allow assessment of whole-body distribution with PET imaging. The radiolabelling of a small molecule analogue of AMD3465 with fluorine-18 is reported for the first time. [18F]MCFB uptake was sensitive to CXCR4, accumulating in the cells in a manner that reflected receptor expression. However, tracer uptake could only be partially blocked by treatment with AMD3465 (>44% decrease), suggesting that [18F]MCFB is not completely CXCR4-specific. About 25% of the total tracer uptake was found inside of the cells at 0oC, indicating that [18F]MCFB can cross the membrane by receptor-mediated endocytosis. In vivo, [18F]MCFB localised in the tumours, and differences in uptake reflected the CXCR4 expression in U2932 and SuDHL8, but not in MDA-MB-231 tumours; the necrotic nature of the latter compromised tracer delivery and was a confounding factor. [18F]MCFB was shown to be stable with favourable pharmacokinetics, but high liver and kidneys uptake was observed. Competitive antagonism with metformin resulted in a small decrease in liver (21%) and kidneys (9%) localisation in U2932-bearing mice, indicating that [18F]MCFB uptake in these organs may be partially mediated by polyspecific organic cation transporters (OCT). A MB contrast agent functionalised with the T140 peptide was subsequently developed for the imaging of CXCR4 in the vasculature using US. The contrast agent, T140-MB, was successfully developed and was specific and sensitive for CXCR4: T140-MB accumulation was lower (43%) in the shRNA-inducible MDA-MB-231 cell line following doxycycline (DOX) treatment, in line with decreased CXCR4 expression, and almost entirely blocked (95%) when co-incubated with T140. In vivo, T140-MB allowed visualisation of the vasculature in U2932 and SuDHL8. T140-MB showed a non-significantly higher residence time compared to non-targeted MB, NT-MB (27% higher for both U2932 and SuDHL8), together with slower wash-out kinetics (21% slower for both U2932 and SuDHL8), suggesting that there is some attachment of T140-MB in the tumour. Kinetic profile of T140-MB was comparable between U2932 and SuDHL8; this was determined to be due to the comparable expression of CXCR4 in the vasculature in these tumours, despite large differences (4-fold) in whole-cell receptor expression. US is an attractive technique due to its cost-effectiveness and safety; however, it does not allow whole-body imaging for assessment of biodistribution of a tracer, which is fundamental for tracer development. Thus, a 68Ga-radiolabelled MB was designed to profit from whole-body imaging capabilities of PET. Radiolabelling was attained by bioorthogonal 'click' inverse electron-demand Diels-Alder (IeDDA) reaction between the trans-Cyclooctene (TCO)-modified MB and a 68Ga-labelled tetrazine, [68Ga]DOTA-Tz- labelled using a sodium chloride (NaCl)-based cationic elution method. Despite challenges in method development due to the need for multiple steps with modest radiochemical yields (typical total RCY: ~4.5%), [68Ga]DOTA-MB was successfully produced that could be used for in vivo imaging. PET data shows that [68Ga]DOTA-MB accumulates mainly in the urine, followed by the spleen, lung and blood (411 ± 72, 65 ± 11 and 48 ± 9 %ID/g, respectively). In all, these data elucidate how the use of two different imaging modalities can provide complementary information due to the non-linearity of biomarker expression between different tumour compartments.
Supervisor: Aboagye, Eric ; Tang, Mengxing Sponsor: Cancer Research UK
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral