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Title: Molecular targeting for clinical cancer imaging
Author: Morley, Nicholas Christopher Donald
ISNI:       0000 0004 5916 2354
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2015
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Modern cancer treatment makes extensive use of clinical imaging methods for diagnosis and response assessment. To this end, there is increasing desire to non-invasively measure various drugs and biomarkers inside a patient on a centimetre scale. Despite undeniable preclinical progress and evaluation of many techniques, few new imaging drugs are emerging into pragmatic clinical cancer imaging. There are many drug targeting strategies, including target-affinity and activation-by-target. Affinity selections can identify binders from combinatorial libraries of heteropolymers such as nucleic-acid sequences and peptides. Using this approach, in combination with next-generation DNA sequencing, I identified sequences as binders of putative cancer biomarkers. In addition, I investigated a target-activated fluorescent probe as a reporter of cancer-associated enzyme activation. Messenger RNA levels for Leucine-rich-repeat containing 15 (LRRC15) are reported to be elevated in human, breast-cancer samples. I analysed a new antibody to LRRC15, which locates this protein in genetically triggered murine breast tumours and in their lysates on Western blot. Antibody staining also showed a distinct pattern in sections of normal murine kidney, and protein expression in human breast-cancer samples. LRRC15 affinity selection of phage peptide and aptamer libraries was performed with immunopurified protein, and this identified consensus sequences. However, specific binding of the peptides or aptamers to the target was not demonstrable. Alpha folate receptor overexpression has been described in many human tumours, particularly ovarian cancer. Cell-lines to enable whole-cell selection of binders to the folate receptor were developed. Specific staining with a folate-fluorophore compound validated these. Selection of peptide and aptamer binders showed early emergence of spurious dominant sequences, triggering abandonment of this approach. The cell-lines were used to test a folate-quantum dot conjugate, with disappointing results. Matrix Metalloproteinase-9 (MMP-9) activity in cancer has previously been described and pursued as a therapeutic target. A novel probe to report activity of MMP-9 was tested using fragments of murine tissue, successfully differentiating normal murine fat pad from pieces of murine mammary tumour. Significant off-target activation was also observed, particularly with kidney. Recombinant proteins based on human MMP-2 and -13 also activated the probe. Expression and activity of equivalent enzymes in the murine tissues and tumours were assessed using RT-PCR, Western blot, immunohistochemistry and zymography, but the basis of spurious activation remains obscure. In conclusion, a new antibody identifies LRRC15 in both human and murine breast cancers, and in the murine kidney. Library affinity selections with LRRC15 and the alpha folate receptor developed consensus sequences, but were unsuccessful. An MMP-9 activated probe successfully differentiated breast tumour from normal tissue but also showed significant off-target activation. Non-invasive detection and measurement of cancer biomarkers remains an important topic, likely to see much progress in coming decades. Some of the practical difficulties in developing reagents to achieve this are discussed.
Supervisor: Brunton, Valerie Sponsor: Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: cancer ; aptamers ; imaging ; LRRC15